Variable immune cell frequencies in peripheral blood of LEW.1AR1-iddm rats over time compared to other congenic LEW strains.

The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes (T1D), which arose through a spontaneous mutation within the major histocompatibility complex (MHC)-congenic background strain LEW.1AR1. The LEW.1AR1-iddm rat is characterized by two phenotypes: diabetes development with a diabetes incidence of 60% and a variable T cell frequency in peripheral blood. In this study the immune cell repertoire of LEW.1AR1-iddm rats was analysed over time from days 30 to 90 of life and compared to the background strain LEW.1AR1 and the LEW rat strain as well as the LEW.1WR1 rat strain. The LEW.1AR1-iddm rats are characterized by a high variability of CD3(+), CD4(+) and CD8(+) T cell frequencies in peripheral blood over time, and the frequency is unique for each animal. The variability within the frequencies resulted in changes of the CD4(+) : CD8(+) T cell ratio. The other three rat strains studied were characterized by a stable but nevertheless strain-specific T cell frequency resulting in a specific CD4(+) : CD8(+) T cell ratio. The frequency of natural killer (NK) cells and B cells in LEW.1AR1-iddm rats was increased, with a higher variability compared to the other strains. Only monocytes showed no differences in frequency and variability between all strains studied. These variabilities of immune cell frequencies in the LEW.1AR1-iddm rats might lead to imbalances between autoreactive and regulatory T cells in peripheral blood as a prerequisite for diabetes development.

Systemic treatment with 4-211Atphenylalanine enhances survival of rats with intracranial glioblastoma.

OBJECTIVE: Increased amino acid transport in brain tumours is used for diagnostic purposes. It has been shown that the α-emitting radionuclide astatine-211 labeled to L-phenylalanine is taken up by glioblastoma cells. We here tested, if systemic treatment with 4-[211At]astatine-phenylalanine (At-Phe) has a beneficial effect on survival of rats with intracranial glioblastoma. ANIMALS, METHODS: The rat glioblastoma cell line BT4Ca was implanted into the prefrontal cortex of female BDIX rats by stereotaxic microinjection (10,000 cells/3 µl; n = 83). 3 days after implantation At-Phe or phosphate buffered saline were injected intravenously. A third group was treated twice, i.e., on day 3 and 10. Health condition was assessed each day by using a score system. Rats were sacrificed on days 6, 10, 13 and 17 after implantation, or when showing premortal health condition to measure tumour volume and necrosis. The proliferation index (PI) was assessed after immunohistochemical staining of Ki-67. RESULTS: Survival time of rats treated twice with At-Phe was significantly prolonged. Additionally, both At-Phe-treated groups remained significantly longer in a better health condition. Rats with poor health status had larger tumours than rats with fair health condition. Overall, irrespective of treatment the PI was reduced in rats with poor health condition. Necrosis was larger in rats treated twice with At-Phe. CONCLUSION: Intravenous treatment with At-Phe enhanced survival time of rats with intracranial glioblastomas and improved health condition. These results encourage studies using local treatment of intracranial glioblastoma with At-Phe, either by repeated local injection or by intracavital application after tumour resection.

ICLAS Working Group on Harmonization: international guidance concerning the production care and use of genetically-altered animals.

Replacement, Reduction and Refinement, the 'Three Rs' of Russell & Burch, are accepted worldwide as fundamental to the ethics of animal experimentation. The production, care and use of genetically-altered animals can pose particular challenges to the implementation of the Three Rs,1 necessitating additional considerations by those responsible for overseeing the ethical use and appropriate care of animals involved in science. The International Council for Laboratory Animal Science brings representatives of the international laboratory animal science community together to recommend acceptance of guidance documents.The harmonization of guidance concerning genetically-altered animals was seen as a priority because of the increasing globalization of research involving these animals.

Treatment of male mice with gonadotropins to improve the fertilization rate and reproduction.

The possibility of modifying the genome in mice has led to an exponential increase in the number of strains that have been developed for biomedical research. This will continue during the next few decades because international programmes plan to develop genetically-modified strains for every known mouse gene. Due to our own experiences and that of colleagues we know that the reproductive performance of many of these modified stains is impeded, despite that the modification is independent from genes that control reproduction. In some cases the spermatogenesis might be disturbed. The reason presumably lies in a defective endocrine function of the testes. This can cause reduced and/or abnormal sperm production. In livestock as well as in humans these disorders can be treated with gonadotropins. One treatment period lasts for the duration of spermatogenesis of the respective species. Up to now, nothing is known about such treatments in laboratory mice to restore or increase reproduction of genetically-modified strains. Spermatogenesis in the mouse lasts approximately 35 days. Therefore, we treated sexually mature male mice of C57BL/6 and BALB/c strains with gonadotropins for this period. The aim of this study was to test the principle suitability of such treatment for the improvement of sperm count, sperm motility, fertilization ability and reproduction.

A safety and toxicity assessment of the administration of multiple intracerebral injections of irinotecan or doxorubicin drug-eluting beads.

OBJECTIVE Previous research in a rat glioma model has shown that the local intratumoral application of polymerbased drug-eluting beads (DEBs) loaded with doxorubicin or irinotecan suppress tumour growth and prolong survival. For translation into a clinical setting, the present experiment investigates in the healthy cat brain the local and systemic toxicity of a multiple injection shot technique. METHODS Three injection shots were placed, each at a 1 cm distance in the frontal lobe. The DEBs were suspended in an aqueous alginate excipient solution, which becomes subject to a sol-gel transition when injected into the Ca(2+)- rich brain tissue environment. Systemic and local side effects were monitored over a period of two weeks. Injection sites were histologically investigated. RESULTS Gelling of the alginate results in the permanent immobilisation of the microspheres at the implantation site. A distinct local cytotoxic effect of doxorubicin was found with intracerebral and intraventricular haemorrhages, and signs of brain tissue necrosis. In cats injected with irinotecan DEBs, such local adverse side effects did not occur. No signs of systemic toxicity were found with both chemotherapeutics. DISCUSSION We conclude that the multiple injection shot technique with irinotecan DEBs meets feasibility criteria and safety requirements for a clinical application.

Successful sanitation of an EDIM-infected mouse colony by breeding cessation.

Despite decreasing prevalence, rotavirus infections still rank among the most important viral infections in colonies of laboratory mice. Although the disease is characterized by low mortality and a relatively short and mild clinical period, the infection has the potential to alter the outcome of experiments substantially. For animal facilities, it is therefore essential to eradicate the virus. Here we report a successful sanitation of a rotavirus-infected mouse colony in an animal facility. Despite a high ratio of transgenic and partially immunodeficient strains, a permanent eradication of the virus was achieved by euthanasia of highly susceptible mice, a prolonged breeding cessation in areas containing immunocompromised mice and a strict hygienic management. The management of a rotavirus infection reported here is a feasible and inexpensive opportunity for sanitation that benefits from maintaining most of the animal population, even in today's mouse colonies comprising mainly transgenic mice with unknown or compromised immune status.

Genetic dissection of granulomatous enterocolitis and arthritis in the intramural peptidoglycan-polysaccharide-treated rat model of IBD.

BACKGROUND: Inflammatory arthropathies are common extraintestinal manifestations of inflammatory bowel diseases (IBD). As genetic susceptibility plays an important role in the etiology of IBD, we questioned how granulomatous enterocolitis and arthritis are genetically controlled in an experimental animal model displaying both conditions. METHODS: Chronic intestinal and systemic inflammation was induced by intramural injection of peptidoglycan-polysaccharide (PG-PS) polymers in the ileocecal region of female F2 progeny derived from susceptible LEW and resistant F344 rats. Animals were followed for 24 days after injection and phenotyped by evaluating gross gut lesions, liver weight and granulomas, hematocrit, white blood cell count, and change in rear ankle joint diameters. Coinheritance of the phenotypic parameters with polymorphic DNA markers was analyzed by genome-wide quantitative trait locus (QTL) analysis. RESULTS: Linkage analysis revealed significant QTLs for enterocolitis and/or related phenotypes (liver granulomas, white blood cell count) on chromosomes 8 and 17. The QTL on chromosome 8 also showed suggestive linkage to arthritis. Significant QTLs for arthritis were detected on chromosomes 10, 13, 15, and 17. Analyses of the modes of inheritance showed arthritogenic contributions by both parental genomes. In addition, several other loci with suggestive evidence for linkage to 1 or several phenotypes were found. CONCLUSIONS: Susceptibility to PG-PS-induced chronic intestinal and systemic inflammation in rats is under complex multigenic control in which the genetic loci regulating arthritis are largely different from those controlling enterocolitis. Possible candidate genes within these QTL (including Tnfrsf11a/RANK, Gpc5, Il2ra, and Nfrkb) are also implicated in the respective human diseases.

Prevention of spontaneous immune-mediated diabetes development in the LEW.1AR1-iddm rat by selective CD8+ T cell transfer is associated with a cytokine shift in the pancreas-draining lymph nodes.

AIMS/HYPOTHESIS: The LEW.1AR1-iddm rat is an animal model of spontaneous type 1 diabetes mellitus. This study analysed how adoptive transfer of selective T cell subpopulations affects the incidence of diabetes. METHODS: CD4(+) or CD8(+) T cells were isolated from diabetic LEW.1AR1-iddm rats or diabetes-resistant LEW.1AR1 rats. Cells were selectively transferred into athymic LEW.1AR1-Whn ( rnu ) or prediabetic LEW.1AR1-iddm rats. The animals were monitored for blood glucose, islet infiltration and immune cell composition of pancreas-draining lymph nodes. RESULTS: After adoptive transfer of CD4(+) T cells from diabetic LEW.1AR1-iddm rats into athymic LEW.1AR1-Whn ( rnu ) rats, 50% of the recipients developed diabetes. Transfer of CD8(+) T cells failed to induce diabetes. Only 10% of the athymic recipients became diabetic after co-transfer of CD4(+) and CD8(+) T cells. Adoptive transfer of CD8(+) T cells from LEW.1AR1 or diabetic LEW.1AR1-iddm rats into prediabetic LEW.1AR1-iddm rats significantly reduced the incidence of diabetes. In protected normoglycaemic animals regulatory CD8(+)/CD25(+) and CD4(+)/CD25(+) T cell subpopulations that were also FOXP3-positive accumulated in the pancreas-draining lymph nodes. In this lymphatic organ, gene expression of anti-inflammatory cytokines was significantly higher than in diabetic rats. CONCLUSIONS/INTERPRETATION: Our results show that adoptive transfer of CD4(+) but not CD8(+) T cells from diabetic LEW.1AR1-iddm rats induced diabetes development. Importantly, CD8(+) T cells from diabetic LEW.1AR1-iddm rats and diabetes-resistant LEW.1AR1 rats provided protection against beta cell destruction. The accumulation of regulatory T cells in the pancreas-draining lymph nodes from protected rats indicates that transferred CD8(+) T cells may have beneficial effects in the control of beta cell autoimmunity.

Experimental middle ear surgery in rabbits: a new approach for reconstructing the ossicular chain.

This experimental animal study aimed at evaluating a new prosthesis to replace the ossicular chain; we developed a new technique for surgical implantation into the middle ear of rabbits. The rabbit middle ear is, owing to the relative anatomical dimensions involved, an ideal environment for implantation procedures involving the ossicles, as the surgical conditions are similar to those of the human middle ear. This study included a total of 34 approximately six-month-old female white rabbits (New Zealand) weighing between 3.2 and 4.4 kg. The implants used were constructed of ceramic materials (titania, TiO(2)) of various pore sizes. Directly prior to implanting the total ossicular reconstruction prostheses (TORPs), as well as at 28, 84 and 300 days after implantation, electric response audiometry was used to determine the hearing thresholds of the animals (bone conduction; click stimulus nHL). An erbium:YAG laser was used to excise the original ossicular chain. Following implantation, we were unable to detect any stenosis of the outer ear canal or perforation of the tympanic membrane. The conductive hearing threshold was in the range of 4.21 +/- 6.68 dB nHL (n = 131). The hearing level showed no significant difference before and after surgery (P < 0.05).

Collection and cryopreservation of preimplantation embryos of Cavia porcellus.

Individual differences and a rather long-lasting reproductive cycle, as well as the relatively small number of oocytes that mature during one reproductive cycle makes it difficult to establish a cryopreserved stock of preimplantation embryos of the guineapig (Cavia porcellus) when compared with other laboratory rodents. Only a few data for superovulation protocols that can be used for routine laboratory use in guineapigs are available. However, a huge number of different strains exist for many purposes and the establishment of a frozen repository makes sense. Here, we describe the successful freezing of preimplatation embryos of the strain 2BS with a two-step freezing protocol in a freezing medium containing 1,2-propanediol as cryoprotectant. Human menopausal gonodotrophin induced superovulation in the embryo donors.

Control of Toxoplasma gondii infection by athymic LEW-Whn rnu rats.

In immunocompetent rats and humans infection with Toxoplasma gondii remains mostly without overt clinical symptoms, but can be fatal, if the T-cell response is impaired. For a better understanding of the lack of control of T. gondii infection under immunosuppressed conditions, congenitally athymic rats were used as the experimental model. Whereas athymic F344-Whn(rnu) (F344 nude) rats die from a generalized infection during the first 3 weeks after peritoneal inoculation with 10(6) tachyzoites of T. gondii strain NTE, LEW-Whn(rnu) (LEW nude) rats and euthymic LEW rats infected with a 10-fold higher number of parasites developed chronic infection. To identify underlying mechanisms of LEW rats resistance to T. gondii infection and to investigate a possible contribution of residual T-cells to LEW-Whn(rnu) rat resistance, we characterized the immune response of LEW rats by determination of cellularity and composition of lymphocyte population, antigen-specific IgG2b response as well as assays of antigen-specific proliferation and production of IL-2, IFN-gamma and TNF-alpha. As only euthymic LEW rats developed production of antigen-specific IgG and cellular in vitro responses, these results strongly suggest that the genetic background of LEW rats permits a control of the infection independent of an adaptive immune response.

Characterisation of electrogenic nutrient absorption in the Cftr TgH(neoim)Hgu mouse model.

Most cystic fibrosis (CF) patients show an exocrine pancreatic insufficiency that results in lower enzyme and bicarbonate secretion. To test whether an altered function of nutrient transporters might additionally attribute to the lower bodyweight of CF patients we investigated electrogenic absorption of alanine, glycyl-glutamine, glucose and the effect of pH on nutrient absorption by Ussing chambers in a CF mouse model carrying the Cftr TgH(neoim)Hgu mutation. The electrogenic transport of all three nutrients was similar between the D2.129P2(CF/3)-Cftr TgH(neoim)Hgu congenic strain and DBA/2J mice as well as between the B6.129P2(CF/3)-Cftr TgH(neoim)Hgu congenic strain and C57BL/6J mice. This indicates that the Cftr TgH(neoim)Hgu mutation does not affect the electrogenic absorption of alanine, glycyl-glutamine and glucose. In contrast, electrogenic nutrient absorption was reduced in the CF/1-Cftr TgH(neoim)Hgu and CF/3-Cftr TgH(neoim)Hgu inbred strains compared to the HsdOla:MF1, D2.129P2(CF/3)-Cftr TgH(neoim)Hgu and B6.129P2(CF/3)-Cftr TgH(neoim)Hgu strains, whereas no difference was found among the wild-type strains. This indicates that not the Cftr TgH(neoim)Hgu mutation but differences in the genetic background of the CF/1-Cftr TgH(neoim)Hgu and CF/3-Cftr TgH(neoim)Hgu strains compared to HsdOla:MF1, D2.129P2(CF/3)-Cftr TgH(neoim)Hgu and B6.129P2(CF/3)-Cftr TgH(neoim)Hgu strains are associated with the differences in electrogenic nutrient absorption. The electrogenic absorption of alanine, glycyl-glutamine and glucose was not influenced by an acidic pH (5.4) compared to absorption at pH 7.4.

FELASA guidelines for the production and nomenclature of transgenic rodents.

The standardized nomenclature of rodent strains, genes and mutations has long been the focus of careful attention. Its aim is to provide proper designation of laboratory animals used in research projects and to convey as much information on each strain as possible. Since the development of different techniques to mutate the genome of laboratory rodents on a large scale, the correct application of current nomenclature systems is of increased significance. It facilitates not only the accurate communication of scientific results but is indispensable in controlling the dramatically increased number of transgenic animal models in experimental units, archives and databases. It is regrettable that many publications, especially on transgenic rodents, use vague and inappropriate strain designation. This situation should definitely be improved, particularly considering the increasingly emphasized importance of genetic background on the phenotype of mutations. The aim of these guidelines is to raise awareness about specific features of production and of the current nomenclature system used for transgenic rodents.

Rederivation of Helicobacter hepaticus-infected Mongolian gerbils by Caesarean section and cross-fostering to rats and mice.

The Mongolian gerbil serves as an animal model for a wide range of diseases. As these animals are extensively used for the study of Helicobacter pylori-induced gastritis, naturally occurring infections with rodent Helicobacter species in gerbils are a possible source of interference in studies of H. pylori-associated disease. The gerbil stock at the Central Animal Facility in Hannover was persistently infected with H. hepaticus. The aim of this study was to derive Helicobacter species-free Mongolian gerbils. Therefore, germfree gerbil pups were obtained by Caesarean section and the pups were transferred to female rats and mice with recently delivered litters. In total, four Ztm:NMRI mice, four Ztm:SPRD rats and one DA/Ztm rat that originated from a specified pathogen-free area were selected to serve as foster mothers. With this approach, it was possible to obtain Helicobacter-free gerbils. Rearing by mice was more successful than by rats, as six of nine gerbils were reared by mice, but only one of 29 gerbils was reared by rats.

Genetic analysis of susceptibility to endometrial adenocarcinoma in the BDII rat model.

Most cancers are genetically complex and heterogeneous, a serious obstacle to identifying specific genes underlying the disease. If inbred animal models are used, then both the genetic constitution and environmental influences can be carefully controlled. Females of the BDII inbred rat strain are genetically predisposed to endometrial cancer; more than 90% of virgin BDII females will develop endometrial adenocarcinoma (EAC) during their life span. BDII females were crossed to males from inbred strains with low EAC incidence (SPRD or BN). When F(1) males were backcrossed to BDII females to generate N(1) populations of offspring, about one fourth of the female progeny developed EAC. With transmission disequilibrium test analysis, significant association was detected in three chromosomal regions (on RNO1, RNO11, and RNO17) in the SPRD crosses and in the short arm of RNO20 in the BN crosses. It appears that several susceptibility genes with minor but cooperating effects are responsible for the susceptibility. Furthermore, it seems clear from the interstrain crosses not only that the onset of tumors depends on the presence of susceptibility alleles from the EAC-prone BDII strain, but also that tumor development is affected by the contribution of a genetic component derived from the nonsusceptible strains.

WKY/Ztm-ter: a new rat inbred strain on the WKY/Ztm genetic background with congenital teratomas.

We describe a new rat model for teratomas (WKY/Ztm-ter) which arose through a spontaneous mutation in the inbred WKY/Ztm rat strain. When the tumours of the gonads became clinically apparent, affected males were 14 to 224 days of age, whereas the females only developed tumours between days 21 and 63. Tumour incidence is not gender-dependent. However, almost all females develop bilateral tumours, while 50% of the males show unilateral tumours. Histologically, all examined tumours (n = 65) represent partially undifferentiated teratocarcinomas.

Orthotopic transplantation of rat ovaries as a tool for strain rescue.

Spontaneous mutations in inbred strains provide valuable resources for new disease models. Unfortunately, these mutations may affect reproduction, which require considerable efforts in breeding management. We transplanted ovaries of such mutant rat strains orthotopically into ovariectomized premature coisogenic recipients. A reproductive cycle was established in each of the recipients within 5 to 6 weeks after transplantation. Moreover, one-third became pregnant and had litters an average of 3 months after transplantation. These experiments demonstrate that orthotopic transplantation of ovaries can be used in the management of subfertile rat colonies.

Does preoperative administration of metamizol (Novalgin) affect postoperative body weight and duration of recovery from ketamine-xylazine anaesthesia in mice undergoing embryo transfer: a preliminary report.

Thirty-two female mice used for embryo transfer or as controls received either metamizol or equal volumes of normal saline administered subcutaneously following induction of anaesthesia with ketamine-xylazine. Body weight was measured immediately before surgery, after 24 and after 48 h. The duration of the surgical anaesthesia was recorded and postoperative behavioural responses were measured. Comparison of the treatment groups revealed no significant differences in body weight and recovery times, nor were other signs of discomfort detected in either treatment group. It was concluded that administration of metamizol did not provide additional analgesia following embryo transfer in mice anaesthetized with ketamine-xylazine.

Human alpha2-macroglobulin: genotype-phenotype relation.

A pentanucleotide deletion polymorphism in the gene of alpha2-macrolgobulin (alpha2-M) is suggested to be associated with late-onset Alzheimer's disease (AD), though controversial results have been reported. The underlying assumption is that the intronic pentanucleotide deletion may affect the biological function and quantity of the inhibitor and thus contribute to the AD pathology. In the present study we have analyzed the distribution of the deletion polymorphism within a group of 227 healthy Caucasians. In parallel studies, we determined the plasma concentrations of total and transformed alpha2-M. A strong correlation of the total concentration of alpha2-M with age was ascertained (r(s) = -0.54, P < 0.001). However, no significant correlation between age and the genotypes (P = 0.68) was detected, and no statistically significant effect of the genotype on the concentrations of total and transformed alpha2-M was found (P = 0.49 and 0.96, respectively). A significant correlation was observed between total and transformed alpha2-M in the genotype groups Ins/Ins (r(s) = 0.56, P < 0.001) and Ins/Del (r(s) = 0.35, P < 0.004). Furthermore, in the entire data set, a significantly elevated concentration of total alpha2-M was found in females as compared to males (P = 0.003). There was a slight but nonsignificant difference in the genotype distributions between males and females (P = 0.14). To test the proposed existence of genotype-specific alterations of functional properties of alpha2-M, we isolated alpha2-M from the plasma of carriers with different genetic background and analyzed the alpha2-M subunit structure as well as the binding of the inhibitor to growth factors/cytokines, to amyloid-beta and to the receptor. The experiments failed to reveal any genotype-specific functional alterations of the alpha2-M. The absence of abnormalities in alpha2-M mRNA and protein suggests that the alpha2-M deletion polymorphism is probably not associated with functional deficiencies important in AD pathology. However, it can be speculated that the observed general age-related alpha2-M deficiency may lead to accelerated accumulation of amyloid-beta, which might be relevant to AD pathology.

Are the effects of different enrichment designs on the physiology and behaviour of DBA/2 mice consistent?

Environmental enrichment is intended to improve the well-being of laboratory animals. Although many researchers have indicated that environmental enrichment may enhance animal well-being, there is some evidence that enrichment differs in its effects on physiology and behaviour between species and strains. The present study focuses on the effects of different enrichment designs on the physiology and behaviour of male and female DBA/2 mice. A total of 48 DBA/2J mice, 24 males and 24 females were used for this experiment. Upon arrival at about 3 weeks of age, the animals were randomly allotted to three experimental groups: NE, non-enrichment; E1, enriched with nest box, wooden climbing bar and nest material according to Scharmann (1993); E2, enriched with horizontal and vertical dividers, modified from Haemisch and Gärtner (1994). Same-sex groups of four mice were housed for 12 weeks in type III Makrolon cages with (E1 or E2) or without (NE) enrichment objects. Behavioural performance (Open Field, Food Drive and Elevated Plus Maze tests) and physiological traits (haematological variables, body weight and organ weights, corticosterone and thyroxine levels) were measured. This study observed that enrichment had significant effects on the mean values of body weight (females), Open Field and Food Drive tests. The most significant housing differences were found between the E2 and NE/E1 groups. Furthermore, sex differences in the NE, E1 and E2 groups were not consistent for several variables (growth rate, relative weights of spleen, kidney and heart, Food Drive and Elevated Plus Maze behavioural performance). There was often a higher coefficient of variation (CV) in the E1 and E2 groups as compared to the NE group, chiefly in physiological traits and in the Open Field and Food Drive tests. The results of this study indicate, that the effects of enrichment designs used in the present study are not consistent, but vary according to sex and the variable studied.