Volume 57, Number 2, April 2007
Rowland. Food or Fluid Restriction in Common Laboratory Animals: Balancing
Welfare Considerations with Scientific Inquiry, pp. 149-160
Species: Mice and Rats
Task: 3; Provide Research Support, Information, and Services
SUMMARY: Deprivation or restricted access to food and/or water is essential to certain
types physiological or behavior research. Deprivation studies withhold food or water for a
set period or time, the animals are then tested, and returned to free excess. Restriction
studies withhold only a portion of the daily food or water intake, usually for a prolonged
period. In the normal lab setting, rodents usually have continual access to food and
water for the convenience of the husbandry staff. Rodents typically become "grazers", in
that they will eat and drink small amounts throughout the daily period, mostly during the
dark cycle. In nature, this is usually not the case, and the animal needs to travel to food
and water source, so does not consume as many meals per day. This free, continual,
access to food often leads to obesity and decreased life longevity.
When water is removed from rats and mice, they will decrease food intake. This results
increased retention of water by less intake of electrolytes and decreased water loss
through defecation. Rats will loss 4% of body weight in the 1st 24 hours and 11% by 72
hours. Activity levels did not decrease until 96 hours. Withholding water for 21 hours led
to increased motivation in a water reward task vs. withholding for 14 hours. Mice will lose
7-9.5% body weight by 24 hours because of their smaller body size and increased water
consumption (25% of body weight/day vs. 10% for rats). The author summarized by
stating both mice and rats can normally have water withheld for 24 hours without overt
signs of distress or abnormalities. Animals on long term restrictions should be weighed
When food is withheld from rats, they lose 7% body weight by 24 hours and 17% by 72
hours. Activity levels actually increase. Rodents will quickly start to use body fat reserves
for energy metabolism. Rats fed at 75% and 48% of ad libitum consumption had lower
weight gain with much lower body fat content and higher survivability compared to rats
fed ad libitum. Housing temperature needs to be considered, as low environmental
temperature will lead to increased physiological metabolism.
1) Rats and mice fed ad libitum will typically consume (2, 4, 6, 8, or 10) meals per day,
mostly during the __________ cycle.
2) Rats deprived of water for 72 hours lost (8, 11, 14, 17%) of body weight and activity
level was (decreased, increased, same).
3) T/F Withholding water for 21 hours vs. 14 hours did not increase water reward
4) T/F Since mice consume more water as a percent of body weight, they can withstand
longer periods of water deprivation.
5) T/F Rats fed at 48% of ad libitum do not survive as long as rats fed ad libitum.
1) 10 and dark cycle
2) 11% activity level did not decrease until 96 hours
Stevens and Balahura. Aspects of Morphine Chemistry Important to Persons
Working with Cold-blooded Animals, Especially Fish, pp. 161-166
SUMMARY: The relative amounts of the different forms of morphine and many other
pharmacologic agents depend on temperature and pH. Some forms are more
efficacious because they are uncharged and can penetrate lipid membranes more easily
than the charged forms. Persons who administer pharmacologic agents to ectotherms
(that is, cold-blooded animals) should consider the effects of temperature on the relative
amounts of the different forms of drugs. For example, the fraction of morphine present
in the uncharged form is twice as high in a fish or frog at 5 C as in a mammal at 37 C.
Moreover, because the pH of blood, plasma, and tissues of ectotherms is higher when
they are held at lower temperatures, the combined effect of temperature and pH on the
speciation of pharmacologic agents also should be considered. In addition, the total
solubility of morphine and other pharmacologic agents depends on temperature and pH.
The purpose of this overview is to describe how temperature and Ph influence the
solubility and speciation of morphine.
1. The common ion effect is?
a. The reduction in solubility of a sparingly soluble salt by the addition of a soluble
salt that has an ion in common with it.
b. An increase in solubility of a soluble salt by the addition of a soluble salt that has
an ion in common with it.
c. An increase in solubility of a soluble salt by the addition of a soluble salt that
does not have an ion in common with it.
2. When administered to mammals about _______ of morphine is bound to plasma
3. True or false: Albumin is present in the plasma of all fish.
4. Total solubility of morphine __________ with ___ __________ in temperature.
a. Increases, a decrease
b. Increases, an increase
c. Decreases, an increase
d. Decreases, a decrease
5. Total solubility of morphine _________ with _______________ in pH.
a. Increases, a decrease
b. Increases, an increase
c. Decreases, an increase
d. Decreases, a decrease
3. False; albumin is present in the plasma of some fish but absent in others.
Mast and Griff. The Effects of Analgesic Supplements on Neural Activity in the
Main Olfactory Bulb of the Mouse, pp. 167-174
Task: Task 2 - Prevent, Alleviate, and Minimize Pain and Distress
Species: Primary - mouse
SUMMARY: Opioid analgesics such as buprenorphine may alter neuronal activity and
therefore may not be the ideal antinociceptive for use during in vivo neurophysiologic
recordings. NSAIDs such as ketoprofen may provide analgesia and reduce the amount
of anesthetic needed during in vivo neurophysiologic recordings, but may also alter
neuronal activity. This group therefore investigated the effects of ketoprofen on main
olfactory bulb (MOB) neuronal spontaneous activity in the mouse under chloral hydrate
ICR and B6 mice were anesthetized with chloral hydrate (400 mg/kg IP plus 30-70
mg/kg IP supplementation as needed). Mice were instrumented to record spontaneous
MOB activity. Mice additionally received buprenorphine (0.02-0.2 mg/kg) or ketoprofen
(100-200 mg/kg) after baseline MOB activity was established.
Buprenorphine decreased the rate of MOB activity in a dose-dependent manner but did
not affect temporal patterning. In contrast, ketoprofen did not affect spontaneous rate or
temporal patterning of MOB activity. Additionally, ketoprofen decreased the amount of
chloral hydrate needed to maintain an adequate anesthetic plane when ketoprofen was
administered at the beginning of an experiment.
In conclusion, ketoprofen deepens the anesthetic plane in chloral hydrate anesthetized
mice but did not significantly alter MOB spontaneous activity. The authors recommend
administering ketoprofen at 100 mg/kg in conjunction with chloral hydrate when
investigating neurophysiology of MOB neurons.
1. True or false: NSAIDs such as ketoprofen cross the blood-brain-barrier and therefore
may have some analgesic properties through central nervous system effects.
2. Which analgesic was shown to deepen the anesthetic plane of mice under chloral
hydrate anesthesia but did not significantly inhibit spontaneous activity of the main
olfactory bulb neurons?
Hariri et al. Ex Vivo Optical Coherence Tomography and Laser-induced
Fluorescence Spectroscopy Imaging of Murine Gastrointestinal Tract, pp. 175-185
Task: 9; Collaborate on the Selection and Development of Animal Models
SUMMARY: Optical coherence tomography (OCT) is a nondestructive, high resolution
imaging modality that has been investigated for a vast array of applications, including
imaging the anterior chamber of the eye and intracoronary stenting.
OCT provides cross-sectional images with short penetration depths (1 to 2 mm) and can
operate with either a water or air interface between the tissue and imaging probe.
Laser-induced fluorescence (LIF) spectroscopy measures the autofluorescence of
biochemical components such as NADH, collagen, and porphyrin excited by ultraviolet to
green wavelengths of light, providing biochemical information about a specimen.
The motivation for the use of OCT and LIF lies in their noninvasive nature.
Mouse models of human pathologies can be used to enhance knowledge of pathologic
processes and test therapeutic and chemopreventative compounds. Mouse models of
colorectal cancer now available include the C57BL/6J-ApcMin strain, which exhibits an
autosomally inherited predisposition to multiple intestinal neoplasia. Mouse models of
inflammatory bowel disease (IBD) also exist, such as the interleukin 2 (B6.129P2-
Il2tm1Hor, IL2)-deficient mouse. Mice deficient for IL2 have a 50% mortality rate by 9 wk
of age. The remaining mice develop IBD that is clinically and histologically similar to
ulcerative colitis and reportedly die within 10 to 25 wk under conventional housing
Authors evaluated 30 normal mice (A/J and 10- and 21-wk-old and retired breeder
C57BL/6J) and 10 mice each of 2 strains modeling colon cancer and IBD (ApcMin and
IL2-deficient mice, respectively). Histology was used to classify tissue regions as normal,
Peyer patch, dysplasia, adenoma, or IBD. Features in corresponding OCT images were
analyzed. Spectra from each category were averaged and compared via Student t tests.
Based on the preliminary data, authors suggest that together, OCT and LIF can
potentially be used to distinguish the categories of normal, Peyer patch, adenoma or
dysplasia, and IBD. Therefore combined OCT-LIF may be another tool for evaluating the
GI tract of mouse models and may provide the capability to monitor the effects of drug
treatment and experimental therapies in mouse models of human disease. Further
studies with more diseased animals are needed to fully assess the significance of our
findings. Both OCT and fluorescence spectroscopy have the capability to be used for in
vivo imaging applications of the GI tract, as they are both readily adaptable to small-
diameter endoscopic packaging with relative ease. Future work includes developing
smaller-diameter endoscopes for endoscopic esophageal imaging and laparoscopic
small intestine and upper colon imaging.
1) Name a mouse model of inflammatory bowel disease (IBD)?
2) What is Optical coherence tomography (OCT)?
3) T/F Mice deficient for IL2 have a 50% mortality rate by 9 wk of age
4) T/F IL/2 deficient mice develop IBD that is clinically and histologically similar to
Crohn's disease and reportedly die within 10 to 25 wk under conventional housing
1) Interleukin 2 (B6.129P2-Il2tm1Hor, IL2)-deficient mouse
2) Optical coherence tomography (OCT) is a nondestructive, high resolution imaging
modality that has been investigated for a vast array of applications, including imaging
the anterior chamber of the eye and intracoronary stenting
4) F (histologically similar to ulcerative colitis)
Funk et al. Effects of Leukotriene Inhibition on Pulmonary Morphology in Rat Pup
Lungs Exposed to Hyperoxia, pp. 186-192
Primary species: rat
ACLAM task #9: Collaborate on selection and development of animal models
SUMMARY: Rat pups are used here as a model for bronchopulmonary dysplasia, a
chronic pulmonary condition associated with prolonged exposure of neonatal humans to
high concentrations of oxygen. Normoxic or hyperoxic groups of pups were treated with
saline, or one of two leukotriene inhibitors, zileuton or zafirlukast. Zileuton but not
zafirlukast significantly ameliorated the histological changes associated with hyperoxia.
1. Which of the following histological changes in the lung are associated with
c. Alveolar macrophage influx
d. Type II pneumocyte proliferation
e. All of the above
2. The results of this study indicate that differences between zileuton and zafirleukast in
effectiveness of decreasing pulmonary lesions of bronchopulmonary dysplasia may
be due to?
a. Differing pharmacodynamics (absorption) of the two drugs
b. Difference in ability to suppress leukotriene B4
3. T/F Leukotrienes are metabolites of arachidonic acid and 5-HPETE (5-
4. T/F Leukotrienes are elevated in broncho-alveolar lavage fluid from humans with
persistent pulmonary hypertension, obstructive pulmonary disease and acute
5. T/F Leukotriene modifiers have both anti-inflammatory and bronchodilator activity.
ANSWERS: 1) E, 2) C, 3) T, 4) T, 5) T
Bauer et al. Breeding Colony Refinement through Phenotypic and Genotypic
Characterization of the SPRD-Pkdr1/Rrrc Rat Model of Polycystic Kidney Disease,
Task 3 - Provide research support, information and services
Task 5 - Execute IACUC veterinary responsibilities ("three Rs")
Primary species - Mus musculus
SUMMARY: Autosomal dominant polycystic kidney disease (ADPKD) causes focal renal
cysts and eventually end-stage renal disease in humans. The SPRD-Pkdr1/Rrrc rat is
used as a model for this disease, and animals that demonstrate the Cy mutation are
affected. Cy/Cy rats never reach sexual maturity while Cy/+ rats demonstrate slow
progression of disease similar to ADPKD (disease is most severe in males). Previously,
random mating was used, and the pups were assessed at 2-3 weeks of age by kidney
palpation; if any of the pups had large kidneys, this confirmed heterozygosity of both
parents. This is an inefficient and expensive method that results in the euthanasia of
many animals due to unwanted genotype. Thus, the purpose of this study was to
evaluate the use of biochemical, ultrasound, or genetic analysis as a means to
determine which animals were Cy carriers, which could result in a more efficient
Breedings were set up to result in either Cy/+ or +/+ offspring. The following tests and
results are as follows:
BUN and creatinine were measured at 4, 6, 9, and 12 weeks of age. Creatinine was not
predictive of heterozygosity in either males or females. In contrast, BUN was elevated in
9-week-old het males but not females. Thus, BUN as early as 8-9 weeks can be used
as a biochemical marker in males.
Ultrasound was also measured at 4, 6, 9, and 12 weeks of age. At 9 weeks, all hets had
enlarged kidneys and males had visible cysts. By 12 weeks, cysts were visible in both
male and female hets.
Genetic analysis was performed using microsatellite analysis and restriction fragment
length polymorphism (RFLP). Microsatellite analysis using three markers did not
correlate with Cy carrier status, while RFLP correlated 100%.
Conclusion: Cy carrier status can be assessed in these rats using BUN (in males),
ultrasonography and genetic testing. Genetic testing can be used to genotype both
males and females as young as 14 days of age, which may be the most effective means
of efficiently managing the breeding colony.
1. BUN can be used to assess Cy carrier status in what sex and age of SPRD-
2. At what age were cysts visible in both males and females?
3. True or False: Microsatellite analysis can be used to determine the Cy carrier status.
4. What does ADPKD stand for?
1. Males over 8-9 weeks of age
2. 12 weeks of age
3. False. Microsatellite analysis was not effective; RFLP assay, however, showed
4. Autosomal dominant polycystic kidney disease
Song et al. Brief Small Intestinal Ischemia Lessens Renal Ischemia-reperfusion
Injury in Rats, pp. 200-205
Task 1 - Prevent, Diagnose, Control, and Treat Disease (Knowledge - Physiology)
Primary Species - Rat
SUMMARY: In humans, renal ischemia is associated with high mortality. Damage of the
renal tissues is a direct result of the ischemic event (due to the lack of available oxygen),
but can also be significantly increased following reperfusion of the tissues - ischemia-
reperfusion (IR) insult. IR injury is one of the leading causes of the early loss of
transplanted organs. “Renal ischemia leads to a series of cellular events, which may end
with organ failure depending on the duration of blood deprivation.” The precise cause of
IR injury is not fully understood. There are two main methods through which it is
hypothesized that the damage occurs.
1) Through the direct effects of lipid peroxidation which is induced by the rapid creation
of reactive oxygen species (ROS) (such as superoxide anion, hydrogen peroxide, and
hydroxyl radical) when oxygen supply is restored
2) Through the indirect effects of inflammatory cells (especially neutrophils) that were
activated during the period of ischemia and then enter the kidney during reperfusion.
The inflammatory cells damage the renal tissues, resulting in the tissue’s production of
ROS that then causes cellular death.
Superoxide dismutase (SOD) and catalase (CAT) are antioxidant enzymes that
participate in the detoxification of ROS.
Ischemic preconditioning (IPC) is a technique whereby a brief episode of nonlethal
ischemia produces protections against a subsequent detrimental ischemia-reperfusion
(IR) insult. IPC has been described in the heart, brain, liver, skeletal muscle, lung, and
small intestine. It has also been found that an ischemic event in one organ can have a
remote protective effect on another organ. This is referred to as ‘remote preconditioning’
The objective of the study was to determine if rIPC of the small intestine protects the rat
against renal IR injury. The ultimate goal is to find more effective ways to prevent IR
A nephrectomy was performed on sixty-four, 250-300g Wistar rats. Fifteen days later,
the anterior mesenteric artery (providing blood supply to the intestines) and/or renal
arteries were occluded for varying lengths of time. After varying amounts of time the
occlusion was removed and the respective tissues reperfused. Some groups of animals
received more than one cycle of ischemia and reperfusion. Plasma creatinine and BUN
were collected. The levels of renal malodialdehyde (MDA) and activities of SOD and
CAT were determined. Renal histopathology was evaluated.
It was shown that “brief small intestinal ischemia attenuates renal IR injury in rat[s], as
shown by decreases in the levels of Cr, BUN, and MDA; attenuation of histopathologic
change; and preservation of SOD and CAT activities…rIPC of the small intestine
protects against renal IR injury via the inhibition of lipid peroxidation due to preservation
of SOD and CAT activities.”
In the case of kidney donation, the study suggests that “pretreatment of the living donor
with small intestinal IPC prior to procurement would protect the kidney against ischemia
and subsequent reperfusion injury.”
1. What is the name of the technique in which a brief episode of nonlethal ischemia
produces protections against a subsequent detrimental ischemia-reperfusion (IR)
a. Ischemia-reperfusion bypass
b. Ischemic preconditioning
c. Reperfusion protection protocol
2. Ischemia-reperfusion of what tissue is increasingly recognized as a primary effector
of multiple-system organ failure?
3. What was the primary finding of this study?
1. b. Ischemic preconditioning
2. a. Intestines
3. It was shown that brief small intestinal ischemia attenuates renal IR injury in rats.
Nonhuman Primate Model
Bentzel and Bacon. Comparison of Various Antihelmintic Therapies for the
Treatment of Trypanoxyuris micron Infection in Owl Monkeys (Aotus nancymae),
Task 1: Prevent, diagnose, control and treat disease
Tertiary species: Owl monkey - Aotus nancymae
SUMMARY: Trypanoxyuris microon is a pinworm that infects multiple New World
nonhuman primates. It is typically clinically insignificant, but may serve as a significant
variable during research data collection due to undetermined physiologic, immunologic
or behavioral effects. In their natural habitat, approximately half of the owl monkeys are
infected with this nematode. The pinworm has a direct life cycle, similar to other
pinworms. This paper compared treatment regimens with pyrantel pamoate, ivermectin
and thiabendazole for effectiveness. Treatments were given on day 0 and day 14. Daily
pinworm checks were done on the 19 animals for a total of 28 days. Effectiveness of
treatments was evaluated using the time-to-event approach with the Kaplan-Meier
method and log-rank test. Pyrantel pamoate was the most efficacious treatment with
100% clearance after one dose. This drug acts as a depolarizing neuromuscular
blocking agent leading to paralysis of the worm. However, it may be ineffective against
developing eggs and immature larval stages in view of a resurgence in egg detection
after successful clearance of eggs. The reason for resurgence in egg detection may also
have been due to reinfection as all animals on study were housed in the same room.
Ivermectin is a suitable alternative however it had 80% clearance after a single dose.
This compound acts as an inhibitory neurotransmitter and blocks the postsynaptic
stimulation of the adjacent neuron in nematodes, causing their paralysis and death.
Thiabendazole was not effective with only a 20% clearance after one dose. This data
indicates that pyrantel pamoate was the most effective and rapidly acting anthelmintic for
T. microon infection and it should be repeated after 1-2 weeks in combination with
effective environmental sanitation.
1. Give the genus and species of the owl monkey.
2. What clinical signs can be associated with pinworm infestations?
3. Describe the life cycle of the pinworm.
4. According to this study, which is the most effective treatment for pinworms in owl
5. What is the mechanism of action of ivermectin?
6. What is the mechanism of action of pyrantel pamoate?
1. Aotus nancymae
2. It is usually asymptomatic, but can be associated with anal pruritis and irritation.
3. Infection occurs through the ingestion of larval eggs, with the adult worms colonizing
the cecum, followed by the migration of gravid female worms to the perianal skin,
where eggs are deposited. The life cycle is direct.
4. Pyrantel pamoate
5. Ivermectin acts as an inhibitory neurotransmitter and blocks the postsynaptic
stimulation of the adjacent neuron in nematodes, causing paralysis and death.
6. Pyrantel pamoate acts as a depolarizing neuromuscular blocking agent leading to
paralysis of the worm.
Cheeseman et al. Spontaneous Exocrine Pancreas Hypoplasia in Specific
Pathogen-free C2HeB/FeJ and 101/H Mouse Pups Causes Steatorrhea and
Runting, pp. 210-216
Task 1 - Prevent, Diagnose, Control and Treat Disease
Primary Species: Mouse
SUMMARY: Spontaneous exocrine pancreas hypoplasia has not been previously
reported in mice. This article is a case study of the pancreatic disorder in inbred SPF
C3HeB/FeJ and 101/H strains. Clinical signs seen in pups included runting, greasy
hindquarters and diarrhea. All microbiologic screenings were negative. On gross
examination affected pups had undigested milk in the intestines. On histopathology, the
exocrine pancreas appeared small, lacked acinar cells and no cytokeratin duct like
structures. Then endocrine pancreas was unremarkable. Focal ulcerations were seen in
small intestine and diffuse hepatocyte glycogen microvacuolation in the liver. Based on
pathology, strain, age of onset and SPF status, a diagnosis of pancreatic hypoplasia
with a complex genetic component is most likely the cause of this occurrence.
1. T or F. Based on the preliminary data, functional islet cell mass is maintained even
with a loss of exocrine pancreas mass.
2. Strains observed with runting and steatorrhea include_______________ and
3. Inherited exocrine pancreatic hypoplasia discussed in this article is also seen in
which other animal:
a. DSH cats
b. German Shepherd Dogs
c. Guinea pigs
d. Nonhuman primates
4. The age of onset of disease seen commonly is:
a. 5-10 days
b. 10-15 days
c. 14-25 days
d. 14-25 weeks
2. C3HeB/FeJ and 101/H mice