Internal Carotid Artery, Middle Cerebral Artery Cerebral Ischemia Tolerance And Tnf-alpha, Of Sgc Expression In Focal Cerebral Ischemia And Effect

Intracranial arterial tumor is a dangerous disease in neurosurgery, in which one third of the patients have died due to lack of imediate treatment after the hemorrhge. With more high-tech methods applied to the treatment in recent years, the percentage of the cases cured largely increased. One of the frequently used methods in the artery clipping operation is the temporary occlusion of the tumor-carrying artery. However, long time occlusion of the artery may cause cerebral infarction and mal-function of the nerosystem. The occurance of the cerebral ischemia due to the temporary parent arterial occlusion after the intracranial aneurysm surgery is between 5.2% to 19%, while cerebral ischema is relavant to the site and the time of the occlusion. This experiment adopts the method of temporary aneurym clipping of intra-carotid artery and MCA of the Sprague-Dawley rtats to establish the focal cerebral ischemia models, which is done completely by the way of aneurym clipping in craniotomy and with the view to find out an ideal animal model of cerebral ischemia caused by the temporay occlusion. Although the temporary parent arterial occlusion is used more often in the operation of aneurym clipping, the comparatively long-time occlusion of the parent artery may cause brain infarction and neurological deficit. It was considered that the secure time span of occlusion was 15 min, while some secientists reported it to be 15～120min with or without the brain proctection measures. This experiment studied the damages to the vessel wall caused by the aneurym clipping. Thus to understand better the possible mechanism of the cerebral ischemia caused by temporary occlusion in the operation of aneurym clipping, which can serve as an instruction in deciding the safe time span of the occlusion. According to Malinski and others, during the reperfusion after cerebral ischemia, the increasing of the expression of TNF-αand the calcium inflow caused by combination of glutamic acid and N-methyl-D-aspartate receptors will induce the endotheliocyte and soomth muscle cell to activate NOS, produce NO and participate in the pathological process of cerebral ischemia and reperfusion injury. Through activating sGC, the increasing focal density of NO will catalyze GTP transformation into cGMP and cause the phomological transformation of endotheliocyte the increase of microvessle penetrability. This experiment aims at exploring the expression of TNF-αand sGC in focal cerebral ischemis and the possible machenism of their mutual action.Tumor necrosis factor-α(TNF-α), as a cell factor with extensive biological vitality, is being increasingly noticed for its role in the cerebral ischemia reperfusion injury. The increased expression of TNF-αin the cerebral ischemia reperfusion injury was considered mainly for its toxigenicity in the cell after cerebral ischemia reperfusion. In the recent years, however, with the researchs in this field go deeper, it has been proved that TNF-αcan also be of use in protecting neuron and promoting its recover. In the course of pathology, its mechanism is relevant to the cause of inflammation, the mechanism of thromboplast, the damage of blood brain barrier, the inducing of cerebral ischemic tolerance and the neuron growth factor, etc. But It is not seen reported until now that the specific time and mechanism of the damage and recover done by TNF-αremain unclear. Through presenting the expression of TNF-αin different time span of cerebral ischemia, this article is to explore the possible mechanism of its double effect. Nitricoxide(NO) was regarded as gaseous messenger molecular,which hold the role of dilating blood vessel and tranferring neuroimpuse. Biological effect was activated by the mechanism of activatingintrocellular Soluble guanylate cyclase (sGC)after entering cells by the way of diffusion,elevating levels of cGMP. sGC is the only receptor of NO in the cell. It exists in the brain tissue in form ofα1,β1 sub-unit. sGC activates GTP to produce cGMP as the second messenger molecule in the cell, and takes part in the dilate-retraction function of the smooth muscle, the regulation of the cellular proliferation and appotosis. According to the research done by Sinnaeve, the balloon injury of carotid artery may result in the decrease of the expression of sGC sub-unit in the smooth muscle cell and the activity of sGC, which in turn induce the vascular retraction and neointima formation. Due to the migration and proliferation of the smooth muscle cell, extracellular matrix production, and the increasing activity of blood platelet and white cell, sGC has an important function in NO/cGMP signal transduction in the self stability of normal vessle and its reaction toward injury. The sGC expression and its meaning in different time span of temporary occlusion induced focal cerebral ischemia is also explored in this article. Part one Experimental study of the endurance after ischemia in ICA/MCA Objectives: to establish stable and reproductive focal cerebral ischemia models in rats. Methods: SD rats were randomly divided into two groups, with the one group of intra-carotid artery (ICA) and the other of middle cerebral artery (MCA). Each group was divided again randomly into control subgroup and ischemia subgroup. The operation on the rats in the control subgroup only expose the ICA and MCA without clipping; while theoperation on the ischemia subgroup clipping the ICA and MCA after their exposure. The time span of the occlusion of 0.5h,1h,2h,2.5h respectively, then reperfusion to 24h before excution. Observe the character behaviour, character of morphology of infarction tissue and character of HE staining. Results: the SD rats in each subgroup largely resumed eating and drinking 24h after the operation. The sensitivity and resistance of rats in the ischemia subgroups is considerablely lower than those in the control subgroups. There are 13 rats in M2 and M2.5 subgroups,Horner's syndrome occurred in left side of rats,that is shrunken distance between lower and upper eyelids,lessened pupil,hemiplegia in left side(upper limbs obviously) . The brain tissue of all the rats underwent 30min of dyeing by 1%T.T.C., ICA blood supply region in group C0.5 and C1 presented red color; the blood supply region in group C2,C2. and M0.5 presented spotty white color; in the rest subgroups of M1,M2,M2.5, the MCA blood supply region presented white and regions at the non-occlusion side presented red. HE dyeing showed the cortex and striatum of ICA group did not present obvious core and penumbra, some of the neuron in the ischimic region swelled or contracted. Except M0.5, the sub-groups in the MCA group all presented core and penumbra in the cortex and striatum, with the infusion of the inflammatory cell observable. Conclusion: through imitating temporary arterial occlusion, establish the model by way of clipping MCA and ICA is a stable and trusty method to reproduce the focal ischemic of MCA and ICA models in rats.Part two Study through electron microscope of focal vascular injury caused by temporary clipping. Objective: to explore the focal vascular injury caused by temporary clipping. Methods: SD rats were randomly divided into two groups, with the one group of intra-carotid artery (ICA) and the other of middle cerebral artery (MCA). Each group was divided again randomly into control subgroup and ischemia subgroup. After being exposed through operation, the MCA or ICA was temporarily clipped for 0.5h, 1h, 2h, and 2.5h respectively before reperfusion to 24h before excution. The clipped segment was taken, observed and photoed through electron microscope. Results: In the 0.5h subgroup, bubbles were occasionally seen in the endotheliocyte plasm, the coarse endoplasmic reticulum could be seen dilating when taking a closer look. In the 1h subgroup, the cytoplasm of these smooth muscular contained slightly swollen mitochondrin; the muscle layer thickened a little, among which newly-formed collagen or elastin may be seen taking shape, with some loose CM; scattered bubbles appeared in the endotheliocyte plasm. In the 2h subgroup, the muscle layer thickened considerably, and mitochondrin swelled in the smooth muscle. Media of smooth muscle had protruding part which penetrated the nearby smooth muscle cells and internal elastic lamina to reach under the endothelium with the newly-formed collagen or elastin around; in some cases, the endothelium was separated from the internal elastic lamina. In the 2.5h group, karyon of endothelium appeared contraction and part of the endothelium had peeled off. Conclusion: The maximum time span of clipping the vessle wall can endure should be 60min. The prolonged clipping time could cause the local vascular cavity stenosis and closure, thus leading to irreversible cerebral ischemia or injury.Part three The expression and function of TNF-αin different time span of cerebral ischemia injury of SD rats Objectives: To explore the possible double functional mechanism through studying the expression and function of TNF-αin different time span of cerebral ischemia and reperfusion injury of SD rats. Methods: SD rats were randomly divided into two groups, with the one group of intra-carotid artery (ICA) and the other of middle cerebral artery (MCA). Each group was divided again randomly into control subgroup and ischemia subgroup. The operation on the rats in the control subgroup only expose the ICA and MCA without clipping; while the operation on the ischemia subgroup clipping the ICA and MCA after their exposure. The time span of the occlusion of 0.5h,1h,2h,2.5h respectively, then reperfusion to 24h before excution. Caculate respectively the infarction volume and the expression of TNF-αin the brain tissue of the rats in each subgroup. Results: In the control subgroup, there was no obvious cerebral infarction and only a little TNF-αmRNA expression. ICA clipping subgroups C0.5, C1, C2, C2.5 each had a infarction size of 2.1±1.3%,5.1±1.2%,6.3±1.8%, and 5.4±1.5%, and the number of TNF-αpositive cells of 7.14±5.28,11.53±6.77,10.71±8.12, and 12.54±7.89 respectively. MCA clipping subgroups M0.5,M1,M2,M2.5 each had a infarction size of 5.2±2.3%,13.5±1.8%,29.75±1.3%, and 35.13±1.9%, and the number of TNF-αpositive cells of 7.29±4.53,12.79±7.53,37.96±9.75, and 43.56±11.9 respectively. M2 had a distinct difference compared with the control subgroup (P<0.01). conclusions: The increase early of the expression of TNF-αmay protect the brain tissue from being injured, but as the time of ischemia expands, the over-expressed TNF-αactives PMN andproduces biological effects of injury. Part four The expression of soluble guanylyl cyclase in the focal cerebral ischemia of ICA ,MCA Objectives: To explore the expression and functional mechanism of α1β1 subunits of the soluble guanylyl cyclase (SGC) in time phase of cerebra ischemia and reprofusion in rats. Methods: SD rats were randomly divided into two groups, with the one group of intra-carotid artery (ICA) and the other of middle cerebral artery (MCA). Each group was divided again randomly into clipping 0.5h,1h,2h,2.5h four groups. After being exposed through operation, the MCA or ICA was temporarily clipped for 0.5h, 1h, 2h, and 2.5h respectively before reperfusion to 24h, then the rats were executed. After the immunal reaction, the mean absorbance (mA) was read by mA analysis software. Caculate the volumne of the sGC α1β1 subunits in the brain vessles of rats in each group. Results: in the control group, the sGC α1β1 subunits in smooth muscle media of the ICA and MCA presented brown as strong immunal reaction (being 0.17±0.13 and 0.18±0.17 respectively). In the ICA clipping groups C0.5,C1,C2,and C2.5, the sGC α1β1 subunits were 0.15±0.11,0.13±0.13,0.14±0.09 and 0.15±0.12 respectively. In the MCA clipping groups M0.5,M1,M2,and M2.5, the figure were 0.15±0.16,0.12±0.09,0.08±0.03 and 0.06±0.02 respectively. M2 had a distinct difference compared with the control group (P<0.01).Conclusions: The decrease of sGC expression and its function will lower the internal and external NO reaction, thus suppress the formation of neointima in the injured vessle, therefore has protection against cerebral injury.