Journal of the American Association for Laboratory Animal Science
Volume 51, Number 5, September 2012
ORIGINAL RESEARCH
Husbandry
Hurst and Litwak. Accelerative Forces Associated with Routine Inhouse Transportation
of Rodent Cages, pp. 544-547
Domain 3
SUMMARY: Transportation of rodents for even small periods of time can affect research
endpoints. This study used a rodent sized 24 g accelerometer inside a mouse cage to measure
accelerative and decelerative forces associated with cage transport on a plastic cart, small or
large metal cart, or by hand, and the effect of placing either a towel or pad between the cage
and the cart. Surprisingly, the plastic cart was associated with highest acceleration forces, and
hand carrying one of the lowest (n.b. the carrying was done with special attention to not moving
the cage in the x-y axis at all, which is not standard for hand carrying). The best method for
ameliorating the accelerative forces was the use of the towel between the cage and the cart.
The route used to measure acceleration in the cage included changes in flooring type, starting
and stopping, and going over the threshold of an elevator. The threshold bump produced the
greatest peaks in acceleration in all cart types. The authors also discovered that housing
methods did not expose the cage to acceleration, but low levels of vibration, and that motion of
the cage correlated with cart type – large metal cart having the lowest acceleration of the cart
types, perhaps due to hollow tube struts and spot welded shelves, that might decrease vibration
transmitted to the cage.
QUESTIONS
1. What obstacle produced greatest accelerative forces within the cage on all cart types?
a. Changing floor types
b. Picking up the cage to put it on the cart
c. Entering an elevator
d. Moving the rack the cages were on
2. What was the best method of decreasing accelerative forces?
a. Padding with a towel
b. Padding with a cotton pad
c. Using a metal cart
d. Hand carrying
ANSWERS
1. c
2. a
Costa et al. Handling of Adolescent Rats Improves Learning and Memory and Decreases
Anxiety, pp. 548-554
Domain 3: Research; T3. Design and Conduct Research
Domain 4: Animal Care; T1 K11. Environmental Causes and Effects on Research
Primary Species: Rat (Rattus norvegicus)
SUMMARY: The investigators were interested in examining the effects of adolescent rat’s
adaptation to handling on experimental procedures and experimental outcomes. In particular
they were concerned with combining behavioral responses to anxiety tests with stress hormone
concentrations. Extensive evidence exists to show that handling of neonatal animals results in
changes in anxiety, cognition, and fear responses, altered physiological parameters, and
changes in plasticity of the CNS. Studies exist for adolescent and adult animals that show the
changes in behavioral responses to anxiety-related test but none have combined behavioral
studies with stress hormone concentrations of the animals. In this study, they promote the use
of the Elevated Plus Maze (EPM) for examining behaviors as it allows for the evaluation of not
only anxiety but also cognition behaviors. The rationale given for this is that transfer latency
(movement from open arms to closed arms) can be used as a measure of learning and memory.
In the experiments 24 (n=12 handled and n=12 control) male Sprague-Dawley rats, 60 days old
when the experiments began. Rats were handled for 5 minutes per day by placing the animal on
the experimenters lap or on a tabletop and stroking its neck and back. This continued 5 days
per week for 6 weeks. Control animals were left undisturbed for the same time period. Animals
were evaluated 2 days after the last handling session in the EPM test. For testing anxiety and
fear, the percentage of time spent in the open arms and latency to first open arm entry were
examined. The number of closed arm entries was used as a measure of general motor activity
and the percentage of open arm entries was used as a mixed index of anxiety and general
motor activity. Cognitive measures in the EPM were measured 48 hours later by recording
latency from the enclosed arm which would indicate learning and this was repeated 24 hours
later to measure retention. Blood collection took place one week post- behavioral tests after
decapitation without anesthesia. Catecholamines and corticosterone were analyzed for.
Compared with the control group, handled rats showed increases in the percentage of time
spent in the open arms, the percentage of entries into the open arms, and the number of entries
into the open arms all of which indicate decreased anxiety. General locomotion was not
affected. These animals also showed decreases in the latency of the first open arm entry in the
second round of testing indicating improved learning. This was reflected in the second trial of
testing escape latency and possibly involved improved memory however the evidence for this is
questionable. These findings were consistent with previous studies in variably aged animals.
Plasma norepinephrine was significantly lower in the handled group but no differences were
found for epinephrine or corticosterone. The authors suggest that this could be a reflection of
decreased anxiety due to decreased basal activity of sympathetic nerves.
In their conclusion, the authors offer this study as evidence that the effects of handling are not
restricted to neonatal periods. Handling should be considered a positive experience in
adolescent rats and is valid as a form of environmental enrichment.
QUESTIONS
1. Handling of rats improves has been found to diminish anxiety when implemented:
a. During Neonatal Periods
b. During Adolescence
c. During Adulthood
d. All of the above
2. Elevated Plus Maze can be used in rats for the study alternations of:
a. Anxiety
b. Food motivation
c. Learning
d. Memory
e. All of the above
f. A, C and D
3. Anxiolytic effect in adolescent rats can be produced with:
a.
b.
c.
d.
5-minutes of handling per day, 5 days per week
20-minutes of handling per day, 5 days per week
One hour of handling per day, 5 days per week
20 minutes of handling per day, once per week
ANSWERS
1. d
2. f
3. a
Management
Glueck et al. Exposure of Laboratory Animal Care Workers to Airborne Mouse and Rat
Allergens, pp. 554-560
Domains 5: Regulatory Responsibilities; T3: Provide advice to OHS programs, K5: OHS
Primary Species: Mouse (Mus musculus) and Rat (Rattus norvegicus)
SUMMARY: Urine of rats and mice is the main source of allergenic proteins that can enter the
respiratory tract of laboratory animal care workers. The proteins from urine can become
aerosolized allowing entry into the respiratory tract of workers and this may explain why
Laboratory Animal Allergy (LAA) is a common occupational hazard for workers exposed to
laboratory animals. Little is known about the levels and determinants of these exposures in the
United States. This study investigated differences between activities in animal facilities and
levels of personal exposure to allergen by collecting personal breathing zone dust samples from
caretakers during full workdays. Mice and rat urinary allergens in inhalable dust were quantified
via immunoassay. The activities of the sampled workers were observed, and the methods of
preventing exposure to allergens were recorded. Washing and cleaning cages and the number
of mice handled daily were the most important determinants of personal exposure to mouse
urinary allergen, whereas those with lower exposures were tasks in the animals rooms that did
involve major animal movements. There was a positive relationship between increased allergen
exposure and working with a greater number of rodents. Personal exposures to mouse urinary
allergen were associated with day-to-day variation of tasks rather than characteristics of
workers. Only persons who handled rats were exposed to rat urinary allergen. The current
findings are valuable for establishing exposure levels against which comparisons of
improvement or deterioration of personal exposures can be made.
QUESTIONS
1. Name the protein product of the mouse Mup17 gene that accounts for much of the
allergenic properties of mouse urine.
2. Exposure and sensitization to rodent Mup proteins is a leading cause of what occupational
disease of laboratory animal care workers and scientists?
3. Workers involved in which of the following activities experience the greatest exposure to
rodent allergens? Select all that apply.
a. Daily health checks without opening cages
b. Dry cage cleaning
c. Washing cages
d. Transferring animals between cages during cage change outs
ANSWERS
1. Mus m 1, Ag1, or MA1 – all names for the same gene product
2. Laboratory Animal Allergy (LAA)
3. b & d – tasks associated with the highest rate of exposure were cleaning cages and
handling of animals; washing cages, which is presumably a wet process that entails dust
suppression, was associated with much lower exposures than cleaning
Weigler et al. Risk-Based Immunization Policies and Tuberculosis Screening Practices
for Animal Care and Research Workers in the United States: Survey Results and
Recommendations, pp. 561-572
Domain 5: Regulatory Responsibilities; T3. Provide advice to occupational health and safety
programs
SUMMARY: Occupational health and safety is especially important for laboratory animal and
research workers (ACRW) due to the increased exposure to animals that may carry zoonotic
disease and the use of infectious disease agents in research. As with any place of employment,
the United Sates Occupational Safety and Health Act requires that the employer ensure a safe
working environment without recognizable hazards. A variety of individuals and committees
have responsibility for assessment of risk in laboratory animal facilities including laboratory
directors, principal investigators, Institutional Biosafety Committees, IACUC, biologic safety
professionals, and laboratory animal veterinarians. It had been previously shown that there is a
low rate of zoonotic disease in ACRW and the authors sought to describe the preventive
practices that may have led to this success.
The authors have conducted a web-based survey of all veterinarians working in laboratory
animal facilities in the United States. The survey consisted of 70 logic based questions allowing
multiple answers and was first tested on a small group of OHS professionals (n=5). The majority
of veterinarians worked in academic institutions, pharmaceutical or biotechnology companies.
Most institutions reported using rodents or rabbits with 51.9% using non-human primates. All
institutions maintained an OHS program and more than 90% enrolled husbandry staff,
veterinarians, veterinary technicians and research investigators. The majority also enrolled
maintenance workers, IACUC members, graduate and undergraduate students. Most OHS
programs were located in the same building. OHS consisted of health questionnaires, animal
species exposure assessment and immunizations with annual re-evaluation. Only a few
institutions stored serum. All institutions that housed NHP's had a TB screening program
including purified protein derivative (PPD) intradermal skin tests and symptom questionnaires.
Other modalities included the interferon-gamma release assay (IGRA) or chest radiographs.
Typical immunizations included tetanus, hepatitis B, influenza, rabies and measles. Note that
there are no licensed vaccines available for B virus, Q fever, Chlamydophila, Pasteurella or Orf.
Overall, they found that OHS programs are well-designed and thorough at the institutions of the
respondents representing 65% of those contacted.
QUESTIONS
1. In the United States, what regulation requires that places of employment be free from
recognizable hazards?
2. The IGRA:
a. Detects antibodies to Mycobacterium tuberculosis
b. Tests for response of leukocytes to M. tuberculosis
c. Requires two visits to OHS
d. Can be performed on serum
3. There are major deficiencies in OHS programs in laboratory animal facilities in the USA. T/F
4. According to this survey, the most commonly administered vaccine as part of an OHS
program is:
a. Rabies
b.
c.
d.
e.
Influenza
Tetanus
Hepatitis B
Measles
ANSWERS
1. Occupational Health and Safety Act
2. b
3. F
4. c
Health Surveillance
Weiss et al. Comparison of a Fur Mite PCR Assay and the Tape Test for Initial and
Posttreatment Diagnosis during a Natural Infection, pp. 574-578
Domain 1; Task 3
Primary Species: Mouse (Mus musculus)
SUMMARY: Effectiveness of the tape test and PCR assay were compared in a small population
of mice at the onset of diagnosis (day 1) and during treatment at 6 wk and 12 wk. Mice were
treated by placing cotton balls impregnated with Mitarrest (7.4% permethrin) in the cage once
weekly for 6 weeks. PCR was performed by passing a sterile polyester swab multiple times
against the grain of the fur over the dorsum, head, abdomen, and inguinal region. It was
hypothesized that the PCR assay would be a more sensitive indicator than microscopic tape
impression examination after treatment compared with initial diagnosis.
Results showed that PCR is a reliable diagnostic method during active fur mite infection
(sensitivity 100% before treatment) but false-negative results are possible after treatment.
Therefore, negative PCR results should be interpreted carefully if mites are still suspected, and
a secondary diagnostic method should be considered.
Side Note: Efficacy of treatment was not calculated – several samples remained both PCR and
tape test positive after treatment.
QUESTIONS
1. How many days does it take Myobia musculi to complete a life cycle of fur-bound egg to
motile nymph to reproductively mature adult in mice?
a. 8
b. 16
c. 23
d. 32
2. Identify the mites shown in the picture. How long is its life cycle?
ANSWERS
1. c
2. Myocoptes musculinus – 8-14 days
Anesthesia
Mert and Gunes. Antinociceptive Activities of Lidocaine and the Nav1.8 Blocker A803467
in Diabetic Rats, pp. 579-585
Domain 3: Research; T3. Design and conduct research; K3. animal models
Primary Species: Rat (Rattus norvegicus)
SUMMARY
Background: The streptozocin-induced diabetic rat is a model of chronic pain that shows signs
of hyperalgesia and allodynia and may replicate signs in diabetic humans. Sodium channels
blockade is one of the best-known treatments for relieving diabetes-induced pain. Recent work
has suggested that A803467 (5-[4-chloro-phenyl]- furan-2-carboxylicacid [3, 5-dimethoxy
phenyl]-amide) is a potent and highly selective blocker of Nav1.8 channels.
The authors investigated the antinociceptive effects of A803467 in diabetic rats with painful
neuropathy.
Methods: They systemically (intraperitoneal) or locally (intraplantar) administered A803467 (or
lidocaine, a nonselective sodium channel blocker, as a control) to diabetic rats (Wistar) with
hyperalgesia and allodynia and then measured thermal latencies and mechanical thresholds. To
assess sensory abnormalities that include hyperalgesia to noxious thermal and allodynia to
innocuous mechanical stimuli, authors used thermal plantar test and a dynamic plantar
aesthesiometer, respectively.
Results: With intraperitoneal administration, A803467 led to 6-fold greater reduction of
hyperalgesia and 2-fold greater reduction of allodynia than did lidocaine.
Whereas the antihyperalgesic effects of lidocaine and A803467 were similar after intraplantar
administration, A803467 (1 mg) was at least 2 times more effective as an antiallodynic than was
lidocaine (0.5 mg). These results suggest that compared with lidocaine, systemic or local
blockade of Nav1.8 channels by A803467 may more effectively relieve hyperalgesia and
allodynia in diabetic neuropathy. These findings, together with previous observations, provide
insight into the functions of Nav1.8 channels in the clinical signs of diabetes. These channels
may be appropriate targets for treating the painful clinical signs of diabetes.
QUESTIONS
1. Lidocaine acts on sodium channels to what effect in STZ-treated rats?
a. Specific blockade of sodium channels in renal excretion
b. Non-specific blockade of sodium channels in pain pathways
c. Reversal of effects of STZ on pancreatic islet beta cells
d. Non-specific blockade of renal excretion of sodium ion
2. A recent study demonstrates the effect of A803467 on specific sodium channel blockage for
allodynia and hyperalgesia in what common model of induced disease?
a. Nerve sheath tumors
b. Spinal cord injury
c. Type I diabetes
d. Envenomation
ANSWERS
1. b
2. c
Experimental Use
Hampton et al. Progression of Ulcerative Dermatitis Lesions in C57BL/6Crl Mice and the
Development of a Scoring System for Dermatitis Lesions, pp. 586-593
Domain 1: Diagnose, Treat, and Prevent Disease
Primary Species: Mouse (Mus musculus)
SUMMARY: Ulcerative dermatitis (UD) is a spontaneous disease commonly associated with
aged C57BL/6 mice or mice engineered on the C57BL/6 background. UD is associated with
environmental factors, diet, season, age at weaning, alopecia, sex, immune complex vasculitis,
follicular dysplasia, lesion location, and deficiencies in vitamin A metabolism. UD results in
significant morbidity in laboratory mice. In the early stages UD is characterized by alopecia,
pruritis, erythema and crusting. Progression of the disease can rapidly lead to erosions and
ulcerations. The authors followed 200 mice up to 19 months observing the progression of UD.
This information was used to develop a scoring system and characterize the linear progression
of clinical signs and how they relate to each other. The anticipated progression of clinical signs
was excoriation, single punctate crust, multiple punctate crusts, coalescing crusts, erosion and
ulceration.
Several parameters were scored with a numerical range of 0-3 including:
A. The number of scratching bouts in a 2 minute period
B. The character of the lesion – this includes the presence of absence of crusts, erosions and
ulceration
C. The length of the lesion – the longest diameter of the largest lesion present
D. The regions affected – including head, thorax and everything distal to the last rib. Heavier
weighting was given to the head region
The total score out of 12 was divided by 12 then multiplied by 100 to give a final number as a
percentage. A survival curve was generated to establish a numerical relationship between
clinical signs and survivability of the mouse.
The results suggested a number of interesting findings. For example, the lesion character,
number of scratching bouts, and lesion size were the only parameters to change significantly
over time. Region 2 (pinna, cervical and thoracic region) was affected in 95% of the mice.
Lymphadenopathy could not be detected via external palpation. Scores over 75 had a
significant chance of being euthanized. General observations suggest that the disease has a
sudden onset and a rapid disease progression. Interestingly, pruritis did not occur in the
absence of dermatitis but was clearly a factor in the rapid progression of UD. Alopecia did not
appear to have any predictive value in the onset or progression of the disease. This scoring
system will have applications relevant to monitoring the progression of the disease, quantifying
the therapeutic benefits of treatment and help to predict end-stage disease.
QUESTIONS
1. List predisposing factors for the development of ulcerative dermatitis.
2. T/F. Alopecia has predictive values for monitoring the progression of UD
3. T/F. Pruritis commonly precedes the onset of dermatitis.
4. List the 4 parameters evaluated in this scoring system.
5. What region of the mouse is most commonly affected?
6. T/F. The substrain of the C57BL/6 may affect the clinical presentation of UD
ANSWERS
1. UD is associated with environmental factors, diet, season, age at weaning, alopecia, sex,
immune complex vasculitis, follicular dysplasia, lesion location, and deficiencies in vitamin A
metabolism
2. F
3. F
4. A. The number of scratching bouts in a 2 minute period
B. The character of the lesion – this includes the presence of absence of crusts, erosions
and ulceration
C. The length of the lesion – the longest diameter of the largest lesion present
D. The regions affected – including head, thorax and everything distal to the last rib.
Heavier weighting was given to the head region
5. 2
6. T
Lofgren et al. Castration Eliminates Conspecific Aggression in Group-Housed CD1 Male
Surveillance Mice (Mus musculus), pp. 594-599
Domain 4: Animal Care
Primary Species: Mouse (Mus musculus)
SUMMARY: As a refinement initiative to reduce overall animal use, the male progeny of CD1
mice bred in house for a transgenic core facility, who would otherwise be euthanized due to
their lack of use, were integrated into the surveillance program. Female pups were used as
recipients for embryo transfer, cycled back into the breeding program, or are introduced into the
surveillance program. However, the male surveillance mice had a high prevalence of intracage
aggression, often necessitating separation or euthanasia of affected mice. Numerous factors
reported to influence aggression in adult male mice were reviewed with the following
conclusions:




Stocking density, cage space, and enrichment had been optimized for both animal welfare
and achieving the goals of the study
Cagemate familiarity – littermates designated for the surveillance program were maintained
as a group
Olfactory cues – unavoidable to expose surveillance mice to exogenous olfactory cues from
dirty bedding from foreign mice
Sex hormone influence – study was designed to test the theory that early castration could
eliminate fighting in the male CD1 sentinels
During the first week of monitoring, 50% of the rooms containing male surveillance mice were
reported for fighting (9% of which had to be separated, and 27% required both separation and
euthanasia of mice due to the severity of wounds). Over the following 3 month period, overall
prevalence of fighting in cages housing intact male mice was 64% (one cage required
separation, whereas 4 cages required both separation and euthanasia of mice). In the 3
months after the addition of castrated surveillance male mice, the prevalence of fighting among
castrated mice was 0% (0 of 16 cages). No fighting among female surveillance mice was
reported at any time during the study.
Conclusion: Castration significantly (P < 0.0001) decreased fighting among male mice CD1
mice.
QUESTIONS
1) T/F – Adequate enrichment was provided to the mice in the form of a single shelter or igloo.
2) Which factor is not considered a major contribution to aggressive behavior in male mice?
a. Interaction with unfamiliar male mice
b. Lack of appropriate enrichment
c. Nesting material
d. Stocking density
e. Influence of sex hormones
ANSWERS
1) False – A single shelter or igloo-running wheel has been reported to increase aggressive
behavior in mice, whereas multiple structures can act as visual barriers for subordinate
escape and prevent dominant mice from controlling a single shared resource. Therefore,
multiple enrichment devices were provided.
2) d. – Nesting material is not a known contributing factor to aggressive behavior in male mice
Helwig et al. Effect of Intraperitoneal Radiotelemetry Instrumentation on Voluntary Wheel
Running and Surgical Recovery in Mice, pp. 600-608
Domain 2; Task 1
Primary Species: Mouse (Mus musculus)
SUMMARY: Radiotelemetry devices are frequently used to allow remote monitoring of
physiologic data, such as temperature, heart rate, activity, blood pressure, etc. There is
evidence that using remote monitoring systems in mice may have a negative effect on the
health of mice, depending on the size of the implanted device. Different studies have
demonstrated that mice implanted with telemetry devices intraperitoneally can have reduced
body weights, decreased activity, transient suppression of grooming behaviors, decreased food
and water intake post-operatively. The goal of this study was to compare the effect of two
different sized telemetry implants on body weight, food and water intake, core body
temperature, daily activity, and running wheel activity in the 16 day post-operative period, as
well as characterize any pathologic changes associated with the larger device in the abdominal
cavity.
C57BL/6 male mice were implanted with either a large (weight= 3.5g; volume = 1.75mL, n = 20)
or small (weight = 1.1g; volume = 0.52mL, n = 14) radiotelemetry device in the peritoneal cavity
(large devices had an attached battery). Mice within each experimental group were further
separated into those with a freely moving running wheel (voluntary running mice) and those with
a locked wheel (sedentary mice). Body temperature, activity counts, body weight, and food and
water intake were monitored for 16 days post-operatively. A separate group of mice were
implanted with the large device and then had gross and microscopic evaluations of selected
abdominal organs 8 weeks after surgery (n= 25). The effect of small transmitters on
surrounding organs was not evaluated because the smaller devices can be sutured to the
peritoneal wall and thus do not move freely around the abdomen.
Mice with large transmitters implanted showed a significantly greater weight reduction after
implantation than the smaller transmitter mice, as well as a longer time to return to presurgical
body weights. Mice with large transmitters did not show the same robust increase in food intake
in response to fresh food and water at cage change (days 0, 7, and 14) as the small transmitter
mice, and the larger transmitter runners were the only group to show a significant reduction in
food intake below baseline of the surgery day (although food intake returned to pre-surgical
levels by day 1 post-op). Larger transmitter running mice had decreased water intake
throughout the 16 day recovery period. Large transmitter mice in both groups showed
significantly less activity and recorded smaller running distances than small transmitter mice.
None of the mice in any group showed outward signs of distress or pain.
84% of mice with large transmitters had evidence of reactive mesothelium with varying degrees
of fibrosis and inflammation of the diaphragm. 20% of the mice had evidence of peritonitis at
the large intestines and 56% at the duodenum. The authors posit that this is due to shifting of
the device with the abdomen during normal movements and activity. None of these mice had
any visual evidence of these changes at necropsy, nor did they show signs of pain and/or
distress antemortem.
Conclusions: Large transmitter mice had decreased running distances for 7 days post-op, as
well as overall decreased activity, indicating that the geometry of the device interferes with
normal activity patterns. Large transmitter running mice had consistently lower food and water
intake compared to the other 3 groups, and the large transmitter is associated with a 6 day
delay in recovery of body weight and food/water intakes. Overall the larger transmitter caused
delayed recovery, peritoneal inflammation, and reduced normal activity and exercise,
suggesting that a large body-to-transmitter weight ratio is important for mice to return to normal
growth, activity, and behavior after telemetry device implantation.
QUESTIONS
1. Mice implanted with larger radiotelemetry devices showed all of the following when
compared to small transmitter mice EXCEPT:
a. Longer time to return of pre-surgical body weight
b. Higher incidence of incision site dehiscence
c. Depressed response to external stimuli during postsurgical recovery
d. Decreased overall activity and voluntary exercise
2. T/F: The smaller radiotelemetry device was associated with more peritoneal inflammation
than the larger device.
3. T/F: Although the small transmitter mice groups recovered more quickly to pre-surgical
levels of body weight, food/water intake, and activity levels, the smaller transmitters do not
record as many parameters and are less accurate than the larger devices.
ANSWERS
1. b
2. F
3. F
Ezell et al. Palatability and Treatment Efficacy of Various Ibuprofen Formulations in
C57BL/6 Mice with Ulcerative Dermatitis, pp. 609-615
Primary Species: Mouse (Mus musculus)
SUMMARY
Aim: This study aimed to determine whether orally administered ibuprofen could be used as an
effective therapy for ulcerative dermatitis (UD) in mice, and if so, which formulation would be
more palatable and efficacious. In addition the authors examined clinical parameters associated
with UD, such as pruritus & skin ulceration, and compared therapeutic outcomes between the 2
different formulations used: a pediatric suspension compared with liquid gel capsules. The
therapeutic response was measured as water and food intake, locomotor activity, grooming
behavior, pruritic activity, lesion repair, and sediment formation of the formulation in water.
Background: UD is a spontaneous syndrome associated with C57BL/6 mice and those of
C57BL/6 background, current studies suggest a multifactorial syndrome with a genetic link and
indicated that a possible factor in the development of ulcerative dermatitis in C57BL/6J mice
and C57BL/6J substrains is related to vitamin A metabolism deficiencies. The clinical
presentation of ulcerative dermatitis may include excoriation, epidermal ulceration, granulation
tissue, serocellular crusts, and degloved skin segments and in severe cases, the lesions may
bleed, expose underlying musculature, or resulting scar contracture. Inflammation is associated
with an increase in free radical production, which accumulation results in pruritus.
Ibuprofen, a nonsteroidal anti-inflammatory drug, is commonly used for its analgesic, antiinflammatory, and antipyretic properties and reduces inflammation and pain by decreasing
leukocyte accumulation and activation and proinflammatory cytokines.
Methods: Adult mice used in this study were C57BL/6 or genetically engineered mice of
C57BL/6 background were from facility mouse colonies and were identified as having ulcerative
skin lesions. These individuals were randomly assigned to receive drinking water containing
either pediatric ibuprofen suspension or the contents of liqui-gel capsules for 9 d. For both
groups, ibuprofen-medicated water was prepared to a final concentration of 1 mg/mL water by
adding either 10 mL of the pediatric ibuprofen suspension (20 mg/mL) or the contents of a
single liqui-gel capsule to 200 mL of tap water. A sample of each medicated water preparation
was collected, placed on blood agar plates placed in an incubator, and examined at 0, 24, 48,
and 72 h for bacterial growth.
The volume of water consumed was determined every third day & food intake monitored.
Grooming, pruritis, and locomotor activity were measured by observing in-cage activity for 10min time intervals twice daily and were scored according to a 5-level scale. Also size of UD
lesions was measured.
Results: Results suggest that mice consumed significantly more ibuprofen-medicated water
when the liqui-gel formulation was used compared with the berry-flavored pediatric suspension.
Thereby the lower consumption of the pediatric suspension (possibly since berry flavoring may
not be highly palatable to mice) may have contributed to a reduced clinical response to therapy.
After 24 to 48 h of treatment, the liquid-gel mice had resumed typical locomotor activity and
grooming behavior and had markedly decreased pruritic behavior. In direct contrast, mice in the
pediatric suspension group were hunched and lethargic, exhibited decreased locomotor activity,
had constant or moderate pruritic behavior, and had unkempt pelage due to decreased
grooming. Also the lesion repair rate over the 9-d course of treatment was significantly better in
the liqui-gel group compared with the pediatric suspension group.
Conclusions: Authors concluded that this difference likely reflected the steady consumption of
the medicated water by the liqui-gel group and resulted into their receiving a higher dose of
ibuprofen. The steady intake of the liqui-gel medicated water resulted in reduced pain,
inflammation, and self-trauma and likely contributed to skin repair.
The study did not prove the hypothesis that the palatability of the ibuprofen formulation was
directly correlated to efficacy. Instead, the efficacy of the liqui-gel formulation of ibuprofen is
likely due to the homogeneous distribution of ibuprofen in the water, as supported by the clinical
response of the treatment groups. Overall the study demonstrated that the concentration of the
medication in the water consumed is paramount to healing.
QUESTIONS
1. Mechanisms of Ibuprofen.
2. Besides genetic predisposition, which other factors are associated with the development of
ulcerative dermatitis?
3. Concerns regarding delivering analgesics and anti-inflammatory components via pediatric
solutions.
4. Which other, non-drug related, treatments may be used for additional prevention of severe
UD?
5. Bacteria associated with UD
ANSWERS
1. Nonselectively inhibits cyclooxygenases 1 and 2, reduces the release of prostaglandins in
the cyclooxygenase pathway, thereby decreasing the release of inflammatory factors.
2. Age, diet, immune complex vasculitis, barbering, environmental changes including relative
humidity, temperature and season, diets with 11% fat, elevated vitamin A levels, and
ectoparasites.
3. Sugar-based flavored components may require frequent shaking to keep the medication in
suspension & more frequent water-bottle changes are necessary since bacteria may grow
more rapidly.
4. Trimming front and back nails of mice on a regular base.
5. Staph aureus
Cora et al. Artifactual Changes In Sprague-Dawley Rat Hematologic Parameters after
Storage of Samples in 3˚C and 21˚C, pp. 616-621
Task 3: Research
Primary Species: Rat (Rattus norvegicus)
SUMMARY: The purpose of this study was to characterize artifactual changes in rat
hematologic parameters after storage of samples at 3 and 21 C and to document the effects of
storage on peripheral blood smear findings. Terminal paired blood samples were collected in
EDTA from Sprague-Dawley rats, both male and female. The blood was analyzed immediately
then stored at either 3 or 21C and then retested at 6, 24, 48, and 72 hours. Tests consisted of
an automated analyzer and blood smears w/ Wright-Giemsa stain. Results were as follows:
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Comparable effects in both male and female rats
RBC count and Hgb unchanged at either temp after 72 hrs.
PCV and Hct both unchanged when stored at 3C but increased when stored at 21C.
Platelet counts, MCV increased at 6 hours for both temps. MCV appears more sensitive to
storage than other lab species. Reason for increased platelet counts unclear.
MPV increased at 24 hours at both temps
WBC count unchanged for 72 hours, higher after at 3C.
Neutrophil/lymphocyte counts unchanged
Abnormalities in the smears were seen as early as 6 hours and included smudge cells,
pyknotic leukocytes, echinocytes, spheroechinocytes. Similar morphologic changes are
observed in stored human blood.
Delayed analysis of Sprague-Dawley rat blood will result in artifactual changes in MCV, Hct,
platelet count, and MPV. The best practice is to analyze the blood within 6 hours. If this is not
possible, refrigeration may mitigate some of the changes. Blood smears should be done within 1
hour of collection to avoid morphologic changes in the RBCs. Care should be taken not to
misinterpret artifactual changes as pathologic findings.
QUESTIONS
1. T or F: Sprague-Dawley blood shows an increase in MCV when stored regardless of
refrigeration.
2. T or F: At 21C, PCV will increase to a greater degree in blood from male rats compared to
that from female rats.
3. Tor F: It takes at least 24 hours before significant morphologic changes are seen in the
RBCs of stored rat blood.
ANSWERS
1. T
2. F. Changes are comparable in both male and female rats.
3. F. Morphologic changes will occur within as little as a few hours.
Lalonde-Robert et al. Electroencephalographic and Physiologic Changes after Tricaine
Methanesulfonate Immersion of African Clawed Frogs (Xenopus laevis), pp. 622-627
Domain 2: Management of Pain and Distress; Task 2 – Minimize or eliminate pain and/or
distress
Secondary Species: African Clawed Frog (Xenopus laevis)
SUMMARY: Tricaine methanesulfonate (MS222) is a commonly used anesthetic and
euthanasia agent for many aquatic animals, including Xenopus spp. The current study
examined electroencephalogram (EEG) and electrocardiogram (ECG) activity of X. laevis frogs
placed into MS222 baths at either 1 g/L or 3 g/L for up to 2 hours to determine effectiveness of
this method as a means of euthanasia. Although different concentrations of MS222 did produce
significantly different result, the study found that EEG activity was suppressed within 30 minutes
of animals being placed into the solution. While EEG data showed suppression of activity, EEG
activity did not cease regardless of the duration of exposure to the MS222 solution, and ECG
activity was not substantially impacted. Final euthanasia of animals required the addition of
sodium pentobarbital injections. This study indicates that MS222 is insufficient as a single
agent for inducing euthanasia in the time frame studied. To achieve euthanasia in Xenopus
frogs, as defined by cessation of cardiac function and/or brain, MS222 must be coupled with a
secondary method.
QUESTIONS
1. What is the mechanism of action of tricaine methanesulfonate (MS222) in inducing
anesthesia/euthanasia in aquatic animals?
2. What are methods are considered acceptable for inducing euthanasia in amphibians, such
as the African clawed frog?
ANSWERS
1. MS 222 is a sodium channel blocker. Blocking sodium channels blocks depolarization and
signal propagation in excitable tissues.
2. Some acceptable euthanasia methods as identified by the AVMA panel on euthanasia
include:
a. Barbiturates (sodium pentobarbital)
b. Inhalant anesthetics
c. CO2
d. Benzocaine hydrochloride
e. MS222 (In spite of this report it is listed in the AVMA guidelines)
f. Double pithing