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.

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.

Viral and bacterial infections interfere with peripheral tolerance induction and activate CD8+ T cells to cause immunopathology.

We studied the impact of various infectious and proinflammatory agents on the induction of peripheral T cell tolerance. Adoptive transfer of CD8+ T cells from lymphocytic choriomeningitis virus (LCMV) T cell receptor transgenic mice into LCMV antigen transgenic mice expressing the LCMV glycoprotein epitope (gp) 33-41 under control of a major histocompatibility complex class I promoter led to efficient induction of peripheral tolerance after a period of transient activation. If, however, the recipient mice were challenged with viral or bacterial infections or proinflammatory agents (lipopolysaccharide or Poly:IC) early after cell transfer, tolerance induction was prevented and instead, CD8+ T cell activation leading to vigorous expansion and generation of cytolytic activity ensued. This became manifest in significant immunopathology mainly involving destruction of the splenic architecture and lysis of antigen-expressing lymphocyte and macrophage populations. Important parameters involved in the activation of host-reactive T cells by nonspecific infectious agents included the presence, localization, and quantity of the specific transgene-encoded self-antigen; in contrast, CD4+ T cells were not required. In mice surviving the acute phase, the transferred CD8+ T cells persisted at high levels in an anergic state; they were unable to generate cytolytic activity in vitro or to control LCMV infection in vivo. These results impinge on our understanding of the role of infectious agents in graft verus host reactions towards minor histocompatibility antigens.

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.

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.

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.

Development and malignant progression of astrocytomas in GFAP-v-src transgenic mice.

We have generated a transgenic mouse model for astrocytoma by expressing the v-src kinase under control of the glial fibrillary acidic protein (GFAP) gene regulatory elements in astrocytes. Abnormal astrogliosis was observed in all transgenic animals already at 2 weeks postnatally, frequently followed by the development of dysplastic changes. Later, small proliferative foci arose, and overt astrocytoma developed in the brain and spinal cord in 14.4% of mice after a follow up time of 65 weeks. While early lesions were histologically consistent with low-grade astrocytoma, at later stages most tumors were highly mitotic and frankly malignant. Vascular endothelial growth factor (VEGF) was expressed by tumor cells already at early stages, suggesting induction by v-src, and it was most pronounced in pseudopalisading cells surrounding necrotic areas, implying additional upregulation by hypoxia. In larger lesions, mitotic activity and expression of flk-1, the cognate receptor of VEGF were induced in endothelial cells. Therefore, end-stage tumors mimicked the morphological and molecular characteristics of human glioblastoma multiforme. Time course and stochastic nature of the process indicate that v-src did not suffice for malignant transformation, and that astrocytomas were the result of a multistep process necessitating co-operation of additional genetic events.

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.

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.

MHC-genotype of progeny influenced by parental infection.

In a previous series of in vitro fertilization experiments with mice we found non-random combination of major histocompatibility complex (MHC) haplotypes in the very early embryos. Our results suggested that two selection mechanisms were operating: (i) the eggs selected specific sperm; and (ii) the second meiotic division in the eggs was influenced by the type of sperm that entered the egg. Furthermore, the proportion of MHC-heterozygous embryos varied over time, suggesting that non-random fertilization was dependent on an external factor that changed over time. As a higher frequency of heterozygous individuals correlated with an uncontrolled epidemic by MHV (mouse hepatitis virus), we suggested that MHV-infection might have influenced the outcome of fertilization. Here, we present an experiment that tests this hypothesis. We infected randomly chosen mice with MHV and sham-infected control mice five days before pairing. We recovered the two-cell embryos from the oviduct, cultured them until the blastocyst stage, and determined the genotype of each resulting blastocyst by polymerase chain reaction. We found the pattern that we expected from our previous experiments: virus-infected mice produced more MHC-heterozygous embryos than sham-infected ones. This suggests that parents are able to promote specific combinations of MHC-haplotypes during fertilization according to the presence or absence of a viral infection.

Neuronal subtype-specific expression directed by the GABA(A) receptor delta subunit gene promoter/upstream region in transgenic mice and in cultured cells.

The promoter of the GABA(A) receptor delta subunit gene was analyzed in transgenic mice and in cultured cells to study sequences involved in neuronal subtype-specific gene expression. A 6.4-kb genomic fragment faithfully directed neuron-specific transcription of a lacZ reporter gene in the central nervous system. The transgene expression pattern in the cerebral cortex, hippocampal formation, thalamus, and brainstem was consistent with the regional and neuronal subtype-specific expression of the endogenous delta subunit protein in both developing and mature brain. In the cerebellum, however, the transgene was ectopically expressed in Purkinje cells and silent in granule cells, where the endogenous delta subunit is abundantly expressed. These mice provide a useful tool for investigating activity-dependent regulation of GABA(A) receptor expression under physiological and pathological conditions. Transfection studies using primary cortical neurons and astroglial cells revealed that the delta subunit gene promoter was selectively active in neurons even when truncated to a 267-bp core fragment. In conclusion, the delta subunit gene promoter/upstream region contains the information for neuronal subtype-specific expression in the entire brain except in the cerebellum and is selectively active in primary cortical neurons in vitro.

Mice homozygous for a modified beta-amyloid precursor protein (beta APP) gene show impaired behavior and high incidence of agenesis of the corpus callosum.

The amyloid precursor protein (beta APP) gene of the mouse was disrupted by homologous recombination; however, contrary to expectation, brain and other tissues still contained beta APP-specific RNA, albeit at a level 5-10 fold lower than wild-type and lacking the disrupted exon, which had been spliced out. The brain contained shortened beta APP-specific protein at a low level. Mutant mice were severely impaired in spatial learning and exploratory behavior and showed increased incidence of agenesis of the corpus callosum.

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.

The use of transgenic mice in the investigation of transmissible spongiform encephalopathies.

The prion, the transmissible agent that causes spongiform encephalopathies such as scrapie, bovine spongiform encephalopathy and Creutzfeldt-Jakob disease, is believed to be devoid of nucleic acid and to be identical to PrPSc (prion protein: scrapie form), a modified form of the normal host protein PrPC (prion protein: cellular form) which is encoded by the single copy gene Prnp. The 'protein only' hypothesis proposes that PrPSc, when introduced into a normal host, causes the conversion of PrPC into PrPSc; it therefore predicts that an animal devoid of PrPC should be resistant to prion diseases. The authors generated homozygous Prnp(o/o) ('PrP knockout') mice and showed that, after inoculation with prions, these mice remained free from scrapie for at least two years while wild-type controls all died within six months. There was no propagation of prions in the Prnp(o/o) animals. Surprisingly, heterozygous Prnp(o/+) mice, which express PrPC at about half the normal level, also showed enhanced resistance to scrapie despite high levels of infectious agent and PrPSc in the brain at an early stage. After introduction of murine PrP transgenes, Prnp(o/o) mice became highly susceptible to mouse--but not to hamster--prions, while the insertion of Syrian hamster PrP transgenes rendered the mice susceptible to hamster prions but much less susceptible to mouse prions. These complementation experiments enabled the application of reverse genetics. The authors prepared animals transgenic for genes encoding PrP with amino terminal deletions of various lengths and found that PrP that lacks 48 amino proximal amino acids (which comprise four of the five octa repeats of PrP) is still biologically active.

Potential of two-cell mouse embryos to develop to term despite partial damage after cryopreservation.

Approximately 18% of cryopreserved 2-cell mouse embryos of 26 different batches showed various degrees of morphological damage after the freeze-thaw process. Normal and damaged morphology were assessed by light microscopy and the ability of an embryo to develop in vitro to a blastocyst, or to develop to term, after transfer to foster mothers. Using vital stains such as Fluorescein-diacetate (FDA) and 4', 6-Diamidino-2-Phenylindole (DAPI) it was found that in approximately 82% of the cases, both of the 2 blastomeres of the cryopreserved embryos survived the freeze-thaw process; in 10% only one cell survived the process; and in 8% none survived. Normally, only intact 2-cell embryos are considered for transfer. Here it was shown that over 60% of the partially damaged embryos developed in vitro to the blastocyst stage and, of those, 26% developed to term after transfer to suitable foster mothers. Although the inner cell mass (ICM) appeared to remain smaller during culture after the transfer of partially damaged 2-cell stage embryos, no difference during gestation period was found compared with intact embryos.

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.

The elimination of mouse hepatitis virus by temporary transplantation of human tumors from infected athymic nude mice into athymic nude rats (rnuN/rnuN).

A nude mouse colony held in an isolation unit was found to harbor MHV despite the fact that all hygienic precautions were taken. The virus spread rapidly causing a high mortality rate predominantly in experimental animals. Moreover, we observed a high percentage of tumor regression in our tumor transplanted mice. Attempts to eliminate the MHV by repeated tumor transplantation into virus-free nude mice were unsuccessful. Since MHV has a limited host range, we transplanted, in parallel, four different lines of embryonic renal tumors (three triphasic nephroblastomas and one malignant rhabdoid tumor of the kidney) from athymic mice into athymic rats and fragments of the same tumors into "fresh" nude mice. All manipulations were performed in isolators. Detection of MHV was done twice by serological examination of six-week-old sentinels. The results showed transmission of MHV infection in the control mice under gnotobiotic conditions as previously found in the normal animal room. On the other hand, there was no evidence of infection, neither in the transplanted nude rats nor after retransplantation of tumors into nude mice. We hypothesize that the virus is harbored in the stromal cells of the murine host but not of the rat host nor in the human tumor cells. Histological comparison showed no alteration of specific tumor morphology in the different hosts.

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'.