To Explore The Effect Of Human Urinary Kallikrein On The Formation Of Collateral Circulation In Focal Cerebral Ischemia-reperfusion Rats

Cerebrovascular disease which harm the aged peope's health and life became the principal disease in our country at present.The death rate and mutilation rate of the patients with acute cerebral infarction were high,the health of human were threaten gravely.The infarction focus was composed by center and ischemic penumbra areas.The functions of nerve cells in ischemic penumbra areas were reversible,so how to establish the collateral circulation in ischemic penumbra as soon as possible to save the nerve cells became the internal and overseas investigative hot spot.Kinin system resided in many system of animal internal widespreadly,and had protection to the acute ischemic cerebrovascular disease.Tissue kininogenase the important ingredient of Kinin system mediated through the kinin B2 receptor had the functions of antioxyen,antiinflammatory,antiapoptosis and to enhance neural regeneration when the brain tissue in ischemia.Human urinary kallikrein that is a proteinase was extracted from healthy masculine urine is a sort of Tissue kininogenase.Having animal experiment proved that can improve nervous function,diminished infarct size,and lessen encephaledema on the acute focal cerebral ischemia-reperfusion rats obviously.But if it has the pharmacodynamic action of Cerebrovascular recanalization,collateral circulation establishment,and function reconstruction still need detailed empirical study.To explain in further,we did research works following.PartⅠEstablishment of focal cerebral ischemia-reperfusion rats modelAIM:To establish focal cerebral ischemia-reperfusion rats(Middle Cerebral Artery Occlusion,MCAO)model using a intraluminal thread technique and study the degree of injury of the nervous function.METHODS:1.To estabalish MCAO model using a intraluminal thread technique.8 health SD rats were choosed,their weight limited between 280～350g.The rats were bought from animal center of Nanfang Medical University.We estabalished MCAO models by a intraluminal thread technique modified by Longa at al.We choosed imported fish line for the models,the diameter was between 2.4～2.6mm.The 8 rats were divided into of operation group and sham operated group.The operated process of sham operated group rats was same with operation group rats excepted not to plant line.2.Five grades and four scores standard to evaluate the degree of injury of the nervous function.We gived mark to the two group rats at 4h,12h,24h,48h,72h,to evaluated the degree of injury of the nervous function.The five grades and four scores standard was choose,0 score meaned no injury of nervous function,1 score meaned the rat couldn't expand front the opposite side leg,2 scores meaned the rat turn round when creeped,3 scores meaned the rat falled to opposite side when creeped,4 scores meaned the rat couldn't creep,and no consciousness. 3.TTC dyeing.The normal rats hemicerebrum tissue was red and the infarction brain tissue was white after dyed by TTC.RESULTS:1.Evaluation of nervous function.We overview the changes of nervous function in two groups'rats at 4h,12h,24h,48h and 72h after ischemic reperfusion.The operation group rats' opposite side limbs could not abduce especially the anterior limbs,and the muscular tension decreased.The opposite side anterior limbs flexed and spontaneous movement decreased when extracted the tails of operation group' rats.The rats circled to the opposite side when walking,and the serious rats tumbled to opposite side.The injury side of experiment rats appearanced Honer syndrome,rima oculi and pupil shrink.The above symptom tapered or disapeared with time.The sham operated group' rats was no injury of nervous function after operated.2.TTC dyeing.The cerebral hemisphere of injury side of the experiment rats appeared white infarct area.And the brain tissue of sham operated group' rat was red after dyed by TTC.CONCLUSION:We successfully established MCAO models using a intraluminal thread technique.Established fundament for the next experiment.PartⅡTo explore the effect of human urinary kailikrein on the formation of collateral circulation in focal cerebral ischemia-reperfusion ratsAIM:To study the effect of human urinary kallikrein on the formation of collateral circulation in focal cerebral ischemia-reperfusion rats.METHODS:1.To estabalish focal cerebral ischemia-reperfusion rats(Middle Cerebral Artery Occlusion,MCAO)model and divide into groups.56 health SD rats were choosed,their weight limited between 280～350g.The rats were bought from animal center of Nanfang Medical University.We estabalished MCAO models by a intraluminal thread technique modified by Longa at al.The 56 rats were divided into sham operated group(SH),human urinary kallikrein group(HUK) and saline group(NS).There were 24 rats in NS group and HUK group,and there were 4 rats in SH group.We injected saline and human urinary kallikrein through vena caudali at 3h,then one time every day after ischemia reperfusion.2.To evaluate the degree of injury of the nervous function.We gived mark to the three group' rats at 4h,12h,24h,48h,72h,to evaluated the degree of injury of the nervous function.The five grades and four scores standard was choose,0 score meaned no injury of nervous function,1 score meaned the rat couldn't expand front the opposite side leg,2 scores meaned the rat turn round when creeped,3 scores meaned the rat falled to opposite side when creeped,4 scores meaned the rat couldn't creep,and no consciousness.3.TTC dyeing and to measure cerebral infarction volume.Used the HPIAS21000 Patho-gram analytical system to measure the infarction area and the area of every lamella area by the following formula.V =Σ(A1+A2)t/2The t meaned thickness,A1 and A2 were piece bouche and caudal infarction area.4.HE dyeing and linght microscopy.5.VEGF and f8 Immunohistochemistry. We use ABC Immunohistochemistry.The VEGF polyclonal antibody was diluted into 1:100,and the f8 antibody was diluted into 1:200.6.statistical analysis.All the data were dealed by SPSS11.0,and demonstrated by means of(?)±s.①Factorial statistical analysis was used to deal the data of expression of VEGF and f8.②Two sample t test statistical analysis was used to deal the date of brain tissue infarction volum of experiment'rats③Non-parameter test statistical analysis was used to deal the date of injury of nervous function score.RESULTS:1.Evaluation of nervous function.There was significant deviation of the degrees of neurologic impairment in the rats between human urinary kallikrein group and saline group.There was no significant deviation in the two groups(P＞0.05),at the time points of 4h,12h,72h after ischemia-reperfusion.The difference of the degrees of neurologic impairment between two groups was significant(P＜0.05),at the time points of 24h,48h.2.Measurement of cerebral infarction volume.There was significant deviation of cerebral infarction volume in rats between human urinary kallikrein group and saline group(P＜0.05).3.Result of linght microscope detection.At the time points of 6 hours and 12h after ischemia-reperfusion the cerebral cortex pyramidal neurons intumesced slightly and endochylema dyed slightly,at the time point of 24h after ischemia-reperfusion some pyramidal neurons shrinked,with nucleus anachromasis and gliacyte hyperplasy.At the time point of 24h after ischemia-reperfusion,neurons died obviously,the tissue of ischemic center looseness looked like sponginess,neurosome dissolved,endochylema dyed slightly,nucelus karyopycnosis,the peripheral glia cells of infarctus focus increased.At the time point of 7d after ischemia-reperfusion,some neurons shrinked,endochylema and nucelus dyed strongly,glial cells proliferation,neogenesis blood capillary appeared.The above were detected in Saline group experiment rats.Sham operated group rats showed no abnormal under the light microscope at five time points.At all time points,the process of cerebral damage was same between human urinary kallikrein group and saline group rats,but the degrees was lighter in human urinary kallikrein group rats.4.VEGF and F8 Immunohistochemistry.①There was expression of VEGF at the center and perimeter of cerebral infarcted areas of Saline group experiment rats.The nerve cells,gial cells and megalophage expressed VEGF.At the time point of 72h after ischemia-reperfusion, VEGF expressed by gial cells mainly at the center infarction.At the time point of 72h and 7d after ischemia-reperfusion,there were many new vascular endothelial cells expressed VEGF.The cells that expressed VEGF were more and more with time,24h was crest-time and 7d expressed cells decreased.There was no cell expressed VEGF in cerebral tissue of Sham operated rats.There was significant difference of the cell population expressed VEGF between human urinary kallikrein group and saline group at all time point(P＜0.05),but the regularity is similar.②The expression of f8 at the perimeter of cerebral infarcted areas of Saline group experiment rats increased at the time point of 72 hours after ischemia-reperfusion,and continue to increase.The difference of the f8 expression between human urinary kallikrein group and saline group was significant(P＜0.05).CONCLUSIONHuman urinary kallikrein can relieve The degrees of neurologic impairment,reduce the cerebral infarction volume,promote the formation of collateral circulation of the acute cerebral ischemia rats.