Pain evaluation and response to buprenorphine in rats subjected to sham middle cerebral artery occlusion. Contemporary Topics 41 (6): 09.
The objective of this study was to evaluate both subjective and objective measures for pain evaluation in male Winstar rats subjected to sham middle cerebral artery occlusion (MCAO) and to determine whether buprenorphine would be an appropriate analgesic in this surgical model. Appropriate and efficacious use of analgesics in rodents must be balanced between the research objectives and the need of the animals especially in protocols involving surgical manipulations or surgically derived disease models. A concern in these type of studies is that pre and/or post operative analgesics will alter study outcomes and thus confound results. Male Wistar rats (n=37) underwent a sham 2-h MCAO by the intraluminal filament technique followed by 22-h of recovery. Animals were divided in three post operative treatment groups and randomly assigned. Group 1: Vehicle only (VH; vehicle subcutaneously [s.c] and plain jello orally). Group 2: Low dose buprenorphine (LB; 0.05 mg/kg s.c and 0.25 mg/kg drug in jello orally). Group 3: High dose buprenorphine (HB 0.5 mg/kg s.c. and 0.25 mg/kg drug in jello orally). Animals received subcutaneous treatments prior to anesthetic recovery and about 18 hr later. Jello treatments were given once at the end of the surgery day. Previously published behavioral scoring system was modified to be used as subjective and objective method for pain evaluation. All animals were evaluated for pain before sham surgery and at 3, 18 and 22-h postoperatively by observers blinded to the treatment groups. Brain tissue was collected to confirm lack of brain injury due to surgical procedure. Result showed sham intraoperative physiologic variables were equivalent among treatment groups. Base line assessment scores were zero for all groups. Postoperatively, all treatment groups showed elevated assessment scores relative to baseline values. Buprenorphine at the tested dose did not markedly reduce total assessment scores postoperatively relative to those in vehicle-treated animals. In conclusion, the authors recommend further evaluation of rodent postoperative pain and response to analgesia is needed to formulate scientific approach for pain assessment and management.

Questions:
1. Name three other conditions that can cause pain/distress in laboratory animals.

Answers:
1. Freud's complete Adjuvant, Ocular and skin irritancy testing, Noxious electrical shock, Paralysis or immobility in a conscious animal etc.. (Animal care resource guide 1997, 11.1)

Butorphanol decreases edema following carrageenan-induced paw inflammation in rats. Contemporary Topics 41 (6): 15.
Opioids and nonsteroidal anti-inflammatory drugs are commonly used in rodents for analgesia. The mu receptor-activating opioid analgesics inhibit anti-inflammatory responses and decrease edema, hyperalgesia, and pain. The kappa receptor-activating opioids, U50488 and tifluadom, decrease carrageenan-induced paw swelling which is a widely accepted test to produce a predictable inflammatory response. This study evaluated the effects of butorphanol on paw swelling with or without a concurrent nonsteroidal anti-inflammatory, indomethacin in varying concentrations.

Sixty-four female rats were divided into 8 groups. Groups 2/6 received 1.0 mg/kg indomethacin(gavage) 1 hour prior to injection of carageenan. Groups 3/7 2.5 Groups 4/8 5.0 Groups 1/5 received only the vehicle without indomethacin

Butorphanol treatments Groups 1/4 received only saline in addition to indomethacin tx above Groups 5/8 2 mg/kg butorphanol sq 1 hour prior to injection with the above indomethacin tx

Carageenan was administered 1 hour after the above treatments at a dosage of 50ul of 0.5% in the right hind foot pad. The animals were euthanized 3 hours after the injection of carageenan with an overdose of isoflurane. Both rear limbs of each animal were severed at the tibiotarsal joint and weighed. Difference in weight was recorded. Histologic cross sections were prepared.

Butorphanol with or without concurrent indomethacin treatment decreased paw edema after the carageenan injections. Conversely indomethacin reduced swelling in a similar manner with or without butorphanol and independent of varying dosages of indomethacin. Both treatments reduced paw edema by 17 to 38%.

Care should be given to the selection of analgesics used in studies where an inflammatory response is monitored as a primary indicator.

Questions:
1. What is the mechanism of action of a mu opioid?
2. What is the mechanism of action a kappa opioid?
Both mu and kappa opioid agonists stimulate the hypothalamo- pituitary-adrenal axis which causes a release of corticosterone from the adrenal glands which may account for the anti-inflammatory properties of opioid drugs but the two act in different ways

Answers:
1. Because catecholamines can inhibit the inflammatory response, mu opioids may work by increases in epinephrine and perhaps coricosterone.
2. Kappa opioid agonists may be explained by elevated levels of only corticosterone.

The effect of stocking density on growth rate and maturation time in laboratory-reared California sea hares. Contemporary Topics 41 (6): 18.
The California sea hare, Aplysia californica, is a saltwater invertebrate that has been utilized as an animal model for neurobiology studies. Prior to the development of techniques for culture of Aplysia spp, these animals were obtained by hand-collection of specimens. The age of wild-caught animals is usually estimated from body size ("notoriously unreliable", according to the authors) or from internal shell diameter. These authors studied the effect of stocking density on growth rate and time to sequal maturity in laboratory Aplysia spp. The study was conducted over a 1-year period, as Aplysia californica completes its life cycle in approximately 1 year. Sea hares were reared from juvenile age (100 days) to a senescent age of 365 daysto dtudy groth rate and maturation at different stocking densities. Temperature, light, and food were controlled, and water parameters were "optimized" by a flowthrough seawater caging system. A total of 6 stocking density groups ranged from 1 to 20 per 16-liter cage. Animals maintained at stocking densities >2/cage reached sexual maturity at the same age; animals maintained at the lowest stocking densities reached sexual maturity at a moderately later age. Most importantly, stocking densities influenced growth rates; growth rates were highest in cages with low stocking densities, and lowest in high-density cages. The authors conclude that stocking density influences growth rates and plays a role in consistent Aplysia rearing environments.

Questions:
1. The genus and species of the California sea hare is:
a. Sepia officinalis
b. Aplysia californica
c. Limulus poyphemus
d. Sepioteuthis lessoniana
e. Sepia californica
2. The length of the life cycle of the California sea hare is:_____________.
3. T/F The authors found that growth rates of sea hares were highest in cages with the lowest stocking densities and lowest in cages with the highest stocking densities.
4. The California sea hare has been an animal model for:
a. Cardiac studies
b. Neoplasia research
c. Neurobiological studies
d. Food sources for other aquatic animals

Answers:
1. b
2. approximately 1 year
3. True
4. c

Use of a novel abdominal wrap for protection of indwelling catheter exit sites in sheep. Contemporary Topics 41 (6): 24.
Due to problems in previous studies with nylon mesh and subsequent site infection with Staphylococcus (both alpha hemolytic and gamma non-hemolytic), coagulase-negative Streptococcus, Enterbacter cloaca, and Klebsiella oxytoca, these authors developed an abdominal wrap for protection of indwelling catheters in the femoral artery and vein. The catheters were placed as part of a chronic drug study. The authors found that the wrap provided protection of the catheter and exit sites, with no problems associated with catheters being chewed, inflammation, or infection for 22 weeks postoperatively.

Questions:
1. What is the genus and species of sheep?
2. What is the estrous cycle of sheep?
3. What is the gestation period of sheep?

Answers:
1. Ovis aries
2. Seasonally Polyestrous 16-17 days (fall/winter only-short-day breeders)
3. 144-151 days

What is your diagnosis? Multifocal subcutaneous tumors in a young male baboon. Contemporary Topics 41 (6): 27.
Signalment: 2-3 year old male baboon (wild caught) Negative for antibodies to simian hemorrhagic fever (SHF) and simian T-cell lymphrotrophic virus type I (STLV-1). Had three soft subcutaneous tissue masses noted on the elbow, wrist, and knee upon physical examination. The masses were firm, freely moveable, and ranged in size from 3 X 5 cm to 2.5 X 1.5 cm. An excisional biopsy of the knee mass was performed. Histogenesis was not able to be determined from the histopathology. The animal was euthanized for a full necropsy. At necropsy, there was no visible involvement of bone or skin on gross examination. All masses were found to infiltrate connective tissue and underlying musculature. On cut surface, the masses were found to have a necrotic core. All organs were within normal limits on gross examination. Histologically, the masses were composed of nests and cords of large uniformly round to slightly angular cells with a vesiculated cytoplasm, distinct cell borders, and a large oval nucleus with finely dispersed or occasionally vesiculated chromatin and a large central magenta nucleolus. Some cells contained one or multiple small, irregular, eosinophilic cytoplasmic inclusion bodies with rare mitotic figures. The cells were supported by abundant fibrovascular stroma and large fibrous septae that compartmentalized the lesions. Immunohistochemistry (IHC) revealed the large cells to be of mesenchymal, nonmacrophage origin because of positive staining for vimentin and negative for cytokeratin and the macrophage maturation marker CD68. Eosinophilic cytoplasmic inclusions were seen on H&E and corresponded to ovoid to brick-shaped viral particles on transmission electron microscopy (TEM). The masses were determined to be Yaba monkey tumor virus (YMTV) induced benign histiocytomas. This is a naturally occurring zoonotic disease of baboons and macaques. Lesions are usually found on the head and limbs.The lesions are regarded as benign and usually regress in 2-3 months. It appears that the lesions may be able to reappear at different locations on the body at some point in the future. Multifocal occurrence without a clear primary metastasis, and the presence of intracytoplasmic inclusion bodies are helpful in regards to the diagnosis of Yaba disease. However, the definitive diagnosis may require TEM or virus detection by IHC, isolation, or molecular diagnostic approaches. Although YMTV-induced disease has been a relatively uncommon finding among nonhuman primates used in research, several cases have been described previously.

Questions:
Q: What is the genus of the baboon?
Q: What type of virus is Yaba?
Q: What type of cells does this virus infect, and how does this differ from most poxviruses?
Q: What is the mode of transmission of Yaba?

Answers:
A: Papio
A: A poxvirus in the genus Yatapoxvirus.
A: Yaba infects subcutaneous mesenchymal cells whereas most poxviruses infect epithelial cells.
A: Unknown, although arthropod vectors and trauma have been suggested as possible mechanisms.

Changes in hepatic and renal enzyme concentrations and heart and respiratory rates in New Zealand white rabbits after anesthetic treatments. Contemporary Topics 41 (6): 30.
This study illustrates the effects of intravenous ketamine-xylazine or ketamine-diazepam on liver and renal enzyme values in New Zealand White rabbits. Blood samples were obtained from the central ear artery before injection of anesthetic agents and at time points of 10, 30, 60, 120 minutes, and 24 hours following administration of anesthesia. Authors found significant elevations of the following enzymes: ALT, AST, BUN, and Creatinine. Authors concluded that both anesthetic combinations induced changes in hepatic and renal biochemical parameters, as well as heart and respiratory rates.

Questions:
1. Which feature distinguishes the sex of a rabbit?
a. urethral orifice
b. inguinal canal
c. inguinal pouch
2. Rabbit urine is normally cloudy due to which type(s) of crystals?
a. calcium carbonate c. both of the above
b. magnesium phosphate d. none of the above
3. Which structure comprises only 7% of total body weight in rabbits?
a. teeth c. GI tract
b. skin d. skeleton
4. Which are comprised of lymphoid tissue in rabbits? 

a. sacculus rotundus c. cecal appendix
b. Peyer's patches d. all of the above

Answers:
1. a 2. c 3. d 4. d

A unique application to the IACUC for studies of wild animals in or from natural settings. Contemporary Topics 41 (6): 33.
Background: The authors intent was to develop an IACUC protocol form that was not only responsive to the policies and guidelines advanced by the Guide for the care and use of traditional laboratory animal species but also applicable for captured wildlife species and field studies. Policies developed from the Health Research Extension Act of l985 ( PL 99-158), the Guide and PHS policy, are explicit for the traditional laboratory animal species but do not mention wild animals that may be captured for laboratory study and/or observed in the field after tagging with various techniques. Within three years of the promulgation of this act, the major biological societies covering vertebrate animals, not traditionally used in biomedical research, had published recommendations for observation as well as capture/restraint/caging of wild life species for studies. The following areas were addressed specifically for additional input when an investigator is proposing field studies on feral species. Response to the guidelines established by various vertebrate societies, the Guide and PHS policy, the IACUC at this institution developed a specialized application titled: Application for the Study of Wild Animals in or from Natural Settings

Wildlife Permits: 1. Knowledge of the regulations pertaining to the specific animals proposed for a study is entrusted to the applicant biologist who must obtain permits necessary for carrying out the study. Copies of said permits are submitted with the Application for the Study of Wild Animals in or from Natural Settings. 2. It was pointed out that not only are there broad range permits related to the capture and tagging of wild animals, but the site on which the study is conducted (often protected State and/or Federal lands) may also require a permit. In some instances there are species specific permits (The American Bald Eagle and the Golden Eagle).

Characteristics and Number of Animals: 1. This institutional IACUC had to come to grips with the fact that the numbers of animals anticipated in these studies was grossly larger than what they had come to normalize in the biomedical research environment. 2. They also had to contend with the idea that the field study is a much more dynamic program than the lab bench experiment requiring some generalization and flexibility where they had once been quite rigid

Capture and Restraint Techniques: 1. Restraint of laboratory species, even large ones, has evolved with development of biomedical research and has become second nature. This IACUC was cautioned that there are restraint techniques that are definitely injurious to certain genera and to be avoided, including: use of gill nets, electrocution, ichthyocides, hooks and spears (fish): trapping and netting (amphibians , birds, amphibians and mammals) and drug darts (mammals). Once in the laboratory, restraint and procedures should involved the use of appropriate sedation and/or analgesia as applied for other laboratory animal species.

Assurances: 1. In the realm of assurance of proper training, field biologists is placed in a "Catch 22" situation. While they are prodded by the IACUC to be firm on the number and kinds of animals to be used, the study itself is often an investigation to further the knowledge of at least one species and perhaps the interactions of multiple species which may dictate that for completeness more or less numbers of any genera/sex/age/hair coat; pillage or skin color etc. may need to be taken-

Marking: 1. Most of the techniques used for laboratory animal species are applicable to some or all of the various bird, amphibian, mammals and reptile genera collected in field studies. In the wild, however, increased effort must be made to prevent markings that put the animal at higher risk for infection; reduce its suitability as a mate; reduce the ability to cope with normal environmental rigors or present high stress of restraint to effect such markings.

Euthanasia/Specimen Collection: 1. Tissue sampling should proceed with the same sterile techniques used for laboratory animal species with the application of antibiotics/analgesics as appropriate. The 2000 AVMA Panel on Euthanasia is a good basis for determining appropriate euthanasia methods for wildlife. Unanesthetized animals are not to be directly placed in preservative fluids.

Health Risks: 1. While the risk from enteric pathogens and parasites is probably not unlike some research facilities dealing with laboratory animal research, the increased risk of rabies and tetanus should be preempted with booster vaccinations and/or at least titer checks for individuals working in the field.

Release: 1. Unless there are laws or policies to the contrary; the wild population is not put at risk and contingent upon unimpaired ability to survive, animals captured for study should be released as soon as the study is completed, in the area from which captured and during a time when most likely to survive

Questions:
1. What are acceptable marking techniques for fish, amphibians, reptiles and birds?
2. With respect to import and export of animals, the acronym CITES stands for _________________________?
3. With respect to Federal Laws, the acronym CFR seen associated with many laws related to the biomedical research community, stands for __________________?
4. What were the six (6) key points of the Health Research Extension Act of 1985?

Answers:
1. Fish: Electrocautery; Radioisotopes; Fin Clipping; Tagging; Freeze-Branding and Radiotelemetry.
Amphibians: Electrocautery; Tattooing; Radioisotopes: Tagging: Banding; Radiotelemetry and Branding.
Reptiles: Electrocautery; Tattooing; Radioisotopes; Scale Clipping; Tagging; Banding; Radiotelemetry; Branding and Shell Marking.
Birds: Tagging; Banding; Dyes; Collars and Radiotelemetry
2. CITES stands for Convention on International Trade in Endangered Species (Wildlife and Fauna). Parenthetical is sometimes added when CITES is written in full.
3. CFR stands for the Code of Federal Regulations.
4. The Health Research Extension Act of 1985 included regulatory power for:
a. Use of Animals
b. Experimental Procedures
c. Administration of Anesthesia
d. Euthanasia
e. Research Techniques
f. Included any proposed scientific study that involved free-living wild vertebrate animals.