铁离子在实验性脑室出血后脑损伤中的作用机制及干预研究

发布时间：2018-04-27 14:42

  本文选题：脑室出血 + 出血后脑积水　； 参考：《第三军医大学》2014年博士论文

【摘要】：脑出血(intracerebral hemorrhage，ICH)是常见的卒中亚型，发病率高居卒中第二位。脑出血发病急骤，病情往往十分凶险，发病后1月内病死率可高达50％，且幸存者多有严重的神经功能障碍。然而，脑出血后脑损伤的机理仍不够清楚，亦缺乏经严格验证有效的治疗措施。在脑出血患者中，合并脑室出血(intraventricular hemorrhage，IVH)的发生率高达50%以上，脑室出血以及继发形成的脑积水将进一步加重脑损伤，是临床脑出血患者预后不良的独立危险因素，近年来逐步受到关注。因此，脑室出血及继发性脑积水的防治研究已成为脑出血领域关注的新方向，明确IVH后脑损伤机制及探索有效的干预措施对提高临床脑出血患者的治疗效果具有重大意义。 目前，，脑室出血后脑损伤及继发脑积水的确切病因及机理还不是很清楚，也缺乏有效的药物治疗措施1。IVH后病理损伤机制可能包括：早期梗阻性脑积水导致颅内压增高、脑室内血肿的占位效应、血液代谢产物的毒性作用和继发慢性脑积水等2。然而，IVH后血液代谢产物与脑损伤和慢性脑积水之间的关系尚缺乏研究。近年来，血肿及其代谢产物的毒性效应作为脑出血后脑损伤的重要研究方向取得了较大进展。ICH后数日内血块开始融解，铁离子作为血红蛋白的主要降解产物过量集聚导致组织铁超载和氧化损伤，造成血脑屏障受损、脑水肿及神经元死亡等。相应的，铁螯合剂去铁敏干预措施在多种ICH动物模型研究中均被证实具有神经保护效应，因此研究ICH后铁超载相关损伤及治疗措施意义重大。 脑室出血后铁代谢紊乱及其在继发脑损伤中的作用尚不清楚，我们分析IVH后溶血产物可广泛播撒至全脑室系统及蛛网膜下腔，与脑室壁及脑组织接触面更为广泛，铁超载及相关损伤可能在IVH后脑损伤及脑积水发生中同样具有重要作用。因此，我们推测：IVH后脑室内积血逐步代谢后有大量铁离子释放，可能导致显著的脑铁超载，并引起脑室壁室管膜纤毛上皮、脑室旁组织等氧化应激损伤，从而参与IVH后脑损伤及慢性脑积水的发生发展。本课题以IVH后铁超载及氧化损伤为主要切入点，观察大鼠实验性IVH后脑损伤、脑积水和铁代谢特征，并分别采用去铁敏、米诺环素和依达拉奉进行干预，观察在铁超载和氧化损伤两个关键环节的干预效应，分三部分研究验证上述推测：第一部分采用不同血液制品行大鼠单侧脑室注射建立IVH损伤模型，明确IVH后脑损伤、脑积水和铁代谢相关特征，观察铁螯合剂去铁敏干预是否能减轻IVH后脑损伤；第二部分采用脑室内铁离子注射模型，观察铁离子在脑室内的直接脑损伤效应，以及米诺环素对脑室内铁损伤的保护效应，进一步证明铁离子可能参与了IVH后继发损伤及脑积水形成；第三部分采用实验性大鼠IVH模型，观察自由基清除剂依达拉奉对IVH后氧化应激损伤及脑积水的干预效应，验证氧化损伤参与IVH后室管膜损伤及脑积水发生的推测。 一、铁离子在大鼠实验性脑室出血后脑损伤中作用及去铁敏的干预研究 目的 通过侧脑室注射自体全血、抗凝血或红细胞建立大鼠脑室出血模型，动态观察出血后脑损伤、脑积水、脑铁沉积和铁代谢相关蛋白表达变化，以及铁螯合剂去铁敏的干预效应，从而评估铁离子超载在脑室出血后脑损伤中的作用及铁螯合治疗的意义。 方法 实验分为3部分，第一部分将实验动物分成生理盐水对照组(Saline)、自体血组(Bloodor IVH)及抗凝血组(Heparinied blood)，分别将200μl生理盐水、自体血及自体抗凝血注入大鼠右侧脑室，分别于1天、3天、7天、14天和28天行MRI扫描，并于上述时象点留取脑标本行组织学、免疫组化和Western blot检测；第二部分实验观察红细胞脑室内注射的效应，将动物分为浓缩红细胞组(Packed RBC)和融解红细胞组(Lysed RBC)，分别将50μl浓缩红细胞和融解红细胞注入大鼠右侧脑室，于24小时行MRI扫描并留取标本供组织学检测；第三部分观察去铁敏的干预效应，实验动物分为去铁敏干预组(IVH+DFX)和溶剂干预组(IVH+Veh)，分别给予右侧脑室内注入200μl自体全血，再于血液注射后2小时、6小时分别给予去铁敏(DFX，100mg/kg)或溶剂，肌肉注射，然后每12小时注射一次，共7天疗程。所有动物分别于1天、3天、7天、14天和28天行MRI扫描，扫描结束后留取标本行组织学检查。 结果 1.单侧脑室注血法成功建立大鼠实验性IVH模型，自体血及自体抗凝血注射均导致明显脑积水，至28天脑室仍显著扩张，但抗凝血组脑积水轻于非抗凝血组；IVH引起大鼠脑铁蓄积和HO-1、Ferritin表达升高，以及后期海马萎缩、海马神经元缺失和脑室壁胶质增生。 2.脑室内注射融解红细胞导致急性脑室扩张、脑水肿，明显重于浓缩红细胞组，脑组织HO-1及OX-42免疫阳性反应均强于浓缩红细胞组。 3.去铁敏可减轻IVH后脑室扩张和铁蓄积，Ferritin表达水平也相应降低，去铁敏干预组于IVH后28天海马萎缩、海马神经元缺失均较溶剂对照组减轻。 结论 大鼠IVH导致持续脑室扩张，并伴有明显的脑铁蓄积和铁代谢相关蛋白表达上调，相应地给予铁螯合剂去铁敏治疗可减轻IVH后脑积水、海马损伤及铁蓄积，提示铁超载可能是IVH后脑损伤和脑积水的重要机制，去铁敏在IVH后继发脑损伤治疗中具有的应用前景。 二、大鼠脑室内铁离子注射模型脑损伤及米诺环素的干预研究 目的 建立大鼠脑室内铁离子注射模型，观察铁离子脑室内注射后的直接脑损伤效应，探讨米诺环素和巨噬细胞/小胶质细胞抑制剂(macrophage/microglial inhibitory factor，MIF)对在体条件下脑室内高铁所致脑损伤的保护效应及机制。 方法 将实验动物分成生理盐水对照组(Saline)、氯化亚铁组(FeCl2)、氯化亚铁+米诺环素组(FeCl2+Minocycline)、氯化亚铁+MIF组(FeCl2+MIF)、三氯化铁组(FeCl3)和三氯化铁+米诺环素组(FeCl3+Minocycline)，各组分别将上述(混合)溶液200μl注入大鼠右侧脑室内，氯化亚铁和三氯化铁浓度为0.5mM，米诺环素和MIF浓度为1.5mM；模型建立后1天分别行MRI扫描、脑水含量测定，留取致伤后1天组织标本行Fluoro-Jade C、PANT和TUNEL检测；分别采用铁三嗪比色法和改良铬天青S法检测米诺环素对二价和三价铁离子的螯合力。 结果 1.氯化亚铁脑室内注入引起急性脑室扩张、脑水肿和神经元变性损伤，米诺环素和MIF可明显抑制氯化亚铁脑室注射后所引起的小胶质细胞活化，但仅有米诺环素能显著降低大鼠死亡率，减轻脑水肿和神经元损伤。 2.三氯化铁脑室内注射引起显著的急性脑室扩张和海马神经元变性，米诺环素能减轻神经元损伤，但未减轻脑室扩张。 3.米诺环素对二价和三价铁离子均具有螯合效应。 结论 铁离子脑室内注射可导致急性脑积水、脑水肿及神经元损伤；米诺环素具有亚铁和三价离子螯合力，并能显著减轻铁离子脑室内注射所致的脑水肿和神经元损伤，其神经保护作用的机理可能与铁螯合效力有关。 关键词：脑室出血铁米诺环素脑积水 三、依达拉奉对大鼠脑室出血后脑损伤的干预研究 目的 观察自由基清除剂依达拉奉对实验性大鼠脑室出血后氧化应激损伤、脑积水和神经行为学损伤的干预效应。 方法 研究分为2部分，第一部分为急性期指标测定，动物分为生理盐水对照组(Saline)、脑室出血+依达拉奉干预组(IVH+Edv)和脑室出血+溶剂干预组(IVH+Veh)，分别给予右侧脑室内注入200μl生理盐水或不抗凝自体全血，注血后分别给予依达拉奉(6mg/kg，IVH+Edv组)或溶剂(IVH+Veh)皮下注射，损伤后24小时取脑组织检测脑含水量、丙二醛(MDA)水平和超氧化物歧化酶(SOD)活性。第二部分实验分组同前，注血组大鼠分别给予依达拉奉(6mg/kg，IVH+Edv组)或溶剂(IVH+Veh)皮下注射共3天(注血后15分钟，1天和2天)，IVH后23天开始行为学检测，28天行MRI扫描并留取标本行组织学检查。 结果： 1.大鼠IVH模型可导致急性脑水肿、氧化应激及后期学习记忆功能缺陷。 2.依达拉奉减轻了IVH后组织氧化应激、脑水肿及脑室壁室管膜纤毛上皮损伤，改善了大鼠后期脑积水和学习记忆功能缺陷。 结论 自由基清除剂依达拉奉可减轻大鼠IVH后氧化应激和急性脑水肿，并改善后期脑室壁损伤、脑积水和学习记忆功能缺陷，氧化应激可能是IVH后脑损伤及脑积水的重要机制和治疗靶点之一。
[Abstract]:Intracerebral hemorrhage (ICH) is a common subtype of Apoplexy with a high incidence of second high incidence of stroke. The incidence of cerebral hemorrhage is urgent and the disease is often very dangerous. The mortality rate in January can be as high as 50%, and the survivors have serious neurological dysfunction. However, the mechanism of brain injury after brain blood is still not clear and strict. In the patients with cerebral hemorrhage, the incidence of intraventricular hemorrhage (IVH) is more than 50%. Cerebral hemorrhage and secondary hydrocephalus will further aggravate the brain damage. It is the independent risk factor of poor prognosis in the patients with clinical cerebral hemorrhage. The prevention and treatment of hemorrhage and secondary hydrocephalus has become a new direction of attention in the field of cerebral hemorrhage. It is of great significance to clarify the mechanism of IVH posterior brain injury and to explore effective intervention measures to improve the therapeutic effect of clinical cerebral hemorrhage.
At present, the exact cause and mechanism of brain injury and secondary hydrocephalus after intracerebral hemorrhage are not very clear, and there is no effective drug treatment for 1.IVH. The mechanism of pathological injury may include increased intracranial pressure caused by early obstructive hydrocephalus, the occupying effect of intraventricular hematoma, toxic effects of blood metabolites and secondary chronic brain Water accumulation and so on 2., however, the relationship between blood metabolites and brain damage and chronic hydrocephalus is still lack of research. In recent years, the toxic effects of hematoma and its metabolites have been the important research direction of brain injury after cerebral hemorrhage, which has made great progress in.ICH after IVH, and the main degradation of hemoglobin is iron ion as the main degradation of hemoglobin. Excessive concentration of products leads to iron overload and oxidative damage, resulting in damage to the blood brain barrier, brain edema and neuronal death. Accordingly, iron chelating agents have been proved to have neuroprotective effects in the study of various ICH animal models. Therefore, it is of great significance to study the related damage and treatment of iron overload after ICH.
Iron metabolism disorder and its role in secondary brain injury after intracerebral hemorrhage are not clear. Our analysis of hemolytic products after IVH can be widely spread to the whole ventricle system and subarachnoid cavity, more extensive with the ventricle wall and brain tissue. Iron overload and related injuries may also be important in the IVH after brain injury and hydrocephalus. Therefore, we speculate that a large number of iron ions are released after the gradual metabolism of blood in the IVH posterior cerebral ventricle, which may lead to significant iron overload, and cause oxidative stress in the ependymal ciliated epithelium and paraventricular tissue in the ventricles of the brain, so as to participate in the injury of the IVH brain and the development of chronic hydrocephalus. This subject takes iron overload and oxidative damage after IVH. The main entry point was to observe the experimental IVH injury, hydrocephalus and iron metabolism in experimental rats, and the intervention effects of iron sensitive, minocycline and edaravone were used respectively to observe the intervention effects of two key links in iron overload and oxidative damage. Three parts of the study verified the above speculation: the first part was the use of different blood products lines. The IVH injury model was established in the unilateral ventricle of the rat, and the brain injury, hydrocephalus and iron metabolism related characteristics were identified. Whether the iron chelating mixture was used to reduce the brain damage after IVH was observed. The second part of the intraventricular iron ion injection model was used to observe the direct brain damage effect of iron ions in the ventricles of the brain and the minocycline in the brain. The protective effect of iron damage further proves that iron ions may be involved in secondary injury and formation of hydrocephalus after IVH; the third part uses experimental rat IVH model to observe the effect of free radical scavenger edaravone on oxidative stress injury and hydrocephalus after IVH, and to verify that oxidative injury participates in ependymal injury and hydrocephalus after IVH. A birth conjecture.
The effect of iron ions on brain injury after experimental intraventricular hemorrhage in rats and the intervention of iron removal
objective
A rat model of cerebral hemorrhage was established by injection of autologous blood, anticoagulant or red blood cells by injection of autologous blood, anticoagulant or red blood cells, and dynamic observation of brain injury after hemorrhage, hydrocephalus, changes in iron deposition and iron metabolism related protein expression, and the interference effect of iron chelating mixture, so as to evaluate the role of iron overload in brain injury after cerebral hemorrhage and iron chelation. The significance of treatment.
Method
The experiment was divided into 3 parts. In the first part, the experimental animals were divided into the normal saline control group (Saline), the autologous blood group (Bloodor IVH) and the anticoagulant group (Heparinied blood). The normal saline, the autologous blood and the autologous anticoagulant were injected into the right ventricle of the rat, and the MRI scan was performed on 1 days, 3 days, 7 days, 14 days and 28 days respectively. Brain labeling, immunohistochemistry and Western blot detection; the second part of the experiment observed the effect of the erythrocyte intraventricular injection, divided the animals into the concentrated red cell group (Packed RBC) and the fusion red cell group (Lysed RBC), and injected the concentrated red blood cells and the fusion red cells into the right ventricle of the rat, respectively, and took the MRI scan and retained for 24 hours. The specimens were tested for histological examination; the third part observed the intervention effect of DFE, the experimental animals were divided into the iron sensitive intervention group (IVH+DFX) and the solvent intervention group (IVH+Veh). The right ventricle was injected into the right ventricle with 200 L autologous whole blood, then 2 hours after the blood injection, and 6 hours respectively to Tie Min (DFX, 100mg/kg) or solvent, muscle injection, and then every 1 After 2 hours of injection, there were 7 days of treatment. All animals performed MRI scan on 1 days, 3 days, 7 days, 14 days and 28 days respectively.
Result
1. the experimental IVH model of rats was successfully established by unilateral ventricle injection. Autologous blood and autologous anticoagulant injection resulted in obvious hydrocephalus, and the ventricles of the brain still expanded significantly on the 28 day, but the accumulation of hydrocephalus in the anticoagulant group was lighter than that in the non anticoagulant group; IVH induced the accumulation of iron in the brain and the increase of HO-1, Ferritin expression in the rats, and the atrophy of hippocampus and the loss of hippocampal neurons in the later period. Glial hyperplasia of the ventricles of the ventricles of the brain.
The intraventricular injection of red blood cells in the 2. ventricle leads to acute ventricular dilatation and brain edema, which is significantly heavier than the concentrated red cell group. The immunoreactive reaction of HO-1 and OX-42 in the brain is stronger than that in the concentrated red cell group.
3. de iron sensitive can reduce the ventricular dilatation and iron accumulation after IVH, and decrease the expression level of Ferritin correspondingly. The hippocampus atrophy in the Tie Min intervention group at the 28 day after IVH, and the loss of hippocampal neurons in the hippocampus is less than that in the solvent control group.
conclusion
IVH induced persistent ventricular dilatation, accompanied by obvious iron accumulation and up-regulated expression of iron metabolism related proteins. The corresponding iron chelating agent in the treatment of iron sensitive treatment can reduce the hydrocephalus, hippocampal damage and iron accumulation in IVH, suggesting that iron overload may be an important mechanism of IVH after brain injury and hydrocephalus, and the treatment of secondary brain injury after IVH The prospect of application.
Two, brain injury and intraventricular injection of minocycline in rats
objective
A rat model of intraventricular iron ion injection was established to observe the effect of direct brain injury after intraventricular injection of iron ion. The protective effect and mechanism of minocycline and macrophage / microglia inhibitor (macrophage/microglial inhibitory factor, MIF) on brain injury induced by high iron in the brain under the condition of body condition were investigated.
Method
The experimental animals were divided into the normal saline control group (Saline), the ferrous chloride group (FeCl2), the ferrous chloride + minocycline group (FeCl2+Minocycline), the ferrous chloride +MIF group (FeCl2+MIF), the iron trichloride group (FeCl3) and the iron trichloride + minocycline group (FeCl3+Minocycline). Each group was injected 200 u l into the right ventricle of the rat's right ventricle, respectively. The concentration of ferrous chloride and ferric chloride was 0.5mM, the concentration of minocycline and MIF was 1.5mM; 1 days after the establishment of the model, the MRI scan was performed, the content of brain water was measured, and the tissue mark was detected by Fluoro-Jade C, PANT and TUNEL at the 1 day after the injury. The iron three azine colorimetric method and the modified chromite S method were used to detect the two valence and trivalent iron ions of minocycline respectively. The chelation force.
Result
Intraventricular injection of 1. ferrous chloride causes acute ventricular dilatation, brain edema and neuronal degeneration. Minocycline and MIF can significantly inhibit the activation of microglia caused by intraventricular injection of ferrous chloride, but only minocycline can significantly reduce the mortality of rats and reduce brain edema and neuron damage.
2. intraventricular injection of iron trichloride caused significant acute ventricular dilation and degeneration of hippocampal neurons. Minocycline reduced neuronal damage but did not relieve ventricular dilatation.
3. minocycline has chelating effect on two valent and trivalent iron ions.
conclusion
Intraventricular injection of iron ions can lead to acute hydrocephalus, brain edema and neuronal damage. Minocycline has a combined force of ferrous and trivalent ion, which can significantly reduce brain edema and neuronal damage caused by intraventricular injection of iron ions. The mechanism of neuroprotective effect may be related to iron chelation.
Key words: ventricle hemorrhage, iron minocycline hydrocephalus
Three, edaravone intervention on brain injury after intraventricular hemorrhage in rats.
objective
Objective To observe the intervention effect of edaravone, a free radical scavenger, on oxidative stress injury, hydrocephalus and neurobehavioral impairment after experimental intraventricular hemorrhage in rats.
Method
The study was divided into 2 parts. The first part was the determination of the acute phase index, the animals were divided into the normal saline control group (Saline), the ventricle hemorrhage + edaravone intervention group (IVH+Edv) and the ventricle hemorrhage + solvent intervention group (IVH+Veh). The right ventricle was injected with 200 L saline or the agaginate autologous whole blood respectively, and edaravone was given respectively after blood injection (6mg/kg The IVH+Edv group) or the solvent (IVH+Veh) was injected subcutaneously, and the brain tissue was taken 24 hours after the injury to detect the brain water content, the level of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD). The second part of the experimental group was given a total of 3 days after the injection of edaravone (6mg/kg, IVH+ Edv group) or the solvent (IVH+Veh) for a total of 3 days (15 minutes, 1 days after blood injection. On the 2 day), behavioral tests were performed on the 23 day after IVH, and MRI scan was performed on the 28 day and histological examination was taken.
Result锛

本文编号：1811143