本文選題：腦室出血 + 出血后腦積水�。� 參考：《第三軍醫(yī)大學(xué)》2014年博士論文

【摘要】：腦出血(intracerebral hemorrhage，ICH)是常見的卒中亞型，發(fā)病率高居卒中第二位。腦出血發(fā)病急驟，病情往往十分兇險(xiǎn)，發(fā)病后1月內(nèi)病死率可高達(dá)50％，且幸存者多有嚴(yán)重的神經(jīng)功能障礙。然而，腦出血后腦損傷的機(jī)理仍不夠清楚，亦缺乏經(jīng)嚴(yán)格驗(yàn)證有效的治療措施。在腦出血患者中，合并腦室出血(intraventricular hemorrhage，IVH)的發(fā)生率高達(dá)50%以上，腦室出血以及繼發(fā)形成的腦積水將進(jìn)一步加重腦損傷，是臨床腦出血患者預(yù)后不良的獨(dú)立危險(xiǎn)因素，近年來逐步受到關(guān)注。因此，腦室出血及繼發(fā)性腦積水的防治研究已成為腦出血領(lǐng)域關(guān)注的新方向，明確IVH后腦損傷機(jī)制及探索有效的干預(yù)措施對(duì)提高臨床腦出血患者的治療效果具有重大意義。 目前，，腦室出血后腦損傷及繼發(fā)腦積水的確切病因及機(jī)理還不是很清楚，也缺乏有效的藥物治療措施1。IVH后病理?yè)p傷機(jī)制可能包括：早期梗阻性腦積水導(dǎo)致顱內(nèi)壓增高、腦室內(nèi)血腫的占位效應(yīng)、血液代謝產(chǎn)物的毒性作用和繼發(fā)慢性腦積水等2。然而，IVH后血液代謝產(chǎn)物與腦損傷和慢性腦積水之間的關(guān)系尚缺乏研究。近年來，血腫及其代謝產(chǎn)物的毒性效應(yīng)作為腦出血后腦損傷的重要研究方向取得了較大進(jìn)展。ICH后數(shù)日內(nèi)血塊開始融解，鐵離子作為血紅蛋白的主要降解產(chǎn)物過量集聚導(dǎo)致組織鐵超載和氧化損傷，造成血腦屏障受損、腦水腫及神經(jīng)元死亡等。相應(yīng)的，鐵螯合劑去鐵敏干預(yù)措施在多種ICH動(dòng)物模型研究中均被證實(shí)具有神經(jīng)保護(hù)效應(yīng)，因此研究ICH后鐵超載相關(guān)損傷及治療措施意義重大。 腦室出血后鐵代謝紊亂及其在繼發(fā)腦損傷中的作用尚不清楚，我們分析IVH后溶血產(chǎn)物可廣泛播撒至全腦室系統(tǒng)及蛛網(wǎng)膜下腔，與腦室壁及腦組織接觸面更為廣泛，鐵超載及相關(guān)損傷可能在IVH后腦損傷及腦積水發(fā)生中同樣具有重要作用。因此，我們推測(cè)：IVH后腦室內(nèi)積血逐步代謝后有大量鐵離子釋放，可能導(dǎo)致顯著的腦鐵超載，并引起腦室壁室管膜纖毛上皮、腦室旁組織等氧化應(yīng)激損傷，從而參與IVH后腦損傷及慢性腦積水的發(fā)生發(fā)展。本課題以IVH后鐵超載及氧化損傷為主要切入點(diǎn)，觀察大鼠實(shí)驗(yàn)性IVH后腦損傷、腦積水和鐵代謝特征，并分別采用去鐵敏、米諾環(huán)素和依達(dá)拉奉進(jìn)行干預(yù)，觀察在鐵超載和氧化損傷兩個(gè)關(guān)鍵環(huán)節(jié)的干預(yù)效應(yīng)，分三部分研究驗(yàn)證上述推測(cè)：第一部分采用不同血液制品行大鼠單側(cè)腦室注射建立IVH損傷模型，明確IVH后腦損傷、腦積水和鐵代謝相關(guān)特征，觀察鐵螯合劑去鐵敏干預(yù)是否能減輕IVH后腦損傷；第二部分采用腦室內(nèi)鐵離子注射模型，觀察鐵離子在腦室內(nèi)的直接腦損傷效應(yīng)，以及米諾環(huán)素對(duì)腦室內(nèi)鐵損傷的保護(hù)效應(yīng)，進(jìn)一步證明鐵離子可能參與了IVH后繼發(fā)損傷及腦積水形成；第三部分采用實(shí)驗(yàn)性大鼠IVH模型，觀察自由基清除劑依達(dá)拉奉對(duì)IVH后氧化應(yīng)激損傷及腦積水的干預(yù)效應(yīng)，驗(yàn)證氧化損傷參與IVH后室管膜損傷及腦積水發(fā)生的推測(cè)。 一、鐵離子在大鼠實(shí)驗(yàn)性腦室出血后腦損傷中作用及去鐵敏的干預(yù)研究 目的 通過側(cè)腦室注射自體全血、抗凝血或紅細(xì)胞建立大鼠腦室出血模型，動(dòng)態(tài)觀察出血后腦損傷、腦積水、腦鐵沉積和鐵代謝相關(guān)蛋白表達(dá)變化，以及鐵螯合劑去鐵敏的干預(yù)效應(yīng)，從而評(píng)估鐵離子超載在腦室出血后腦損傷中的作用及鐵螯合治療的意義。 方法 實(shí)驗(yàn)分為3部分，第一部分將實(shí)驗(yàn)動(dòng)物分成生理鹽水對(duì)照組(Saline)、自體血組(Bloodor IVH)及抗凝血組(Heparinied blood)，分別將200μl生理鹽水、自體血及自體抗凝血注入大鼠右側(cè)腦室，分別于1天、3天、7天、14天和28天行MRI掃描，并于上述時(shí)象點(diǎn)留取腦標(biāo)本行組織學(xué)、免疫組化和Western blot檢測(cè)；第二部分實(shí)驗(yàn)觀察紅細(xì)胞腦室內(nèi)注射的效應(yīng)，將動(dòng)物分為濃縮紅細(xì)胞組(Packed RBC)和融解紅細(xì)胞組(Lysed RBC)，分別將50μl濃縮紅細(xì)胞和融解紅細(xì)胞注入大鼠右側(cè)腦室，于24小時(shí)行MRI掃描并留取標(biāo)本供組織學(xué)檢測(cè)；第三部分觀察去鐵敏的干預(yù)效應(yīng)，實(shí)驗(yàn)動(dòng)物分為去鐵敏干預(yù)組(IVH+DFX)和溶劑干預(yù)組(IVH+Veh)，分別給予右側(cè)腦室內(nèi)注入200μl自體全血，再于血液注射后2小時(shí)、6小時(shí)分別給予去鐵敏(DFX，100mg/kg)或溶劑，肌肉注射，然后每12小時(shí)注射一次，共7天療程。所有動(dòng)物分別于1天、3天、7天、14天和28天行MRI掃描，掃描結(jié)束后留取標(biāo)本行組織學(xué)檢查。 結(jié)果 1.單側(cè)腦室注血法成功建立大鼠實(shí)驗(yàn)性IVH模型，自體血及自體抗凝血注射均導(dǎo)致明顯腦積水，至28天腦室仍顯著擴(kuò)張，但抗凝血組腦積水輕于非抗凝血組；IVH引起大鼠腦鐵蓄積和HO-1、Ferritin表達(dá)升高，以及后期海馬萎縮、海馬神經(jīng)元缺失和腦室壁膠質(zhì)增生。 2.腦室內(nèi)注射融解紅細(xì)胞導(dǎo)致急性腦室擴(kuò)張、腦水腫，明顯重于濃縮紅細(xì)胞組，腦組織HO-1及OX-42免疫陽(yáng)性反應(yīng)均強(qiáng)于濃縮紅細(xì)胞組。 3.去鐵敏可減輕IVH后腦室擴(kuò)張和鐵蓄積，F(xiàn)erritin表達(dá)水平也相應(yīng)降低，去鐵敏干預(yù)組于IVH后28天海馬萎縮、海馬神經(jīng)元缺失均較溶劑對(duì)照組減輕。 結(jié)論 大鼠IVH導(dǎo)致持續(xù)腦室擴(kuò)張，并伴有明顯的腦鐵蓄積和鐵代謝相關(guān)蛋白表達(dá)上調(diào)，相應(yīng)地給予鐵螯合劑去鐵敏治療可減輕IVH后腦積水、海馬損傷及鐵蓄積，提示鐵超載可能是IVH后腦損傷和腦積水的重要機(jī)制，去鐵敏在IVH后繼發(fā)腦損傷治療中具有的應(yīng)用前景。 二、大鼠腦室內(nèi)鐵離子注射模型腦損傷及米諾環(huán)素的干預(yù)研究 目的 建立大鼠腦室內(nèi)鐵離子注射模型，觀察鐵離子腦室內(nèi)注射后的直接腦損傷效應(yīng)，探討米諾環(huán)素和巨噬細(xì)胞/小膠質(zhì)細(xì)胞抑制劑(macrophage/microglial inhibitory factor，MIF)對(duì)在體條件下腦室內(nèi)高鐵所致腦損傷的保護(hù)效應(yīng)及機(jī)制。 方法 將實(shí)驗(yàn)動(dòng)物分成生理鹽水對(duì)照組(Saline)、氯化亞鐵組(FeCl2)、氯化亞鐵+米諾環(huán)素組(FeCl2+Minocycline)、氯化亞鐵+MIF組(FeCl2+MIF)、三氯化鐵組(FeCl3)和三氯化鐵+米諾環(huán)素組(FeCl3+Minocycline)，各組分別將上述(混合)溶液200μl注入大鼠右側(cè)腦室內(nèi)，氯化亞鐵和三氯化鐵濃度為0.5mM，米諾環(huán)素和MIF濃度為1.5mM；模型建立后1天分別行MRI掃描、腦水含量測(cè)定，留取致傷后1天組織標(biāo)本行Fluoro-Jade C、PANT和TUNEL檢測(cè)；分別采用鐵三嗪比色法和改良鉻天青S法檢測(cè)米諾環(huán)素對(duì)二價(jià)和三價(jià)鐵離子的螯合力。 結(jié)果 1.氯化亞鐵腦室內(nèi)注入引起急性腦室擴(kuò)張、腦水腫和神經(jīng)元變性損傷，米諾環(huán)素和MIF可明顯抑制氯化亞鐵腦室注射后所引起的小膠質(zhì)細(xì)胞活化，但僅有米諾環(huán)素能顯著降低大鼠死亡率，減輕腦水腫和神經(jīng)元損傷。 2.三氯化鐵腦室內(nèi)注射引起顯著的急性腦室擴(kuò)張和海馬神經(jīng)元變性，米諾環(huán)素能減輕神經(jīng)元損傷，但未減輕腦室擴(kuò)張。 3.米諾環(huán)素對(duì)二價(jià)和三價(jià)鐵離子均具有螯合效應(yīng)。 結(jié)論 鐵離子腦室內(nèi)注射可導(dǎo)致急性腦積水、腦水腫及神經(jīng)元損傷；米諾環(huán)素具有亞鐵和三價(jià)離子螯合力，并能顯著減輕鐵離子腦室內(nèi)注射所致的腦水腫和神經(jīng)元損傷，其神經(jīng)保護(hù)作用的機(jī)理可能與鐵螯合效力有關(guān)。 關(guān)鍵詞：腦室出血鐵米諾環(huán)素腦積水 三、依達(dá)拉奉對(duì)大鼠腦室出血后腦損傷的干預(yù)研究 目的 觀察自由基清除劑依達(dá)拉奉對(duì)實(shí)驗(yàn)性大鼠腦室出血后氧化應(yīng)激損傷、腦積水和神經(jīng)行為學(xué)損傷的干預(yù)效應(yīng)。 方法 研究分為2部分，第一部分為急性期指標(biāo)測(cè)定，動(dòng)物分為生理鹽水對(duì)照組(Saline)、腦室出血+依達(dá)拉奉干預(yù)組(IVH+Edv)和腦室出血+溶劑干預(yù)組(IVH+Veh)，分別給予右側(cè)腦室內(nèi)注入200μl生理鹽水或不抗凝自體全血，注血后分別給予依達(dá)拉奉(6mg/kg，IVH+Edv組)或溶劑(IVH+Veh)皮下注射，損傷后24小時(shí)取腦組織檢測(cè)腦含水量、丙二醛(MDA)水平和超氧化物歧化酶(SOD)活性。第二部分實(shí)驗(yàn)分組同前，注血組大鼠分別給予依達(dá)拉奉(6mg/kg，IVH+Edv組)或溶劑(IVH+Veh)皮下注射共3天(注血后15分鐘，1天和2天)，IVH后23天開始行為學(xué)檢測(cè)，28天行MRI掃描并留取標(biāo)本行組織學(xué)檢查。 結(jié)果： 1.大鼠IVH模型可導(dǎo)致急性腦水腫、氧化應(yīng)激及后期學(xué)習(xí)記憶功能缺陷。 2.依達(dá)拉奉減輕了IVH后組織氧化應(yīng)激、腦水腫及腦室壁室管膜纖毛上皮損傷，改善了大鼠后期腦積水和學(xué)習(xí)記憶功能缺陷。 結(jié)論 自由基清除劑依達(dá)拉奉可減輕大鼠IVH后氧化應(yīng)激和急性腦水腫，并改善后期腦室壁損傷、腦積水和學(xué)習(xí)記憶功能缺陷，氧化應(yīng)激可能是IVH后腦損傷及腦積水的重要機(jī)制和治療靶點(diǎn)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錛

本文編號(hào)：1811143