There has been increasing interest in the use of selected genetically modified (GM) mouse models for the testing of chemicals to determine their carcinogenic potential. GM mouse models are believed to be useful tools that offer mechanistically relevant insights for understanding and predicting the human response to chemical exposure. They have been proposed as alternatives to the traditional 2-year mouse oncogenicity bioassay. In this overview we will review the GM mouse models that have been proposed as bioassay alternatives and present some of the key laboratory animal science challenges that need to be considered when using these unique animals.

Disease-susceptible (C3H) and -resistant (B6) immunocompetent and immunodeficient (C3H- scid and B6- rag1) mice were examined up to 10 weeks after inoculation with Helicobacter bilis (a prototype species of proven virulence). Infection was monitored weekly by use of fecal culture, polymerase chain reaction (PCR) nucleic acid amplification, membrane extract enzyme-linked immunosorbent assay (ELISA), and histologic examination. All mice became infected by three to five weeks after inoculation, on the basis of results of culture and PCR analysis of feces. The PCR analysis was more sensitive than culture at determining infection status, particularly during early infection. None of the mice had evidence of disease by week 10. Immunoglobulin G seroconversion was detectable in C3H mice by week eight and in B6 mice by week nine. Results indicated that culture and PCR analysis are more sensitive than is membrane extract ELISA serologic testing for detecting early infection in individual mice, regardless of genotype or immune status. Results underscore the need for improved seroassays for this important group of murine pathogens.

Helicobacter hepaticus infection causes hepatitis in A/JCr mice but mild or no disease in C57BL/6 mice. Colonization of H. hepaticus in the cecum of experimentally infected A/JCr and C57BL/6 mice was quantified by use of real-time polymerase chain reaction (PCR) analysis with primers for the H. hepaticus cdtB gene and mouse 18srRNA. Eight-weekold mice were experimentally (n = 48) or sham (n = 24) infected with H. hepaticus, then were necropsied 6 months after infection. Liver specimens from experimentally infected mice had negative results of PCR analysis for H. hepaticus ; thus, real-time quantification was not attempted. Quantitative PCR analysis of H. hepaticus in cecal specimens indicated that C57BL/6 mice were colonized to a greater extent than were A/JCr mice (P < 0.006). Appreciable typhlitis was not observed, but was consistent with that of previous reports; A/JCr mice developed more severe parenchymal necrosis, portal inflammation, and phlebitis in the liver (P < 0.0001), with mild disease observed in infected C57BL/6 mice. Thus, hepatitis in A/JCr mice caused by H. hepaticus infection is associated with significantly lower colonization levels of H. hepaticus in the cecum, compared with those of hepatitis-resistant C57BL/6 mice. Host responses of A/JCr mice that limit cecal colonization with H. hepaticus may have important roles in the pathogenesis of hepatic lesions.

Purpose: Helicobacter pylori is a human gastroduodenal pathogen associated with type-B gastritis and gastric cancer. Low gastric tissue antioxidant levels are believed to increase the risk of developing gastric cancer. We investigated whether dietary antioxidant levels protect against infection and type-B gastritis in H. pylori-infected guinea pigs. Methods: Dunkin-Hartley guinea pigs infected for 6 weeks with H. pylori were fed diets with various antioxidant levels. Stomach specimens were cultured, and gastritis was graded from 0 to 3. Results: Supplementation with vitamins A, C, and E and with selenium yielded H. pylori recovery from 17% of challenged animals, compared with 43% of those fed a control diet. Gastritis was scored at 0.33 and 0.93, respectively. Supplementation with only vitamin C or astaxanthin had less effect on gastritis and recovery rate. In a second experiment, gastritis score in a group given vitamins A, C, E, and selenium and β -carotene was 2.25 and in a control group, it was 2.57. The H. pylori recovery rate was 75 and 100%, respectively, with fewer colonies from animals given antioxidant supplementation (P < 0.05). Conclusions: A combination of antioxidants can protect against H. pylori infection in guinea pigs. In animal studies, antioxidant intake should be low to optimize development of H. pylori-associated disease. Furthermore we established that H. pylori causes severe gastritis in guinea pigs.

Purpose: The objective of the study reported here was to explore whether a nonhuman primate model could be developed for chemoprevention of ovarian cancer. Methods: An initial feasibility trial was done with three monkeys to determine tolerance for these drugs and for acquisition of surgical ovarian biopsy specimens. In the study, 19 female adult Macacca mulatta (rhesus macaques) were given fenretinide (4HPR) oral contraceptive (OCP), the combination of 4HPR+OCP, or no medication for three months. Laparotomy was performed before and after drug administration, and ovarian biopsy specimens were obtained to evaluate the potential for this animal as a model for ovarian cancer chemoprevention, as well as evaluating fluorescence spectroscopy and other potential biomarkers for ovarian cancer prevention studies. Results: The monkeys tolerated the drugs, surgeries, and acquisition of multiple ovarian biopsy specimens with resultant minimal morbidity. On initial data analysis, fluorescence spectroscopy was the marker that appeared the most promising. Conclusions: On the basis of results of this study, this model merits further investigation. The rhesus monkey is an excellent candidate for a nonhuman primate model for ovarian cancer chemoprevention.

Purpose: To investigate the potential activity of cyclosporin A (CsA) to induce mammary hyperplasia in New Zealand White (NZW) rabbits. Methods: Female NZW rabbits were used throughout experiments. To simulate the conditions of immunosuppression, CsA (10 mg/kg of body weight/d) was administered intravenously on a daily basis for 14 days and methylprednisolone (5 mg/kg/d) was administered on the first two days. The CsA (10 mg/kg/d) also was administered without methylprednisolone for 14 days to another cohort of rabbits. Mammary tissue of each rabbit was palpated and serially measured during this treatment period. The CsA was discontinued, and rabbits were monitored for 14 more days during the washout period. Sequential plasma concentrations of prolactin, 17 β-estradiol, and progesterone in each blood sample were determined by use of radioimmunoassay. Results :All NZW rabbits treated with CsA and methylprednisolone for immunosuppression consistently developed striking mammary tissue hyperplasia. At the end of treatment with CsA and methylprednisolone, mammary glands had extensive changes consistent with actively lactating glands. Similar but less extensive hyperplasia developed in response to CsA alone. Plasma concentration of prolactin increased during treatment and decreased during the washout period. Plasma concentration of 17 β-estradiol increased during treatment and continued to increase during the washout period. Plasma progesterone concentration decreased at the end of treatment. On discontinuation of CsA, mammary hyperplasia regressed. Conclusions: Cyclosporine A, with or without methylprednisolone, induces mammary hyperplasia and hyperprolactinemia in NZW rabbits. This rabbit model may be a reliable in vivo system by which to study immunosuppressantinduced structural and functional changes of mammary glands similar to those observed in humans.

The pig is useful as a model for human physiology and pathophysiology and could be an important supplement to the many available rodent models of diabetes mellitus. Due to their small size, Göttingen minipigs are especially suitable for long-term studies. The aim of the study reported here was to establish reference values for a range of glucose and lipid homeostasis parameters of interest that could be used to identify possible diabetes-prone male Göttingen minipig individuals, families, or age groups. Plasma samples from nonfed animals were analyzed for glucose, leptin, fructosamine, insulin, C-peptide, triglyceride, free fatty acids, and total cholesterol values. Breeding family had significant effects only on plasma triglyceride concentrations (P < 0.001). Plasma concentrations of glucose (P = 0.012), fructosamine ( P < 0.001) and triglycerides (P < 0.001) increased significantly with age, whereas total cholesterol concentration decreased significantly (P = 0.001) with age. Age did not influence other parameters. In conclusion, glycemia and insulinemia increased with age and body weight, possibly indicating a small deterioration in insulin sensitivity with age. It is, therefore, hypothesized that older, compared to younger animals may be more useful in the development of a model of type-2 diabetes mellitus. Furthermore, on the basis of decrease in cholesterol concentration with age, animals fed ad libitum with possibly a high calorie diet might be even more useful in the development of a type-2 diabetes mellitus model.

Purpose: The goals of the study were to find a safe intraperitoneal injection anesthesia protocol for medium-duration surgery in mice (e. g., embryo transfer/vasectomy) coupled with a simple method to assess anesthesia depth under routine laboratory conditions. Methods: Eight anesthetic protocols consisting of combinations of dissociative anesthetics (ketamine, tiletamine), α₂-agonists (xylazine, medetomidine), and/or sedatives (acepromazine, azaperone, zolazepam) were compared for their safety and efficacy (death rate, surgical tolerance), using observations and reflex tests. The four best protocols were further evaluated during vasectomy: physiologic measurements (respiratory rate, electrocardiogram, arterial blood pressure, body temperature, blood gas tensions, and acid-base balance) were used to characterize the quality of anesthesia. The reactions of physiologic parameters to surgical stimuli were used to determine anesthesia depth, and were correlated with reflex test results. Results: The protocol with the highest safety margin and the longest time of surgical tolerance (54 min) was ketamine/ xylazine/acepromazine. Three further anesthetic combinations were associated with surgical tolerance: ketamine/ xylazine, ketamine/xylazine/azaperone, and tiletamine/xylazine/zolazepam (Telazol/xylazine). The protocols consisting of ketamine/medetomidine and ketamine/azaperone were not associated with clearly detectable surgical tolerance. The most reliable parameter of surgical tolerance under routine laboratory conditions was the pedal withdrawal reflex. Conclusions: The best intraperitoneal injection anesthesia regimen consisted of ketamine/xylazine/acepromazine. The dose must be adapted to the particulars of each experimental design (mouse strain, sex, age, mutation). This is best done by measuring surgical tolerance, using the pedal withdrawal reflex.

Guinea pigs (GP; Cavia porcellus) are used extensively as an experimental animal model in a wide range of disciplines including respiratory physiology. Guinea pigs are difficult to anesthetize, and many investigators use paralytic agents to eliminate spontaneous respiratory movements; however, strict federal regulations and institutional policies governing use of paralytic agents are few. We report an anesthesia protocol, using the injectable anesthetic agents sodium pentobarbital (SP) and xylazine (XYL) for the GP that induces consistent anesthesia while eliminating use of paralytic agents. Sixty percent of the calculated SP dose (45 mg/kg of body weight) was given for anesthesia induction, followed by 50% of the calculated XYL dose (7 mg/kg) 15 min later. Depth of anesthesia was monitored by response to toe pinch, ECG, and spontaneous respiratory movements. The animals were given additional boosts of SP (5 to 15% of the original dose, i. p. or i. v.) if a change in anesthesia depth was noted. Thirty-one animals completed the hyperpnea-induced bronchoconstriction (HIB) study with no fatalities. Using this protocol, we collected consistent, repeatable, and reliable data without use of propranolol or skeletal muscle paralytics. We believe that this protocol is not restricted to the GP and could be adapted for use in other terminal experiments.

Male PD strain rats are sterile in the homozygous condition (pd/pd) due to abnormal spermatogenesis detectable at around nine weeks of age. Previous studies have indicated electron microscopic abnormalities in the Sertoli cells of pd/pd males at three and 12 weeks of age. Since spermatogenesis and Sertoli cell function depend on gonadotropins (luteinizing and follicle-stimulating hormones [LH and FSH]) and testosterone, production and/or secretion of these hormones might be altered in pd/pd males. The aim of the study reported here was to investigate the hormonal status of pd/pd males at three, six, and nine weeks of age. Although alteration was not evident in the LHimmunoreactive cells, FSH-immunoreactive cells in pd/pd males were small in size with scant cytoplasm and were reduced in number and area (73 and 51% of phenotypically normal pd/+ males, respectively) at three weeks of age, although serum FSH concentration was similar to that in pd/+ males. At six and nine weeks of age, percentages of the areas occupied by LH- and FSH-immunoreactive cells in pd/pd males were higher than those in pd/+ males. Serum FSH concentration in pd/pd males was significantly high at nine weeks of age, although a difference in serum LH and testosterone concentration was not evident. These results suggest that FSH production in pd/pd males is decreased at three weeks of age. This might be associated with the Sertoli cell abnormalities and subsequent abnormal spermatogenesis seen in adult life.

Background and History: An adult Macaca mulatta was examined because of a history of multiple episodes of conjunctivitis and an acute, pruritic, dermatitic eruption that affected the axillary and inguinal regions, forearms, thorax, and neck. Methods and Results: Results of corneal staining, examination of skin scrapings and feces, fungal culture, CBC, and a thyroid profile (thyroxine/triiodothyronine concentrations) were negative or normal, with the exception of eosinophilia (1,040/mm 3). Examination of a punch biopsy specimen of the skin indicated chronic, nonsuppurative eosinophilic dermatitis. Skin patch testing against 25 contact allergens was negative for a delayed-type hypersensitivity reaction. Allergen-specific IgE testing, using six monkey chow additives, also yielded negative results, but testing against latex revealed a strong positive result (0.74 KU/L) consistent with a latex allergy. A skin prick test performed by use of a latex supernatant revealed significant inflammation at the latex site at 72 h and one week. Vinyl gloves were substituted for latex gloves, and that resulted in a marked decrease in erythema, pruritus, and lichenification with no flares of dermatitis for four years. Repeat skin biopsy fourteen weeks after the original biopsy revealed normal epidermis; however, mild chronic active nonsuppurative, perifolliculitis persisted. Conclusion: Latex can induce allergic dermatitis in nonhuman primates and should be included in the differential diagnosis for atopic dermatitis.