Unsafe abortion after legalisation in Nepal: a cross-sectional study of women presenting to hospitals.

OBJECTIVE: To investigate abortion practices of Nepali women requiring postabortion care. DESIGN: Cross-sectional study. SETTING: Four tertiary-care hospitals in urban and rural Nepal. SAMPLE: A total of 527 women presenting with complications from induced abortion in 2010. METHODS: Women completed questionnaires on their awareness of the legal status of abortion and their abortion-seeking experiences. The method of induction and whether the abortion was obtained from an uncertified source was documented. Multivariable logistic regression was used to identify associated factors. MAIN OUTCOME MEASURES: Induction method; uncertified abortion source. RESULTS: In all, 234 (44%) women were aware that abortion was legal in Nepal. Medically induced abortion was used by 359 (68%) women and, of these, 343 (89%) took unsafe, ineffective or unknown substances. Compared with women undergoing surgical abortion, women who had medical abortion were more likely to have obtained information from pharmacists (161/359, 45% versus 11/168, 7%, adjusted odds ratio [aOR] 8.1, 95% confidence interval 4.1-16.0) and to have informed no one about the abortion (28/359, 8% versus 3/168, 2%, aOR 5.5, 95% CI 1.1-26.9). Overall, 291 (81%) medical abortions and 50 (30%) surgical abortions were obtained from uncertified sources; these women were less likely to know that abortion was legal (122/341, 36% versus 112/186, 60%, aOR 0.4, 95% CI 0.2-0.7) and more likely to choose a method because it was available nearby (209/341, 61% versus 62/186, 33%, aOR 2.5, 95% CI 1.5-4.3), compared with women accessing certified sources. CONCLUSIONS: Among women presenting to hospitals in Nepal with complications following induced abortion of pregnancy, the majority had undergone medically induced abortions using unknown substances acquired from uncertified sources. Women using medications and those accessing uncertified providers were less aware that abortion is now legal in Nepal. These findings highlight the need for continued improvements in the provision and awareness of abortion services in Nepal.

Neuronal defects and posterior pituitary hypoplasia in mice lacking the receptor tyrosine phosphatase PTPsigma.

The LAR-family protein tyrosine phosphatase sigma (PTPsigma, encoded by the gene Ptprs) consists of a cell adhesion-like extracellular domain composed of immunoglobulin and fibronectin type-III repeats, a single transmembrane domain and two intracellular catalytic domains. It was previously shown to be expressed in neuronal and lung epithelial tissues in a developmentally regulated manner. To study the role of PTPsigma in mouse development, we inactivated Ptprs by gene targeting. All Ptprs+/- mice developed normally, whereas 60% of Ptprs-/- mice died within 48 hours after birth. The surviving Ptprs-/- mice demonstrated stunted growth, developmental delays and severe neurological defects including spastic movements, tremor, ataxic gait, abnormal limb flexion and defective proprioception. Histopathology of brain sections revealed reduction and hypocellularity of the posterior pituitary of Ptprs-/- mice, as well as a reduction of approximately 50-75% in the number of choline acetyl transferase-positive cells in the forebrain. Moreover, peripheral nerve electrophysiological analysis revealed slower conduction velocity in Ptprs-/- mice relative to wild-type or heterozygous animals, associated with an increased proportion of slowly conducting, small-diameter myelinated fibres and relative hypomyelination. By approximately three weeks of age, most remaining Ptprs-/- mice died from a wasting syndrome with atrophic intestinal villi. These results suggest that PTPsigma has a role in neuronal and epithelial development in mice.

Differential sensitivity of skeletal and fusimotor neurons to Bcl-2-mediated apoptosis during neuromuscular development.

Proper development of the nervous system requires that a carefully controlled balance be maintained between both proliferation and neuronal survival. The process of programmed cell death is believed to play a key role in regulating levels of neuronal survival, in large part through the action of antiapoptotic proteins, such as Bcl-2. Consistent with this, Bcl-2 has been shown to be a key regulator of apoptotic signaling in post-mitotic neurons. However, we still know remarkably little regarding the role that Bcl-2 plays in regulating the survival of specific motor neuron populations. In the present study, we have examined somatic motor neurons of the lumbar spinal cord, and branchiomotor neurons of the facial nucleus in bcl-2-null mice to determine the differential dependence among motor neuron populations with respect to Bcl-2-mediated survival. Examination of neuronal and axon number, axonal area, and the distribution of axonal loss in bcl-2-null mice demonstrates that, in contrast to the great majority of alpha motor neurons, gamma motor neurons exhibit a unique dependence upon bcl-2 for survival. These results demonstrate, for the first time, the connection between Bcl-2 expression, motor neuron survival, and the establishment of different motor populations.

Excitatory tonus is required for the survival of granule cell precursors during postnatal development within the cerebellum.

In addition to protective effects within the adult central nervous system (CNS), in vivo application of N-methyl-d-aspartate inhibitors such as (+) MK-801 have been shown to induce neurodegeneration in neonatal rats over a specific developmental period. We have systematically mapped the nature and extent of MK-801-induced neurodegeneration throughout the neonatal murine brain in order to genetically dissect the mechanism of these effects. Highest levels of MK-801-induced neurodegeneration are seen in the cerebellar external germinal layer; while mature neurons of the internal granule layer are unaffected by MK-801 treatment. Examination of external germinal layer neurons by electron microscopy, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) and bromodeoxyuridine (BrdU) labeling, and caspase-3 activation demonstrate that these neurons die through the process of programmed cell death soon after they exit from the cell cycle. Significantly, ablation of caspase-3 activity completely inhibited the MK-801-induced (and developmental) programmed cell death of external germinal layer neurons. Similar to caspase-3, inactivation of muscarinic acetylcholine receptors in vivo using scopolamine inhibited MK-801-induced programmed cell death. By contrast, the GABAergic agonist diazepam, either alone or in combination with MK-801, enhanced programmed cell death within external germinal layer neurons. These data demonstrate that, in vivo, cerebellar granule neurons undergo a dramatic change in intracellular signaling in response to molecules present in the local cellular milieu during their first 24 h following exit from the cell cycle.

The mammalian ShcB and ShcC phosphotyrosine docking proteins function in the maturation of sensory and sympathetic neurons.

Shc proteins possess SH2 and PTB domains and serve a scaffolding function in signaling by a variety of receptor tyrosine kinases. There are three known mammalian Shc genes, of which ShcB and ShcC are primarily expressed in the nervous system. We have generated null mutations in ShcB and ShcC and have obtained mice lacking either ShcB or ShcC or both gene products. ShcB-deficient animals exhibit a loss of peptidergic and nonpeptidergic nociceptive sensory neurons, which is not enhanced by additional loss of ShcC. Mice lacking both ShcB and ShcC exhibit a significant loss of neurons within the superior cervical ganglia, which is not observed in either mutant alone. The results indicate that these Shc family members possess both unique and overlapping functions in regulating neural development and suggest physiological roles for ShcB/ShcC in TrkA signaling.

The receptor tyrosine kinase EphB2 regulates NMDA-dependent synaptic function.

Members of the Eph family of receptor tyrosine kinases control many aspects of cellular interactions during development, including axon guidance. Here, we demonstrate that EphB2 also regulates postnatal synaptic function in the mammalian CNS. Mice lacking the EphB2 intracellular kinase domain showed wild-type levels of LTP, whereas mice lacking the entire EphB2 receptor had reduced LTP at hippocampal CA1 and dentate gyrus synapses. Synaptic NMDA-mediated current was reduced in dentate granule neurons in EphB2 null mice, as was synaptically localized NR1 as revealed by immunogold localization. Finally, we show that EphB2 is upregulated in hippocampal pyramidal neurons in vitro and in vivo by stimuli known to induce changes in synaptic structure. Together, these data demonstrate that EphB2 plays an important role in regulating synaptic function.

Forward genetic screen of mouse reveals dominant missense mutation in the P/Q-type voltage-dependent calcium channel, CACNA1A.

Dominant mutations of the P/Q-type Ca(2+) channel (CACNA1A) underlie several human neurological disorders, including episodic ataxia type 2, familial hemiplegic migraine 1 (FHM1) and spinocerebellar ataxia 6, but have not been found previously in the mouse. Here we report the first dominant ataxic mouse model of Cacna1a mutation. This Wobbly mutant allele of Cacna1a was identified in an ethylnitrosourea (ENU) mutagenesis dominant behavioral screen. Heterozygotes exhibit ataxia from 3 weeks of age and have a normal life span. Homozygotes have a righting reflex defect from postnatal day 8 and later develop severe ataxia and die prematurely. Both heterozygotes and homozygotes exhibit cerebellar atrophy with focal reduction of the molecular layer. No obvious loss of Purkinje cells or decrease in size of the granule cell layer was observed. Real-time polymerase chain reaction revealed altered expression levels of Cacna1g, Calb2 and Th in Wobbly cerebella, but Cacna1a messenger RNA and protein levels were unchanged. Positional cloning revealed that Wobbly mice have a missense mutation leading to an arginine to leucine (R1255L) substitution, resulting in neutralization of a positively charged amino acid in repeat III of voltage sensor segment S4. The dominance of the Wobbly mutation more closely resembles patterns of CACNA1A mutation in humans than previously described mouse recessive mutants (tottering, leaner, rolling Nagoya and rocker). Positive-charge neutralization in S4 has also been shown to underlie several cases of human dominant FHM1 with ataxia. The Wobbly mutant thus highlights the importance of the voltage sensor and provides a starting point to unravel the neuropathological mechanisms of this disease.

Systemic administration of ciliary neurotrophic factor induces cachexia in rodents.

Ciliary neurotrophic factor (CNTF) has previously been shown to promote the survival of several classes of neurons and glial. We report here that in addition to its effects on the nervous system, CNTF can induce potent effects in extra-neural tissues. Implantation of C6 glioma cells engineered to secrete CNTF either subcutaneously or into the peritoneal cavity of adult mice, or systemic injections of purified rat or human recombinant CNTF, resulted in a rapid syndrome of weight loss resulting in death over a period of 7-10 d. This weight loss could not be explained by a reduction in food intake and involved losses of both fat and skeletal muscle. CNTF also induced the synthesis of acute phase proteins such as haptoglobin. Implantation of C6 lines expressing a nonsecreted form of CNTF, or the parental C6 line itself, did not result in wasting effects. Analysis of this CNTF-induced wasting indicates similarities with the previously described cachectins, tumor necrosis factor, interleukin 6, and leukemia inhibitory factor, but does not involve the induction of these cytokines.

Development of a high resolution three-dimensional surgical atlas of the murine head for strains 129S1/SvImJ and C57Bl/6J using magnetic resonance imaging and micro-computed tomography.

The mouse has emerged as a major experimental model system for examining the functional properties of the mammalian CNS; both during development and following CNS injury. Histologic procedures currently used to determine the relative position of structures within the CNS are presently limited in their ability to take full advantage of this system for surgical and morphometric procedures. We present here the first three-dimensional interactive digital atlas of the murine brain and skull for two genetically important strains of mice; 129S1/SvImJ and C57Bl/6J. The final resolution of these digital atlases is 54 micro m(3). These representations of the murine brain and skull, in conjunction with our development of a new, more dynamic master coordinate system, provide improved accuracy with respect to targeting CNS structures during surgery compared with previous systems. The interactive three-dimensional nature of these atlases also provide users with stereotactic information necessary to perform accurate "off-axis" surgical procedures, as is commonly required for experiments such as in vivo micro-electroporation. In addition, three-dimensional analysis of the brain and skull shape in C57Bl, 129Sv, CD1, and additional murine strains, suggests that a stereotactic coordinate system based upon the lambda and rostral confluence of the sinuses at the sagittal midline, provides improved accuracy compared with the traditional lambda-bregma landmark system. These findings demonstrate the utility of developing highly accurate and robust three-dimensional representations of the murine brain and skull, in which experimental outputs can be directly compared using a unified coordinate system. The aim of these studies is to enhance comparative morphometric analyses and stereotactic surgical procedures in mice.

Reducing maternal mortality due to elective abortion: Potential impact of misoprostol in low-resource settings.

Over 99% of deaths due to abortion occur in developing countries. Maternal deaths due to abortion are preventable. Increasing the use of misoprostol for elective abortion could have a notable impact on maternal mortality due to abortion. As a test of this hypothesis, this study estimated the reduction in maternal deaths due to abortion in Africa, Asia and Latin America. The estimates were adjusted to changes in assumptions, yielding different possible scenarios of low and high estimates. This simple modeling exercise demonstrated that increased use of misoprostol, an option for pregnancy termination already available to many women in developing countries, could significantly reduce mortality due to abortion. Empirical testing of the hypothesis with data collected from developing countries could help to inform and improve the use of misoprostol in those settings.

Rad54 and Mus81 cooperation promotes DNA damage repair and restrains chromosome missegregation.

Rad54 and Mus81 mammalian proteins physically interact and are important for the homologous recombination DNA repair pathway; however, their functional interactions in vivo are poorly defined. Here, we show that combinatorial loss of Rad54 and Mus81 results in hypersensitivity to DNA-damaging agents, defects on both the homologous recombination and non-homologous DNA end joining repair pathways and reduced fertility. We also observed that while Mus81 deficiency diminished the cleavage of common fragile sites, very strikingly, Rad54 loss impaired this cleavage to even a greater extent. The inefficient repair of DNA double-strand breaks (DSBs) in Rad54(-/-)Mus81(-/-) cells was accompanied by elevated levels of chromosome missegregation and cell death. Perhaps as a consequence, tumor incidence in Rad54(-/-)Mus81(-/-) mice remained comparable to that in Mus81(-/-) mice. Our study highlights the importance of the cooperation between Rad54 and Mus81 for mediating DNA DSB repair and restraining chromosome missegregation.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) deficiency results in exencephaly and is required for apoptosis within the developing CNS.

Tumor necrosis factor receptor-associated factors (TRAFs) are adaptor proteins important in mediating intracellular signaling. We report here that targeted deletion of traf6 greatly increases the frequency of failure of neural tube closure and exencephaly in traf6 (-/-) mice. The penetrance of this defect is influenced by genetic background. Neural tube fusion requires the coordination of several biological processes, including cell migration invoked by contact-dependent signaling, cell proliferation, and programmed cell death (PCD). To gain greater insight into the role of TRAF6 in these processes, neural development and migration within the CNS of traf6 (-/-) mice and controls were assessed through temporal examination of a number of immunohistochemical markers. In addition, relative levels of cellular proliferation and PCD were examined throughout embryonic development using bromodeoxyuridine (BrdU) and in situ terminal deoxynucleotidyl transferase-mediated dUTP biotinylated nick end labeling (TUNEL), respectively. The data suggest that loss of TRAF6 does not significantly alter the level of cellular proliferation or the pattern of neural differentiation per se, but rather regulates the level of PCD within specific regions of the developing CNS. Substantial reductions in TUNEL were observed within the ventral diencephalon and mesencephalon in exencephalic traf6 (-/-) embryos. Our results demonstrate a novel and prominent role for TRAF6 in the regional control of PCD within the developing CNS.

Assignment of the aromatic 1H-NMR resonances of myotoxin a isolated from the venom of Crotalus viridis viridis.

The 400 MHz 1H-NMR spectrum of myotoxin a from the venom of Crotalus viridis viridis is described. The identification of spin systems in the aromatic region corresponding to the six aromatic residues of myotoxin a was completed using both one- and two-dimensional NMR spectroscopy and the pH dependence of chemical shifts. Assignments of these spin systems to specific residues was possible for the singly occurring amino acids Tyr-1 and Phe-12. Resonances from Tyr-1, His-5 and His-10 were shifted significantly from their random coil values in a pH-dependent manner. These shift perturbations were deemed evidence of a helical arrangement of the amino terminal region which placed these residues in close proximity to each other.

Blood-brain barrier: an impediment to neuropharmaceuticals.

The blood-brain barrier (BBB) serves as a highly selective barrier separating the central nervous system from the systemic circulation. Although contributing to neurological health, the BBB restricts the ability of drugs to reach their site of action and thus presents a major challenge to the treatment of neurological disorders. Advances in our understanding of the complexity of the BBB have fostered development of novel pharmacometric models and drug delivery strategies to better predict and improve therapeutic access.

Smoking and risk factors in deep vein thrombosis.

The incidence of deep vein thrombosis (DVT) as diagnosed by the 125I fibrinogen test (125IFT) was determined in a series of 300 newly admitted medical and 201 surgical patients. 6 medical patients died before 125IFT screening could be completed. The incidence of DVT was 14% in medical patients and 18% in surgical patients. Increasing age, a malignant condition and a past history of thromboembolism all increased the risk of DVT. Increasing levels of cigarette smoking were found to be associated with a reduced incidence of DVT. Although statistical significance was achieved at only the 10% level for this finding it is in agreement with the results from studies on patients with myocardial infarction. The protective effect of cigarrette smoking was observed at all ages, and in both medical and surgical patients.

Localization of CNTF immunoreactivity to neurons and astroglia in the CNS.

Species specific antibodies were raised to a peptide of rat ciliary neurotrophic factor (CNTF-amino acids number 131-147). Following affinity purification, these antibodies were used to determine the pattern of CNTF immunoreactivity in adult rat and mouse brain, spinal cord, and sciatic nerve. Alternate sections stained using neurofilament and the affinity purified anti-CNTF antibody (HARC-1) demonstrate that CNTF immunoreactive neurons are present within the facial nucleus, dentate gyrus, olfactory bulb, basal forebrain, locus coeruleus, cortex and substantia nigra. In addition, neurons throughout the hippocampus, and Purkinje cells within the cerebellum also exhibit CNTF immunoreactivity. CNTF immunopositive neurons demonstrate a preponderance of nuclear staining, with some staining present in the cytoplasm. Alternate sections incubated with glial fibrillary acidic protein (GFAP) antibody also demonstrate glia which are positive for CNTF. In the peripheral nervous system, Schwann cells of the sciatic nerve exhibit strong immunoreactivity for CNTF, however staining is confined to the cytoplasm and is absent from the cell nucleus. These data demonstrate that CNTF immunoreactivity is broadly distributed throughout neurons and glia of the adult rodent nervous system.

Multiple behavioral anomalies in GluR2 mutant mice exhibiting enhanced LTP.

We have previously disrupted the ionotropic glutamate receptor type 2 gene (GluR2) using gene targeting in embryonic stem cells and generated mice which lacked the GluR2 gene product. Neurophysiological analyses of these mice showed a markedly enhanced long-term potentiation (LTP) and a 9-fold increase in kainate induced Ca2+ permeability in the hippocampus. Here, we analyze the behavioral and neuroanatomical consequences of GluR2 deficiency in homozygous null mutant and age-matched littermate control mice. We show that despite unaltered gross brain morphology, several aspects of behavior were abnormal in the mutants. Object exploration, rearing, grooming and locomotion were altered in the novel arena. Eye-closure reflex, motor performance on the rotating rod and spatial and non-spatial learning performance in the water maze were also abnormal in the mutants. These abnormalities together with the widespread expression pattern of GluR2 in most excitatory CNS pathways suggest that the absence of GluR2 leads to neurological phenotypes associated with not only the hippocampus but several other brain regions potentially including the cortex and cerebellum. We speculate that GluR2 mutant mice suffer from an overall non-specifically increased excitability that may alter cognitive functions ranging from stimulus processing to motivation and learning.

Ciliary neurotrophic factor promotes the terminal differentiation of v-myc immortalized sympathoadrenal progenitor cells in vivo.

Survival and differentiation of a sympathoadrenal progenitor cell line (termed MAH), transduced with a v-myc oncogene, was studied subsequent to transplantation in the peripheral and central nervous system of adult rats. In the brain, MAH cell survival depended on the secretion of ciliary neurotrophic factor (CNTF) by co-grafts of genetically modified glioma cells. No trophic factor supplement was required for development of the MAH cells in the peripheral nerve environment. Transplanted progenitor cells withdrew from the cell cycle within 48 h and differentiated into a prominent population of large sympathetic-like neurons. The neurons expressed the alpha subunit of the CNTF receptor and appropriate spatial distributions of cytoskeletal proteins and catecholamine related enzymes. The results identify a role for CNTF in the development of the sympathoadrenal cell lineage and support the concept of immortalized progenitor cells as alternatives to primary cells for cell replacement strategies in the nervous system.

Glial overexpression of NGF enhances neuropathic pain and adrenergic sprouting into DRG following chronic sciatic constriction in mice.

Adrenergic sprouts within axotomized dorsal root ganglia (DRG) may contribute to neuropathic pain, and may arise under the influence of nerve growth factor (NGF). We investigated effects of chronic constriction injury (CCI) on behavior and sprouting in mice in which NGF overexpression is driven by a glial protein (GFAP) promotor. GFAP-NGF mice were naturally hyperresponsive to radiant heat, and had enhanced ipsilateral responses to thermal and mechanical stimulation following CCI compared to wild-type mice. Sympathetic axons were already present in intact DRG of GFAP-NGF mice. Following CCI, sprouting in ipsilateral and to a lesser extent contralateral DRG occurred in both genotypes, but the sprout density 2 weeks post-lesion was much greater in GFAP-NGF mice. These results demonstrate a connection between the endogenous ectopic overexpression of NGF and (1) neuropathic pain behaviour and (2) sympathetic sprouting in the DRG.

Antigenic relationships between Mojave toxin subunits, Mojave toxin and some crotalid venoms.

Immunochemical responses of a number of pit viper venoms to antibodies derived separately from the acidic and basic subunits were investigated by enzyme linked immunosorbent assay (ELISA) and Ouchterlony immunodiffusion. The polyclonal antisera to the basic subunit were generated in rabbits, whereas mouse hybridoma cell cultures were used to produce antibodies to the acidic subunit. The immunochemical response of a venom correlated well with published values for LD50 dose for the test venom. Many venoms that elicited a positive response with antiserum to the basic subunit also reacted strongly with the hybridoma derived antibodies to the acidic subunit. The data support the conclusion that crotalid venoms which are more lethal have in common a potent venom component that is immunochemically related to Mojave toxin.