Effects And Mechanism Of Hypertonic Saline Against Focal Cerebral Ischemia-reperfusion Injury In Rats

Partâ… Effects of hypertonic saline on cerebral water content water following focal cerebral ischemia-reperfusionObjective 1.To investigate the effects of hypertonic saline on cerebral water content following focal cerebral ischemia-reperfusion injury in rats.2.To investigate the safety of intravenous bolus infusion of 7.5%saline 4ml/kg.Methods 50 adult male Sprague-Dawley(SD)rats were divided into 5 groups in random.Rats were anesthetized with chloral hydrate and ventilated spontaneously.The right common carotid artery,internal carotid artery and external carotid artery were exposed.The external artery and the proximal end of the common artery were ligated.A nylon thread with globular head was inserted into the beginning part of the anterior cerebral artery to block the blood flow of the middle cerebral artery through the incision of the common carotid artery.The thread was only inserted into the internal carotid artery in sham-operated rats(group P,n=10).The right femoral vein were exposed and cannulated for drug infusion and femoral artery cannulated for blood sampling.After 2 hours ischemia,7.5%saline(group HS-A,n=10), 4.2%saline(group HS-B,n=10),or normal saline(group NS,n=10) 4ml/kg was injected intravenously within 5 minutes.Sham-operated rats and simple ischemia rats(group IR,n=10)were not given any drugs. The thread was drawn back to the internal artery following drugs administration.Arterial pH,arterial pressure of carbon dioxide(PaCO2), arterial pressure of oxygen(PaO2),blood glucose,hemocrit(Hct), arterial sodium concentration and potassium concentration were measured at 5 minutes before reperfusion,and at 30 minutes,60 minutes and 90 minutes of reperfusion.After 22 hours of reperfusion,rats were killed by decapitation under deep anesthesia.Then the brain was quickly removed and dissected into ipsilateral and contralateral hemispheres. Brain edema was assessed by comparing wet-to-dry weight ratios.The wet weight of the hemispheres was determined by a scale.The dry weight of the hemispheres was determined after heating the brain tissue for 24 hours at 100℃in a drying oven.Brain water content was then calculated as H2O=(1-dry wt/wet wt)×100%.Results pH value in HS-A and HS-B showed slight reduce at 30minutes of reperfusion.Howevere there were no differences in pH values,PaCO₂,PaO₂,Hct and glucose among ischemic groups(IR, HS-A,HS-B,NS).Infusion of 7.5%saline resulted in elevated sodium level for longer than 90 minutes.Administration of 4.2%saline resulted in elevated sodium level for 60 minutes.Potassium level and Hct were decreased temporarily in hypertonic saline treated groups.Water content was increased in both ipsilateral and contralateral hemispheres and more obviously in ipsilateral part in rats of IR and NS groups.There were no differences between these two groups.Water content was decreased following hypertonic saline treatment compared to normal saline and vehicle treatments and more obviously in ipsilateral hemisphere.Water content was decreased significantly in ipsilateral hemisphere treated with 7.5%saline than that treated with 4.2%saline.Conclusion1.Hypertonic saline reduces pH value slightly and temporarily.2.Serum sodium level is increased significantly by hypertonic saline treatment.3.Serum potassium level and Hct are temporarily decreased by hypertonic saline treatment.4.Water content is decreased in both ipsilateral and contralateral hemispheres by hypertonic saline treatment and is more obvious in rats treated with saline of higher concentration. Partâ…¡Effects of hypertonic saline on inflammatory cytokine level induced by focal cerebral ischemia-reperfusionObjective To investigate the effects of hypertonic saline on brain tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)levels and neurological function score following focal cerebral ischemia-reperfusion in rats,as well as ultrastructural change in brain.Methods SD male adult rats were anesthetized with chloral hydrate.A nylon thread was inserted into the entrance of anterior cerebral artery to block the blood flow of the middle cerebral artery.The thread was only inserted into the internal carotid artery in sham-operated rats(group P,n=10).After 2 hours ischemia,7.5%saline(group HS-A, n=10),4.2%saline(group HS-B,n=10)or normal saline(group NS, n=10)4ml/kg was injected intravenously within 5 minutes. Sham-operated rats and simple ischemia rats(group IR,n=10)were not given any drugs.The thread was drawn back to the internal artery following drugs administration to restore cerebral blood supply. Neurological deficit score was assessed at 22 hours of reperfusion with the Zea longa standard.Then rats were killed by decapitation under deep anesthesia.Homogenate was made and centrifuged with brain tissue in the ischemic hemisphere.The supernatant was measured for TNF-αand IL-1βlevels.Pathological examination was conducted under electron microscope to detect ultrastructural change in cerebral neuron.Results Neurological function was improved by either 7.5%saline or 4.2%saline treatment and not improved by normal saline treatment. TNF-αand IL-1βlevels in brain were increased significantly by focal cerebral ischemia-reperfusion and decreased by hypertonic saline treatment and were decreased more obviously by 7.5%saline.Normal saline had no effects on cerebral inflammatory cytokine levels.Cerebral ultrastructure was disrupted by ischemia-reperfusion and improved by hypertonic saline.Conclusion inflammatory reaction and cerebral ultrastructure are greatly attenuated and neurological function is improved by hypertonic saline. Partâ…¢Effects of hypertonic saline on neuron apoptosis following focal cerebral ischemia-reperfusionObjective To investigate the effects of hypertonic saline on neuron apoptosis,bcl-2 and bax protein expression and intracellular free calcium level in order to search for the mechanism of brain protection by hypertonic saline following cerebral focal ischemia-reperfusion.Methods Focal cerebral ischemia was made by insertion of a nylon thread into the entrance of anterior cerebral artery.The thread was only inserted into the internal carotid artery in sham-operated rats(group P,n=10).After 2 hours ischemia,7.5%saline(group HS-A,n=10),4.2% saline(group HS-B,n=10)or normal saline(group NS,n=10)4ml/kg was injected intravenously within 5 minutes.Sham-operated rats and simple ischemia rats(group IR,n=10)were not given any drugs.The thread was drawn back to the internal artery following drugs administration to recover cerebral blood supply.At 22 hours of reperfusion,5 rats in every group were killed by decapitation under deep anesthesia.Brain was quickly removed.Intracellular free calcium content in the ischemic hemisphere was determined by flow cytometry. Thoracotomy was carried and normal saline and buffered paraformaldehyde was perfused transcardially in the rest 10 rats.The ischemic hemisphere was removed and paraffin-embedded. Immunohistochemistry was conducted to determine the expression of bcl-2 and bax protein.Apoptotic cells were counted by TUNEL.Results apoptotic cells were increased by focal cerebral ischemia-reperfusion injury and decreased by hypertonic saline treatment.Bcl-2 protein expression was enhanced while bax was nhibited by hypertonic saline.Intracellular free calcium content was decreased by hypertonic saline,which is increased by ischemic stress. Normal saline had no effects on either apoptotic cells counts or apoptotic protein expression.Conclusion Hypertonic saline inhibits neuronal apoptosis by enhancing bcl-2 protein expression,suppressing bax protein expression and decreasing intracellular calcium overload following focal cerebral ischemia-reperfusion.