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MURDOCH RESEARCH REPOSITORY

This is the author’s final version of the work, as accepted for publication following peer review but without the publisher’s layout or pagination.

The definitive version is available at http://dx.doi.org/10.1016/j.neuroscience.2014.09.046

Drummond, E.S., Dawson, L.F., Finch, P.M., Li, W., Guo, T-Z, Kingery, W.S. and Drummond, P.D. (2014) Increased bilateral expression of α1-adrenoceptors on peripheral nerves, blood

vessels and keratinocytes does not account for pain or neuroinflammatory changes after distal tibia fracture in rats.

Neuroscience, 281 . pp. 99-109.

http://researchrepository.murdoch.edu.au/24332/

Copyright: © 2014 Elsevier BV.

It is posted here for your personal use. No further distribution is permitted.

http://dx.doi.org/10.1016/j.neuroscience.2014.09.046

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Please cite this article in press as: Drummond ES et al. Increased bilateral expression of a1-adrenoceptors on peripheral nerves, blood vessels and keratinocytes does

not account for pain or neuroinflammatory changes after distal tibia fracture in rats. Neuroscience (2014), http://dx.doi.org/10.1016/j.neuroscience.2014.09.046

NSC 15719 No. of Pages 11

3 October 2014

Neuroscience xxx (2014) xxx–xxx

INCREASED BILATERAL EXPRESSION OF a1-ADRENOCEPTORSON PERIPHERAL NERVES, BLOOD VESSELS AND KERATINOCYTESDOES NOT ACCOUNT FOR PAIN OR NEUROINFLAMMATORYCHANGES AFTER DISTAL TIBIA FRACTURE IN RATS

E. S. DRUMMOND, a L. F. DAWSON, a P. M. FINCH, a

W. LI, b,c T.-Z. GUO, b W. S. KINGERY b ANDP. D. DRUMMOND a*

aCentre for Research on Chronic Pain and Inflammatory

Diseases, Murdoch University, Perth, Western Australia, AustraliabPhysical Medicine and Rehabilitation Services, VAPAHCS,

Palo Alto, CA, USA

cDepartment of Anesthesia, Stanford University, Stanford, CA, USA

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Abstract—In certain forms of nerve injury and inflammation,

noradrenaline augments pain via actions on up-regulated

a1-adrenoceptors (a1-ARs). The aim of this study was to

use immunohistochemistry to examine a1-AR expression

on peripheral neurons, cutaneous blood vessels and kerati-

nocytes after distal tibia fracture and cast immobilization, a

model of complex regional pain syndrome type 1. We

hypothesized that there would be increased a1-AR expres-

sion on neurons and keratinocytes in the injured limb in

comparison to the contralateral unaffected limb after distal

tibia fracture, in association with inflammatory changes

and pain. a1-AR expression was increased on plantar kerat-

inocytes, dermal blood vessels and peripheral nerve fibers

at 16 weeks after injury both in the fractured and contralat-

eral uninjured limb. Similar changes were seen in controls

whose limb had been immobilized in a cast for 4 weeks but

not fractured. Neurofilament 200 (NF200), a marker of mye-

linated neurons, and calcitonin gene-related peptide

(CGRP), a neuropeptide involved in neuro-inflammatory sig-

naling, decreased 4 weeks after fracture and casting but

then increased at the 16-week time point. As some of these

changes were also detected in the contralateral hind limb,

they probably were triggered by a systemic response to frac-

ture and casting. Soon after the cast was removed, intrapl-

antar injections of the a1-AR antagonist prazosin released

local vasoconstrictor tone but had no effect on pain behav-

iors. However, systemic injection of prazosin inhibited

behavioral signs of pain, suggesting that fracture and/or

casting triggered an up-regulation of a1-ARs in central noci-

ceptive pathways that augmented pain. Together, these find-

ings indicate that a1-AR expression increases in the hind

http://dx.doi.org/10.1016/j.neuroscience.2014.09.0460306-4522/� 2014 Published by Elsevier Ltd. on behalf of IBRO.

*Corresponding author. Address: School of Psychology and ExerciseScience, Murdoch University, 6150 Western Australia, Australia. Tel:+61-8-93602415; fax: +61-8-93606492.

E-mail address: [email protected] (P. D. Drummond).Abbreviations: a1-AR, a1-adrenoceptor; CGRP, calcitonin gene-relatedpeptide; CRPS, complex regional pain syndrome; IL, interleukin;NF200, neurofilament 200; PBS, phosphate-buffered saline; PGP9.5,protein gene product 9.5; SMA, smooth muscle actin; TNF, tumornecrosis factor-a.

1

limbs after distal tibia fracture and cast immobilisation.

However, these peripheral increases do not contribute

directly to residual pain. � 2014 Published by Elsevier Ltd.

on behalf of IBRO.

Key words: distal tibia fracture, alpha-1 adrenoceptor,

immunohistochemistry, calcitonin gene-related peptide,

neurofilament 200, sympathetically maintained pain.

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INTRODUCTION

Distal limb fracture is the most common trigger of complex

regional pain syndrome (CRPS) type 1 (de Mos et al.,

2007), a condition associated with acute neuro-

inflammatory changes and chronic sensory, motor and

autonomic disturbances (Marinus et al., 2011). Investiga-

tion of the neuro-inflammatory changes in an animal model

of distal tibia fracture/cast immobilisation has shown that

pain after fracture is associatedwith signaling of neuropep-

tides such as substance P and calcitonin gene-related

peptide (CGRP), and key inflammatory mediators such

as tumor necrosis factor-a (TNF), interleukin-1b (IL-1b),IL-6 and nerve growth factor (Guo et al., 2004;

Sabsovich et al., 2008a,b; Li et al., 2009, 2010, 2012;

Wei et al., 2009a,b). Many of these neuro-inflammatory

changes after fracture are reduced in transgenic mice defi-

cient in CGRP receptors or substance P, and are inhibited

by neurokinin-1 receptor blockade (Wei et al., 2009b; Guo

et al., 2012). Thus, distal limb fracture appears to trigger a

neuro-inflammatory cascade that might contribute to the

development of CRPS.

We recently reported that noradrenaline, released

from sympathetic vasomotor neurons or produced

locally in the epidermis, may augment inflammation after

distal tibia fracture by activating keratinocytes or

vascular endothelial cells (Li et al., 2013). In particular,

intradermal injection of the b2-adrenoceptor agonist ter-

butaline provoked mechanical allodynia and triggered

the production of IL-6 in the skin. Conversely, chemical

sympathectomy with guanethidine and selective blockade

of b2-adrenoceptors inhibited edema, vascular distur-

bances and behavioral signs of pain. Together, these

findings suggest that sympathetic efferent signaling

contributes to nociceptive sensitization after distal limb

fracture by acting on b2-adrenoceptors.


mailto:[email protected]



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3 October 2014

In certain forms of nerve injury and inflammation,

noradrenaline may also augment pain via actions on

a1-adrenoceptors (a1-AR) (Hong and Abbott, 1996; Ali

et al., 1999; Nam et al., 2000; Hord et al., 2001; Xie

et al., 2001; Kim et al., 2005; Dogrul et al., 2006; Maruo

et al., 2006; Kopp et al., 2007; Meisner et al., 2007;

Trevisani et al., 2007; Gibbs et al., 2008; Dawson et al.,

2011; Donello et al., 2011). Notably, we recently reported

that a1-AR expression increased in dermal nerve bundles

4–28 days after partial sciatic nerve lesion (Drummond

et al., 2014b), and on nociceptive nerve fibers and kerati-

nocytes 7 days after chronic constriction injury of the sci-

atic nerve (Drummond et al., 2014a). This is consistent

with an aberrant involvement of these receptors in CRPS

(Drummond et al., 1996, 2014b; Finch et al., 2014), neu-

roma pain (Chabal et al., 1992; Lin et al., 2006), post-her-

petic neuralgia (Choi and Rowbotham, 1997; Schmidt

et al., 2003) and other neuropathic pain conditions (Lee

et al., 2000). Thus, the first aim of this study was to look

for signs of nerve injury and to examine a1-AR expression

on peripheral neurons, cutaneous blood vessels and kerat-

inocytes after distal tibia fracture and cast immobilization.

We hypothesized that a1-ARs would be expressed more

strongly on neurons and keratinocytes in the injured than

contralateral limb, in association with signs of inflammation

and pain (Li et al., 2013). Likewise, we hypothesized that

a1-AR up-regulation would be greater after distal tibia

fracture and cast immobilization than in a cast-only group.

Our second aim was to determine whether

sympathetic signaling in the skin might contribute to

pain after distal tibia fracture by activating a1-ARs on

neurons or keratinocytes. As local administration of

a1-AR agonists intensifies pain behaviors associated

with acute inflammation (Hong and Abbott, 1996), we

hypothesized that injection of the a1-AR antagonist prazo-

sin into the hind paw skin of the injured limb would allevi-

ate pain associated with distal tibia fracture.
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EXPERIMENTAL PROCEDURES

Animals

These experiments were approved by the Veterans

Affairs Palo Alto Health Care System Institutional Animal

Care and Use Committee (Palo Alto, CA, USA) and the

Murdoch University Animal Research Ethics Committee,

and followed the guidelines of the International

Association for the Study of Pain. Adult (9-month-old)

male Sprague–Dawley rats (Simonsen Laboratories,

Gilroy, CA, USA) were housed individually in isolator

cages with solid floors covered with 3 cm of soft

bedding and were given food and water ad libitum.

During the experimental period the animals were fed

Lab Diet 5012 (PMI Nutrition Institute, Richmond, IN,

USA), which contains 1.0% calcium, 0.5% phosphorus,

and 3.3 IU/g vitamin D3, and were kept under standard

conditions with a 12-h light–dark cycle.

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Surgery

Tibia fracture was performed under 2–4% isoflurane to

maintain surgical anesthesia (Guo et al., 2004). The right

Please cite this article in press as: Drummond ES et al. Increased bilateral expression o

not account for pain or neuroinflammatory changes after distal tibia fracture in rats. Ne

hind limb was wrapped in a stockinet (2.5 cm wide) and

the distal tibia was fractured using pliers with an adjust-

able stop (Visegrip, Petersen Manufacturing, Dewitt, NE,

USA) that had been modified with a three-point jaw. The

hind limb was wrapped in casting tape ([delta]-Lite,

Johnson & Johnson, New Brunswick, NJ, USA) so that

the hip, knee, and ankle were flexed. The cast extended

from the metatarsals of the hind paw up to a spica formed

around the abdomen. The cast over the paw was

applied only to the plantar surface; a window was left

open over the dorsum of the paw and ankle to minimize

constriction when post-fracture edema developed. To pre-

vent the animals from chewing at their casts, the cast

material was wrapped in a galvanized wire mesh. The rats

were given subcutaneous saline and buprenorphine

immediately after the procedure (0.03 mg/kg) and on

the first day after fracture for postoperative hydration

and analgesia. At 4 weeks the rats were anesthetized with

isoflurane and the cast was removed with a vibrating cast

saw. All rats used in this study had union at the fracture

site after 4 weeks of cast immobilization.

As only one limb was fractured, values recorded from

the contralateral limb were used as an internal control. A

cast-only group served as an additional external control

as the limb was immobilized but not fractured.

Behavioral and physiological measures

Behavioral measures were obtained the day after the cast

was removed at 4 weeks, and again at 16 weeks in a

subgroup of animals. To measure mechanical allodynia,

an up-down von Frey testing paradigm was used as

previously described. In addition, an incapacitance

device (IITC Inc. Life Science, Woodland Hills, CA, USA)

was used to measure hind paw unweighting. The rats

were manually held in a vertical position over the

apparatus with the hind paws resting on separate metal

scale plates, and the entire weight of the rat was

supported on the hind paws. The duration of each

measurement was 6 s, and 10 consecutive

measurements were taken at 60-s intervals. Eight

readings (excluding the highest and lowest) were

averaged to calculate the hind paw weight-bearing

values (Kingery et al., 2003; Guo et al., 2004, 2006).

Room temperature was maintained at 23 �C and

humidity ranged between 25% and 45%. The

temperature of the hind paw was measured using a fine

wire thermocouple (Omega, Stamford, CT, USA) applied

to the paw skin, as previously described (Kingery et al.,

2003; Guo et al., 2004, 2006). In addition, a laser sensor

technique was used to determine the dorsal-ventral thick-

ness of the hind paw, as previously described (Kingery

et al., 2003; Guo et al., 2004, 2006).

Administration of a1-AR agonists and antagonists4–6 weeks after distal tibia fracture

All drugs were dissolved in normal saline. Baseline

behavioral testing was performed prior to drug injection

and 1 h after intraperitoneal injection of the a1-AR

agonists phenylephrine (3 mg/kg) and cirazoline (1 mg/

kg), or the a1-AR antagonist prazosin (2 mg/kg). This

f a1-adrenoceptors on peripheral nerves, blood vessels and keratinocytes does

uroscience (2014), http://dx.doi.org/10.1016/j.neuroscience.2014.09.046


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E. S. Drummond et al. / Neuroscience xxx (2014) xxx–xxx 3


3 October 2014

time point was chosen based on our previous experience

with intraperitoneal injection of these drugs (Guo et al.,

1991; Kingery et al., 2002). In addition, von Frey allodynia

and skin temperature were measured bilaterally before

and after unilateral injection of prazosin (20 lg in 25 llsaline) into the plantar hind paw. Prazosin was injected

into the right paw (ipsilateral to the cast and fracture) on

day one and into the left paw 3 days later. Von Frey allo-

dynia was investigated in both hind paws at 0.5, 1, 2 and

3 h after each injection, and skin temperature was mea-

sured at 0.5, 1, 2, 3, 4, 6 and 24 h. These time points were

selected based on preliminary tests.

Tissue processing and immunohistochemistry

Animals were euthanized and immediately perfused with

Zamboni’s fixative (FD NeuroTechnologies, Inc., Ellicott,

MD, USA), via the ascending aorta; the hind paw dorsal

and plantar skin including sub-dermal layers, and

approximately 10 mm of both sciatic nerves and tibia

nerves were collected from intact controls (n= 5), week

4 (n= 5) and week 16 (n= 5) cast only rats, and week

4 (n= 8) and week 16 (n= 7) post-fracture rats.

Tissue samples were post-fixed in Zamboni’s fixative for

4 h at 4 �C, then rinsed with 0.1 M phosphate buffer (pH

7.4) and 50% ethanol followed by embedding in

TissurePrep2 paraffin (Fisher Scientific, Fair Lawn, NJ,

USA). Some results were not available in the hind paw

skin for the 4-week cast-only group because of technical

difficulties with the fixation of these samples.

Ten-micrometer sections were collected onto silane-

coated slides (Hurst Scientific). Sections were

deparaffinized through xylene and a descending series

of ethanol washes to 0.1 M phosphate- buffered saline

(PBS). Antigen retrieval was performed by incubation

with 1 mg/ml porcine trypsin (Sigma–Aldrich) for 30 min

at 37 �C. Sections were then treated with 0.5% Triton

X-100 for 5 min and incubated with blocking solution for

2 h (10% normal donkey serum in 0.1 M PBS). The

following combinations of primary antibodies were then

applied: a1-AR/protein gene product 9.5 (PGP9.5) to

examine a1-AR expression on all nerve fibers; a1-AR/

CGRP to examine a1-AR expression on peptidergic

nerve fibers; a1-AR/ neurofilament 200 (NF200) to

examine a1-AR expression on myelinated fibers; and

Table 1. Primary and secondary antibodies used in rat tissues: dilutions and s

Antigen and host species Dilution – skin D

Primary antibodies

Anti a1-AR, rabbit polyclonal 1:200 1

Anti CGRP, goat polyclonal 1:400 1

Anti NF200, chicken polyclonal 1:3000 1

Anti SMA, mouse monoclonal 1:1000 1

Anti PGP9.5, mouse monoclonal 1:500 1

Secondary antibodies

Donkey anti-rabbit Dylight 488 1:600 1

Donkey anti-goat Cy3 1:750 n

Donkey anti-goat Dylight 549 n/a 1

Donkey anti-chicken Dylight 647 1:1000 1

Donkey anti-mouse Dylight 647 1:1000 1

Abbreviations: a1-AR, a1-adrenoceptor; CGRP, calcitonin gene related peptide; SMA, sm



a1-AR/smooth muscle actin (SMA) to examine a1-AR

expression on blood vessels (details and dilutions are

shown in Table 1). Antibodies were co-incubated for

48 h at 4 �C, diluted in blocking solution. Sections were

then washed with PBS (3 � 15 min) and incubated with

the appropriate species- specific secondary antibodies

(Table 1) diluted in 5% donkey serum (Sigma) in PBS

for 4 h at room temperature. Sections were washed with

PBS (3 � 15 min) and coverslipped with Prolong Gold

anti-fade mounting media (Invitrogen). The pattern of

staining produced by the a1-AR antibody on blood

vessels, nerves and epidermal cells in rat skin tissue

resembles the staining pattern produced by BODIPY

FL-prazosin, a fluorescent a1-AR antagonist (Dawson

et al., 2011). In addition, staining was eliminated following

pre-adsorption of the anti-sera with an a1-AR-specific

peptide, indicating that the antibody binds specifically to

a1-ARs (Dawson et al., 2011). In this study, no staining

was observed on negative control sections that had all

primary antibodies omitted.

Confocal imaging and quantification ofimmunohistochemistry

Images of immunostained sections were collected using a

Nikon Eclipse Ti multiphoton confocal microscope. All

images were collected at 200� magnification and

consisted of a stack of optical sections (Z-series)

acquired at 2-lm intervals throughout the depth of the

tissue section. Each label was collected sequentially at

the appropriate excitation and emission spectra to

prevent bleed-through between channels, and imaging

settings were consistent for all sections in each staining

run. For sciatic and tibial nerve sections, two images

were collected per sample for each staining combination.

For skin sections, two images containing the epidermis

and underlying papillary dermis and two images of the

deeper dermis containing large nerve bundles were

collected per sample for each staining combination.

Quantification of immunohistochemistry was performed

using ImageJ software (ImageJ, version 1.47t, National

Institute of Health, USA) on the maximum projection

image of each z-stack. The investigator who performed

the quantification was blinded to the experimental group

and to the laterality of each slide.

ource

ilution – sciatic/tibial nerve Product code and source

:200 A270, Sigma–Aldrich

:400 1720-9007, AbD Serotec

:3000 Jackson ImmunoResearch

:1000 A2547, Sigma–Aldrich

:500 MCA2084, AbD Serotec


/a Jackson ImmunoResearch




ooth muscle actin; NF200, neurofilament 200; PGP9.5, protein gene product 9.5.




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3 October 2014

To examine cutaneous CGRP+ nerve fiber

populations, a standardized threshold level that

identified CGRP+ pixels was applied to all images of

dermal nerve bundles. The number of CGRP+ pixels in

each nerve bundle above this threshold was expressed

as a percentage of the total nerve bundle area to allow

for comparison between nerve bundles of different

sizes. This same method was also used to determine

the expression of NF200+ fibers in nerve bundles. The

expression of CGRP and NF200 in sciatic and tibial

nerve fibers was examined using a similar method in a

standard rectangular area that was applied to all images.

a1-AR expression was quantified in the sciatic nerve,

tibial nerve and epidermis, and in dermal nerve fibers

and blood vessels. The average a1-AR staining intensity

in sciatic and tibial nerves was quantified in a standard

rectangular area that was consistent across all images.

This area was representative of the whole nerve with

regard to a1-AR staining intensity and only contained

nerve fibers. In skin sections, the epidermis was

identified by morphology and the average a1-AR

staining intensity in this region was quantified. To

identify a1-AR expression on nerve fibers, PGP9.5

staining was used to create a mask of all nerve fibers,

allowing a1-AR staining intensity to be measured only

on these nerve fibers. Similarly, SMA staining was used

to identify dermal blood vessels and a1-AR staining

intensity was measured on these identified regions.

The average a1-AR staining intensity was also

quantified in CGRP+ and NF200+ fiber populations.

Fig. 1. Behavioral responses in week 4 (n= 5) and week 16 (n= 5) cast onl

the behavioral measures differed between the ipsilateral and contralateral hin

In A–D, #represents a significant difference between the ipsilateral and contra

the ipsilateral hind paw denote significant differences between groups tha

differences were detected among the four groups for the contralateral hind p



CGRP or NF200 staining was used to create a mask of

nerve fiber location, which was then applied to the

corresponding a1-AR image and the average a1-AR

staining intensity in this defined region was measured.

The average a1-AR staining intensity in CGRP+ and

NF200+ fibers was also determined in the sciatic and

tibial nerves using the same method.

Statistical approach

Differences between the fracture and cast-only groups in

hind paw allodynia, unweighting, temperature and

swelling were investigated for the ipsilateral versus the

contralateral hind paw in repeated measures analyses

of variance. A similar approach was used to investigate

differences in PGP, CGRP, NF200 and a1-AR

expression across the experimental and control groups.

Significant main effects and interactions were explored

across groups in a one-way analyses of variance and

post hoc Newman–Keuls tests, and within each group

with paired t-tests. Results are reported as the

mean ± standard error, and the criterion of statistical

significance was p< 0.05.

RESULTS

Behavioral and physiological changes after distaltibia fracture

At 4 weeks, allodynia and unweighting were greater in the

ipsilateral than contralateral hind paw in the fracture and

cast-only groups (p< 0.001) (Figs. 1A, B). These

y, and week 4 (n= 8) and week 16 (n= 7) post-fracture rats. Each of

d paws across the four groups (Group � Side interactions, p< 0.01).

lateral hind paw (p< 0.05). The letters a, b and c above the bars for

t do not share the same letter (p< 0.05, Newman–Keuls test). No

aw (Newman–Keuls test). Error bars represent standard errors.




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3 October 2014

behavioral signs of pain persisted at 16 weeks in the

fracture group but had resolved in the cast-only group.

The temperature of the ipsilateral hind paw was

elevated in both groups at 4 weeks (p< 0.001)

(Fig. 1C), but this response had disappeared by

16 weeks. In addition, the dorsal-ventral thickness was

greater in the ipsilateral than contralateral hind paw of

the fracture group at 4 weeks (p< 0.001), but the

swelling had gone by 16 weeks (Fig. 1D).

Four weeks after fracture, intraperitoneal injection of

prazosin inhibited allodynia and unweighting in the

ipsilateral hind paw (Fig. 2). In addition, temperature

increased in both hind paws after the prazosin injection;

this increase was greater in the contralateral than

ipsilateral hind paw possibly because temperature was

already elevated in the ipsilateral hind paw at the

baseline (Fig. 2). Allodynia did not change in either hind

paw after intraplantar injections of prazosin; however,

increases in temperature were greater when prazosin

was injected into the ipsilateral than contralateral

hind paw (Fig. 3). Allodynia did not increase after

intraperitoneal injection of phenylephrine or cirazoline,

perhaps due to ceiling effects as allodynia was already

pronounced before the injections (data not shown).

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Changes in PGP, CGRP and NF200 expression afterdistal tibia fracture

The percentage of PGP+ pixels was not affected either by

fracture or casting, either in the ipsilateral or contralateral

sciatic nerve. However, the percentage of CGRP+ pixels

in sciatic nerves was greater in the 16-week fracture

group than in the other four groups (p< 0.05) (Fig. 4).

CGRP staining also differed among groups in the

Fig. 2. Effect of intraperitoneal injection of prazosin (2 mg/kg) on behavior

allodynia and unweighting were greater in the ipsilateral than contralateral h

swollen than the contralateral hind paw (⁄p< 0.05). Allodynia and unweigh

p< 0.05). In addition, increases in skin temperature were greater for the cont

thereby offsetting differences at baseline. #represents a significant difference

the prazosin injection (p< 0.05). Error bars represent standard errors.



plantar dermal nerves, primarily in the contralateral hind

paw. Increases were most pronounced in the 16-week

cast group; in addition, the percentage of CGRP+ pixels

was lower on the ipsilateral than contralateral side in the

4- and 16-week fracture groups (p< 0.05) (Fig. 4).

However, CGRP expression did not after fracture or

cast-immobilisation in the tibial nerve or in dorsal dermal

nerve bundles (data not shown).

The percentage of NF200+ pixels in sciatic nerves

also differed among groups (Fig. 5). Specifically,

NF200+ expression decreased in the 4-week fracture

group, particularly on the contralateral side (p< 0.001

compared with the ipsilateral side), but then increased in

the 16-week fracture group such that levels were

greater on the ipsilateral than contralateral side

(p< 0.05). Similar trends in the tibial and dermal nerves

did not achieve statistical significance.

Changes in a1-AR expression after distal tibiafracture

a1-AR expression increased significantly in the sciatic

nerve in all groups (Fig. 6). In the 16-week cast and

fracture groups, a1-AR expression also increased

significantly in plantar dermal blood vessels and

keratinocytes in both hind paws (Figs. 7 and 8).

a1-AR expression was also examined in individual

nerve populations (Fig. 9). a1-AR staining intensity on

CGRP+ and NF200+ axons trended upward in the hind

limbs of the 16-week cast and fracture groups. For

CGRP+ fibers, a1-AR expression differed among

groups for the tibial (p< 0.05) and plantar dermal

nerves (p< 0.05). For NF200+ fibers, a1-AR

al responses 4 weeks after distal tibia fracture (n= 7). At baseline,

ind paw; in addition, the ipsilateral hind paw was warmer and more

ting decreased after the prazosin injection (Time � Side interactions,

ralateral than ipsilateral hind paw (Time � Side interaction, p< 0.05),

between levels at baseline and levels in the same hind paw 1 h after




Fig. 3. Effect of intraplantar injection of prazosin (20 lg in 25 ll saline) on allodynia (n= 7) and hind paw temperature (n= 4) 6 weeks after distal

tibia fracture. Allodynia did not change after injections into the ipsilateral or contralateral hind paw. However, shortly after prazosin administration,

increases in skin temperature on the injured side were greater after ipsilateral than contralateral injections (Side Injected � Time � Side Injured

interaction, p< 0.01; ⁄p< 0.05 for differences between the ipsilateral and contralateral sides). Error bars represent standard errors.

Fig. 4. CGRP expression (in terms of % total area) in peripheral nerve fibers. The images illustrate CGRP expression in nerve bundles (outlined in

white) in plantar skin (Scale = 20 lm). The main effect for Group was statistically significant for the sciatic nerve (p< 0.001), and main effects for

Group (p< 0.001) and Side (p< 0.05) were statistically significant for dermal nerves in plantar skin. For the sciatic nerve, the ⁄denotes a significant

difference in CGRP expression, averaged across the ipsilateral and contralateral sides, between the 16-week fracture group and each other group;

for plantar dermal nerves, the ⁄denotes a significant difference in CGRP expression, averaged across sides, between the 16-week cast-only group

and each other group (p< 0.05, Newman–Keuls test). In addition, #represents a significant difference between the ipsilateral and contralateral hind

paw in plantar dermal nerves (p< 0.05). Error bars represent standard errors.



3 October 2014




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Fig. 5. NF200 expression in sciatic nerves. The main effect for Group

(p< 0.001) and the Group � Side interaction (p< 0.01) were

statistically significant, due primarily to a decrease in NF200 expres-

sion 4 weeks after fracture followed by an increase above baseline on

the injured side at 16 weeks (p< 0.05, Newman–Keuls test). Error

bars represent standard errors, and #represents a significant differ-

ence between sides.

Fig. 6. a1-AR expression on PGP+ neurons in the sciatic nerves

(main effect for Group, p< 0.01). The ⁄above the bars (averaged

across the ipsilateral and contralateral sides) denote significant

differences between each experimental group and the control (naı̈ve)

group (p< 0.05, Newman–Keuls test). Error bars represent standard

errors.

Fig. 7. a1-AR expression in the epidermis. The images illustrate a1-AR exp

Group was statistically significant in plantar skin (p< 0.001). The ⁄above the

greater expression than bars without the ⁄(p< 0.05, Newman–Keuls tes

references to color in this figure legend, the reader is referred to the web ve



3 October 2014



expression differed among groups for the sciatic

(p< 0.05) and tibial nerves (p< 0.01).

DISCUSSION

Distal tibia fracture and casting produced a range of

behavioral, physiological and anatomical changes,

including an increase in a1-AR expression on nerve

fiber populations at 4 and 16 weeks, and on plantar

keratinocytes and dermal blood vessels at the 16-week

time point (Table 2). The increase in a1-AR expression

involved not only the fractured limb but also the

contralateral uninjured limb; furthermore, a1-AR

expression was elevated in the cast-only group

12 weeks after the cast had been removed, when pain

had resolved. Four to six weeks after the fracture,

systemic injection of the a1-AR antagonist prazosin

inhibited behavioral signs of pain; however, intraplantar

injections of prazosin did not. Thus, it seems unlikely

that an increase in a1-AR expression on peripheral

nociceptive neurons or other dermal tissues was the

primary source of pain after distal tibia fracture.

Behavioral and physiological changes

As in previous studies (Guo et al., 2004), both distal tibia

fracture and cast immobilisation generated behavioral

signs of pain which resolved more quickly after cast

immobilisation than distal tibia fracture. In addition, the

hind paw was swollen in the fracture group and hind

paw temperature was elevated in both groups at 4 weeks.

Pain in the distal tibia fracture model can be attenuated by

LY303870 (a substance P antagonist) (Guo et al., 2004),

soluble TNF receptor type 1 (an antagonist for TNF)

(Sabsovich et al., 2008a), anti-nerve growth factor anti-

bodies (Sabsovich et al., 2008b), the cytokine inhibitor

pentoxifylline (Wei et al., 2009b), the IL-6 antagonist

ression (green) in plantar skin (Scale = 50 lm). The main effect for

bars (averaged across the ipsilateral and contralateral sides) indicates

t). Error bars represent standard errors. (For interpretation of the

rsion of this article.)




Fig. 8. a1-AR expression in blood vessels. The images illustrate a1-AR expression (green) in blood vessels in plantar skin identified by smooth

muscle actin (SMA, blue) (Scale = 50 lm). The main effect for Group was statistically significant in plantar skin (p< 0.01). The ⁄ and ^ symbols

above the bars (averaged across the ipsilateral and contralateral sides) denote significant differences between groups that do not share the same

symbol (p< 0.05, Newman–Keuls test). Error bars represent standard errors. (For interpretation of the references to color in this figure legend, the

reader is referred to the web version of this article.)

Fig. 9. a1-AR expression on individual nerve fiber populations. The nerve bundle shown is from 16 week cast (ipsilateral) plantar skin.

Colocalization of a1-AR and CGRP or a1-AR and NF200 was identified using the colocalization plugin for ImageJ, and colocalized pixels are shown

in white (Scale = 20 lm). Differences among groups are illustrated graphically for the tibial nerves. For a1-AR expression on CGRP+ fibers, the

main effect for Group was statistically significant for the tibial nerve (p< 0.05) and plantar dermal nerves (p< 0.05). For a1-AR expression on

NF200+ fibers, the main effect for Group was statistically significant for the tibial (p< 0.01) and sciatic nerves (p< 0.05). The ⁄, ^ and @ symbols

above the bars (averaged across the ipsilateral and contralateral sides) denote significant differences between groups that do not share the same

symbol (p< 0.05, Newman–Keuls test). Error bars represent standard errors.



3 October 2014




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Table 2. Summary of findings

4-Week cast 4-Week fracture 16-Week cast 16-Week fracture

Allodynia and

unweighting

" Ipsilaterally " Ipsilaterally No abnormality detected " Ipsilaterally

Hind paw temperature " Ipsilaterally " Ipsilaterally No abnormality detected No abnormality detected

Edema No abnormality

detected

" Ipsilaterally No abnormality detected No abnormality detected

CGRP+ pixels No abnormality

detected

; Ipsilaterally in plantar

dermal nerves

" Most prominent in

contralateral plantar

dermal nerves

" In sciatic nerve and

greater in contralateral

than ipsilateral plantar

dermal nerves

NF200+ pixels No abnormality

detected

; Bilateral trend in the

sciatic and tibial nerves

No abnormality detected " Ipsilaterally in sciatic

nerve

a1-AR staining intensity

in the sciatic nerves

" Bilaterally " Bilaterally " Bilaterally " Bilaterally

a1-AR staining intensity

in the epidermis and

dermal blood vessels

Not measured No abnormality detected " Bilaterally in plantar

skin

" Bilaterally in plantar

skin



3 October 2014

TB-2-081, chemical sympathectomy with 6-OHDA or gua-

nethidine, and butoxamine (a b2-adrenoceptor antagonist)

(Li et al., 2013). Together, these findings suggest that

neuropeptides released from nociceptive afferents and

noradrenaline secreted by sympathetic nerve fibers trig-

ger the production of inflammatory mediators (e.g., from

keratinocytes, vascular endothelial cells, mast cells or

immune cells); in turn, these inflammatory mediators sen-

sitize nociceptors and cause pain to persist (Li et al.,

2013).

In the present study, systemic administration of the

a1-AR antagonist prazosin inhibited behavioral signs of

pain 4 weeks after fracture whereas intraplantar

injections two weeks later did not. One possibility is that

fracture and/or casting triggered an up-regulation of

a1-ARs in central nociceptive pathways that augmented

pain (Holden et al., 1999; Brightwell and Taylor, 2009;

Jeong and Holden, 2009). Alternatively, the intraplantar

dose might not have blocked local a1-AR signaling com-

pletely. However, this seems unlikely as hind paw temper-

ature increased significantly after the intraplantar

injections, implying release of noradrenergic vasocon-

strictor tone. This increase was more prominent in the

ipsilateral than contralateral hind paw, possibly because

release of noradrenergic vasoconstrictor tone unmasked

underlying inflammatory vasodilator influences on the

plantar microvasculature in the injured limb.

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Neural markers

To determine whether distal tibia fracture-cast

immobilisation evoked signs of nerve injury, we

investigated changes in expression of the pan-neuronal

marker PGP9.5 and the selective markers NF200 and

CGRP. PGP9.5 staining did not differ significantly from

control levels after fracture or casting indicating that, on

the whole, nerve fiber populations remained stable.

NF200 is a marker for neurofilaments, which provide a

cytoskeleton for myelinated neurons and regulate

axonal diameter. Not only do they influence conduction

velocity, but they are also involved in neuronal

differentiation, axon outgrowth and regeneration (Perrot

et al., 2008; Shea et al., 2009). Axonally-transported neu-



rofilament proteins decrease in injured neurons whereas

the expression of neurofilament subunits is strongly up-

regulated during regeneration (Perrot et al., 2008). Thus,

the bilateral decrease in NF200 in the sciatic nerves

4 weeks after distal tibia fracture, succeeded at 16 weeks

by heightened NF200 expression, is consistent with nerve

injury followed by repair. However, this perturbation was

relatively minor because it did not involve dermal nerves

in the hind paw. Possibly a systemic response to fracture

temporarily suppressed the production or transport of

neurofilaments in myelinated sciatic neurons.

Four weeks after distal tibia fracture, expression of the

neuropeptide CGRP had decreased in plantar dermal

nerves in the injured hind paw but 12 weeks later this

expression had increased in the contralateral hind paw

both in the cast-only and fracture groups. Although this

might indicate a cycle of denervation and reinnervation,

these changes could also reflect a temporary reduction

in CGRP due to a lag in replenishing depleted neural

stores. In particular, the increase in CGRP in

contralateral plantar dermal neurons both after fracture

and cast immobilisation suggests that a systemic

response to immobilisation triggered an increase in

CGRP production. This would be consistent with

previous findings that distal tibia fracture increased

CGRP gene expression in the dorsal root ganglia

supplying the ipsilateral hind limb without a significant

change in epidermal neurite counts (Wei et al., 2009a).

Neuropeptide signaling is actively involved in the neuro-

inflammatory response triggered by distal fracture and

immobilisation, as blocking this signaling inhibits post-

fracture allodynia, vascular changes and production of

inflammatory mediators such as IL-1b, TNF and nerve

growth factor (Wei et al., 2009b; Guo et al., 2012).

a1-AR expression

After peripheral nerve injury, heightened expression of

a1-ARs on nociceptive afferents contributes to an

increase in neural excitability and pain (Ali et al., 1999;

Xie et al., 2001; Maruo et al., 2006). Similar effects on

keratinocytes could stimulate the production and/or

release of inflammatory mediators that sensitize




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3 October 2014

nociceptive afferents, whereas up-regulation of a1-ARs on

cutaneous blood vessels might cause pain by altering

microvascular blood flow (Coderre and Bennett, 2010).

Thus, it is tempting to speculate that an increase in expres-

sion of a1-ARs on neurons, blood vessels and keratino-

cytes contributed to pain in the fracture group. However,

it seems unlikely that this was the primary source of pain

as a1-AR expression increased bilaterally in plantar hind

paw skin both in the 16-week fracture group (associated

with signs of residual pain in the affected limb) and in the

16-week cast-only group where pain had resolved. Fur-

thermore, a1-AR expression did not increase in dorsal

hind paw skin either on the fractured/immobilized or con-

tralateral side. It might be relevant that constriction due

to post-fracture edema was minimized by providing a win-

dow in the cast over the dorsum of the paw and ankle.

Together, these findings suggest that a non-specific

effect of the procedure (e.g., psychological stress or

changes in activity or weight distribution on the hind

paws after fracture/immobilisation) induced circulatory or

systemic neural or inflammatory changes that altered

a1-AR expression. In particular, inflammatory mediators,

growth factors or hormones released after fracture and

cast immobilisation (Li et al., 2013) might initiate a1-AR

up-regulation. For example, the inflammatory mediators

TNF and IL-1b trigger the expression of the a1A-AR sub-

type in the THP-1 human monocyte cell line (Heijnen

et al., 2002). In addition, glucocorticoids and the b2-AR

agonist terbutaline increase the expression of messenger

RNA (mRNA) for a1B-AR and a1D-AR on THP-1 cells

(Rouppe van der Voort et al., 1999), and nerve growth

factor increases levels of a1B-AR mRNA and protein in

cultured dorsal root ganglion cells (Zhang and Tan, 2011).

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Limitations and conclusions

Several limitations apply to this study. In particular,

additional quantitative methods will be required to more

precisely delineate the triggers and sequence of change

in a1-AR expression after fracture and cast

immobilisation. Furthermore, effects of a1-AR blockade

were examined at only one time point, and only a single

concentration of antagonist was used.

Despite these limitations, our findings clearly show

that distal tibia fracture and cast immobilisation

produced transient reductions followed by increases in

the expression of the neural markers NF200 and CGRP,

and non-specific increases in a1-AR expression in

peripheral neurons, blood vessels and keratinocytes.

These changes are unlikely to account for persistent

pain after limb fracture. Nevertheless, further

examination of the systemic responses that generated

these changes seems warranted, to determine whether

they aggravate symptoms (e.g., by compromising neural

structure or function) or delay recovery (e.g., by

augmenting the neuroinflammatory responses that

mediate symptoms).

Acknowledgment—This work was supported by grants from the

National Health and Medical Research Council of Australia (grant

numbers APP1030379, 437205), the Australian and New Zea-

land College of Anaesthetists (grant number 10/021), and the



Department of Veterans Affairs Rehabilitation and Research

Development grant F7137R.

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(Accepted 19 September 2014)(Available online xxxx)




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