Difference between two hybrid stocks of mice in the incidence of congenital abnormalities following X-ray exposure of stem-cell spermatogonia

Unbalanced (duplication/deficiency) sperm from balanced reciprocal translocations induced in spermatogonial stem cells of mice generally lead to embryonic lethality around the time of implantation. In a recent study (Generoso et al., 1985), it was found that the incidence of X-ray-induced embryonic lethality differed markedly between two hybrid stocks of irradiated male mice. A parallel difference in the frequencies of reciprocal translocations was observed cytologically in the meiocytes of irradiated males. In the present report, which is an adjunct to the study by Generoso et al. (1985), it was determined whether or not similar differences between the two stocks exist for congenital defects resulting from genetic damage to stem-cell spermatogonia. The results indicate not only an association between the frequencies of induced reciprocal translocations and congenital abnormalities, but also a parallel greater frequency of induced malformations in the (C3H × 101)F1 stock versus the (SEC × C57BL)F1 stock of males.     

Genetic mapping of an insertional hydrocephalus-inducing mutation allelic to hy3

The transgenic mouse line OVE459 carries a transgene-induced insertional mutation resulting in autosomal recessive congenital hydrocephalus. Homozygous transgenic animals experience ventricular dilation with perinatal onset and are noticeably smaller than hemizygous or non-transgenic littermates within a few days after birth. Fluorescence in situ hybridization (FISH) revealed that the transgene inserted in a single locus on mouse Chromosome (chr) 8, region D2-E1. Genetic crosses between hemizygous OVE459 mice and mice heterozygous for the spontaneous mutation hydrocephalus-3 (hy3) produced hydrocephalic offspring with a frequency of 22%, demonstrating that these two mutations are allelic. A genomic library was made by using DNA from homozygous OVE459 mice, and genomic DNA flanking the transgene insertion site was isolated and sequenced. A PCR polymorphism between C57BL/6 DNA and Mus spretus was used to map the location of the transgene insert to 1.06 cM ± 0.75 proximal to D8Mit152 by using the Jackson Laboratory Backcross DNA Panel Mapping Resource. Furthermore, sequence analysis from a mouse bacterial artificial chromosome (BAC) clone, positive for unique markers on both sides of the transgene insertion site, demonstrated that the genomic DNAs flanking each side of the transgene insertion are physically separated by approximately 51 kb on the wild-type mouse chromosome.     

Lipopolysaccharide aggravates cerebral pathology in B10.PL-derived CD1-/-, beta2m-/-, TCRalpha-/-, and TCRdelta-/- knockout mice

Adult B10.PL-derived immunological genes knockout mice injected with 100 microg lipopolysaccharide (LPS) showed severe hydrocephalus and meningitis. A consequence of the hydrocephalus is pineal hyperplasia, sponginess of periventricular parenchyma, gliosis and, at the last stage of hydrocephalus formation, disappearance of the ependymal layer and the Gomori-positive subependymal astrocytes. Possible mechanisms for the aggravation of cerebral pathology induced by LPS are discussed.     

Chronic Hydrocephalus after Experimental Subarachnoid Hemorrhage

Chronic communicating hydrocephalus is a significant health problem affecting up to 20% of survivors of spontaneous subarachnoid hemorrhage (SAH). The development of new treatment strategies is hampered by the lack of well characterized disease models. This study investigated the incidence of chronic hydrocephalus by evaluating the temporal profile of intracranial pressure (ICP) elevation after SAH, induced by endovascular perforation in rats. Twenty-five adult male Sprague-Dawley rats (260–320g) were subjected to either endovascular perforation or sham surgery. Five animals died after SAH induction. At 7, 14 and 21 days after surgery ICP was measured by stereotaxic puncture of the cisterna magna in SAH (n=10) and SHAM (n=10) animals. On day 21 T-maze test was performed and the number of alterations and latency to decision was recorded. On day 23, samples were processed for histological analyses. The relative ventricle area was evaluated in coronal Nissl stained sections. On day 7 after surgery all animals showed normal ICP. The absolute ICP values were significantly higher in SAH compared to SHAM animals on day 21 (8.26±4.53 mmHg versus 4.38±0.95 mmHg) but not on day 14. Observing an ICP of 10mmHg as cut-off, 3 animals showed elevated ICP on day 14 and another animal on day 21. The overall incidence of ICP elevation was 40% in SAH animals. On day 21, results of T-maze testing were significantly correlated with ICP values, i.e. animals with elevated ICP showed a lower number of alterations and a delayed decision. Histology yielded a significantly higher (3.59 fold increased) relative ventricle area in SAH animals with ICP elevation compared to SAH animals without ICP elevation. In conclusion, the current study shows that experimental SAH leads to chronic hydrocephalus, which is associated with ICP elevation, behavioral alterations and ventricular dilation in about 40% of SAH animals.     

Prenatal Exposure to Fenugreek Impairs Sensorimotor Development and the Operation of Spinal Cord Networks in Mice

Fenugreek is a medicinal plant whose seeds are widely used in traditional medicine, mainly for its laxative, galactagogue and antidiabetic effects. However, consumption of fenugreek seeds during pregnancy has been associated with a range of congenital malformations, including hydrocephalus, anencephaly and spina bifida in humans. The present study was conducted to evaluate the effects of prenatal treatment of fenugreek seeds on the development of sensorimotor functions from birth to young adults. Pregnant mice were treated by gavage with 1g/kg/day of lyophilized fenugreek seeds aqueous extract (FSAE) or distilled water during the gestational period. Behavioral tests revealed in prenatally treated mice a significant delay in righting, cliff avoidance, negative geotaxis responses and the swimming development. In addition, extracellular recording of motor output in spinal cord isolated from neonatal mice showed that the frequency of spontaneous activity and fictive locomotion was reduced in FSAE-exposed mice. On the other hand, the cross-correlation coefficient in control mice was significantly more negative than in treated animals indicating that alternating patterns are deteriorated in FSAE-treated animals. At advanced age, prenatally treated mice displayed altered locomotor coordination in the rotarod test and also changes in static and dynamic parameters assessed by the CatWalk automated gait analysis system. We conclude that FSAE impairs sensorimotor and coordination functions not only in neonates but also in adult mice. Moreover, spinal neuronal networks are less excitable in prenatally FSAE-exposed mice suggesting that modifications within the central nervous system are responsible, at least in part, for the motor impairments.     

Cross-talk between thyroid hormone receptor and liver X receptor regulatory pathways is revealed in a thyroid hormone resistance mouse model

Hypercholesterolemia is found in patients with hypothyroidism and resistance to thyroid hormone. In this study, we examined cholesterol metabolism in a thyroid hormone receptor beta (TR-beta) mutant mouse model of resistance to thyroid hormone. Whereas studies of cholesterol metabolism have been reported in TR-beta knock-out mice, generalized expression of a non-ligand binding TR-beta protein in this knock-in model more fully recapitulates the hypothyroid state, because the hypothyroid effect of TRs is mediated by the unliganded receptor. In the hypothyroid state, a high cholesterol diet increased serum cholesterol levels in wild-type animals (WT) but either did not change or reduced levels in mutant (MUT) mice relative to hypothyroidism alone. 7alpha-Hydroxylase (CYP7A1) is the rate-limiting enzyme in cholesterol metabolism and mRNA levels were undetectable in the hypothyroid state in all animals. triiodothyronine replacement restored CYP7A1 mRNA levels in WT mice but had minimal effect in MUT mice. In contrast, a high cholesterol diet markedly induced CYP7A1 levels in MUT but not WT mice in the hypothyroid state. Elevation of CYP7A1 mRNA levels and reduced hepatic cholesterol content in MUT animals are likely because of cross-talk between TR-beta and liver X receptor alpha (LXR-alpha), which both bind to a direct repeat + 4 (DR+4) element in the CYP7A1 promoter. In transfection studies, WT but not MUT TR-beta antagonized induction of this promoter by LXR-alpha. Electromobility shift analysis revealed that LXR/RXR heterodimers bound to the DR+4 element in the presence of MUT but not WT TR-beta. A mechanism for cross-talk, and potential antagonism, between TR-beta and LXR-alpha is proposed.     

Susceptibility of the developing brain to acute hypoglycemia involving A1 adenosine receptor activation

It has been suggested that the developing brain is less vulnerable to the adverse effects of hypoglycemia than the mature brain; however, this issue remains controversial. We also do not know the magnitude or duration of hypoglycemia needed to trigger hypoglycemic brain injury during development. To address this issue a series of in vivo and in vitro studies were performed. First, we established an acute model of insulin-induced hypoglycemia in mice by administering 3 U/kg of neutral-protamine Hagadorn insulin subcutaneously. When we examined degenerating neurons in hippocampus and striatum by TUNEL labeling, injury was observed after 4 h of hypoglycemia in postnatal day (P)7 mice, and we observed more cell injury in animals rendered hypoglycemic at P7 than at P21. Studies of hippocampal slice cultures revealed that reduction in glucose concentration induced more neuronal injury in slices prepared from P3 and P7 than from P14 and P21 mice. Treatment of slices with an adenosine A(1) receptor (A(1)AR) antagonist reduced the hypoglycemic damage, whereas agonists increased damage, particularly in slices prepared from very young pups. This suggests a critically important role for A(1)ARs, which was further demonstrated by the reduction of hypoglycemic damage in hippocampal slices prepared from A(1)AR(-/-) mice. Furthermore, insulin-induced hypoglycemia in P7 A(1)AR(-/-) mice did not increase TUNEL-positive cells, but a major increase was seen in A(1)AR(+/-) mice. These observations show that the developing nervous system is indeed sensitive to acute hypoglycemic injury and that A(1)AR activation contributes to damage induced by hypoglycemia, particularly in immature mouse brain.     

Larsen syndrome associated with severe congenital hydrocephalus

Larsen syndrome associated with severe congenital hydrocephalus: Larsen syndrome (LS) is characterized by the association of flattened facies with a prominent forehead, a depressed nasal bridge and hypertelorism, dislocation of hips, elbows and knees, equinovarus or valgus deformities of the feet, long and tapering fingers, normal intelligence. Hydrocephalus has rarely been reported in association with LS. In this article, a newborn infant with the classical features of LS and severe congenital hydrocephalus is presented. Our purpose was to emphasize the importance of congenital hydrocephalus in infants with LS and to highlight the management of cases of LS associated with congenital hydrocephalus.     

Herpes simplex virus type 1-induced hydrocephalus in mice.

Adult ICR/Slc or BALB/c mice developed hydrocephalus when attenuated herpes simplex virus type 1 (HSV-1) (strain Ska) was injected intracerebrally 2 to 4 weeks earlier and then after mice were challenged with the same virus or virulent HSV-1. Initial inoculation of the Ska strain elicited acute meningitis and ependymitis with transient mild hydrocephalus. Viral antigen was seen in the meninges and subependymal areas, and the virus was titrated during the acute phase of infection. After the second virus inoculation, more prominent inflammation was evoked in the same area, and the animals developed hydrocephalus, although viral antigen and infectious virus were hardly detected. When the mice were immunosuppressed with cyclophosphamide, they ceased to develop hydrocephalus. BALB/c nude mice did not show the same pathology, even though they were treated in the same way. When irradiated mice, which had been infected with the Ska strain intracerebrally 2 weeks earlier, received syngeneic immune spleen cells, they developed hydrocephalus. The T-cell nature of the effector cells was confirmed by the elimination of the pathology after treatment of the donor cells with anti-Thy-1.2 plus complement. No hydrocephalic mice were observed after treatment of the donor cells with anti-Lyt-1.2 plus complement, which gave further evidence of the T-cell nature of the effector cells as the Lyt-1+.2+ antigen-bearing subsets. Intervals between priming and challenge virus inoculation could be more than 18 months. The presence of purified HSV-1 envelope protein was feasible for the development of the hydrocephalic animals.     

The role of T-lymphocytes in the development of a humoral immune response to the corpuscular antigen of Staphylococcus aureus

The dependence of humoral immune response and the formation of immunological memory to corpuscular staphylococcal antigen (CSA) on the T-system of immunity was studied in experiments on B-mice and on mice with the congenital absence of the thymus (nude). Primary and secondary immune response to CSA in athymic mice was found to be considerably less than in normal animals. After the repeated immunization of genetically athymic mice the pronounced secondary reaction of the formation of antibodies to CSA was observed. As shown in this investigation carried out with the use of adoptive transfer techniques, the induction of memory B-cells to CSA may occur in animals with congenital or experimentally induced T-immunodeficiency. The conclusion was made on the T-dependence of humoral immune response to CSA, the formation of immunological memory to this antigen being relatively T-independent.     

The relationship between hydrocephalus and Chiari type II malformation in the experimental rat fetuses with Arnold-Chiari malformation

Spina bifida, Chiari type II malformation, cerebral aqueduct stenosis and hydrocephalus are the most frequent association anomalies in the congenital malformation of the central nervous system (Warkany et al., 1958). They are potentially treatable and of clinical importance. But the relationship between hydrocephalus and Chiari type II malformation is still a controversial subject. A single oral dose of 240 mg/kg of ethylenethiourea (ETU) was given to Sprague Dawley (SD) rats on the 11th day of gestation. Fetuses were removed in the 20th day of gestation by cesarean sections; high incidence of spinal dysraphism associated with hindbrain crowding was found in these fetuses. They are similar to Arnold-Chiari malformation in humans. We used these experimental models to analyze the relationship between hydrocephalus and Chiari type II malformation. From the present investigation, no hydrocephalus or cerebral aqueduct stenosis was found in the experimental rat fetuses with the Arnold-Chiari malformation. So we do not consider the hydrodynamic theory that Chiari type II malformation was induced by increasing intracranial pressure in hydrocephalus. Hydrocephalus in the Arnold-Chiari malformation may not be the primary disorder but seems to be caused by plugging the foramen magnum in Chiari type II malformation. So the cerebrospinal fluid in the spinal subarachnoid space can not move upward to the cranial subarachnoid space for absorption to venous return. Cerebral aqueduct stenosis may be secondarily compressed by hydrocephalus and not be the primary development anomaly or acquired occlusion due to gliosis. This is in accord with the theory proposed by Russell and Donald (1935).(ABSTRACT TRUNCATED AT 250 WORDS)     

NK cytotoxic activity and relative distribution of NK cells in 3-acetylpyridine influenced mice

NK-cell cytotoxic activity and their relative distributions were studied in the spleen of female Lurcher mice with spontaneous olivopontocerebellar degeneration (C3H) and female athymic nu/nu mice (BALB/c) influenced by 3-acetylpyridine (the neurotoxin causing selective degeneration of cerebellar and inferior olive neurons in some rodent species). The congenital olivopontocerebellar degeneration in Lurcher mice is followed by only an insignificant increase of NK-cell cytotoxic activity (1.2 times). On the other hand, the congenital thymic dysgenesis in nu/nu mice is compensated by a substantial increase in cytotoxic activity (19.4-fold). The administration of 3-acetylpyridine (including prevalent neuronal destruction particularly in Lurcher mutants) caused a decrease of NK-cell cytotoxic activities in all groups of mice (in Lurcher and C3H controls to 60 and 50%, respectively, and in nu/nu and BALB/c controls to 25 and 60%). Relative distributions of NK-cells in spleens of non-influenced and influenced animals were not significantly changed. Some fundamental immune mechanisms, such as the NK-cell cytotoxic activity, were demonstrated to be controlled by congenitally determined or artificially induced changes in both the nervous and the immune systems.     

Protection against radiation oxidative damage in mice by Triphala

Protection against whole body gamma-irradiation (WBI) of Swiss mice orally fed with Triphala (TPL), an Ayurvedic formulation, in terms of mortality of irradiated animals as well as DNA damage at cellular level has been investigated. It was found that radiation induced mortality was reduced by 60% in mice fed with TPL (1g/kg body weight/day) orally for 7 days prior to WBI at 7.5 Gy followed by post-irradiation feeding for 7 days. An increase in xanthine oxidoreductase activity and decrease in superoxide dismutase activity was observed in the intestine of mice exposed to WBI, which, however, reverted back to those levels of sham-irradiated controls, when animals were fed with TPL for 7 days prior to irradiation. These data have suggested the prevention of oxidative damage caused by whole body radiation exposure after feeding of animals with TPL. To further understand the mechanisms involved, the magnitude of DNA damage was studied by single cell gel electrophoresis (SCGE) in blood leukocytes and splenocytes obtained from either control animals or those fed with TPL for 7 days followed by irradiation. Compared to irradiated animals without administering TPL, the mean tail length was reduced about three-fold in blood leukocytes of animals fed with TPL prior to irradiation. Although, similar protection was observed in splenocytes of TPL fed animals, the magnitude of prevention of DNA damage was significantly higher than that observed in leukocytes. It has been concluded that TPL protected whole body irradiated mice and TPL induced protection was mediated through inhibition of oxidative damage in cells and organs. TPL seems to have potential to develop into a novel herbal radio-protector for practical applications.     

Reactive microglia and mitochondrial unfolded protein response following ventriculomegaly and behavior defects in kaolin-induced hydrocephalus

Ventriculomegaly induced by the abnormal accumulation of cerebrospinal fluid (CSF) leads to hydrocephalus, which is accompanied by neuroinflammation and mitochondrial oxidative stress. The mitochondrial stress activates mitochondrial unfolded protein response (UPRmt), which is essential for mitochondrial protein homeostasis. However, the association of inflammatory response and UPRmt in the pathogenesis of hydrocephalus is still unclear. To assess their relevance in the pathogenesis of hydrocephalus, we established a kaolin-induced hydrocephalus model in 8-week-old male C57BL/6J mice and evaluated it over time. We found that kaolin-injected mice showed prominent ventricular dilation, motor behavior defects at the 3-day, followed by the activation of microglia and UPRmt in the motor cortex at the 5-day. In addition, PARP-1/NF-κB signaling and apoptotic cell death appeared at the 5-day. Taken together, our findings demonstrate that activation of microglia and UPRmt occurs after hydrocephalic ventricular expansion and behavioral abnormal-ities which could be lead to apoptotic neuronal cell death, providing a new perspective on the pathogenic mechanism of hydrocephalus.     

Deficiency of immunoreactive somatostatin in the median eminence of Snell dwarf mice

Snell dwarf mice (dw/dw) are characterized by a genetically determined, congenital lack of pituitary GH, TSH and prolactin. Given that hypothalamic somatostatin is involved in the regulation of pituitary GH and TSH release, it was decided to investigate the content of immunoreactive somatostatin (IRS) in the median eminence of dw/dw and phenotypically normal mice of the same strain. The content of IRS in the pyloric antrum and pineal gland of these animals was also examined. The effects of ovariectomy and of hyperprolactinemia (induced by a pituitary graft under the kidney capsule) on the median eminence content of IRS were also studied in both normal and dwarf mice. Median eminence IRS content was significantly lower in the dw/dw (23.6 +/- 1.8 ng) than in normal mice (57.4 +/- 7.1 ng); no difference was found in the pyloric IRS content of dw/dw (16.9 +/- 1.6 ng/mg of protein) and normal animals (13.8 +/- 1.9 ng/mg of protein), nor in the pineal content of IRS (639.4 +/- 64.4 pg/gland in the dw/dw; 732 +/- 265 pg/gland in normals). Neither ovariectomy nor hyperprolactinemia were found to affect the IRS content in the tissues studied in normal or dwarf mice. Treatment of an additional group of 9 dwarf mice with L-thyroxine (L-T4 2 micrograms/48 h. s.c. for 2 weeks) significantly increased the animals weight (10.2 +/- 0.4 g versus 7.4 +/- 0.3 g) and produced maturation of facial features; however, it did not change the IRS content in any of the tissues studied. It is concluded that the content of IRS in the median eminence of mice with a congenital lack of GH, TSH and prolactin is significantly reduced and that this is unlikely to be related to the deficiency of thyroid hormones in these animals.     

Effect of cage population density on plasma corticosterone and peripheral lymphocyte populations of laboratory mice

The effect of different population densities of mice per cage on plasma corticosterone, peripheral lymphocytes and specific lymphocyte subpopulations was investigated. The animals were housed in groups of 2, 4 or 8 mice per cage and the blood samples were taken from each animal of these groups on days one, 7 and 14. A significant elevation (P less than 0.05) in plasma corticosterone concentration was observed in the group of 8 mice per cage on days one and 7 as compared with those of 2 or 4 mice per cage. The number of peripheral lymphocytes was significantly decreased in the groups of 2 (P less than 0.01) and 8 (P less than 0.05) mice per cage as compared with the group of 4 mice per cage on day one. A significantly decreased number of lymphocytes (P less than 0.01) in the group of 8 mice per cage continued to day 7. There were no significant differences in specific lymphocyte subpopulations observed among these groups. The results of this study suggest that a population density of 4 mice per cage induced minimal stress compared to that induced by the population densities of 2 or 8 mice per cage. Since stress is known to induce alteration in a variety of biological functions, the population density of mice per cage should be considered in the interpretation of research data.     

MiR-21 in Extracellular Vesicles Leads to Neurotoxicity via TLR7 Signaling in SIV Neurological Disease

Recent studies have found that extracellular vesicles (EVs) play an important role in normal and disease processes. In the present study, we isolated and characterized EVs from the brains of rhesus macaques, both with and without simian immunodeficiency virus (SIV) induced central nervous system (CNS) disease. Small RNA sequencing revealed increased miR-21 levels in EVs from SIV encephalitic (SIVE) brains. In situ hybridization revealed increased miR-21 expression in neurons and macrophage/microglial cells/nodules during SIV induced CNS disease. In vitro culture of macrophages revealed that miR-21 is released into EVs and is neurotoxic when compared to EVs derived from miR-21^-/- knockout animals. A mutation of the sequence within miR-21, predicted to bind TLR7, eliminates this neurotoxicity. Indeed miR-21 in EV activates TLR7 in a reporter cell line, and the neurotoxicity is dependent upon TLR7, as neurons isolated from TLR7^-/- knockout mice are protected from neurotoxicity. Further, we show that EVs isolated from the brains of monkeys with SIV induced CNS disease activates TLR7 and were neurotoxic when compared to EVs from control animals. Finally, we show that EV-miR-21 induced neurotoxicity was unaffected by apoptosis inhibition but could be prevented by a necroptosis inhibitor, necrostatin-1, highlighting the actions of this pathway in a growing number of CNS disorders.     

Antigen recognition and IL-2 receptor gene expression as evidence against clonal deletion in mice with neonatally induced transplantation tolerance.

Neonatal transplantation tolerance was induced in B10.A mice by the injection of spleen and bone marrow cells from semiallogeneic [C57BL/10(B10) x B10.A] F1 donors. The neonatally treated mice accepted skin grafts from B10 donors. Spleen cells from tolerant animals did not respond by proliferation to tolerated B10 antigens in vitro. However, spleen cells from tolerant mice recognized specific (B10) antigens and synthesized mRNA for the inducible 55-kDa interleukin-2 receptor (IL-2R) as did cells from normal animals. Maintenance of this early phase of cell activation upon contact with tolerated antigens is direct evidence against clonal deletion as a mechanism, in this particular model of neonatally induced transplantation tolerance.