Warsaw Summer School 2015, OSU Study Abroad Program Regression.
2014 OSU ASLAP Summer Student€¦ · “The open-field test A critical review”. Psychological...
Transcript of 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
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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
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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
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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
Laboratory Animal Care and Use Program and Facilities
Agenda
• Background
• Materials & Methods
• Results
• Discussion
• Future Directions
Laboratory Animal Care and Use Program and Facilities
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
Laboratory Animal Care and Use Program and Facilities
Why do Mice Build Nests?
Laboratory Animal Care and Use Program and Facilities
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
Laboratory Animal Care and Use Program and Facilities
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
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Laboratory Animal Care and Use Program and Facilities
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)
Laboratory Animal Care and Use Program and Facilities
Temperature Influences Nests
Fig 2. Effects of different factors on thermoneutral zone (Gordon 1998)
Higher quality nests minimize
heat loss
Laboratory Animal Care and Use Program and Facilities
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
Laboratory Animal Care and Use Program and Facilities
Housing Influences Nests
• IVCs: air flow enters cage through port, exits through filter lid– We control air changes per hour
Laboratory Animal Care and Use Program and Facilities
Housing Influences NestsIVC Benefits
• stocking density
• contamination
• cage ∆ freq
• humidity,CO2,NH3
• Protection
• Sterile
IVC Cons
stress?
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Laboratory Animal Care and Use Program and Facilities
Housing Influences Nests
• Nests counteract negative effects of high intracage ventilation rates (Baumans 2001)
• Less stress at lower rates (30 vs 60 ACH)
Laboratory Animal Care and Use Program and Facilities
Housing Influences Nests
• 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)
Laboratory Animal Care and Use Program and Facilities
Housing Influences Nests: Our Hypothesis
• Mice in IVCs will build more complex nests at the front of the cage, furthest from air intake
Laboratory Animal Care and Use Program and Facilities
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
Laboratory Animal Care and Use Program and Facilities
The Nest Study
Laboratory Animal Care and Use Program and Facilities
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
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Laboratory Animal Care and Use Program and Facilities
Compare IVC and static housing
IVCs Static Caging
ACH= 34 avg (20-40 range)
ACH= 16-19 ACH (Reeb et al. 1997)
Laboratory Animal Care and Use Program and Facilities
Data Collection
• Nest complexity, location
• Enrichment Type
• Temperature
• Air changes per hour (ACH)
Laboratory Animal Care and Use Program and Facilities
Data Collection
Laboratory Animal Care and Use Program and Facilities
Timelines
Laboratory Animal Care and Use Program and Facilities
MaterialsEnviro-Gard™ Cage Monitor Control Unit
iPad
Books on Tape
Thermometers
Nestlets (5 cm square)
Laboratory Animal Care and Use Program and Facilities
Enrichment Options
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Laboratory Animal Care and Use Program and Facilities
Nest Location
• Cage dimensions 29.8 cm x 15.6 cm x 14.1 cm
B M F
Laboratory Animal Care and Use Program and Facilities
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
Laboratory Animal Care and Use Program and Facilities
0 = untouched
Laboratory Animal Care and Use Program and Facilities
1 = dispersed
Laboratory Animal Care and Use Program and Facilities
2 = flat, gathered
Laboratory Animal Care and Use Program and Facilities
3=cup
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Laboratory Animal Care and Use Program and Facilities
4= ½ dome
Laboratory Animal Care and Use Program and Facilities
5= full dome
Laboratory Animal Care and Use Program and Facilities
IE = In Enrichment
Laboratory Animal Care and Use Program and Facilities
Results
Laboratory Animal Care and Use Program and Facilities
BRT Survey Results
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Mice Nest Location in IVCs
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ice
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The majority of mice cages surveyed in the BRT were located in the back third of the individually ventilated cage. B M F
Laboratory Animal Care and Use Program and Facilities
BRT Survey Results
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ice
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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?
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Laboratory Animal Care and Use Program and Facilities
BRT Survey Results
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Nest Scores for Male, Female, Breeding Mice
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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
Laboratory Animal Care and Use Program and Facilities
BRT Survey Results
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Enrichment Devices in BRT
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Bottles were the most common enrichment device found in the BRT.
Laboratory Animal Care and Use Program and Facilities
BRT Survey Results
10%
90%
Cages with Enrichment
In Enrichment Total Enrichment
~10% of cages with enrichment had nests inside the enrichment
Laboratory Animal Care and Use Program and Facilities
IVC vs Static Results
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• IVC nests: most in back third of cage.
• Static nests: most in middle third.
B M F
Laboratory Animal Care and Use Program and Facilities
IVC vs Static Results
Mean nest scores were significantly higher for IVCs than static
Mea
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Laboratory Animal Care and Use Program and Facilities
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
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Laboratory Animal Care and Use Program and Facilities
Discussion
• Provide alternative nesting material?– Average nest score all mice in BRT: 2.34
• Need baseline nesting data for different mice– Lots of variables!
Laboratory Animal Care and Use Program and Facilities
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
Laboratory Animal Care and Use Program and Facilities
Future Studies
• Vary time of nest scoring
• Temperature monitoring
• Vary amount/type of nesting material
• Change feed hoppers
• Determine lighting effects
Laboratory Animal Care and Use Program and Facilities
Challenges
Laboratory Animal Care and Use Program and Facilities Laboratory Animal Care and Use Program and Facilities
Questions?
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Laboratory Animal Care and Use Program and Facilities
Special Thanks!
• Stephanie Lewis*
• Judy Hickman-Davis
• Tara Martin*
• Matt Hogan
• Candace Hendrick*
• Pete McKinley
• Charlie Martin
• Mike Rowley*My study team
Laboratory Animal Care and Use Program and Facilities
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.