Expression And The Significance Of INOS,nNOS,eNOS On The SD Rats Brain After Their Skeletal Muscle Be Ischemia/reperfusion Injury

BackgroundLimb ischemia/reperfusion injury(LIRI)is the recurrent patho-process in clinic.We can meet it in these conditions,such as the replantation of severed limb,using tourniquet for trauma patients in emergency treatment, severe crush injury and recanalization of great vessels embolism et al and in these conditions more organs would be damaged,even multi-organ dysfunction syndrome the one main reason that cause patients be mutilated and lethal.The pathomechanism of the injury to remoteness organs by LIRI have not be identified.We have known it is confirmed that heterophil granulocyte and oxygen free radical(ODFR)mediated inflammatory and oxidative stress had close relationship with remoteness organs after LIRI. Among them,ODFR can not only make cells and tissue to be lipid peroxidation,but also make cell injury and inflammatory reaction.Nitric oxide(NO)has relationship with inflammatory reaction and oxidative stress, as one of the important messenger molecules and effecter molecules in biosystem.Nitric oxide synthase(NOS)is the key enzyme of NO.It is confirmed that NOS-NO participate in the brain injury process after limb long time ischemia/reperfusion and inducible NOS can be changed with the extension of I/R time.Whether neuronal NOS and endothelial NOS would be changed like iNOS? We want to find the answer.ObjectiveTo observe the injury of the cerebral by hind limb ischemia/reperfusion, to study the time-quantitative change feature and the rule of the different type of NOS in the process of the cerebral by hind limb ischemia/rerperfusion and to approach the trend effect of the different thpe of NOS in the process of oxidative stress.MethodsFifty six healthy male SD rats,two months old,of clean grade,and 226.90±8.44g average weight,were randomly divided into two groups: normal control group(N)and limb ischemia/reperfusion group(I/R).Normal rats(n=8)were anesthesia after be directly intraperitoneal injected with chloral hydrate,their corresponding head regional be shearing,disinfected with complex iodine,directly stripping the side of the brain hemisphere craniotomy and cryopreservated.The experimental group rats(n=48)were randomly divided into six groups,eight rats each group.Rat models of hind limb ischemia/reperfusion were established,and than rats were divided into six groups:2 hour,4 hour,6 hour,8 hour,16 hour and 24 hour to directly stripping the side of the brain hemisphere craniotomy and cryopreservated. After the experiment,we observe the cell morphological changes using light microscopy in brain tissue,and measure the semi-quantitative expressions of different types of NOS and the content of MDA,SOD at different time points after limb ischemia/reperfusion injury,and spproach the dependability between SOD and MDA,iNOS,nNOS,eNOS.Results1,Three types of NOS were detected in brain from control rats.Two hours after reperfusion,the expressions of iNOS,nNOS and eNOS increased with the elongation of time of reperfusion,nNOS and eNOS were highly expressed within 6 hours to 24 hours after reperfusion in the brain,and iNOS was highly expressed within 4 hours to 24 hours after reperfusion,iNOS expression increasing and decreasing occurred earlier than nNOS and eNOS. But the expression of three kinds of NOS all reached peak on the 24 hours after reperfusion.The significant correlation was found between each time point and control group.(P＜0.05)2,The MDA content of brain in I/R group were significantly larger than those in normal group and it reached peak on the 4 hours after reperfusion.The SOD activity of brain in I/R group were also significantly lower than those in normal group and it reached peak on 4 hours after reperfusion.3,The level of SOD activity in brain negtive correlated with the wariety of MDA(r=-0.475,P=0.000),the expression of iNOS mRNA(r=-0.304,P =0.001),nNOS(r=-0.415,P=0.000)and eNOS(r=-0.31,P=0.001).4,Light microscopy findings the brain tissue dropsy on the 4 hours and 24 hours after reperfusion.The pericaryon and nucleus accreted,the endochylema and core dens decreased,some nerve cell core disappeared on the cerebral cortex of the rats in I/R 4 hours and 24 hours groups.Conclusion1,It is related to oxidative stree after brain injury,by limb long time ischemia/ reperfusion and after 4 hours I/R the brain injury is the best severity.2,NOS is the key enzyme of NO.The content express of different types of NOS in brain has been changed at different time after limb ischemia/ reperfusion,iNOS is faster than eNOS and nNOS.We can consider that it is the best severity at I/R 4h and 24h to brain after limb ischemia/reperfusion.3,Three nitric oxide synthases is inverse correlation with SOD.It is consider that the high express of three nitric oxide synthases can make oxidative stress damage.Among them,nNOS is the best and eNOS is better.