| Objectives: A strong glial reaction typically surrounds the affected upper and lower motor neurons and degenerating corticospinal tracts of ALS patients. Reactive astrocytes in ALS contain protein inclusions, express inflammatory makers such as the inducible forms of nitric oxide synthase (iNOS) and cyclooxygenase (COX-2), and downregulate the glutamate transporter EAAT2.Available evidence supports the view that glial activation could be initiated by proinflammatory mediators secreted by motor neurons in response to injury, axotomy or muscular disease. In turn, reactive astrocytes produce nitric oxide and peroxynitrite, which cause mitochondrial damage in cultured neurons and trigger apoptosis in motor neurons. Astrocytes may also contribute to the excitotoxic damage of motor neurons by decreasing glutamate transport or actively releasing the excitotoxic amino acid. In addition, reactive astrocytes secrete pro-apoptotic mediators, such as nerve growth factor (NGF) or Fas-ligand, which is a mechanism that may serve to eliminate vulnerable motor neurons. The comprehensive understanding of the interactions between motor neurons and glia in ALS may lead to a more accurate theory of the pathogenesis of the disease. This study was aimed at developing a reproducible model for selective motor neuron death in cultured organotypic spinal cord slices to mimic a feature of ALS, which is selective motor neurons loss and sensory neurons surviving. This model was based on specific inhibition of glutamate transport carrier by exposing organotypic spinal cord cultures to threohydroxyaspartate(THA), an inhibitor of glutamate transporter, which continuously raised the concentration of glutamate in the culture medium and then on the base of the model, we did astrocyte staining to analyzing the role of astrocyte in the pathogenisis of ALS. Methods: 1 Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old SD rat pups. Nerve roots were cut out. Lumbar spinal cord were quickly collected under sterile conditions and sliced into 350μm-thick sections with a Mcllwain tissure chopper. Sections were then transferred to sterile GBSS and gently separated at room temperature. The slices were carefully placed on the surface of membrane insert with 5 –6 slices per insert. These inserts were placed in 35 mm(6 wells) culture dishes containing 1ml of culture medium and kept in a 5%CO2+95% air incubator at 370C.Culture medium was changed twice weekly. On the basis of the Organotypic spinal cord cultures, we added 100μmol/l THA into culture medium to establish ALS Organotypic spinal cord cultures. 2 Experimental groups: ①normal control group:1mlculture medium was put into per insert ,and every insert was kept 1 week, 2 weeks, 3 weeks and 4 weeks. ②THA effect group: 1ml culture medium+1ml 100μmol/l THA was put into per insert, and every insert was kept 1 week, 2 week, 3 week and 4 week. 3 Immunohistochemistry : After 4 weeks in culture, tissues were taken out and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). α-motorneurons survival was evaluated by culture morphology and nonphosphorylated neurofilament H (SMI-32) immunohisto chemical staining and by GFAP staining. Under low power microscopy total astrocyte counts were performed and averaged for each experiment. Results are presented as means ±SEM. Analysis of any astrocyte counts was performed by comparison of THA effect compared with the normal control group by variance analysis of SPSS (α=0.05). Results: The results showed that the spinal cord explants in control group could maintain excellent organotypic cellular organization and a stable population of ventral motor neurons. According the results of our laboratory that 100μmol/l THA resulted in significantly decrease of motor neuron and ultrastructural alternations after cultured 4 weeks while the inter-neurons in the dorsal horn were less affected. These changes were same as changed in ALS. As compared to control group, there was significantly astrogliosis in the anterior ho...
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