Neuronal NMDA Injury: Brain Slice And Primary Neuronal Injury Models,Protective Effect Of Drugs And Characteristics Of Signal Transduction

Cerebral ischemia is one of common CMS diseasie with high incidence and is the cause leading to death and disablement. Its prevention and treatment is becoming more and more important in the modern society because of the increasing of old people. Effective treatments at early stage of this disease may decline the rate of death and disablement to a minimum. So, it is very important to study the damage/anti-damage mechanisms and to explore the preventive ways. Over the past 10-15 years, substantial evidence has accumulated implicating excitotoxicity in the pathogenisis of ischemic brain injury. NMDA receptors are crucial in acute glutamate neurotoxicity, NMDA-antagonists could reduce neuronal death in animal models of ischamic or hypoglycemic brain injury. Many laboratories havedemonstrated that NMDA-receptor blockade reduces infarct volume in both lissencephalic and gyrencephalic animal models of transient or permanent focal ischemia.Permanent focal cerebral ischemia and global ischemia models have been established in our laboratory recently. Also, we have found that ONO-1078, a selective cysteinyl-leukotriene receptor antagonist, is protective against ischemic brain injury due to the permanent focal cerebral ischemia. To study the mechanisms of ischemic brain injury the excitotoxicity more thoroughly, and the therapeutic approach of ischemic brain injury, we in this research established the rational in vitro models of brain slice and primary neuronal cultures, searched new NMDA receptor antagonists, and analyzed the characteristics of signal transduction triggered by NMDA. We hope to elucidate the characteristics of injury induced by NMDA in vitro and find potential important targets for therapeutic invention for cerebral ischemia.1. In vitro injury models of brain slice (OGD and NMDA insult) and primary neuronal cultures(NMDA insult)Oxygen/glucose deprivation (OGD)-induced injury of rat hippocampal slice in vitroThe rat hippocampal slices prepared were allowed to recover in the normal artificial cerebrospinal fluid(ACSF) bubbled with gas mixture of 95% O2+5% CO2 for 1 h, then they were thansfered to glucose-free nACSF which was bubbled with gasmixture of 95%N2+5%CO2. After treatment with OGD, the slices were placed into 2% TTC solution in dark and incubated at 37*Cfor 1h. The slices were weighted and a 50 : 50 mixture of ethanol/dimethyl sulfoxide was then added to extract the formazan in dark for 24 h. For analysis, 200 y I aliquots of the red solvent extract were placed in 96 well plate. Average absorbance was read at x 490nm in Automated Microplate Reader. In a second experiment, the released LDH due to OGD insult was measured. We found that with the time prolongation of OGD, TTC staining of the hippocampal slices was reduced and LDH release rate was increased consistently. The percent decrease of TTC staining was significantly correlated (r = -0.933) to the percent increase of LDH release due to OGD insults. Our results indicate that the solvent extraction and spectrophotometric quantification of formazan has potential utility as an objective way to index OGD-induced injury of brain slices, it is a simple and reliable method for screening neuroprotective drugs.OGD and NMDA-induced injury of mouse corticostriatum slice in vitro The mouse corticostriatum slices prepared were allowed to recover for 1 h, then subjected to OGD or NMDA. After treatment, the slices were incubated in 2% TTC solution in dark at 37"Cfor 1h, then fixed in fomaldehyde, taken photographs with CCD camera under microscope, the computer imaging was utilized to assess the intensity and area of TTC staining. We found that with the time prolongation of OGD, the average intensity and area of TTC staining decreased consistently. The average area of TTC staining also decreased consistently after NMDA insult, and inmixture of 95%N2+5%CO2. After treatment with OGD, the slices were placed into 2% TTC solution in dark and incubated at 37*Cfor 1h. The slices were weighted and a 50 : 50 mixture of ethanol/d...