Effects Of Lamotrigine On Expression Of NR1 And GluR2 In Rat Forebrain After Intracerebral Hemorrhage

Background and objectiveThe pathophysiology changes of brain hemorrhagic is a quite complicated process,which involves the space occupying lesion of hematoma ,the toxicity damage of blood constituent and its split product and the cascade reaction caused by excitatory amino acids.This reaction begined with exitotoxicity, exitotoxicity is the trigger of downstream event ,which is the most important and crucial link .The activation of NR1 which is the subunite of excitatory amino acids recepters NMDA is the core of exitotoxicity,because NR1 can activate channel which depend on voltage directly or indirectly and cause the imput of Ca²⁺ .In the end that induce the delayed degeneration and necrotic of neuro.But GluR2 which is the subunite of of AMPA can protect brain through inhibiting the imput of Ca²⁺.Lamotrigine is a drug which is used to treat refractoriness epileptic, Now we have found that it has protective effects to the damage of exitotoxicity caused by excitatory amino acids,but the specific mechanism is not very clear. The recent research indicated that the research in EAAS is main concentrated in ischemic cerebrovascular diseases,and we seldom see the articles in ICH in this aspects,especially the articles about Lamotrigine in this aspects.This experiment proceed by the establishment of rats ICH model,and use immunohistochemical method to detect the expression of NR1,GluR2 in hippocamp , cortex and perihaematoma in deffrient times,and detect the edema of brain tissue through dry-humid weight method.we see the influence of Lamotrigine to the expression of NR1,GluR2 and the edema of brain tissue. we approach mechanism of EAAs to the damage of neuro and the protective mechanism of Lamotrigine to ICH,So we can supply some method for the therpy of ICHMaterials and methods1. experimental animal and grouping72 healthy Wister rats were divided into 3groups randomly . That are sham operated group,control group and medication administration team, All animals are supplied by the expremiental animal center of zhenggzhou university,and the weight of all animals is between 200-250g.The number of certification is SYK2006-011. except sham operated group ,each group has four different times,each has 8 rats. HE staining, Immunohistochemical technique was used to study the expression of excitatory amino acids receptors during different periods after ICH for 8 rats. The changes of brain water content were measured too.2. The make of the model of ICH and the sign of sucessAll the rats were narcotized and fixed to stereo orientation instrument in prostration, and anesthetized by 10% chloral hydrate. The rats were infused self body blood 50μl which were took out from the right thigh artery . Needles of trivial syringe were inserted into the right caudate nuclear. The infused speed was very slow and detained for 15 min. False operation group was infused NS 50μl . Rats were classified when sobered according to the method of Zea Longa, exceed 1 record were successful model.3. The preparation of histological sectionRemoved the operation side from cerebral hemisphere, paraffin imbedding, brain tissue centered with the needles passage approximately 3mm thick was sacrificed to investigate HE coloration and the expression of NR1,GluR2. the slices were 4μm thick. 4. The detection of indexesHE staining :observe the pathological change of brain tissue under light microscope.Immunohistochemistry method was used to detected the expression of NR1and GluR2,and we used the SP method to detect,we operated strictly as the direction of kit.The colour of cell membrane and kytoplasm of positive cells is buffy yellow .5. Image analysisEach rat has 6 slices, and selected 5 different regions every slice at random to observe. Using Biosens Digital Imaging System to analyze the positive area rate.6. The determine of the water content of brain tissueThe slices behind needle passages about were sacrificed to investigate the content of brain water. First, weigh the wet heft with electronic balance and put them into 100°C to desiccated chest for 48h, took out them to weigh the dry left. The content of brain water = (wet heft - dry heft)/ wet heft×100%.7. Method of statisticsThe data was handled with SPSS 10.0statistic software, the difference between groups were handled with analysis of variance , the content of brain water was handled with analysis of variance, significant disparity is P<0.05.Results1. The change of histomorphology of brainHE staining : neural cell body around hematoma, hippocamp , cortex was bullated 12h—1d after ICH, parts of glial cell proliferated 1-3d were the obviously period. Cells were deformed and necrotic. The nucleuses were concentrated, fragmentary and dissolved, the red dying of cytoplasm was more obviously than contrast group. The tumefaction of cell was relieved obviously 7d after ICH, only little glial cells proliferated.the control group did not chang obviously.2. The dynamic change of NR1,GluR2sham operated group:there was weakly to moderately positive expression in the cortex , the positive cell ,which' s membrane which has a positive expression is buffy yellow and mottling and the cytoplasm which has a less positive expression is yellow and the nucleus has a weakly positive or negative expression and the dendrite has no expression,has adistinctness layer and doesn't chang obviously during 12h to 7d.Control group: The expression of NR1,GluR2 around hematoma, hippocamp , cortex increased gradually at 12hrs and reached a peak between 1-3 days after ICH. It remained higher than normal level at 7days.Medication administration team: The expression of NR1 around hematoma, hippocamp , cortex decreased obviously. The expression of GluR2 around hematoma, hippocamp , cortex increased gradually at 12hrs and reached a peak between 1-3 days after ICH. It remained higher than normal level at 7days.3.The variation of the water content of brain tissueThe brain water content increased from 6hrs and peaked between 1-3 days following ICH. It was obviously reduced at 7days and returned to normal at 14days basically following ICH.Conclusions1. Lamotrigine has the ability of reducing the brain water content of expremiental rats.2. Lamotrigine can decrease the expression of NR1 around hematoma, cortex. But it can increase the expression of GluR2 around hematoma, cortex.3. The expression of NR1,GluR2 around hematoma is dynamic after ICH, suggesting that NR1 is participated in the course of hemorrhagic cerebral edema and injury after ICH . It is one of the important factors to cause hemorrhagic cerebral edema and injury after ICH.4. Lamotrigine has protective effects after ICH .The mechanism of that may be that it can increase the expression of GluR2 and decrease the expression of NR1.5. It will be an effective way to control the excessive expression of NR1 after ICH early and actively in order to alleviate the degree of hemorrhagic cerebral edema and injury after ICH.