| Objective: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that selectively involving the upper and lower motor neurones. The disease is characterized by progressive muscle weakness,atrophy, which ultimately leads to respiratory failure and death occurring 3-5 years from the first appearance of symptoms .The cause of ALS is not known. Several hypotheses have been proposed about the aetiology of the disease, including excitotoxicity, autoimmunity, oxidative stress, neuron apoptosis, mitochondria dysfunction et al. Glutamate-induced excitotoxicity is implicated as playing a key role in the pathogenesis of ALS. Glutamate is the principal excitatory neurotransmitter in the central nervous system. Over activity of the glutamatergic neurons and excessive extracellular accumulation of Glu surrounding the synaptic cleft may cause exicitotoxic damage in the postsynaptic neurons and other surrounding tissues through over activation of glutamate receptors. Organotypic spinal cord culture can be reliably maintained for a long time and have well preserved organotypic morphology, which provides a more effctive way to study spinal cord diseases than dissociated cell culture. We used organotypic spinal cord cultures to develop an in vitro model of ALS by chronically blocking the glutamate carriers. Topiramate is a novel anticonvulsant and structurally distincts from other known anti-convulsants. A number of independent studies suggest that the actions of topiramate depend, at least in part, on its ability to inhibit kainite-induced, but not NMDA-induced seizures. Furthermore, studies of ion channel activity indicate that topiramate can influence the activity of the AMPA/kainate subtype of glutamate receptors. Therefore due to these pharmacological properties we tested whether topiramate was neuro-protective for the motor neuron in an organotypic spinal cord culture system that had been developed as a model for long-term glutamate-mediated excitotoxicity. Methods: 1. Organotypic spinal cord cultures. Organotypic spinal cord cultures were prepared from 8 day old rat pup lumbar spinal cords. Spinal cords were collected under sterile conditions and sectioned transversely at 350 mm intervals with a McIl wain tissue chopper. Sections were then transferred to sterile Gey's balanced saltsolution (Gibco) containing glucose (6.4mg/ml) and gently separated at room temperature. Slices were carefully placed on the surface of membrane insert with five slices per insert. These inserts were placed in 35mm(6 wells) culture wells containing 1ml of culture medium and incubated at 37℃in a 5% CO2/25% air humidified environment .The culture medium was changed twice weekly. 2. The cultures were divided into four groups. ⑴control group: Every well was added 1ml culturemedium. ⑵T HA group: Every well was added 1ml culture medium containing THA achieved a final concentration 100μmol/L. ⑶THA+ topiramate A group: Every well was added 1ml culture medium containing topiramate achieved a final concen-tration 10μmol/L and 100μmol/L THA. ⑷THA+ topiramate B group: Every well was added 1ml culture medium containing topiramate achieved a final concentration 100μmol/L and 100μmol/L THA. All the groups were treated with normal culture medium for the first week. Tissues were added THA 100μmol /L, Topiramate A,Topiramate B respectively in the ⑵⑶⑷group from the second 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 non-phosphorylated neurofilament H (SMI-32) immunohistochemical staining. Under low power microscopy total motor neuron counts were performed and averaged for each experiment. Results are presented as means±SEM. Analysis of any neuroprotective effect was performed by comparison of topiramate treatment in the presence of THA compared with the THA effect alone by Student's t-test (α=0.05). Results: 1. The spinal cord explants had well-preserved organo... |
PDF Full Text Request