Influence of graft size, histocompatibility,and cryopreservation on reproductive outcome following ovary transplantation in mice.

PURPOSE: Transplantation of ovarian tissue is a valuable method to rescue mouse strains with fertility problems and to revitalize archived strains. The purpose of this study was to investigate the effect of (i) different sizes of transplanted ovary pieces on reproductive outcome, (ii) use of immunodeficient recipients to overcome the limitation of histocompatibility, and (iii) to compare different protocols for cryopreservation of ovarian tissue. METHODS: Halves, quarters, and eights of mouse ovaries were transplanted. Half ovaries from B6 donors were transferred into immunodeficient mice. Halves of ovaries were frozen according to four different protocols, thawed and transferred. RESULTS: Pregnancy rate after transplantation of ovarian tissue was high (90-100%) independent of the transplant size. Although, the average litter size was significantly lower for recipients of quarters and eights (4.4 and 4.6 vs. 6.5), the total number of offspring produced per donor ovary was higher compared with recipients of halves. Pregnancy rate of immunodeficient recipients was 40% (mean 4.7 offspring per litter). All four cryopreservation protocols used were able to preserve functionality of the ovarian tissue. CONCLUSIONS: Transplantation of ovarian tissue smaller than halves resulted in reduced litter sizes. The distribution of ovarian tissue of one donor female to 4 or 8 recipients will therefore yield in a higher total number of offspring in a certain time period. The use of immunodeficient recipients is an option for non-histocompatible donors. Cryopreservation of ovarian tissue is generally feasible but the function of frozen-thawed ovary halves after transplantation differs depending on the freezing protocol used.

Welfare assessment in rabbits raised for meat and laboratory purposes in enclosures with two floor types: Perforated plastic with holes versus slats.

Due to welfare concerns and legal restrictions in certain countries, alternatives to wire net floors must be developed in rabbit husbandries. Also, there is a difference in regulations in Europe for laboratory rabbits vs. rabbits bred and kept for meat production. While there are regulations concerning floor design of enclosures for rabbits bred for meat production in many European countries, the European Directive 2010/63 lacks regulations for rabbits used for scientific purposes. This study compares two floors, which meet the Austrian legal requirements for growing rabbits intended for consumption as well as the requirements for laboratory rabbits. The dual use of rabbits bred for meat production and applicable for scientific purposes would avoid the problem of surplus animals of specialized producers for laboratory rabbits. A noryl floor with 12 mm circular holes was compared to a 10 mm slatted plastic floor. Parameters were soiling of cages and animals, parasitic burden, clinical health, and losses using objective scoring. Soiling of cages and animals and coccidial oocytes were significantly higher on the floors with circular holes. Obvious signs of disease showed a non-significant trend to be more frequent in the group with circular holes. This was linked with significantly higher losses. In conclusion, our study clearly shows that the floor with circular hole design cannot be endorsed, although it meets legal requirements. The slatted floor type can be cautiously recommended; however, to assure animal welfare in laboratory rabbits, legal authorities in Europe should take on the responsibility of regulating floor design in this sector.

Assessment and refinement of intra-bone marrow transplantation in mice.

Intra-bone marrow transplantation (IBMT) may improve the seeding efficiency of transplanted hematopoietic stem cells compared to the routinely used intravenous injection. Current IBMT protocols are optimized for ease of use and to improve experimental results. However, there have been no investigations to assess the impact of IBMT on animal welfare. Here, we report the results of pain assessment after IBMT and the effects of refinements to the current standard procedure. IBMT was performed in either the tibia or the femur of a recipient mouse under general anesthesia. Impact was determined using clinical scoring of different parameters (lameness, grip capacity, body weight loss, footprint analysis), behavioural tests (burrowing, open-field), monitoring of stress hormones and post-mortem histology. The results revealed that IBMT definitely induces severe post-operative distress. Although IBMT in the tibia is technically easier, the degree of impairment and the distress observed were consistently higher than for transplantation in the femur. A refinement for IBMT in the tibia was achieved by using 30- instead of 26-gauge needles and by sparing the patellar tendon. Consequently, for IBMT, we recommend either using the femur, or if the tibia is required due to its better feasibility, using our refined protocol. Furthermore, IBMT should definitely be limited to one leg per animal.

NI-1: a novel canine mastocytoma model for studying drug resistance and IgER-dependent mast cell activation.

BACKGROUND: Advanced mast cell (MC) disorders are characterized by uncontrolled growth of neoplastic MC in various organs, mediator-related symptoms, and a poor prognosis. Kit mutations supposedly contribute to abnormal growth and drug resistance in these patients. METHODS: We established a novel canine mastocytoma cell line, NI-1, from a patient suffering from MC leukemia. RESULTS: NI-1 cells were found to form mastocytoma lesions in NOD/SCID IL-2Rgamma(null) mice and to harbor several homozygous Kit mutations, including missense mutations at nucleotides 107(C→T) and 1187(A→G), a 12-bp duplication (nucleotide 1263), and a 12-bp deletion (nucleotide 1550). NI-1 cells expressed several MC differentiation antigens, including tryptase, Kit, and a functional IgE receptor. Compared to the C2 mastocytoma cell line harboring a Kit exon 11 mutation, NI-1 cells were found to be less responsive against the Kit tyrosine kinase inhibitors (TKI) masitinib and imatinib, but were even more sensitive against proliferation-inhibitory effects of the mammalian target of rapamycin (mTOR) blocker RAD001 and PI3-kinase/mTOR blocker NVP-BEZ235. The Kit-targeting multikinase inhibitors PKC412 and dasatinib were also found to override TKI resistance in NI-1 cells, and produced growth inhibition with reasonable IC(50) values (<0.1 µM). CONCLUSION: NI-1 may serve as a useful tool to investigate IgE-dependent reactions and mechanisms of abnormal growth and drug resistance in neoplastic MC in advanced mastocytosis.

The Hsp32 inhibitors SMA-ZnPP and PEG-ZnPP exert major growth-inhibitory effects on D34+/CD38+ and CD34+/CD38- AML progenitor cells.

Heat shock protein 32 (Hsp32), also known as heme oxygenase 1 (HO-1), has recently been identified as a potential target in various hematologic malignancies. We provide evidence that Hsp32 is constitutively expressed in primary leukemic cells in patients with acute myeloid leukemia (AML) and in various AML cell lines (HL60, U937, KG1). Expression of Hsp32 mRNA was demonstrable by qPCR, and expression of the Hsp32 protein by immunocytochemistry and Western blotting. The stem cell-enriched CD34+/CD38+ and CD34+/CD38- fractions of AML cells were found to express Hsp32 mRNA in excess over normal CD34+ progenitor cells. Two Hsp32-targeting drugs, pegylated zinc-protoporphyrin (PEG-ZnPP) and styrene-maleic-acid-copolymer-micelle-encapsulated ZnPP (SMAZnPP), were found to inhibit cytokine-dependent and spontaneous proliferation in all 3 AML cell lines as well as in primary AML cells. Growth inhibitory effects of SMA-ZnPP and PEG-ZnPP were dose-dependent with IC50 values ranging between 1 and 20 µM, and were accompanied by apoptosis as evidenced by light- and electron microscopy, Tunel assay, and caspase-3 activation. Finally, we were able to demonstrate that SMA-ZnPP inhibits cytokine-dependent proliferation of CD34+/CD38+ and CD34+/CD38- AML progenitor cells in vitro in all patients as well as leukemiainitiation of AML stem cells in NOD-SCID IL-2Rγ(-/-) (NSG) mice in vivo. Together, our data suggest that Hsp32 plays an important role as a survival factor in leukemic stem cells and as a potential new target in AML.

Early maternal investment in mice: no evidence for compatible-genes sexual selection despite hybrid vigor.

Confronting a recently mated female with a strange male can induce a pregnancy block ('Bruce effect'). The physiology of this effect is well studied, but its functional significance is still not fully understood. The 'anticipated infanticide hypothesis' suggests that the pregnancy block serves to avoid the cost of embryogenesis and giving birth to offspring that are likely to be killed by a new territory holder. Some 'compatible-genes sexual selection hypotheses' suggest that the likelihood of a pregnancy block is also dependent on the female's perception of the stud's and the stimulus male's genetic quality. We used two inbred strains of mice (C57BL/6 and BALB/c) to test all possible combinations of female strain, stud strain, and stimulus strain under experimental conditions (N(total) = 241 mated females). As predicted from previous studies, we found increased rates of pregnancy blocks if stud and stimulus strains differed, and we found evidence for hybrid vigour in offspring of between-strain mating. Despite the observed heterosis, pregnancies of within-strain matings were not more likely to be blocked than pregnancies of between-strain matings. A power analysis revealed that if we missed an existing effect (type-II error), the effect must be very small. If a female gave birth, the number and weight of newborns were not significantly influenced by the stimulus males. In conclusion, we found no support for the 'compatible-genes sexual selection hypotheses'.

Higher heart rate of laboratory mice housed individually vs in pairs.

Many studies have shown that housing mice individually over a long period significantly alters their physiology, but in most cases measurement has required human interference and restraint for sampling. Using a radio-telemetry system with implantable transmitters, we recorded heart rate (HR), motor activity (ACT) and body temperature (BT) of freely moving male mice (NMRI) housed either individually or in pairs with an ovarectomized female. Data for each parameter were collected at 5 min intervals for two consecutive 24 h periods. Even after several weeks of habituation to the social conditions, HR was increased in mice housed individually compared with mice housed in pairs, although their measured ACT did not differ. Additionally, BT tended to be reduced in individually-housed mice. When the data were analysed according to different ACT levels, HR was increased in individually-housed mice during phases of low and high, but not intermediate, motor activity. Furthermore, individually-housed mice had more, but shorter, resting bouts, indicating disruption of the normal circadian sleep pattern. Enhanced HR in individually-housed mice does not necessarily indicate stress, but might be an important physiological indicator of discomfort. The fact that individual housing alters basic physiological parameters in laboratory mice highlights the need to control for housing-dependent variation, especially in experiments that are sensitive to changes in these parameters.

Prevention of scrapie pathogenesis by transgenic expression of anti-prion protein antibodies.

Variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy are initiated by extracerebral exposure to prions. Although prion transmission from extracerebral sites to the brain represents a potential target for prophylaxis, attempts at vaccination have been limited by the poor immunogenicity of prion proteins. To circumvent this, we expressed an anti-prion protein (anti-PrP) mu chain in Prnp(o/o) mice. Transgenic mice developed sustained anti-PrP titers, which were not suppressed by introduction of Prnp+ alleles. Transgene expression prevented pathogenesis of prions introduced by intraperitoneal injection in the spleen and brain. Expression of endogenous PrP (PrP(C)) in the spleen and brain was unaffected, suggesting that immunity was responsible for protection. This indicates the feasibility of immunological inhibition of prion disease in vivo.

Differential effects of TNF and LTalpha in the host defense against M. bovis BCG.

Signaling via TNF receptor type 1 (TNFR1) was shown to be crucial in host defense against the intracellular pathogens L. monocytogenes, M. tuberculosis and M. bovis. To investigate the function of TNF and LTalpha in host defense against M. bovis, mice double deficient for TNF and LTalpha (TNF / LTalpha (- / -)), TNF / LTalpha (- / -) mice complemented with a murine LTalpha transgene (TNF(- / -)) and LTalpha (- / -) mice were infected with BCG and the ensuing pathology was investigated. Control mice showed a normal host defense with early clearance of bacteria. The granulomatous reaction in the liver was accompanied by recruitment of activated macrophages characterized by their acid phosphatase positivity and differentiation into epithelioid cells as well as a coordinated expression of proinflammatory transcripts. In contrast, TNF / LTalpha (- / -) mice showed no comparable recruitment of activated macrophages in the liver. Furthermore, these mice showed extensive necrotic pulmonary lesions with massive growth of acid fast bacilli. Reintroduction of LTalpha as a transgene into TNF / LTalpha (- / -) mice prolonged survival but did not restore resistance to BCG. This, at least partially protective role of LTalpha was further supported by data demonstrating that LTalpha -deficient mice as well were susceptible to BCG infection. In contrast to the deleterious effect of TNF / LTalpha deficiency in BCG infection, BCG-infected TNF / LTalpha (- / -) mice were tolerant to LPS-induced shock. These results demonstrate that TNF as well as LTalpha are involved in murine host defense against BCG and that absence of TNF / LTalpha protects BCG-infected mice from LPS mediated shock.

B lymphocyte-restricted expression of prion protein does not enable prion replication in prion protein knockout mice.

Prion replication in spleen and neuroinvasion after i.p. inoculation of mice is impaired in forms of immunodeficiency where mature B lymphocytes are lacking. In spleens of wild-type mice, infectivity is associated with B and T lymphocytes and stroma but not with circulating lymphocytes. We generated transgenic prion protein knockout mice overexpressing prion protein in B lymphocytes and found that they failed to accumulate prions in spleen after i.p. inoculation. We conclude that splenic B lymphocytes are not prion-replication competent and that they acquire prions from other cells, most likely follicular dendritic cells with which they closely associate and whose maturation depends on them.

Onset of ataxia and Purkinje cell loss in PrP null mice inversely correlated with Dpl level in brain.

PrP knockout mice in which only the open reading frame was disrupted ('Zürich I') remained healthy. However, more extensive deletions resulted in ataxia, Purkinje cell loss and ectopic expression in brain of Doppel (Dpl), encoded by the downstream gene, PRND: A new PrP knockout line, 'Zürich II', with a 2.9 kb PRNP: deletion, developed this phenotype at approximately 10 months (50% morbidity). A single PRNP: allele abolished the syndrome. Compound Zürich I/Zürich II heterozygotes had half the Dpl of Zürich II mice and developed symptoms 6 months later. Zürich II mice transgenic for a PRND:-containing cosmid expressed Dpl at twice the level and became ataxic approximately 5 months earlier. Thus, Dpl levels in brain and onset of the ataxic syndrome are inversely correlated.

Neuroserpin, a neuroprotective factor in focal ischemic stroke.

Because recent studies have indicated that tissue plasminogen activator (tPA) aggravates neurodegenerative processes in many neural pathologies, we studied whether the endogenous tPA antagonist neuroserpin has a neuroprotective effect in an animal model of focal ischemic stroke. After induction of a focal ischemic stroke in the mouse by occlusion of the middle cerebral artery, we found that microglial cells accumulated in the marginal zone of the infarct are the most important source for both plasminogen activators, tPA and uPA. To investigate the effect of neuroserpin on the size and the histology of the infarct we produced transgenic mice overexpressing neuroserpin approximately sixfold in the nervous system. In the brain of these mice the total tPA activity in the uninjured tissue was strongly reduced. After induction of a focal ischemic stroke in the transgenic mice by a permanent occlusion of the middle cerebral artery (MCA), the infarcts were 30% smaller than in the wild-type mice. Immunohistochemical analyses and in situ hybridization revealed an attenuation of the microglial activation in the reactive zone. Concomitantly, the microglial production of tPA and uPA, as well as the PA-activity in the infarct region was markedly reduced. Thus, our results indicate that neuroserpin reduces microglial activation and, therefore, the PA activity and has a neuroprotective role after focal ischemic stroke.

Optimization of intraperitoneal injection anesthesia in mice: drugs, dosages, adverse effects, and anesthesia depth.

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), alpha2-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/xylazinelazaperone, 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.

[A comprehensive form for the standardized characterization of transgenic rodents: genotype, phenotype, welfare assessment, recommendations for refinement]. / Umfassendes Formular zur strukturierten Charakterisierung gentechnisch veränderter Tierlinien.

When "creating" transgenic mouse strains it is not possible to predict their phenotype with certainty, particularly not with respect to welfare. The generation of models for (human) diseases deliberately implies a compromised health ranging from minor (clinically inapparent) to lethal. To ensure animal welfare requirements and apply the criteria of the 3R, a careful phenotype characterisation and welfare assessment has to be done routinely for each newly produced strain, at individual and strain level, starting by the standardised monitoring of founders and their consequent generations. A comprehensive form has been developed for a standardised characterisation of transgenic mouse strains. It is subdivided into basic and detail information. It can be kept up to date continuously in the form of a computerised database, incorporating growing knowledge and experience of the strain. Basic information mainly serves the requirements of housing and breeding facilities as well as the authorities in view of animal welfare measures, detail information mainly serves the interests of research and efficiency.

Nitric oxide prevents cardiovascular disease and determines survival in polyglobulic mice overexpressing erythropoietin.

Nitric oxide (NO) induces vasodilatatory, antiaggregatory, and antiproliferative effects in vitro. To delineate potential beneficial effects of NO in preventing vascular disease in vivo, we generated transgenic mice overexpressing human erythropoietin. These animals induce polyglobulia known to be associated with a high incidence of vascular disease. Despite hematocrit levels of 80%, adult transgenic mice did not develop hypertension or thromboembolism. Endothelial NO synthase levels, NO-mediated endothelium-dependent relaxation and circulating and vascular tissue NO levels were markedly increased. Administration of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) led to vasoconstriction of peripheral resistance vessels, hypertension, and death of transgenic mice, whereas wild-type siblings developed hypertension but did not show increased mortality. L-NAME-treated polyglobulic mice revealed acute left ventricular dilatation and vascular engorgement associated with pulmonary congestion and hemorrhage. In conclusion, we here unequivocally demonstrate that endothelial NO maintains normotension, prevents cardiovascular dysfunction, and critically determines survival in vivo under conditions of increased hematocrit.

Mice with combined gene knock-outs reveal essential and partially redundant functions of amyloid precursor protein family members.

The amyloid precursor protein (APP) involved in Alzheimer's disease is a member of a larger gene family including amyloid precursor-like proteins APLP1 and APLP2. We generated and examined the phenotypes of mice lacking individual or all possible combinations of APP family members to assess potential functional redundancies within the gene family. Mice deficient for the nervous system-specific APLP1 protein showed a postnatal growth deficit as the only obvious abnormality. In contrast to this minor phenotype, APLP2(-/-)/APLP1(-/-) and APLP2(-/-)/APP(-/-) mice proved lethal early postnatally. Surprisingly, APLP1(-/-)/APP(-/-) mice were viable, apparently normal, and showed no compensatory upregulation of APLP2 expression. These data indicate redundancy between APLP2 and both other family members and corroborate a key physiological role for APLP2. This view gains further support by the observation that APLP1(-/-)/APP(-/-)/APLP2(+/-) mice display postnatal lethality. In addition, they provide genetic evidence for at least some distinct physiological roles of APP and APLP2 by demonstrating that combinations of single knock-outs with the APLP1 mutation resulted in double mutants of clearly different phenotypes, being either lethal, or viable. None of the lethal double mutants displayed, however, obvious histopathological abnormalities in the brain or any other organ examined. Moreover, cortical neurons from single or combined mutant mice showed unaltered survival rates under basal culture conditions and unaltered susceptibility to glutamate excitotoxicity in vitro.

Progressive segregation of unmyelinated axons in peripheral nerves, myelin alterations in the CNS, and cyst formation in the kidneys of myelin and lymphocyte protein-overexpressing mice.

Myelin and lymphocyte protein (MAL) is a putative tetraspan proteolipid that is highly expressed by Schwann cells and oligodendrocytes as a component of compact myelin. Outside of the nervous system, MAL is found in apical membranes of epithelial cells, mainly in the kidney and stomach. Because MAL is associated with glycosphingolipids, it is thought to be involved in the organization, transport, and maintenance of glycosphingolipid-enriched membrane microdomains. In this report, we describe the generation and analysis of transgenic mice with increased MAL gene dosage. Immunohistochemical analysis revealed that the localization of MAL overexpression in the transgenic animals corresponded closely to the MAL expression pattern observed in wildtype animals, indicating correct spatial regulation of the transgene. Phenotypically, MAL overexpression led to progressive dissociation of unmyelinated axons from bundles in the PNS, a tendency to hypomyelination and aberrant myelin formation in the CNS, and the formation of large cysts in the tubular region of the kidney. Thus, increased expression of MAL appears to be deleterious to membranous structures in the affected tissues, indicating a requirement for tight control of endogenous MAL expression in Schwann cells, oligodendrocytes, and kidney epithelial cells.

Molecular and neuronal substrate for the selective attenuation of anxiety.

Benzodiazepine tranquilizers are used in the treatment of anxiety disorders. To identify the molecular and neuronal target mediating the anxiolytic action of benzodiazepines, we generated and analyzed two mouse lines in which the alpha2 or alpha3 GABAA (gamma-aminobutyric acid type A) receptors, respectively, were rendered insensitive to diazepam by a knock-in point mutation. The anxiolytic action of diazepam was absent in mice with the alpha2(H101R) point mutation but present in mice with the alpha3(H126R) point mutation. These findings indicate that the anxiolytic effect of benzodiazepine drugs is mediated by alpha2 GABAA receptors, which are largely expressed in the limbic system, but not by alpha3 GABAA receptors, which predominate in the reticular activating system.

T1-deficient and T1-Fc-transgenic mice develop a normal protective Th2-type immune response following infection with Nippostrongylus brasiliensis.

The IL-1 receptor-related protein T1 is expressed on the surface of Th2, but not Th1 cells. Studies with anti-T1 monoclonal antibodies have suggested that T1 is critical for development of normal Th2-type responses. To elucidate the role of T1 in vivo, we generated T1-deficient mice and a T1-transgenic strain which secretes soluble T1-Fc fusion protein into the serum. These were analyzed for the Th2 immune response induced by infection with the parasitic nematode Nippostrongylus brasiliensis. Although Th2 cytokine production by lymph node cells was similar in all groups of N. brasiliensis-infected mice, a decrease in IL-5 production by lung lymphocytes was detected in both T1-deficient and T1-Fc-transgenic mice compared to control littermates. This difference in IL-5 production did not influence blood eosinophilia, but recruitment of eosinophils into lung tissue, especially in T1-Fc-transgenic mice was slightly decreased. However, induction of all other immune parameters was normal and both T1-deficient and T1-Fc-transgenic mice were able to clear the parasite infection within 12 days with kinetics similar to those in control mice. Therefore, in contrast to previous suggestions, we conclude that the T1 protein is not obligatory for normal development of Th2 immune responses.