The HIF-1α's Effect Of Neuroprotection During The Brain Injuries Induced By Ischemia

Brain injuries are common diseases in department of neurosurgery, Such as cerebral hemorrhage, stroke and traumatic brain injury (TBI). Its morbility and multilation rate were very high. The neuron injuries after acute brain injuries mainly have two categories. One is the primary injuries induced by physical and chemical reasons; another one is second brain injury, mainly induced by ischemia caused by primary brain injuries.It is very important to remedy brain injuries patients as soon as possible. But it is more important to prevent second brain injuries from primary brain injuries. Many studies have shown that the production of oxygen free radical after primary brain injuries was the main reason causing the second brain injury. But currently still have no enough evidence on clinical application of free radical scavenger.To today, scientist all of the world, physicians and pharmacology factories are working hard to transfer many medicine which were very effective to remedy the neuron after brain injuries to clinical application. But we still have no one medicine can be used in clinic. Even free radical scavenger did not show satisfied result.Hypoxia inducible factor 1-a is a recent study field, and some researches have shown that HIF-la was expressed after brain stroke and brain hemorrhage. It is the key gene when response to ischemia. And it has been proven that its regulation of downstream genes up to as many as 70. We used MCAO method to produce a stable model to study the cerebral protection role of HIF-1αin the brain injury. We found that application of NAC, Edaravone such as free radical scavengers can indeed reduce the brain damage after the ischemia. But the brain protective effect was blocked by the HIF-1αinhibitor YC-1,2ME2. This result showed that HIF-1αpathway plays a key role in protection effect after brain injury.After acute brain injury, HIF-1αsignal transduction pathway is closely related to improve cerebral blood flow which was the key steps to reduce brain damage. So HIF-1αmay be a new molecular target site of treatment for the protection of brain after acute brain injury. It opened a new research direction of great theoretical and clinical significance for treatment of brain injury use cerebral protective medicine. We also conducted a research on HIF-1αprotective time windows and we carried out a preliminary study on HIF-1αprotective effect on BBB during brain injury. The experiment was divided into five main parts:PartⅠEstablishment of brain injury model of rat in vivoObjective To introduce a relatively simple model of cerebral ischemia injury model andestablish a novel, practical, and reproducible brain injury model of the rat in vivo.Methods Take 270-310g SD rats induced with 4% Isoflurane and oxygen and maintained with 2% Isoflurane throughout all procedures. A midline ventral cervical incision is made after the neck has been surgically prepared with betadine solution. Undering the operating microscope, the right common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) are isolated. The CCA and ICA are then temporarily occluded using microvascular clips, cutting a small mouth in the external carotid artery. A monofilament nylon suture.3.5 mm in diameter with a previously rounded and polished tip. is advanced via the ECA 19-20 mm from the CCA bifurcation into the ICA in order to occlude middle cerebral artery (MCA). After ischemia 90 min, the suture was removed under the microscope for reperfusion.24 h late, the neurological scores was evaluated and progress a MRI scan to evaluate the brain injury and brain edema. After MRI scan, the brain was taken out for TTC staining.Results This method produced brain damage area is basically the same. SD value is small. Neurological scores evaluation after MCAO have a normality of distribution. Rats with brain injury making a consistent neurological deficit. Mortality in group was 0. And after 24 h, the MRI scan showed marked cerebral edema. The infarct size and area of brain damage were consistent with the reported in the literature. Similarly. TTC staining also showed that the success of obstruction in middle cerebral artery occlusion range.Conclusions This in vivo model of cortical neurons injury can be easily repeated and can simulate the damage mechanism of brain injury ischemia. This model can be used in the further research of neuroprotection in brain injury.Part II HIF-la inhibitor on neuroprotection effect of HIF-la in the animal experiments.Objective To study the HIF-lagene expression in the acute brain injury induced by ischemia and the effect of HIF-1αinhibitor YC-1 and 2ME2 on the ischemic brain injury.Methods SD rats were randomly divided into MCAO group, HIF-1αinhibitor treatment group total of 3 groups. Using middle cerebral artery occlusion method to make ischemia model, DMEO or YC-1 2mg/kg.2ME2 5mg/kg were intravenously injection into femoral vein at 30 min before MCAo. Neurological scores were evaluated after reperfusion 24 h in each group. After MRI scan, the brain tissue was taken out for TTC staining and Western Blot analysis.Results In pure brain injury group and DMEO treatment group and HIF-1αinhibitor MRI scans showed cerebral edema, in which the brain damage area was largest in HIF-1αinhibitor group. Compared to pure MCAO group the infarct size and cerebral ischemia were significantly increased (p<0.05). In YC-1 treated group and 2ME2 treated group the brain ischemia area increased significantly (p<0.05). No significant difference between two HIF-1αinhibitor groups. About western blot result, in MCAO group, the HIF-la expression level was no difference. In HIF-1αinhibitor groups, no HIF-1αexpression was detected by western blot.Conclusions HIF-1αexpression level in brain injury tissue significantly increased. It showed that HIF-1αinvolved in brain injury secondary neuronal injury protection. The using of inhibitor YC-1, and 2ME2 can significantly increase the injury induced by ischemia, showed that HIF-1αhave a protective therapeutic effect in acute brain injury.PartⅢExpression of HIF-1αin MCAO model of rat and the effect of Antioxidant in the ischemia-induced brain injuryObjective To investigate the expression level of HIF-1αafter brain injury induced by ischemia and the neuroprotection effect of antioxidant NAC and Edaravone.Methods SD rats were divided into three groups, one for the simple MCAO group, one for the NAC treatment group. NAC 2mg/kg i.v. through the femoral artery at 30 min before MCAO. Edaravone treated group using Edaravone 5mg/kg intravenous injectiong via femoral artery at the 30 min before MCAO 30 min. Each group of rats was under cerebral ischemia of 90 min and reperfusion for 24 h. After 24 h the neurological score was evaluated and MRI scan was finished to evaluate the cerebral injury area. The brain was taken out after scan and was stained with TTC to evaluate the brain ischemia area. We also using Western Blot method to evaluate the HIF-1αexpression level in the cortex of rat brain injury part after ischemia.Results MRI scan and TTC staining both showed after using NAC and Edaravone antioxidants the brain injury area significantly reduced. Compared with control single MCAO group, the infarct size and ischemia area obviously reduced (p<0.05). Neuorlogical score also have obviously improvement in group treated with antioxidant. Western Blot show no HIF-1αexpression in brain cortex without injury, in the cortex with ischemia 90 min following 24 reperfusion the HIF-1αexpression level increased significantly(p<0.05). In the group treated with antioxidant, HIF-1αexpression level increased more than 42% compared to the control group(p<0.05).Conclusions HIF-1αexpression level in the cortex part of ischemia brain improved significantly after cerebral ischemia. After using antioxidant NAC and Edaravone the brain injury area in the rat reduced obviously and HIF-1αexpression level increased significantly compared with control group. Antioxidant show a obviously neuroprotection effect in the MCAO rat model.Part IVTiming study of HIF-la protective effect in ischemic brain damageObjective Using cerebral ischemic injury model for HIF-1αneuroprotective role time window study. In order to further establish a foundation to guide clinical treatment.Methods MRI scan have the advantage to detect brain damage in vivo level. We using MRI to scan same rat with 90 min middle cerebral artery occlusion after reperfusion 0 h, 3h,6h,12h and 24 h respectively. To analysis brain tissue water content and timing of changes in the size of cerebral infarction.270-310g SD rats were randomly divided into control group, or HIF-1αinhibitor (YC-1) treated group. The control group, middle cerebral artery occlusion group, injected through femoral vein catheterization equal DMEO at 24 h and 30 min before the middle cerebral artery occlusion. YC-1 treatment group injected YC-1 2mg/kg twice times at 24 h and 30 min before the middle cerebral artery occlusion through femoral vein.Results In the control group, rats were occluded the cerebral middle artery for 90 min. the brain edema and cerebral infarct area was slowly to develop. After reperfusion 0 h.3 h. almost no sign of brain edema and cerebral infarction. At 6 h after reperfusion. there was some subcortical infarct area. To 12 h. the infarct outline has emerged, and maintained to 24 h, their MRI scans of the infarct density continued to deepen. This result showed that the protective effect of HIF-1αwas time-sensitive, and the treatment time window should be 12 h in advance. After the cerebral ischemia-reperfusion 12 h the development of cerebral infarction was irreversible, and from 12 to 24 h the infarct size basically was formed totally. In the HIF-1αinhibitor YC-1 treatment group, after ischemia-reperfusion 0-3 h, the cerebral infarction outline already emerged and remained until 24 h late. This result showed that when using HIF-la inhibitor to block the protective effect of HIF-1αcerebral ischemic injury occurred rapidly. In the middle cerebral artery area the brain tissue was more sensitive to ischemia and hypoxia. and brain injury happened faster and more damage occurred. In the MRI scan images showed that infarct outline appeared in 0-3 h. and the size was the most part of the cerebral middle artery area. Followed the necrosis of neurons in the infarct area of the rat. the density of imagin of the infarct area gradually enhanced to the deepest density at 24 h.Conclusions The expression of the HIF-1αafter middle cerebral artery occlusion leading to cerebral ischemia injury could protect the secondary brain injury, and its protective effect have a short time window, from after reperfusion 0-6 h there was a certain protective effect.12 h late, the brain damage formed basically, and would keep to 24 h. When using the HIF-1αinhibitor YC-1 inhibited HIF-la expression in ischemic brain tissue, the ischemic brain tissue was more sensitive, prone to secondary injury occurring after ischemia. Infarct size was largest from start, and the infarct contour appeared earlier. At 12 h after reperfusion. in the middle cerebral artery occlude zone the brain ischemic infarction lesion could be formed. This result showed that HIF-1αhas an important role in cerebral ischemia protection.PartⅤThe effect of HIF-la on BBB injury induced by focal cerebral ischemiaObjective Application of Evans blue method and MRI methods to detect the role of HIF-la on the BBB injury induced by ischemia.Methods 270-310g SD rats were randomly divided into control group, and YC-1 treated group. The control group injected Evans blue 100mg/kg through femoral vein after MCAO 90 min. and measured brain tissue content of Evans blue. In HIF-1αinhibitor YC-1 treated group. YC-1 was injected through femoral vein at 24 h and 30 min before MCAO. and injected Evans blue 100mg/kg just after removing the suture. Reperfusion for 24 h late, the brain was taken out and Evans blue detected by augmented concentrations of this substances in the cerebrum. Another 270-310g SD rats were randomly divided into control group and the HIF-la inhibitor treated group. After 24 h of reperfusion, Gd-DTPA was injected through femoral vein to enhance MRI scan. The BBB permeability was calculated after MRI scan.Results Pretreatment with HIF-la inhibitor (YC-1.2mg/kg i.v. before MCAO 24 h and 30 min) decreased permeability of the BBB to Evans blue dye detected by augmented concentrations of this substance in the cerebrum. By contrast. HIF-1αinhibitor did not alter BBB permeability to Gd-DTPA, as defined by MRI assay.Conclusions These data demonstrate the potential utility of HIF-la inhibitor for pharmacological modulation of the BBB. and indicate that the increase in BBB permeability mediated by HIF-1αis limited by the size of the delivered substance.