| Recent years, whether in animal experimental research or in clinical study, ischemic preconditioning performed by transient brain ischemia or transient ischemic attacks will induce ischemic tolerance (IT) , which posseses the neuroprotective effect. Models of global and focal cerebral ischemia have recently provided evidence for the induction of IT in animals previously subjected to short ischemic episodes, rendering the brain more tolerant to subsequent occurences of persistent cerebral ischemia. Hitherto, mechanism of IT has not been clear. There were various theories which could presume the induction of IT, while each of them failed in one way or other aspects.It is very known that nitric oxide has played an important role in ischemic procedure. There is a lot of evidence in patients or animals to support the double - edge swords effect of nitric oxide on neurons. Those experimental findings suggested that the production of nitric oxide increased after brain ischemia, and nitric oxide is related closely with neuronal apoptosis. The effect of inhibitor of nitric oxide synthase ( NOS) could reduce the production of nitric oxide, delay the progress of cell apoptosis and attenuate effectively the volume and size of in-farction after brain ischemia. In the present study, we aimed to research for the effect of neuronal nitric oxide synthase on IT and to study the possible mechanism of IT's formation and the IT's meaning in clinical practice.MethodsA total of 40 adult Wistar rats of both sexes, weighing 280 -320g, were used in the present study. They were kept at room temperature (20 -25℃ ). Each rat was lightly anesthetized with 10% chloral hydrated in normal saline (35mg/100g, intraperitoneally ). Briefly , the right common carotid artery ( CCA) was exposed through a midline incision. After the vagus nerve was carefully separated from the CCA, the external carotid artery and the pterygopalatine artery were ligated. At this point, the internal carotid artery is the only remaining extracranial branch of the common carotid artery. Next, a 4 -0 silk suture was tied loosely around the CCA and another suture was ligated the point towards heart of CCA. An incision in the CCA was performed and a 4 - 0 monofilament nylon suture was inserted in to the CCA. The suture was gently advanced. After about 17 -22mm length of nylon suture had been inserted, the respiratory rhythm of rat changed immediately, indicating that the blunted tip of the suture had passed the middle cerebral artery origin and reached the proximal segment of the anterior cerebral artery, which has a smaller diameter. At this point, the intraluminal suture must have caused the middle cerebral artery occlusion (MCAO). After various ischeinic times, the intraluminal suture was backdrawn about 8 - 10mm to reperfusion. The sole ischemic groups rats were sacrificed in 3 days. After reperfusion3 days, the rat was anesthetized, opened the previous incision and advanced the suture to reach the previous point. After various ischemic time, the suture was backdrawn and the rat was sacrificed in 3 days. The criteria of MCAO are as followed. The rats presented the right Homers sign and/or left limb paralysis or circling towards lift in original site. The rat was anesthetized again after reperfusion 3 days. Tho-racotomy was performed and the heart perfusion was performed firstly with 0. 9% normal saline and then with 0. 4% paraformalin 200ml. The brain was removed and kept in 0. 4% paraformalin. After dehydrate and postfixation of the brains, the coronary sections (7 um thickness) were cut in the frontal edge of optic chiasm. The adjacent sections were stained with HE and immunohistochemistry ( nNOS antibody ). The neuronal pathological changes were demonstrated under light microscope on ( 10 x 20) these sections stained with HE. The numbers of positive nNOS neurons were counted under microscope ( 10 x20) in the hippocampal CA, region. The numbers of positive nNOS neurons in various ischemic groups were analyzed statistically a using one - way a... |
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