trpv1 activation and gastric vagal afferent …

The Pennsylvania State University

The Graduate School

College of Medicine

TRPV1 ACTIVATION AND GASTRIC VAGAL

AFFERENT DYSFUNCTION FOLLOWING

EXPERIMENTAL SPINAL CORD INJURY

A Dissertation in

Anatomy

by

Emily M. Besecker

©2015 Emily M. Besecker

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Doctor of Philosophy

May 2015

ii

The dissertation of Emily M. Besecker was reviewed and approved* by the following:

Gregory M. Holmes

Associate Professor of Neural and Behavioral Sciences

Dissertation Advisor

Chair of Committee

Kirsteen N. Browning

Associate Professor of Neural and Behavioral Sciences

Charles H. Lang

Distinguished Professor of Cellular and Molecular Physiology

Ann Ouyang

Professor of Medicine, Division of Gastroenterology and Hepatology

Patricia J. McLaughlin

Professor of Neural and Behavioral Sciences

Director of the Graduate Program in Anatomy

*Signatures are on file in the Graduate School.

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ABSTRACT

Spinal cord injury (SCI) currently affects over 1.7 million Americans and this

number continues to rise as approximately 12,000 new injuries occur each year. While

deficits seen in quadriplegic and paraplegic individuals clearly include impaired motor

control of the limbs, autonomic nervous system function is severely impaired, as well.

Clinical and experimental reports indicate that high thoracic or cervical spinal trauma

often leads to pathologically depressed gastrointestinal (GI) transit accompanied by

bacterial translocation. It is estimated that 10% of all mortalities in the SCI population are

attributed to GI dysfunction. The vagus nerve (cranial nerve X), is the source of

parasympathetic control to the stomach The sensory limb (afferent vagus) of gastric

reflexes is via bipolar neurons in the nodose ganglia which extend from the stomach to

the nucleus tractus solitarius (NTS). This sensory signal is then integrated within the

NTS second order neurons. The motor limb (efferent vagus) begins with neurons in the

dorsal motor nucleus of the vagus (DMV) in the caudal brainstem (medulla) which

synapse onto enteric neurons within the stomach to elicit gastric contraction or relaxation,

thus completing the vago-vagal circuit. While the vagus nerve remains anatomically

intact following a SCI, derangements in GI function suggest that the vago-vagal signaling

is disrupted. The pathophysiology of post-SCI gastric dysmotility remains to be fully

understood, however, previous data have suggested that diminished vagal signaling

within the afferent limb of the gastric vagal reflex may play a significant role in the

observed pathophysiological changes.

Using an acute rat model of T3-SCI combined with molecular in vivo and

pharmacological approaches this research tested the hypothesis that following spinal cord

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injury, gastric inflammation initiates gastric dysmotility through increased expression of

transient receptor potential vanilloid type 1 receptor (TRPV1).

Using a strain gage transducer, of our own construction, the first experiments

assessed the anatomical, neurophysiological, and functional integrity of gastric-projecting

DMV neurons (efferents) in T3-SCI rats. Immunohistochemical, whole cell in vitro

recordings, and in vivo functional assessment of gastric efferent integrity in both control

and T3-SCI rats revealed no significant differences between groups. These data suggest

that the acute, 3-days to 1-week post-SCI, dysfunction of vagally-mediated gastric

reflexes do not include derangements in the efferent DMV motoneurons. This

observation extends our previous conclusions that dysfunction of gastric vago-vagal

reflexes following T3-SCI may be due, in part, to compromised vagal sensory input

affecting the gain of vagally-mediated reflexes.

The next experiments were designed to identify a possible mechanism for the

initiation of vagal afferent dysfunction. High-thoracic or cervical SCI produces marked

deficits in sympathetically-mediated vascular control leading to chronic hypotension. The

systemic pooling of blood may limit blood flow through the superior mesenteric artery

(SMA) perfusing a majority of the GI tract. Vascular hypotension can result in visceral

hypoperfusion, leading to ischemia, the initiation of an inflammatory cascade, and end-

organ dysfunction. Following T3-SCI, rats displayed significantly reduced SMA blood

flow under all experimental conditions. The postprandial elevation of SMA blood flow

following duodenal infusion was delayed or diminished after injury. These data suggest

that arterial hypotension diminishes mesenteric blood flow and that the resulting GI

ischemia may be an underlying pathology leading to gastric dysfunction seen following

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SCI. Subsequent experiments employed molecular techniques to test the hypothesis that

T3-SCI triggers GI inflammation. As a basis for comparison, we further analyzed the

expression of inflammatory markers following mesenteric ischemia/reperfusion injury in

age-matched animals.

The final series of experiments tested the hypothesis that the reduction in gastric

blood flow and increase in gastric inflammation lead to an up-regulation in TRPV1

expression within vagal afferent neurons. Quantitative assessment of CCK, CCK-1r and

TRPV1 expression by qRT-PCR revealed significant elevation of TRPV1 and CCK-1r

within the nodose ganglion after acute T3-SCI. In conclusion, these data suggest that

derangements in gastric reflex function are related to dysfunction of vagal afferent

signaling. Targeted reduction of inflammatory mechanisms which occur post-injury may

offer therapeutic strategies to reduce such alterations, thereby improving the short- and

long-term GI function of individuals with SCI.

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TABLE OF CONTENTS

List of Figures xi

List of Tables xii

List of Abbreviations xiii

Acknowledgements xiv

Chapter 1: Introduction: Spinal Cord Injury, Blood Flow, Inflammation, 1

and TRPV1 Neurophysiology

1.1 Spinal Cord Injury Overview 2

1.1.1 Cascade of Events in Spinal Cord Injury 3

1.1.2 Classification of Human Spinal Cord Injury 6

1.1.3 Incidence 8

1.1.4 Traumatic vs. Non-traumatic Spinal Cord Injury 9

1.1.5 Current Therapies for Spinal Cord Injury 9

1.1.6 Animal Models 12

1.1.6.1 Injury Devices 13

1.2 Autonomic Nervous System Overview 14

1.2.1 Enteric Autonomic Nervous System 15

1.2.2 Sympathetic and Parasympathetic Nervous Systems 16

1.2.3 Overview of Autonomic Nervous System Dysfunction 17

1.2.3.1 Gastric Dysfunction 18

1.2.3.2 Cardiovascular Dysfunction 19

1.2.3.3 Gastrointestinal Hypoperfusion 20

1.3 The Emerging Role of Transient Receptor Potential Vanilloid Type 1 21

Receptor in Gastrointestinal Dysfunction

1.4 Summary 24

Reference List 26

Chapter 2: Fabrication and Implantation of Miniature Dual-element 33

Strain Gages for Measuring In Vivo Gastrointestinal Contractions in

Rodents

2.1 Abstract 34

2.2 Introduction 35

2.3 Protocol 37

2.3.1 Procedures for fabrication of strain gage 37

2.3.1.1 Preparation and Bonding of two single element strain gages 37

2.3.1.2 Sizing and wiring dual element strain gages 38

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2.3.1.3 Testing and epoxying dual element strain gages 39

2.3.1.4 Encapsulating dual element strain gages in silicone 40

2.3.1.5 Completion of wire connector and calibration 40

2.3.2 Surgical Procedures for Acute Implantation of Strain Gage 41

2.3.2.1 Animal Care and Preparation 41

2.3.2.2 Tracheal intubation for terminal experiments 41

2.3.2.3 Strain gage instrumentation to gastrointestinal surface 42

2.3.2.4 Gastric Motility recordings 44

2.3.2.5 Representative Measurement of Gastric Contractions Following 44

Brainstem Stimulation

2.3.2.6 Exposure of brainstem and fourth ventricle 44

2.3.2.7 Administering fourth ventricle thyrotropin releasing hormone or 45

intravenous sodium nitroprusside

2.4 Representative Results 45

2.5 Discussion 47

2.6 Acknowledgements 49

Reference List 53

Chapter 3: Gastric vagal motoneuron function is maintained following 55

experimental spinal cord injury

3.1 Abstract 56

3.2 Introduction 57

3.3 Materials and Methods 59

3.3.1 Surgical procedures and animal care 60

3.3.2 Neuronal tracing 62

3.3.3 Histological Processing 62

3.3.4 Immunohistochemistry 64

3.3.5 Electrophysiology 65

3.3.6 Morphological reconstructions and analysis 66

3.3.7 Gastric Motility recordings 68

3.3.8 Drugs and chemicals 69

3.3.9 Statistical Analysis 69

3.4 Results 70

3.4.1 Histological assessment of T3-SCI severity 70

3.4.2 Assessment of post-injury weight loss and reduction of spontaneous 70

oral intake of food

3.4.3 The number of CTB-immunoreactive DMV neurons projecting to 71

the stomach is unaffected by T3-SCI

3.4.4 T3-SCI does not alter DMV neuron electrophysiological or 72

morphological properties

3.4.5 T3-SCI does not reduce brainstem sensitivity to TRH 72

3.5 Discussion 74


Reference List 91

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Chapter 4: Mesenteric vascular dysregulation accompanies acute 97


4.1 Abstract 98

4.2 Introduction 99

4.3 Methods 100

4.3.1 Surgical Procedures and Animal Care 101

4.3.1.1 SCI/Control Surgery 101

4.3.2 In vivo studies 102

4.3.3 Blood Flow Analysis 105

4.3.4 Histological Processing 105


4.4 Results 106

4.4.1 Histological assessment of T3-SCI severity 106

4.4.2 Assessment of the post-injury reduction of spontaneous feeding and 107

bodyweight

4.4.3 Basal arterial blood pressure and mesenteric blood flow are decreased 107

in SCI rats

4.4.4 Postprandial mesenteric blood reflexes are reduced in SCI rats 108

4.5 Discussion 109


Reference List 122

Chapter 5: Acute experimental spinal cord injury evokes low-grade 127

gastrointestinal inflammation

5.1 Abstract 128

5.2 Introduction 129

5.3 Methods 131

5.3.1 Spinal Cord Injury Surgery and Post-Operative Care 131

5.3.2 Superior Mesenteric Artery Occlusion 133

5.3.3 Gastrointestinal Tissue Harvest 133

5.3.4 Histopathological Processing 134

5.3.5 RNA Isolation, Reverse Transcription Reaction and q-PCR 134


5.4 Results 136

5.4.1 T3-SCI provokes gastrointestinal tissue necrosis and reduction in 136

mucosal villi

5.4.2 T3-SCI increases mRNA expression of mucosal inflammatory markers 137

5.4.3 T3-SCI alteration of mRNA expression of nitric oxide isoforms 138

5.5 Discussion 139


Reference List 159

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Chapter 6: Diminished gastric motility following experimental 162

spinal cord injury in the rat is accompanied by changes in receptor

expression in the nodose ganglia

6.1 Abstract 163

6.2 Introduction 164

6.3 Materials and Methods 167

6.3.1 Spinal Cord Injury Surgery and Post-Operative Care 167

6.3.2 Superior Mesenteric Artery Occlusion 169

6.3.3 Nodose Ganglia Isolation and Harvest 169

6.3.4 Fluorescent Immunohistochemistry for Nodose TRPV1 and CCK-1r 170

6.3.5 RNA Isolation from Nodose Ganglia 171

6.3.6 Reverse Transcription and q-PCR of Nodose Ganglia 172


6.4 Results 173

6.4.1 T3-SCI increases Nodose TRPV1 and CCK-1r immunofluorescence 173

6.4.2 T3-SCI increases mRNA expression of TRPV1 and CCK-1r in Nodose 173

Ganglia

6.5 Discussion 174


Reference List 186

Chapter 7: Summary of this Dissertation, Short Perspective, and 192

Future Directions

7.1 Summary of this Work 193

7.1.1 Spinal Cord Injury does not affect gastric vagal efferent sensitivity 193

7.1.2 Reduced mesenteric blood flow occurs following T3-SCI 193

7.1.3 Gastrointestinal inflammation up-regulates gastrointestinal 195

inflammatory markers and TRPV1 receptors in nodose ganglia

7.2 Perspective for Vagal Afferent Functional Experiments 196

7.2.1 Introduction 196

7.2.2 Proposed Materials and Methods 197

7.2.2.1 Spinal Cord Injury Surgery and Post-Operative Care 197

7.2.2.2 In Vivo Studies 197

7.2.2.3 Electrode Placement for Neurophysiology 198

7.2.2.4 Vagal Parasympathetic Nerve Recording 199

7.2.3 Proposed Outcomes 200

7.3 Future Directions 202

7.3.1 qRT-PCR of Labeled Gastric Nodose 202

7.3.2 Test additional GI peptides with Neurophysiology 203

7.3.3 Test tensile strength of SCI vagus nerve 203

7.3.4 Anatomical comparison of DMV neurons and nodose neurons 204

7.3.5 Calcium Measurements 204

7.3.6 Therapeutic targeting of post-SCI gastric dysfunction 205

7.3.7 Obesity, Aging, & Inflammation 206

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7.4 Acknowledgments 207

Reference List 211

xi

LIST OF FIGURES

1.1 Schematic representation of the gastric vago-vagal reflex circuit 25

2.1 Principal stages of strain gage fabrication 51

2.2 Representative motility traces generated with fabricated dual 52

element strain gages

3.1 T3-SCI does not induce neuronal degeneration within the dorsal motor 87

nucleus of the vagus (DMV)

3.2 T3-SCI does not alter basic membrane properties or neuronal morphology 88

of DMV neurons

3.3 Representative original polygraph traces of gastric corpus contractions 89

from control and T3-SCI

3.4 Microinjection of thyrotropin-releasing hormone into the dorsal vagal 90

complex, including the DMV, induces gastric contractions following both

control and T3-SCI

4.1 Average percent change in body weight after SCI expressed as the 115

difference from pre-injury weight

4.2 T3-SCI provokes a reduction in the spontaneous intake of standard 116

laboratory chow

4.3 T3-SCI diminishes basal SMA blood flow at 3 days following injury 118

4.4 Post-prandial hyperemia is significantly lower in T3-SCI animals 119

4.5 Duodenal tissue blood flow does not increase in T3-SCI animals during 121

enteral feeding

5.1 T3-SCI provokes gastrointestinal tissue pathology 146

5.2 T3-SCI provokes gastrointestinal mucosal histopathology 147

5.3 Expression levels of gastric MCP-1 mRNA after T3-SCI 149

5.4 Expression levels of gastric MIP-1α mRNA after T3-SCI 150

5.5 Expression levels of gastric ICAM-1 mRNA after T3-SCI 151

5.6 Expression levels of duodenal MCP-1 after T3-SCI or SMA occlusion 152

5.7 Expression levels of duodenal MIP-1α after T3-SCI or SMA occlusion 153

5.8 Expression levels of duodenal ICAM-1 after T3-SCI or SMA occlusion 154

5.9 Expression levels of gastric iNOS after T3-SCI 155

5.10 Expression levels of gastric nNOS after T3-SCI 156

5.11 Expression levels of duodenal iNOS after T3-SCI or SMA occlusion 157

5.12 Expression levels of duodenal nNOS after T3-SCI or SMA occlusion 158

6.1 T3-SCI provokes increased TRPV1 1-day post-injury 181

6.2 T3-SCI provokes increased CCK-1r 1-day post-injury 182

6.3 Expression levels of nodose TRPV1 after T3-SCI 183

6.4 Expression levels of nodose CCK-1r after T3-SCI 184

6.5 Expression levels of nodose CCK after T3-SCI 185

7.1 Summary figure of the overall work of this dissertation 208

7.2 Schematic diagram of potential mechanisms of gastrointestinal 209

pathophysiology following T3-SCI

7.3 Representative image of vago-vagal recording reflex circuit 210

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LIST OF TABLES

2.1 Parts and tooling for strain gage fabrication 50

3.1 White matter expressed as a percent of the total spinal cord 82

cross-sectional area

3.2 Body weight change expressed as percentage of pre-operative weight 83

is reduced following T3-SCI

3.3 T3-SCI decreases normalized food intake 84

3.4 Basic membrane properties of DMV neurons 85

3.5 Basic morphological properties DMV neurons remain unchanged 86

4.1 Mean arterial pressure is not altered by 60 minutes of Ensure™ 117

infusion in 3 day T3-SCI and surgical controls

5.1 Semi-quantitative measurements of inflammation scoring for 144

gastrointestinal tissue

5.2 Forward and reverse primer sequences for quantitative real time PCR 145

5.3. T3-SCI provokes an inflammatory response and blunting of mucosal 148

villi in duodenal tissue

6.1 Forward and reverse primer sequences for quantitative real time PCR 180

xiii

LIST OF ABBREVIATIONS

AD: autonomic dysreflexia

AIS: ASIA Impairment Scale

ANS: autonomic nervous system

AP: area postrema

ASIA: American Spinal Injury

Association

Ca2+

: calcium

CB1: cannabinoid 1

CCK: cholecystokinin

CCK-1r: cholecystokinin receptor 1

CNS: central nervous system

CN X: cranial nerve X (vagus nerve)

DMV: dorsal motor nucleus of the vagus

DVC: dorsal vagal complex

ENS: enteric nervous system

GI: gastrointestinal

GLP-1: glucagon-like peptide 1

ICAM-1: intercellular adhesion

molecule-1

IGLEs: intraganglionic laminar endings

IHC: immunohistochemistry

IL-10: Interleukin-10

IMAs: intramuscular arrays

ISCoS: International Spinal Cord

Society

ISNCSCI: International Standards for

the Neurological Classification of Spinal

Cord Injury

MASCIS: Multicenter Animal Spinal

Cord Injury Study

MCP-1α: monocyte chemoattractant

protein-1 alpha

MIP-1: macrophage inflammatory

protein

mmHg: millimeters Mercury

NANC: non-adrenergic non-cholinergic

NPY: neuropeptide Y

NSCISC: National Spinal Cord Injury

Statistical Center

NTS: nucleus tractus solitarius

PCR: polymerase chain reaction

qRT-PCR: quantitative real time-

polymerase chain reaction

T3: thoracic level 3

T3-SCI: thoracic level 3 spinal cord

injury

TRH: thyrotropin-releasing hormone

TRP: transient receptor potential

TRPV1: transient receptor potential

vanilloid type 1

SCI: spinal cord injury

SNS: sympathetic nervous system

PNS: parasympathetic nervous system

US: United States

VIP: vasoactive intestinal peptide

WHO: World Health Organization

xiv

ACKNOWLEDGEMENTS

I am so very grateful to everyone whom has helped me along the way.

I would like to thank my thesis advisor and mentor Dr. Gregory M. Holmes. Thank you

for your guidance, encouragement, and support. Thank you for always believing in me.

I would like to thank the members of my thesis committee: Dr. Kirsteen Browning, Dr.

Charles Lang, Dr. Ann Ouyang, and Dr. Patricia McLaughlin. I appreciate your time,

your comments, your suggestions, and your questions which have helped guide me and

this work. Thank you all for your encouragement and support.

I would also like to express my sincerest gratitude to Dr. Sean Stocker and his laboratory

technicians, Sarah Simmonds and Jenny Lay, for teaching me neurophysiological

recordings. I cannot thank them enough for their patience, kindness, and endless hours of

experimental support and advising.

I would be remiss not to express a gracious thank you to Gina Deiter, Margaret McLean,

and Nicole Pironi for their contributions and support in my work.

I would like to thank the members of the anatomy program. The friendships, the laughter,

and the support have made this journey memorable.

To my family, the unending love and support has made me who I am today. I sincerely

thank you all for being there for me to listen and to provide encouraging words. Thank

you all so much for your boundless patience and support.

To my loving husband, I am so grateful for all that you have done, from just listening to

editing, your support has never wavered. You pushed me through the tough times and

celebrated with me in the good times. Thank you for everything. I love you.

1

Chapter 1:

Introduction

Spinal Cord Injury, Blood Flow, Inflammation, and

TRPV1 Neurophysiology

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Spinal cord injury (SCI) is one of the most devastating and debilitating human

conditions. Traumatic SCI produces an instantaneous disconnection of the bidirectional

neural communication between the body and the brain. In a complete SCI, the sensory

neural impulses ascending within the spinal tracts below the level of injury are unable to

reach the brain. Likewise, descending motor impulses located within the same vicinity of

the spinal cord are no longer able to pass beyond the level of the injury in order to

regulate the neuronal cell bodies that form the central gray matter. Finally, at the level of

the lesion center, neuronal damage results in a loss of all final output to the periphery

normally innervated by that region of the spinal cord.

1.1 Spinal Cord Injury Overview

Worldwide, approximately 250,000- 500,000 SCIs occur each year and represents

an annual incidence of 15 to 52.5 cases per million of population (Lee et al., 2014). The

greatest proportion of cases remains young males, whereby 80% of injuries are between

the ages of 15 and 35 and 5% are children. In countries with aging populations, males

and females over 65 represent an increasing injury demographic. Improvements in acute

care of SCI have extended the post-injury life expectancy of young individuals with SCI

to approximately 40 years post-injury (Shavelle et al., 2014). Despite improvements in

care, the chronic SCI population remains at a higher risk for injury-related complications

and they incur greater health care burdens. Post-injury care of the SCI individual

necessitates significant financial costs that are incurred throughout the remainder of life.

These costs include initial hospitalization and acute rehabilitation, home and vehicle

modification, and recurring costs for durable medical equipment, medications, supplies,

and personal assistance (Cristante et al., 2012). Recent reports (Devivo et al., 2011) place

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the average acute care costs from $334,170 to $1,023,924 and the annual chronic care

costs at $40,589 - $177,808. Additionally, there was loss of wages, fringe benefits, and

productivity related to unemployment due to the SCI. Costs related to unemployment are

estimated to be more than $57,000/year but vary according to job (Priebe et al., 2007).

1.1.1 Cascade of Events in Spinal Cord Injury

The mechanisms by which traumatic SCI occurs can be organized into four

categories according to how the trauma occurred and the degree of tissue damage.

Contusion injuries consist of an impact leading to bruising of the neural tissue with

relatively minimal physical severing of axons. Contusion injuries may be accompanied

by the presence of persistent compression whereby vertebrae, or fragments of vertebrae,

remain in contact with the neural tissue. Transection and hemi-section of the cord result

most often from foreign objects, such as knives or bullets, but also include bone

fragments penetrating the vertebral canal and severing axonal tracts. Distraction injuries

are the result of axial stretch of the neural tissue (Dumont et al., 2001).

Accumulating evidence over the last two decades has revealed that the

pathophysiological progression of SCI occurs in two phases: primary injury and

secondary injury (reviewed in Tator & Fehlings, 1991,Dumont et al., 2001). The primary

injury is the result of the actual mechanical damage caused by the injury, causing

immediate tissue destruction and cell death of the white and gray matter, depending on

severity of injury.

Primary injury is easily conceptualized in the context of transection or

hemisection of the cord, whereby axonal processes are severed. However, the most

common type of human SCI is a compression/contusion injury due to dislocation or

4

fracture of the vertebral column; the spinal cord is crushed rather than transected. This

results in mechanical injury to spinal cord seen as a contusion/bruise and comparatively

fewer severed axonal processes. Regardless of the mechanism of injury, the primary

phase of SCI consists of an immediate onset of spinal shock. Spinal shock is a

physiological disruption of spinal cord functioning and reflexes below the level of injury

due to an interruption in communication between the supraspinal central nervous system

(CNS) and segmental spinal somatic and autonomic nervous system (ANS) circuits. The

term “spinal shock” dates back to 1750 where it was first describe by Whytt and then

introduced into the literature by Hall in 1841 (cited in Nacimiento & Noth, 1999). Spinal

shock is characterized by sensory deficits, flaccid paralysis, absence of deep tendon

reflexes, abolishment of reflex somatic activity, and thermoregulatory disturbances below

the level of injury. Spinal shock varies according to the level of the injury with high

cervical injuries including acute respiratory failure, tetraplegia, anesthesia, lack of all

reflexes below injury site, neurogenic shock, detrusor and anal sphincter areflexia. High

cervical injury also presents ipsilateral constriction of the pupil, weakness of the

ipsilateral eyelid, and anhidrosis (decreased sweating over the forehead). This

constellation of symptoms associated with high cervical injury is also known as Horner

syndrome. Following low cervical SCI there is less risk of respiratory failure. By

contrast, low thoracic SCI reduces the risk for arterial hypotension and no neurogenic

shock (severe arterial hypotension, bradycardia, and hypothermia). Spinal shock signs

and symptoms are caused by the absence of the sympathetic nervous system (SNS)

through loss of supraspinal control and unopposed parasympathetic tone via the intact

vagus nerve. The condition of spinal shock typically lasts for days-weeks (Garstang &

http://en.wikipedia.org/wiki/Anhidrosis

5

Miller-Smith, 2007) with the average time frame ranging from 4-12 weeks. The typical

signs indicating that the spinal shock phase has ended are the appearance of the

bulbocavernosus reflex, recovery of deep tendon reflexes, or return of reflex detrusor

functions. Others define spinal shock in a series of four phases: areflexia/hyporeflexia (0-

24 hours), initial reflex return (1-3 days), early hyperreflexia (4 days-1 month), and

spasticity (1-12 months; Popa et al., 2010).

Secondary injury is represented by a cascade of vascular, biochemical, and

inflammatory changes that develop over minutes to hours following the initial

mechanical injury and promote further tissue degeneration and neurological dysfunction

(Kwon et al., 2004,Rowland et al., 2008). The progression of secondary injury is often

the most critical phase that ultimately determines the outcome for the individual and the

severity of the injury. Interventions designed to attenuate the secondary

pathophysiological events are generally considered to be the most promising means of

treatment for improving functional recovery following SCI (Wilson et al., 2013,Bowes &

Yip, 2014).

Specifically, secondary injury is initiated by a multifactorial interaction of

petechial hemorrhage, ischemia, hypoxia, oxidative stress, calcium dysregulation,

excitatory amino acid release, inflammation, cytokine up-regulation, release of

degradative enzymes, free-radical release, and edema occur as time progresses after

injury (reviewed in Tator & Fehlings, 1991,Beattie, 2004). Specifically, the secondary

phase of SCI begins in which further damage to potentially viable neural cells and axonal

processes within the spinal cord is sustained and systemic complications begin to develop

throughout the body. Petechial hemorrhages occur in the gray matter, a capillary-rich

6

region, of the spinal cord leading to neuronal cell death and then the lesion center spreads

radially causing edema in the white matter leading to swelling of the spinal cord

spreading rostrally and caudally affecting levels superior and inferior to the injury site.

Accompanying this cascading injury, oligodendrocytes, the cells responsible for

myelination of central nervous system axons, die which ultimately leads to dysfunction of

otherwise intact axonal processes. Monocytes are recruited to the area and infiltrate the

spinal cord upon which they transform into macrophages and begin removing the dead

cells and debris from the injury (Popovich et al., 2001). Additionally, there is

myelin disruption, axonal degeneration, and ischemic endothelial damage at just 4-hours

following a contusion SCI. Blood flow to the spinal cord is markedly reduced as the

vessels surrounding the hemorrhagic region clot to prevent further blood loss (Satake et

al., 2000). The injury continues to progress as a result of the ischemia that is caused by

the reduced blood to the spinal cord; the hypoperfusion is a result of a change in the

spinal canal due to the significant edema and hemorrhage or the overall reduced systemic

blood pressure. The ischemia leads to a chain of events ultimately resulting in further cell

death. Inflammatory cells and macrophages are recruited to the lesion center contributing

to the duration of the chronic phase, lasting 1-4 weeks, upon which proliferation and

hypertrophy of astrocytes form a glial scar or a cyst (Cristante et al., 2012).

1.1.2 Classification of Human Spinal Cord Injury

Following SCI, even casual observation reveals a loss of motor control and

sensory detection below the level of the injury. When viewed from the neck, downward

(i.e. cranial to caudal), the spinal cord is divided into four regions: a) cervical; b)

thoracic; c) lumbar; and d) sacral; all of which are vulnerable to SCI. Each region is

7

comprised of a spinal segment, or level, which is defined by the paired spinal nerves that

enter or exit the spinal cord. The level of the spinal injury determines the loss of

functionality sustained by the individual. A SCI which is sustained above the first

thoracic spinal cord segment results in tetraplegia (formerly known as quadriplegia) and

affects the cervical spinal nerves and results in paralysis of all four limbs as well as

autonomic dysfunction. Injury to the spinal cord that occurs below the first thoracic

spinal segment results in paraplegia. Paraplegia is defined as paralysis of the lower limbs

and is accompanied by variable degrees of autonomic dysfunction. The level at which the

spinal cord is most severely injured is typically referred to as the lesion epicenter and is

readily observed clinically as the region below which sensory loss is determined. Taken

as a whole, the SCI population is comprised of 53% tetraplegic and 42% paraplegic

(Sinescu et al., 2010).

Clinically, each type of trauma presents variable degrees of sensory and motor

deficits such that the completeness of the injuries are categorized based upon specific

motor and sensory criteria scored according to the American Spinal Injury Association

(ASIA) scale (Waring et al., 2010). In 1982, the National SCI Statistical Center Database

requested an improved classification system to provide greater precision in defining the

extent of neurologic damage and severity of injury to obtain more consistent and reliable

data for the National Database. The ASIA Standards were superseded by the more

encompassing International Standards for the Neurological Classification of Spinal Cord

Injury (ISNCSCI) database (Waring et al., 2010). The ISNCSCI further updated their

classifications to include a defined set of procedures for clinicians and researchers to use

when assessing SCI. The standards focus on identifying and adopting key muscles and

8

sensory points in the neurologic assessment. This new standard of examination is referred

to as the International Standards as well as the ASIA Impairment Scale (AIS; Burns et al.,

2012). The overall grading system established by the AIS remains the most commonly

used scale and categorizes injury severity as follows (Biering-Sorensen et al., 2009): A =

complete, no sensory or motor function is preserved in the sacral segments S4-S5; B =

sensory incomplete, sensory but not motor function is preserved below the neurological

level and includes the sacral segments S4-S5 (light touch, pin prick at S4-S5: or deep anal

pressure, AND no motor function preserved more than three levels below the motor level

on either side of the body); C = motor incomplete, motor function is preserved below the

neurological level, and more than half of key muscle functions below the single

neurological level of injury have a muscle grade less than 3; D = motor incomplete,

motor function is preserved below the neurological level and at least half of key muscle

functions below the neurological level of injury have a muscle grade ≥3; and E = normal,

if sensation and motor function as tested with the ISNCSCI are graded as normal in all

segments, and the patient had prior deficits, then the AIS grade is E as someone without

an initial SCI does not receive an AIS grade (Kirshblum et al., 2014).

1.1.3 Incidence

The incidence of SCI is increasing worldwide. According to the National Spinal

Cord Injury Statistical Center (NSCISC), there is an estimated 12,000 new cases of SCI

that occur each year, representing an incidence of 40 cases per million population per

year, with approximately 270,000 persons living with SCI in the U.S. (Selvarajah et al.,

2014). The overall mortality of all patients with SCI is 9.2% and the mortality of patients

under 60 is 7.7%. The fact that cervical SCI also predicts mortality confirms findings that

9

higher level of SCI predicts mortality (Claxton et al., 1998). A study by (Catz et al.,

2002) demonstrated 81% survival after 10 years, 75% after 20 years, 50% after 35 years

in contrast to the World War I era whereby 80% of American and British soldiers who

sustained SCI died within a few weeks to 3 years (Catz et al., 2002). Recent

epidemiological evidence conducted in the United States revealed that the leading cause

of SCI is motor vehicle accidents, followed by falls, acts of violence, sports, and disease.

SCI is most commonly seen in a youthful population (16-30 years of age) and the

prognosis is such that these individuals (whom survive) face lifelong effects of aftermath

due to the injury. Following SCI there is an increased risk of depression, sleep disorders,

spasticity, bladder and gastrointestinal changes, bedsores, sexual dysfunction, involuntary

movements, obesity, and vascular and respiratory diseases (Cristante et al., 2012). The

comorbidity of depression leads to an attempted suicide rate of approximately 40% (Fann

et al., 2011).

1.1.4 Traumatic vs. Non-traumatic Spinal Cord Injury

SCI is divided into 2 categories: traumatic and non-traumatic. Traumatic SCI is

far more common and results from injuries sustained in military service, auto accidents,

falls, sports injuries, hemorrhage, stroke, vertebral fracture, or violence. Non-traumatic

SCI is due to arthritis, cancer, vascular lesions, spinal stenosis, nerve root entrapment,

Multiple Sclerosis, developmental etiologies, infections, or disc degeneration of the

spine.

1.1.5 Current Therapies for Spinal Cord Injury

Researchers and clinicians seek to improve the neurological outcome following

SCI through treatment strategies. The immediate emergency action to be taken following

10

a SCI, is to prevent further damage to the spinal cord therefore the person must be

carefully stabilized to prevent movement. The secondary injury begins within minutes to

hours after injury. Subsequently, researchers and clinicians are most interested in treating

the secondary injury with antioxidants and anti-inflammatory agents with the aim of

preventing further inflammatory damage to the spinal cord. For example, nicotine has

recently shown some promise in preventing neurotoxicity by inhibiting formation of the

inducible isoform of nitric oxide synthase (iNOS; Lee et al., 2009). Additional

interventions have been categorized into 4 main modalities: corrective surgery,

biological, physical, and pharmacological treatment methods.

Surgical techniques for repairing SCI damage have been utilized for over 40

years. The most commonly used surgical approach is decompression surgery and it is

intended to realign the spine, decompress any nerves and the spinal cord, stabilize the

spine, and prevent additional neurologic injury. This procedure has limited success and

does not fully correct functional outcomes. Specifically, only 1-1.8% of patients with

cervical and thoracic SCI can walk after surgical decompression. When employed, the

earlier the decompression surgery is done (within 24 hours of the injury), the better the

functional outcome for the SCI patient (Dvorak et al., 2014).

The second treatment method used in SCI is biological therapy consisting of

factors which promote neuronal regeneration (i.e. tissue growth factors, stem cells, and

precursor cell transplantations). Stem cell therapies and cell transplantations are relatively

new treatment modalities and have only been studied for a little over 10 years, but offer

promise for cell regeneration, re-innervation, and improving functional outcomes after

SCI (Tetzlaff et al., 2010).

11

The third type of commonly used treatment is defined as physical means or

approaches which are employed to minimize secondary spinal cord damage. The most

frequently applied approaches include hypothermia (Dietrich et al., 2011), hyperbaric

oxygen, and exercise such as treadmill training (reviewed in Cristante et al., 2012).

Hypothermia treatments seek to lower the temperature of the CNS tissues to prevent the

hypoxic and ischemic effects; however, hypothermia is unable to prevent electrolyte

imbalances (potassium loss) as the extracellular fluid surrounding the spinal cord

becomes very acidic. The rationale for hyperbaric oxygen is based upon overcoming the

hypoperfusion of the injured spinal cord. Treadmill training attempts to facilitate central

program generators by re-training the injured neural circuits.

The final type of therapy for treatment of SCI utilizes pharmacological

interventions. Experimental and clinical trials have shown conflicting results in treating

the secondary injury following SCI (reviewed in Baptiste & Fehlings, 2006). Based upon

promising initial results, corticosteroids are most commonly administered for treating

SCI (Bracken et al., 1990). Corticosteroids come in the form of dexamethasone and

methylprednisolone which are both used for their anti-inflammatory actions and their

effectiveness in treating cerebral edema. Additionally, methylprednisolone has been

shown to increase blood flow AND stabilize plasma membranes; yet no improvement in

functional outcomes has been documented (see Schroeder et al., 2014). However, the

problem still persists that the therapies are largely focused on restoration of motor

function and result in minimal functional recovery in clinical application. There are

limited therapies currently available for re-gaining sensory or autonomic or visceral

functions and this creates considerable quality-of-life challenges for the SCI population.

12

1.1.6 Animal Models

Beginning with the early studies of Allen (Allen, 1911) numerous species have

been used as animal models of SCI. The most commonly utilized animal model has

employed laboratory rodents. While the use of mice in SCI research offers the advantage

of advances in genetic models with the rise in knockout and transgenic strains, rats offer

distinct advantages. A rat SCI develops a large fluid-filled cystic cavity at the lesion

center (mimicking a human SCI) whereas a cystic cavity does not form in murine SCI

models. Additionally, the lesion center in mice becomes densely packed with cells and

actually decreases in size. However, the presence of a cavity, or lack thereof, has no

influence on axonal regeneration and only becomes important when looking to study the

cellular cascade present at the lesion center or the composition of the glial scar.

Additionally, transplantation strategies differ significantly between rats and mice due to

the presence and absence of the cyst at the lesion center. Furthermore, rats are the

preferable choice for translational studies investigating transplant treatments. Another

important factor to consider is strain differences; strains of mice and rats respond

differently to SCI. For instance, the C57BL/6 mouse, the most commonly used strain in

axon regeneration studies, has been reported to present poor locomotor outcome, the

greatest immunological response, and the least amount of measurable axonal

growth compared to other inbred mouse strains. In rats, clear strain differences exist

between Sprague-Dawley, Long-Evans, and Wistar rats (Mills et al., 2001).

The use of large animals (cats, dogs, pigs, and non-human primates) has seen a

recent resurgence in SCI research. Cats are frequently used among spinal cord

electrophysiologists (Jordan et al., 2014). Porcine models, especially mini-pigs, have

13

been proposed as an intermediary animal model for translational studies to closely

resemble human physiological response to injury (Lee et al., 2013). Non-human primates

remain the most closely anatomically- and physiologically-related to humans. These

larger-animal models are not nearly as common as rodent models due to size, cost,

availability, housing facilities, medical care, and ethics; yet, they provide useful insight

on our understanding of human SCI and they have served as preclinical models for

testing new treatments and therapies.

1.1.6.1 Injury Devices

Clinically relevant contusion injury models and injury devices have been

continuously developed since the original weight-drop method (Allen, 1911). Dorsal

hemisection models are often used to study the regeneration of the corticospinal tract

axons (Gensel et al., 2006). For this model, only the dorsal columns of the spinal cord are

cut to cause the axons of the corticospinal tracts to be lesioned. A complete transection

injury model is utilized for researchers wanting to investigate the most severe type of

injury (Holmes et al., 1998). This injury model is also the most rigorous test of axon

regeneration since the white matter tracts are bilaterally sectioned and no axons are

spared. As discussed previously, a contusion model is the preferred injury model for best

replicating the pathophysiology that occurs in a human SCI (Biering-Sorensen et al.,

2009). Injury devices to model contusion injury range from blunt trauma, epidural

balloons, circumferential cuffs or aneurism clips, to weight drop (Sledge et al., 2013).

One essential feature of injury models concerns the ability to experimentally produce a

range of injury severities. Early weight drop models relied upon the drop height in order

to vary injury severity, often with low reproducibility (Onifer et al., 2007). The New

14

York University Weight Drop (Gruner, 1992) device was one of the first to record

biomechanical properties (acceleration, displacement, etc.) of the impact and rapidly

became the standard model following its use in the Multicenter Animal Spinal Cord

Injury Study (MASCIS) studies (Basso et al., 1996). Like most models prior, the New

York University Weight Drop device involves performing a laminectomy to expose the

spinal cord while leaving the dura mater intact, stabilizing the rat by dorsal processes of

the vertebral column just superior and inferior to the laminectomy segment, and then

dropping a 10-gram weight from 12.5 -50 millimeters onto the exposed spinal cord

segment. This injury device results in a range of injuries (Basso et al., 1996). A contusion

injury device developed at The Ohio State University, employed an electromagnetic

solenoid impactor that could vary the depth and duration of the injury (Behrmann et al.,

1992). More recently, the Infinite Horizons Impactor was developed and uses a high

speed motor with force transducer feedback to generate a consistent graded SCI (Scheff

et al., 2003). The Infinite Horizons Impactor device shares many procedural similarities

to the New York University and The Ohio State University devices (e.g., laminectomy,

vertebral clamps) but with greater inter-user consistency.

1.2 Autonomic Nervous System Overview

The intact spinal cord carries impulses of motor and sensory modalities to and

from the peripheral nervous system. Although the CNS regulation of sensory and motor

function is severely altered in a SCI, effects of the injury involve autonomic functioning,

as well (Inskip et al., 2009,Krassioukov, 2009). The ANS is composed of the

sympathetic and parasympathetic nervous systems and is responsible for regulating the

so-called involuntary functioning of the body, including the viscera. The ANS regulates

15

all major organ functions including cardiovascular functions (i.e. systemic blood flow,

cardiac contractility, heart rate, peripheral vasomotor responses), GI functions (GI

emptying, motility/contractions, transit), urinary and bowel functions (sensations of

distension, control of the smooth muscle sphincters necessary for continence), and

reproductive functions (sensory components of blood flow, arousal, menses, child birth).

1.2.1 Enteric Autonomic Nervous System

The ANS consists of three anatomically distinct divisions: the enteric,

sympathetic, and parasympathetic. The enteric nervous system (ENS) innervates the

gastrointestinal tract and is capable of quasi-autonomous control throughout most of the

GI tract. This network of neurons is capable of homeostatic functions of the GI tract but

is insufficient to fully meet the homeostatic needs of the organism. Subsequently, the

ENS is modulated by the sympathetic and parasympathetic divisions. The ENS mediates

digestion through localized control over the specialized cells and individual reflex

systems for the smooth musculature, secretory glands, and microvasculature of much of

the digestive tract (reviewed in Furness, 2012). Briefly, functional units of the ENS are

formed by a polysynaptic circuit consisting of sensory neurons, interneurons, and motor

neurons all of which serve in a manner that is similar to many reflexive or integrative

neural circuits (Wood, 2008). For example, the ENS maintains the constant slow wave

contractions of the GI tract, beginning with activation of sensory neurons that respond to

either specific chemical cues; mechanical distortion of the mucosa; or distortion of

sensory processes embedded within the muscle layers. These afferent neurons terminate

onto excitatory or inhibitory interneurons that terminate on excitatory or inhibitory motor

neurons innervating the smooth musculature.

16

1.2.2 Sympathetic and Parasympathetic Nervous Systems

Despite the intrinsic ENS circuitry, extrinsic ANS modulation is imperative for

proper functioning of the GI tract. In contrast to the intestines, the sympathetic control of

the upper gastrointestinal tract is primarily restricted to local regulation of vascular

supply. Cranial nerve X, the vagus nerve, is well known for its significant involvement in

the parasympathetic control of the viscera (see Browning & Travagli, 2010). The

parasympathetic innervation of the viscera consists of sensory and motor pathways that

integrate to generate precise output to the visceral effector organ, in particular the

stomach. Specifically, this so-called gastric vago-vagal circuit (Figure 1.1) consists of the

vagus nerve, the dorsal vagal complex (DVC), and the stomach. The vagus nerve bundle

is composed of sensory afferent fibers and motor efferent fibers. The DVC includes the

nucleus tractus solitarius (NTS), dorsal motor nucleus of the vagus (DMV), and the area

postrema (AP) all within the caudal extent of the medulla oblongata of the brainstem

(Browning & Travagli, 2010). Focusing primarily on the vagal innervation originating

within the circular smooth muscle found just interior to the myenteric plexus (a

component of the ENS that is modulated by the ANS); the gastric vagal afferent fibers

arise from bipolar cell bodies within the nodose ganglion that extend to the NTS.

Vagally-mediated mechanical and chemical gastric sensory stimuli signals from

the lumen of the stomach are transmitted to the NTS through a glutamatergic synapse.

Vagal afferent response to mechanical stimulation (i.e. post-prandial stretch) is due to

mechanosensitive receptors within the smooth muscle layers of the stomach. These

mechanoreceptors have been identified as intraganglionic laminar endings (IGLEs,

Powley & Phillips, 2002) and, possibly, intramuscular arrays (IMAs; Berthoud &

17

Powley, 1992). From the NTS, second order neurons respond to glutamate and transmit

the integrated sensory signal from the NTS to the DMV for motor output to the stomach.

The DMV is comprised of parasympathetic vagal cholinergic preganglionic

motorneurons. DMV neurons release acetylcholine (ACh) onto nicotinic receptors of the

postganglionic cholinergic neurons within the ENS of the stomach. A population of these

postganglionic neurons releases ACh onto cholinergic muscarinic receptors within the

myenteric plexus of the stomach which cause the circular smooth muscle to contract. A

separate population of the cholinergic preganglionic DMV neurons release ACh onto

nicotinic receptors of the postganglionic neurons within the ENS, as well (Grundy et al.,

2000). These postganglionic neurons are frequently categorized as non-adrenergic non-

cholinergic (NANC) neurons that release nitric oxide (NO) and vasoactive intestinal

peptide (VIP) to act on the circular smooth muscle of the stomach eliciting gastric

relaxation.

1.2.3 Overview of Autonomic Nervous System Dysfunction

As a result of the interruption of descending supraspinal inputs following SCI, the

ANS undergoes significant alterations which affect physiological outcomes. Depending

upon the level of the injury, the spinal autonomic neurons are either directly damaged, or

indirectly impacted by the loss of descending modulation. Of particular clinical interest is

the post-injury dysregulation of vascular reflexes. The ANS is responsible for controlling

blood flow to skeletal muscle, kidneys, the splanchnic circulation, and the skin

(thermoregulation). Individuals with injuries at the cervical or high thoracic level are

often faced with unstable blood pressure regulation and do not respond appropriately to

orthostatic challenge (Claydon & Krassioukov, 2006). Furthermore, arterial hypotension,

18

due to loss of supraspinal control of the sympathetic nervous system, leads to visceral

hypoperfusion which may provoke end-organ dysfunction or failure (Popa et al., 2010).

Post-SCI ANS dysfunction differentially manifests across the acute and chronic

injury periods. In the acute phase of ANS dysfunction, spinal shock (as previously

described in 1.1.2) occurs and presents as hypothermia, hypotension, and bradycardia.

Cardiovascular dysfunction in the spinal shock phase may last 2 to 6 weeks and is often

accompanied by arrhythmias. The chronic phase may result in long-term orthostasis but

also includes impaired temperature regulation and impaired cardiovascular functions

(Laird et al., 2006,Hou & Rabchevsky, 2011).

The chronic phase is also noted by the episodic occurrence of autonomic

dysreflexia (AD); a life-threatening condition characterized by overactive sympathetic

outflow which includes vasoconstriction in muscular, skin, renal, and GI vascular beds.

Noxious afferent stimuli below the level of the lesion (i.e. distended bladder, decubitus

ulcer; (Karlsson, 2006) is the most common triggering stimulus. Pathophysiological

sprouting of segmental afferents below the lesion has been proposed as one causative

mechanism (Hou, 2009). In addition to altered visceral control following SCI, ANS

dysfunction may also have an effect on immune function. Emerging evidence suggests

that pathophysiological changes within the spinal autonomic circuitry that triggers AD

may suppress immune function (Zhang et al., 2013).

1.2.3.1 Gastric Dysfunction

As previously described, reflexive gastric motility and emptying is mediated

predominantly by vago-vagal neural circuits in the caudal medulla (Rogers et al., 2006).

Previous research has validated the model in which T3-SCI affects gastric emptying via

19

13C-breath test experiments (Qualls-Creekmore et al., 2010). This study revealed that

there is diminished gastric emptying following SCI. This delay in gastric emptying has

been shown to extend out to 6-weeks; and is therefore, a chronic condition of SCI. The

mechanisms underlying the delayed gastric emptying remain to be elucidated. One

possible explanation for the delayed gastric emptying is impaired contractions or motility

of the stomach. It should be noted that contractions and motility are often used

interchangeably in the literature. Clinically, “contractions” is the proper term but

“motility” is frequently used to globally describe these same gastric movements.

Additional experiments using SCI rats revealed significantly diminished gastric motility

through an unspecified vagal mechanism (Tong & Holmes, 2009). Subsequent studies

utilized central administration of a well-characterized GI peptide, cholecystokinin (CCK),

which acts via vagal afferents to evoke gastric relaxation. Medullary application of CCK

demonstrated that rats with T3-SCI are not responsive, presumably through derangements

in afferent processing; though the precise mechanism of this dysfunction remains obscure

(Tong et al., 2011).

1.2.3.2 Cardiovascular Dysfunction

Following SCI, the most common cause of death (contributing to 40.5% of the

deaths), after neoplasms, are cardiovascular disturbances. Some of the leading causes of

death due to cardiovascular dysfunction include cardiac failure, atrial fibrillation,

atherosclerosis, and coronary heart disease. All patients categorized as ASIA A and B

(complete and sensory incomplete) develop bradycardia and over half develop arterial

hypotension (Popa et al., 2010). Dysfunction of the sympathetic nervous system occurs

within the efferent pathways, with loss of supraspinal control of spinal centers. Both

20

inhibitory and excitatory control is effected resulting in absent or decreased sympathetic

tone below the level of the lesion. Parasympathetic outflow is preserved since the vagus

nerve remains anatomically intact; but the balance between the sympathetic and

parasympathetic systems is lost in cervical or high thoracic SCI. In response to the

systemic hypotension, unopposed vagal drive to the heart provokes bradycardia in a

compensatory attempt to maintain systemic blood pressure. Ultimately, any SCI above

the 6th

thoracic spinal level disrupts the sympathetic efferent pathways and diminishes

systemic vascular perfusion, including the splanchnic vascular bed.

1.2.3.3 Gastrointestinal Hypoperfusion

With a resting blood flow of approximately 20-25% of the total cardiac output,

the GI tract is one of the most highly perfused organ systems in the body. In addition to

marked deficits in gastric motility SCI produces vascular hypotension, especially

following cervical or high thoracic injury. Vascular hypotension can result in visceral


organ dysfunction. The superior mesenteric artery (SMA) perfuses the midgut from the

lower part of the duodenum through two-thirds of the transverse colon, as well as the

pancreas. Post-SCI inflammation can recruit other organs into an injured-state via the

release of multiple inflammatory mediators from macrophages, dendritic cells, and T-

cells (Hotchkiss & Karl, 2003). The celiac artery perfuses the upper GI tract including the

stomach. High-thoracic injuries that isolate the sympathetic preganglionic neurons below

the lesion epicenter will similarly affect tissues perfused by the celiac artery. Recently

reports regarding gastric hypoperfusion indicate that diminished vascular supply is an

important inciting event in the pathogenesis of organ failure (De Winter & De Man,

21

2010). In a model of postoperative ileus, peritoneal mast cells were shown to contribute

to the subsequent gastric hypoperfusion. The mast cells found adjacent to mesenteric

blood vessels appeared to be activated by neuropeptides, such as substance P (SP) and

calcitonin gene related peptide (CGRP), and released from adjacent afferent neurons

(Boeckxstaens & de Jonge, 2009). Such mast cells are able to release pro-inflammatory

mediators into the peritoneal cavity diffusing into the blood vessels and increasing

mucosal permeability. The increased permeability allows easy transit of bacterial

products into the GI wall which activate resident macrophages triggering intracellular

signaling pathways. The end result is transcription of inflammatory molecules,

cytokines, chemokines, and adhesion molecules (i.e. iNOS, iCAM; Boeckxstaens & de

Jonge, 2009,De Winter & De Man, 2010).

1.3 The Emerging Role of Transient Receptor Potential Vanilloid Type 1 Receptor

in Gastrointestinal Dysfunction

Transient receptor potential vanilloid type 1 receptor (TRPV1), an inflammatory-

related channel, has gained recent attention. First cloned in 1997, TRPV1 has most

recently gained recognition in the vagus nerve in association with visceral inflammatory

conditions. TRPV1 is known as ‘a molecular gateway’ to nociceptive sensation in

somatic and visceral tissues (Caterina et al., 1997,Caterina & Julius, 2001). TRPV1 is a

member of the transient receptor potential (TRP) channel family, which is the largest

group of noxious stimulus detectors (Furuta et al., 2012). There are over 20 ion channel

proteins encoded by the TRP channel gene and they are widely distributed in various

tissues (Clapham et al., 2001). The TRP superfamily is a large group of phylogenetically

22

similar, non-selective cation channels that respond to changes in the local environment

(Moran et al., 2004).

TRPV1 is a polymodal transducer activated by capsaicin, noxious heat, and low

pH (a common consequence of inflammation from SCI; Nilius et al., 2007b,Furuta et al.,

2012). TRPV1 is a membrane protein with six transmembrane-spanning domains which

form a nonselective cation channel. This channel has a very high permeability to Na+ and

Ca2+

(Lee & Gu, 2009). TRPV1 is expressed throughout the entire cell of the sensory

afferent neurons; on sensory nerve terminals of the vagus nerve as well as within the

soma of the neurons within the nodose ganglion, particularly during times of tissue injury

or inflammation (Lee & Gu, 2009,Zhao et al., 2010,Jia & Lee, 2007,Caterina & Julius,

2001). Inflammation enhances the sensitivity of TRPV1 channels by lowering the

threshold for activation (Jia & Lee, 2007,De Schepper et al., 2008). Peripheral

inflammatory mediators activate intracellular pathways which phosphorylate TRPV1

altering its trafficking to the membrane (Furuta et al., 2012). Tissue inflammation, as

seen post-SCI, leads to increased temperature within the inflamed area (Lee & Gu, 2009).

Furthermore, the inflammation leads to a multitude of endogenous inflammatory

mediators which can sensitize TRPV1 resulting in nociceptor hypersensitivity and

hyperalgesia (Carr et al., 2003).

In pulmonary vagal sensory neurons, hyperthermic temperatures activate TRPV1

channels (Ni et al., 2006) leading to an influx of cations, mostly Na+ and Ca

2+ (Montell,

2005). The influx of cations may trigger several cascading events such as Ca

2+-induced

transport of vesicles to the plasma membrane (Clapham, 2003). It has also been proposed

that the protein kinase A- and protein kinase C-mediated phosphorylation of the TRPV1

23

channel induces TRPV1 sensitization caused by endogenous inflammatory mediators

such as prostaglandin E2 and bradykinin (Gu & Lee, 2006,Moriyama et al.,

2005,Premkumar & Ahern, 2000,Ni & Lee, 2008).

Recent studies have shown efferent-like functions of primary sensory afferent

neurons in the presence of tissue injury or inflammation (Richardson & Vasko, 2002).

The efferent-like functions include the release of neurogenic mediators such as SP and

CGRP (Schwartz et al., 2011). A pancreatitis-induced inflammatory model has

demonstrated that attenuation of inflammation leads to positive disease outcomes.

Specifically, exacerbation of inflammation tends to worsen the prognosis potentially

through up-regulation of endogenous TRP channels in sensory neurons (Xu et al.,

2007,Nilius et al., 2007a,Nilius et al., 2007b).

In addition to increased sensitivity of TRPV1 channels in states of inflammation,

TRPV1 plays a role in satiety and GI motility (Zhao et al., 2010). TRPV1 channels act

centrally, within the DVC, to modulate synaptic input to the motor neurons of the DMV

(Derbenev et al., 2006,Zsombok et al., 2011,De Man et al., 2008). This modulation has

significant outcome on all visceral function, particularly the motility of the stomach

(Zsombok et al., 2011). TRPV1 is found abundantly throughout the DVC and has been

shown to be specific to GABAergic neuron activation (Derbenev et al., 2006). When

TRPV1 channels of DMV neurons are activated by capsaicin, increased temperature, or

decreased pH, there is an increase in action potential-independent inhibitory input onto

DMV neurons. The amount of glutamate from the NTS does not determine the specific

DMV neuron activation; rather the glutamate release is mediated by TRPV1 and is then

specific for inhibitory actions on DMV neurons. The inhibitory action reduces firing of

24

the excitatory DMV neurons causing a withdrawal of excitatory input to gastric motility.

This effect is easily blocked by TRPV1 antagonists (Derbenev et al., 2006) indicative of

TRPV1-specific inhibitory effects responsible for early satiety and gastric dysmotility

seen post-SCI.

1.4 Summary

Previous work in our laboratory indicates that gastric functioning is compromised

post-SCI, but the exact mechanism responsible for this remains obscure. Our limited

mechanistic understanding of GI dysfunction following SCI precludes any evidence-

based development of effective therapies to treat individuals with SCI. Our review of

literature suggests that post-SCI GI dysfunction may be due to a combination of factors

including vascular hypotension that provokes visceral hypoperfusion and tissue ischemia,

systemic inflammation, diminished sensitivity to GI peptides, and increased TRPV1

expression. It is known that TRPV1 channels are activated during times of tissue injury

or inflammation, which we propose is a possible cellular mechanism involved in gastric

dysfunction post-SCI. Further, we suggest that the relationship between receptors for GI

peptides, such as CCK, and TRPV1 within the afferent vagus nerve is intimately involved

in the gastric dysfunction post-SCI. This thesis sets forth the novel hypothesis that post-

SCI dysmotility is due to GI inflammatory processes which hypersensitize vagal afferent

signaling through TRPV1 ion channel expression. The research design and techniques

incorporated into this work will be the first time these techniques are applied to

investigate the role of GI inflammation leading to the unique derangements in gastric

vagal afferents following SCI.

25

Figure 1.1 Schematic representation of the gastric vago-vagal reflex circuit

Gastric vagal sensory signals (green) are transmitted to the brainstem via vagal afferent fibers.

Cell bodies for these vagal afferents reside within the nodose ganglion. Vagal afferents enter the

brainstem by way of the tractus solitarius (ts) and terminate onto second order neurons (blue)

within the nucleus tractus solitarius (NTS) principally as a glutamatergic (Glu) synapse. The

parasympathetic preganglionic neurons (black) of the dorsal motor nucleus of the vagus (DMV)

relay the motor output back to the stomach. Preganglionic DMV motor neurons innervate gastric

enteric neurons through two competing pathways: ACh (gastric contraction) and NANC (gastric

relaxation). GABA = gamma-aminobutyric acid; NE = norepinephrine; AP = area postrema;

ACh = acetylcholine; NO = nitric oxide; VIP = vasoactive intestinal peptide

26

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33

Chapter 2:

Fabrication and Implantation of Miniature Dual-

element Strain Gages for Measuring In Vivo

Gastrointestinal Contractions in Rodents

This paper was published in Journal of Visualized Experiments (JoVE)

HOLMES,G.M., SWARTZ,E.M., and MCLEAN,M.S. (2014). Fabrication and Implantation of Miniature

Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents. J. Vis. Exp. 91:

51739.

34

2.1 ABSTRACT

Gastrointestinal dysfunction remains a major cause of morbidity and mortality.

Indeed, gastrointestinal (GI) motility in health and disease remains an area of productive

research with over 1,400 published animal studies in just the last 5 years. Numerous

techniques have been developed for quantifying smooth muscle activity of the stomach,

small intestine, and colon. In vitro and ex vivo techniques offer powerful tools for

mechanistic studies of GI function, but outside the context of the integrated systems

inherent to an intact organism. Typically, measuring in vivo smooth muscle contractions

of the stomach has involved an anesthetized preparation coupled with the introduction of

a surgically placed pressure sensor, a static pressure load such as a mildly inflated

balloon or by distending the stomach with fluid under barostatically-controlled feedback.

Yet many of these approaches present unique disadvantages regarding both the

interpretation of results as well as applicability for in vivo use in conscious experimental

animal models. The use of dual element strain gages that have been affixed to the serosal

surface of the GI tract has offered numerous experimental advantages, which may

continue to outweigh the disadvantages. Since these gages are not commercially

available, this video presentation provides a detailed, step-by-step guide to the fabrication

of the current design of these gages. The strain gage described in this protocol is a design

for recording gastric motility in rats. This design has been modified for recording smooth

muscle activity along the entire GI tract and requires only subtle variation in the overall

fabrication. Representative data from the entire GI tract are included as well as discussion

of analysis methods, data interpretation and presentation.

35

2.2 INTRODUCTION

Experimental studies that record in vivo gastrointestinal (GI) motility across a

number of experimental conditions remain a powerful tool for understanding the

underlying normal and pathophysiological processes necessary for nutrient homeostasis.

Traditionally, numerous experimental methodologies, some with similarities to those

found in clinical practice (Szarka & Camilleri, 2009), have been employed to directly

quantify changes in GI contraction rate (Pascaud et al., 1978,Gourcerol et al.,

2011,Américo et al., 2010,Fujitsuka et al., 2012), intraluminal pressure (Monroe et al.,

2004,Herman et al., 2008), or the GI transit of non-absorbable markers (Gondim et al.,

1999,Van Bree et al., 2013) or stable isotopes (Qualls-Creekmore et al., 2010b,Qualls-

Creekmore et al., 2010a,Choi et al., 2007). Each of these techniques has unique

advantages and disadvantages, which have been addressed previously in the literature.

For example, the utility of balloon manometry to quantify pressure changes has been

questioned due to the inherent compliance of the balloon material while gastrointestinal

recovery of non-absorbable markers requires euthanizing the experimental animal for a

single data point. Recently, the application and validation of a miniaturized arterial

pressure catheter has been reported that offers a non-surgical method for monitoring

gastric contractility in rats and mice (Gourcerol et al., 2011). While an orogastrically

placed pressure transducer effectively eliminates confounding variables on

gastrointestinal function by avoiding invasive surgical procedures, such an approach is

only suitable for anesthetized preparations. Furthermore, the lack of visual guidance does

not permit consistent placement of the transducer within specific regions of the stomach.

36

As such, this application is restricted to the stomach or colon since visualization, coupled

with the relatively stiff transducer wire, within the duodenum or ileum is not an option.

Similarly, the bio-magnetic alternate current biosusceptometry (ACB) technique

has been validated for GI contraction analysis (Américo et al., 2010). While the ACB

technique provides a non-invasive approach for measuring gastrointestinal contractions,

ACB suffers from a similar limitation in that the use of ingested magnetic detection

media does not permit precise recording of specific regions of the GI tract. This

limitation can be overcome through the surgical implantation of magnetic markers.

Nonetheless, the ACB technique necessitates that the animal be anesthetized for data

collection.

Ultrasonomicrometry has been employed in some GI studies (Adelson et al.,

2004,Xue et al., 2005) in order to take advantage of the small size, spatial, and temporal

advantages of piezoelectric crystal transmitter/receivers. Waves of gastric smooth muscle

contraction are not a high-frequency event and occur at a rate of approximately 3-5

cycles/min. Therefore, the temporal advantages of sonomicrometry may be unnecessary

to justify the cost. Furthermore, while linear motion is accurately measured with

sonomicrometry, limitations have been presented regarding accurate gastrointestinal data

interpretation that may result from implanting an insufficient number of crystals (Xue et

al., 2005).

Based upon the original designs of Bass and colleagues (Bass & Wiley,

1972,Pascaud et al., 1978) this visualized protocol more fully documents the step-by-step

fabrication and experimental application of miniature, dual element strain gages that

possess high sensitivity and flexibility for recording smooth muscle contractions along

37

the entire GI tract. The dimensions of the strain gage elements are suitable for any rodent

application since sensitivity and size of the finished strain gage are most dependent upon

the silicone sheets encapsulating the elements. These strain gages are readily adapted for

acute and chronic application in anesthetized and freely behaving laboratory animal

models thereby providing a single technique for quantifying smooth muscle contractions.

2.3 PROTOCOL

All procedures followed National Institutes of Health guidelines and were

approved by the Institutional Animal Care and Use Committee at the Penn State Hershey

College of Medicine. Rats were housed using common vivarium practices. Note: This

protocol uses male Wistar rats ≥8 weeks of age and initially weighing 175-200 g.

2.3.1 Procedures for fabrication of strain gage

Most tooling and components remain available from the original or successor

companies and are summarized in Table 2.1.

2.3.1.1 Preparation and Bonding of two single element strain gages

Always handle the strain gage elements (EA-06-031-350) carefully with clean

Dumont #5 forceps. To limit unwanted movement of elements, use a small, clean, self-

adhesive piece of paper with the adhesive side facing up to secure elements to the work

surface without the risk of contamination or excessive adhesion.

Bond two single strain gage elements back-to-back, to form a dual element. Clean

the back of each element film with Isopropyl alcohol and allow drying by evaporation

(drying with gauze often introduces fiber contaminants that are difficult to remove).

Under stereomicroscope guidance (1-3X), and using a clean artist brush (10-0 camel

hair), apply a thin film of epoxy-phenolic adhesive to the back of one element and

38

immediately place the opposing back of the second element in contact and align the foil

grids. (Fig. 2.1A).

Do not clamp the bonded elements since excess epoxy may contaminate the

elements and pressure may cause misalignment of the grids. Place the bonded elements in

a 50-60°C oven overnight to fully cure the epoxy.

Note that two-part epoxy has a usable refrigerated-life of only 6 weeks after

mixing. Bond and cure a sufficient supply of elements at one time and store them in a

clean, dust-free, environment for later use.

2.3.1.2 Sizing and wiring dual element strain gages

Trim bonded dual elements to a final size of 3X3mm with a #11 scalpel blade.

Delay trimming the topmost portion of the dual elements at this time in order to have an

area for safely handling the element. (Figure 2.1A).

Each element requires a four-conductor wire fabricated from three-conductor,

bondable, Teflon insulated wire (P/N 336-FTE). Disassemble one 30cm braided strand of

three-conductor wire into three constituent wires

To make a four wire cable, pair one of the resulting single wires with the like-

colored wire contained within a second 30cm length of three-conductor wire. In the

following steps, these matching colored wires will be joined at the terminal end to form a

common wire for the final strain gage (Figure 2.1B).

Remove approximately 1mm of Teflon insulation from both ends of each wire

with thermal wire strippers. Using activated rosin soldering flux and low temperature

solder (melting point 183º C) tin the wire ends with a soldering pencil.

39

For the next stage, a micro-soldering tip is needed to form a more discrete solder

joint to prevent heat damage to the film layer of the element (Figure 2.1C). To fabricate

a smaller micro-soldering tip, wrap a small piece of copper wire (~0.25mm diameter)

once around the standard soldering tip, ensuring that the copper wire extends beyond the

length of the standard soldering tip.

Flux just the solder pads on one side of the bonded dual element with a clean 10-0

brush and solder one single lead and one of the paired common leads to the solder pad

(Figure 2.1D). Residual flux can be removed afterward with a clean brush dipped in resin

solvent

Repeat the process on the opposite side, ensuring that the remaining common wire

lead is soldered to the pad opposite the original common lead.

2.3.1.3 Testing and epoxying dual element strain gages

Solder gold socket connectors (E363/0) to the free ends of the wire leads. At this

point, connect the strain gage to a recording amplifier (described below) to test the

integrity of the dual element assembly.

Measure the resistance with a good-quality volt-ohm meter. Elements register a

resistance of approximately 350 Ω. Re-solder inadequate connections at this point with

fresh solder.

If the solder connections and the dual element assembly are deemed satisfactory,

trim off any remaining element film.

Insulate the solder joints on the element solder pads with a thin layer of two-part

silicone-rubber epoxy resin (P/N E211). Best results are achieved if the resin is allowed

to partially cure for 20-30 min prior to application (Figure 2.1E).

40

2.3.1.4 Encapsulating dual element strain gages in silicone

Cut three pieces of 0.5mm thick silicone sheet (P/N 20-20) to 15mm2 and clean

the silicone with distilled water. Cut one piece of silicone sheet into a U-shape in order to

accommodate the final dual element assembly without deforming the encapsulating

silicone (Figure 2.1F).

Coat the inner surfaces of the notch-free silicone sheets with clear silicone

adhesive.

Sandwich the dual element assembly within the notch and the aligned outer

sheets, then gently press out any excess silicone, as well as air bubbles, from the center

outward. Carefully clamp the encapsulated assembly between two blocks of metal bar

stock for 24 hr to ensure uniform thickness and that no deformations occur.

Allow the excess silicone to remain along the boundaries of the assembly and

cure. This excess will be removed when the sheet silicone is trimmed to the desired final

dimensions (commonly 6mmX8mm; Figure 2.1G).

2.3.1.5 Completion of wire connector and calibration

Reinforce the solder joint of the gold socket connectors on the individual terminal

wire leads with 3mm (1/8 inch) shrink tubing and align within a plastic electrode pedestal

(MS363, Figure 2.1H). Secure the electrode pedestal and wires with 0.125- and 0.25-inch

diameter shrink tubing to prevent disconnection during the experiment (Figure 2.1I).

Strain gage signals are processed through a high gain bridge amplifier (P/NAMP-

01-SG). Connect the strain gage to the amplifier using a cable with a mated plug (363-

SL/6) to match the electrode pedestal. The threaded cap provides additional security to

maintain uninterrupted signals during the experiment.

41

Adjust the Bridge, Balance and Gain settings on the amplifier to a dedicated strain

gage per manufacturer instructions. Affix the end of the strain gage where the wires exit

horizontally to a rigid clamp and calibrate by placing a 1g static load on the opposite end

as originally described by Pascaud and colleagues (Pascaud et al., 1978,Holmes et al.,

2009,Tong et al., 2011).

2.3.2 Surgical Procedures for Acute Implantation of Strain Gage

2.3.2.1 Animal Care and Preparation:

Food deprive experimental animals the night before surgical implantation (water

may be provided ad libitum).

Deeply anesthetize the animal. Thiobutabarbital (100–150 mg/kg; i.p. for rats) is

preferred for terminal (i.e., non-survival) strain gage implantation and experimentation

due to sustained anesthetic effect and minimal alteration of gastric reflexes in the rat

(Qualls-Creekmore et al., 2010b). Test for absence of paw pinch reflex to determine

depth of anesthesia.

Prepare the rat for aseptic surgery as dictated by the experimental design and

approved IACUC guidelines including sterilizing surgical tools, shaving incision sites,

applying vet eye ointment and disinfecting all surgical areas.

2.3.2.2 Tracheal intubation for terminal experiments:

For long-duration, terminal, experiments the rat must be intubated with a tracheal

tube to maintain an open airway. Make a 1-2-cm midline incision on the ventral side of

the neck from the inferior border of the mandible to the sternal notch.

42

Separate the underlying strap muscles using blunt dissection at the midline to

expose the trachea. Isolate the trachea from the underlying esophagus and place a loop of

3-0 ethilon suture between the trachea and esophagus to form a ligature.

Open the trachea anteriorly by making a small cut in the membrane between two

of the cartilaginous rings of the trachea just distal to the thyroid gland. Insert a small

piece of polyethylene tubing (P/N PE-270), 5mm in length and beveled at one end) into

the trachea and secure it into place with the ligature.

Put the strap muscles back in place and suture the overlying skin with 3-0 ethilon.

2.3.2.3 Strain gage instrumentation to gastrointestinal surface:

Thread the four corners of the strain gage with 4-5cm lengths of 4-0, or smaller,

sterile silk suture using a #14 taper point 3/8 circle needle prior to surgery. Silk suture

provides a high level of flexibility and is less likely to damage the silicone encapsulating

the strain gage element.

Note that silk thread is acceptable for non-survival surgeries and for internal

applications, where the wicking of bacteria across an epithelial barrier is not a risk. In

applications requiring survival surgery, a prolene suture is necessary in order to reduce

the risk of infection inherent in the braided cloth fibers of the silk suture.

Perform a laparotomy by incising the abdominal skin along the midline. Section

the rectus abdominus musculature along the connecting linea alba (avascular) to prevent

bleeding. Then make a very superficial midline incision in the parietal peritoneum to

avoid lacerating underlying abdominal viscera.

43

Exteriorize the stomach with the aid of saline-soaked cotton tipped applicators.

Keep the stomach in its anatomical position by carefully placing it on a saline-soaked

gauze pad at the caudal end of the abdominal incision.

Align the grid of the encapsulated strain gage in parallel with the circular smooth

muscle fibers. Using the previously threaded sutures (step 2.3.1), attach the corners of the

gage to the ventral serosal surface of the gastric corpus using a #14 taper point 3/8 circle

needle. In order to minimize tissue damage and potential bleeding, do not use cutting-

edged needles and do not perforate any superficial blood vessels on the surface of the

stomach.

Begin the suture pattern of the gage along the greater curvature of the stomach

near the fundus/corpus boundary and proceed next along the fundus/corpus boundary

toward the lesser curvature. The serosa underlying the strain gage should neither be slack

nor overly stretched in order to obtain the best results.

Carefully return the stomach to its anatomical position using saline-soaked cotton

tipped applicators.

In an acute model, exteriorize the strain gage leads at the caudal end of the

midline incision before closure of the abdominal incision. Secure the free wires to the

animal (e.g., hind foot) in order to provide strain relief during manipulation of the animal

or terminal wire connector. Close the rectus abdominus muscles and the abdominal skin

separately with 3-0 nylon suture. In a chronic model, secure the leads subcutaneously

along the dorsal side of the rat and exteriorize them above the skull (Miyano et al., 2013).

After surgical instrumentation, place animals in a stereotaxic frame to support the

head and elevate the upper torso. The latter step helps to reduce respiration artifact during

44

recording. Monitor rectal temperature and maintain at 37±1°C using a feedback-

controlled heating pad.

At the conclusion of terminal experiments utilizing thiobutabarbital anesthesia,

the animal must be euthanized in a manner consistent with American Veterinary Medical

Association (AVMA) Guidelines on Euthanasia.

2.3.2.4 Gastric Motility recordings

Amplify the strain gage signal with any commercially available DC bridge

amplifier. Record the DC output signal on a computer using the chart recorder function of

any commercially available data acquisition system. Note: A hardcopy of the amplifier

output can be generated through a polygraph chart recorder.

2.3.2.5 Representative Measurement of Gastric Contractions Following Brainstem

Stimulation

2.3.2.6 Exposure of brainstem and fourth ventricle

After surgical instrumentation, and placement of the Thiobutabarbital-

anesthetized animal in a stereotaxic frame, make a 1.5-2 cm midline skin incision from

the occipital bone toward the base of the neck.

Separate the connective tissue joining the bilateral muscle bellies of the

underlying neck muscles along the midline (muscles from superficial to deep are levator

outis longus cranial portion, levator outis longus caudal portion, and platysma cranial

portion).

Detach the levator outis longus from the occipital bone once the midline is clearly

defined and exposed.

45

Carefully expose the caudal region of the skull by using blunt dissection to detach

platysma muscle from the underlying dura mater.

Use a new 25 gauge needle to carefully detach the dura mater along the foramen

magnum extending bilaterally to the occipital condyles.

Use #5 Dumont forceps to remove the pia and arachnoid meninges overlying the

fourth ventricle and expose the brainstem.

2.3.2.7 Administering fourth ventricle thyrotropin releasing hormone or intravenous

sodium nitroprusside

Weigh and dissolve thyrotropin releasing hormone (TRH) in sterile saline to reach

a final concentration of 5 µM TRH.

Weigh and dissolve sodium nitroprusside (SNP) in sterile saline to reach a final

concentration of 150 µM SNP.

Using a 10 µl syringe, administer 2µl of TRH (final dose equals 100 pmol) to the

dorsal surface of the brainstem fourth ventricle to facilitate recording of gastric

contractions.

Using a sterile syringe and 27 gauge needle, administer 150 µmol/kg of SNP

through the tail vein to facilitate recording of gastric relaxation.

2.4 REPRESENTATIVE RESULTS

Representative data from a Thiobutabarbital -anesthetized rat are shown in Figure

2.2. The top trace represents the gastric corpus contractions from the rat during the

brainstem administration of thyrotropin releasing hormone (TRH, 100 pmol), a known

motility-enhancing peptide (Gourcerol et al., 2011,Holmes et al., 1995). It shows baseline

contractions prior to the increase in phasic gastric smooth muscle activity. Note: Analysis

46

of these peaks in gastric contractions follow the original formula devised by Ormsby and

Bass (Ormsbee & Bass, 1976):

Motility Index= (N1x1) + (N2x2) + (N3x4) + (N4x8)

Based upon this formula, N equals the total number of peaks in a particular

milligram range. Therefore, presuming that a 0 mg signal is indicative of no gastric

motility, the grouping of peak-to-peak sinusoidal signals may be calculated as 25-50 mg,

60-100 mg, 110-200 mg and signals greater than 210 mg for N1 through N4, respectively.

This formula is less sensitive to baseline tone fluctuations that naturally occur across

several seconds or minutes. Such fluctuations would have to be subtracted in order to

generate valid area using under the curve measurements (Gourcerol et al., 2011).

The second trace demonstrates a reduction in baseline gastric smooth muscle tone

from the same animal in response to the nitric oxide donor, sodium nitroprusside

(20mg/kg i.v.). Data representing an inhibition of gastric smooth muscle activity are

readily analyzed by the reduction in signal voltage between baseline and maximal

response. This voltage signal can then be used to derive the equivalent static load, in

grams, if the strain gage was calibrated prior to the experiment. These representative data

demonstrate the bidirectional capabilities of a dual element strain gage that has been

properly attached to the gastric serosa.

The third trace represents basal smooth muscle contractions recorded by a sub-

miniature strain gage sutured to the serosal surface of the duodenum of a fasted rat. The

orientation of the strain gage elements were also in parallel with the circular muscle of

the duodenum.

47

2.5 DISCUSSION

The procedures presented here allow individual laboratories to fabricate sensitive

miniature strain gages for biological applications including, but not limited to,

gastrointestinal motility in small laboratory animals. Since the commercial manufacture

of these strain gages has ceased, laboratories investigating gastrointestinal function are

limited to other techniques which may not permit the full range of experimental

applications that are available. This report provides an updated and more detailed

description of previously described techniques (Bass & Wiley, 1972). The text and

accompanying video specifically address solutions to common pitfalls that we recognized

during development and mastery of the fabrication process.

Each step, as described, presents techniques to successful fabrication. Careful

attention to cleanly and securely soldering all connections as well as avoiding damage to

the element with excessive heat from the soldering process are the most frequent

challenges to success. The fine gauge wire is prone to breaking if it is not properly

reinforced with shrink tubing or silicone epoxy and will result in an absence of signal

when the gage is gently flexed. A strain gage with a broken or disconnected wire in the

vicinity of the gold connectors within the plastic terminal pedestal is the most common

failure of a previously functional gage. Individual gages can be carefully disassembled by

removing the shrink tubing in order to expose the broken wire. After re-soldering the

wire to the gold connector, the entire gage is reassembled with new shrink tubing.

With a bit of practice and careful attention to fabricating strain gages of uniform

dimensions, affixing strain gages relative to clear landmarks (e.g. Greater gastric

48

curvature, fundus/corpus boundary), and avoiding damage to the vasculature, novice

users will rapidly develop the ability to achieve consistent results.

Encapsulating the dual element in three layers of silicone creates a durable and

flexible yet highly sensitive strain gage that will last over repeated use with proper care.

The high sensitivity of an unencapsulated strain gage is minimally affected by any

resistance that is imparted by the silicon laminate. Thinner silicone sheets (P/N 20-05) are

recommended in order to modify the gage for intestinal applications or for fabricating

smaller gages for mice and discrete gut regions such as sphincters and esophagus. Extra

caution is required since thinner gages have diminished resistance to tearing of the

silicone sheet during implantation.

Surgical difficulties with the use of these gages often result from excessive

manipulation of the visceral organs or misalignment of the gage during implantation. The

former likely initiates neural and inflammatory processes that directly lead to impaired GI

motility, (Fukuda et al., 2005,Van Bree et al., 2013) though both pitfalls are easily

remedied by refinement of surgical technique. This may include altering the length and

starting point of the midline incision into the abdomen as well as minimizing the

manipulation of the viscera during exteriorization and replacement of the stomach.

The validity and fidelity of these strain gages have been discussed previously

(Bass & Wiley, 1972,Pascaud et al., 1978). We, and others, routinely measure gastric

smooth muscle activity in acute, anesthetized preparations (Holmes et al., 2009,Browning

et al., 2013). With adequate instrumentation, a single investigator can instrument and

acquire data from up to four animals in a single day. Additionally, implantation of

49

multiple gages within the same animal allows one to measure the relationship between

adjacent, or distant, regions of the gastrointestinal tract.

In summary, the fabrication of these subminiature strain gages allows for a wider

range of studies utilizing a common array of implantation techniques, instrumentation

and data analysis. Among applications across the entire gastrointestinal tract, these gages

allow for cross comparison of data collected from A) acute and/or chronic experimental

designs; B) multiple (simultaneous) recording sites from within a single animal; and C) a

wider range of experimental interventions.

2.6 Acknowledgements

Research funding was received through the National Institute of Neurological Disorders

and Stroke (NS049177). The authors wish to acknowledge the intellectual contributions

of the late Dr. Paul Bass and his colleagues to the original design of the strain gages; and

Carol Tollefsrud for the fabrication and marketing of the strain gages until the cessation

of production in 2010 as well as for her insightful correspondence.

50

Table 2.1. Parts and tooling for strain gage fabrication.

51

Figure 2.1. Principal stages of strain gage fabrication.

A) Dual bonded elements that have been trimmed on three of four sides to final

dimensions. B) Representative ends of wires configured for attachment to gage elements

(left) and terminal connectors (right). Note that dual read leads are joined only at the

terminal end (arrowhead). C) Representative placement of a strand of copper wire in

proximity to fine (1.5mm) soldering tip. Maintaining fresh solder along this junction

(arrowhead) ensures sufficient heat transfer through the micro tip to melt 63%

Tin:36.65% Lead:0.35% Antimony solder. D) Representative extent of solder joints

between wire leads and solder pads on the gage element. E) Properly potted solder joints.

F) Representative notch in the internal silicone laminate sheet to accommodate strain

gage element without deforming completed element. G) Bonded layers of silicone sheets

(three in total) forming a completed strain gage prior to final sizing. H) Wire connections

to gold plated sockets are reinforced with layers of succeedingly larger diameter shrink

tubing before insertion into electrode pedestal. I) Final shrink wrap affixing of terminal

connectors and electrode pedestal.

Calibration bars: A-D, 5mm; E, 2mm; & F-I, 5mm.

52

Figure 2.2. Representative motility traces generated with fabricated dual element

strain gages.

Recordings made from the anterior gastric corpus during an increase in gastric

contractions (top trace) and during an inhibition of gastric contractions (middle trace) and

duodenum (bottom trace) of fasted rats (200-250g).

53

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sphincter motility evoked by intravenous CCK-8 as monitored by ultrasonomicrometry in

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Américo MF,ques RG, Zandoná EA, Andreis U, Stelzer M, Corá A, Oliveira RB,

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Bass P, Wiley JN. Contractile force transducer for recording muscle activity in

unanesthetized animals. J Appl Physiol 1972; 32: 567-70.

Browning KN, Babic T, Holmes GM, Swartz E, Travagli RA. A critical re-evaluation of

the specificity of action of perivagal capsaicin. J Physiol 2013; 591: 1563-80.

Choi KM, Zhu J, Stoltz GJ, Vernino S, Camilleri M, Szurszewski JH, Gibbons SJ,

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Fukuda H, Tsuchida D, Koda K, Miyazaki M, Pappas TN, Takahashi T. Impaired gastric

motor activity after abdominal surgery in rats. Neurogastroenterol Motil 2005; 17: 245-

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Gondim FA, de Alencar HM, Rodrigues M, da Graca L, dos Santos R, Rola FH.

Complete cervical or thoracic spinal cord transections delay gastric emptying and

gastrointestinal transit of liquid in awake rats. Spinal Cord 1999; 37: 793-9.

Gourcerol G, Adelson DW, Million M, Wang L, Tache Y. Modulation of gastric motility

by brain-gut peptides using ael non-invasive miniaturized pressure transducer method in

anesthetized rodents. Peptides 2011; 32: 737-46.

Herman MA, Niedringhaus M, Alayan A, Verbalis JG, Sahibzada N, Gillis RA.

Characterization of noradrenergic transmission at the dorsal motor nucleus of the vagus

involved in reflex control of fundus tone. Am J Physiol Regul Integr Comp Physiol 2008;

294: R720-R729.

Holmes GM, Rogers RC, Bresnahan JC, Beattie MS. Thyrotropin-releasing hormone

(TRH) and CNS regulation of anorectal motility in the rat. J Auton Nerv Syst 1995; 56: 8-

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Holmes GM, Browning KN, Tong M, Qualls-Creekmore E, Travagli RA. Vagally

mediated effects of glucagon-like peptide 1: in vitro and in vivo gastric actions. J Physiol

2009; 587: 4749-59.

Miyano Y, Sakata I, Kuroda K, Aizawa S, Tanaka T, Jogahara T, Kurotani R, Sakai T.

The role of the vagus nerve in the migrating motor complex and ghrelin- and motilin-

induced gastric contraction in suncus. PLoS ONE 2013; 8: e64777.

Monroe MJ, Hornby PJ, Partosoedarso ER. Central vagal stimulation evokes gastric

volume changes in mice: ael technique using a miniaturized barostat. Neurogastroenterol

Motil 2004; 16: 5-11.

Ormsbee HS, Bass P. Gastroduodenal motor gradients in the dog after pyloroplasty. Am J

Physiol 1976; 230: 389-97.

Pascaud XB FAU, Genton MJ FAU, Bass P. A miniature transducer for recording

intestinal motility in unrestrained chronic rats. Am J Physiol Endocrinol Metab

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and motility in rats with experimental spinal cord injury. Neurogastroenterol Motil

2010a; 22: 62-e28.

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liquid meal in conscious rats and during sustained anaesthesia. Neurogastroenterol Motil

2010b; 22: 181-5.

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Van Bree SHW, Cailotto C, Di Giovangiulio M,sen E, van der Vliet J, Costes L,

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55

Chapter 3:

Gastric vagal motoneuron function is maintained

following experimental spinal cord injury

This paper was published in Neurogastroenterology and Motility

SWARTZ,E.M. and HOLMES,G.M. (2014). Gastric vagal motoneuron function is maintained following

experimental spinal cord injury. Neurogastroenterol. Motil. 26(12): 1717-29.

56

3.1 ABSTRACT

Background: Clinical reports indicate that spinal cord injury (SCI) initiates

profound gastric dysfunction. Gastric reflexes involve stimulation of sensory vagal fibers,

which engage brainstem circuits that modulate efferent output back to the stomach,

thereby completing the vago-vagal reflex. Our recent studies in a rodent model of

experimental high thoracic (T3-) SCI suggest that reduced vagal afferent sensitivity to

gastrointestinal (GI) stimuli may be responsible for diminished gastric function.

Nevertheless, derangements in efferent signals from the dorsal motor nucleus of the

vagus (DMV) to the stomach may also account for reduced motility.

Methods: We assessed the anatomical, neurophysiological and functional

integrity of gastric-projecting DMV neurons in T3-SCI rats using: 1) retrograde labeling

of gastric-projecting DMV neurons; 2) whole cell recordings from gastric-projecting

neurons of the DMV; and, 3) in vivo measurements of gastric contractions following

unilateral microinjection of thyrotropin releasing hormone (TRH) into the DMV.

Key Results: Immunohistochemical analysis of gastric-projecting DMV neurons

demonstrated no difference between control and T3-SCI rats. Whole cell in vitro

recordings showed no alteration in DMV membrane properties and the neuronal

morphology of these same, neurobiotin-labeled, DMV neurons were unchanged after T3-

SCI with regard to cell size and dendritic arborization. Central microinjection of TRH

induced a significant facilitation of gastric contractions in both control and T3-SCI rats

and there were no significant dose-dependent differences between groups.

57

Conclusions: Our data suggest that the acute, 3 day to 1 week post-SCI,

dysfunction of vagally-mediated gastric reflexes do not include derangements in the

efferent DMV motoneurons.

3.2 INTRODUCTION

Spinal cord injury (SCI) imparts immediate, and long-term, changes to motor,

sensory and autonomic function. In addition to cardiovascular dysregulation (Weaver et

al., 2006,Inskip et al., 2009,Krassioukov, 2009), gastric dysmotility occurs following SCI

(reviewed in Holmes, 2012). Specifically, upper gastrointestinal (GI) dysfunction

following SCI includes impairment of gastric emptying as well as gastric and intestinal

motility (Wolf & Meiners, 2003,Kirshblum et al., 2002,Park & Camilleri, 2006).

Individuals with SCI also present reflux, abdominal pain, bacterial translocation,

bloating, and prolonged/delayed colonic transit (Fynne et al., 2012). One-third of patients

with complete quadriplegia for more than 1-year continue to experience GI symptoms

(Lu et al., 1998). Based upon previous reports, fatalities in SCI patients who initially

survived the injury may partially be caused by GI tract complications and depressed

immune responses that lead to sepsis (DeVivo et al., 1989,Miller et al., 1975,Riegger et

al., 2009). In brief, acute failure to regulate GI function following SCI may induce

greater morbidity and mortality through eventual bacterial overgrowth and translocation

(Bohm et al., 2013,Liu et al., 2004).

The reflex functions of the upper gastrointestinal organs, most notably the

stomach, are modulated by medullary neurons within the dorsal vagal complex (DVC).

The DVC is comprised of the area postrema (AP), the nucleus tractus solitarius (NTS),

and the dorsal motor nucleus of the vagus (DMV) and is located at the transition from the

58

open to closed medulla (Browning & Travagli, 2010). The DVC finely regulates the

coordinated delivery of nutrients to the duodenum by integrating GI stimuli that are

conveyed through the sensory vagus (Iggo, 1955,Blackshaw et al., 1987,Raybould,

1992), from spinosolitary inputs (Menetrey & Basbaum, 1987,Gamboa-Esteves et al.,

2001), as well as inputs arriving from higher CNS nuclei (Blevins et al., 2004,Morton et

al., 2005,Blevins & Baskin, 2010). Furthermore, the DVC has been shown to possess the

characteristics of a circumventricular organ (Gross et al., 1990,Cottrell & Ferguson,

2004) as well as active transport mechanisms for circulating cytokines. As a result, the

DVC is positioned to integrate the presence of circulating factors with neural input.

Additionally, passive permeability to blood-borne agents increases following

inflammatory and traumatic insults (Pan & Kastin, 2004) thus rendering the DVC

vulnerable to pathophysiological conditions. For example, in animal models of

inflammatory bowel disease (IBD), circulating cytokines (IL-1β, IL-6 and TNF-α),

presumptively entering the DVC through the permeable blood-brain barrier , initiate an

apoptotic cascade resulting in an in vitro decrease in DMV proliferation and an in vivo

decrease in gastric projecting DMV neurons (Ammori et al., 2008). Furthermore,

peripheral injections of IL-1β, systemic activation of TNF-α production, and central

administration of TNF-α rapidly suppress gastric motor activity (Tsuchiya et al.,

2012,Hermann et al., 1999,Emch et al., 2002). The presence of circulating inflammatory

cytokines, including IL-6 and IL-1βhave also been reported following experimental and

clinical SCI (Pan & Kastin, 2001,Hayes et al., 2002) and may compromise DMV

neuronal function and integrity as part of a larger systemic inflammatory response (Gris

et al., 2008).

59

Persistent gastroparesis has been reported in animal models of SCI (Leal et al.,

2008,Tong & Holmes, 2009). In particular, we have demonstrated that rats with

experimental high thoracic (T3-) SCI show a rapidly-developing, and prolonged, delay in

gastric emptying of a [13

C]-labeled solid meal (Qualls-Creekmore et al., 2010). The

neurocircuitry comprising the gastric vago-vagal reflex remains anatomically intact after

T3-SCI.While our previous report suggests that T3-SCI diminishes vagal afferent

sensitivity (Tong et al., 2011), derangements of gastric efferent signaling may play a role

in post-SCI dysmotility that has not been investigated.

The aims of the present study were to use an acute rat model of experimental SCI

to investigate 1) if experimental SCI induces rapid degeneration of gastric projecting

vagal motoneurons; 2) if the in vitro biophysical properties of DMV neurons demonstrate

reduced excitability; and 3) if in vivo gastric motility continues to respond to brainstem

microinjection of thyrotropin releasing hormone (TRH).

3.3 MATERIALS AND METHODS



College of Medicine. Male Wistar rats ≥8 weeks of age, upon entrance into the

experiment, and initially weighing 175-200 g (Harlan, Indianapolis, IN, USA) were used

and double housed in a room maintained at 21-24ºC and a 12:12-h light-dark cycle with

food and water provided ad libitum. After surgery animals were single housed, observed

daily, and food and body weight were recorded.

60

3.3.1 Surgical procedures and animal care

Rats were randomly assigned for SCI or surgical control and each group was

further divided into 3-day and 1-week post-surgical study groups. Prior to surgery, rats

were anesthetized with isoflurane (2-3%, 1 L min-1

O2) as necessary to achieve areflexia

(absence of palpebral reflex). All animals were administered ophthalmic ointment to

both eyes, buprenorphine (0.01mg kg-1

, s.c., Reckitt Benckiser Pharmaceuticals Inc.,

Richmond VA) to alleviate post-operative pain, and antibiotics (Baytril, 2.5 mg/ml

concentration at 1ml kg-1

s.c., Bayer, Shawnee Mission KS) to reduce post-surgical

infection prior to any surgical manipulation (Qualls-Creekmore et al., 2010). Once a

deep plane of anesthesia was achieved, the skin overlying vertebral thoracic levels 1-3

(T1-T3) was shaved and cleaned with three alternating applications of Nolvasan

(Chlorhexidine Acetate, Fort Dodge Animal Health, Fort Dodge, IA) and ethanol. The

surgical site was incised 3-4mm along the midline and the underlying spinous processes

were cleared of all musculature. Using fine tipped rongeurs, spinal T3 was exposed via

laminectomy of the T2 spinous process which extended laterally to the T2 transverse

processes as described previously (Tong & Holmes, 2009,Primeaux et al., 2007). The rats

were placed in the Infinite Horizon controlled impact device (Precision Systems and

Instrumentation, LLC, Lexington, KY) and clamped via the T1 and T3 spinous processes.

Once secure, a rapid 300 kDyne displacement of the cord and overlying dura was

performed. Procedures for the control animals were the same as for spinal injury except

that the spinal cord and surrounding dura mater was not disturbed following

laminectomy. Upon completing the surgical procedure, the muscle tissue overlying the

lesion site was closed in anatomical layers with Dexon II suture and the skin closed with

61

9mm wound clips. Animals were administered warmed supplemental fluids (5 cc lactated

Ringer’s solution, s.c.) and placed in an incubation chamber maintained at 37 C until the

effects of anesthesia had subsided.

Post-operative chronic care of both injured and control animals involved placing

corn cob filled animal housing tubs on a warming unit (Gaymar T-pump, Stryker,

Kalamazoo, MI) to maintain a warmed environment (ca. 25°C) and received

subcutaneous supplemental fluids (5-10 cc lactated Ringer’s solution) twice daily,

analgesics (buprenorphine 0.01mg kg-1

, IP) twice daily for 3 days and antibiotics (Baytril,

2.5 mg kg-1

) twice daily for 5 days after surgery. Bladder expression was performed at

least twice daily in animals with T3-SCI until the return of spontaneous voiding. The

ventrum of control animals was inspected daily without need for manual compression of

the bladder. Due to the reduction in locomotor capacity after T3-SCI, a reservoir of chow

was placed at head level in order to facilitate ease of access for feeding. All T3-SCI rats

ingested a measureable amount each day, thereby confirming that access to chow was

available. Bodyweight and food intake were measured daily for all animals. Since

animals were fasted overnight prior to euthanasia, only bodyweight and food intake for

day 2 (all animals) and for day 6 (those animals surviving 7-9 days) were selected for

analysis. In order to normalize the raw measurements of bodyweight and food intake

across animals, mean energy intake (MEI) was calculated by dividing the daily average

of kcal consumed per 100 g of bodyweight (bw) for each 2-day period of monitored

feeding.

62

3.3.2 Neuronal tracing

To identify DMV neurons projecting to the stomach, the retrograde neuronal

tracer, cholera toxin B subunit (CTB), was injected into the muscular layers of the gastric

corpus in one subset of animals (n=18) six days prior to T3-SCI surgery. The rats were

anesthetized as above with isoflurane, the abdomen was shaved and prepared for aseptic

surgery as described above. The stomach was isolated via an abdominal laparotomy, and

CTB (0.5% solution, 5 µl total volume) was injected via a glass micropipette (50 µm

diameter) in 4-6 sites so as to encompass a 5 mm2 region of the anterior gastric corpus

following the greater curvature of the stomach. The incision was closed in layers using

Dexon II suture and the skin closed with 9mm wound clips. Six days later, wound clips

were removed and these animals were randomly assigned to T3-SCI or control surgery.

Nine days after T3-SCI or control surgery, rats were euthanized for

immunohistochemistry.

3.3.3 Histological Processing

At the conclusion of every experiment, deeply anesthetized rats were

transcardially perfused with heparinized phosphate-buffered saline (PBS) until fully

exsanguinated and followed immediately with PBS containing 4% paraformaldehyde.

The brainstem and spinal cord at the lesion level were removed and refrigerated

overnight in PBS containing 20% sucrose and 4% paraformaldehyde. For

immunohistochemical processing, the entire rostrocaudal extent of the DVC was

sectioned on a freezing stage microtome and alternating coronal slices (40μm thick) were

saved in PBS-filled microplate wells for free-floating immunolabeling. For histological

63

staining of brainstem injection sites or T3-SCI lesion extent, tissue was sectioned (40μm

thick) and alternating sections were mounted on gelatin coated slides.

To verify lesion severity or control, spinal cord sections were stained with luxol

fast blue (LFB) to visualize myelinated fibers. LFB-stained slides were digitally imaged

on a Zeiss Axioscope light microscope and Axiocam CCD camera, imported into Adobe

Photoshop and contrast digitally enhanced to allow identification and threshold

measurements of LFB-stained (i.e., spared) white matter. For individual images, the

boundaries of the tissue slice were outlined to determine cross-sectional area. A separate

threshold histogram was generated and the pixels corresponding to LFB staining above

background were selected. These pixels were quantified and expressed per unit cross-

sectional area (Noble & Wrathall, 1985). The proximity of the T3 lesion center to the

cervical enlargement precluded an appropriate determination of spinal cord cross-

sectional area in undamaged tissue rostral to the injury (i.e., damaged tissue extended into

the cervical enlargement as described in (Tong & Holmes, 2009). Therefore, it was

necessary that the cross-sectional area of the intact spinal cords at T3 of comparably sized

animals be determined for normalization purposes. LFB-stained myelin in injured tissue

was then expressed as a percent of the total spinal cord cross-sectional area as would be

predicted by the intact tissue. Based upon our previous report (Tong et al., 2011) we

determined a priori that animals with white matter sparing ≤ 25% were categorized as

severe injury while those ≥ 25% were categorized as moderate injury.

To verify microinjection sites for in vivo recordings, brainstem sections were

stained with cresyl violet to verify the placement of the microinjection pipette tip. Stained

slides were digitally imaged on a Zeiss Axioscope light microscope, imported into Adobe

64

Photoshop and injection sites were mapped with the aid of a rat stereotaxic atlas.

(Paxinos & Watson, 1986)

3.3.4 Immunohistochemistry

After sectioning, free-floating brainstem sections were washed for 30min in a 1:1

pretreatment solution of Triton + PBS (TPBS; 1:200) and hydrogen peroxide. Between

each incubation step, sections were rinsed 3x5 minutes in PBS. Sections were blocked

for 1 hour in 10% normal donkey serum (NDS) in PBS. Sections were removed from

blocking solution and placed directly into primary antibody for incubation at room

temperature (Goat α-CTB; List Biologicals, Campbell, CA, USA; 1:40,000 in PBS).

Following 48 hours of antibody incubation, sections were removed, washed and then

incubated in biotinylated secondary antibody (donkey α goat; 1:500; Jackson

ImmunoResearch, West Grove, PA) for 2 hours. The Avidin-Biotin Complex (ABC)

Solution (Vectastain Elite ABC kit, Vector Labs, Burlingame, CA) was prepared

according to kit directions and sections were incubated for 1 hour. Sections were

exposed to a peroxidase reaction (Vector SG SK-4700; Vector Laboratories, Inc.

Burlingame, CA 94010) for as long as necessary to reveal immuno-reactive structures

(blue CTB-labeled neurons) against a light background. Sections were washed in PBS

and mounted onto gelatin-coated glass slides and air-dried overnight. Slides were placed

in Clear Rite and coverslipped with Permount. Slides were digitally imaged on a Zeiss

Axioscope light microscope and Axiocam CCD camera, imported into Adobe Photoshop

for analysis. Cell counts followed a highly-conservative protocol for inclusion. Non-

adjacent sections were randomly selected for analysis and only cells with a threshold

density in the ≥95th

percentile above baseline were counted as immunopositive.

65

3.3.5 Electrophysiology

Brainstem slices were prepared as described previously (Browning et al.,

2013,Swartz et al., 2014). Briefly, T3-SCI or control rats (n=5 each) were anesthetized

deeply (Isoflurane, 5%, 1 L min-1

O2) and euthanized via administration of a bilateral

pneumothorax. The brainstem was removed and cut into 3-4 coronal slices ( 300m thick)

encompassing the entire rostro-caudal extent of the DVC. Slices were incubated at

301°C in Krebs’ solution (in mM: 126 NaCl, 25 NaHCO3, 2.5 KCl, 1.2 MgCl2, 2.4

CaCl2, 1.2 NaH2PO4, and 11 dextrose, maintained at pH 7.4 by bubbling with 95% O2-5%

CO2) for at least 60-90 minutes before use. A single slice was then transferred to a

perfusion chamber (volume 500l) which was placed on the stage of a Nikon E600FN

microscope and kept in place with a nylon mesh. Brainstem slices were maintained at

351°C by perfusion with warmed Krebs’ solution at a rate of 2.5-3.0 ml min

-1. DMV

neurons were identified by their soma size and location (ventral to the smaller NTS

neurons and dorsal to the larger, heavily myelinated hypoglossal neurons). Whole cell

patch clamp recordings were made from DMV neurons using patch pipettes of 2-4MΩ

resistance when filled with a potassium gluconate solution (in mM: K gluconate 128, KCl

10, CaCl2 0.3, MgCl2 1, Hepes 10, EGTA 1, ATP 2, GTP 0.25 adjusted to pH 7.35 with

KOH) and a single-electrode voltage-clamp amplifier (Axopatch 1D, Molecular Devices,

Sunnydale, CA). Data were sampled at 10kHz, filtered at 2kHz, digitized via a Digidata

1320 interface and analyzed using pClamp 9 software (Molecular Devices). The liquid

junction potential was compensated at the beginning of the experiment; recordings were

discarded if the series resistance was >20 MΩ.

66

Basic membrane properties were assessed as described previously. (Browning et

al., 1999) Briefly, to calculate the membrane input resistance (Rin), the instantaneous

current displacement was measured after the voltage-clamped membrane was stepped

from -50mV to -60mV. To measure the action potential firing characteristics, DMV

neurons were current clamped at approximately -60mV and injected with depolarizing

current pulses (15ms duration) of intensity sufficient to evoke the firing of a single action

potential at the current pulse offset. The action potential duration at threshold was

measured, as was the amplitude of the afterhyperpolarization amplitude; the duration of

the afterhyperpolarization (decay constant, ) was fitted to a single exponential equation

and measured. To measure the action potential firing frequency, DMV neurons were

current clamped at approximately -60mV before being injected with depolarizing current

pulses (400ms duration) of increasing intensity (30-270pA). The number of action

potentials fired were counted and expressed as pulses per second (p.p.s.).

3.3.6 Morphological reconstructions and analysis

At the end of the electrophysiological recording, Neurobiotin© (2.5%; Vector

Laboratories, Burlingame, CA) included in the recording pipette was injected into the

neuron via the passage of subthreshold depolarizing current (400ms duration; 0.8Hz for

20min). The neuronal membrane was allowed to reseal for 10-20min following removal

of the pipette before the brainstem slice was fixed in Zamboni’s fixative (in mM: 1.6%

paraformaldehyde, 19mM KH2PO4 and 100mM Na2PO4 in 240ml saturated picric acid

and 1600ml water, adjusted to pH7.4 with NaOH) at 40C for at least 24hr.

Brainstem slices were cleared of fixative by repeatedly washing in PBS

containing Triton X-100; in mM: 115 NaCl, 75 Na2HPO4, 7.5 KH2PO4 and 0.15% Triton

67

X-100). Neurobiotin filled neurons were visualized as described previously (Browning et

al., 2013). Briefly, brainstem slices were incubated with avidin-D- horseradish peroxidase

solution (Vector Laboratories; 0.002% avidin D-horseradish peroxidase in PBS

containing 1% Triton X-100) for 2hr. The slices were then washed repeatedly in PBS-

Triton X before incubation in PBS containing diaminobenzidine, cobalt chloride and

nickel sulfate (0.05% diaminobenzidine in PBS containing 0.025% cobalt chloride and

0.02% nickel sulfate) for 30min. Slices were then exposed to 3% H2O2 for a period of

time sufficient for the adequate visualization of the Neurobiotin-filled neurons. Brainstem

slices were mounted on gelatin—subbed slides, air dried and dehydrated through a

graded series of alcohols and xylene before being mounted in Permount (Fisher

Scientific, Pittsburgh, PA).

The morphological characteristics of Neurobiotin-filled neurons were assessed

using Neurolucida software (MBF bioscience, Willison, VT) attached to a Nikon E400

microscope at a final magnification of X400. Three-dimensional reconstructions of

individual neurons were made and morphological properties assessed included soma area,

form factor (a measure of soma circularity where 1=perfect circle and 0=straight line),

dendritic branching (including number of segments and branch order) and dendritic

length in both X- and Y-axes. In order to be accepted, the neuronal reconstruction had to

have a mediolateral and rostrocaudal branch extension of at least 200m with no major

branches being severed during the initial sectioning of the brainstem slice and a soma

with no obvious damage from pipette retrieval. A subroutine of the Neurolucida software

was used to rescale the brainstem slice to 300m (the thickness of original slicing) to

68

correct for any optical and physical compression that may have occurred during fixation

and processing.

3.3.7 Gastric Motility recordings

In vivo gastric motility recordings and intracerebroventricular (ICV)

microinjections of TRH in the DVC were performed in control and T3-SCI rats (n=60).

Rats, which received only a T3-SCI or control surgery, were randomly divided into 3-day

and 1 week post-injury or post-surgical control study groups. Brainstem microinjections

of TRH and PBS-control were done in a dose-dependent manner to experimentally test

gastric motility. For the following pharmacological experiment, all rats were prepared as

follows: Following an overnight fast (water ad libitum), rats were deeply anesthetized

with thiobutabarbital (Inactin®, Sigma; 100–150 mg/kg; IP) and Dexamethasone (1

mg/kg s.c., Sigma, St. Louis, MO) was administered to prevent cerebral edema. Rats

were then intubated with a tracheal tube to maintain an open airway and a laparotomy

was performed. The stomach was isolated and a 6 X 8-mm encapsulated sub-miniature

strain gage of our own fabrication was aligned with the circular smooth muscle fibers and

sutured to the ventral surface of the gastric corpus (Holmes et al., 2014). The strain gage

leads remained exteriorized before closure of the abdominal incision. After surgical

instrumentation, animals were placed in a stereotaxic frame and rectal temperature was

monitored and maintained at 37±1°C (TCAT 2LV, Physitemp Instruments, Clifton, NJ).

After a midline incision and removal of the overlying dorsal neck musculature, the head

of the animal was oriented such that the floor of the fourth ventricle was exposed and the

brainstem surface was horizontally oriented in a manner that prevented washout of the

solution(s) applied. The pial membrane overlying the vagal trigone was dissected and the

69

exposed tissues were covered with a warm, saline-infused cotton patch. The strain gage

signal was amplified (QuantaMetrics EXP CLSG-2, Newton, PA) and recorded on a

polygraph (model 79, Grass, Quincy, MA) or on a computer using Experimenter’s

Workbench software (Datawave Technologies, Loveland, CO). After 1 hour of

stabilization, 10 min of baseline motility was recorded before any experimental

manipulation. The effects of TRH, (0, 3, or 10 pmoles/60nl) microinjected in the left

DVC (at coordinates from calamus scriptorius: +0.1–0.3mm rostro-caudal, 0.1–0.3mm

medio-lateral and −0.3-0.5mm dorso-ventral) adjacent to the area postrema, were

observed as recorded peaks that increased in frequency, height, and/or rose above

baseline (Holmes et al., 2014).

3.3.8 Drugs and chemicals

Inactin and all other salts were purchased from Sigma (St. Louis, MO). All

drugs were dissolved in sterile isotonic phosphate buffered saline (PBS; in mM: 147.6

NaCl, 83.3 NaH2PO4, 12.9 KH2PO4).

3.3.9 Statistical Analysis

Results are expressed as means ± S.E.M. with significance defined as P < 0.05.

Between group results from anatomical or in vivo studies, between pre- and post-

treatment motility values, were compared by one-way ANOVA and Tukey post hoc

analysis or paired t-test as appropriate (SPSS Inc, Chicago, IL). Results from in vitro

studies were compared using the Student’s grouped t-test.

70

3.4 RESULTS

3.4.1 Histological assessment of T3-SCI severity

Harvest of the brainstem for in vitro electrophysiology precluded processing of

the spinal cord for histological analysis. In animals receiving a spinal cord injury for

anatomical or in vivo studies, the range of total white matter sparing was 2 – 64% of

control. Based upon our criteria, animals were categorized as severe injury (range 2 –

24%; n=40) or moderate injury (range 27 – 67%; n=12) and segregated into their

respective groups for further behavioral, anatomical and physiological analysis. Due to

injury variability and subsequent low sample numbers for the immunohistochemical and

motility studies, animals categorized as moderate injury were excluded from further

analysis.

Analysis of total white matter of the T3 thoracic spinal cord from control animals

that had been prepared for immunohistochemistry or that were tested for motility at 3

days or 1 week after surgery revealed no damage to the spinal cord as a result of the

spinal laminectomy (Table 3.1; P > 0.05). Comparisons of control and T3-SCI rats,

however, demonstrated a significant reduction of white matter (P < 0.05) while there

were no group differences between T3-SCI rats prepared for immunohistochemistry or

that were tested for motility (Table 3.1; P > 0.05).

3.4.2 Assessment of post-injury weight loss and reduction of spontaneous oral intake of

food

There were no significant experimental group differences between the post-

operative bodyweights of animals designated as control, nor were there significant

experimental group differences between the post-operative bodyweights of animals

71

designated as severe T3-SCI (n = 40, P > 0.05). Therefore, the bodyweight and food

intake data were collapsed within surgical groups. On the second day after surgery, the

bodyweight was significantly lower between T3-SCI and surgical control animals (Table

3.2; P < 0.05). In animals prepared for CTB-immunohistochemistry or tested for gastric

motility 1 week after surgery, the mean bodyweight prior to fasting remained

significantly lower between T3-SCI and surgical control animals as the bodyweight of

surgical controls began to increase while T3-SCI animals remained consistently reduced

(Table 3.2; P < 0.05). At this same time point, the MEI displayed significant differences

between T3-SCI and surgical control (Table 3.3; P < 0.05). By 1 week, MEI remained

significantly reduced in T3-SCI animals compared to surgical controls despite a

significant increase from 3 day values in the T3-SCI animals (Table 3.3; P < 0.05).

Furthermore, MEI significantly increased from 3 day values for the surgical control

animals (Table 3.3; P < 0.05).

Taken together, our histological, bodyweight and feeding data verify the profound

severity, effectiveness and reproducibility of our surgical procedures for T3-SCI and

surgical control animals. All animals in these groups were selected for further data

analysis.

3.4.3 The number of CTB-immunoreactive DMV neurons projecting to the stomach is

unaffected by T3-SCI

Labeling of CTB-IR neurons in surgical control and T3-SCI animals revealed

corpus-projecting neurons in the DMV projecting to the anterior gastric corpus (Figure

3.1). These CTB-IR neurons extended from throughout the caudal, intermediate (sections

that included the AP), and rostral DMV. There were no significant differences between

72

T3-SCI and surgical controls in the number of CTB-IR gastric-projecting DMV neurons

in the intermediate DMV (control: 34.7±3.4, T3-SCI 39.2±6.7; P > 0.05). The

intermediate region of the DMV corresponds to our TRH microinjection site. No DMV

neurons from T3-SCI neurons displayed signs of necrosis/degeneration (e.g., vacuoles,

pyknosis, and/or axonal swelling).

These results suggest that T3-SCI does not induce any overt loss of DMV

neurons projecting to the stomach and that the number and staining intensity of

parasympathetic preganglionic neurons is similar to surgical controls.

3.4.4 T3-SCI does not alter DMV neuron electrophysiological or morphological

properties

Recordings were made from 25 control and 17 T3-SCI neurons. Of these,

complete electrophysiological and morphological properties were assessed in 25 and 20

control neurons, respectively, and 13 and 17 T3-SCI neurons, respectively. The

membrane properties of DMV neurons from control (n=25 cells) and T3-SCI (n=13 cells)

rats were assessed under current clamp or voltage clamp conditions as described

previously (Browning et al., 1999). As detailed in Table 3.4, T3-SCI did not affect either

the membrane input resistance or action potential firing properties of DMV neurons

(Figure 3.2). Complete morphological properties were assessed in 20 control and 17 T3-

SCI DMV neurons and are summarized in Table 3.5. T3-SCI did not alter any of the

morphological features of the recorded DMV neurons (Figure 3.2).

3.4.5 T3-SCI does not reduce brainstem sensitivity to TRH

Representative raw data traces demonstrate a rapid onset for gastric corpus

contractions following microinjection of TRH in all experimental groups (Figure 3.3).

73

Histologically verified microinjection of PBS in the left DVC did not induce any

significant percent change in gastric motility index above baseline in either control (n =

5) or T3-SCI rats (n = 5; P > 0.05; Figure 3.4).

In animals tested 3 days after surgery, microinjection of 3 pmol of TRH into the

left DVC of control animals induced a significant percent increase in gastric motility

index compared to baseline values (n = 5; P < 0.05; Figure 3.4). Similarly, microinjection

of 3 pmol TRH into the left DVC of T3-SCI animals produced a significant percent

increase in gastric motility index compared to individual baseline values (n = 5; P < 0.05;

Figure 3.4). Between subjects comparisons revealed there were no significant differences

between control (n=5) and T3-SCI (n=5) animal groups in the percent change of motility

index nor the duration of response to 3 pmol TRH (P > 0.05; Figure 3.4).

In a separate group of animals that were tested 3 days after surgery,

microinjection of 10 pmol of TRH into the left DVC of both control and T3-SCI animals

induced a significant percent increase in gastric motility index compared to baseline

values (n = 5/group; P < 0.05; Figure 3.4). Despite a trend toward an increase from the 3

pmol dose, there was not a significant difference in the percent change in motility index

between control animals receiving 3 or 10 pmol TRH (n = 5/group; P < 0.05; Figure 3.4).

In addition, there was no significant difference in both the percent change of motility

index and response duration to 10 pmol TRH between control and T3-SCI animals

(n=5/group; P > 0.05; Figure 3.4).

A separate cohort of animals was tested 1 week after surgery in order to more

closely approximate the post-injury time frame of our animals prepared for histology.

Microinjection of 3 pmol of TRH into the left DVC of control animals induced a

74

significant percent increase in gastric motility index compared to baseline values (n = 5;

P < 0.05; Figure 3.4). Similarly, microinjection of 3 pmol TRH into the left DVC of T3-

SCI animals significantly increased gastric motility index compared to baseline values (n

= 5; P < 0.05; Figure 3.4). There was no significant difference in both the percent

increase in motility index or response duration to 3 pmol TRH between control and T3-

SCI animals (n=5/group; P > 0.05; Figure 3.4). In a separate group of animals that were

tested 1 week after surgery, microinjection of 10 pmol of TRH into the left DVC of both

control and T3-SCI animals induced a significant percent increase in gastric motility

index compared to baseline values (n = 5/group; P < 0.05; Figure 3.4). The trend toward

an increase between the 3 and 10 pmol dose did not reach significance (n=5/group; P >

0.05; Figure 3.4). In a similar manner, the response duration between control and T3-SCI

animals following microinjection of 10 pmol TRH also demonstrated a non-significant

trend (P > 0.05; Figure 3.4).

3.5 DISCUSSION

The present study demonstrates that the gastroparesis induced during the acute

T3-SCI period does not involve morphological changes of gastric-projecting vagal

motoneurons or reduction in motor responses mediated by vagal motoneurons innervating

the gastric corpus. Our experimental data indicate that: 1) retrogradely-labeled gastric-

projecting DMV neurons demonstrated no difference in the number of CTB-labeled cells

between control and T3-SCI rats; 2) whole cell in vitro electrophysiological recordings

showed similar membrane properties in the DMV of control and T3-SCI rats; 3) the cell

size and dendritic arborization of Neurobiotin-labeled DMV neurons were unchanged

after T3-SCI; and 4) in vivo central microinjection of TRH induced a significant

75

facilitation of gastric contractions in both control and T3-SCI rats with no significant

dose-dependent differences between groups. These data suggest that, given the

appropriate afferent signals, the vagal efferent limb likely remains fully capable of

eliciting gastric motility following T3-SCI and lends further support that diminished

vagal afferent input to the brainstem neurocircuitry may be solely responsible for

diminished gastric reflexes after T3-SCI (Holmes, 2012). The mechanism leading to this

impairment remains to be determined but does not include DMV neuron loss.

Anatomical and physiological changes of DMV neurons have been recently

reported within 1 week following perivagal capsaicin (Browning et al., 2013). Those

studies demonstrated a loss of CTB-labeled, gastric-projecting DMV neurons; decreased

input resistance and excitability of DMV neurons as well as a decrease in number of

neurons responding to TRH with an increase in action potential firing; and decreased in

vivo response to brainstem microinjection of TRH. Therefore, we are confident that the

similar techniques employed within this study are robust enough to demonstrate any

reduction in the efferent limb of vagally-mediated gastric reflexes following SCI.

While perivagal application of a TRPV1 agonist, capsaicin, at a

supraphysiological dose (>30mM) does not physiologically reflect endogenous processes

resulting in vagal damage (Browning et al., 2013), emerging evidence in other animal

models suggests that vagal afferent and efferent fibers are susceptible to rapid

degenerative processes across several disease states. Vagal neuropathies following

ischemic insults have been demonstrated within hours following experimental apnea

(Zhang et al., 2010) and hypobaric hypoxia (Luo et al., 2012,Adak et al., 2014) whereby

vagal neuropathy has been proposed as a causal mechanism for cardiac arrhythmias.

76

(Aydin et al., 2011) Vagal neuronal degeneration has also been implicated within hours

after the experimental induction of blood-brain barrier dysfunction or trauma (Wu et al.,

2011), diabetes (Tay & Wong, 1994,Yan et al., 2009), and experimentally-induced colitis

(Ammori et al., 2008).

Comorbidities associated with SCI share features with the above pathologies and

would, hypothetically, predict DMV degeneration or vago-vagal reflex dysfunction.

Specifically, diminished sympathetic tone and reduced cardiovascular reflex function

below the level of the lesion may place the GI tract at risk for hypoxia with resulting

inflammation. The principal nutritive functions of the GI tract are critically dependent

upon adequate blood flow to GI tissues. Not surprisingly, the GI tract is one of the most

highly perfused organ systems in the body, and resting GI blood flow is approximately

20-25% of the total cardiac output (Chou, 1983). Thus, the intestine is one of the most

sensitive tissues to hypoxic insult and even brief periods of GI hypoxia induce the

production of inflammatory mediators and dysmotility. Furthermore, GI hypoxia may

potentially promote oxidative stress associated with inflammation and result in

mitochondrial dysfunction within neuronal processes. While our data do not support the

hypothesis that acute T3-SCI adversely affects DMV efferent neuronal survival or

functional properties, the effects on vagal afferent fibers originating within the GI tract

remains to be determined.

Decades of experimental evidence has elucidated the physiological role of TRH

(reviewed in Tache et al., 2006) including the vagally-mediated augmentation of gastric

contractions (McTigue et al., 1992,Hornby et al., 1989,Garrick et al., 1987,Krowicki &

Hornby, 1994,Gourcerol et al., 2011,Travagli et al., 1992). Briefly, microinjection of

77

TRH within the dorsal vagal complex (DVC) excites the firing of DMV neurons, thereby

stimulating efferent outflow of gastric-projecting vagal neurons. In turn, vagal efferents

drive gastric myenteric cholinergic neurons to increase in gastric motility. (Rogers et al.,

1996) In fact, thyrotropin releasing hormone (TRH)-containing projections from the

medullary raphe serve as an example of how a single agonist can appropriate the function

of a vagal gastric control reflex by acting at multiple sites within the reflex circuit. In

vitro evidence has demonstrated that TRH activates DMV neurons directly by

modulating calcium-modulating calcium-dependent potassium conductance (Travagli et

al., 1992). In addition, TRH disinhibits DMV neurons by acting to inhibit neurons in the

NTS directly (McCann et al., 1989,Browning & Travagli, 2001). These NTS neurons

provide GABAergic input to the DMV (Travagli et al., 1991) thereby resulting in a net

excitation. Finally, TRH potentiates the effects of neurotransmitters that also act to inhibit

NTS neurons through the modulation of NTS transduction pathways (Browning &

Travagli, 2001). The end result is that TRH fully engages medullary gastric reflex control

circuitry to produce maximal cholinergic activation of gastric motility. As such, TRH

serves as a robust pharmacological tool to study the gastric efferent vagus.

The DVC is regarded as possessing the characteristics of a circumventricular

organ (Gross et al., 1990,Cottrell & Ferguson, 2004). Additionally, dendritic

arborizations of DMV neurons extend into the area postrema and fourth ventricle. As a

result, the DVC is positioned to integrate the presence of circulating factors with neural

input (Price et al., 2008,Babic & Browning, 2014). Additionally, passive permeability to

blood-borne agents increases following inflammatory and traumatic insults (Pan &

Kastin, 2004) thus likely rendering the DVC vulnerable to pathophysiological conditions

78

following SCI. A rapidly-developing systemic inflammatory response has been reported

as early as 2 hours following SCI (Gris et al., 2008) and circulating inflammatory

cytokines, including IL-6, IL-1β and TNF-α have been reported following SCI (Pan &

Kastin, 2001,Hayes et al., 2002) that may compromise gastric-projecting DVC

neurocircuitry centrally (Emch et al., 2002,Tsuchiya et al., 2012). The specific time

course of elevating cytokine levels within the DVC remains obscure and the present

study did not monitor circulating levels of inflammatory cytokines following SCI.

However, the actions of sub-femtomolar administration of TNF-α within the DVC

include a profound and immediate suppression of TRH-stimulated DVC neuronal firing

and gastric motility (Hermann & Rogers, 1995,Emch et al., 2002). If circulating levels of

TNF-α were sufficiently high in the acute phase of T3-SCI, our microinjection

experiments with TRH would have been expected to reveal a blunted gastric motility

response. While the reported doses of TNF-α (Hermann & Rogers, 1995,Emch et al.,

2002) have been considered to be within the range of circulating levels following

systemic infection (Hermann et al., 2002) the interpretation of pro-inflammatory cytokine

expression and physiological effects need to be thoroughly addressed in our particular

experimental model.

While the post-injury use of prophylactic antibiotic therapy across all groups

might affect GI status, previous reports of systemic inflammatory response indicate that

prophylactic enrofloxacin (Baytril™) treatment does not appear to influence the

cytokine-mediated local and systemic inflammatory response cascade to spinal cord

injury (Gris et al., 2008). Preliminary data from our own laboratory reveal that local

inflammatory responses within GI tissues are detectable despite this standard antibiotic

79

regimen (Swartz et al., 2013). Our present data suggests that acute cytokine infiltration

into the DVC may not be as extensive as what occurred in previous studies, however, this

requires further investigation.

Studies on the rate and extent to which normal aging diminishes extrinsic

innervation of the GI tract reveal slowly progressing changes to vagal afferent (Phillips et

al., 2010) and sympathetic innervation (Phillips & Powley, 2007), while changes to

gastric-projecting vagal neurons remain obscure. Clinically, the progression of SCI-

related GI symptoms have been reported to fully develop over a period of several years

(Stone et al., 1990). Our previous study demonstrated that gastric dysfunction, in the

form of diminished gastric emptying of a solid meal and gastric contractions following

experimental SCI, developed quickly and persisted over a six week time period (Qualls-

Creekmore et al., 2010). In the present study, we selected time points which overlapped

previous demonstrations of DMV pathophysiology (Ammori et al., 2008,Browning et al.,

2013). One limitation to this approach was the relatively short time course after SCI.

Insufficient literature exists with which to determine whether SCI accelerates aging of the

GI tract (Hitzig et al., 2011), especially with regard to extrinsic input to the stomach.

Premature aging associated with chronic SCI may exert long-term effects on the

conductance and overall health of the DMV neurons while peripheral factors play a

significant role in adding to the premature degeneration. In a clinical context it is

tempting to speculate how the ensuing lack of vagal efferent drive following SCI might

ultimately impact the SCI patient. In addition to a well-established modulation of GI

function, numerous studies have produced compelling experimental evidence that the

DMV is a potent inhibitor of inflammation via what has been coined the cholinergic anti-

80

inflammatory pathway (Pavlov & Tracey, 2012). Briefly, activation of vagal

parasympathetic fibers attenuates the systemic inflammatory response to a variety of

insults. This effect is mediated via the α7 subunit of the nicotinic acetylcholine receptor,

which is expressed on macrophages (Yoshikawa et al., 2006) and other non-neuronal cell

types (see Pavlov & Tracey, 2012). The afferent signaling for this reflex has both neural

and humoral components and has received considerable clinical attention (Boeckxstaens,

2013). Experimental interruption of this anti-inflammatory reflex, through vagotomy or

pharmacological blockade, has a pro-inflammatory effect (The et al., 2011). Furthermore,

indirect vagal activation of the spleen has been shown to be an important component of

this reflex (Cailotto et al., 2014,Ji et al., 2014). Therefore, our observation of diminished

vagal efferent signaling to the GI tract may be of particular importance not only to our

model of gastric dysfunction following high-thoracic SCI but also contribute to the

chronic suppression of immune function.

In conclusion, our data suggest that the gastric dysfunction that immediately

accompanies SCI 3 days to 1 week following injury does not include a reduction in the

functionality of DMV motoneurons. This observation extends our previous conclusions

that dysfunction of gastric vago-vagal reflexes following T3-SCI may be due, in part, to

compromised vagal sensory input affecting the gain of vagally-mediated reflexes (Tong

et al., 2011,Holmes, 2012). Clearly, anatomical and functional changes in the integrative

neurocircuitry at the level of the NTS remain largely unexplored in acute and chronic

animal models of neurotrauma. Understanding the long-term consequences of alterations

in vago-vagal neurocircuitry, which remains anatomically intact following SCI, remains

crucial for the development of effective therapeutic strategies. Our data identify a

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potential therapeutic target, the efferent loop of the vago-vagal circuit, for relieving post-

SCI gastric dysfunction.

3.6 ACKNOWLEDGEMENTS

Support: NS 49177 (GMH), NS 87834 (EMS). Dr. Kirsteen N. Browning contributed to

the in vitro study design, data generation and analysis. The authors would also like to

express their gratitude to Samuel R. Fortna for his effort assisting with the morphological

analysis, Gina Deiter, Kristy Pugh, and Margaret McLean for their assistance in multiple

capacities. The authors have no competing interests to disclose.

EMS acquired and analyzed in vivo experimental data, performed histological

processing, and contributed to writing the manuscript. GMH designed the study and

protocol, acquired and analyzed in vivo experimental data, and contributed to writing the

manuscript.

82

Table 3.1

White matter expressed as a percent of the total spinal cord cross-sectional area.

(*P<0.05 vs control).

Experimental Groups

CTB

immunohistochemistry

Motility Studies

1 week post-op 3 day post-op 1 week post-op

3pmol

TRH

10pmol

TRH

3pmol

TRH

10pmol

TRH

Control 76 ± 1% 77 ± 3% 69 ± 3% 70 ± 2% 70 ± 2%

T3-SCI 15 ± 3%* 8 ± 2%* 14 ± 3%* 14 ± 2%* 7 ± 4%*

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Table 3.2

Body weight change expressed as percentage of pre-operative weight is reduced

following T3-SCI. (*P<0.05 vs control).

Experimental Groups

CTB 3 day post-op 1 week post-op

6d weight 2d weight 6d weight

Control 102 ± 2 % 101 ± 1 % 108 ± 2 %

T3-SCI 87 ± 3 %* 91 ± 1 %* 89 ± 3 %*

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Table 3.3

T3-SCI decreases normalized food intake (MEI, kcal/day/100g BW)

(*P<0.05 vs control, § P<0.05 vs 2d T3-SCI, P<0.05 vs 2d control).

Experimental Groups

CTB 3 day post-op 1 week post-op

6d MEI 2d MEI 6d MEI

Control 24.1 ± 1.1 % 25.8 ± 2.6 % 33.6 ± 3.0 %

T3-SCI 16.4 ± 3.1 %* 4.9 ± 2.1 %* 25.7 ± 3.1 %*§

85

Table 3.4

Basic membrane properties of DMV neurons

Experimental Groups

Control

(n=20)

T3-SCI

(n=13)

Input resistance (MΩ) 324±28 336±46

Action potential duration (ms) 2.6±0.1 2.4±0.1

Afterhyperpolarization amplitude (mv) 18.9±0.9 18.8±1.0

Afterhyperpolarization duration (ms) 144±32.6 125±20.3

Action potential firing rate (pps) - 30pA 1.8±0.4 1.0±0.4





86

Table 3.5

Basic morphological properties DMV neurons remain unchanged

Experimental Groups

Control

(n=20)

T3-SCI

(n=17)

X-axis 333±39 332±41

Y-axis 190±26 233±46

Soma area 223±12 227±16

Soma diameter 23±1.0 22±0.7

Form factor 0.5±0.07 0.6±0.67

Segment length 210±23 213±18

87

Figure 3.1. T3-SCI does not induce neuronal degeneration within the dorsal motor

nucleus of the vagus (DMV).

Representative low- (10X, A & B) and high- (20X, a & b) power photomicrographs of

choleratoxin B-immunopositive dorsal motor nucleus neurons at the level of the area

postrema. Region of higher magnification of each corresponding section is indicated

within inset. Injection of CTB into the gastric corpus revealed that both control (A, a) and

T3-SCI (B, b) rats displayed a comparable number of gastric projecting neurons with no

evidence of degeneration. Scale: 500µM in A & B; 50µM in C & D.

88

Figure 3.2. T3-SCI does not alter basic membrane properties or neuronal

morphology of DMV neurons.

Representative traces illustrating the effects of T3-SCI on action potential properties. (A)

DMV neurons were current clamped at -60mV prior to injection of a short (15ms)

depolarizing current pulse of intensity sufficient to evoke the firing of a single action

potential at current pulse offset. Note that T3-SCI had no effect on action potential

duration or afterhyperpolarization amplitude or duration. (B) Control (left) and T3-SCI

(right) neurons were current clamped at -60mV prior to injection of long (400ms)

depolarizing current pulses of increasing magnitude. Note that the number of action

potentials fired was unaffected by T3-SCI. (C) Graphical representation of the frequency

of action potential firing (expressed as pulses per second, p.p.s.) in DMV neurons from

control (black) and T3-SCI (red) DMV neurons. Note that T3-SCI did not have any

significant effect upon the number of action potentials fired in DMV neurons.

Representative computer-aided reconstructions of DMV neurons from control (D) and

T3-SCI (E) brainstems reveals that T3-SCI did not alter the soma size or dendritic

arborization of DMV neurons. Scale: 100µM in A & B.

89

Figure 3.3. Representative original polygraph traces of gastric corpus contractions

from control and T3-SCI.

Gastric corpus contractions following microinjection of vehicle (phosphate buffered

saline, PBS), 3 pmol or 10 pmol of TRH into the left DVC of animals tested 3 days or 1

week after surgery. Arrows depict the initiation of microinjection for each respective

dose. Selected traces are interrupted (denoted by parallel bars) to provide examples of

gastric contractions following a return to baseline levels.

90

Figure 3.4. Microinjection of thyrotropin-releasing hormone into the dorsal vagal

complex, including the DMV, induces gastric contractions following both control

and T3-SCI.

Schematic representation of effective injection areas from 3 days (top left) and 1 week

(bottom left) control (marked by ○) or T3-SCI rats (marked by ♦). For clarity, doses of

TRH were pooled across surgical treatment groups and only a few examples of the

distribution of injection sites are depicted. AP, area postrema; NTS, nucleus tractus

solitarius; DMV, dorsal motor nucleus of the vagus; CC, central canal; XII, hypoglossus.

Graphic summary of the increase in gastric corpus motility induced by microinjection of

PBS or TRH (3 or 10 pmol, expressed as percent increase from baseline) in the left DVC

of 3 day (top center) control and T3-SCI (bottom center) subjects (n = 5 per group;

*p < 0.05 vs pre-microinjection baseline). Duration of the response to microinjection of

TRH (top and bottom right) was not significantly different between surgical treatment

and dose at any time point (p>0.05).

91

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Chapter 4:

Mesenteric vascular dysregulation accompanies acute


98

4.1 ABSTRACT

Spinal cord injury (SCI) drastically impairs autonomic nervous system function.

Many SCI individuals, especially those with injuries at spinal level T5 or higher, present

with gastrointestinal and cardiovascular disorders that include arterial hypotension.

Despite the clinical implications, systematic investigation of GI blood flow following

experimental SCI has not been performed. The aim of this study was to test the

hypothesis that T3-SCI induces visceral hypoperfusion and diminished postprandial

vascular reflexes. We measured in vivo arterial blood pressure and superior mesenteric

artery (SMA) blood flow in anesthetized T3-SCI rats 3-days post-injury under the

following experimental conditions: fasted and following enteral feeding by duodenal

infusion of a liquid mixed-nutrient meal (Ensure™). Our results show that T3-SCI rats

displayed significantly reduced SMA blood flow under all experimental conditions.

Specifically, the anticipated postprandial elevation of SMA blood flow in response to

duodenal infusion was either delayed or lower after T3-SCI. The dysregulation of SMA

blood flow in acutely-injured T3-SCI rats coincides with our previous observations of

diminished gastric motility and gastric emptying. Our data suggest that arterial

hypotension consequently diminishes mesenteric blood flow necessary to meet mucosal

demands at rest and during digestion. The resulting GI ischemia may be an underlying

pathology leading to gastric dysfunction seen following SCI. Our model may represent an

intermediate stage in a continuum of gastrointestinal ischemia that ultimately plays a role

in GI co-morbidities following SCI.

99

4.2 INTRODUCTION

In addition to the catastrophic sensory and motor losses following SCI, ANS

dysfunction is also widely recognized (Krassioukov, 2009). Cardiovascular instability,

arterial hypotension, and pooling of blood in the extremities have been documented

clinically (West et al., 2013) or experimentally (Laird et al., 2006). Furthermore, GI

dysmotility is observed clinically immediately after SCI (Kirshblum et al., 2002,Wolf &

Meiners, 2003) and may persist for years after the initial injury (Cosman et al.,

1991,Berlly & Wilmot, 1984,Fealey et al., 1984,Nino-Murcia & Friedland, 1991,Segal et

al., 1995,Stinneford et al., 1993,Williams et al., 2011). GI system dysfunction following

experimental SCI includes reduced gastric motility and gastric emptying, abnormal

response to GI peptides and reduced nutrient absorption. Each of these co-morbidities

contributes to diminished long-term quality of life after SCI (Primeaux et al., 2007).

In recent human studies, a SCI individual with a lesion above T5 may develop

autonomic dysreflexia (AD) two to three months following the injury. This AD is

marked by vasoconstriction across skeletal muscles, integumentary, renal and GI vascular

beds that is caused by a sympathetically-mediated afferent peripheral stimulation caudal

to the lesion level. Ultimately, AD can lead to cerebrovascular complications and have a

drastic effect on metabolism (Karlsson, 2006,Teasell et al., 2000,Wan & Krassioukov,

2013). Conversely, reduced GI blood flow over an extended period of time deprives GI

tissues of the oxygen needed to maintain organ integrity (Chou & Coatney, 1994).

Vascular hypotension and pooling of blood in the extremities may predispose the GI tract

to hypoperfusion following SCI.

100

The principal functions of the GI tract, the digestion and absorption of nutrients

and the maintenance of proper fluid balance, require adequate blood flow to GI tissues.

The principal vascular perfusion occurs through the celiac, superior mesenteric, and

inferior mesenteric arteries (Matheson et al., 2000). The GI tract is one of the most highly

perfused organ systems in the body, and resting GI blood flow can reach approximately

20-25% of the total cardiac output (Chou, 1983). Postprandial hyperemia, the increase in

blood flow to the GI tract following a meal, is a critical reflex for adequate GI function

and has been demonstrated to result from the exposure of the intestinal mucosa to

nutrients in concert with the release of GI peptides (Chou & Coatney, 1994).

In the present work, we employed our established rodent model of experimental

SCI to investigate 1) if T3-SCI leads to reduced mean arterial pressure; 2) if T3-SCI

reduces resting blood flow within the superior mesenteric artery supplying the mesenteric

bed; 3) if T3-SCI diminishes postprandial vascular reflexes; and 4) if local duodenal

tissue perfusion increases in response to nutrient infusion.

4.3 METHODS

Male Wistar rats ≥8 weeks of age, initially weighing 175-200 g (Harlan,

Indianapolis, IN, USA), were used for all experimental procedures.

All procedures were performed following National Institutes of Health guidelines

and under the approval of the Institutional Animal Care and Use Committee at the Penn

State University College of Medicine. Rats (n = 49) were housed in a temperature-

controlled room (23◦C) on a 12:12-h light-dark cycle with unlimited access to food and

water. Following surgical manipulation, rats were housed singly and observed twice a

day. Each rat was randomly assigned to one of two surgical manipulations; surgical

101

controls in which the T3 spinal cord was exposed by laminectomy or T3-SCI. Following

surgery, SCI rats were housed on warming pads to maintain an adequate body

temperature of 37◦C.

4.3.1 Surgical Procedures and Animal Care

4.3.1.1 SCI/Control Surgery

Animals were anesthetized with a 3-5% mixture of Isoflurane with oxygen (400-

600ml/min) and surgery for T3 contusion SCI using the Infinite Horizons device was

performed using established aseptic surgical techniques. When the rat was no longer

responsive to toe pinch or palpebral reflex, the surgical site overlying the vertebrae from

the interscapular region to mid-thorax was shaved and cleaned with three alternating

scrubs of Nolvasan and alcohol. Animals were maintained at 35.5–37.5°C on a feedback-

controlled heating block, and rectal temperature was monitored continuously. The

location of the elongated T1 and T2 spinous processes were determined by midline

palpation. A 3-5cm midline incision of the skin overlying the T1-T3 vertebrae was

performed and the muscle attachments to the T1-T3 vertebrae were cleared by blunt

dissection, taking care not to damage the vasculature supply to the dorsal nuchal adipose

tissue. Using fine-tipped rongeurs, the spinous process and the dorsal surface of the T2

vertebra was removed laterally to the superior articular process.

The rat was transferred to the Infinite Horizons spinal contusion injury device

(Precision Systems and Instrumentation, Lexington KY). The adjacent T1 and T3

vertebrae were clamped into the device and the torso of the animal was suspended

slightly above the platform. After centering the exposed spinal cord underneath the

impactor tip a 300 kDyne impact (15 sec dwell time) was initiated. This level of injury

102

produces a consistent and reliable neurological and histological outcome whereby

animals exhibit a residual, chronic, locomotor deficit and severe loss of integrity to the

spinal cord white matter (≤ 25% of white matter). After removal from the contusion

device, all surgical incisions were closed in reverse anatomical order with absorbable

suture (Vicryl 4-0) for internal sutures and skin closure with wound clips. Wound clips

are removed 5-7 days following surgery. Surgical controls undergo all procedures except

for the weight drop.

Post-operatively, rats were administered supplemental fluids by injection of 5cc

warmed lactated Ringer’s solution (s.c.) and stabilized in an incubator (37ºC) until fully

recovered from anesthesia. Afterward, animals were monitored daily for any signs of

infection or complications from surgery. Rats received extended-release analgesics

(buprenorphine SR, 1mg/kg IP, Pfizer Animal Health, Lititz, PA) once and antibiotics

(Baytril, 2.5 mg/kg) and subcutaneous supplemental fluids (5-10 cc lactated ringers),

twice daily for five days after surgery. Due to the reduction in locomotor capacity after

T3-SCI, a reservoir of chow was placed at head level in order to facilitate ease of access

for feeding. All T3-SCI rats ingested a measureable amount each day, thereby confirming

that access to chow was available. Bodyweights and food weights were recorded once

daily. SCI rats received bladder expression and ventrum inspection twice daily until the

return of spontaneous voiding occurred.

4.3.2 In vivo studies

Three days following the initial surgery, T3-SCI or control animals were fasted

overnight, water provided ad libitum, prior to being deeply re-anesthetized with

Isoflurane (3-5%, 400-600ml/min flow rate) for physiological instrumentation. Animals

103

were placed on a feedback-controlled warming pad (TCAT 2LV, Physitemp Instruments,

Clifton, New Jersey) maintained at 37±1 °C for the duration of the experiment.

Tracheal cannulation - Once prepared for surgery, the animal was tracheally

intubated by way of a 1-2-cm midline incision on the ventral side of the neck from the

inferior border of the mandible to the sternal notch. The underlying strap muscles were

separated using blunt dissection at the midline to expose the trachea. The exposed trachea

was isolated from the underlying esophagus in order to place a loop of 3-0 ethilon suture

between the trachea and esophagus to form a ligature. The trachea was opened anteriorly

by making a small cut in the membrane between two of the cartilaginous rings of the

trachea just distal to the thyroid gland. A small piece of polyethylene tubing (PE-270,

5mm in length and beveled at one end) is inserted into the trachea and secured in place

with the ligature. The strap muscles are returned to their proper anatomical location and

the overlying skin was secured around the tracheal tube with 3-0 ethilon.

Femoral arterial catheter - Following intubation, the femoral artery and adjacent

vein or tributaries are exposed via a small incision of the upper thigh which exposes the

quadriceps and associated adductors. Connective tissue is cleared from the femoral artery

and vein proximally to the inguinal ligament. The proximal and distal extremes of the

exposed artery are gently ligated with 4-0 silk suture. The artery is hemisected and an

aseptic PE-50 catheter is inserted in the direction of the abdominal aorta. The proximal

artery and catheter are fully secured with the 4-0 silk suture and exteriorized. The wound

margin is closed with wound clips.

Transonic flow probe - Animals were weaned off Isoflurane inhalation and deeply

anesthetized with thiobutabarbital (Inactin; Sigma, St. Louis, MO; 75-150 mg/kg iv)

104

which does not affect cardiovascular (Buelke-Sam et al., 1978) or gastrointestinal

(Qualls-Creekmore et al., 2010b) autonomic function. The rate of Inactin infusion was

monitored in conjunction with the resulting temporary drop in arterial blood pressure

(SYS-BP1, World Precision Instruments, Sarasota, FL). Once a deep plane of anesthesia

was achieved, a midline laparotomy was made and the intestines were gently displaced

laterally to allow the exposure of the aorta at the level of the left renal artery and vein.

The superior mesenteric artery (SMA) was carefully cleared of connective tissue

immediately distal to where it passed over the inferior vena cava to allow for the

perivascular flow probe (1PR, Transonic Systems, Inc. Ithaca, NY) to be positioned

alongside the artery.

Prior to positioning the perivascular flow probe around the SMA of animals that

were to receive duodenal infusion of Ensure, a PE-90 catheter was inserted into the

proximal duodenum through a small incision in the stomach adjacent to the pylorus and

secured with a purse-string suture. The Ensure-filled syringe was driven by a syringe

pump set at an infusion rate of 1 ml/hr.

Laser Doppler Flow Probe - The retracted viscera were returned to the original

location. In a subset of animals, a laser Doppler flow probe (BLF22, Transonic Systems,

Inc. Ithaca, NY) was positioned in close contact with the mesenteric border of the

duodenum immediately distal to the region where the tip of the implanted catheter

terminated. Once a stable reading was achieved from the flow probe, the incision was

closed around the implanted flow probes and the skin loosely secured with stainless steel

wound clips. Animals were allowed to recover for 1 hour before data collection was

initiated.

105

4.3.3 Blood Flow Analysis

At the initiation of the stabilization period, the femoral arterial catheter was

attached to a pressure transducer (BP-1, World Precision Instruments, Sarasota, FL). Data

from the flowmeter (T206, Transonic Systems, Inc. Ithaca, NY), blood pressure monitor

and laser Doppler flow probe was continuously recorded to computer (Spike 2,

Cambridge Electronic Design, Cambridge, UK). Flow probe signals were filtered at

0.1Hz and converted to blood flow in ml·min-1

and was normalized for bodyweight. The

mean percent change in Doppler output from baseline was calculated for each

experimental manipulation. The effect of duodenal infusion was compared to the average

blood flow rate of the 10 min preceding the infusion. Peak flow rate was calculated as the

highest achieved value during the 1 h following the infusion.

4.3.4 Histological Processing.

At the conclusion of the experiment, deeply anesthetized rats were transcardially

perfused with heparinized phosphate-buffered saline (PBS) until fully exsanguinated and

followed immediately with PBS containing 4% paraformaldehyde. The spinal cord

encompassing the lesion level was removed and refrigerated overnight in PBS containing

20% sucrose and 4% paraformaldehyde. For histological staining of T3-SCI lesion

extent, tissue was sectioned (40μm thick) and alternating sections were mounted on

gelatin coated slides.

To compare lesion severity with the spinal cords of control animals, spinal cord

sections were stained with luxol fast blue (LFB) to visualize myelinated fibers. LFB-

stained slides were digitally imaged on a Zeiss Axioscope light microscope and Axiocam

CCD camera, imported into Adobe Photoshop and contrast digitally enhanced to allow

106

consistent identification of LFB-stained (i.e., spared) white matter. For individual images,

the boundaries of the tissue slice were outlined to determine cross-sectional area. A

separate threshold histogram was generated and the pixels corresponding to LFB staining

above background were selected. These pixels were quantified and expressed per unit

cross-sectional area (Noble & Wrathall, 1985). The proximity of the T3 lesion center to

the cervical enlargement precluded an appropriate determination of spinal cord cross-

sectional area in undamaged tissue rostral to the injury (i.e., damaged tissue extended into

the cervical enlargement as described in (Tong & Holmes, 2009). Therefore, it was

necessary that the cross-sectional area of the intact spinal cords at T3 of comparably sized

animals be determined for normalization purposes. LFB-stained myelin in injured tissue

was then expressed as a percent of the total spinal cord cross-sectional area as would be

predicted by the intact tissue. Based upon a previous report (Tong et al., 2011) we

determined a priori that animals with white matter sparing ≤ 25% were categorized as

severe injury while those ≥ 25% were excluded from further analysis.



Between groups results from in vivo studies were compared by one-way ANOVA and

Tukey post hoc analysis or paired t-test as appropriate. Statistical analysis was performed

using SigmaPlot for Windows (SPSS Inc, Chicago, IL).

4.4 RESULTS

4.4.1 Histological assessment of T3-SCI severity

The severity of experimental SCI was verified based upon the reduction of LFB-

stained white matter at the T3 spinal segment. The mean area of white matter at the lesion

107

epicenter of T3-SCI rats (n=24) was significantly reduced in comparison to T3 control

(n=25) animals (2.66 ± 0.11 mm2 vs. 0.62 ± 0.07 mm

2; P < 0.05). When white matter is

expressed as percentage of spared tissue, the lesion epicenter of T3-SCI rats retained

~14% of white matter (range 5 – 24%). These data are comparable to the injury extent

reported previously and indicate the severity of our injury model (Tong & Holmes,

2009,Qualls-Creekmore et al., 2010a,Tong et al., 2011,Swartz & Holmes, 2014).

4.4.2 Assessment of the post-injury reduction of spontaneous feeding and bodyweight

The bodyweight change between T3-SCI and control animals was -24.8 ± 2.8g vs.

2.8 ± 1.9g, respectively. When normalized as percent of preoperative weight, T3-SCI rats

were significantly lower than surgical controls (Figure 4.1; P < 0.05). The mean energy

intake as a result of spontaneous feeding (MEI; defined as kcal/day/100 g body weight)

for T3-SCI animals was significantly lower than controls (Figure 4.2; P < 0.05).

As demonstrated in our previous studies, T3-SCI reduced the area of intact white

matter, feeding and bodyweight. These data further verify the profound severity,

effectiveness and reproducibility of our surgical procedures for T3-SCI and surgical

control animals. Based upon these criteria, all animals in these groups were selected for

further data analysis.

4.4.3 Basal arterial blood pressure and mesenteric blood flow are decreased in SCI rats

Prior to experimental manipulation, the mean arterial pressure (MAP) of Inactin-

anesthetized T3-SCI rats was significantly lower than the MAP of surgical control

animals (Table 4.1; P<0.05). Following normalization for body weight, basal SMA blood

flow in fasted T3-SCI rats was significantly lower than controls (Figure 4.3; P<0.05).

These results confirm that T3-SCI in the rat produces vascular hypotension of the

108

skeletal and splanchnic vascular beds that is similar to that which is observed in high-

thoracic or cervical spinal cord-injured patients.

4.4.4 Postprandial mesenteric blood reflexes are reduced in SCI rats

Following duodenal infusion of a liquid mixed nutrient meal (Ensure™ delivered

at 1ml/hr), SCI rats fail to exhibit the typical phasic increase in SMA blood flow that is

demonstrated by control animals (Figure 4.4). During infusion of Ensure™ into the

duodenum, the MAP remained significantly different between T3-SCI and control rats

(Table 4.1; P<0.05), however, in both T3-SCI and control rats, duodenal infusion of

Ensure™ did not significantly change MAP from pre-infusion baseline values (Table 4.1;

P>0.05).

During intra-duodenal infusion of Ensure™, blood flow within the SMA

demonstrated a significantly lower peak blood flow of T3-SCI rats following the nutrient

challenge (Figure 4.5A; P<0.05). Additionally, the elevation of SMA blood flow relative

to percent baseline flow is reduced in T3-SCI rats compared to controls (Figure 4.5B;

P<0.05).

Laser Doppler analysis of the local duodenal perfusion in the region of Ensure™

infusion demonstrated that the percent change in blood flow within the duodenal serosa

of surgical control rats is significantly elevated from baseline in comparison to T3-SCI

rats which did not increase during enteral feeding (Figure 4.5; P<0.05).

These results indicate that T3-SCI in the rat diminishes the sympathetically-

mediated mesenteric vascular reflexes in response to feeding. Local, enterically-

mediated, changes in duodenal blood flow are also diminished in these same animals.

109

4.5 DISCUSSION

The present study demonstrates that systemic and mesenteric vascular

competence is reduced following a severe experimental T3-SCI. Specifically, these

experimental data indicate that: 1) baseline mean arterial pressure is significantly reduced

in our experimental model of high thoracic SCI; 2) basal flow rate through the SMA is

reduced in rats 3d after T3-SCI; 3) mean arterial pressure remains at baseline levels in

response to enteral administration of a liquid mixed nutrient meal in both control and T3-

SCI rats; 4) T3-SCI rats have a significantly reduced post-prandial mesenteric response

following a liquid mixed-nutrient meal; and 5) enterically-mediated vascular reflexes do

not demonstrate any local, compensatory, response to duodenal nutrient supplementation.

The level of tissue loss at the lesion epicenter, coupled with the observed reduction in

feeding and weight loss, is consistent with our previous findings in severe T3-SCI rats

that demonstrated gastroparesis and delayed gastric emptying (Tong & Holmes,

2009,Qualls-Creekmore et al., 2010a,Swartz & Holmes , 2014). These data suggest that

clinically-recognized vascular reflex deficits in the SCI population may extend to the

mesenteric bed and the local gastrointestinal tissues as demonstrated in our experimental

model of high thoracic SCI.

A common consequence of SCI is systemic vascular dysfunction (Popa et al.,

2010). Human studies have shown that high-level (cervical) SCIs are accompanied by

severe hypotension and bradycardia (Furlan et al., 2003,Furlan & Fehlings,

2008,Garstang & Miller-Smith, 2007,Krassioukov et al., 2007). Often times, such

rampant sympathetic responses following an SCI can last up to 5 weeks following an

injury and are most likely due to unopposed vagal parasympathetic inputs (Furlan et al.,

110

2003). Furthermore, cardiovascular disturbances, following SCI, are the leading causes of

morbidity and mortality (Popa et al., 2010). The sympathetic preganglionic neurons

within the spinal cord normally receive descending efferent inputs from the medullary

cardiovascular centers. These supraspinal fibers are interrupted by SCI and the

sympathetic nervous system becomes hypoactive, resulting in low resting blood pressure,

low cardiac output, low venous return, loss of ability to regulate blood pressure, and

disturbed reflex control (Krassioukov, 2009,Popa et al., 2010). The parasympathetic

nervous system remains anatomically intact following SCI and is unopposed by the

appropriate sympathetic neurocircuitry in cervical and high thoracic SCIs. This would

lead to the prediction that the dysregulation of sympathetic vascular regulation results in

a net increase of blood flow to the GI tract. However, evidence from these experiments as

well as that gathered from Doppler blood flow studies of the liver suggests that this is not

the case since hepatic microvascular blood flow was diminished in mid thoracic (T5)

injured rats 24h after SCI (Vertiz-Hernandez et al., 2007).

While the GI mucosa is a richly perfused vascular bed, it is directly juxtaposed

with the anaerobic and nonsterile lumen of the gut. As such, intestinal epithelial cells,

which line the mucosa, experience a uniquely steep physiologic oxygen gradient in

comparison with other cells of the body. Thus, the intestine is one of the most sensitive

tissues to hypoxic insult and even brief periods of GI hypoxia induce the production of

inflammatory mediators and dysmotility. Furthermore, there is evidence that hypoxia

may be more deleterious to cells than complete anoxia (Dawson et al., 1993).

Experimental in vitro studies in which mitochondrial or glycolytic metabolism has been

disrupted pharmacologically (thereby depleting ATP) have shown that minor reduction in

111

ATP which is maintained for 12-24 hours, is sufficient to induce epithelial monolayer

dysfunction. From a clinical standpoint, visceral hypoperfusion in the critically ill patient

leads to hypoxia and initiates an inflammatory cascade with consequent end-organ

dysfunctions and cervical SCI patients are, indeed, susceptible to multiple organ

dysfunctions.

Postprandial hyperemia in healthy subjects consists of a profound increase (ca.

200%) in regional gastrointestinal blood flow in response to nutrients (Matheson et al.,

2000). This redistribution of blood flow is compensated by reflex increase in cardiac

output and a redistribution of flow to other tissues. (Chou & Coatney, 1994)

In particular, there is substantial evidence that postprandial hyperemia is locally

mediated within the intestinal microvasculature through a complex, and not completely

understood, interplay of local oxygen titers, adenosine levels, prostaglandins, sodium-

induced hyperosmolarity and the degree of muscle deformation (Nowicki, 2006). In

addition, these changes in microvasculature are under the influence of the hemodynamics

of upstream mesenteric arteries. These larger caliber supply arteries and arterioles are

under greater influence from extrinsic, sympathetic, sources (reviewed in Holzer 2006).

One mechanism involves the release of GI peptides that have been demonstrated to exert

a role in regulating postprandial hemodynamic demand through a centrally mediated

reflex. (Sartor & Verberne, 2002,Sartor et al., 2006) Specifically, intestinal

cholecystokinin (CCK) and gastric leptin activate subdiaphragmatic vagal afferents that,

ultimately, terminate in the nucleus tractus solitarius (NTS). In addition to the role of the

NTS toward the modulation of gastric-projecting preganglionic motorneurons in the

vagal dorsal motor nucleus (DMN; Rogers et al., 2006) CCK-sensitive afferents

112

terminate upon a subpopulation of NTS neurons that directly project to select

cardiovascular neurons in the rostral ventrolateral medulla (RVLM). Activation of these

RVLM neurons provokes an elevation of sympathetic drive and vasoconstriction within

skeletal muscle. In this proposed model, simultaneous input by NTS neurons that project

to caudal ventrolateral medulla (CVLM) provokes a reduction in splanchnic sympathetic

tone resulting in vasodilation within the mesentery (Sartor & Verberne, 2008). With

particular emphasis on the rat, the segmental distribution of identified cardiovascular

sympathetic preganglionic neurons begins principally at the second spinal thoracic

segment and progresses caudally (Gonsalvez et al., 2010).

It is generally recognized that the intestinal tract is acutely sensitive to traumatic

events (Bai et al., 2011). The relationship of properly regulated gastrointestinal blood

flow with patient morbidity or mortality is well recognized in many instances of

advanced aging (Luciano et al., 2010), trauma and critical illness (Yang et al.,

2014,Casaer & Van den Berghe, 2014). The implications of severely diminished blood

flow to the GI tract following SCI are likely to mirror some aspects of these other clinical

situations. Therefore, if post-SCI hypoperfusion leads to ischemia, tissue damage and

necrosis are likely to occur whereby the walls of the GI tract may become permeable

allowing bacteria to proliferate and translocate through the gut wall and into lymph nodes

and blood vessels. (Bohm et al., 2013,Liu et al., 2004) With inadequate splanchnic

perfusion, multiple organ failure and death may ensue (Jakob et al., 2012). While the

mechanism remains inadequately understood, the impaired gastrointestinal blood flow we

have observed may contribute to the gastric dysfunction. Other models have shown that

ischemic GI tissue reacts by releasing lactate as the mucosal-arterial pCO2 gradient

113

increases indicating the initiation of anaerobic metabolism in the gut (Jakob et al., 2001)

and recruitment of pro-inflammatory cytokines and inflammatory markers.

While there is a general consensus regarding the benefit of nutrient

supplementation in the critically ill, emerging evidence suggests that the timing and

composition of supplementation is not universally applicable across all critical care

scenarios. Specifically, there is an increased risk of challenging critically ill patients with

vascular instability by increasing mesenteric metabolic demands through enteral nutrient

supplementation (Rokyta, Jr. et al., 2003,Kles et al., 2001,Lucchini et al., 1996). The

metabolic requirements of the GI tract have been described in detail in animal models

(Kles et al., 2001), but there is no evidence-based guide regarding when to feed a human

with SCI following injury. Many researchers have documented the protective effects of

enteral nutrition (Kudsk, 2002,Matheson et al., 2002,Moore, 1994). Determining the

proper time of enteral nutrition is beyond the scope of this paper; however, we would like

to point out that the GI tract is severely hypoperfused following SCI and vascular reflexes

are not responding properly to liquid nutrient meals which further exacerbates the GI

tract gut-barrier function and promotes further insult leading to systemic inflammation.

In conclusion, our novel data reveal that basal mesenteric blood flow is severely

diminished following injury. Furthermore, postprandial vascular reflexes are blunted

following experimental SCI. We propose that changes in nutrient-vascular relationships

may render the post-SCI gut susceptible to ischemic and inflammatory events that lead to

AP-1 mediated iNOS up-regulation and GI dysmotility. We further propose that these

changes in nutrient-vascular relationships last for weeks after the original spinal cord

injury.

114

We have shown in this study that high-level (T3-) SCI results in diminished

splanchnic perfusion. The resulting intestinal hypoperfusion and hypoxia/reperfusion

may contribute to the pathogenesis of multiple end-organ dysfunction (Swank & Deitch,

1996,O'Boyle et al., 1998,Kale et al., 1998). Brief periods of mesenteric ischemia have

been demonstrated to increase vagal and spinal afferent firing that may be mediated by

mast cell degranulation and histamine release (Bulmer et al., 2005,Jiang et al., 2011).

These studies, however, did not investigate the long term effects of mesenteric ischemia

on GI immune response or function. Furthermore, SCI exerts profound changes to the

physiology of the affected individual, thereby limiting the comparisons that may be

drawn from the ischemia/reperfusion literature. It is clear that the competence of

mesenteric blood flow is intimately tied to the overall health of the GI tissues and

function of the afferent nerves terminating near the gastrointestinal mucosa. We propose

that a chronic reduction of GI blood flow triggers a parallel GI inflammation following

SCI. Each of these adverse events may contribute to the GI dysfunction observed in the

SCI population.


Nicole Pironi contributed a subset of the SMA blood flow experiments. Dr. Sean Stocker

provided invaluable guidance for performing and analyzing femoral MAP recording.

Margaret McLean provided assistance with animal care, data entry and analysis in Excel.

Preliminary protocols and data supporting this experimental protocol were generated by

Emily Qualls-Creekmore.

115

Figure 4.1. Average percent change in body weight after SCI expressed as the

difference from pre-injury weight.

Control animals (n=25) did not lose weight following surgery while T3-SCI animals

(n=24) displayed a significant weight reduction immediately after surgery and did not

return to pre-operative weight until 5 weeks after surgery (* P<0.05 vs. control). The

weekly difference in weight between groups was significantly different throughout the

entire course of the study.

Values expressed as mean ± SEM.

116

Figure 4.2. T3-SCI provokes a reduction in the spontaneous intake of standard

laboratory chow.

Daily mean energy intake, calculated as kcal consumed/day/100g of body weight (BW)

for each day that food intake was measured, is significantly lower in T3-SCI rats

compared to controls (* P<0.05 vs. control).


117

Table 4.1

Mean arterial pressure (mmHg) is not altered by 60 minutes of Ensure™ infusion in 3

day T3-SCI (n=17) and surgical controls (n=18).

Baseline 30 min infusion 60 min infusion

Control 112.9 ± 4.3 109.2 ± 2.4 105.2 ± 5.0 %

T3-SCI 74.8 ± 4.9* 69.9 ± 5.7* 65.9 ± 7.0 %*

Values presented as mean ± SEM. *P<0.05 vs control

118

Figure 4.3. T3-SCI diminishes basal SMA blood flow at 3 days following injury.

The average SMA blood flow of T3-SCI rats (expressed as ml/min/100g body weight) is

significantly lower than surgical controls (* P<0.05 vs. control).


119

A

B

C

120

▲Figure 4.4. Post-prandial hyperemia is significantly lower in T3-SCI animals.

Representative traces (A) illustrating the normal post-prandial hyperemia from a control

rat (top trace) while post-prandial SMA blood flow from T3-SCI rats (bottom trace) did

not demonstrate a response to duodenal perfusion of a mixed nutrient meal (Ensure™;

infusion of rate was 1ml/hr). Arrows depict the initiation of Ensure™ administration for

each representative subject. (B) The peak volume of SMA blood flow reached during the

intra-duodenal infusion period was also significantly reduced in T3-SCI rats (* P<0.05 vs

control). (C) The percent change from baseline SMA blood flow was also significantly

lower in T3-SCI rats (* P<0.05 vs. control).


121

Figure 4.5 Duodenal tissue blood flow does not increase in T3-SCI animals during

enteral feeding

Local tissue perfusion was measured by Laser Doppler Flow of the duodenal serosa.

Compared to controls (n=6), the percent change in Doppler signal vs. baseline flow was

significantly lower in T3-SCI rats (n=8; * P<0.05 vs. control).


122

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Chapter 5:

Acute experimental spinal cord injury evokes low-grade

gastrointestinal inflammation

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5.1 ABSTRACT

Significant autonomic nervous system (ANS) dysfunction follows higher thoracic

level spinal cord injury (T3-SCI) whereby gastrointestinal and cardiovascular functions

are impaired. The ramifications of post-SCI vascular hypotension, especially following

cervical or high thoracic injury, and visceral hypoperfusion remain underexplored. Our

previous data showed a significant reduction in mesenteric blood flow, specifically within

the superior mesenteric artery (SMA), following experimental SCI. Therefore, we

employed quantitative molecular techniques to test the hypothesis that high-thoracic SCI

triggers gastrointestinal inflammation. We further analyzed the expression of

inflammatory markers in age-matched animals following mesenteric

ischemia/reperfusion injury evoked by 60 minute ligation of the SMA. Similar to

previous reports, mesenteric ischemia/reperfusion elicited a significant elevation of

gastric iNOS and nNOS. By contrast, gastric and small intestinal MCP-1, MIP-1α and

ICAM-1 and were significantly elevated acutely following T3-SCI.

This study suggests a relationship between the GI dysfunction and the increased

GI inflammation resulting from the visceral hypoperfusion. Visceral hypoperfusion is

further supported by our histopathology, at 1-day post-SCI, revealing a gastric ulcer and

necrosis in the small intestine. Following SCI there are marked deficits in motility,

transit, and emptying. Understanding the inflammatory cascade that is triggered within

the GI tract following SCI will ultimately permit evidence-based strategies to manage the

acute SCI patients and prevent the onset of GI inflammation and organ dysfunction.

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5.2 INTRODUCTION

Gastrointestinal (GI) function is significantly altered following spinal cord injury

(SCI) and presents in various forms of dysphagia, gastroparesis, gastric dilation, delayed

GI transit, nausea and emesis, among other symptoms, which may persist for a lifetime

(reviewed in Holmes, 2012). The mechanisms for this persistent dysfunction remain

obscure, however, there is accumulating evidence that significant traumatic events

involving reduction in mesenteric blood flow provoke gastrointestinal tissue ischemia

that, in turn, initiates a dysfunctional inflammatory cascade (Hierholzer et al., 2004).

Acute GI inflammation is multifactorial, with the involvement of neutrophils,

macrophages, reactive oxygen species, and cytokines all contributing to the development

of pathophysiology (Mawe et al., 2009a,MacNaughton, 2006). Because of the nonsterile

lumen of the gut, the GI tract is widely considered to exist in a state of controlled

inflammation typified by a constant trafficking of leukocytes in lymphoid and non-

lymphoid mucosal tissues. Numerous cell adhesion molecules have been implicated in

the specific interactions between vascular endothelial cells and the GI tract (Granger et

al., 2006). Among this molecular cascade, pro-inflammatory cytokines such as

interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) as well as chemotactic

molecules such as macrophage inflammatory protein 1 alpha (MIP-1α) and monocyte

chemoattractant protein-1 (MCP-1) constitute part of the early inflammatory response

that also includes the up-regulation of adhesion molecules (e.g., intracellular adhesion

molecule-1; ICAM-1) that are responsible for leukocyte adhesion (Granger et al.,

2006,Snoek et al., 2010). The firm adhesion of leukocytes through ICAM-1 is amplified

through a feed-forward mechanism by subsequent chemokine release (Granger et al.,

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2006). The timing of molecular events occurs on the order of minutes and the temporal

pattern of activation remains contradictory in the literature, however, the fundamental

role and the duration of leukocyte migration (described in Snoek et al., 2010) and

tethering in inflammatory processes has led to ICAM-1 serving as a principal marker of

gut inflammation. It is important to acknowledge that GI inflammatory responses are

further mediated by the activation of transcription factors such as NF- B and

activator

protein-1 (AP-1; reviewed in Delerive et al., 2001). The transcription factor AP-1, is a

homo- or heterodimeric transcription factor composed of members of the Jun and Fos

families of DNA-binding proteins and are implicated in cellular stress responses (Johnson

& Nakamura, 2007).

Three isoforms of nitric oxide synthase catalyze the formation of the signaling

molecule, nitric oxide, from L-arginine. One principal role of the endothelial isoform

(eNOS) involves the regulation of vascular tone. The neuronal isoform (nNOS) serves to

relax smooth muscle, as described previously for the gastrointestinal tract. The inducible

isoform (iNOS) serves to generate prodigious amounts of nitric oxide as part of a host

defense response. In models of gastrointestinal ischemia/reperfusion, AP-1 has been

specifically proposed as the key upstream mediator of inducible nitric oxide synthase

(iNOS) which, in turn, exacerbates GI dysmotility and increase barrier permeability (Ban

et al., 2011). Ultimately, this inflammatory response may result in end-organ dysfunction

including reduced GI motility, transit, and emptying. Left unchecked, this cascade may

rapidly progress to bacterial translocation and sepsis (Evans et al., 2009). Specifically,

blood borne infections occur with a high incidence of mortality in SCI clinical settings

(Evans et al., 2009).

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Based upon the observations that vascular hypotension can result in visceral


organ dysfunction and our experimental observation that mesenteric blood flow is

significantly reduced following T3-SCI we used qRT-PCR to investigate the expression

of select inflammatory markers along the entire length of the GI tract to test the

hypothesis that T3-SCI GI dysfunction is due, in part, to GI inflammation following SCI.

5.3 METHODS






food and water provided ad libitum. After surgery animals were single housed in cages

mounted on warming pads to maintain an adequate body temperature of 37◦C. All

experimental animals were observed daily, and food and body weight were recorded.

5.3.1 Spinal Cord Injury Surgery and Post-Operative Care

Animals (n=11) were anesthetized with a 3-5% mixture of Isoflurane with oxygen

(400-600ml/min) and surgery for T3 contusion SCI using the Infinite Horizons device

was performed using established aseptic surgical techniques. When the rat was no longer

responsive to toe pinch or palpebral reflex, the surgical site overlying the vertebrae from

the interscapular region to mid-thorax was shaved and cleaned with three alternating

scrubs of Nolvasan and alcohol. Animals were maintained at 35.5–37.5°C on a feedback-

controlled heating block, and rectal temperature was monitored continuously. The

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location of the elongated T1 and T2 spinous processes were determined by midline

palpation. A 3-5cm midline incision of the skin overlying the T1-T3 vertebrae was

performed and the muscle attachments to the T1-T3 vertebrae were cleared by blunt

dissection, taking care not to damage the underlying nuchal vasculature. Using fine-

tipped rongeurs, the spinous process and the dorsal surface of the T2 vertebra was

removed laterally to the superior articular process. The rat was transferred to the Infinite

Horizons spinal contusion injury device (Precision Systems and Instrumentation,

Lexington KY). The adjacent T1 and T3 vertebrae were clamped into the device and the

torso of the animal was suspended slightly above the platform. After centering the

exposed spinal cord underneath the impactor tip a 300 kDyne impact (15 sec dwell time)

was initiated. This level of injury produces a consistent and reliable neurological and

histological outcome whereby animals exhibit a residual, chronic, locomotor deficit and

severe loss of integrity to the spinal cord white matter (≤ 25% of white matter). After

removal from the contusion device, all surgical incisions were closed in reverse

anatomical order with absorbable suture (Vicryl 4-0) for internal sutures and skin closure

with wound clips. Wound clips are removed 5-7 days following surgery. Surgical

controls undergo all procedures except for the injury-inducing impact.







133




that access to chow was available. Body weights and food weights were recorded once

daily. SCI rats received bladder expression and ventrum inspection twice daily until the

return of spontaneous voiding occurred.

5.3.2 Superior Mesenteric Artery Occlusion

Age-matched rats (n= 8) were fasted with free access to water overnight.

Following Isoflurane-anesthesia, a midline laparotomy was made and the intestines were

gently displaced laterally to allow the exposure of the aorta at the level of the left renal

artery and vein. The superior mesenteric artery (SMA) was carefully cleared of

connective tissue immediately distal to where it passed over the inferior vena cava a

midline laparotomy was performed and occluded with 3-0 silk suture for 60 minutes and

then released to re-establish blood flow. The muscle and skin layers were sutured (3-0

Vicryl and 3-0 nylon monofilament, respectively) and the animals were housed

individually and continuously monitored for 6 hours of reperfusion.

5.3.3 Gastrointestinal Tissue Harvest

Rats were deeply re-anesthetized with Isoflurane until non-responsive to toe

pinch. Quickly, the rats were decapitated and the abdomen was opened via a midline

incision. Gastrointestinal (GI) tissue (stomach and proximal duodenum) was taken at 6-

hours (SMA occlusion only), 1-day, 3-days, and 7-days post-SCI or control (n = 8 per

group). Following isolation, a small tissue sample, weighing ~200 milligrams, was

removed and placed in aluminum foil and immediately frozen in liquid nitrogen then

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transferred to a -80°C freezer until used for qRT-PCR. In the same animals, an adjacent

section of GI tissue was removed (as above) and placed in room temperature 10% neutral

buffered formalin (NBF) for histological processing.

5.3.4 Histopathological Processing

Formalin fixed tissues were processed in an automated Tissue-Tek VIP processor

and paraffin-embedded with a Tissue-Tek TEC embedding station (Sakura Finetek USA,

Torrance, CA). Sections were cut at 6 µm for routine hematoxylin and eosin (H&E)

staining or were mounted on charged plus slides for staining.

All tissues were examined by an American College of Veterinary Pathologists

diplomate blinded to treatment/genotype/intervention. All images were obtained with an

Olympus BX51 microscope and DP71 digital camera using cellSens Standard 1.6

imaging software (Olympus America, Center Valley, PA).

Multiple (3-6) random tissue samples were quantified as described previously

(Gulbinowicz et al., 2004) and the following measures determined: 1) Villus height and

width; 2) Crypt depth and width; and 3) Villus:Crypt height ratio was calculated. In each

case, 10 independent measurements for each variable were collected from at least 3

different intestinal sections. Semi-quantitative measurements of inflammation scoring

were made on a modified scale (Table 5.1; adapted from Berg et al., 1996 and Bleich et

al., 2004).

5.3.5 RNA Isolation, Reverse Transcription Reaction and q-PCR

Quantitative reverse transcriptase PCR (qRT-PCR) was used to quantify the level

of inflammatory mediators present at the assigned time points. Tissue sections from the

anterior gastric corpus and proximal duodenum were analyzed for induced nitric oxide

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synthase (iNOS), neuronal nitric oxide synthase (nNOS), cholecystokinin (CCK),

intercellular adhesion molecule (iCAM-1), macrophage inflammatory protein (MIP-1α),

and monocyte chemotactic protein (MCP-1) post-SCI and control surgery.

Whole GI tissue sections were used for RNA isolation. A small section of GI

tissue, weighing 50-100 milligrams was cut away from the whole tissue section and used

for RNA isolation. RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) and

RNeasy Microkit procedures (Qiagen, Valencia, CA). Briefly, frozen tissue was

homogenized in TRIzol using a glass homogenizer and Teflon pestle on ice, chloroform

was added to lysate, and the mixture was centrifuged in microcentrifuge tubes to separate

RNA. Ethanol was added to the upper aqueous phase, the mixture was applied to an

RNeasy spin column and filtered by centrifugation. After several washes, the samples

were subjected to an elution step using RNase-free water. Reverse transcription (RT) was

conducted using the High Capacity cDNA Reverse Transcription Kit (Applied

Biosystems, Foster City, CA). For RT, ~1 μg of RNA from each sample was added to

random primers (10×), dNTP (25×), MultiScribe reverse transcriptase (50 U/μl), RT

buffer (10×) and RNase Inhibitor (20U/μl) and incubated in a thermal cycler (Techne TC-

412, Barloworld Scientific, Burlington, NJ) for 10 min at 25°C, then for 120 min at 37°

C. Primers for B-actin were a QuantiTect Primer Assay (Qiagen). Primers for iNOS,

nNOS, CCK, iCAM, MIP-1α, and MCP-1 were designed using Primer Express (Applied

Biosystems). The forward and reverse primer pairs used for these studies are shown in

Table 5.2.

For real-time PCR, SYBR Green 2× Master Mix (Qiagen), forward and reverse

primers (100 μM), and RT product (1μl of a 1:16 dilution) were added to a 384-well

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plate. The cycling parameters consisted of an initial 2-min incubation at 50°C, followed

by 10 min at 95°C, then 15 s at 95 °C, a 30 sec annealing step at 55°C and a 30 sec

extension step at 72°C (55 cycles). A dissociation step (15 s at 95°C) was added

following 55 cycles to determine specificity of primers. In this assay, the dissociation

step confirmed the absence of nonspecific amplifications. Quantity of iNOS, nNOS,

CCK, iCAM, MIP-1α, and MCP-1 mRNA was based on a standard curve and normalized

to B-actin mRNA (ABI QuantStudio 12KFlex with available OpenArray block, Applied

Biosystems).



Group results from qRT-PCR were compared by two-way ANOVA and Tukey post hoc

analysis or paired t-test as appropriate (SPSS Inc, Chicago, IL).

5.4 RESULTS

5.4.1 T3-SCI provokes gastrointestinal tissue necrosis and reduction in mucosal villi

The SMA occlusion model was used to represent the maximal pathophysiology

resulting from reduced blood flow to the GI tract. We found that our SMA occlusion

significantly reduced blood flow to the midgut and caused the GI tissue to present

considerable edema by the time of euthanasia. The qualitative inspection of the GI tissue

was considered sufficient for including all animals for qRT-PCR analysis.

Upon removal of the GI tract from T3-SCI animals, it was grossly observed that

GI tissue was compromised compared to controls (Figure 5.1). Necrosis was noticed as

sections of the GI tract which were atrophic, darkly pigmented and excised tissue was

friable. Histopathological processing and analysis of the GI tissue further revealed

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damage in the form of reduced mucosal villus structure and gastric ulceration (Figure

5.2); profound reduction in duodenal integrity; small intestine revealed necrosis of

mucosa and submucosa, neutrophils and macrophages infiltration, and fibroplasia of

serosa and submucosa accompanied by mild autolysis. The duodenum was unremarkable

at 1-day but at 3-days T3-SCI revealed a significant reduction in mucosal villi height and

width (Table 5.3, P < 0.05). The inflammatory score of randomly analyzed tissue

segments was significantly elevated in the duodenum of T3-SCI rats (Table 5.3,

P < 0.05).

These data demonstrate a continuum of gastrointestinal tissue health immediately

following T3-SCI. Taken together, our anatomical and histological verify the profound

severity, produced by our surgical procedures for T3-SCI and surgical control animals.

5.4.2 T3-SCI increases mRNA expression of mucosal inflammatory markers

To quantify the upper GI inflammatory state, total RNA was isolated to analyze

expression of inflammatory markers. Despite a non-significant trend, gastric MCP-1

expression was not significantly different between T3-SCI and control (Figure 5.3;

P>0.05). Gastric expression of the chemokine MIP-1α demonstrated a significant

increase of MIP-1α (Figure 5.4; P<0.05) at 1-day and 3-days following T3-SCI. After 1-

week, MIP-1α expression was not different between T3-SCI and control (Figure 5.4;

P>0.05). The post-SCI expression of ICAM-1 demonstrated a significant increase at 1-

day and 3-days following T3-SCI (Figure 5.5; P<0.05).

Since superior mesenteric artery irrigates the intestines, only duodenal tissues

were analyzed from animals with SMA occlusion. After six hours of reperfusion,

duodenal MCP-1 expression was significantly elevated in SMA occlusion rats compared

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to control (Figure 5.6; P<0.05). Similarly, duodenal expression of the chemokine MIP-1α

demonstrated a significant increase in SMA occlusion rats compared to control (Figure

5.7; P<0.05). Lastly, the post-reperfusion expression of ICAM-1 demonstrated a

significant increase (Figure 5.8; P<0.05). By contrast, duodenal MCP-1 expression was

only significantly different between T3-SCI and control at 3-days after T3-SCI (Figure

5.6; P<0.05). Duodenal expression of MIP-1α demonstrated a significant increase in T3-

SCI rats at 1-day after injury (Figure 5.7; P<0.05). The significant differences between

T3-SCI rats are interpreted to reflect that MIP-1α returned to low levels beginning at 3-

days onwards. The post-SCI expression of ICAM-1, however, demonstrated a significant

increase at 1-day and 3-days following T3-SCI (Figure 5.8; P<0.05) and returned to low

levels by 1-week following T3-SCI.

In contrast to SMA ischemia reperfusion injury, these data in animals with T3-

SCI demonstrate a lower-grade of GI inflammation immediately following injury.

5.4.3 T3-SCI alteration of mRNA expression of nitric oxide isoforms

The gastric expression of iNOS was significantly different from controls only at

1-week following T3-SCI in which levels were lower (Figure 5.9; P < 0.05). As expected

from the literature, gastric nNOS was significantly reduced at 1-day and 3-days following

T3-SCI (Figure 5.10; P<0.05).

Following acute SMA occlusion/reperfusion injury, duodenal expression of both

iNOS and nNOS was significantly elevated (Figures 5.11 and 5.12 respectively; P <

0.05). By contrast, iNOS and nNOS did not demonstrate any overexpression in T3-SCI

rats at 1-day and 3-days after injury and only nNOS demonstrated any significant

elevation at 1-week following injury (Figure 5.12; P < 0.05).

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These data indicate that in a manner similar to SMA ischemia reperfusion injury,

animals with T3-SCI demonstrate a lower-grade of gastrointestinal NOS immediately

following injury. The low level of gastrointestinal injury in the T3-SCI rats may reflect

differential temporal characteristic of the constraints inherent to the time points chosen

for the experimental design.

5.5 DISCUSSION

The present study demonstrates that severe experimental T3-SCI initiates a low-

grade inflammatory cascade. Specifically, these experimental data indicate that: 1)

Similar to previous reports, mesenteric ischemia/reperfusion elicited a significant

elevation of duodenal iNOS, nNOS, MCP-1, MIP-1α and ICAM-1; 2) T3-SCI induced a

brief, but significant elevation in the gastric expression of MIP-1α and ICAM-1; and 3)

duodenal expression of ICAM-1 was most profoundly elevated after T3-SCI. These data

suggest that mesenteric vascular reflex deficits following T3-SCI may trigger low grade

inflammation in gastrointestinal tissues. This SCI-induced low grade inflammation does

not appear to be as profound as that observed following frank ischemia/reperfusion of the

mesentery.

Persistent hypotension often accompanies high-level SCI as a result of

decentralized autonomic control. Previous studies experimental T3-SCI in the rat

(Chapter 4) demonstrated that one manifestation of this loss of supraspinal modulation of

cardiovascular tone is the resultant significant reduction of resting blood flow to the

mesenteric bed. Additionally, the anticipated postprandial elevation of mesenteric blood

flow in response to duodenal infusion is significantly impaired after experimental T3-

SCI. The dysregulation of mesenteric blood flow in acutely-injured T3-SCI rats suggests

140

that arterial hypotension consequently diminishes mesenteric blood flow necessary to

meet mucosal demands at rest and during digestion. The resulting GI ischemia may be an

underlying pathology leading to gastric dysfunction through the generalized mechanism

of reduction in energy homeostasis and the initiation of cell damage, destruction, and

death due to ischemia (Oruckaptan et al., 2009,Temiz et al., 2013). Furthermore, it is

recognized that ischemia initiates an inflammatory cascade (Zhu et al., 2013). In the

present study, the histopathology and necrotic tissue seen at 1-day to 3-days post-SCI

accurately reflects the most severe examples of the injury accompanying visceral

hypoperfusion-induced ischemia. Caution must be exercised when extrapolating the data

from ischemia/reperfusion models and our model of SCI. The acute period of high-level

SCI presents severe hypotension requiring vasopressor therapy (reviewed in Weaver et

al., 2012). It is unclear, however, whether this period of so-called neurogenic shock

produces a level, and duration, of mesenteric hypoperfusion that is comparable to the

approximate 90% reduction of flow seen after SMA occlusion.

The reperfusion of ischemic tissues involves a known, biochemically mediated,

event involving the increased expression of adhesion molecules and chemokines (Oz et

al., 2008). Beginning with early mast cell degranulation and histamine release (Bulmer et

al., 2005,Jiang et al., 2011), the up-regulation of adhesion molecules and chemokines

forms the early line of defense in the intestinal mucosa and leads to an inflammatory

pathway which promotes neurotoxicity, leukocyte (including lymphocytes, neutrophils,

and monocytes), macrophage, and astrocyte recruitment (Mautes et al., 2000), endothelial

damage, hypoperfusion, and apoptosis (Temiz et al., 2013,Zhu et al., 2013,Blight, 1992).

Utilizing our model of acute T3-SCI in rats, we demonstrated the effects of SCI upon

141

iCAM-1, MIP-1α, and MCP-1 expression within the upper gastrointestinal tract which

suggests the initiation of a low grade inflammatory cascade following SCI. Furthermore,

we investigated the downstream inflammatory mediator nitric oxide synthase (NOS).

Nitric oxide (NO), acting as a neurotransmitter, plays significant detrimental and

beneficial roles in the central nervous system following SCI. NO is produced by three

types of nitric oxide synthase (NOS) enzymes: iNOS, nNOS, and eNOS; the inducible

calcium-independent isoform (iNOS); the constitutive calcium/calmodulin-dependent

neuronal NOS (nNOS); and endothelial NOS (eNOS) isoform. Soon after a SCI, NOS

isoforms would be expected to produce significant amounts of NO which may contribute

to the primary phase of the inflammatory response both through increased vasodilation as

well as diminished smooth muscle contractility. Our understanding of NOS isoform

expression and interactions after SCI is limited to that which occurs within the injured

spinal tissue. Interestingly, nNOS and eNOS show maximal enzyme expression 6 hours

after SCI and nNOS mRNA has been shown to be elevated as early as 30 minutes after

SCI and remains elevated for only the next 1-4 hours (Diaz-Ruiz et al., 2005). The short

lived increase in nNOS expression in the profoundly inflammatory milieu of the lesion

epicenter may explain why we did not observe an increase in nNOS mRNA 1-day, 3-

days, or 7-days following SCI. Indeed, our data indicated a significant reduction in nNOS

mRNA at 3-days, or 7-days following SCI. These specific findings are consistent with the

reduced optical density of immunocytochemically labeled nNOS following T9-SCI in

female rats (Kabatas et al., 2008). The reduction of nNOS is significant in that NO, along

with VIP, is a major inhibitory neuroeffector within the GI tract (see Chapter 1).

Furthermore, NO plays a significant part in the regulation of vascular smooth muscle

142

tone. In a study utilizing a murine model of less severe mesenteric ischemic injury, iNOS

inhibitors were protective against the pathological changes caused by ischemia-

reperfusion injury to the gut by preventing the formation of nitric oxide (NO) (Cuzzocrea

et al., 2002). This same study proposed that large amounts of NO, produced by iNOS,

were responsible for diminished local blood flow. The implications of these particular

findings remain to be determined in our model of T3-SCI.

The release of NO from activated astrocytes and microglia, has been reported to

cause neuronal cell death (Bal-Price & Brown, 2001,Venkataramana et al.). The

administration of 1400W, a selective inhibitor of iNOS (Garvey et al., 1997) was found

to serve as a protector against the pathology caused by diminished blood flow (Squadrito

et al., 2000). The role, and timing, of iNOS following T3-SCI in rats requires further

investigation before adapting therapeutic interventions.

The inflammatory chemokines MIP-1α and MCP-1 have been shown to be

partially co-localized with TRPV1 (transient receptor potential vanilloid 1; capsaicin

receptor VR1) in the dorsal horn of spinal cord lesioned animals (Knerlich-Lukoschus et

al., 2011). The specific correlation between chemokines and pain-sensitive TRPV1

receptors in the above model may be linked to neuropathic pain following SCI (Knerlich-

Lukoschus et al., 2011). In our model, we have demonstrated that there is a low-grade

inflammation of the GI tract following a severe T3-SCI. Activation and up-regulation of

TRPV1 has been shown to be involved in hypersensitivity in the inflamed gut (Mawe et

al., 2009b). We propose that similar up-regulation of TRPV1 occurs within vagal

afferents following experimental T3-SCI. We propose that the relationships between

143

inflammatory cells, TRPV1, and GI function will provide the next step in gaining greater

insight into post-SCI pathophysiology of the GI tract.


Gina Deiter provided invaluable instruction and assistance for performing and analyzing

the qRT-PCR experiments. Margaret McLean provided assistance with animal care,

tissue harvesting, data entry and analysis in Excel.

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Table 5.1

Semi-quantitative measurements of inflammation scoring for gastrointestinal tissue

Grade Description

Grade 0: no change from normal tissue

Grade 1: one or a few multifocal mononuclear cell infiltrates in the lamina

propria

Grade 2: lesions involve more of the intestine than grade 1 lesions, and/or are

more frequent. Typical changes include several multifocal, mild

inflammatory cell infiltrates in the lamina propria composed primarily

of mononuclear cells with a few neutrophils. Inflammation rarely

involves the submucosa

Grade 3: lesions involve a large area of the mucosa or are more frequent than

grade 2 lesions. Inflammation is moderate and involves the submucosa

but is not transmural. Inflammatory cells are a mixture of mononuclear

cells as well as neutrophils, and crypt abscesses are sometimes

observed. Small epithelial erosions are occasionally present.

Grade 4: lesions involve most of the intestinal section and are more severe than

grade 3 lesions. Inflammation is severe, including mononuclear cells

and neutrophils, and can be transmural. Crypt abscesses and ulcers are

present.

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Table 5.2

Forward and reverse primer sequences for quantitative real time PCR (qRT-PCR)

Gene Forward Primer Reverse Primer

iNOS 5’-GCCCCAACCGGAGAAGGGGA-3’ 5’-CCTTGCACCAGGGCCGTCTG-3’

nNOS 5′-GCGTCTCCACACCAACGGGA-3′ 5′-GTGCAGGGTGGGAGGCGAGA-3′

CCK 5′-AGCTGAGGGCTGTGCTCCGA-3′ 5′-TTCGAGGCGAGGGGTCGTGT-3′

ICAM-1 5’-TGCCAGCCCGGAGGATCACA-3’ 5’-CGGGAGCTAAAGGCACGGCA-3’

MIP-1α 5’-TGACACCCCGACTGCCTGCT-3’ 5’-TGACACCCGGCTGGGACCAA-3’

MCP-1 5’-TCTCTGTCACGCTTCTGGGCCT-3’ 5’-TAGCAGCAGGTGAGTGGGGCA-3’

146

Figure 5.1. T3-SCI provokes gastrointestinal tissue pathology.

Post-SCI pathophysiology occasionally presents as tissue necrosis as seen in small

intestine of control (A) vs T3-SCI (B) 3-days following surgery.

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Figure 5.2. T3-SCI provokes gastrointestinal mucosal histopathology.

Compared to controls (A, C) T3-SCI rats (B, D) reveal a significant histopathology of

duodenal villi after SCI. Occasional gastric ulceration is observed 1-day post-SCI (E).

(Calibration bar: A-B = 1mm; C-D = 100μM; E = 500μM).

E

148

Table 5.3

T3-SCI provokes an inflammatory response and blunting of mucosal villi in

duodenal tissue (*P<0.05 vs. control).

Experimental Groups

Control

(n=5)

T3-SCI

(n=7)

Average inflammatory score 0.4 ± 0.2 0.9 ± 0.1 *

Average villus height (µm) 435 ± 24 341 ± 11 *

Average villus width (µm) 122 ± 4 102 ± 2 *

Average crypt depth (µm) 149 ± 6 147 ± 10

Average crypt width (µm) 52 ± 2 52 ± 2

Villus:crypt ratio 3 ± 0.1 2 ± 0.2

Villus height:width ratio 4 ± 0.2 3 ± 0.2

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Figure 5.3. Expression levels of gastric MCP-1 mRNA after T3-SCI.

Measurement of gastric MCP-1 mRNA expression does not demonstrate any significant

difference between control and T3-SCI animals for the three post-injury time points

(n=8/group).


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Figure 5.4. Expression levels of gastric MIP-1α mRNA after T3-SCI.

Measurement of gastric MIP-1α mRNA expression demonstrated a significant elevation

in T3-SCI rats at 1-day and 3-days compared to control animals matched for the same

post-operative time point.

Bars with lower case letters reflect significant effects, P<0.05, based on ANOVA,

followed by Tukey post hoc test. a = T3-SCI vs same day control; b = 1-day control vs 3-

days control; c = 3-days control vs 1-week control; d = 1-day T3-SCI vs 3-days T3-SCI;

e = 3-days T3-SCI vs 1-week T3-SCI (n=8/group).


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Figure 5.5. Expression levels of gastric ICAM-1 mRNA after T3-SCI.

Measurement of gastric ICAM-1mRNA expression demonstrated a significant elevation

in T3-SCI rats at 1-day and 3-days compared to control animals matched for the same

post-operative time point. ICAM-1 levels remained significantly elevated until 1-week

post-injury.


followed by Tukey post hoc test. a = T3-SCI vs same day control; b = 3-days T3-SCI vs

1-week T3-SCI; c = 1-day T3-SCI vs 1-week T3-SCI (n=8/group).


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Figure 5.6. Expression levels of duodenal MCP-1 after T3-SCI or SMA occlusion.

Measurement of duodenal MCP-1mRNA expression demonstrated a significant elevation

in T3-SCI rats at 3-days compared to control animals matched for the same post-

operative time point. In animals that received a 60-minute occlusion of the SMA, MCP-1

mRNA was significantly elevated after 6-hours of reperfusion.


followed by Tukey post hoc test. a = T3-SCI vs same day control; * P<0.05 vs control

(n=8/group).


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Figure 5.7. Expression levels of duodenal MIP-1α after T3-SCI or SMA occlusion.

Measurement of duodenal MIP-1α mRNA expression demonstrated a significant

elevation in T3-SCI rats only after 1-day post-injury compared to control animals

matched for the same post-operative time point. In animals that received a 60-minute

occlusion of the SMA, MIP-1α mRNA was significantly elevated after 6-hours of

reperfusion.


followed by Tukey post hoc test. a = T3-SCI vs same day control; b = 1-day T3-SCI vs 3-

day T3-SCI; c = 1-day T3-SCI vs 1-week T3-SCI; * P<0.05 vs control (n=8/group).


154

Figure 5.8. Expression levels of duodenal ICAM-1 after T3-SCI or SMA occlusion.

Measurement of duodenal ICAM-1 mRNA expression demonstrated a significant

elevation in T3-SCI rats as early as 1-day post-injury and continuing through 3-days post-

injury compared to control animals at the same post-operative time point. In animals that

received a 60-minute occlusion of the SMA, ICAM-1 mRNA was significantly elevated

after 6-hours of reperfusion.


followed by Tukey post hoc test. a = T3-SCI vs same day control; c = 1-day T3-SCI vs 3-

day T3-SCI; d = 3-day T3-SCI vs 1-week T3-SCI; e = 1-day T3-SCI vs 1-week T3-SCI; f

= 3-day control vs 1-week control; * P<0.05 vs control (n=8/group).


155

Figure 5.9. Expression levels of gastric iNOS after T3-SCI.

Measurement of gastric iNOS mRNA expression demonstrated a significant difference in

T3-SCI rats only at 1-week post-injury compared to control animals at the same post-

operative time point (* P<0.05 vs control; n=8/group).


156

Figure 5.10. Expression levels of gastric nNOS after T3-SCI.

Measurement of gastric nNOS mRNA expression demonstrated a significant reduction in

T3-SCI rats beginning at 3-days and continuing 1-week post-injury compared to control

animals at the same post-operative time point (* P<0.05 vs control; n=8/group).


157

Figure 5.11. Expression levels of duodenal iNOS after T3-SCI or SMA occlusion.

Measurement of duodenal iNOS mRNA expression did not demonstrate any significant

difference in T3-SCI rats compared to control animals at the same post-operative time

point. Data for 1-day post-operative animals were not analyzable and are not shown. In

animals that received a 60-minute occlusion of the SMA, iNOS mRNA was significantly

elevated after 6-hours of reperfusion (* P<0.05 vs control; n=8/group).


158

Figure 5.12. Expression levels of duodenal nNOS after T3-SCI or SMA occlusion.

Measurement of duodenal nNOS mRNA expression demonstrated a significant elevation

in T3-SCI rats beginning at 1-week post-injury compared to control animals at the same

post-operative time point (* P<0.05 vs control). In animals that received a 60-minute

occlusion of the SMA, nNOS mRNA was significantly elevated after 6-hours of

reperfusion (* P<0.05 vs control; n=8/group).


159

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Chapter 6:

Diminished gastric motility following experimental

spinal cord injury in the rat is accompanied by changes

in receptor expression in the nodose ganglia

163

6.1 ABSTRACT

Background: In addition to dysfunction of gastrointestinal motility following

high-thoracic experimental spinal cord injury (T3-SC), previous observations of T3-SCI

rats have identified a dramatic reduction in mean arterial pressure that also manifests as

mesenteric hypoperfusion at rest and during the postprandial phase of digestion. In

addition, experimental animals with T3-SCI express elevated inflammatory markers

within the stomach and duodenum during the same post-injury time period that

gastrointestinal dysfunction and mesenteric hypoperfusion occur. Transient receptor

potential vanilloid-1 (TRPV1) has been linked to gastrointestinal afferent function in

health and disease states including inflammation. The cell bodies of vagal afferent fibers

are contained within the nodose ganglion and display receptor plasticity to the

gastrointestinal peptide, cholecystokinin (CCK) in other experimental models. The

present study tested the hypothesis that vagal afferent cell bodies express changes in

TRPV1 and CCK receptor expression following experimental T3-SCI.

Methods: We investigated the expression of CCK receptor type 1 (CCK-1r) and

TRPV1 in vagal afferent cell bodies from T3-SCI or surgical control rats using

immunohistochemical and quantitative molecular techniques.

Key Results: Qualitatively, immunohistochemical expression of CCK and CCK-

1r was elevated as was expression of TRPV1. Quantitative assessment of mRNA

expression by qRT-PCR revealed significant elevation of CCK, CCK-1r and TRPV1

following T3-SCI.

Conclusions: These data suggest a relationship between the GI dysfunction,

increased GI inflammation and CCK, CCK-1r and TRPV1receptor expression within

vagus nerve afferents following T3-SCI. These post-injury changes may contribute to

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gastric dysmotility following T3-SCI since CCK expression may reflect up-regulation

that is similar to what has been observed following vagotomy. The increase in CCK-1r

and TRPV1 may include alterations in the functional properties of vagal afferent fibers in

response to the physiological signals associated with digestion.

6.2 INTRODUCTION

The vagus is a bilateral mixed sensory and motor nerve, with approximately 80%

of the nerve being sensory fibers. The cell bodies of these vagal sensory afferent fibers

reside within the nodose ganglia. In the rat, these ganglia are immediately inferior to the

foramen magnum adjacent to the common carotid artery. The vagus nerve is the

predominant autonomic nerve supply to the upper-GI and mid-colon at approximately the

hepatic flexure (reviewed in Chapter 1). The parasympathetic innervation to the stomach,

through the vagus which then modulates the enteric nervous system (ENS), provides the

dominant extrinsic control of the stomach. Gastrointestinal vagal fibers originate within

different layers of the stomach with morphological specializations known as IGLEs,

(Powley & Phillips, 2002) and IMAs (Berthoud & Powley, 1992). Gastric vagal afferents

ascend centrally into the caudal medulla via the tractus solitarius and terminate within the

nucleus tractus solitarius (NTS), dorsal motor nucleus of the vagus (DMV), and the area

postrema (AP) by way of a glutamatergic synapse (see Browning & Travagli, 2010). This

glutamate release in the NTS acts upon one of three (GABA-ergic, glutamatergic, or

noradrenergic) second order neurons projecting to the dorsal motor nucleus of the vagus

(DMV). The DMV consists of parasympathetic vagal cholinergic preganglionic

motorneurons. Either at rest as a slow pacemaker cell (Browning et al., 1999,Travagli et

al., 1991,Marks et al., 1993), or in response to excitatory synaptic input, DMV neurons

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release ACh onto nicotinic receptors of the postganglionic cholinergic neurons within the

ENS of the stomach. A subset of these postganglionic neurons release ACh onto

cholinergic muscarinic receptors within the myenteric plexus of the stomach causing

gastric contraction. A separate subset of the cholinergic preganglionic DMV neurons

release ACh onto nicotinic receptors of the postganglionic neurons within the ENS

(Grundy et al., 2000) causing gastric relaxation. This subset of postganglionic neurons

are categorized as the NANC neurons that release NO and VIP to act on the circular

smooth muscle of the stomach.

As stated previously, the GI tract is a highly perfused organ system, receiving 20-

25% of the total cardiac output. Persistent vascular hypotension is especially likely

following cervical or high thoracic injury in patients and similar deficits have been

demonstrated for experimental SCI in rats (see Chapter 4). Specifically, our previous

data shows that experimental SCI significantly impairs superior mesenteric artery blood

flow at rest and in response to nutrient ingestion. Vascular hypotension resulting in

visceral hypoperfusion elevates the risk of GI ischemia, the initiation of an inflammatory

cascade, and end-organ dysfunction. These diminished vascular reflexes in the rat model

of experimental SCI render the GI tract susceptible to elevation of markers associated

with inflammatory injury (see Chapter 5).

As previously described in Chapter 1, TRPV1 has received attention in

association with inflammatory conditions and nociceptive sensation in somatic and

visceral tissues (Caterina et al., 1997,Caterina & Julius, 2001). TRPV1, a member of the

TRP channel family (Furuta et al., 2012,Clapham et al., 2001) is activated by noxious

stimuli (i.e. capsaicin, noxious heat, and low pH; all of which have been proposed as

166

common consequences of SCI-induced inflammation) (Nilius et al., 2007a,Nilius et al.,

2007b,Furuta et al., 2012). Furthermore, this membrane protein forms a nonselective

cation channel with a very high permeability to Na+ and Ca

2+ (described in Lee & Gu,

2009). Expression of TRPV1 has been revealed in many sensory afferent neurons;

specifically on sensory nerve terminals of the vagus nerve and within the soma of the

neurons within the nodose ganglion, particularly during times of tissue injury or

inflammation (Lee & Gu, 2009,Zhao & Simasko, 2010,Zhao et al., 2010,Jia & Lee,

2007,Caterina & Julius, 2001). Upon peripheral detection of noxious stimuli, nociceptor

C-fibers transduce signals (i.e. mechanical, chemical, or thermal) into action potentials

and transmit this information to the NTS (Caterina & Julius, 2001). Emerging evidence in

other sensory neuronal fibers suggest that in response to injury, afferent C-fibers express

injury-induced markers such as P2X3, SP, tropomyosin receptor kinase A and CGRP

(Snider & McMahon, 1998,Banerjee et al., 2009,Arms & Vizzard, 2011).

As seen in the pancreatitis-induced inflammatory model, attenuating

inflammation lead to positive disease outcomes; whereas, exacerbation of inflammation

worsened the prognosis via up-regulation of endogenous TRP channels in sensory

neurons (Xu et al., 2007,Nilius et al., 2007a,Nilius et al., 2007b). This increased

sensitivity of TRP channels to inflammatory conditions coincides with role TRPV1 plays

in satiety and GI motility (Zhao et al., 2010). Emerging evidence suggests that TRPV1

channels are functional centrally, within the DVC, modulating synaptic input to the DMV

motor neurons (Derbenev et al., 2006,Zsombok et al., 2011,De Man et al., 2008) thus

altering visceral function, particularly the motility of the stomach (Zsombok et al., 2011).

167

Based upon the previous observations of post-SCI hypoperfusion and elevated

inflammatory markers, the present work used immunohistochemistry and qRT-PCR to

measure transient receptor potential vanilloid type 1 receptor (TRPV1), cholecystokinin

(CCK), and cholecystokinin-1 receptor (CCK-1r) in the vagal afferent cell bodies of the

nodose ganglia to test the hypothesis that T3-SCI diminished gastric motility is associated

with receptor changes within the vagal afferent cell bodies.

6.3 MATERIALS AND METHODS






food and water provided ad libitum. After surgery animals were single housed in cages

mounted on warming pads to maintain an adequate body temperature of 37◦C. All

experimental animals were observed daily, and food and body weight were recorded.

6.3.1 Spinal Cord Injury Surgery and Post-Operative Care

Animals (n=96) were randomly assigned to the control or SCI group. After initial

groups were assigned, the groups were further divided into 1-day, 3-day, and 7-days post-

control or post-SCI study groups (n=8/group). Due to the small tissue volume of the

nodose ganglia, each experimental study, immunohistochemistry (IHC) or qRT-PCR,

used the same total number of animals. Following group assignments, SCI surgeries

surgery for T3 contusion SCI was performed using established aseptic surgical

techniques. When the rat was no longer responsive to toe pinch or palpebral reflex, the

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surgical site overlying the vertebrae from the interscapular region to mid-thorax was

shaved and cleaned with three alternating scrubs of Nolvasan and alcohol. Animals were

maintained at 35.5–37.5°C on a feedback-controlled heating block, and rectal

temperature was monitored continuously. The location of the elongated T1 and T2

spinous processes were determined by midline palpation. A 3-5cm midline incision of the

skin overlying the T1-T3 vertebrae was performed and the muscle attachments to the T1-

T3 vertebrae were cleared by blunt dissection, taking care not to damage the underlying

nuchal vasculature. Using fine-tipped rongeurs, the spinous process and the dorsal

surface of the T2 vertebra was removed laterally to the superior articular process. The rat

was transferred to the Infinite Horizons spinal contusion injury device (Precision Systems

and Instrumentation, Lexington KY). The adjacent T1 and T3 vertebrae were clamped

into the device and the torso of the animal was suspended slightly above the platform.

After centering the exposed spinal cord underneath the impactor tip a 300 kDyne impact

(15 sec dwell time) was initiated. This level of injury produces a consistent and reliable

neurological and histological outcome whereby animals exhibit a residual, chronic,

locomotor deficit and severe loss of integrity to the spinal cord white matter (≤ 25% of

white matter). After removal from the contusion device, all surgical incisions were closed

in reverse anatomical order with absorbable suture (Vicryl 4-0) for internal sutures and

skin closure with wound clips. Wound clips are removed 5-7 days following surgery.

Surgical controls undergo all procedures except for the weight drop.




169







that access to chow was available. Body weights and food weights were recorded once

daily. SCI rats received bladder expression and ventrum inspection twice daily through

the duration of the experiment.

6.3.2 Superior Mesenteric Artery Occlusion

Age-matched rats (n= 8) were fasted with free access to water overnight.

Following Isoflurane-anesthesia, a midline laparotomy was made and the intestines were

gently displaced laterally to allow the exposure of the aorta at the level of the left renal

artery and vein. The superior mesenteric artery (SMA) was carefully cleared of

connective tissue immediately distal to where it passed over the inferior vena cava a

midline laparotomy was performed and occluded with 3-0 silk suture for 60 minutes and

then released to re-establish blood flow. The muscle and skin layers were sutured (3-0

Vicryl and 3-0 nylon monofilament, respectively) and the animals were housed

individually and continuously monitored for 6 hours of reperfusion.

6.3.3 Nodose Ganglia Isolation and Harvest

Following the appropriate time-course (1d, 3d, or 7d), animals were anesthetized

with 5% Isoflurane until non-responsive to toe pinch. Once hind-paw reflex was absent, a

rat was immediately placed dorsally and an incision was made in the skin from the notch

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of the sternum towards the midline of the mandible. The strap muscles of the neck were

quickly separated and both carotid arteries were exposed. The vagus nerve was located

bilaterally and following rostrally until the nodose ganglia were visualized and isolated.

The rat was then rapidly exsanguinated and the nodose were quickly excised and placed

in liquid nitrogen-cooled-microcentrifuge tubes. The microcentrifuge tubes were stored in

-80C freezer until time for PCR. Nodose for IHC were isolated and harvested in the

same way but stored in 5mL glass vials containing 4% paraformaldehyde and 20%

sucrose. These vials were stored overnight in the 4C refrigerator and then sectioned on

the microtome (10µm) prior to IHC.

6.3.4 Fluorescent Immunohistochemistry for Nodose TRPV1 and CCK-1r

After sectioning, nodose ganglia sections were immediately placed on gel-subbed

slides and allowed to dry overnight. Excess embedding compound (Optimal cooling

temperature, Thermo-Fisher Scientific, Waltham, MA) was carefully removed to apply

liquid paraffin (PAP pen, Thermo-Fisher Scientific, Waltham, MA) to create a well to

hold fluid around the tissue sections. The slides were rehydrated with PBS for 10min at

RT. After the PBS was removed, the slides were blocked with 10% NDS + PBS + triton

and incubated for one hour. The blocking solution was removed and TRPV1 primary

antibody was added to the slides (polyclonal rabbit anti-TRPV1 + 1% NDS; 1:500;

Millipore, Billerica, MA) and the slides were incubated for 2 hours. Primary antibody

was removed and the slides were rinsed in PBS. Secondary antibody (Alexa 488 donkey

anti-rabbit IgG, Life Technologies, Thermo-Fisher Scientific, Waltham, MA) was placed

on slides and slides were incubated for 2.5 hours. The slides were then rinsed in PBS.

The slides were then incubated with the primary antibody for CCK-AR (Goat anti-CCK-

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AR; 1:500; polyclonal Santa Cruz Biotechnology, Dallas, TX) in 1% NDS for 2 hours.

Primary CCK-AR was vacuumed off the slides, the slides were rinsed with PBS and

incubated in Alexa 594 secondary (donkey anti-goat IgG, Life technologies, Thermo-

Fisher Scientific, Waltham, MA) for 2.5 hours. The slides were rinsed with PBS, PAP

pen was wiped off, and the slides were immediately mounted with vectashield “Hard Set”

for view under the fluorescent microscope. Slides were digitally imaged on a Zeiss

Axioscope fluorescent microscope and Axiocam CCD camera, imported into Adobe

Photoshop for analysis.

6.3.5 RNA Isolation from Nodose Ganglia

Microcentrifuge tubes containing the harvested nodose ganglia were removed

from the -80C freezer. Each pair of nodose ganglia were placed in a new tube

containing 400µL TRIzol and immediately homogenized with a Teflon pestle until

nodose were uniformly homogeneous. The homogenate was then transferred to a 1.5mL

microcentrifuge tube and incubated at room temperature (RT) for 5 minutes. Next, 80µL

chloroform was added, to the tubes and homogenate, and the tubes were shaken

vigorously for 15 seconds (0.2mL chloroform/1mL TRIzol). The tubes were again

incubated at RT for 2-3 minutes. The tubes were then centrifuged at 14,000rpm for 15

minutes at 4C. The upper aqueous portion was transferred to a new 1.5mL tube and 1

volume (~150µL) of 70% EtOH was added to each tube and vortexed. Each sample was

then pipetted into Rneasy Mini Spin Columns in a 2mL collection tube. The collection

tubes were centrifuged at 10,000rpm for 15 seconds at RT and flow through was

discarded. Columns were placed in new 2mL tubes and 350µL Buffer RW1 was added

to Rneasy mini spin columns and centrifuged for 15 seconds at 10,000rpm at RT; flow

172

through was discarded. Then 350µL Buffer RW1 was pipetted into the RNeasy MinElute

Spin Column and centrifuged for 15 seconds at 10,000rpm at RT; flow through was

discarded. Then 500µL Buffer RPE was pipetted into the column and centrifuged for 15

seconds at 10,000rpm at RT; flow through discarded. The column was placed in a new

2mL tube and the cap of the spin column was opened and the column was centrifuged at

14,000rpm for 3 minutes at RT. The column was transferred to a new 1.5mL tube and

14µL of Rnase-free water was pipetted directly onto a silica-gel membrane. The

membrane was incubated for 10 minutes at RT then centrifuged for 1minute at

14,000rpm at RT. The RNA was lastly checked on the nanodrop in preparation for qRT-

PCR.

6.3.6 Reverse Transcription and q-PCR of Nodose Ganglia

For the Reverse transcription (RT) procedure, the High Capacity cDNA Reverse

Transcription Kit (Applied Biosystems, Foster City, CA) was used at RT. Approximately

1 μg of RNA from each sample was added to random primers (10×), dNTP (25×),

MultiScribe reverse transcriptase (50 U/μl), RT buffer (10×) and RNase Inhibitor

(20U/μl) and incubated in a thermal cycler (Techne TC-412, Barloworld Scientific,

Burlington, NJ) for 10 min at 25°C, then for 120 min at 37° C. Primers for β-actin were a

QuantiTect Primer Assay (Qiagen). The forward and reverse primer pairs used for these

studies were designed using Primer Express (Applied Biosystems) and are shown in

Table 6.1.

SYBR Green 2× Master Mix (Qiagen), forward and reverse primers (100 μM),

and RT product (1μl of a 1:16 dilution) were added to a 384-well plate for real-time PCR.

The cycling parameters were comprised of initial 2-min incubation at 50°C, followed by

173

10 min at 95°C, then 15 s at 95 °C, a 30 sec annealing step at 55°C and a 30 sec extension

step at 72°C (55 cycles). A dissociation step (15 s at 95°C) was added following 55

cycles to determine specificity of primers. In this assay, the dissociation step confirmed

the absence of nonspecific amplifications. Quantity of, the above listed primers, mRNA

was based on a standard curve and normalized to B-actin mRNA (ABI QuantStudio

12KFlex with available OpenArray block, Applied Biosystems).


All results are expressed as means ± S.E.M. with significance defined as P < 0.05.

The group results from qRT-PCR were compared by two-way ANOVA and Tukey post

hoc analysis or paired t-test as appropriate (SPSS Inc, Chicago, IL).

6.4 RESULTS

6.4.1 T3-SCI increases Nodose TRPV1 and CCK-1r immunofluorescence

Our fluorescent IHC images suggested a generalized increase in TRPV1 (Figure

6.1) and CCK-1r (Figure 6.2) expression throughout the nodose ganglia cell bodies at 1-

day post-SCI compared to 1-day controls. These data led us to pursue TRPV1 and CCK-

1r gene expression quantification using RT-PCR.

6.4.2 T3-SCI increases mRNA expression of TRPV1 and CCK-1r in Nodose Ganglia

To quantify the post-injury phenotypic changes in the vagal afferent neurons, total

RNA was isolated to analyze expression of TRPV1 and CCK-1r in nodose ganglia.

Following acute SMA occlusion/reperfusion injury, nodose expression of TRPV1 mRNA

was significantly elevated compared to 1-day surgical control (Figure 6.3; P < 0.05). Our

data further demonstrated that TRPV1 mRNA significantly increased in nodose ganglia

at 1-day and 3-days following SCI when compared to same day controls (Figure 6.3; P <

174

0.05). There were also significant changes within the T3-SCI group over time whereby

TRPV1 mRNA expression was significantly lower after 1-week, compared to both 1-day

and 3-days controls (Figure 6.3; P < 0.05).

Following acute SMA occlusion/reperfusion injury, nodose expression of CCK-1r

mRNA was significantly elevated compared to 1-day surgical control and T3-SCI (Figure

6.4; P < 0.05). At 1-day, T3-SCI animals were not different from control (P > 0.05).

These data further demonstrate CCK-1r mRNA expression to be significantly increased

at 3-days and 7-days post-SCI when compared to same-time controls (Figure 6.4; P <

0.05). There were also significant changes within the T3-SCI group over time whereby

CCK-1r mRNA expression was significantly higher at 3-days, compared to both 1-day

and 1-week (Figure 6.4; P < 0.05). Lastly, CCK mRNA expression was increased within

the nodose ganglia at 1-day following SCI. (Figure 6.5; P < 0.05).

6.5 DISCUSSION

The present data demonstrate that during the acute phase of severe T3-SCI injury,

vagal afferent neurons express alteration in receptor coding. Specifically, these

experimental data indicate that: 1) T3-SCI induced a significant up-regulation of nodose

TRPV1 over surgical control that returned to low levels by 1-week after injury; 2) T3-

SCI also induced a significant up-regulation of nodose CCK-1r mRNA beginning at 3-

days and continuing through the first week; 3) a significant up-regulation of TRPV1 and

CCK-1r mRNA was also observed in the nodose ganglia of animals that received SMA

ischemia/reperfusion injury.

The majority of reports in the clinical literature describe derangements in upper

gastrointestinal reflex emptying and motility, especially after spinal cord injuries

occurring above the mid-thoracic spinal segments (Berlly & Wilmot, 1984,Fealey et al.,

175

1984,Kao et al., 1999,Kewalramani, 1979,Nino-Murcia & Friedland, 1991,Rajendran et

al., 1992,Segal et al., 1995,Stinneford et al., 1993,Williams et al., 2011). Persistent,

vagally-mediated gastroparesis has been reported in an animal model of SCI (Tong &

Holmes, 2009,Rodrigues et al., 2001).The mechanism for this dysfunction remains

obscure, however, accumulating evidence suggests that SCI triggers impairments in the

vagal afferent signaling necessary for proper gastrointestinal reflexes (Tong et al.,

2011,Swartz & Holmes, 2014,Holmes, 2012).

Evidence of adaptive and pathophysiological neuronal plasticity is beginning to

accumulate in numerous organ systems innervated by the vagus nerve (see Browning &

Travagli, 2010,Browning, 2010,Mawe et al., 2009,Demir et al., 2013). The terminal

projections of the gastric vagal afferent fibers, detect mechanical signals (e.g., gastric

distension) as well as chemical signals (e.g., increased acidity, increased pH,

inflammatory molecules). Nociceptor cell bodies have been isolated to three primary

locations, the dorsal root ganglia, trigeminal ganglia, and nodose ganglia (Caterina &

Julius, 2001). The principal neural circuit for conducting visceral nociceptive signals is

recognized to pass through the dorsal root ganglia and spinal cord (see Christianson et al.,

2009,Christianson & Davis, 2010). There is some emerging evidence that has led

investigators to propose that vagal afferents transmit nociceptive information (see

Surdenikova et al., 2012).

Accumulating evidence is beginning to support the relationship that following

SCI there is a sudden increase in GI and systemic inflammation in conjunction with a

decrease in GI blood flow. While it is appreciated that vascular dysregulation leads to

visceral hypoperfusion, the response of vagal afferents remains obscure. Experimental

176

evidence has revealed that 42% of primary gastric vagal afferent neurons express TRPV1

(Banerjee et al., 2007), and TRPV1 channels have been identified and are expressed on

the vagal C-fiber afferents of the vagus nerve (Hayes, Jr. et al., 2013). Additionally,

TRPV1 channels are polymodal transducers known to be activated during conditions that

are likely to be present after SCI, including acidity (Strain & Waldrop, 2005),

endogenous inflammatory mediators (Hayes, Jr. et al., 2013) and as set forth in this thesis

which has demonstrated that T3-SCI initiates immediate local and systemic inflammation

manifested as low grade inflammation in the stomach and duodenum.

More specifically, the onset of inflammation causes a release of arachidonic acid

derivative, anandamide, lipoxygenase derivatives, and N-acyl dopamines known as

endovanilloids (Banerjee et al., 2007) which have been shown to activate TRPV1

channels and further enhance the inflammatory process. In addition to inflammation

increasing the expression of TRPV1, physiological levels of cations also contribute to

activation of TRPV1 (Ahern et al., 2005) specifically high levels of gastric acid (H+) and

Ca2+

(Kishimoto et al., 2011). Individuals with SCI have a profoundly transformed

physiology which will preclude them being compared against the scientific literature

which addresses a more focused derangement (such as ischemia reperfusion). For

example, one highly speculative mechanism may be drawn based upon the observation

that SCI individuals have been shown to have a high degree of bone mineralization loss

and elevated levels of Ca2+

in their urine (Ohry et al., 1980). This indicates a greater

concentration of free Ca2+

available to activate the Ca2+

-selective TRPV1 receptors.

TRPV1 has been shown to share functional homology with a G-protein-coupled receptor,

the Ca2+

-sensing receptor (Hofer & Brown, 2003). Both receptors are directly activated

177

by Ca2+

and this suggests that TRPV1 may transduce a subset of hypertonic nociception

(Ahern et al., 2005). Additionally SCI causes severe tissue damage and inflammation-

induced spinal tissue injury, causing the systemic release of chemical mediators (Hayes et

al., 2002). Various chemical mediators further stimulate the activation of TRPV1

(Chuang et al., 2001) and TRPV1 activation leads to the release of neuropeptides

including substance P (SP) and calcitonin-gene-related peptide (CGRP) from the primary

sensory afferent nerve terminals (Banerjee et al., 2007). Vagal afferent injury has also

been shown to induce changes in TRPV1 and cholecystokinin (CCK) within the nodose

ganglion. We demonstrate that SCI acutely leads to increased mRNA expression of

TRPV1 along the afferent vagus nerve, and specifically within the nodose ganglia. The

increased TRPV1 (inflammatory-induced receptor) indicates GI inflammation and the

increased CCK suggests that CCK increases as per vagal afferent injury. Our data present

similarities to experimental ischemia-reperfusion of the GI tract, in which SMA occlusion

causes a dramatic inflammatory response within the GI tract. We propose that our model

of SCI represents a continuum of GI ischemia and that the increase in inflammatory

mediators and TRPV1 activation may play a role in the post-SCI pathophysiology of

gastric vagal afferents.

The neurochemistry of the nodose ganglia has been identified to include

glutamate, catecholamines, serotonin, and acetylcholine as well as a considerable number

of neuropeptides including substance P, neurokinin A, vasoactive intestinal peptide,

calcitonin gene-related peptide, galanin, encephalin, somatostatin, cholecystokinin,

neuropeptide Y, calcium-binding proteins, and other neuroactive molecules (i.e. nitric

oxide) found within the neurons of the nodose ganglia (reviewed in Zhuo et al., 1997).

178

Furthermore, evidence with various neurotransmitters, neuropeptides, and neuroactive

molecules in other models has revealed that the intracellular environment of the nodose

ganglia is not static (Burdyga et al., 2006,Burdyga et al., 2008,Burdyga et al., 2010). The

alterations are also likely to result from injured and inflammatory conditions.

Cholecystokinin (CCK) is a brain-gut peptide released from duodenal epithelium

in response to luminal nutrients (Dockray, 2006). Type 1 CCK receptors (CCK-1r,

previously known as CCK-A receptors) are present on vagal afferent neurons (Zhuo et

al., 1997). Release of CCK activates vagal afferent terminals and central DMV neurons

via CCK-1r causing a suppression of gastric contractions and food intake (Holmes et al.,

2009,Ritter et al., 1994,Moran & Kinzig, 2004,Woods, 2004,Raybould, 2007,Baptista et

al., 2005,Baptista et al., 2006,Zheng et al., 2005). Following SCI, alterations in the

synthesis and release of CCK in response to nutrients remains obscure though the

peripheral and central sensitivity to exogenous CCK is diminished (Tong et al., 2011). In

addition to satiety, CCK-1r activation may also play a role in chronic inflammation

(Miyamoto et al., 2012).

In cultured vagal afferents, CCK acts via low-affinity binding sites and increases

non-selective cationic conductances (Kinch et al., 2012). The non-selective conductances

include TRP channels and suggest that CCK acts via pathways that involve TRPV

channels. Subsequently, this study proposes that reduced food intake by CCK is initiated

by activation of capsaicin- and CCK-sensitive subdiaphragmatic vagal afferents (Kinch et

al., 2012). There is increased membrane excitability where CCK acts at vagal afferent

neurons and this activation is completely independent of voltage-gated calcium current.

179

However, extracellular calcium influx is required further suggesting involvement of a

TRP channel.

The increased expression of TRPV1 and CCK-1r mRNA following a T3-SCI

suggests that receptor alteration is occurring post-SCI. To further understand the

functional implications of these receptor expression changes, whole nerve recordings are

required. Based upon previous observations that vagal afferent neurons respond to CCK

application by showing an increase in spike rate in capsaicin-sensitive single vagal fibers

(Schwartz & Moran, 1994,Okano-Matsumoto et al., 2011), we propose that an interaction

between TRPV1 and CCK-1r following SCI may contribute to dysfunction soon after

injury onset.


Gina Deiter provided invaluable assistance for modifying the qRT-PCR experiments for

nodose tissue samples, performing the assays and analyzing the data. Amanda Troy

provided initial guidance for nodose extraction. Margaret McLean provided assistance

with animal care, tissue harvest, data entry and analysis in Excel.

180

Table 6.1

Forward and reverse primer sequences for quantitative real time PCR (qRT-PCR)

Gene Forward Primer Reverse Primer

TRPV1 5’-CCCAGGCAACTGTGAGGGCG-3’ 5’-GGCAGGCACAGAGTGGACCC-3’

CCK 5’-AGCTGAGGGCTGTGCTCCGA-3’ 5’-TTCGAGGCGAGGGGTCGTGT-3’

CCK-1r 5’-TTCTCAGTGTGCTGGGGAAC-3’ 5’-AGGTTGAAGGTGGAAACGCT-3’

181

Figure 6.1. T3-SCI provokes increased TRPV1 1-day post-injury.

Increased TRPV1 immunofluorescence suggests an increase in expression of

receptors throughout the nodose ganglion including, but not limited to, afferent

neuronal bodies of the gastric-vagus.

Scale bar = 200µm

182

Figure 6.2. T3-SCI provokes increased CCK-1r 1-day post-injury.

Increased CCK-1r immunofluorescence suggests an increase in expression of

receptors throughout the same nodose ganglia including, but not limited to,

afferent neuronal bodies of the gastric-vagus that also express increased TRPV1.

Scale bar = 200µm

183

Figure 6.3. Expression levels of nodose TRPV1 after T3-SCI.

Measurement of nodose TRPV1mRNA expression (n=8/group) demonstrated a

significant elevation in T3-SCI rats beginning at 1-day and returning to baseline 1-week

post-injury (P<0.05 vs control). Bars with lower case letters reflect significant effects,

P<0.05, based on ANOVA, followed by Tukey post hoc test. a = T3-SCI vs same day

control; b = SMA occlusion vs control; c = 1-day T3-SCI vs 1-week T3-SCI; d = 3-day

T3-SCI vs 1-week T3-SCI; e = 3-day control vs 1-week control.


184

Figure 6.4. Expression levels of nodose CCK-1r after T3-SCI.

Measurement of nodose CCK-1r mRNA expression (n=8/group) demonstrated a

significant elevation in T3-SCI rats beginning at 3-day and returning to baseline 1-week

post-injury (P<0.05 vs control). Bars with lower case letters reflect significant effects,

P<0.05, based on ANOVA, followed by Tukey post hoc test. a = T3-SCI vs control; b =

SMA occlusion vs T3-SCI; c = SMA occlusion vs control; d= 1-day T3-SCI vs 3-day T3-

SCI; e = 3-day T3-SCI vs 1-week T3-SCI.


185

Figure 6.5. Expression levels of nodose CCK after T3-SCI.

Measurement of nodose CCK mRNA expression (n=8/group) demonstrated a significant

elevation in T3-SCI rats beginning at 1-day and returning to baseline 3-days post-injury

(* P<0.05 vs control).


186

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192

Chapter 7:

Summary of this Dissertation,

Short Perspective,

and Future Directions

193

7.1 SUMMARY OF THIS WORK

7.1.1 Spinal Cord Injury does not affect gastric vagal efferent sensitivity

As described above, gastric reflexes are known to be predominantly controlled by

the parasympathetic vagus nerve. Recalling that the enteric nervous system (ENS)

maintains the pacemaker activity of the majority of the GI tract, the vagus is still

critically important in regulating the ENS and coordinating the ENS with the CNS. The

afferent and efferent limbs of the vagus nerve require attention to both sensory and motor

modalities individually within the vagus nerve bundle. Previous evidence (Tong et al.,

2011) led us to speculate that the afferent vagus was compromised following SCI, though

the functional implications of SCI on the efferent limb were unexplored. Using

established techniques (Chapter 2), data within this thesis (Chapter 3) validated the

anatomical and functional integrity of the DMV neurons and efferent fibers (see Figure

7.1). We were able to excite DMV neurons and stimulate in vivo gastric contractions in

control and SCI rats, thereby revealing that the efferent limb of the vagus nerve remained

anatomically and functionally intact following a T3-SCI. This refocused our hypothesis

that the post-SCI gastric dysfunction may be due to afferent dysfunction of the vagus

nerve. Identifying the mechanism of gastric vagal afferent dysfunction becomes more

difficult due to the peripheral terminals lying within the linings of gastric viscera, the

peripherally located cell bodies in the nodose ganglia, and the central projections to the

NTS.

7.1.2 Reduced mesenteric blood flow occurs following T3-SCI

The link between vascular dysfunction and compromise of vagally-innervated

gastrointestinal tissues is now well recognized (reviewed in Bhattacharyya et al., 2014).

194

Cardiovascular disease is the leading cause of morbidity and mortality in SCI (Phillips et

al., 2012) due to significant cardiovascular and autonomic dysfunction following the

injury. SCI patients may also suffer severe orthostatic hypotension and autonomic

dysreflexia due to a blocked sympathetic pathway (lesion center of SCI blocks

sympathetic fibers from ascending/descending the spinal cord) and unopposed

parasympathetic tone and activity. However, sympathetic responses may still occur but

will be undetected until a serious complication arises; autonomic dysreflexia (i.e.

paroxysmal hypertension in response to below-level noxious stimuli). Following SCI at

or above T5, the absence of descending input to the sympathetic nervous system

permanently diminishes the proper regulation of blood flow, blood pressure, cardiac

contractility, or cardiac rate. Additionally, venous return fails to be sufficient due to

lower limb paralysis (skeletal muscles are unable to assist venous return to the heart),

renal hypoperfusion (prevents return of vascular volume in the advent of low blood

pressure). These secondary complications may ultimately contribute to compromised

mesenteric perfusion and initiation of a pathophysiological cascade. Using the blood flow

within the superior mesenteric artery as a model system, the findings of this thesis

identified the reduction of vascular perfusion to the GI tract at rest and in response to

duodenal nutrient administration (itself, an experimental model of enteric

supplementation). Vascular hypotension and the resulting visceral hypoperfusion can lead

to end-organ failure and dysfunction. The visceral hypoperfusion incites an ischemic

emergency for the tissues and an inflammatory cascade is recruited in an effort to repair

or rescue the tissue.

195

7.1.3 Gastrointestinal inflammation up-regulates gastrointestinal inflammatory markers

and TRPV1 receptors in nodose ganglia

The study of low-grade or systemic inflammation following SCI had previously

not been heavily investigated in our model of T3-SCI, but has since begun to gain

popularity both in research and treatments. Mast cells are of crucial importance to the

immune system and the GI tract. The majority of mast cells reside within the gut wall and

function as an important part of the immunological barrier between the internal milieu

and luminal content. But as it has been shown with SCI, gut barrier dysfunction is

prevalent and the walls begin to break down. This break down in the intestinal barrier

permits the transit of unauthorized cells in and out of the gut; particularly bacteria. Once

pathogens begin to enter the gut, muscosal mast cells recognize the invaders and mount

an immediate immune response consisting of instant release of biologically active

chemokines, cytokines, vasoactive amines, and proteases. Mucosal mast cells are crucial

in the recruitment of neutrophils and the development of increased vascular and mucosal

permeability. There is now substantial evidence that these local intestinal events

contribute to the development of systemic inflammatory complications (de Haan et al.,

2013). This thesis confirmed that T3-SCI provokes a low-grade inflammatory state

(Chapter 5) although not as profound as that observed following superior mesenteric

artery occlusion. The later occlusion model was incorporated to provide a comparison

between our model and the more fully developed ischemia/reperfusion literature (e.g.,

Kozar et al., 2002, Ban et al., 2011).

The transient receptor potential (TRP) channel superfamily has been implicated in

numerous processes in health and disease. In particular, the vanilloid 1 receptor (TRPV1)

196

is an ion channel which is activated during times of tissue injury or inflammation.

TRPV1 is a non-selective cation channel that has been shown to be upregulated in other

models of inflammation. We proposed that inflammation associated with our model of

T3-SCI would cause an increase in TRPV1 expression in nodose ganglia and might be

one mechanism leading to gastric vagal afferent dysfunction. The mechanisms are still

unclear as to how this happens in the SCI model, but data from this thesis revealed

nodose upregulation of receptors (CCK-1r and TRPV1, Chapter 6). Based upon the

findings of this thesis and the existing literature, a tentative model for identifying future

directions can be developed (Figure 7.2).

7.2 PERSPECTIVE FOR VAGAL AFFERENT FUNCTIONAL EXPERIMENTS

7.2.1 INTRODUCTION

Ultimately, in vivo physiological experiments are necessary to demonstrate the

functional response to CCK-8s, the TRPV1 agonist, capsaicin, and their respective

blockers (lorglumide and capsazepine, respectively) following T3-SCI. Based upon the

findings of this thesis, the following experiments are a logical extension of this research.

In models of superior mesenteric artery (SMA) occlusion, the artery which

perfuses the midgut, GI tissue undergoes changes related to inflammatory insult (i.e.

decreased villi height and width, decreased crypt to villi ratio, ulceration, and in extreme

cases necrosis) and mRNA expression of inflammatory markers is significantly elevated.

In our model of T3-SCI, SMA blood flow to the midgut is significantly reduced. The

diminished blood flow and increased mRNA expression of inflammatory markers

following T3-SCI represent two pathological sources of functional aggravation which

may be sources of parasympathetic vagal dysfunction.

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Based upon the observations in this thesis that following T3-SCI, a shift in

receptor expression for CCK-1r and TRPV1 may signal a mechanism leading to gastric

dysfunction, the use of neurophysiological techniques are warranted to verify the

functional deficits implied by our molecular findings. Specifically, it is crucial to test the

gastric vagal afferent response to CCK-1r and TRPV1 agonists to test the hypothesis that

T3-SCI gastric dysfunction is due, in part, to changes in receptor expression following an

acute injury.

7.2.2 PROPOSED MATERIALS AND METHODS

7.2.2.1 Spinal Cord Injury Surgery and Post-Operative Care

Male Wistar rats would be anesthetized with a 3-5% mixture of Isoflurane with

oxygen (400-600ml/min) and prepped for T3-SCI or control surgery using established

aseptic surgical techniques as previously described within this thesis.

7.2.2.2 In Vivo Studies

Three days following the initial surgery, T3-SCI or control animals would be

fasted overnight, water provided ad libitum, prior to being deeply re-anesthetized with 3-

5% mixture of Isoflurane with oxygen (400-600ml/min) for physiological

instrumentation. Animals would be placed on a feedback-controlled warming pad (TCAT

2LV, Physitemp Instruments, Clifton, New Jersey) and rectal temperature monitored and

maintained at 37±1 °C for the duration of the experiment.

Tracheal cannulation - Once prepared for surgery, the animal would be tracheally

intubated as described in Chapter 2.

Femoral arterial and venous catheters - Following intubation, the femoral artery

and adjacent vein or tributaries would be exposed via a small incision of the upper thigh

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which exposes the quadriceps and associated adductors. Both the femoral artery and vein

would be cannulated as described in Chapter 4. The vein would next be hemisected and

an aseptic PE-50 catheter inserted in the direction of the abdominal vena cava for drug

administration. The venous catheter would be secured adjacent to the exteriorized venous

catheter and the wound margin closed with wound clips. The rat would slowly be weaned

from Isoflurane to thiobutabarbital (Inactin 80-100mg/kg, i.v.) prior to preparation for

vagal afferent nerve recording.

7.2.2.3 Electrode Placement for Neurophysiology

A midline abdominal laparotomy would be performed to expose the stomach and

distal esophagus. Under higher power stereoscopic magnification (45-60X), the

subdiaphragmatic anterior vagus would be isolated from the esophageal surface with

fine-tipped Dumont forceps by removing the adjacent connective tissue from the nerve

and esophageal surface while keeping the area moist with heparinized saline. The isolated

gastric vagus nerve would then be carefully placed on bipolar recording electrodes

(Figure 7.3). The electrodes connected to a differential amplifier (1700, A-M Systems,

Inc., Sequim, WA), and whole-nerve recordings would be filtered (low pass, 1-5 kHz;

and high pass, 50-100 Hz with a 60-Hz notch filter) and the output signal distributed to

the 1400 Micro3 A/D converter , and audio monitor (AM-8, Grass Instruments,

Cambridge, MA). The vagal nerve signal would be verified and the nerve and electrodes

secured to the bipolar electrode using KwikSil (World Precision Instruments, Sarasota,

FL). Prior to closure of the wound margin around the exteriorized electrode wires, the

portion of the gastric vagus proximal to the bipolar electrodes would be crushed to

eliminate recording vagal efferent action potentials.

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7.2.2.4 Vagal Parasympathetic Nerve Recording

Femoral arterial pressure would be measured continuously throughout all nerve

recording experiments, and heart rate data could be extrapolated by the Spike2 software

from the blood pressure recording channel. Baseline recordings would be established

after the rat had stabilized for at least 60 minutes following surgical procedures. Infusions

of PBS or pharmacological agents would be administered separated by a minimum of 20

minute recovery period.

Specifically, baseline recordings would be established and then PBS i.v. infusions

performed as a vehicle control recording. In order to establish a consistently effective

dose, CCK-8s i.v. infusions would be given at the following doses: 0.06 µg/kg, 0.12

µg/kg, 0.18 µg/kg, 0.40 µg/kg, 0.60 µg/kg, 1.8, and 3.0 µg/kg) for vagal parasympathetic

activity (anticipated n = 10). Similarly, capsaicin i.v. infusions would be given at the

following doses: 0.05 µg/kg, 0.10 µg/kg, 0.15 µg/kg, 0.33 µg/kg, 0.50 µg/kg, 0.74 µg/kg,

and 1.2 µg/kg to measure gastric vagal afferent responses to the TRPV1 agonist

(anticipated n = 10). At the conclusion of respective agonist administration, lorglumide

(CCK-1r agonist, 10mg/kg i.v.) or capsazepine (TRPV1 agonist, 3mg/kg i.v.) would be

administered to demonstrate antagonism of a subsequent individual dose determined to

evoke a significant response.

At the conclusion of each experiment, background noise would be quantified then

subtracted from the nerve recordings post-hoc to ensure that the nerve recording voltage

changes represented nerve activity.

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7.2.3 PROPOSED OUTCOMES

It is hypothesized that the data from the presently described study would

demonstrate a differential sensitivity to both CCK-8s and the TRPV1 agonist capsaicin.

Specifically, the experimental data would indicate that: 1) T3-SCI provokes a significant

increase in the sensitivity of gastric vagal afferents to CCK-8s; and 2) T3-SCI provokes a

significant amplification in the sensitivity of gastric vagal afferents to the TRPV1

agonist, capsaicin. Functional alterations would suggest that clinically-recognized

vascular reflex deficits in the SCI population provoke hypoperfusion and inflammation

within the mesenteric bed and lead to local inflammation of gastrointestinal tissues. This

inflammation may be responsible for altering the receptor expression of vagal afferent

fibers, and as a result, the physiological sensitivity in our experimental model of high

thoracic-SCI.

These hypotheses are based upon the following reports. Pharmacologically, CCK

binding occurs through two receptor subtypes, CCK-1r and CCK-2r. Vagal afferent

neuron activation by CCK involves the CCK-1r in the central effect of gastric

neurocircuitry (see Holmes et al., 2009). The nodose ganglion expresses both of these

CCK receptors (Broberger et al., 2001) and in a model of vagal axotomy CCK-2r and

CCK are upregulated. The elevated expression of CCK-1r by afferent cell bodies within

the nodose ganglia after T3-SCI is in contrast to the effects of vagotomy, though the

pathophysiology of the two models is by no means similar. Despite this dissimilarity

between these models of vagal pathophysiology, the elevated expression of CCK-1r

would predict a potential increase in vagal afferent sensitivity to systemic CCK-8s.

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Previous neurophysiological recordings of the effects of systemic CCK-8s on

distinct gastric vagal afferents or efferents revealed greater complexity of the afferent and

efferent response (Okano-Matsumoto et al., 2011). The present study proposes to record

from a location on the gastric vagus proximal to the division of the gastric vagus into

proximal and distal branches innervating the fundus and corpus, respectively. These two

components of the stomach serve different roles in the coordinated process of digestion.

Clinical and experimental SCI results in delayed gastric emptying, but individuals with

SCI also report rapid satiety in response to a small volume. Behavioral studies to

investigate a similar process in our T3-SCI model have not been performed. Therefore, it

would be beneficial to these detailed studies with a goal of developing an effective

therapeutic intervention.

Our proposed experiment is based upon the observed increase in nodose ganglia

TRPV1 mRNA expression. We propose that the systemic inflammation provokes satiety-

mediated alterations in vagal afferent signaling. However, TRPV1 is only one of a family

of TRP channels, others of which were not investigated in the present study, yet have

been proposed to play a role in CCK signaling (Zhao & Simasko, 2010). It has also been

reported that cannabinoid (CB1) receptors are expressed by the same vagal afferent

neurons, and the CB1 receptor expression is increased in fasted rats (Burdyga et al.,

2006). CB1 agonists are currently being used clinically to stimulate appetite and the

potential for therapeutic uses of CB1 receptor agonists includes inflammatory disorders

and SCI (Singh & Budhiraja, 2006). The possibility exists that the increased expression

of inflammatory mediators dampens or blocks the effect of the endogenous receptors

(CCK-1r, TRPV1, CB1) known for exciting the gastric vagal afferent neurons and may

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diminish the therapeutic potential of these receptors. Recent findings indicate that CCK

acts through a pathway which involves TRPV channels. When CCK binds to CCK-1, the

resulting afferent vagus signal serves to mediate the reduction of gastric emptying,

increase pancreatic secretion, and facilitate the process of satiation (Kinch et al.,

2012,Duca et al., 2013). The potential exists that our T3-SCI model, with delayed gastric

emptying, stops producing CCK since nutrients fail to leave the stomach at a rate to

stimulate duodenal release of CCK. With a reduction in CCK release, CCK-1r are

activated by the other putative humoral agents which cause a different effect than what

we see with CCK activation of CCK-1r. Therefore, the baseline gastric vagal afferent

response may be diminished following T3-SCI.

7.3 FUTURE DIRECTIONS

7.3.1 qRT-PCR of Labeled Gastric Nodose

Approximately 80% of nerves in the vagal bundle are sensory afferent fibers. Yet

there is no clear way to tease apart the nerve bundle and clearly identify an afferent fiber

from an efferent fiber. Anterograde neuronal tracers, however, may be used via injection

into the gastric corpus and allowed transit time to reach the cell bodies in the nodose

ganglia. By isolating the nodose neurons specific to gastric projecting neurons, we could

better test and confidently define gastric nodose neurons in receptor expression changes

via IHC. With that said, it is critical to prove that the IHC-positive changes reported in

this thesis correspond to the gastric functional outcomes we anticipate seeing with the

neurophysiological data. We previously tried Fast Blue anterograde labeling but the

imaging was not clear and the neurons were not vivid making identification very difficult.

Further refinement in order to optimize the technique of anterograde tracing and

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fluorescent microscopy is required. Alternatively, confocal microscopy would greatly

enhance the level of magnification and allow cross-section images to be taken. The

increased resolution would allow us to better visualize the plasma membrane of the

afferent neurons to determine if receptor expression (namely TRPV1 and CCK-1r) is

indeed being externalized to the plasma membrane. The option to take cross-sectional

images of the neurons would further allow internal visualization to see if receptor

expression (TRPV1 and CCK-1r) is being internalized within the nucleus or cytoplasm. I

would also be interested in experimenting with cultured neurons. The labeling technique

would allow us to again, identify and isolate only gastric vagal afferent neurons.

Colchicine treatment would help us identify microtubule transport changes with receptor

trafficking to the membrane. Colchicine prevents microtubules from transporting parts

across a cell; if TRPV1 or CCK-1r expression is still externalized post-colchicine, then

another mechanism is at work in transporting these receptors.

7.3.2 Test additional GI peptides with Neurophysiology

Additionally, it would be advantageous to test the other unexplored GI peptides

(e.g. GLP-1, NPY) with the proposed neurophysiological design to see how the

parasympathetic vagus responds to various feeding peptides. This could help identify the

relationship between CCK-1r and TRPV1, if a relationship even exists. Other peptides

such as Ghrelin or Neuropeptide Y may also present interesting findings as that they also

activate the CNS and ANS.

7.3.3 Test tensile strength of SCI vagus nerve

An interesting anatomical study would be to test the tensile or fibro-strength of

the vagus nerve. During pilot neurophysiological experiments, in which handling of the

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vagus nerve was imperative, it was noted that the vagus in the SCI rats was much more

delicate, friable, and easily severed. This qualitative observation could represent some

morphological derangements that would have system-wide implications for the SCI

patient.

7.3.4 Anatomical comparison of DMV neurons and nodose neurons

Another anatomical property to investigate would be the soma size of the neurons

of the DMV with those of the nodose ganglion. We demonstrated that gastric vagal

efferent function is maintained following injury but are there mechanisms in place which

are promoting cell survival? It would be interesting to compare the soma of gastric vagal

afferent neurons both in SCI rats and controls; particularly if soma size is increasing in

response to an increase in receptor (TRPV1 or CCK-1r) production. This could be a very

nice incorporation into the IHC data we have collected.

7.3.5 Calcium Measurements

Calcium has long been known for its cytotoxic properties to a cell when received

in excess. TRPV1 is a non-selective cation channel which will allow the influx of

calcium ions to occur unregulated. This unregulated event could potentially prove

harmful to the cell; consequently killing the cell and contributing the gastric vagal

afferent dysfunction. Calcium measurements are relatively easy to conduct via labeling

calcium, labeling the gastric afferent neurons, and visualizing via confocal microscopy.

An increase in calcium would be a strong piece of evidence for hyperpolarization of the

SCI vagal afferent neurons; eliciting a gastric dysfunction. Additionally, with the

abundance of calcium channel blockers we could locally implement a calcium block at

the level of the nodose ganglia via injection or flooding the area. By using a TRPV1

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antagonist we would prove or disprove the action of TRPV1 in calcium influx. By using a

less specific calcium channel blocker, we could further prove or disprove the action of

TRPV1 in calcium loading of the nodose neurons.

7.3.6 Therapeutic targeting of post-SCI gastric dysfunction

Ideally, I would be interested in treating rats with anti-inflammatory agents from

the time of SCI or control surgery to the day of the neurophysiological experiment. If

CCK-1r truly does offer inflammatory protective measures, I would like to see if CCK-1r

are more readily available in SCI rats after treatment with anti-inflammatories.

Particularly, does the diminished CCK-8s response (Tong et al., 2011) persist following

anti-inflammatory treatment? With anti-inflammatory agents on board, I would also be

interested in seeing how TRPV1 is affected upon i.v. infusion of capsaicin. TRPV1

mRNA expression is upregulated in times of inflammation, but does the influx of calcium

cause the afferent fibers to become hyperpolarized or would anti-inflammatory treatment

maintain a normal level of TRPV1 ion channels and elicit a vagal response similar to

controls.

Our first series of experiments provided considerable evidence that the efferent

limb of the gastric vago-vagal reflex remained functionally capable of modulating gastric

contractility and our subsequent data suggest that diminished signaling within the afferent

limb may result in a net inhibition of the efferent vagus (Holmes, 2012). In a broader

sense, there is growing interest in an anti-inflammatory circuit which involves the

afferent and efferent vagus nerve and α7 nicotinic acetylcholine receptor- (nAChR)

expressing macrophages. This circuit has become known as the cholinergic anti-

inflammatory pathway (CAP) and results in a finely tuned pro-inflammatory response

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(Pavlov & Tracey, 2012,Ji et al., 2014,Martelli et al., 2014). Recent evidence links

activation of CCK-1r and the nAChR-mediated vagal reflex reduce inflammation and

protect the intestinal damage (de Haan et al., 2013). The exact mechanism of CCK-1r and

nAChR anti-inflammatory properties are not well understood. Electrical stimulation of

the vagus nerve suppresses activation of NF-κB and diminished the production of

proinflammatory cytokines in α7 nAChR- expressing macrophages (Wu et al., 2014).

This pathway may be involved in the neurochemical changes in our model of T3-SCI

injury. We’ve shown that there is an increase in inflammatory markers post-SCI;

therefore the CAP pathway may be activated; however, the long-term anti-inflammatory

effects may not be visible in our acute model (>7 days post-SCI).

7.3.7 Obesity, Aging, & Inflammation

Finally, it is important to recognize SCI as a disease. As with most chronic

diseases, SCI has the tendency to significantly age the individual afflicted with the

disease. This prompts the investigation at a comparative study across disciplines in which

obesity, aging, and inflammation would be studied. Increased visceral adiposity

following SCI is one common denominator and it is known that adipocytes release pro-

inflammatory cytokines; suggesting that inflammation may be an underlying culprit in

the disease process leading to gastric dysfunction. Future studies should focus on the

commonality among the inflammatory cascades across diseases and compare the

similarities. For example, recently published data is beginning to emerge regarding the

pathophysiological changes within the liver (Sauerbeck et al., 2014). Again, treatment for

SCI has focused primarily on restoration of motor function, but now we know that the

leading cause of morbidity and mortality is due to cardiovascular complications

207

exacerbated by inflammation-induced ischemia. To move towards a treatment in SCI, we

must figure out how to prevent the inflammatory cascade which takes over the body

following an injury.


Dr. Sean Stocker, and his laboratory technicians Sarah Simmonds and Jenny Lay, have

provided invaluable guidance in demonstrating the techniques of the proposed

experiments for subdiaphragmatic vagal nerve recordings, data entry, and analysis in

Excel.

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Figure 7.1. Summary figure of the overall work of this dissertation.

The dysfunction within the vago-vagal reflex, following an experimental T3-SCI,

has yet to be fully understood. This dissertation has helped answer some questions

in regard to the circuit as follows: Chapters 2 and 3 data suggest that the gastric

vagal efferent limb remains capable of regulating gastric reflexes. Diminished vagal

afferent input to the brainstem neurocircuitry may be solely responsible for

diminished gastric reflexes after SCI; Chapter 4 data suggest that chronic reduction

of GI blood flow triggers a parallel GI inflammation following T3-SCI; Chapter 5

data suggest that T3-SCI results in low-grade inflammation of the GI tract. Previous

research has linked GI inflammation with up-regulation of transient receptor

potential vanilloid type 1 receptor (TRPV1). Similar changes may occur within

gastric vagal afferents following T3-SCI; Chapter 6 data suggest that increased

expression of TRPV1 and CCK-1r mRNA following T3-SCI may reflect more

widespread changes in receptor expression; Chapter 7 perspective proposes T3-SCI

gastric vagal afferent hypersensitivity to capsaicin which may contribute to the

gastric dysfunction post-SCI.

209

Figure 7.2. Schematic diagram of potential mechanisms of gastrointestinal

pathophysiology following T3-SCI.

Sensory and motor loss is well-recognized following SCI. Additional co-morbidities exist

and contribute, in concert or individually, to elevate reactive oxygen species (ROS) or

reactive nitrogen species (RNS) within the gastrointestinal tract. In addition to the

mechanisms investigated in this thesis, multiple pathways are likely affected by the likely

ROS/RNS cascade following SCI and reflect the knowledge gaps that limit therapeutic

interventions. This transformed physiology of the GI tract of the SCI individual

collectively present the recognized co-morbidities of diminished vago-vagal reflexes,

luminal barrier permeability and nutrient malabsorption.

210

Figure 7.3. Representative image of vago-vagal recording reflex circuit.

The image indicates the relative location of the bipolar recording electrodes

when recording gastric vagal response. (Courtesy of Dr. Holmes; Adapted

from: Horn & Friedman, 2005)

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Vita

Emily Mae Besecker

Education

2015 Doctor of Philosophy in Anatomy Pennsylvania State University - College of Medicine, Hershey, PA

2010 Bachelor of Science in Biology Shippensburg University, Shippensburg, PA

Teaching Experience, Training, & Professional Development

2014 -Visiting Assistant Professor at Gettysburg College, Gettysburg, PA

-Penn State Course in College Teaching, Penn State Harrisburg

2013 -TARGETTing Student Motivation Training at Penn State Harrisburg

-Lecturer for Neuroanatomy Residents Penn State Hershey, College of Medicine,

2011-2014 -Teaching Assistant & Tutor, Human Gross Anatomy in medical school

curriculum, Penn State Hershey, College of Medicine

2012-2013 -Graduate Research Mentor, Undergraduate Student Summer Research

Programs, American Heart Association-Summer Undergraduate Fellowship

2012-2013 -Cadaver Prosector for the Penn State Hershey Anesthesiology Department

Resident Training Program

2012-2013 -Co-director of the Central Pennsylvania Chapter of Society for Neuroscience

and the Penn State Institute of the Neurosciences’ 4th Annual Local Brain Bee

Competition Laboratory Portion

Honors and Awards

2014 -Gettysburg College Research and Professional Development Grant

-National Institutes of Health- National Institute of Neurological Disorders and

Stroke (NIH-NINDS) F-31 Pre-doctoral Fellowship Award

-College of Medicine Endowed Scholarship

-Anthony Marmarou Research Excellence Award

-American Association of Anatomists (AAA) Travel Award

2013 -College of Medicine Class of 1971 Endowed Scholarship

-Michael Goldberger Research Excellence Award

-National Neurotrauma Society Annual Meeting Travel Award

-American Association of Anatomists (AAA) Travel Award

Publications

Swartz EM, Holmes GM (2014). Gastric vagal motoneuron function is maintained following

experimental spinal cord injury. Neurogastroentrol Motil. 26(12):1717-29

Holmes GM, Swartz EM, & McLean MS (2014). Fabrication and implantation of miniature

dual-element strain gages for measuring in vivo gastrointestinal contractions in rodents. J Vis

Exp. 91:51739

Swartz EM, Browning KN, Travagli RA, & Holmes GM (2014). Ghrelin increases vagally

mediated gastric activity by central sites of action. Neurogastroentrol Motil. 26(2):272-82.

Browning KN, Babic T, Holmes GM, Swartz E, & Travagli RA (2013). A critical re-evaluation

of the specificity of action of perivagal capsaicin. J Physio. 591(Pt 6):1563-80.

trpv1 activation and gastric vagal afferent …

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