| Thc role of dexamethasone on ncuronalapoptosis after reperfusion folIowing middlecerebral artery occlusion in ratsObjectiveTO investigate the effect of dexamethas one (DXM), a pureglucocorticoid, on infarction size and aPoPtosis after transientfocal ischemia with middle cerebral artery occlusion in rats.Materials and MethodsReagents: Dexamethasone 5mg/ml(XINCHANGPharmaceutical comPany), TTC(sigma); aPoPtosis kit(ROCHEcomPany); All other chemicals were AR.Animal preparation: Male SD rats (n=60, weighing250-295g) were randomly divided into 6 grouPs:sham-operated grouP (n=6), non-operated DXM grouP (n=6),ischemia-control grouP, three ischemia-DXM grouPs (DXM0.5mg/kg.bw l.5mg/kg.bw 5mg/kg.bw respectively)(eachn=12). DXM with three different doses was injectedilltraPeritoneally 3 days before operation, once a day Briefly,after deprived of foods for at least 6 hours, rats wereanesthetized with 350mg/kg.bw hydrodehydrede.Approximately 20 mm of 3-0 surgical nylon suture (with its tiprounded by heating near a flame ) was inserted from the righteXternal carotid artery and adVanced iflto the intemal carOtidartery until it blocked the origin of the MCA for 40 min. Insham --opended grouP and non-opefated DXM grouP the nylonsuture was insefted less than 5mm, which is too short toocclude the MCA.Measurement of infarct volume' All rats were killed at24 hours of reperfusion after withdrawing the suture. About 5coronal slides (2rnm thick )of brain were obtained frOm eachrat and then were incubated for 30 min with 2%TTC in 0.l Mphosphate buffer at 37'C.TTC is reduced by dehydrogenase,Which exist in viable cells, thereby tUming tissue deeP red,while nonviable cells are pale. Brain infarCion was photed andquantified by comPuter software with the use of the formula:RI=LT-KN(RI=infarCt area in the right hemisphere, LT= totalarea in the left hemisphere, KN=noninfarcted area in the righhemisphere). The infarct volumes were derived fromsummation of multiplation of slice thick (2mm)with averageareas of two sides in all slices.Histological examination: Brans were stored informalin and a coronal section at the level of the aneriorcommissure was embedded in paraffin and stained withhaematoXylin and eosin.Detection of aPoPtosis: The adacent sections wereStained with the terminal deoxyncleotidyl trallsferase-mediated dUTP nick end-labeling (TUNEL) method,fOllowng the procedure Which was provided by the kits. TheaPoPosis index was measured with ligh microscoPicanalysis(400 field) by couning the positive cells per l000cells.Statistical methods: Vaues were analyzed withSPSS8.0,presented as xi s, and comPared by one-wayANOVA and SNK analysis.ResultslnfarCt volume: TTC staining showed no infarction insham-operated rats and non-operation DXM grouP. InfarCtionwas primarily located in parietal cortex and lateral striatum.The infarCt vo1ume was increased distinctively in the grouPsthat received DXM than in the animals that received saline, butthe difference of infaret volume was not distinct with theincreasing of DXM dose.HistO1ogical outcome' With HE staining and lighmicroscopic analysis, aPoPtOic cells are scattered in corticalpyramidal cells both in ischemic control and ischemic DXMgrouPs and showed as following:cell bodies were contraCted,presenting triangular or linear shaPe, nuclei wee stainedintensely purple-blue with nucleolus disaPpeared, and thespaces between cells were widened. The amoun of aPoPtoticcells in ischemic DXM grouPs was more than in ischemiccontrol. There were not injured cells in sham-operated rats andnon-operated DXM grouP.Detection of aPoPtosis: Wth the use of TorL method,aPoPtotic cells were stained dark for DNA breaks with nucleisM. Round or oval aPoptotic bodies were occasionallyrecognized. The nuclei were stained red by nuclear fast red innegative cells. Sham-operated rats and non-operated DXM...
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