The Research Of Brain Protection Mechanism About The Preconditioning Focal Cerebral Ischemia Towards Brain Damage After Ischemia/reperfusion In Rats

Stroke is a commen disease that threatens the health of the older and happens frequently. With the population becomes older and older, the incidence rate of stroke increases clearly. Stroke threatens the older s health and survival quality seriously. Within the population of stroke, the proportion of cerebral infarction lack of blood is about 80% , bleeding cerebral infarction is only about 10% , while focal cerebral ischemia is 90% in cerebral infarction lack of blood . Cerebral ischemia can result in serious delay neural cells'damage and effect the sufferers'function recover. After cerebral infarction (middle cerebral artery occlusion) , the neural cells within that areas can be lack of blood and oxygen, and then necrosis and apoptosis step by step. There are lots of factors that can influence cells'necrosis and apoptosis. Through study these factors , we hope that neural cells'necrosis and apoptosis can be reduced mostly and the degree of cerebral ischemia damage can be decreased. This has great significances to instruct anti - cerebral ischemia therapy program in clinic.Many studys show that anti - damage mechanism which body owns has important roles at reducing cerebral function damage. Preconditioning cerebral ischemia is to perform temporary , one time or many time sub - fatal cerebral ischemia towards brain , which can reduce the followed damage caused by fatal ische-mia/reperfusion and protect brain. Preconditioning cerebral ischemia is one of the focal points of cerebral ischemia at present .This experiment uses suture - occluded method blocking middle cerebral artery of rats to establish the model of preconditioning cerebral ischemia. Throughtemporary N focal preconditioning cerebral ischemia for 20min and then reperfusion for some time to study the followed cerebral infarction volume caused by is-chemia/reperfusion, the density of neural cells at Hippocampi CA1, the changing expression law of heat shock protein ( HSP) 70 and anti - apoptosis gene protein Bel -2and Bax ,the distribution law of hypoxia imaging agent "T cm -HL91 at cerebral ischemia tissues. To study brain protection mechanism of preconditioning focal cerebral ischemia towards ischemia/reperfusion brain damageMATERIALS1 . Animals for experiment : 42 SD rats, male or female, weighed 260- ~ 320g, were obtained from the China Medical University Animal care facility.2 . Drugs and reagents : TTC, atropine, amobarbital sodium, rabbit anti -mouse HSP70 multi - clone antibody ( first antibody ) , HSP70 immunohisto-chemistry Kit, SABC Kit, DAB reagent (first part); rabbit anti mouse Bel - 2 and Bax multi - clone antibody ( first antibody) , Bel - 2 and Bax immunohisto-chemistry Kit, SABC Kit, bcl - 2 mRNA and bax mRNA in situ hybridization Kit, SABC Kit, DAB reagent (second part); hypoxia imaging agent "T cm -HL91, Nuclear Emulsion (N -4) (third part).3 . Instrument: Olympus substantial microscope, OlympusDPIO digital camera, computer image analysis software ( Metamorph Imaging System) , constant temperature incubate box, Olympus optics microscope, Nuclear Emulsion imbrue box.METHODSUsing suture - occluded method to establish the model of single middle cerebral artery occlusion (MCAO) in rats: were forbidden to have food but permit to drink water last night before experiment. Rats were injected in the abdominal cavity with atropine lOmin before anaesthesia with 1% amobarbital sodium. Cut rats at the center of their necks to expose the common carotid arterys, then sepa-rated and deligated the offset and main trunk of external carotid artery . Separated internal carotid artery and deligated yie artery - the only offset of internal carotid artery outside skull. Deligated common carotid artery, put inside the diameter about 0. 25 ~ 0. 30mm nylon thread from the fork of external carotid artery and internal carotid artery , when the depth is about 19 ~ 20mm, the nylon thread would arrive at right cerebra front artery. Blocked all blood sources of middle cerebral artery for 20min then reperfused for 1 ~ 3 days to eatablish preconditioning focal cerebral ischemia model. Ischemia /reperfusion brain damage model is made through cerebral ischemia for 2 hour then reperfusion for 24 hour.Part One:Establish right MCAO model in rats, divided rats into 4 groups: (1) control group of imitational operation(n =6) , only separated internal carotid artery and external carotid artery not put thread in; (2) control group of preconditioning focal cerebral ischemia ( n = 8 ) , preconditioning focal cerebral ischemia for 20min; ( 3) preconditioning focal cerebral ischemia group ( n = 24 ) , preconditioning focal cerebral ischemia for 20min then divided the rats into 3 smaller groups according to different reperfusion time ( 1 ^ 2 N 3 days ) , every smaller groups cerebral ischemia once again for 2 hours; (4) focal cerebral ischemia group ( n = 8 ) , cerebral ischemia for 2 hours. All rats in these groups were killed after reperfusion for 24 hours. All the 4 groups were divided into 2 sub -groups , one group was stained with TTC (n = 3 ) , then the results were analysed by computer image analysis software to calculate the volume of cerebral infarction through echelon rule; the other group was stained with immunohistochemis-tiy (n=3 ~5)to observe the expression of hot shock protein ( HSP70) ; HE stain was performed to observe the density of neural cells at Hippocampi CA1 s changing expression law under optics microscope.Part Two:Establish right MCAO model in rats, divided rats into 4 groups: (1) control group of imitational operation(n =3) , only separated internal carotid artery and external carotid artery not put thread in; (2) control group of preconditioning focal cerebral ischemia ( n = 5 ) , preconditioning focal cerebral ischemia for 20min; (3) preconditioning focal cerebral ischemia group (n = 15) , precondi-tioning focal cerebral ischemia for 20min then divided the rats into 3 smaller groups according to different reperfusion time ( 1 N 2N 3days ), every smaller groups cerebral ischemia once again for 2 hours; (4) focal cerebral ischemia group (n =5) , cerebral ischemia for 2 hours. All rats in these groups were killed through 4% formamint heart filled after reperfusion for 24 hours. Take brain slices from behand eye cross lmm to 4mm, then perform routine dehydration N transperence^olefin embeded,cut slices from coronal (slices' thick is 5jxm ). Immunohistochemistry and in situ hybridization were performed to observe the expression law of apoptosis control gene and protein bcl - 2 mRNA and bax mR-NA at the area of cortices and basal nuclei .Part Three.-Establish right MCAO model in rats, divided rats into 2 groups: ( 1) preconditioning focal cerebral ischemia group (n =21) , preconditioning focal cerebral ischemia for 20min then reperfusion for 3days, cerebral ischemia for 2 hours. (2) focal cerebral ischemia group (n =21), cerebral ischemia for 2 hours. ALL the rats were injected hypoxia imaging agent"Tcm - HL91 through caudal vein after cerebral ischemia for 2h. The two groups were divided into7 smaller groups according to defferent reperfusion time (5min,30min,lh,2h,4h, 8h,12h. ) ( n = 3 ). The method of 7 - ray measurement and autoradiography were used to observe the dynamic distribution of Tcm - - HL91 .RESULTSPart One: TTC stain showed that healthy brain tissue was red , while infarction tissue was white. The pictures under substantial microscope showed that control group of imitational operation and control group of preconditioning focal cerebral ischemia had no infarction areas. The infarction areas, of preconditioning focal cerebral ischemia group and focal cerebral ischemia group were in cerebral cortex of temporal lobe and parietal lobe and caudate nucleus at the ischemia side. The results of computer image analysis showed that preconditioning focal cerebral ischemia for 20min reperfusion for 1^3 days, then cerebral ischemia once again for 2 hours; the volume of cerebral infarction in preconditioningischemia group reduced significantly compared with ischemia group, (all p <0. 01 ). HE stain showed that The pictures under substantial microscope showed that control group of imitational operation and control group of preconditioning focal cerebral ischemia had no evident pathological changes. The neural cells of ischemia groups at the center of infarction decreased clearly, nucleus karyopy-knosis.karyolysis ,cells shrinked,neural felts collapsed, blood vessel endoder-mis cells fell off; The neural cells and neural colloid cells around the infarction areas ( half - dark zone) swelled, part nucleus karyopyknosis ? stained dark, clearances around neural cells and blood vessels expanded, part red cells leaked around blood vessels . Infarction center of preconditioning ischemia group decreased distinctively compared with cerebral ischemia group , structure integrated neural cells around infarction areas increased cleared. The density of neural cells at Hippocampi CA1 showed that compared with ischemia group , the three sub - groups of preconditioning ischemia group neural cells density increased significantly (all p <0.01). The result of immunohistochemistry of HSP70 showed that compared with ischemia group , expression of HSP70 protein in the three sub - groups of preconditioning ischemia group increased significantly ( all p <0.01).Part Two: The result of immunohistochemistry showed that Bel - 2 and Bax protein expressed little at the area of cortices and basal nuclei in ischemia side of the control group of imitational operation. Control group of preconditioning focal cerebral ischemias Bel -2 and Bax protein partial expressed at cortices and basal nuclei in ischemia side. Compared with the control group of imitational operation, the cells expressed Bel -2 increased clearly (p <0. 01) ,the cells expressed bax had no significant changes. (p >0.05) In preconditioning ischemia group (three sub - groups) Bel - 2 and Bax protein expressed a great deal in neural cells, compared with ischemia group the cells expressed Bel - 2 in preconditioning ischemia group increased clearly (p<0.01),the cells expressed bax decreased greatly ( p < 0. 01). The result of in situ hybridization showed that bcl - 2 mRNA and bax mRNA expressed little at the area of cortices and basal nuclei in ischemia side of the control group of imitational operation. Control group of preconditioning focal cerebral ischemia's bcl - 2 mRNA and baxmRNA partial expressed at cortices and basal nuclei in ischemia side. Compared with the control group of imitational operation, the cells expressed bcl - 2 mRNA increased clearly (p <0. 01) ,the cells expressed bax mRNA had no significant changes. ( p > 0. 05 ) In preconditioning ischemia group and ischemia group bcl - 2 mRNA and bax mRNA expressed a great deal in neural cells, compared with ischemia group the cells expressed bcl - 2 mRNA in preconditioning ischemia group ( three sub - groups) increased clearly (p <0. 001 ) ,the cells expressed bax mRNA decreased greatly ( p <0. 01) All healthy side had no bcl -2 mRNAand bax mRNA expressed.Part Three: The result of 7 - ray measurement: From 5min to 2h after injection of Tcm - - HL91, the radioactivity ratios per weight cerebrum indicated that the ischemia side and the healthy side had no significant differences in both groups(p >0.05) , T/NT was about 1 . After 4h of injection, the radioactivity ratios per weight cerebrum increased significantly in both groups. T/NT was 1. 57 0.13,1.93 ±0.06,2.25 ±0. 17 (p<0. 01) separately after 4 h,8hJ2h of injection in preconditioning cerebral ischemia group, the radioactivity ratios showed significant differences compared with the ratios of 2h. (p <0. 01) From 4h to 12h after injection, T/NT was 1.22 ± 0. 12,1.59 ± 0.07,1.94 ±0.09 the radioactivity ratios also showed significant differences compared with the ratios of 2h. (p <0. 05 ~0. 001 ) The radioactivity ratios per weight cerebrum between preconditioning cerebral ischemia group and cerebral ischemia group showed significant differences after 4h,8h,12h of injection (p <0. 05) . From the photographs of autoradiography we can see that from 5min to 2h after injection , silver granules distributed mainly in intercellular matrix and capillary, only a few inside neuronal cells in both ischemia side and healthy side of the two groups. From 4h to 12h silver granules distributed mainly inside neuronal cells of ischemia side and only a few in intercellular matrix and capillary. The density of silver granules of ischemia side in preconditioning group was higher than in ischemia group.CONCLUSION Part One:...