Genetic Influences on the Amount of Cell Death in the Neural Tube of BXD Mice Exposed to Acute Ethanol at Midgestation.

BACKGROUND: Fetal alcohol spectrum disorders (FASD) have a strong genetic component although the genes that underlie this are only beginning to be elucidated. In the present study, one of the most common phenotypes of FASD, cell death within the early developing neural tube, was examined across a genetic reference population in a reverse genetics paradigm with the goal of identifying genetic loci that could influence ethanol (EtOH)-induced apoptosis in the early developing neural tube. METHODS: BXD recombinant inbred mice as well as the parental strains were used to evaluate genetic differences in EtOH-induced cell death after exposure on embryonic day 9.5. Dams were given either 5.8 g/kg EtOH or isocaloric maltose-dextrin in 2 doses via intragastric gavage. Embryos were collected 7 hours after the initial exposure and cell death evaluated via TUNEL staining in the brainstem and forebrain. Genetic loci were evaluated using quantitative trait locus (QTL) analysis at GeneNetwork.org. RESULTS: Significant strain differences were observed in the levels of EtOH-induced cell death that were due to genetic effects and not confounding variables such as differences in developmental maturity or cell death kinetics. Comparisons between the 2 regions of the developing neural tube showed little genetic correlation with the QTL maps exhibiting no overlap. Significant QTLs were found on murine mid-chromosome 4 and mid-chromosome 14 only in the brainstem. Within these chromosomal loci, a number of interesting candidate genes were identified that could mediate this differential sensitivity including Nfia (nuclear factor I/A) and Otx2 (orthodenticle homeobox 2). CONCLUSIONS: These studies demonstrate that the levels of EtOH-induced cell death occur in strain- and region-dependent manners. Novel QTLs on mouse Chr4 and Chr14 were identified that modulate the differential sensitivity to EtOH-induced apoptosis in the embryonic brainstem. The genes underlying these QTLs could identify novel molecular pathways that are critical in this phenotype.

Molecular pathways underpinning ethanol-induced neurodegeneration.

While genetics impacts the type and severity of damage following developmental ethanol exposure, little is currently known about the molecular pathways that mediate these effects. Traditionally, research in this area has used a candidate gene approach and evaluated effects on a gene-by-gene basis. Recent studies, however, have begun to use unbiased approaches and genetic reference populations to evaluate the roles of genotype and epigenetic modifications in phenotypic changes following developmental ethanol exposure, similar to studies that evaluated numerous alcohol-related phenotypes in adults. Here, we present work assessing the role of genetics and chromatin-based alterations in mediating ethanol-induced apoptosis in the developing nervous system. Utilizing the expanded family of BXD recombinant inbred mice, animals were exposed to ethanol at postnatal day 7 via subcutaneous injection (5.0 g/kg in 2 doses). Tissue was collected 7 h after the initial ethanol treatment and analyzed by activated caspase-3 immunostaining to visualize dying cells in the cerebral cortex and hippocampus. In parallel, the levels of two histone modifications relevant to apoptosis, γH2AX and H3K14 acetylation, were examined in the cerebral cortex using protein blot analysis. Activated caspase-3 staining identified marked differences in cell death across brain regions between different mouse strains. Genetic analysis of ethanol susceptibility in the hippocampus led to the identification of a quantitative trait locus on chromosome 12, which mediates, at least in part, strain-specific differential vulnerability to ethanol-induced apoptosis. Furthermore, analysis of chromatin modifications in the cerebral cortex revealed a global increase in γH2AX levels following ethanol exposure, but did not show any change in H3K14 acetylation levels. Together, these findings provide new insights into the molecular mechanisms and genetic contributions underlying ethanol-induced neurodegeneration.

Use of the expanded panel of BXD mice narrow QTL regions in ethanol-induced locomotor activation and motor incoordination.

BACKGROUND: Alcohol-related responses are under strong genetic regulation. A wealth of alcohol-related data from recombinant inbred (RI) mouse strains enables genetic correlation and mapping of these traits. Previous studies using RI strains have identified numerous chromosomal locations that underlie differential alcohol sensitivity, although the regions identified are typically large. One means to improve power and precision for genetic analysis is to use a larger genetic reference population. The expanded panel of BXD RI mice was used to identify quantitative trait loci (QTLs) associated with sensitivity to locomotor stimulatory and motor incoordinating effects of alcohol. The goals of this study were to determine whether previously reported QTLs were replicated and refined and to determine whether novel QTLs would be identified. METHODS: Following an i.p. dose of 2.25 g/kg of ethanol (EtOH) or saline control, locomotor activation was assessed using an activity chamber and motor incoordination was assessed using the accelerating rotarod. Male and female BXD mice from over 55 strains were tested. Two treatment paradigms were utilized to evaluate the effects of EtOH versus saline treatment-order. RESULTS: Activity chamber measures showed significant differences in strain, sex, and treatment-order whereas rotarod measures showed significant differences in strain and treatment-order. Significant QTLs for various measures of EtOH-induced locomotor activation were identified on chromosomes 2 and 5 that narrowed QTL regions previously identified from 19 to < 2 Mb. Further, a novel significant QTL for EtOH-induced motor incoordination on chromosome 7 was identified. CONCLUSIONS: Using the expanded RI BXD panel, along with a high precision marker map, several novel QTLs were found and several previously identified QTL regions were confirmed and narrowed. The isogenic nature of the population facilitated detection of treatment-order and sex-specific differences. Smaller QTL regions reduced the number of positional candidates thereby increasing the efficiency with which polymorphisms underlying the QTL will be identified.

Effect of shear force on intervertebral disc (IVD) degeneration: an in vivo rat study.

Mechanical stress on the intervertebral disc (IVD) may contribute significantly to IVD degeneration, although its pathomechanism has not been fully understood. The purpose of this study was to test the hypothesis that sustained application of static shear force would result in IVD degeneration with minimum injury. We applied shear force on the rat lumbar spine (L5-L6) using a custom-designed loading device for 1 or 2 weeks. Degenerative changes such as nucleus pulposus cavity loss and border disruption were observed from the histology sections, indicating that the application of sustained dorsoventral shear force on the L6 vertebra induced degeneration of the IVDs in L5-L6 and adjacent levels of motion segment in 1 and 2 weeks. The findings of the present study could be useful for gaining a more relevant understanding of the biomechanical load factors of IVD degeneration not only for enabling better therapeutic interventions but also reducing the risk of low back injury.