Home > * * Journal of the American Association for Laboratory Animal Science Volume 51, Number 2, March 2012 ORIGINAL RESEARCH Biology Urasoko et al. Changes in

* * Journal of the American Association for Laboratory Animal Science Volume 51, Number 2, March 2012 ORIGINAL RESEARCH Biology Urasoko et al. Changes in

Journal of the American Association for Laboratory Animal Science

Volume 51, Number 2, March 2012

ORIGINAL RESEARCH

Biology

Urasoko et al. Changes in Blood Parameters and the Expression of Coagulation-Related Genes in Lactating Sprague-Dawley Rats, pp. 144-149

Domain 3

Primary Species: Rat (Rattus norvegicus)

SUMMARY: This study looks at the changes of blood coagulation parameters during lactation in Sprague-Dawley rats. Liver sections were analyzed by DNA microarray analysis to determine changes in expression of blood coagulation-related genes. Blood was collected, by terminal exsanguination, at lactation day (LD) 1, LD 7, LD 14, LD21 and 1 week after weaning. Hematological and blood chemistry values were then assessed. Liver sections were taken post mortem at LD0 and LD 14. Blood and liver samples were also collected from non-pregnant rats as controls. Differences in blood coagulation parameters were found and attributed to physiologic changes undergone during pregnancy either to prevent prolonged bleeding at delivery (ex. increased platelets and fibrinogen on LD1) or to prevent thrombosis (increased anti-thrombin III on LD1). By LD7, only fibrinogen levels differed in the two groups. Liver analysis showed increased expression of blood coagulation genes in lactating rats. Hematologic differences were found in the two groups on LD1 and the decreases in RBC count, hemoglobin and hematocrit and MHCH were attributed to bleeding at delivery. Neutrophils were elevated in the lactating group from LD1 to LD14, elevations were also found in eosinophils and monocytes 1 week after weaning. Total WBC counts though did no differ at any time points between the 2 groups. Liver enzymes were elevated (LD 7, 14, and 21) possibly due to increased food consumption and milk production. Energy consumption during delivery and during fetal development were likely causes of decreased values of glucose and triglyceride values on LD1. In conclusion, this study provided data for altered blood parameters that occurred during lactation in rats. Although not all the mechanisms for these changes could be conclusively determined, most were attributed to either the delivery process or to physiologic changes undergone during lactation.

QUESTIONS:

  1. Milk production in rats peaks:
    1. At the beginning of lactation
    2. Towards mid lactation
    3. Towards the end of lactation
  2. Albumin in the mother’s milk originates from plasma in rats. T or F

ANSWERS:

1.������������� b

2. ������������� T

Shukan et al. Normal Hematologic and Serum Biochemical Values of Cotton-Top Tamarins (Saguinus Oedipus), pp. 150-154

Domain 1

Secondary Species: Marmoset/Tamarins (Callitrichidae)

SUMMARY: This article provides baseline hematological and serum biochemical reference ranges for cotton-top tamarins. PCV, Hct, Hgb RBC count and creatinine were lower in female than male tamarins. Variations in hematological ranges existed between species of tamarins.

QUESTIONS:

1.������������� True or False. Saguinus oedipus are listed on the critically endangered species list.

2. ������������� Cotton-top tamarins are animal models of:

3.������������� Serum glucose levels in Saguinus oedipus were (higher/lower) when compared to other adult Old and New World nonhuman primates.

ANSWERS:

  1. True
  2. Spontaneous ulcerative colitis, colonic adenocarcinoma, Epstein-Barr Virus, early Alzheimer disease
  3. Higher

Husbandry

Nagamine et al. Carbon Dioxide and Oxygen Levels in Disposable Individually Ventilated Cages after Removal from Mechanical Ventilation, pp. 155-161

SUMMARY: Disposable individually ventilated cages (dIVC) are an appealing option for housing rodents for several reasons, including substantial reduction in labor costs; savings in capital expenditure otherwise allocated for tunnel cage washers, rack washers, and bulk autoclaves; reduction in electrical and water consumption; enhanced biosecurity due to the availability of irradiated, pre-bedded cages, irradiated food, and UV-irradiated and acidified water; and a healthier and ergonomically better working environment for the caretakers due to decreased exposure to allergens, lighter cages, and elimination of the need to install stoppers on water bottles. dIVCs have lids that restrict air exchange when the cage is not mechanically ventilated. This design feature may cause intra-cage CO2 to increase and O2 to decrease (hypercapnic and hypoxic conditions, respectively) when the electrical supply to the ventilated rack fails, the ventilated rack malfunctions, cages are docked in the rack incorrectly, or cages are removed from the ventilated rack for extended periods of time. The authors investigated how quickly hypercapnic and hypoxic conditions developed within dIVC after removal from mechanical ventilation. The results were compared with intra-cage conditions from the same cages with mechanical ventilation, and intra-cage conditions from disposable static cages (dSC), nondisposable static cages (ndSC), and nondisposable IVC (ndIVC) after removal from mechanical ventilation.

(A)������������� Bottom view of the IVC cage lid for the dIVC, showing air inflow and outflow ports, receptacle for the water bottle, and the filter surrounding the outflow port.

(B)������������� The plastic piece that holds the 6.2 � 7.3 cm, rectangular, Reemay spun-fiber filter surrounding the outflow port has been removed, showing the filter and the 3.0 � 5.6 cm trapezoid-shaped opening in the lid (large red arrow).

(C)������������� Two of 14 approximately 3-mm-diameter holes in the plastic holder (small red arrows) that allow air exchange through the filter.

(A)������������� Top view of cage lid for the dSC, showing the 2 raised filters.

(B)������������� The plastic pieces that hold the Reemay spun filters have been removed.

(C)������������� Larger plastic filter holder, showing the grid with 286 9-mm2 square holes through which air exchange occurs. Note that the holes in the plastic filter holder (small red arrows) of the dSC are more numerous per square area than are those in the plastic filter holder of the outflow port of dIVC lids (Figure 1 C).

Intra-cage CO2, O2, and NH3 levels were measured at the start of the experiment (0 h) and then hourly for 6 consecutive hours by using a battery-operated, multi-gas analyzer. In dIVC the probe was placed through the water bottle hole and extended approximately 8 cm into each cage to the approximate level of the mice. For ndIVC and ndSC, sampling was through a 0.75-cm diameter hole drilled 5 cm above the cage floor in the front of the cage. When not sampling, the hole was covered with clear adhesive tape.

Results:

Intra-cage air quality with 5 mice per cage: NH3 levels were undetectable within any of the experimental cages during the 6-h time frame. When dIVC were removed from mechanical ventilation, CO2 concentrations increased from less than 1% at 0 h to approximately 5% at 3 h. In contrast, intra-cage mean CO2 remained below 0.6% and O2 above 20.1% for the entire 6-h time period in ndSC. For dSC, intra-cage CO2 levels were slightly higher and intra-cage O2 levels were slightly lower than those of ndSC. ndIVC removed from mechanical ventilation showed similar changes in intra-cage CO2 and O2 concentrations as those in unventilated dIVC.

Intra-cage air quality with fewer than 5 mice per cage: Housing 4 adult mice in each cage resulted in a slower but similar deterioration of intra-cage air quality in unventilated dIVC, as seen for 5 mice per cage.

Hypercapnia together with hypoxia, which develops in unventilated dIVC, may lead to more severe effects than hypercapnic or hypoxic conditions alone. The data and clinical signs in the current study suggest that dIVC with 5 adult mice per cage quickly result in hypercapnic and hypoxic conditions when removed from mechanical ventilation. These results are consistent with previous data for unventilated ndIVC housing adult mice and rats.

QUESTIONS:

1. ������������� T/F: Hypoxic and hypercapnic conditions develop more quickly in disposable than nondisposable individually ventilated cages.

2. ������������� T/F: NH3 levels did not reach detectable levels at any time point in this study.

ANSWERS:

1. ������������� F

2. ������������� T

Domer et al. Processing and Treatment of Corncob Bedding Affects Cage-Change Frequency for C57BL/6 Mice, pp. 162-169

Domain 4:Task1 (Animal care: animal husbandry)

Primary Species: Mouse (Mus musculus)

SUMMARY: The current study hypothesized that intracage ammonia levels would be comparable between PCC (a novel, proprietary processed corncob bedding product) and autoclaved corncob beddings and that both housing materials would result in an increased cage-change interval. Cages were assigned into 4 groups (n=5) containing 300mL bedding of the following substrates: �-in corncob, autoclaved �-in corncob, �-in PCC, or autoclaved �-in PCC bedding. Some aspects of the study design included 1/8-in bedding to determine the effects of particle size on absorption and bacterial content. While the maximal acceptable concentration of ammonia in rodent housing has not been determined, the 8-h exposure limit for humans (25 ppm) was used as an endpoint. Nonpopulated cages for each bedding group were prepared as controls for bacterial counts.

For static cages, all types of bedding reached 25ppm ammonia in at least 5-7d, demonstrating that static cages should be changed at least weekly to control ammonia levels. Ammonia levels in ventilated cages with PCC or autoclaved corncob bedding took twice as long to reach 25ppm compared with those containing autoclaved PCC or corncob. In all bedding groups, the 1/8-in bedding size was significantly more absorptive than was �-in bedding. For �-in beddings, corncob was significantly more absorptive than PCC. Autoclaving decreased the absorptive capacity of corncob and increased the absorptive capacity of PCC. Bacteria isolated from unused, nonautoclaved bedding were similar in species to those isolated after exposure to mice and were considered to be nonpathogenic commensals. Nonpopulated PCC material contained less bacteria than did all other nonautoclaved beddings; however, autoclaved PCC cultured from populated ventilated cages contained significantly more bacteria than did nonautoclaved PCC and autoclaved corncob. Results from the current study suggest that the use of nonautoclaved PCC may be a feasible substitute for autoclaved corncob bedding for immunocompetent animals.

QUESTIONS:

1.������������� Short answer: List some disadvantages of autoclaving bedding material.

2.������������� Which governing body has set the human exposure limit for ammonia?

a. ������������� Occupational Safety and Health Administration

b. ������������� The United States Department of Labor

c. ������������� The American Conference of Governmental Industrial Hygienists

3.������������� What is the human exposure limit used as a guideline in this study?

ANSWERS:

1.������������� Disadvantages of autoclaving bedding material include: personnel time, autoclave energy consumption and maintenance costs, and potential loss of absorptive capacity of the bedding material.

2.������������� c. The American Conference of Governmental Industrial Hygienists

3.������������� 25ppm ammonia for an 8-h limit

Moons et al. The Effect of Different Working Definitions on Behavioral Research Involving Stereotypies in Mongolian Gerbils (Meriones unguiculatus), pp. 170-176

Domain 1: K9

Secondary Species: Gerbil (Meriones unguiculatus)

SUMMARY: Differences in observational methods represent a source of variation between behavioral studies, in addition to animal related elements, housing conditions, and environmental and observational factors. Not recognizing different descriptions of the same behavior can lead to divergent findings that can be incorrectly interpreted.

Working definitions should be clear and unambiguous when studying stereotypic behavior. Stereotypy characteristics must involve morphologically identical movements that are repeated regularly and must seem purposeless.

Digging behavior in Mongolian gerbils has been observed in the lab and in nature. In the wild, gerbils burrow to protect themselves from predation and temp flux, and burrowing behavior has been noted in the context of territoriality, grooming, and foraging-exploring. Digging in lab gerbils is not often identified as a stereotypy. A detailed working definition was described when the digging behavior lasted longer than 12s in one paper. Shorter instances were considered nonstereotypic digging. This >12s digging has been adopted by other groups for the threshold of stereotypic digging.

This paper’s goal was to compare quantification of stereotypic digging using 2 working definitions: WDmor (morphologic definition) and WD12 (a digging bout more than 12s long). AND, to provide an account of a combined digging-bar gnawing stereotypy.

The WDmor was developed by observations of nonstereotypic digging vs. stereotypic digging not related to burrowing. The WD12 considered digging to be stereotypic when it lasted longer than 12s. The current study used video footage to count and quantify the behaviors. The other stereotypy covered here is combined bar-gnawing and digging (1second each and alternating). The statistics were largely descriptive.

The 24 animals were observed for 6 days during the dark cycle via B+W CCTV cameras. Fur was marked by clipping to ID different gerbils. Total number, total duration, frequency, and average duration of stereotypic digging bouts were calculated. The data were analyzed in SAS. Using the WD12 instead of WDmor to quantify digging decreased the total duration of the bouts by 22% and the total number of bouts by 63%. But using the Pearson correlation test statistic, all parameters showed strong, positive correlation between data from WDmor and WD12.

All 24 gerbils exhibited stereotypic digging but only n=13 in bar gnawing and n=13 in combined stereotypy. Using different definitions for the same behavior lead to different results.

QUESTIONS:

  1. What is an example of a common stereotypy in Meriones unguiculatus?
    1. Digging
    2. Back flips
    3. Self –injurious behavior
    4. Circling to the right

ANSWERS:

        1. a

Management

Martinewski et al. Mouse Housing System Using Pressurized Cages Intraventilated by Direct-Current Microfans, pp. 177-180

Domain 4: Animal Care; Task 1: Develop animal husbandry programs

Primary Species: Mouse (Mus musculus)

SUMMARY: This group of investigators performed an initial assessment of a prototype intraventilated caging (PIV) system for laboratory mice that uses a direct current microfan to achieve cage pressurization and ventilation. The microfans are inexpensive fans similar to those used as cooling fans for conventional desktop computers and are adaptable to voltage fluctuations allowing for precise control of airflow. Room air is moved into the cage via the microfans and then released through exhaust ports toward the back of the rack. These modified cages were compared with standard static filter top (FT) cages. The cages were populated with mice and wet and dry-bulb temperature, relative humidity, intracage pressure, intracage ammonia concentration, and air speed at exhaust ports (used to determine air changes/hour) were measured. Wet and dry-bulb temperatures and relative humidity in both caging systems reached and equilibrium with the room. The air changes per hour decreased in PIV cages with time. An increase in the pressure within the PIV caging system was also noted with time. This decrease in air changes/hour and increased intracage pressure were attributed to fouling of the filter with debris such as hair, bedding dust, and chow residue at the exhaust port over time. The authors’ opinion is that pressurization and microenvironment ventilation of individual mouse cages with a microfan is a simple, reliable system, with low cost, reduced maintenance requirements, and a rare occurrence of failure

QUESTIONS:

  1. T/F. In addition to their low implementation costs, microfans are readily commercially available, require little to no maintenance, and have an expected mean lifespan of 20,000 hours under constant operation.
  2. Define ASHRAE

ANSWERS:

  1. True
  2. American Society of Heating, Refrigerating and Air-Conditioning Engineers

Hickman-Davis et al. Effectiveness of Shoe Covers for Bioexclusion within an Animal Facility, pp. 181-188

Domain 4: Animal Care; T1. Develop animal husbandry programs; K9. Pathogen-free barriers (exclusion)

SUMMARY: Shoe covers are required in many barrier facilities without much research to prove or disprove their effectiveness in excluding pathogens from an animal room. This study used UV-visible powder to assess contamination with and without shoe covers into the animal room, onto the animals, and transport of contaminants from outside into the vivarium and animal room.

Floor “contamination” from just inside the animal room door (after all PPE was donned) resulted in contamination of the floor, as expected, but no contamination onto the cages or onto the inanimate animal surrogates even after a wide variety of simulated husbandry procedures were performed, with or without shoe covers.

Donning PPE in a “contaminated” area just outside the door resulted in no contamination of PPE if shoe covers were not used, but contamination of gloves and gowns when shoe covers were used (gloves were donned last in 100% of cases).

“Contamination” outside the building was only tracked a maximum of 32.3 ft, shorter than the distance to the vivarium from outside or to a rodent room door from outside the vivarium.

Thus, the authors suggest that shoe covers do not contribute to the effectiveness of bioexclusion when rodents are housed in microisolation or individually ventilated cages and shoe cover use may be discontinued unless a specific personnel risk is involved.

QUESTIONS:

1. ������������� True/False: Both in human operating rooms and rodent vivaria, use of shoe covers provides useful protection for the patient or animal.

2. ������������� “Contamination” powder was found on which of the following in the rodent room when simulated husbandry tasks were complete?

a. ������������� Mouse cage

b. ������������� Biosafety cabinet

c. ������������� Floor

d. ������������� Step stool

e. ������������� Bottom rung of cage rack

ANSWERS:

1. ������������� False

2. ������������� c, d, e

Health Surveillance

Burr et al. Corynebacterium bovis: Epizootiologic Features and Environmental Contamination in an Enzootically Infected Rodent Room, pp. 189-198

Domain 4: Animal Care; Task 1: Develop animal husbandry programs

Primary Species: Mouse (Mus musculus)

SUMMARY: Corynebacterium bovis is a potential pathogen of athymic nude mice, causing a hyperkeratosis condition often called scaly skin disease.� A colony of athymic mice spread across multiple rooms and occasionally housed in the same room as immunocompetent mice were found to be contaminated with C. bovis.� Various methods of control were attempted, primarily investigating different cage changing protocols.� The study also evaluated personnel as possible means for transmission.� Animals were housed in individually ventilated cages with HEPA filtration.� In spite of strict efforts to control transmission of C. bovis, no method of cage changing was able to prevent transmission, however high temperature cage wash (180 F) was able to effectively eliminate contamination of caging and equipment.� One animal caretaker was found to a persistent carrier by nasal swab.

Efforts to control C. bovis contamination in a colony must include thorough surface decontamination, strict cage changing protocols, and consideration of airborne transmission as a source of contamination.

QUESTIONS:

  1. What disease can be caused by Corynebacterium bovis in mice, and what strain of mice is it observed in?

ANSWERS:

1.������������� Hyperkeratosis or scaly skin disease which occurs in athymic nude mice.

Roble et al. Total IgE as a Serodiagnostic Marker to Aid Murine Fur Mite Detection, pp. 199-208

Domain 1: Management of spontaneous and experimentally induced diseases and conditions

SUMMARY: Current detection methods for mites (Myobia, Myocoptes, and Radfordia)� have suboptimal sensitivity, are time-consuming, and are costly. Current diagnostic methods include superficial skin scrapes, tape tests, and postmortem pelt exams which all provide high specificity and low sensitivity (a lot of false negatives). A new PCR assay is available but is expensive and gives a positive result even for mite parts and nonviable eggs (false positives). This paper aimed to develop a sensitive serodiagnostic technique that would facilitate detection and ease workload. They evaluated total IgE levels in mice in association with experimental mite infestations.� Mite antigen exposure occurs through microabrasions in the skin. The production of IgE results from sensitization of CD4+ cells resulting in a Th2 cytokine mediated response. Elevation of total IgE is nonspecific, but there are no reports of naturally occurring IgE increases in laboratory mice.

C56/BL6 and Swiss Webster mice were used. Infestation was via either contact with a known infected animal (positive by skin scrape) or exposure to dirty bedding. New infestations were determined by skin scrape and postmortem pelt examinations. Various combinations of mite species were used and various ages of mice were used. IgE levels were assessed before infestation, during, as well as post treatment with ivermectin. IgE was also evaluated in animals infected with pinworms.

Total IgE was measured in all animals (they were unable to develop an assay for mite specific IgE). Results showed significant increases in IgE levels within 2-4 weeks after infestation with mites by direct contact and a delayed but significant increase in mice exposed to dirty bedding. Values decreased within 2 weeks after treatment with ivermectin. Age, strain, and parasite caused variability in the IgE response, but sex did not. Animal infected with pinworms showed elevations in IgE above controls at some time points but were significantly lower than mite infested animals.

They determined a threshold (specifically for Swiss Webster mice) by using a receiver-operating characteristic curve and recommended that animals with values above the threshold be subject to further diagnostic assays. The threshold was not determined for C57/BL6 mice, but levels were generally lower than the Swiss Webster mice, an expected finding as C57/BL6 tend to have a Th1-type cytokine response.

QUESTIONS:

1.������������� Which type of screening method is this paper advocating?

a. ������������� High-sensitivity, low specificity followed by low-sensitivity, high-specificity for those that come up positive in the initial screen?

b. ������������� Low-sensitivity, high specificity followed by high-sensitivity, low-specificity for those that come up positive in the initial screen?

2. ������������� What is one reason that C57/BL6 mice and Swiss Webster mice have different IgE responses to mite infestations?

3. ������������� How long is the life cycle of each of the following mites: Myobia, Myocoptes, Radfordia?

ANSWERS:

1.������������� a

2.������������� C57/BL6 mice have primarily a Th1-type cytokine response, therefore� the rise in IgE is less than in Swiss Webster mice

3.������������� Myobia 23 days, Myocoptes 8 days, Radfordia thought to be close to Myobia

Anesthesia

Cesarovic et al. Combining Sevoflurane Anesthesia with Fentanyl-Midazolam or S-Ketamine in Laboratory Mice, pp. 209-218

Domain 2: Management of Pain and Distress; T2. Minimize or eliminate pain and/or distress

Primary Species:� Mouse (Mus musculus)

SUMMARY: Laboratory mice typically are anesthetized by either inhalation of volatile anesthetics or injection of drugs.� The authors of this article studied whether the synergistic and additive interactions of injectable drugs and volatile anesthetics would result in decreased dosages of each component while inducing sufficient depth for general anesthesia with fewer side effects.�

Female C57BL/6J (free of all viral, bacterial, and parasitic pathogens listed in the FELASA recommendations) mice, were pair housed.� 72 mice were used to determine minimal alveolar concentration (MAC), acid-base balance, and blood gas concentration.� 26 mice were implanted with telemetric transmitters to measure heart rate, core body temperature, and locomotor activity.�

Sevoflurane (5%) alone (S) was compared to S-ketamine (30 mg/kg SC) and sevoflurane (5%) together (KS) and fentanyl-midazolam (0.04 mg/kg and 4.0 mg/kg SC respectively)� and sevoflurane (3.3%) together (FMS).� Injections were given as a premedication� 5-7 minutes prior to sevoflurane induction.� Mice were induced in an induction chamber for 1.5 minutes with sevoflurane (8%) and then transferred to a nose cone at the maintenance concentration of sevoflurane .� Minimal alveolar concentration (MAC) was measured 12 minutes after sevoflurane was started.�

Tail pinch, pedal withdrawal, and abdominal skin pinch reflexes were each tested at 5 minute intervals over a 50 minute period of gas anesthesia.� Blunt forceps with a spacer between its arms to allow uniform application of pressure was used to test for any motor response (head jerking, movement of tail or an extremity) to one or more of the 3 forms of noxious stimuli.� The mouse’s MAC was determined by using these responses.� The MAC was significantly lower (2.2% versus 3.3%) with the FMS premedication when compared to KS and S.� This represents a 33% gas savings when fentanyl-midazolam is used as a preanesthetic.� The time to immobilization was significantly different between all 3 protocols.� FMS < KS < S.

All mice showed signs of sedation approximately 2 minutes after injection with FM.� All mice exhibited symptoms of tremor, ataxia, and dizziness approximately 5 minutes after injection with KS.� Almost all S mice showed urination, defecation, shaking the head or limbs, or jumping and locomotion behaviors when placed in the induction chamber.� These behaviors were less frequent in the KS group and almost complete absent in the FMS group.� Mice maintained their core body temperature and heart rate in the normal range during anesthesia for all 3 groups.� The respiratory rate declined immediately after gas induction and remained markedly depressed during the entire 50 minutes of gas anesthesia compared to baseline levels.� Blood gas and pH measurements of arterial blood showed prominent acidosis, hypercapnia, and hypoxia for all three groups with values markedly exceeding the physiologic range.� All mice in the KS group displayed cardiac arrhythmia and episodes of apnea followed� by tachypnea.� Two KS mice died, one shortly after being placed in the induction chamber and one about 15 minutes into anesthesia.� The mice in all three groups returned to ventral recumbency and were able to move within about 2 minutes after sevoflurane withdrawal.

Baseline values for heart rate, core body temperature, locomotor activity, and body weight were determined for 3 days immediately preceding anesthesia.� Postanesthetic effects for these values were measured for the 3 days following the procedure.� Heart rate was significantly elevated for the first 12 hours postanesthesia in all 3 groups with the S group rate being significantly higher than the FMS group rate.� Core body temperatures significantly increased for the first 12 hours postanesthesia for the KS and S groups.� Locomotion and body weights were unchanged in all 3 groups.�

QUESTIONS:

1. ������������� T/F.� All 3 protocols provided reliable anesthesia in mice with a short recovery phase.�

2. ������������� T/F. Both premedication groups regimens shortened the time required to reach immobilization after exposure to S when compared to S alone.

3. ������������� During the first 12 hours after anesthesia which of the following was true?

a.� ������������� Heart rate increased significantly over baseline in 2 of 3 groups.

b.� ������������� Body weight decreased moderately from baseline values.

c.� ������������� Core body temperature was significantly lower than baseline values.

d.� ������������� Locomotion was unchanged from baseline values in all 3 groups.

4.� ������������� T/F. Two mice died during the study, one KS and one FMS.

5.� ������������� Which of the following reflexes was not tested as noxious stimuli in this study?

a.� ������������� Pedal withdrawal reflex

b.� ������������� Palpebral reflex

c.� ������������� Skin pinch reflex

d.� ������������� Tail pinch

ANSWERS:

1. ������������� T

2. ������������� T

3. ������������� d

4. ������������� F. Both were KS mice

5. ������������� b

Experimental Use

Kuster et al. Voluntary Ingestion of Antiparasitic Drugs Emulsified in Honey Represents an Alternative to Gavage in Mice, pp. 219-223

Primary Species: Mouse (Mus musculus)

SUMMARY: Oral gavage requires a strong technical skill and still posses the risk of injury to the animal as well as stress from handling. Alternative techniques for oral administration have been studied in rats, but little has been studied in mice. This study emulcified Albendazole in honey to create a compound that would be voluntarily ingested by the mice. 100 �L of honey is equivalent to 0.2556 kcal. Study 1 – Female BALB/c mice were infected IP with E. multilocularis. Treatment began 6weeks post infection with control receiving 100 �L of honey and test group receiving 100 �L of honey with Albendazole. Parasite burden was also measured after 8 weeks of treatment. It was found that the addition of honey did not notably affect the efficacy of Albendazole. Study 2- Female BALB/c were dosed via oral gavage of honey+Albendazole, oral gavage of Albendazole+Carboxymethyl cellulose salt, voluntary consumption of honey+Albendazole, voluntary consumption of honey. Metabolites, albendazole sulfoxide (ABZSO) & Albendazole sulfone (ABZSO2), serum concentrations were measured (Via HPLC) 60min post dosing and found to be in similar concentration in all test groups that received Albendazole.

QUESTIONS:

1. Benzimidazoles are an extensively studied drug often requiring the use of laboratory rodents for toxicology studies. Which of the following has NOT been linked with this drug?

    1. Antiparasitic compound
    2. Antifungal compound
    3. Alzheimer treatment
    4. None of the above

2. Which of the following is an advantage of using honey as a carrying compound?

    1. Cheap and easily available
    2. Antimicrobial properties
    3. Viscosity allows easily emulsification of drugs
    4. All of the above

ANSWERS:

1. d.

2. d.

Duan et al. Use of Fenbendazole-Containing Therapeutic Diets for Mice in Experimental Cancer Therapy Studies, pp. 224-230

Domain 1

Primary Species: Mouse (Mus musculus)

SUMMARY: Pinworm infection (oxyuriasis) is a common problem even in well-run rodent colonies. Facility-wide prophylactic treatment of all mice with a diet containing therapeutic levels of fenbendazole for several weeks is often used to control pinworm outbreaks. A general assumption relevant to use of these treatment regimens is that food containing fenbendazole at therapeutic concentrations will not affect the outcomes of the experiments being performed in research facilities. The experiment used adult female BALB/cRw mice that were implanted with EMT6 mouse mammary tumors. The goal of the experiment was to examine the effect of feeding a therapeutic diet containing 150 ppm fenbendazole on the growth of the mammary tumors. One week after the mice were randomized and began receiving either the fenbendazole-containing or control diets, they were injected with tumor cells. Tumor growth was monitored by serial measurements of tumor diameters from the time tumors became palpable until they reached 1000 mm3

The experiments tested 2 possible effects of fenbendazole: alteration of tumor growth, through a cytotoxic effect of the drug on cells within these solid tumors, and alteration of tumor radio-sensitivity. They saw no evidence that either effect occurred when mice were placed on a standard fenbendazole-containing diet appropriate for the treatment of pinworms in rodent colonies. The data for the tumor growth study support the notion that this intervention can be used safely in mice being used in experimental cancer therapy studies. However, the cell culture data show that fenbendazole inhibits the growth of EMT6 tumor cells in vitro when given continuously at doses not far above those expected in the tissues of rodents fed this diet. Recent data from other laboratories also have demonstrated effects of fenbendazole that could complicate experiments. Because of these potential problems, caution is advised when fenbendazole-containing diets are used to treat rodent colonies actively used in research on experimental cancer therapy.

QUESTIONS:

1. Infections with pinworms in rodent colonies can

    1. Be asymptomatic
    2. Cause rectal prolapse and intussusceptions
    3. Hematologic changes
    4. Hair loss
    5. a, b, c
    6. All of the above

2. T or F: In rodents, more than 90% of fenbendazole is excreted in the feces unchanged

3. T or F: In rodents colonies, only the animals that test positive for pinworms should be treated

ANSWERS:

1. e.

2. True

3. False; diagnostic tests for pinworms often yield false-negative results in low-level infections, and treating only those mice proven to be infected can leave undetected pockets of infected animals that can serve as a source of recurrent infections within the facility

Rey et al. Simultaneous Pulmonary and Systemic Blood Pressure and ECG Interval Measurement in Conscious, Freely Moving Rats, pp. 231-238

Domain 3: Research: K1: biomethodology techniques

Primary Species: Rat (Rattus norvegicus)

SUMMARY: Dual pulmonary and systemic blood pressure measurements in freely moving small mammals has historically been unavailable because only one of the two measurements could be collected in conscious, freely moving small animals at any given time. A newly developed dual blood-pressure telemetry transmitter was tested by implantation into both normotensive and monocrotaline-induced pulmonary hypertensive Wistar rats. Sensing catheters were placed in both the pulmonary artery and the descending aorta. Between 2 and 12 weeks after implantation, blood pressure and ECG waveforms were recorded for each animal. During the recording time period, pulmonary arterial pressure gradually increased in rats treated with monocrotaline versus those treated with saline. Accuracy of the blood pressure data obtained from the telemetry transmitters was verified against 2 Miller high-fidelity blood pressure catheters at the end of the study. Post-study histological examination of the tissues revealed that the catheters associated with the telemetry device were tolerated locally.

QUESTIONS:

1. What substance was used to induce pulmonary hypertension in the rats used in this study?

2. The telemetry device tested allows for the simultaneous collection of pulmonary and systemic blood pressures in conscious animals. (T or F)

ANSWERS:

  1. Monocrotaline
  2. True

Ayers et al. Alternatives to Retroorbital Blood Collection in Hispid Cotton Rats (Sigmodon hispidus), pp. 239-245

Tertiary Species: Other Rodents

SUMMARY: Cotton Rats are valuable in the study of many human viral diseases for example polio, measles, RSV and herpes simplex. Despite their widespread use there are few reports on sampling techniques in these animals hence the aim of this study to assess blood sampling methods and trial a novel subzygomatic site. Five blood collection methods used in rodent species were initially trialled; the tail vein, sublingual vein, submandibular, lateral saphenous, and retro-orbital sinus. The volume of blood collected from each site and the number of attempts to collect that volume were recorded for each site. Anaesthesia was used for all procedures. Necropsy was performed at the end of the study and histopathology of the collection site area was performed on a subset of the animals. A new method was also suggested – rostral and proximal to the lateral canthus with landmarks including the ventral side of the caudal rim of the zygomatic arch and the ramus of the mandible. The angle just rostral to this intersection was the puncture site and this technique was named the “subzygomatic” approach.

No blood was yielded from the submandibular approach suggesting a different vascular anatomy in this species. The lateral saphenous yielded little blood flow and was ruled out. The tail vein proved to be problematic due to the dark pigmentation of the tail (thus hard to visualise the vein) and the concern about potential for tail slip. The retroorbital route led to concern due to the already prominent globe and the pressure exerted possibly leading to proptosis. The sublingual approach was difficult due to blood collecting in the cheek and the short tongue making it difficult to hold necessitating two handlers. However, success was achieved with a 22 gauge needle and haemostasis was achieved quickly.

The subzygomatic method proved to be the most viable. The vein was visible after clipping and landmarks easily palpated. This technique produced 0.3-0.5ml blood and flow was best achieved by use of an 18 gauge needle. Necropsy of this site showed minimal haemorrhage on the surface of the masseter muscle. A venous sinus was present under the muscle which appeared to connect to the retro-orbital sinus although histopath could not confirm this. Histopathological examination varied but ranged from linear haemorrhage tracts with myocyte necrosis to fibrosis and foci of granulation.

In conclusion, it seems that blood sampling from cotton rats is best achieved by use of the sublingual or subzygomatic approach. The former is technically more difficult and the authors recommend the latter approach which reliably yields a useable sample.

QUESTIONS:

  1. T/F. The submandibular approach is a reliable method of collecting blood samples in the cotton rat.
  2. Use of which gauge needle is recommended for the subzygomatic method.
  3. T/F. Landmarks for the subzygomatic arch include the intersection of the zygomatic bone and the maxilla.
  4. Which other blood collection site may also be suitable in the cotton rat?

ANSWERS:

  1. F
  2. 18 G
  3. F. Landmarks are the zygomatic bone and the mandibular ramus.
  4. The sublingual vein.

Lee et al. Effects of Various Anesthetic Protocols on 18F-Flurodeoxyglucose Uptake into the Brains and Hearts of Normal Miniature Pigs (Sus scrofa domestica), pp. 246-252

Domain 3: Swine

Primary Species: Pig (Sus scrofa)

SUMMARY: This paper evaluated the effect of various anesthetic protocols on the uptake of 18F-Flurodeoxyglucose (FDG) uptake in the brain and heart of normal miniature pigs.� 18F-flurodeoxyglucose is a glucose analog used as a positron emission tomography radiotracer and is used for detecting abnormal glucose metabolism.� SPF miniature pigs were evaluated in a cross over study design to see the effects of 1) propofol-isoflurane 2) propofol 3) ketamine and 4) tiletamine-zolazepam on the FDG uptake in the brain and heart to establish baseline conditions for PET imaging of this animal model.

This study found that the accumulation of FDG varied greatly according to the type of anesthetic agents used and between the brain and heart.� In the heart isoflurane increased FDG accumulation, whereas tiletamine-zolazepam decreased accumulation.� In the brain propofol increased FDG accumulation compared to other anesthetics.� There was also considerable variability depending on which part of the brain was evaluated.� The conclusion of this study was that the anesthetic regimen should be selected very carefully when performing these studies and in order to compare results the same anesthetic needs to be used across studies.

QUESTIONS:

1.������������� 18F-flurodeoxyglucose is a glucose analog so it useful for evaluating glucose metabolism in various tissues.� T/F

2.������������� The anesthetic regimen is not relevant when comparing results from PET scans with radiotracers.� T/F

ANSWERS:

1.������������� T - this radiotracer is used for evaluating glucose metabolism in heart and brain.

2.������������� F - the anesthetic regimen needs to be standardized in order to compare results when evaluating PET with radiotracers since anesthetics have an effect on how the radiotracer is metabolized and blood flow and pressure to the various organs differs with different anesthetic regimens.

CASE REPORT

Felt et al. Mortality and Morbidity in African Clawed Frogs (Xenopus laevis) Associated with Construction Noise and Vibrations, pp. 253-256

Domain 1

Secondary Species: African Clawed frog (Xenopus spp.)

SUMMARY:

Study Objectives: To assess the effects of construction related vibrations in the well-being of African Clawed frogs. The lateral line sensory systems of Xenopus, comprised of hair cell receptors, are found along the sides of the body and around the head and eyes. The lateral line functions as mechanoreceptors that respond to water movement and vibrations and is a means of detecting the movements of prey. Indiscriminate feeders, such as Xenopus have a unique ability to regurgitate and evert the stomach and expel the contents if they accidently eat a noxious substance.� Activation of this noxious feeding stimulus is believed to occur via the lateral line and over stimulation of this sensory organ by repeated strong vibrations can result in stomach regurgitation with resultant partial airway obstruction, resulting in over inflated lungs and death.

Hence, because of their unique nervous system anatomy and their fully aquatic status, Xenopus are susceptible to adverse health effects resulting from strong vibrations, especially those related to constructions. However, there are currently no published threshold recommendations for vibrations and noise exposure levels for Xenopus. Aquatic species especially Xenopus should be relocated whenever possible before noise and vibration generating construction begins in a vivarium.

QUESTIONS:�������������

1.������������� T/F. The African Clawed Frog may not be housed in an area where a device emitting a sound pressure level of more than 95 dB is in use?

2. ������������� T/F. The lateral lines of the African Clawed frog can be used for sexual dimorphism.

ANSWERS:

1. ������������� F. There are currently no published threshold recommendations for vibrations and noise exposure levels for Xenopus.

2. ������������� F

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