2014 osu aslap summer student€¦ · “the open-field test a critical review”. psychological...

2014 OSU ASLAP Summer Student

Extern: Amanda Darbyshire, ASLAP Foundation Summer Fellowship Currently a student at The Ohio State University, Columbus OH Projected graduation 2016 Involved to present with: American Society of Laboratory Animal Practitioners, 2013,

Ohio Veterinary Medical Association, 2013, Student Chapter of the American Veterinary Medical Association, 2013, American Association of Zoo Veterinarians, 2013, American Association for Laboratory Animal Science, 2007.

Mentor: Dr Dondrae Coble, DVM, DACLAM, Director Experimental Surgery Core ULAR, Assistant Professor Clinical, Veterinary Preventive Medicine Dr Coble is Clinical Veterinarian for OSU facilities and as the Director of the Experimental Surgery Core which provides clinical and didactic support for the Laboratory Animal Residency Program. He is a member of ASLAP and AALAS. Research Project: “An Assessment of the Safety of Recuvyra following Topical

Administration in Mice”

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Amanda DarbyshireAugust 5, 2014

Hypothesis: Topical administration of Recuvyra is safe for use in mice.

Most currently used pain relievers require frequent dosing◦ Time commitment◦ Potentially stressful to postoperative mice◦ May allow for a drop in analgesic efficacy

Recuvyra may allow multiple days of uninterrupted analgesia after one application

Buprenorphine Q12h SC Partial mu agonist Most common opioid used in mice

Tramadol Q24h SC Weak mu opioid

Meloxicam Q24h SC or PO NSAID, COX-2 inhibitor May provide better analgesia than Buprenorphine

Buprenorphine, tramadol, and various NSAIDs can be added to drinking water for continuous administration◦ Mice must consume water to receive analgesia◦ Weight loss and dehydration

Buprenorphine in food?

Transdermal solution of fentanyl (50mg/ml) Approved for control of postoperative pain in

dogs◦ One application provides analgesia for 4 days

1000x more potent than injectable Fentanyl in humans!

One application! No need for CRI, or multiple injections Provides prolonged analgesia Avoids first-pass metabolism in intestines

and liver

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Dermal Patch◦ Provides pain relief for 2-3 days◦ Affected by heat and blood flow rate◦ Cannot be accurately dosed for use in rodents

Injectable◦ Short-acting◦ Used as a CRI or perioperative injection

Approved for use in dogs No need to clip hair No worries about adhesion to skin Not altered by temperature No need for re-administration No potential for abuse by clients

Dosed at 2.7mg/kg 2-4 hours prior to surgery in dogs

Applied topically to dorsal scapular area Wear gloves, eye protection, and a lab coat

during application

Rapid dermal absorption and sequestration in stratum corneum

Absorbed into the bloodstream through the stratum corneum

Activates µ(mu) opioid receptors

Cytochrome P450 metabolism◦ Biotransformation of other drugs via oxidation

P-glycoprotein membrane efflux transporter◦ P-glycoprotein deficient mice may have

prolonged and increased analgesic effect Excreted in feces and urine

Supplied in 10ml vials (50mg/ml) $300/kit ◦ Includes Recuvyra, needleless adaptor, 15 3ml

syringes, 15 applicator tips Store at 20-25ºC (68-77ºF) for 30 days once

open

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Class II opioid Avoid contact with application site for 72

hours Antidote is naltrexone or naloxone Must take online training course prior to use◦ http://www.recuvyratraining.com

• GI stasis• Pronounced sedation• Decreased food and water intake• Hypothermia• Bloody stool• Diarrhea

Do not apply to broken or diseased skin Do not administer to animals that are

hypovolemic, debilitated, or suspected to have paralytic ileus

Do not administer to animals that will experience mild to absent pain

Do not administer a second dose for the same procedure

Do not administer other opioids within 7 days

Male, C57BL/6 mice, 8 weeks of age Singly housed Administered Recuvyra to dorsal tail base

with a pipette while anesthetized (5 minutes) with Isofluorane

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3 Male C57BL/6 mice◦ Low dose, (20mg/kg)◦ Middle dose, (200mg/kg)◦ High dose, (500mg/kg)

Middle and high dose mice were euthanized the day after application due to adverse effects

Doses were lowered for safety assessment study

Groups (n=5)◦ Low dose, 2.5µl (5mg/kg)◦ Middle dose, 6.3µl (12.5mg/kg)◦ High dose, 10µl (20mg/kg)◦ Control, 2.5µl (saline)

Mice were dosed on Day 1 and followed through Day 5

Survival Body weight ◦ Day 1-5

Behavior and nociception◦ Nest Complexity Scoring Day 1-5◦ Time-to-integrate-to-nest test (TINT) Days 2-5◦ Open field test Days 1-5◦ Tail-flick test Days 1-5

Purpose: Nest building is a sign of health and welfare in mice

Assessment: Nests are scored 7-9 hours after Recuvyra application, and at 24, 48, 72, and 96 hour time points◦ Mice build nests at the end of a dark phase◦ By scoring initially at 7-9 hours post-application,

nest scoring can be a short-term indicator of impairment

>90% of the Nestlet is intact 50-90% of the Nestlet remains intact

5

The Nestlet is mostly shredded but there is no identifiable nest: < 50% of the Nestlet remains

intact

> 90% of the Nestlet is torn up into a flat nest with walls higher than mouse body height on less than

50% of its circumference

> 90% of the Nestlet is torn up, the nest is a crater, with walls higher than mouse body

height on more than 50% of its circumference

Purpose: Mice should be motivated to quickly perform nesting behavior when given fresh nesting material◦ Most mice integrate within 2 minutes◦ Singly housed mice are more likely to test negative◦ Mice must have a nest complexity score of 2 or

greater to test

Assessment: ◦ Nest complexity is scored at 24, 48, 72, and 96

hour time points◦ ¼ Nestlet is then added to each cage◦ If Nestlet is moved from the original position within

10 minutes, the test is positive

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Purpose: Assesses emotionality in a novel environment◦ Mice should eagerly explore a new environment and

exhibit thigmotaxis◦ Locomotor activity◦ Anxiety behavior

Assessment: Mouse is placed in the center square of the chamber and allowed to explore for 5 minutes◦ A video camera records data

Center square duration◦ Exploration◦ Anxiety

Rearing◦ Locomotion◦ Exploration◦ Anxiety

Defecation◦ Anxiety◦ Emotionality

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Purpose: Assesses nociception by application of radiant heat to the tail◦ Measures the reflex latency of the withdrawal

reaction◦ Increased latency suggests analgesia

Assessment: ◦ The mouse is restrained and a point at the middle

of the tail is marked◦ The marked area is heated with the apparatus until

the tail is withdrawn, or 15 seconds ◦ The latency is recorded to the nearest 0.1s◦ The test is repeated in areas proximal and distal to

the initial site with 5 minutes between each reading, and the latency times are averaged

One mouse in the high dose group was euthanized on Day 1 due to adverse effects◦ Liver Values Increased ALT 303U/L (17-77U/L) AST 1296U/L (54-298U/L) ALP 114U/L (35-96U/L)◦ Phosphorus Increased 20.9mg/dL (5.7-9.2mg/dL)

Day 1 Day 2 Day 3 Day 4 Day 5 EndControl 5 5 5 5 5 5Low 5 5 5 5 5 3Middle 5 5 5 5 4 1High 5 4 4 4 1 0

Four mice were euthanized on Day 4 due to tail necrosis

Six mice were euthanized on Day 5 after completion of the study due to tail necrosis

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Necrosis of Tail from Days 4 and 5

Gram

s

20

21

22

23

24

25

26

27

28

Day 1 Day 2 Day 3 Day 4 Day 5

Control

Low

Middle

High

***

* P<0.05** P<0.01

0

1

2

3

4

5

6

7

Baseline 7-9 hours Day 2 Day 3 Day 4 Day 5

Control

Low

Middle

High

*

* P<0.05** P<0.001

Nest

Sco

re

** ** **

** % Po

sitiv

e

0

20

40

60

80

100

Control Day 2 Day 3 Day 4 Day 5

Control

Low

60% of the middle dose group had not built a nest on day 1

25% of the high dose group hadnot built a nest on day 1 20% of the middle dose group had not built a nest on day 2

These mice were not tested

Tim

e (s

)

0

2

4

6

8

10

12

14

16

Baseline Day 1 Day 2 Day 3 Day 4 Day 5

Control

Low

Middle

High

****

* P<0.05** P<0.001

*

9

Tim

e (s

)

0

20

40

60

80

100

120

140

Day1 Day 2 Day 3 Day 4 Day 5

Control

Low

Middle

High

* P<0.05

*

Rear

s

0

5

10

15

20

25

30

35


Control

Low

Middle

High

*

* P<0.01

*

0

1

2

3

4

5

6

7


Control

Low

Middle

High

Num

ber o

f Fec

al P

elle

ts

All mice survived dosing with Recuvyra, except one from the high dose group

Mice initially lost weight, but regained it by the end of the study

Nest complexity decreased with dosing initially, and then returned to normal

Mice were less likely to integrate new nesting material after dosing

Mice initially had higher tail-flick latencies according to dose, which gradually decreased over 4 days

Mice showed less exploratory behavior initially

This study did not include a surgical pain model, however is important because the clinical safety of opioids may be greater in the presence of pain◦ Humans in severe pain may tolerate doses 3-4

times higher◦ The adverse effects of opioids can be antagonized

by pain Number of mice Tail-flick assay

The optimal dose of 12.5mg/kg is safe for use in mice, however mice are depressed initially

One application lasts multiple days No break in analgesic efficacy Less handling of post-operative mice Convenient for staff

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Repeat the study with more mice Recuvyra in mice using a surgical pain model

to determine analgesic efficacy Pharmacokinetic fentanyl study Test blood for signs of organ toxicity

Dr. Dondrae Coble Dr. Matthew Hogan Dr. Valerie Bergdall Dr. Hickman-Davis Dr. Popovich’s Lab Dr. Nelson’s Lab ULAR Staff-Psychology QA Staff

Bailey, K. and Crawley, J. “Methods of Behavior Analysis in Neuroscience.” Anxiety-Related Behaviors in Mice, 2nd Ed., Chapter 5.

Barrot, M. “Tests and models of nociception and pain in rodents.” Neuroscience, 2012 Jun. Volume 211, Pages 39-50.

Bölcskei, K. “ Methods to Study Thermonociception in Rodents “TRP Channels in Drug Discovery : Volume II. 2012 Oct: 401-417.

Bourque SL1, Adams MA, Nakatsu K, Winterborn A. “Comparison of buprenorphine and meloxicam for postsurgical analgesia in rats: effects on body weight, locomotor activity, and hemodynamic parameters.” J Am Assoc Lab Anim Sci. 2010 Sep;49(5):617-22.

Brown, R. E., Corey, S. C., Moore, A. K. 1999. “Differences in measures of exploration and fear in MHC-congenic C57BL/6J and B6-H-2K mice”. Behavior Genetics, 26, 263-271.

Deacon, R. “Assessing nest building in mice.” Nature Protocols 1: 1117 -1119 ,2006.

Gaskill, B. N., Karas, A. Z., Garner, J. P., Pritchett-Corning, K. R. “Nest Building as an Indicator of Health and Welfare in Laboratory Mice”. J. Vis. Exp. (82), (2013).

Ide, S. et al. “Buprenorphine antinociception is abolished, but naloxone-sensitive reward is retained, in μ-opioid receptor knockout mice.”

Jirkof, P., Fleischmann, T., Cesarovic, N., Rettich, A., Vogel, J. and Arras, M. “Assessment of postsurgical distress and pain in laboratory mice by nest complexity scoring”.

Kukanich, B., Clark,T.P. “The history and pharmacology of fentanyl: relevance to a novel, long-acting transdermal fentanyl solution newly approved for use in dogs”. J. vet. Pharmacol. Therap. 35 (Suppl. 2), 3–19 2012

Le Bars, D., et al. “Animal Models of Nociception” Pharmacological Reviews 53:597–652, 2001.

Molina-Cimadevila, MJ. et al. “Oral self-administration of buprenorphine in the diet for analgesia in mice.” Lab Animal 2014 Apr; published online before print.

Recuvyra Pamphlet

Rock,ML. “The Time-to-Integrate-to-Nest Test as an Indicator of Wellbeing in Laboratory Mice”. JAALAS 53(1): 24-28.

Walsh, RN., and Cummins, RK. “The open-field test A critical review”. Psychological Bulletin. 83: 482-504, 1976.

2014 OSU CASS Summer Student

Extern: Kelsey Cornelius Cass Summer Fellowship • Currently a student at The Ohio State University, Columbus OH • Projected graduation 2016 • Involved to present with: Student Chapter of the American Veterinary Medical Association,

2012, American Society of Laboratory Animal Practitioners, 2012, Ohio Veterinary Medical Association, 2012, American Association of Zoo Veterinarians, 2012, The Ohio State Veterinary Surgery Club, 2012, The Ohio State Veterinary Pathology Club, 2013,The Ohio State Veterinary Oncology Club, 2013

Mentor: Stephanie Lewis, DVM, MS, DACLAM, Director, Large Animal Quality Assurance and Clinical Medicine, Principle Investigator Training, ULAR, Associate Professor Clinical, Veterinary Preventive Medicine Dr. Lewis is a Clinical Veterinarian for OSU facilities and as the Director of Large Animal Clinical Medicine provides clinical and didactic support for the Laboratory Animal Residency Program. She is the faculty advisor and student liaison for ASLAP, a member of AALAS and the OSU Institutional Biosafety Committee, and serves on the IACUC at OSU and the Veterinary Technician Institute at Bradford School, Columbus, Ohio. Research Project: “Nest location preference in mice housed in individually ventilated and static caging systems”

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Laboratory Animal Care and Use Program and Facilities

Nest location preference in mice housed in individually ventilated and static caging systems

Kelsey C. Cornelius

Stephanie Lewis, DVM, MS, DACLAM

Tara Martin, DVM

Candace Hendrick


Agenda

• Background

• Materials & Methods

• Results

• Discussion

• Future Directions


Mice In Research

• Most commonly used lab animal model

• Many studies observe mice for changes in behavior

• Important to define normal behavior

• Ex of normal behavior: building nests


Why do Mice Build Nests?


Why do Mice Build Nests?

• Help rear young, better breeding performance

• Less stress

• Less aggressive, less cannibalism

• Greater weight gain, Increase feed efficiency

• Eye irritation in hairless strains

More uniform data!

Nest Side Effects


How do Nests Help Researchers? • We can evaluate nests

– Behavioral, neuro studies

– Pain or distress in mice

• How evaluate:– Present or absent

– Walls

– Similar to wild mice nests

2


Factors Influence Nest Quality

• More recently….– Pain, Distress= WELFARE!

• But also!– Environment (macro and micro):

housing (temperature, air flow), etc

– Genetics

– Sex

– Age

– Nest materials

– Research side effects

– Breeding, Pups Laboratory Animal Care and Use Program and Facilities

Temperature Influences Nests

Fig 1. Affect of Temperature on Nest Score (Gaskill 2013)


Temperature Influences Nests

Fig 2. Effects of different factors on thermoneutral zone (Gordon 1998)

Higher quality nests minimize

heat loss


Housing Influences Nests

• Little published data: – does cage ventilation affect nest

complexity, nest location?

• Types of housing: Static caging (SC) or individually ventilated caging (IVC)

• Static caging relies on air flow reaching cage to provide ventilation



• IVCs: air flow enters cage through port, exits through filter lid– We control air changes per hour


Housing Influences NestsIVC Benefits

• stocking density

• contamination

• cage ∆ freq

• humidity,CO2,NH3

• Protection

• Sterile

IVC Cons

stress?

3



• Nests counteract negative effects of high intracage ventilation rates (Baumans 2001)

• Less stress at lower rates (30 vs 60 ACH)



• IVC more air changes/hr than static

• What does the Guide say about air changes/hour?

• Why do we keep IVCs at ~35 ACH?– At least 30 ACH to keep cage

free of NH3 (Reeb-Whitaker et al., 2001)


Housing Influences Nests: Our Hypothesis

• Mice in IVCs will build more complex nests at the front of the cage, furthest from air intake


Why do we care?• Potential confounding effect in research

• Husbandry

• Determine comfort level in IVCs

• Baseline for behavioral, neurologic studies– Recognize deviations from normal behavior


The Nest Study


The Nest Study1. Comparison of IVC and static housing

– BALB/c, C57BL/6 mice

– 14 cages (n= 56)

– Male, Female

2. Survey of IVC housed mice in BRT– 1769 cages (n = ~8500)

– Male, Female, Breeding

4


Compare IVC and static housing

IVCs Static Caging

ACH= 34 avg (20-40 range)

ACH= 16-19 ACH (Reeb et al. 1997)


Data Collection

• Nest complexity, location

• Enrichment Type

• Temperature

• Air changes per hour (ACH)


Data Collection


Timelines


MaterialsEnviro-Gard™ Cage Monitor Control Unit

iPad

Books on Tape

Thermometers

Nestlets (5 cm square)


Enrichment Options

5


Nest Location

• Cage dimensions 29.8 cm x 15.6 cm x 14.1 cm

B M F


Nest Scoring

• 0= untouched

• 1= dispersed

• 2= flat

• 3= cup

• 4= half dome

• 5= full dome

• IE= In Enrichment

Naturalistic nest scoring system from Hess et al., 2008


0 = untouched


1 = dispersed


2 = flat, gathered


3=cup

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4= ½ dome


5= full dome


IE = In Enrichment


Results


BRT Survey Results

0

200

400

600

800

1000

1200

Back Middle FrontLocation in Cage (10 cm lengths)

Mice Nest Location in IVCs

Nu

mb

er o

f M

ice

Cag

es

The majority of mice cages surveyed in the BRT were located in the back third of the individually ventilated cage. B M F


BRT Survey Results

0

200

400

600

800

0 1 2 3 4 5Nu

mb

er o

f M

ice

Cag

es

Nest Score

Mice Nest Scores in IVCs

The majority of cages surveyed had nest scores of 2 (flat nests) or greater.

54 3 2

What about single housed mice?

7


BRT Survey Results

0

10

20

30

40

50

60

Nest Scores for Male, Female, Breeding Mice

Nest Score0 1 2 3 4 5 IE%

of

Tota

l M

ales

/Fem

ales

/Bre

eder

s

The majority of nests in all 3 groups (males, females, or breeders) had a score of 2 (flat nests).

0 1 2 3 4 5


BRT Survey Results

0

50

100

150

200

250

Nu

mb

er o

f C

ages

Enrichment Devices in BRT

Nu

mb

er o

f C

ages

Bottles were the most common enrichment device found in the BRT.


BRT Survey Results

10%

90%

Cages with Enrichment

In Enrichment Total Enrichment

~10% of cages with enrichment had nests inside the enrichment


IVC vs Static Results

0

2

4

6

8

10

# o

f C

ages

Back Middle Front

• IVC nests: most in back third of cage.

• Static nests: most in middle third.

B M F


IVC vs Static Results

Mean nest scores were significantly higher for IVCs than static

Mea

n N

est

Sco

re

54 3 2

*


Discussion

• Better to use cage wires with less of a slope?– Mice in IVCs build nests in back of cage

– More room for enrichment, higher nest

8


Discussion

• Provide alternative nesting material?– Average nest score all mice in BRT: 2.34

• Need baseline nesting data for different mice– Lots of variables!


Ways to Improve Study

• Allow mice to acclimate to static cages

• Nestlets in center of all cages

• Score all nests at same time: start of light phase next morning (6 am)

• Pictures of nests then blind scoring by 2 scorers

• Limit mouse disturbances


Future Studies

• Vary time of nest scoring

• Temperature monitoring

• Vary amount/type of nesting material

• Change feed hoppers

• Determine lighting effects


Challenges

Laboratory Animal Care and Use Program and Facilities Laboratory Animal Care and Use Program and Facilities

Questions?

9


Special Thanks!

• Stephanie Lewis*

• Judy Hickman-Davis

• Tara Martin*

• Matt Hogan

• Candace Hendrick*

• Pete McKinley

• Charlie Martin

• Mike Rowley*My study team


References1. Baumans V, Schlingmann F, Vonck M, Van Lith HA. 2002. Individually ventilated cages: beneficial for mice and men? Contemp Top Lab Anim Science 41: 13 - 19.

2. Gaskill BN, Gordon CJ, Pajor EA, Lucas JR, Davis JK. 2012. Heat or insulation: behavioral titration of mouse preference for warmth or access to a nest. PLoS ONE 7: e32799.

3. Gaskill BN, Gordon CJ, Pajor EA, Lucas JR, Davis JK, Garner JP. 2013. Impact of nesting material on mouse body temperature and physiology. Physiology & Behavior 110 - 111: 87 - 95.

4. Hess SE, Rohr S, Dufour BD, Gaskill BN, Pajor EA, Garner JP. 2008. Home improvement: C57BL/6J mice given more naturalistic nesting materials build better nests. Journal of the Amer Assoc for Lab Anim Sci 47: 25 - 31.

5. Jirkof P, Fleischmann T, Cesarovic N, Rettich A, Vogel J, Arras M. 2013. Assessment of postsurgical distress and pain in laboratory mice by nest complexity scoring. Lab Anim 47: 153 - 161.

6. Kokolus KM, Capitano ML, Lee CT, Eng JWL, Waight JD, Hylander BL, Sexton S, Hong CC, Gordon CJ, Abrams SI, Repasky EA. 2013. Baseline tumor growth and immune control in laboratory mice are significantly influenced by subthermoneutral housing temperature. Proc of National Academy of Sciences, doi: 10.1073/pnas.1304291110.

7. Lynch CB. 1980. Response to divergent selection for nesting behavior in Mus musculus. Genetics 96: 757 - 765.

2014 osu aslap summer student€¦ · “the open-field test a critical review”. psychological...

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