HIF-1α在脑损伤中的神经保护作用研究
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摘要
脑损伤是神经外科常见疾病,包括脑出血,脑梗塞,脑创伤等。其发病率以及致死、致残率均较高,是危害人民健康的主要疾病。急性脑损伤后的神经元损伤可有两种,一种是物理,化学等因素直接作用于神经元导致的原发性神经元损伤。另一种为迟发性脑损伤,主要是原发性损伤导致的缺血缺氧引起。
我们知道,脑损伤病人救治过程中除了抢救时间要快之外,更重要的工作是如何保护损伤后的脑组织,阻止继发性脑损害的进一步发生发展。以往研究认为脑损伤后的缺血缺氧产生大量的氧自由基是造成继发性脑损伤的主要原因。但是目前对自由基清除剂的临床应用尚无充足的证据证明有效。
迄今为止,全世界神经科学家、临床医师和药厂都在通力合作,力争把通过长期实验研究发现的大量能够促进脑损伤后神经功能恢复的药物,逐步过渡到临床应用研究。国外学者已经采用医学循证方法,将200多种脑损伤后的脑保护药物用于治疗急性脑损伤病人,但未能发现任何一种临床有效的药物,其中包括现今临床常用的自由基清除剂。
低氧诱导因子1α(HIF-1α)是近年来研究的热门,其在脑梗塞和脑出血时均有表达。是脑损伤时对缺血缺氧反应的关键基因,已经证明其调控的下游基因多达70个之多。
我们应用MCAO方法制作稳定的脑损伤模型来研究HIF-1α在脑损伤后脑保护中的作用。我们发现,应用NAC, Edaravone等自由基清除剂确实可以减轻脑损伤,但是该脑神经保护作用可被HIF-1α抑制剂YC-1.2ME2等所阻断。这说明自由基清除剂NAC等的脑神经的保护作用机制与HIF-1有关,HIF-1α通路在脑损伤后的脑保护方面起关键作用。
脑损伤时,HIF-1α信号转导通路与改善脑组织血供、减轻脑组织缺血缺氧损伤密切相关,可能是脑损伤后脑保护治疗的新分子靶位,这为寻找治疗脑损伤后脑保护药物开辟了新的研究方向,具有十分重要的理论价值和临床意义。同时我们也进行了HIF-1α脑保护作用时间窗的研究,并对HIF-1α在脑损伤后BBB保护作用方面进行了初步研究。本实验主要分五个部分：
第一部分大鼠MCAO脑损伤模型的建立
目的介绍一种较为简单的脑缺血损伤模型的建立方法并在此基础上进行脑损伤的评价。
方法取270-310gSD大鼠,应用异氟醚(Isoflurane)气体吸入麻醉,在手术显微镜下,经颈部正中切口,暴露右侧颈内动脉,颈外动脉及颈总动脉。暂时阻断颈总动脉和颈内动脉血流,在颈外动脉上切一小口,将末端直径为0.35mm的线栓插入颈内动脉离颈外动脉分叉处18-20mm,阻塞大脑中动脉90分钟后再次麻醉并显微镜下取出线栓。24小时后进行神经功能评分并进行MRI扫描评价脑水肿和脑损伤面积。磁共振扫描后取脑组织并超薄切片进行TTC染色。
结果该方法制作的脑损伤面积基本一致。大鼠MCAO后的神经功能障碍评分表明存在明显的神经功能障碍。损伤的大鼠均出现较为一致的神经功能缺损,大鼠的死亡率为0,24小时后的MRI扫描发现脑水肿明显,脑损伤面积与文献报道一致。TTC染色同样表明成功阻塞大脑中动脉供血范围,大脑中动脉供血范围内有明显缺血表现
结论大鼠大脑中动脉阻塞损伤模型操作简便,重复性好,能模拟脑损伤后神经元缺血损伤过程,可控制损伤程度;是一种较好的动物神经元缺血损伤模型。
第二部分Hif-1α抑制剂在动物实验中对脑损伤的作用
目的研究大鼠急性脑损伤后HIF-1α抑制剂对大脑皮层HIF-1α的基因表达变化与其抑制神经保护的作用。
方法SD大鼠随机分为单纯损伤组、HIF-1α抑剂YC-1处理组2ME2处理组共3组,大脑中动脉阻塞法制作MCAO脑缺血损伤模型,处理组大鼠在损伤前30分钟经股静脉插管注射等量DMEO或YC-1 2mg/kg、2ME2 5mg/kg;单纯MCAO组大鼠、处理组大鼠在缺血再灌注24小时后进行神经功能评分。MRI扫描并断头取脑进行脑组织TTC染色测定脑缺血面积和HIF-1α基因表达的研究。
结果单纯脑损伤组和HIF-1α抑制剂组MRI扫描均有脑水肿,其中脑损伤面积以HIF-1α抑制剂组为最大,相对于单纯手术组其脑缺血脑梗死面积和脑组织水含量均明显增加(P<0.05)。TTC染色进行的脑缺血面积检测也显示在单纯手术组同HIF-1α抑制剂处理组相比较,YC-1处理组和2ME2处理组脑缺血面积有明显的增加(P<0.05)。神经功能评分在单纯手术组和HIF-1α抑制剂处理组比较有显著性差异(P<0.05)。HIF-1α抑制剂组间无明显差异。伤后24 h HIF-1α基因的表达在单纯手术组HIF-1α均有表达。在HIF-1α抑制剂应用组,HIF-1α的表达均未检测到。说明HIF-1α抑制剂的应用很好的抑制了脑损伤后HIF-1α的表达。
结论HIF-1α在脑损伤后表达水平明显增加,说明HIF-1α参与脑损伤后的继发性神经元损伤保护,其表达抑制剂YC-1和2ME2可明显加重损伤大鼠预后,说明HIF-1α对急性脑损伤具有保护性治疗作用。
第三部分：抗氧化剂上调HIF-1α的表达并减少缺血导致的脑损伤
目的研究大鼠脑皮层神经元缺血性损伤后HIF-1α的表达及氧自由基清除剂NAC和Edaravone的保护作用。
方法SD大鼠分三组,一组为单纯的MCAO组,一组为NAC治疗组,NAC150mg/kg经腹腔注射于MCAO前30分钟注射,一组为Edaravone治疗组,Edaravone 5mg/kg经腹腔在MCAO前30分钟注射。每一组大鼠脑缺血90分钟后取出线栓再灌注24小时。24小时后行神经功能评分(Neurological Scores)和MRI扫描检查脑损伤程度。然后取脑组织进行TTC染色监测脑缺血面积。并应用Western Blot方法检测脑缺血损伤后大鼠大脑皮质的HIF-1α的表达水平。
结果MRI和TTC染色结果均显示应用NAC和Edaravone等自由基清除剂后脑损伤程度明显的减轻。和对照组单纯MCAO模型相比较,其梗塞面积和缺血面积均明显的减少(P<0.05)。神经功能评分在应用自由基清除剂组也有明显的改善。Western blot检测结果显示HIF-1α在未损伤皮层神经元无可显示的表达;但是在大脑中动脉阻塞侧HIF-1α损伤后24小时表达明显升高(P<0.05),而在应用了抗氧化剂组,HIF-1α的升高更加明显,差异具有统计学意义(P<0.05)。
结论大鼠MCAO脑缺血损伤后HIF-1α在脑皮层表达明显增强,应用抗氧化剂NAC或Edravone后脑损伤面积明显减少,HIF-1α表达较对照组蛋白表达水平增加明显。抗氧化剂在大鼠缺血脑损伤模型中有显著的神经保护作用,其保护作用与HIF-1α的表达增加有关。
第四部分HIF-1α对缺血导致的脑损伤的保护作用时序性研究
目的利用脑缺血损伤模型来进行HIF-1α神经保护性作用时间窗的研究。为进一步指导临床治疗建立基础。
方法利用MRI可以活体检测脑损伤程度的优势,于大鼠大脑中动脉阻塞90分钟后再灌注0小时,3小时,6小时,12小时和24小时分别进行同一只大鼠的MRI扫描,分析脑组织水含量和脑梗塞的面积时序性变化。取270-310gSD大鼠,随机分入对照组或HIF-1α抑制剂(YC-1)应用组。对照组为大脑中动脉阻塞组,在大脑中动脉阻塞前30分钟经股静脉插管注射等量DMEO。YC-1处理组为MCAO加YC-1 2mg/kg于大鼠大脑中动脉阻塞前30分钟经股静脉注射。
结果在对照组,大鼠经大脑中动脉阻塞90分钟后脑水肿和脑梗塞面积是缓慢发展的,在0小时,3小时时基本无明显脑水肿和脑梗塞。缺血90分钟再灌注6小时时在大脑中动脉阻塞一侧的皮质下出现了少许梗塞灶,到12小时时,梗塞灶的轮廓已经出现,并保持至24小时,其MRI扫描的梗塞灶的密度不断加深。这说明HIF-1α的保护作用是有时效性的,其作用的时间窗应该是在12小时之前。当脑缺血再灌注12小时后脑梗死的发展已经是不可逆转的了,12小时时的梗死面积基本上就是24小时后的梗塞面积。而在HIF-la抑制剂YC-1处理组,从0-3小时开始,脑梗塞轮廓就已经出现,并保持到24小时后。这说明当没有了HIF-1α的保护作用后,脑缺血损伤很快发生,在大脑中动脉供血区的脑组织由于对缺血缺氧更加敏感而更快和更多的发生了损伤。在MRI扫描图像上表现为梗塞灶轮廓在0-3小时即出现并且梗塞灶轮廓为大脑中动脉供血区的绝大部分。随后随着梗塞区脑神经的坏死,脑梗塞灶的密度在MRI图像上逐渐的增强。
结论大鼠大脑中动脉阻塞导致脑缺血损伤后HIF-1α的表达可以保护继发性脑损伤,且其起保护作用的时间窗较短,0-6小时最佳,6-12小时尚有一定的保护作用。12小时后脑损伤灶基本形成,并保持到24小时。当应用HIF-1α抑制剂YC-1抑制HIF-1α的表达,则脑组织对缺血变得更加敏感,容易在缺血后发生继发性损伤。表现为梗塞面积一开始就很大,并且梗塞轮廓出现早,在12小时时大脑中动脉供血区缺血的脑组织即可形成脑梗塞灶。说明HIF-1α脑缺血保护方面具有重要的作用。
第五部分脑缺血损伤时HIF-1α对BBB的作用
目的应用Evans blue方法和MRI方法来检测脑损伤时HIF-1α对BBB的作用。
方法取270-310gSD大鼠,随机分为对照组和YC-1应用组。对照组给与MCAO90分钟后拔出线栓时给予Evans Blue 100mg/kg,经股静脉插管缓慢注射,24小时后测量脑组织内Evans blue含量。HIF-1α抑制剂应用组在MCAO前24小时及半小时给与YC-1 2mg/kg,经股静脉插管注射。MCAO90分钟后拔出线栓时经股静脉插管注射Evans Blue 100mg/kg。再灌注24小时后给与取脑测梗塞侧大脑半球Evans blue含量。另外取270-310gSD大鼠,随机分入对照组和HIF-1α抑制剂应用组。对照组行MCAO90分钟再灌注24小时,在MCAO前30分钟经股静脉插管注射DMSO。24小时后经股静脉插管注射增强药物进行MRI增强扫描,计算血脑屏障通透性(BBB permeability)。HIF-1α抑制剂组在MCAO前24小时和前30分钟经股静脉插管注射YC-1 2mg/kg,24小时后行MRI增强扫描,计算血脑屏障的通透性(BBB permeability)。
结果Evans Blue法检测发现,HIF-1α抑制剂应用组的BBB通透性明显的低于对照单纯MCAO组,这说明HIF-1α抑制剂在抑制了HIF-1α的表达后有效的保护了血脑屏障。但是有趣的是在用磁共振增强扫描方法计算的血脑屏障的通透性在HIF-1α抑制剂应用组是增加的,高于单纯MCAO组
结论脑损伤后HIF-1α的表达增加可促进大鼠血脑屏障通透性增加。HIF-1α抑制剂由于抑制了脑损伤后HIF-1α的表达,从而使血脑屏障通透性减低,起到了保护血脑屏障的作用。另一方面,应用MRI方法测得BBB通透性在HIF-1α抑制剂应用组是增加的。这与脑水肿在HIF-1α应用组是增加的是一致的。造成这两种BBBpermeability监测结果不一致的原因很多,其中一种是大分子通透性的方法,MRI实际是测验的是小分子的通透性。另一方面,BBB的开放可能存在时序性,用Evans Blue测得是BBB通透性的整体24小时内的结果,而MRI测得的是脑损伤24小时后的BBB通透性时间点上结果。如果HIF-1α对BBB起损伤作用,则可能是由于HIF-1α在不同细胞中的表达水平和所诱导的基因的不同所引起。在神经元中,HIF-1α起一定得保护作用,但在血管内皮细胞和胶质细胞中,HIF-1α的表达增加可能是有害的。这需要进一步的研究。
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 vivo
Objective 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 injury
Objective 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 damage
Objective 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 ischemia
Objective 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.
我们知道,脑损伤病人救治过程中除了抢救时间要快之外,更重要的工作是如何保护损伤后的脑组织,阻止继发性脑损害的进一步发生发展。以往研究认为脑损伤后的缺血缺氧产生大量的氧自由基是造成继发性脑损伤的主要原因。但是目前对自由基清除剂的临床应用尚无充足的证据证明有效。
迄今为止,全世界神经科学家、临床医师和药厂都在通力合作,力争把通过长期实验研究发现的大量能够促进脑损伤后神经功能恢复的药物,逐步过渡到临床应用研究。国外学者已经采用医学循证方法,将200多种脑损伤后的脑保护药物用于治疗急性脑损伤病人,但未能发现任何一种临床有效的药物,其中包括现今临床常用的自由基清除剂。
低氧诱导因子1α(HIF-1α)是近年来研究的热门,其在脑梗塞和脑出血时均有表达。是脑损伤时对缺血缺氧反应的关键基因,已经证明其调控的下游基因多达70个之多。
我们应用MCAO方法制作稳定的脑损伤模型来研究HIF-1α在脑损伤后脑保护中的作用。我们发现,应用NAC, Edaravone等自由基清除剂确实可以减轻脑损伤,但是该脑神经保护作用可被HIF-1α抑制剂YC-1.2ME2等所阻断。这说明自由基清除剂NAC等的脑神经的保护作用机制与HIF-1有关,HIF-1α通路在脑损伤后的脑保护方面起关键作用。
脑损伤时,HIF-1α信号转导通路与改善脑组织血供、减轻脑组织缺血缺氧损伤密切相关,可能是脑损伤后脑保护治疗的新分子靶位,这为寻找治疗脑损伤后脑保护药物开辟了新的研究方向,具有十分重要的理论价值和临床意义。同时我们也进行了HIF-1α脑保护作用时间窗的研究,并对HIF-1α在脑损伤后BBB保护作用方面进行了初步研究。本实验主要分五个部分：
第一部分大鼠MCAO脑损伤模型的建立
目的介绍一种较为简单的脑缺血损伤模型的建立方法并在此基础上进行脑损伤的评价。
方法取270-310gSD大鼠,应用异氟醚(Isoflurane)气体吸入麻醉,在手术显微镜下,经颈部正中切口,暴露右侧颈内动脉,颈外动脉及颈总动脉。暂时阻断颈总动脉和颈内动脉血流,在颈外动脉上切一小口,将末端直径为0.35mm的线栓插入颈内动脉离颈外动脉分叉处18-20mm,阻塞大脑中动脉90分钟后再次麻醉并显微镜下取出线栓。24小时后进行神经功能评分并进行MRI扫描评价脑水肿和脑损伤面积。磁共振扫描后取脑组织并超薄切片进行TTC染色。
结果该方法制作的脑损伤面积基本一致。大鼠MCAO后的神经功能障碍评分表明存在明显的神经功能障碍。损伤的大鼠均出现较为一致的神经功能缺损,大鼠的死亡率为0,24小时后的MRI扫描发现脑水肿明显,脑损伤面积与文献报道一致。TTC染色同样表明成功阻塞大脑中动脉供血范围,大脑中动脉供血范围内有明显缺血表现
结论大鼠大脑中动脉阻塞损伤模型操作简便,重复性好,能模拟脑损伤后神经元缺血损伤过程,可控制损伤程度;是一种较好的动物神经元缺血损伤模型。
第二部分Hif-1α抑制剂在动物实验中对脑损伤的作用
目的研究大鼠急性脑损伤后HIF-1α抑制剂对大脑皮层HIF-1α的基因表达变化与其抑制神经保护的作用。
方法SD大鼠随机分为单纯损伤组、HIF-1α抑剂YC-1处理组2ME2处理组共3组,大脑中动脉阻塞法制作MCAO脑缺血损伤模型,处理组大鼠在损伤前30分钟经股静脉插管注射等量DMEO或YC-1 2mg/kg、2ME2 5mg/kg;单纯MCAO组大鼠、处理组大鼠在缺血再灌注24小时后进行神经功能评分。MRI扫描并断头取脑进行脑组织TTC染色测定脑缺血面积和HIF-1α基因表达的研究。
结果单纯脑损伤组和HIF-1α抑制剂组MRI扫描均有脑水肿,其中脑损伤面积以HIF-1α抑制剂组为最大,相对于单纯手术组其脑缺血脑梗死面积和脑组织水含量均明显增加(P<0.05)。TTC染色进行的脑缺血面积检测也显示在单纯手术组同HIF-1α抑制剂处理组相比较,YC-1处理组和2ME2处理组脑缺血面积有明显的增加(P<0.05)。神经功能评分在单纯手术组和HIF-1α抑制剂处理组比较有显著性差异(P<0.05)。HIF-1α抑制剂组间无明显差异。伤后24 h HIF-1α基因的表达在单纯手术组HIF-1α均有表达。在HIF-1α抑制剂应用组,HIF-1α的表达均未检测到。说明HIF-1α抑制剂的应用很好的抑制了脑损伤后HIF-1α的表达。
结论HIF-1α在脑损伤后表达水平明显增加,说明HIF-1α参与脑损伤后的继发性神经元损伤保护,其表达抑制剂YC-1和2ME2可明显加重损伤大鼠预后,说明HIF-1α对急性脑损伤具有保护性治疗作用。
第三部分：抗氧化剂上调HIF-1α的表达并减少缺血导致的脑损伤
目的研究大鼠脑皮层神经元缺血性损伤后HIF-1α的表达及氧自由基清除剂NAC和Edaravone的保护作用。
方法SD大鼠分三组,一组为单纯的MCAO组,一组为NAC治疗组,NAC150mg/kg经腹腔注射于MCAO前30分钟注射,一组为Edaravone治疗组,Edaravone 5mg/kg经腹腔在MCAO前30分钟注射。每一组大鼠脑缺血90分钟后取出线栓再灌注24小时。24小时后行神经功能评分(Neurological Scores)和MRI扫描检查脑损伤程度。然后取脑组织进行TTC染色监测脑缺血面积。并应用Western Blot方法检测脑缺血损伤后大鼠大脑皮质的HIF-1α的表达水平。
结果MRI和TTC染色结果均显示应用NAC和Edaravone等自由基清除剂后脑损伤程度明显的减轻。和对照组单纯MCAO模型相比较,其梗塞面积和缺血面积均明显的减少(P<0.05)。神经功能评分在应用自由基清除剂组也有明显的改善。Western blot检测结果显示HIF-1α在未损伤皮层神经元无可显示的表达;但是在大脑中动脉阻塞侧HIF-1α损伤后24小时表达明显升高(P<0.05),而在应用了抗氧化剂组,HIF-1α的升高更加明显,差异具有统计学意义(P<0.05)。
结论大鼠MCAO脑缺血损伤后HIF-1α在脑皮层表达明显增强,应用抗氧化剂NAC或Edravone后脑损伤面积明显减少,HIF-1α表达较对照组蛋白表达水平增加明显。抗氧化剂在大鼠缺血脑损伤模型中有显著的神经保护作用,其保护作用与HIF-1α的表达增加有关。
第四部分HIF-1α对缺血导致的脑损伤的保护作用时序性研究
目的利用脑缺血损伤模型来进行HIF-1α神经保护性作用时间窗的研究。为进一步指导临床治疗建立基础。
方法利用MRI可以活体检测脑损伤程度的优势,于大鼠大脑中动脉阻塞90分钟后再灌注0小时,3小时,6小时,12小时和24小时分别进行同一只大鼠的MRI扫描,分析脑组织水含量和脑梗塞的面积时序性变化。取270-310gSD大鼠,随机分入对照组或HIF-1α抑制剂(YC-1)应用组。对照组为大脑中动脉阻塞组,在大脑中动脉阻塞前30分钟经股静脉插管注射等量DMEO。YC-1处理组为MCAO加YC-1 2mg/kg于大鼠大脑中动脉阻塞前30分钟经股静脉注射。
结果在对照组,大鼠经大脑中动脉阻塞90分钟后脑水肿和脑梗塞面积是缓慢发展的,在0小时,3小时时基本无明显脑水肿和脑梗塞。缺血90分钟再灌注6小时时在大脑中动脉阻塞一侧的皮质下出现了少许梗塞灶,到12小时时,梗塞灶的轮廓已经出现,并保持至24小时,其MRI扫描的梗塞灶的密度不断加深。这说明HIF-1α的保护作用是有时效性的,其作用的时间窗应该是在12小时之前。当脑缺血再灌注12小时后脑梗死的发展已经是不可逆转的了,12小时时的梗死面积基本上就是24小时后的梗塞面积。而在HIF-la抑制剂YC-1处理组,从0-3小时开始,脑梗塞轮廓就已经出现,并保持到24小时后。这说明当没有了HIF-1α的保护作用后,脑缺血损伤很快发生,在大脑中动脉供血区的脑组织由于对缺血缺氧更加敏感而更快和更多的发生了损伤。在MRI扫描图像上表现为梗塞灶轮廓在0-3小时即出现并且梗塞灶轮廓为大脑中动脉供血区的绝大部分。随后随着梗塞区脑神经的坏死,脑梗塞灶的密度在MRI图像上逐渐的增强。
结论大鼠大脑中动脉阻塞导致脑缺血损伤后HIF-1α的表达可以保护继发性脑损伤,且其起保护作用的时间窗较短,0-6小时最佳,6-12小时尚有一定的保护作用。12小时后脑损伤灶基本形成,并保持到24小时。当应用HIF-1α抑制剂YC-1抑制HIF-1α的表达,则脑组织对缺血变得更加敏感,容易在缺血后发生继发性损伤。表现为梗塞面积一开始就很大,并且梗塞轮廓出现早,在12小时时大脑中动脉供血区缺血的脑组织即可形成脑梗塞灶。说明HIF-1α脑缺血保护方面具有重要的作用。
第五部分脑缺血损伤时HIF-1α对BBB的作用
目的应用Evans blue方法和MRI方法来检测脑损伤时HIF-1α对BBB的作用。
方法取270-310gSD大鼠,随机分为对照组和YC-1应用组。对照组给与MCAO90分钟后拔出线栓时给予Evans Blue 100mg/kg,经股静脉插管缓慢注射,24小时后测量脑组织内Evans blue含量。HIF-1α抑制剂应用组在MCAO前24小时及半小时给与YC-1 2mg/kg,经股静脉插管注射。MCAO90分钟后拔出线栓时经股静脉插管注射Evans Blue 100mg/kg。再灌注24小时后给与取脑测梗塞侧大脑半球Evans blue含量。另外取270-310gSD大鼠,随机分入对照组和HIF-1α抑制剂应用组。对照组行MCAO90分钟再灌注24小时,在MCAO前30分钟经股静脉插管注射DMSO。24小时后经股静脉插管注射增强药物进行MRI增强扫描,计算血脑屏障通透性(BBB permeability)。HIF-1α抑制剂组在MCAO前24小时和前30分钟经股静脉插管注射YC-1 2mg/kg,24小时后行MRI增强扫描,计算血脑屏障的通透性(BBB permeability)。
结果Evans Blue法检测发现,HIF-1α抑制剂应用组的BBB通透性明显的低于对照单纯MCAO组,这说明HIF-1α抑制剂在抑制了HIF-1α的表达后有效的保护了血脑屏障。但是有趣的是在用磁共振增强扫描方法计算的血脑屏障的通透性在HIF-1α抑制剂应用组是增加的,高于单纯MCAO组
结论脑损伤后HIF-1α的表达增加可促进大鼠血脑屏障通透性增加。HIF-1α抑制剂由于抑制了脑损伤后HIF-1α的表达,从而使血脑屏障通透性减低,起到了保护血脑屏障的作用。另一方面,应用MRI方法测得BBB通透性在HIF-1α抑制剂应用组是增加的。这与脑水肿在HIF-1α应用组是增加的是一致的。造成这两种BBBpermeability监测结果不一致的原因很多,其中一种是大分子通透性的方法,MRI实际是测验的是小分子的通透性。另一方面,BBB的开放可能存在时序性,用Evans Blue测得是BBB通透性的整体24小时内的结果,而MRI测得的是脑损伤24小时后的BBB通透性时间点上结果。如果HIF-1α对BBB起损伤作用,则可能是由于HIF-1α在不同细胞中的表达水平和所诱导的基因的不同所引起。在神经元中,HIF-1α起一定得保护作用,但在血管内皮细胞和胶质细胞中,HIF-1α的表达增加可能是有害的。这需要进一步的研究。
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 vivo
Objective 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 injury
Objective 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 damage
Objective 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 ischemia
Objective 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.
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