Download Comparative Medicine - Laboratory Animal Boards Study Group

Comparative Medicine
Volume 55, Issue 4 (August 2005)
NIH Swiss and black Swiss mice have retinal degeneration and performance deficits in
cognitive tests (pages 310-316)
Summary: The authors tested two strains of Swiss mice after noticing poor performance in cognitive
tests. They postulated that poor vision may have been the cause of the reduced activity in tests.
Additional cognitive testing as well as PCR analysis of genotype and histopathologic examination of
the eyes revealed that the mice are homozygous recessive for the 1 mutation on the Pde6b gene and
that they have retinal degeneration after about 9 weeks of age. The mice did not perform in sight
based Morris water maze cognitive tests as well as sighted control animals. The author noted that the
mice also did not perform as well in a contextual fear test that should not have had a large sightdependent component. They speculated that this may be a genetic problem unrelated to vision and
recommended that the mice are not good subjects for fear conditioning experiments. The paper is a
good one for boards review of the Morris water maze test and histopathology of the layers of the
retina.
Questions:
1 Are Swiss mice inbred or outbred strains. How do NIH Swiss differ from Black Swiss?
2 What does the Morris water maze test evaluate?
3 What does a contexual fear conditioning test evaluate?
Answers
1 Outbred. NIH Swiss are an albino strain, derived from the Black Swiss strain.
2 The Morris water maze is a spatial navigation test that tests an animals ability to recognize and
remember the position of a submerged platform based on experience using visual cues to locate the
platform while swimming in the maze tanks. Mice should be able to use visual cues to more quickly
locate the platform after several practice sessions.
3 The contextual fear conditioning test exposes an animal to a noxious stimuli (usually shock). This
stimuli is paired with multiple cues which may be visual, olfactory, tactile or auditory. The test
measures emotional learning based on the animal ability to associate an anticipated noxious stimulus
based on past cues from the environment.
Use and limitation of magnetic resonance phase-contrast assessment of coronary flow
reserve in a model of collateral dependence (pages 317-325)
Phase Contrast-Magnetic Resonance Imaging (PC-MRI) is utilized in assessing coronary artery flow
reserves (CFR) in man and animal models with intermediate coronary lesions. This minimal to noninvasive fast acquisition cardiac MRI is proven beneficial for cardiac function and structural
assessment in man. CFR is defined as the ratio of maximal hyperemic to basal flow. There are two
approaches of assessment of CFR in patients with coronary disease. The first is to measure
coronary artery flow distal to the stenosis in epicardial vessels and the second is to measure the
regional tissue perfusion. Microsphere techniques remain the standard for the assessment of tissue
blood flow while the PET (positron emission tomography) is the clinical standard for evaluating
myocardialperfusion ( and perfusion reserve). Assessment of CFR regardless of approach must take
in consideration hemodynamic factors, hypertophy, small vessel disease, and collaterial
dependence. Phase-contrast MRT offers an alternative to invasive procedures in assessing epicardial
flows in med-sized to large coronary arteries. In this study the use of PC-MRI for assessing CFR in
the setting of progressive coronary constriction was examined in an established model of collateral
dependence. Epicardial coronary artery PC-MRI CFR was compared with mycocardial perfusion
reserves, angiographic flows scores, histopathological changes and visual assessments of
myocardial perfusion during a course of constriction of the proximal left circumflex (LCX) coronary
artery over a 6 week period. A comparison between epicardial arterial by PC-MRI and invasive
myocardial perfusion reserve assessed by microsphere technique was made. In thirteen Yorkshire
swine (Sus scrofa) a titanium-shelled ameroid constrictor on the LCX. PC-MRI measured baseline
and hyperemic CFR in LAD and LCX coronary artery just distal to the constriction (at time of
implantation, 3-4 week post-implantation (PI) and 6 weeks PI. Hypermia was induced by the infusion
of adenosine (140mg/kg/min). As with other approaches the accuracy of the PC-MRI depends on a
number of factors including optimal image plane acquisition, sufficient target vessel size, and diastolic
sampling rates to estimate mean flow coronary flow velocity. Histological, angiographic and
postmortem assessments of occlusion at the ameroid were consistent with collateral dependence.
Although PC-MRI offers a less invasive alternative in assessing CRF, caution in interpretation was be
used when using this approach in a model of progressive coronary constriction. The study found a
discrepancy between PC-MRI epicardial CFR and mycocardial perfusion reserves under conditions in
which collaterial flow dominates tissue perfusion.
Questions:
1). Coronary Flow Reserve is defined as a ratio of maximum hyperemic flow/basal Flow. True or
False?
2). Collaterial Dependence is a factor that should be taken into consideration when interpretating
assessment of CFR regardless of the approach used. True or False?
3). Name the drug used to produce maximum hyperemia in this study.
4). Name the genus and species of the animal model used in this study.
Answers:
1) True.
2). True.
3). Adenosine 140mg/kg/min by infusion.
4). Sus scrofa
Behavioral differences among fourteen inbred mouse strains commonly used as disease
models (pages 326-334)
The behavioral traits of 14 inbred strains of mice were collected and analyzed (129P3/J,
129S1SvImJ, 129S6/SvEvTac, 129T2SvEmsJ, 129X1SvJ, C56BL/6J, C57BL/6Tac, DBA/2NTac,
FVB/NTac, NOD/MrkTac, SJL/JCrNTac, AND B6129S6F1Tac) Each strain was tested in three
behavioral tests (rotorod, open-field activity-habituation, and contextual and cued fear conditioning).
Each test showed particular stain differences, in particular on the rotorod test SJL/JCrNTac mice had
the shortest latencies to fall on the first day and DBA/2NTac mice showed impaired motor learning.
In the open field activity, 129T2/EvEmsJ and A/CCrTac were least active and NOD/MrKTac were
most active. When comparing the C56BL/6J and C57BL/6Tac there were no differences except for a
higher open-field activity in C56BL/6J female mice. These differences play an important role in strain
selection in all research areas.
Questions
1) What is the chromosome number of the mouse?
2) What does contextual and cued fear conditioning test?
3) T or F There were no differences between the strains of mice on their behavioral assessments?
Answers
1) 2n = 40
2) Learning and memory.
3) F
Canine transmissible venereal tumor: a large-animal transplantable tumor model (pages 335343)
Summary: The canine transmissible tumor (cTVT) is a naturally occurring transplantable round cell
tumor that typically is propagated and maintained in the dog population by coitus and such behaviors
as social smelling and licking of the external genitalia. The cTVT is an allograft: it is referred to as
Sticker’s sarcoma in Europe, and in some references, as transplantable histocytoma. The origin of
cTVT is still in question. Worldwide the cTVT cells typically have 58-59 chromosomes compared to
the normal canine number of 78. Tumors typically begin as nodules in the submucosa or subcutis
located on the external genitalia however, they have also been found in the skin, lips, nasal and
buccal mucosa. Tumors characteristically regress spontaneously in 4 to 6 months, but extension into
adjacent tissues has been reported. Metastasis is rare. The authors state that although rodent and
rabbit experimental transplantable tumor models are available, a reliable transplantable tumor in a
large animal that resembles the physical dimensions of humans is not available.
Purpose: This study was designed to characterize the experimental use and gross and
histopathologic characteristics of cTVT in the canine brain, skin, muscle, prostate, lung and, bone.
Research techniques used to characterize the tumors included the following;
X-ray computed tomography (CT), magnetic resonance (MR), and histopathology.
Methods: Sixty –four clinically normal, mongrel hound dogs (Canis familiaris) were used in this study.
Dogs were immunosuppressed with cyclosporine, 10 mg/kg, PO. Dogs were dosed twice daily for 14
days starting 4 to 10 days prior to tumor inoculation and once a day dosing until end of study. Freshly
harvested cTVT tumor was minced into 1 to 4-mm tumor fragments, prepped (see methods section
pp. 335-337), and inoculated into selective tissue and organ sites. At the end of study, dogs were
humanely euthanized. The authors also used a Severe Combined Immune Deficient (SCID) mouse
model of cTVT in comparison to the canine animal model (pp. 336-337).
Results: The tumor allograft in dogs grew reliably in 89% of the inoculated tumor fragments producing
viable tumors. Developing tumors in all tissues formed well-circumscribed, un-encapsulated, firm,
expansile, white, homogenous nodules (Note figures 1 through 3). Note histological interpretation of
canine cTVT in figures 4 and 5. The time to clinical establishment of the tumors were tissue and
organ dependant with palpable tumors in the subcutaneous, mammary, and muscle sites noticeable
in 3 to 4 weeks. Tumor in the lugs could be detected by CT in 5 weeks post inoculation, while those
in the brain, prostate, and liver could be detected in 7 weeks. The canine cTVT animal model was
expensive due to 1), cost of dogs and their maintenance, and 2), cost of immunosuppressive therapy
with cyclosporine. The mouse model of cTVT was cost effective and resulted in a 100% tumor
transplantation success if inoculated either in the subcutaneous tissue or the mammary fat pad.
Review gross Figure 6, MR Figures 7 through 9, and CT Figure 10 and 11.
Conclusion: cTVT animal model is reliable (dogs 89% and mice 100% transplantation success).
Canine model is very expensive. Mouse animal model is cost effective. Non-invasive imaging
protocols (MR and CT) are valuable tools that are continued to be developed.
Questions
1.
Define the acronym cTVT
2.
Define the acronym CT
3.
Define the acronym MR
4.
T or F: Cyclosporine is an immunosuppressive agent.
5.
T or F: The canine cTVT model is cost effective.
Answers
1.
canine transmissible tumor
2.
X-ray computed tomographic
3.
MR-magnetic resonance imaging
4.
T
5.
F
Duration of effects on clinical parameters and referred hyperalgesia in rats after abdominal
surgery and multiple doses of analgesic (pages 344-353)
The authors sought to determine the efficacy and duration of analgesia effected by twice daily
intramuscular administration of low and high dose buprenorphine, (0.01 mg/kg and 0.1 mg/kg), and
ketoprofen, (3 mg/kg), in male Sprague Dawley rats after abdominal surgery, (exposure,
exteriorization, and manipulation or retraction of abdominal viscera). Clinical parameters assessed
include food and water consumption and body weight change. Referred hyperalgesia was
hypothesized to be present following abdominal surgery, and the tail flick assay was performed to
assess the effect of analgesia on this phenomenon. No-surgery and no-analgesic controls were
included in the analysis. There were significant decreases in food consumption and body weight in
no-analgesic controls for the first 24 hours following surgery when compared with no-surgery
controls. Reductions in these effects with analgesia were not statistically significant, although
nonsignificant improvements were noted. Referred hyperalgesia, as measured with the tail flick
assay, did not appear to be present following this abdominal surgery; increased tail flick latency was
noted 4 hours following high dose buprenorphine administration. A number of additional effects were
seen in the buprenorphine-treated groups, (that were not noted following ketoprofen administration),
including increased water consumption, increased activity, decreased food consumption, pica,
tendency to bite the handler, staple removal, and red-tinged periocular discharge.
Questions:
1. What are two potential advantages of using a tonic analgesiometric assay, (e.g. formalin or
writhing test), vs. one using a brief phasic stimulus, (e.g. tail flick test), to determine the duration of
action of analgesics?
2. Define primary, secondary, and referred hyperalgesia, and allodynia.
3. Several studies, including this one, have concluded that the majority of painful effects leading to
decreased food consumption and body weight change in animals occur within the first _________
hours after abdominal surgery
a. 12 b. 24 c. 36 d. 48 e. 72
Answers:
1. Tonic assays detect analgesic effects at lower drug dosages, and stimulate a broader range of
central nervous system mechanisms and structures; tests using brief phasic stimuli only assess the
spinal reflex arc.
2. Decreased pain threshold:
-at the surgical site if the stimulus is normally painful=primary hyperalgesia
-at sites adjacent to the surgical site=secondary hyperalgesia
-at sites distant from the surgical site=referred hyperalgesia
-at the surgical site if stimulus not normally painful=allodynia
3. b. 24
Urolithiasis in rats consuming a fl bitartrate form of choline in a purified diet (pages 354-367)
Animal models of urolithiasis have focused on calcium, oxalate, phosphate, magnesium, ammonia,
urinary pH and citrate as contributors to urolith formation. This paper describes circumstances in
which purified rodent diets containing the /dl/ (racemic) bitartrate isomer to stabilize choline was
implicated in urolithiasis development in rats subjected to a long-term study of the effects of mercury
on growth and development. Long-Evans rats were used for this study, however, similar increased
instances of urolithiasis were reported in other colonies of Sprague-Dawley, Long-Evans, and Wistar
rats fed similar AIN-93 purified diets over a similar time period in 2001-2002. Animals on cholinedeficient diets or diets containing the /l/ tartaric acid isomer of bitartaric acid used to stabilize choline
were uneffected. Clinical signs consisted of hematuria, red-stained bedding, red-stained or wet
pelage, sensitivity to touch in the abdominal area, swollen or palpable kidney or bladder, unkempt fur,
anorexia, reduced urination, reduced water intake and unexpected weight loss or gain. Uroliths were
present on necropsy. Other pathologic findings included: lower urinary tract obstruction resulting in
distension of the urinary bladder and ureters, hydronephrosis, pyelonephritis and hemorrhagic
cystitis. All affected animals consumed the suspect diet for at least 1 month prior to development of
symptoms. Males were more frequently effected than females (64% vs 22%). Chronic
methylmercury exposure increased the risk of urolithiasis.
QUESTIONS:
1) Calcium to Phosphorus (Ca:P) ratios are critical in the diet and an imbalance can cause renal
calcium in the kidney to precipitate into uroliths. What ratio of Ca:P is currently used in the AIN-93
synthetic rodent diet to minimize this problem?
A) 0.75
B) 1:1
C) 1.3:1
D) 2:1
2) Urolithiasis in this report was associated with which of the following ingredients in a purified rodent
diet:
A) Choline cloride
B) Racemic (/dl/) bitartaric acid
C) Neutral levo (/l/) tartaric acid
3) True/False Male animals are more predisposed than females to urolithiasis.
ANSWERS:
1) C
2) B
3) True
Effects of housing density and cage floor space on three strains of young adult inbred mice
(pages 368-376)
These studies compared parameters of the microenvironment associated with housing, using floor
space recommended by the Guide to those at various higher cage densities. The effects of varying
amounts of floor space and cage types on male and female BALB/cJ, NOD/LtJ, and FVB/NJ strains
of mice were assessed over an 8-week period. Three week old mice of the first two strains and 3-5
week mice of the latter strain were used in these studies. Housing densities from 5.6 to 13.5 in2 (The
Guide recommends 5 in cage height and floor space 6 in2 for mice <10 g and 15 in2 for mice > 25 g
for mice) were used for the mice in duplex, weaning, and shoebox polycarbonate cages on positively
ventilated racks. The following parameters were evaluated: survival, aggressive behavior or injuries,
body weight, food and water consumption, cage microenvironment (ammonia and carbon dioxide
levels, temperature, and relative humidity), and urinary testosterone concentrations. Data generated
in these studies were compared to previously reported studies using C57BL/6J (Comp. Med. 54:656663, 2004). The mortality rates affecting BALB and NOD mice were minimal at all housing densities
(0.2%). Levels of urinary testosterone decreased with increasing density in samples from BALB mice.
The variation of the housing densities in all cage types did not affect most of the remaining
parameters studied. However, fighting was noted in male FVB mice (known as aggressive mice)
housed in duplex and weaning cages within the first 7 to 10 days of the study. Several had to be
euthanized for humane reasons. Therefore the FVB male mice data was shown for shoebox cages
only. The health and well-being of the mice in the 8-week study were not affected by the cage type
and housing density, with the notable exception of FVB male mice. Therefore, housing at higher
densities than recommended in the Guide was not deleterious.
Questions:
1) Which are the height and floor space parameters recommended by the Guide for mice >25 g?
a. 6 in and 10 in2
b. 5 in and 10 in2
c. 6 in and 15 in2
d. 5 in and 15 in2
2) Which inbred strain of male mice are the most aggressive?
a. BALB/cJ
b. NOD/LtJ
c. FVB/NJ
d. C57BL/6J
3) Which inbred strain of male mice secretes less urinary testosterone with increasing cage density?
a. BALB/cJ
b. NOD/LtJ
c. FVB/NJ
d. C57BL/6J
4) Which inbred strain mice is model used to study diabetes mellitus?
a. BALB/cJ
b. NOD/LtJ
c. FVB/NJ
d. C57BL/6J
Answers
1. d
2. c
3. a
4. b
Toxic epidermal necrolysis in two rhesus macaques (Macaca mulatta) after administration of
rituximab (pages 377-381)
This is a case report describing two rhesus macaques developing toxic epidermal necrolysis after
administration of rituximab. Rituximab is a human-murine chimeric monoclonal antibody directed
against the CD20 antigen on B lymphocytes, and is used to treat certain B cell neoplasias. The
macaques were part of a gene therapy study involving the use of an adeno-associated viral vector
encoding human factor IX (hFIX). Because the animals developed antibody against hFIX, rituximab
was administered to deplete the population of B cells producing anti-hFIX antibodies. Presenting
clinical sign in both animals was multifocal erythemic skin lesions that rapidly progressed in severity,
resulting in epidermal sloughing and ulceration. The lesions on the first animal were unresponsive to
aggressive analgesics, antibiotics, and corticosteroid treatments; animal was euthanized 5 days after
administration of rituximab. The second animal did recover with chronic aggressive supportive
treatment, which included oral and topical empirical antibiotics, prednisone, and furosamide when
subcutaneous edema and mild lung crackles were noted.
Toxic epidermal necrolysis is a severe life-threatening condition closely related to erythema
multiforme and Stevens-Johnson syndrome. It usually associated with an adverse drug reaction (i.e.
allopurinol, anticonvulsants, anti-inflammatory drugs, antimalarials, and sulfonamides), or less
commonly, to infection. Diffuse erythematous lesions with bullous necrosis of the epidermis
characterize the disease condition. Ulceration and secondary pyoderma are often present. Lesions
are usually apparent on haired skin, mucocutaneous junctions, and mucous membranes, and a
positive Nikolsky sign (epidermal detachment) is usually present. Histopathology shows full-thickness
coagulative necrosis of epidermis, sometimes involving hair follicles. Subepidermal vesiculation as
result of separation of necrotic dermis at dermoepidermal junction is common, with minimal dermal
inflammation.
In any case where mucosal or skin lesions or both are noted in an animal after drug
administration, toxic epidermal necrolysis should be placed on the differential diagnosis list. Please
also review the four colored figures In this article as they provide good visualization of gross and
histopathology lesions of toxic epidermal necrolysis.
Questions:
1.
Rituximab is:
a) a protein subunit involved in molecular transport across epidermis and dermis
b) an exfoliatin toxin produced by staphylococcal organisms
c) a chimeric monoclonal antibody used to treat non-Hodgkins lymphomas
d) an organic solvent that is commonly found in cleaning agents
e) a broad spectrum antibiotic drug
2.
The most common cause of toxic epidermal necrolysis is:
a) a history of herpesvirus infection
b) sensitivity to necrolytic toxin produced by pathogenic staphylococcal species
c) a response to previous drug administration
d) prolonged or repeated contact with organic caustic chemicals
e) accidental ingestion of methyl mercury
Answers:
1.
C
2.
C
Frequent Harderian gland adenocarcinomas in inbred white-footed mice (Peromyscus
leucopus) (pages 382-386)
This report documents the frequent occurrence of Harderian adenocarcinomas in two inbred P.
leucopus strains. P. leucopus are notoriously difficult to inbreed. There was no sex predilection in
ocular tumor occurrence. Benign lesions were not observed in this study. Tumors were identified as
glandular in origin. There are good gross and histo. pictures presented in the article.
Strain GS109A had the highest prevalence of tumors at 22.7%. Random bred P. leucopus
had a prevalence of only 0.07% for ocular tumors.
Inbreeding results in homozygosity and fixed loci at 98.7% by the 20th generation. By the 30th
generation 99.8% of all loci are fixed. Chromosomal variation among peromyscine species
predominately involves pericentric inversions. This contrasts with Mus in which centric (Robertsonian)
fusions are the most common karyotypic variation among species.
Questions:
1. What is the genus and species name for the White footed mouse?
2. After 20 generations of inbreeding loci are fixed at what percent?
a.
99.8%
b.
98.9%
c.
97.8%
d.
98.7%
3. Peromyscine species have chromosomal variation that predominately involves _______
inversions?
a.
pericentric
b.
centric
c.
distal
d.
Robertsonian
Answers:
1. Peromyscus leucopus
2. d
3. a
The efficacy of diazepam treatment for the management of acute wounding episodes in
captive rhesus macaques (pages 387-392)
The spontaneous development of self-injurious behavior (SIB) in singly housed monkeys poses an
ongoing challenge for their management and well-being in captivity. Existing treatment strategies for
this disorder are limited, which in more severe cases necessitates the removal of the subject from
active study protocols or even euthanasia. Although varying degrees of success in reducing SIB in
macaques have been reported by socialization or treatment with pharmacological agents such as
serotonin precursor L-tryptophan, the antiandrogen cyproterone acetate, or the adrenergic 2 receptor
agonist guanfacine, the efficacy of these treatments have not yet been explored fully.
This study examined treatment effects of diazepam (Valium) on the incidence of self-wounding and
self-biting in 8 individually housed adult male rhesus monkeys. The authors also examined the effect
of diazepam treatment on aggression and stereotypic behavior. A within-subject design was used, in
which the incidence of self-wounding and self-biting in the presence of anxiolytic doses of diazepam
was compared to the incidence of these behaviors during drug-free periods. Diazepam treatment
was instituted within 24 h of a reported self-wounding episode. Because the response to treatment
appeared to be dichotomous, the data were analyzed differentiating "positive responders" (PR
monkeys) from "negative responders" (NR monkeys).
Diazepam treatment did not significantly alter the mean number of self-wounding episodes observed
per week when the results were analyzed across all subjects. Whereas 4 of the 8 monkeys
responded with a significant reduction in self-wounding (PR group), the remaining subjects exhibited
a trend towards and increase in self-wounding (NR group). Treatment with diazepam did not
significantly alter the the frequency of self-directed biting when examined across all subjects.
However, PR but no NR monkeys showed significant reduction in the frequency of self-directed
biting. Diazepam treatment significantly reduced the rates of externally directed aggression, but did
not significantly alter the frequency of either whole-body or fine motor stereotypies when examined
across subjects or within individual treatment response groups. PR monkeys were significantly less
likely to have been assessed as self-biters compared with NR monkeys.
4 possible explanations for differential response between PR and NR groups:
1. the 2 groups suffer from distinct types of anxiety-related disorders
2. the groups suffer from the same type of anxiety-related disorder but were at different stages in the
development of the disorder at the time of treatment
3. the groups differ in their threshold for responding to drug treatment
4. self-injurious behavior is not elicited by anxiety in the NR group.
The finding of this study implies the existence of at least 2 subtypes of monkeys with SIB that differ in
their responsiveness to benzodiazepine treatment.
QUESTIONS:
1. Diazepam:
a. is a phenothiazine tranquilizer
b. is a benzodiazepine anxiolytic
c. is soluble in water
d. causes hyperthermia in monkeys
2. Regarding self-injurious behavior (SIB) in macaques:
a. It is more common in older males than younger males.
b. It can be easily cured by simple interventions such as environmental enrichment and relocation
to larger cages.
c. Reports suggest that 5-14% of individually housed macaques in biomedical research facilities
spontaneously engage in self-inflicted wounding at some point in their life.
d. It can be treated with pharmaclolgical agents such as the serotonin precursor L-tryptophan, the
antiandrogen cyproterone acetate, or the adrenergic 2 receptor agonist guanfacine.
Answers:
1. b
2. c