论文标题:人骨髓间充质干细胞移植治疗脑出血的实验研究及脑室出血微创治疗的临床研究
Application of Intravenous Administration of Human Bone Marrow Stromal Cells in the Treatment of Intracerebral Hemorrhage in Rats & the Clinical Study in Minimally Invasive Treatment of Intraventricular Hemorrhage
论文作者
论文导师 李新钢,论文学位 博士,论文专业 外科学
论文单位 山东大学,点击次数 211,论文页数 163页File Size9873K
2007-04-16论文网 http://www.lw23.com/lunwen_125773112/
mesenchymal stem cells; separation; cell culture;mesenchymal stem cell; intracerebral hemorrhage; neural regeneration; rat;Neuroendoscope; Intraventricular hemorrhage; Minimally invasive surgery; External ventricular drainage
目的骨髓间充质干细胞是存在于骨髓中的一种具有跨胚层分化潜能的干细胞。近年来研究发现其可在中枢神经系统内存活并能分化为神经样细胞，在体外也可通过诱导向神经细胞转化，并对脑缺血、脑外伤等疾病的神经功能的缺损有改善作用。其取材方便、体外扩增迅速且能以多种途径移植包括静脉移植、脑内移植。故骨髓间充质干细胞在中枢神经系统损伤修复中的临床应用前景广阔。本研究目的就是探讨人骨髓间充质干细胞的分离和体外培养扩增的方法，通过观察细胞形态、贴壁生长特性、细胞表型等对骨髓间充质干细胞进行鉴定，研究骨髓间充质干细胞的生物学特性。 方法采集健康成人骨髓血，通过密度梯度离心结合贴壁培养得到较纯化的MSCs，使用含有10％胎牛血清的低糖DMEM继续培养扩增MSCs。通过观察细胞形态、贴壁生长特性，使用流式细胞仪检测细胞CD29、CD34、CD45的表达，对培养的MSCs进行鉴定。观察MSCs的细胞形态、生长速度、绘制MSCs的生长曲线。冻存复苏MSCs后观察其存活率和生长状态等。 结果应用国产淋巴细胞分离液及胎牛血清，通过密度梯度离心结合贴壁培养法可以获得较纯化的MSCs；经原代和传代培养得到扩增，早期生物性状稳定。通过本方法培养的MSCs细胞呈现典型的梭形形状，旋涡状排列、贴壁生长；流式细胞仪检测细胞CD29表达阳性，CD34、CD45表达阴性，符合MSCs的特征。观察细胞生长特性发现，MSCs呈贴壁生长，原代培养9-12d可以达到90％融合，传代后的MSCs增殖速度较原代培养明显增快。第3代的MSCs已基本纯化，细胞呈典型的梭形形状，旋涡状排列。随着传代的增加，细胞增殖分化能力减弱。冻存1个月和6个月后复苏的MSCs的生存率分别为90％和85％，复苏后的细胞生长状态良好。 结论应用国产淋巴细胞分离液及胎牛血清，通过密度梯度离心结合贴壁培养法可以获得较纯化的MSCs；经原代和传代培养得到扩增，早期生物性状稳定。冻存对MSCs生长特性无明显影响。 目的 自发性脑出血约占所有脑卒中的10％-20％，但目前尚未找到有效和标准化的临床治疗方法。按照其自然的病理生理学特征，自发性脑出血可产生多种神经损伤机制，包括直接机械性损伤、局部缺血、毒素、凋亡。目前还没有一种内科治疗方法能对脑出血的功能恢复产生明确的效果。近年来，人们把注意力集中在未分化多潜能的干细胞具有能改善包括脑外伤、缺血性脑卒中、脊髓损伤的实验动物神经功能的能力上。特别是，人胚胎神经干细胞被应用于胶原酶脑出血模型中，能改善神经功能状态，证实了细胞可以迁移到脑出血区。 骨髓中含有能充当组织干细胞的细胞亚群，它们是非造血组织的祖细胞。这些骨髓来源的多潜能细胞被称为骨髓间充质干细胞或骨髓基质细胞(MSCs)。他们在各种非血液组织中有自我更新和分化的能力，具有被用做细胞治疗的潜能。在合适的微环境中，MSCs能产生间充质组织，如纤维组织、骨、软骨、肌肉，又能特异性地分化成脂肪细胞、成骨细胞、软骨细胞。有重要意义的是，在实验条件下，MSCs能通过血脑屏障到达脑损伤靶区而治疗神经疾病。在新生的小鼠中，MSCs能广泛地迁移到未发育成熟的脑组织中，并显示出具有分化成神经元和星形胶质细胞的能力。被系统地注射到大鼠体内的骨髓来源的细胞能优先地迁移到局部缺血的脑皮质。在最近的研究中证明，hBMSCs对缺血性脑卒中和闭合性颅脑损伤动物的神经功能恢复有明显的促进作用。在这些神经损伤动物模型中，MSCs似乎能诱导产生内源性的脑源性细胞(可能是来源于脑室下区的细胞)，以参与神经功能恢复过程。普遍认为，血管内注射MSCs能治疗神经损伤，干细胞技术能用于治疗脑出血，本研究的目的就是探索hBMSCs能否改善脑出血动物的神经功能和减小神经细胞的损伤。 方法 1．动物准备实验动物选用成年雄性Wistar大鼠80只，体重270-320g。10％水合氯醛腹腔麻醉(3ml／kg)，俯卧于动物头颅立体定向仪上，取前囟为原点，向前0.5mm，向右3.5mm，深度5.5mm为注射点(尾状核)，骨钻钻开颅骨(直径1mm)，经立体定向导向针注射Ⅳ型胶原酶／肝素／生理盐水溶液2.0ul(每1.0ul含Ⅳ型胶原酶0.2U及肝素2U)。 2．实验分组将制备好的脑出血模型大鼠随机分为四组，每组20只。 组1：对照组，脑出血后第一天尾静脉注射PBS 1ml。 组2：脑出血后第一天尾静脉注射PBS细胞溶液1ml(含1×10~6hBMSCs)。 组3：脑出血后第一天尾静脉注射PBS细胞溶液1ml(含3×10~6hBMSCs)。 组4：脑出血后第一天尾静脉注射PBS细胞溶液1ml(含6×10~6hBMSCs)。 所有大鼠在脑出血后第一天开始，腹腔内注射BrdU(100mg／kg／d)，持续13天，以检测体内新合成的DNA。 3．神经学功能状态评估Wistar大鼠神经状态的评估是依据神经学缺陷程度分数表，在大鼠脑出血前及后1天、7天、14天，根据大鼠的运动、感觉、平衡、及反射等方面进行评估。分值为1-18；得分越高表示神经学缺陷程度越高。 所有动物脑出血后14天处死。断头取脑，置于10％福尔马林内浸泡固定。在脑出血中心部位，相当于以前囟为中心，冠状面+0.1mm～-0.86 mm，连续做6 um厚冠状切片，随机取6张用于HE及免疫组化染色。 应用影像分析系统计算尾状核组织损失的百分率。尾状核组织损失的百分率=(对侧正常尾状核区面积—出血侧残留的尾状核面积)／对侧正常尾状核区面积。 应用TUJ1、BrdU、mAb1281、突触素对所有动物进行免疫组化分析。TUJ1是发育中的神经元的标志物。BrdU是新合成DNA的标志物，被普遍认为是细胞分裂和新细胞生长的表达。突触素是突触前再生及突触发生的标志物。mAb1281(鼠抗人细胞核单克隆抗体)只对所有人类细胞有特异性反应，常用于鉴定hBMSCs。阴性对照的免疫组化染色省略了一抗。 4．TUJ1、BrdU、mAb1281、突触素的定量分析 在脑出血中心部位，相当于以前囟为中心，冠状面+0.1mm～-0.86mm，连续做6 um厚冠状切片，随机取6张用于TUJ1、BrdU、mAb1281、突触素的半定量分析。突触素测量尾状核区，TUJ1测量脑室下区，应用MCID影像分析系统测量TUJ1和突触素阳性区域面积的百分比。 TUJ1和突触素阳性区域面积的百分比=每个视野阳性区域面积／每个视野总面积(628×480um~2)。 对每张切片脑出血灶周围区域BrdU阳性细胞进行计数。 对每张切片的对侧与同侧区域的mAb1281免疫反应阳性细胞进行计数。 5．统计学分析 应用t检验对神经学功能计分、脑出血组织损伤面积、组织化学检测结果进行统计学分析。 结果 1．神经学功能状态评估 所有动物在14天内全部存活。脑出血后1天，根据神经学缺陷程度分数表进行评分，四个实验组间神经功能评估差异无统计学意义。脑出血后7天和14天，与对照组比较，注射细胞的三个实验组的神经功能明显改善，神经功能评估差异有统计学意义。 2．尾状核区组织损失评估应用显微影像分析系统计算尾状核组织损失的百分率。脑出血后14天，处死所有动物，断头取脑，HE染色显示14天后注射细胞的三个实验组的尾状体组织损失较小，其结果(用均数±标准误表示)如下： 组1(对照组)：30±1.1％； 组2(1×10~6 hMBSCs组)：23±2.7％(p=0.002)； 组3(3×10~6 hMBSCs组)：23±2.5％(p=0.003)； 组4(6×10~6 hMBSCs组)：23±3.9％(p=0.002)。 从以上结果可以看出，脑出血后14天注射细胞的三个治疗组的尾状体组织损失面积无明显差异；而与对照组比较则有明显改善。 3．TUJ1、BrdU、mAb1281、突触素的免疫组化分析结果 其结果说明，与对照组比较，注射细胞的三个治疗组的脑出血区的阳性染色细胞数明显增加，尤其是TUJ1和突触素的阳性染色细胞面积明显增加，意味着与对照组比较，注射细胞治疗组的脑出血区突触再生和发育中的神经元明显增多。mAb1281阳性染色出现在注射细胞的治疗组的脑出血区，证实了与对侧大脑半球比较，注射的hBMSCs优先地到达了脑损伤区。BrdU阳性染色细胞在脑出血周围区明显增多，意味着与对照组比较，注射细胞的治疗组的脑出血区有新生的细胞形成。阴性对照的免疫组化染色，由于省略了一抗而没有阳性染色细胞。 结论 1．在实验性脑出血后1天，即经尾静脉注射1×10~6、3×10~6、6×10~6的人骨髓间充质干细胞。hBMSCs能明显改善脑出血大鼠的神经功能，尾状核区脑组织损失也明显减小。 2．注射细胞治疗后14天，发现注射的hBMSCs聚集在脑出血周围区；在尾状核区和脑出血区附近的脑室下区，发育中的神经元、突触发生和新细胞的形成明显增多和增强。 3．hBMSCs能明显改善脑出血大鼠的神经功能，这与其尾状体神经组织损失较小、脑出血区局部人骨髓间充质干细胞和不成熟神经元增多、神经突触再生明显、细胞有丝分裂活动增强有关。 目的 自发性脑室出血是一种发作不频繁但有严重并发症的出血性卒中，据报道死亡率高达42.6％-83.3％，而手术死亡率达35.7％-100％。当脑室出血量太大阻碍了正常脑脊液循环时，在脑室出血亚急性期和慢性期可发生急性梗阻性脑积水；当基底池软脑膜发生纤维化或者脑脊液重吸收因蛛网膜绒毛纤维化而受损害时，可发生交通性脑积水。尽管已经有内科或外科方法治疗脑室出血，但均不尽人意。常用的方法有：①侧脑室引流术(External ventricular drainage，EVD)结合尿激酶治疗；②骨窗或骨瓣开颅血肿清除术；③立体定向脑内血肿穿刺吸除和引流术。脑室体外引流术结合尿激酶治疗，手术操作简单，脑皮质损伤小，不加重深部核团损伤，术后病人恢复快，尤其适合于年龄较大，多脏器功能不全者。它也能用于治疗孕妇患有的脑室出血。但存在着置管准确性差，引流时间长，引流管易阻塞，减压效果不满意，可继发感染和再出血等。骨瓣开颅能清除深部血肿及脑室内积血，减压彻底。但该手术方法皮质牵拉较重，深部止血困难，易加重深部核团损伤。小骨窗开颅，简化了手术操作，可在直视下吸除、钳取血肿，如不强行吸除血肿腔周壁及底部血块，一般不会引起活动性出血。但因难以精确确定和限制手术范围，不利于把手术损伤减少到最小。立体定向脑内血肿穿刺吸除和引流术强调皮质及深部核团的保护，但由于非直视下操作，再出血率约4％～10％，且吸除凝固血块较困难，减压效果有时不满意；不能完全避免抽吸时负压对脑组织造成的损伤。本研究的目的就是探讨微创治疗脑室出血的方法，即应用神经内镜治疗脑室出血。 方法 1一般资料在2002年12月～2004年2月期间，我们对入院的42例脑室出血的病人进行手术治疗。术前全部病例均做头颅CT扫描。脑内血肿体积按公式计算血肿量=1／2×长轴×短轴×层面。当怀疑患有颅内动脉瘤和动静脉畸形时，应做脑血管造影检查。 本研究选择病人的标准是：伴有或不伴有脑出血的脑室出血被临床和CT确诊的病人，脑实质内血肿量均＜30ml，发病时间＜48小时。另外，继发于颅内动脉瘤、动静脉畸形、小脑和脑干出血的脑室出血排除在该研究之外。 该研究是前瞻性和随机的。所有病人被随机分为2组，分别采用脑室外引流术(脑室引流组)和神经内镜下血肿清除引流术(内镜组)治疗。 2手术治疗 内镜组：患者在发病48小时内手术，采用全麻，取仰卧位。 对于原发性或继发性脑室出血(脑实质出血灶出血量＜20ml)的病人，以额入法或枕入法穿刺进入侧脑室。头皮切开3～4cm，形成直径约3cm的骨窗，十字型剪开硬膜，向单或双侧脑室置入可变换角度、外径6mm的硬质神经内镜。 如果脑室内血肿较小，脑室内剩余空间较大，血肿有部分液化者，采用EN(内窥镜神经外科)技术。单纯应用神经内镜完成手术操作。神经内镜进入脑室腔后，吸除血肿与生理盐水冲洗轮流进行，见到呈白色脑室壁时吸除血肿立即停止。 如果脑室内血肿较大，脑室内剩余空间较小，血肿与脑室壁有粘连者，采用ECM(内窥镜控制的显微神经外科)技术。显微神经外科技术与内窥镜结合应用，在神经内镜协助下完成手术。通过上述途经置入神经内镜后，使用观察镜提供良好的照明及清晰和放大的视野，从观察镜旁伸入吸引器清除血肿。如有出血，可用双极电凝止血。 一旦发现脉络丛和室间孔，神经内镜就进入三脑室清除血肿。 最后神经内镜弯向脑室的额角或枕角和三角区清除血块。将内镜留置血肿腔内5～10分钟，确定无出血后，撤去内镜。 手术结束时，所有病人放置脑室引流管做单侧或双侧脑室造瘘术以监测颅内压和引流血肿(持续引流梯度为15mmHg)。 术后24小时和1周分别复查颅脑CT。 对于脑室内无明显血肿残留的病人，脑室外引流放置2-3天。脑压稳定后即可拔管。对残存血肿量大于10ml者，行血肿腔内注射尿激酶(UK)。由天津生化药物公司生产的尿激酶是一种无菌的冻干制剂，呈粉末状，每支含250，000IU，用无菌生理盐水配制成25，000IU／ml溶液。每次给病人注射25，000IU-30，000IU。注射后夹管1小时以防UK流失并保证有充足时间与血肿相互作用。夹管1小时后，脑室引流管以适当引流梯度重新开放。每日3次，持续1周，直至病人能够耐受夹管24小时(即持续颅内压升高不超过15mmHg)才拔除脑室引流管。 对于继发性脑室出血(脑实质内出血量＞20ml)的病人，根据CT图像，选择血肿在脑表面的投影作为靶点，据此选择手术入路和穿刺点。按上述步骤清除脑内血肿和循破入脑室通道清除脑室内血肿。术后处理同前。 脑室引流组；采用传统的脑室外流术结合尿激酶(UK)溶解血肿的方法。患者均在发病48小时内手术。采用局麻，取仰卧位。自额部常规穿刺进入侧脑室。术后处理同前。 3疗效评价、随访及统计学处理 治疗2个月后，参照1975年Jennett和Bond提出的格拉斯哥预后分级(Glasgow outcome scale，GOS)评分为：5分：优，恢复工作或学习；4分：良，有轻度神经系统症状，可自理生活或轻工作；3分：中，有严重神经系统症状，生活需他人照顾；2分：差，植物生存状态；1分：死亡。 临床随访为2个月。 统计学处理：用x~2检验和四格表精确检验法。 结果 内镜组：术后24小时复查头颅CT，脑内或／和脑室内血肿绝大部分清除者(90％以上)者15例，90％以下者7例；未发现继发出血；术后无颅内感染。 脑室引流组：术后24小时复查颅脑CT，仅有3例脑室内血肿大部分清除(60％以下)，脑室内血肿几乎没有清除者有17例；未发现继发出血；术后颅内感染2例。 术后随访2个月对所有患者进行GOS评分，随访结果被列在表1。 神经内镜组死亡2例(分别死于肺部感染和急性心肌梗塞)，脑室引流组死亡2例(1例死于肺炎合并应激性溃疡，1例死于肺炎)。 脑室引流组与内镜组比较，优良率低(x~2值=4.752，P＜0.05)，差异有统计学意义；死亡率差异无统计学意义(x~2值=0.010，P＞0.05) 结论 应用神经内镜清除脑室内血肿，具有直视下操作、术后疗效好等优点，是脑室出血较佳的外科治疗方法。
Background Mesenchymal stem cells (MSCs) are stem cells that lying in marrow .The research showed that the cells could survive and were induced to divide so as to be confirmed as neural stem cells (NSCs) in vivo and ex vivo as so. It could be used to replace the neural cells or repair the nervous system, such as ischemic stroke, brain injury and so on. MSCs was easily grained and cultured, and there were various method of such as vein transplantation, directed transplantation. It provides a hopeful method for central nervous system diseases. The objective of this study is to probe the separation in vitro culture and expanding method of mesenchymal stem cells (MSCs), and investigate the biological characteristics of MSCs by observing the morphous and growth velocity of MSCs, and identification of the phenotype of MSCs. Method MSCs were isolated and purified from human being using density centrifugation and anchoring culture, then cultured in low-glucose DMEM supplement with 10% fetal bovine serum (FBS) for amplification. MSCs were identified by observing the morphous and growth velocity of MSCs, identification of the phenotype of MSCs. Draw the growth curve of MSCs and determine the mitotic index of MSCs. Observe the survival rate and growth state of MSCs underwent freeze thawing.Result MSCs with stable biological properties can be efficiently isolated and purified by density centrifugation and adhering to the culture plastic flask and expanded satisfactory in L-DMEM medium with 15% fatal calf serum. MSCs got together at 9th-12th day .The passage MSCs proliferated fast. MSCs were purified at 3rd generation. MSCs grew in whirlpool. Through detection by flow cytometry, the expressions of CD29 were positive; the exprssions of CD34 and CD45 were negative. As the passage increases, the cloning efficiency and proliferative ability of MSCs decrease. The survival rates of MSCs thawed after being frozen 1 month and 6 months were 90% and 85% respectively.Conclusion hBMSCs with stable biological properties can be efficiently isolated and purified by density centrifugation and adhering to the culture plastic flask and expanded satisfactory in L-DMEM medium with 15% fatal calf serum. Freeze thawing has no obvious influence on MSCs. BackgroundSpontaneous ICH causes 10 to 20% of all strokes, but effective and standardized clinical treatment remains elusive. By the very nature of its pathophysiological features, spontaneous ICH results in a variety of neural injury mechanisms: direct mechanical injury, ischemia, toxicity, and apoptosis. No currently available medical therapy has shown a consistent or unambiguous benefit in terms of functional outcome. In recent years attention has been focused on the ability of undifferentiated pluripotent stem cells to improve experimental neurological conditions, including ischemic stroke, brain trauma, and spinal cord injury. Specifically, human embryonic neural stem cells have been used in a collagenase model of ICH to restore neurological function and demonstrate migration of the cells to the site of hemorrhage.Bone marrow contains a subpopulation of cells that can serve as tissue stem cells because they can be used as precursors of nonhematopoietic tissue. These pluripotent cells of bone marrow origin are referred to as MSCs. The MSCs have a capacity for self-renewal and differentiation in a variety of nonhematological tissues, and have the potential to be used for cell therapy. In the appropriate cellular microenvironments, MSCs are able to produce mesenchymal tissues, such as fibrous tissue, bone, cartilage, and muscle, and can differentiate specifically into adipocytes, osteoblasts, and chondrocytes. Of significance for the treatment of neurological disorders, MSCs pass through the blood-brain barrier to target sites of brain lesions under experimental conditions. In the neonatal mouse, MSCs migrate widely throughout the developing brain and have shown the capacity to differentiate into neurons and astrocytes. Cells of bone marrow origin infused systemically into rats preferentially migrate to ischemic cortex. In recent studies, hBMSCs have shown significant benefit in animal models of ischemic stroke and closed head injury. In these models of neural damage, the MSCs appear to have the capacity to induce endogenous brain-derived cells, likely derived from the SVZ, to participate in the restorative process. In light of the recognized ability of intravascularly delivered MSCs to treat neural injury and the potential application of stem cell technology to treat ICH, this experiment was conducted to test the hypothesis that hBMSCs improve functional outcome and reduce cellular injury after experimental ICH. Methods Animal PreparationEighty adult male Wistar rats weighing between 270 and 320g were used for this study. Stereotactic stabilization and localization were used after general anesthesia was induced with 3 ml/kg chloral hydrate. A 1-mm craniectomy was performed and the stereotactically guided needle was placed at coordinates 0.5 mm anterior, 3.5mm lateral, and 5.5 mm deep relative to the bregma. The ICH was induced by injecting 2 ul saline solution (consisting of IV type collagenase 0.4U and heparin 4U) into the right striatum, with a steady infusion rate of 0.5 ul/minute. Experimental GroupsThe rats were randomly divided into four experimental groups. Group 1, which consisted of 20 animals, was given 1 × 10~6 hBMSCs in 1 ml PBS carrier solution, slowly injected into the rats" tail veins; Group 2, which consisted of 20 animals, was given 3 ×10~6 hBMSCs in PBS solution; Group 3, which consisted of 20 animals, was given 6×10~6 hBMSCs in PBS solution; and Group 4, control group, which was given the 1 ml PBS vehicle solution as a placebo. For mitotic labeling of newly formed DNA, all rats also received daily intraperitoneal injections of 100 mg/kg BrdU starting 24 hours after ICH and continuing for the next 13 days. Assessment of Neurological FunctionNeurological function was evaluated using the NSS, which were performed before and at 1, 7, and 14 days after ICH. The NSS is a composite score in which motor, sensory, balance, and reflex measures are used to calculate a value ranging from 1 to 18, with the higher score implying greater neurological injury. All animals were killed after 14 days, and their brains were fixed in formalin and sliced into 2-mm-thick sections. Every 40th coronal section (cut at a thickness of 6 am between the bregma +0.1 mm to -0.86 mm in each rat, for a total of six sections) was used for H & E and immunochemical staining.The percentage of striatal tissue loss in one section was calculated using an image analysis system.The area of preserved striatum on the side of the hemorrhage was subtracted from the area of the contralateral striatum, thus reckoning the degree of encephalomalacia or tissue loss from the injury in that brain section. A percentage value was then calculated by dividing the amount of cell loss by the total area of the contralateral striatum.Well-established immunohistochemical analytical methods were used, which consisted of staining both control and treatment groups with synaptophysin, TUJ1, mAb 1281, and BrdU. Synaptophysin is a marker of presynaptic plasticity and synaptogenesis; TUJ1 is a developmental neuronal marker, whereas mAb 1281 is specific for all human cell types and is used to identify hBMSCs. On the other hand, BrdU (100 mg/kg) is a marker for newly formed DNA and is generally accepted as an expression of cell division and new cell growth. Control experiments consisted of staining coronal brain tissue sections as outlined earlier, but omitted the use of primary antibodies. Quantification of TUJ1, BrdU, mAb 1281and SynaptophysinFor semiquantitative measurements of TUJ1, and Synaptophysin, six slides from the block (bregma +0.1 mm to — 0.86 mm) were used. Synaptophysin was measured in the striatum, and TUJ1 were measured at the SVZ. Synaptophysin,and TUJ1 were digitized under a 20 xobjective lens by using a 3-CCD color video camera interfaced with an MCID image analysis system. Data are presented as a percentage of area, in which the TUJ1- and synaptophysin-immunopositive areas in each field were divided by the total areas in the field (628 ×480 um~2). The BrdU-positive cell number was measured in the boundary around the lesion. Quantitative data for mAb 1281 are presented as the total number of mAb 1281-immunoreactive cells within contralateral and ipsilateral areas of each slide. Statistical AnalysisStatistical evaluations of functional scores, area of ICH-related tissue damage, and histochemical results were performed using the independent Student t-test. ResultsAll 80 animals survived the 14-day experimental period. There was no apparent difference between the control group and any of the experimental groups in the results of NSS and corner turn tests 1 day after ICH. Nevertheless, after 7 days both tests showed significant improvement in results for the rats injected with hBMSCs compared with controls.The area of tissue loss as a percentage of the normal hemisphere was as follows (given as the mean±standard error of the mean for all values): control, 30±1.1%; 1 million hBMSCs,23±2.7% (p = 0.002); 3 million hBMSCs, 23 ±2.5% (p = 0.003); and 6 million hBMSCs, 23± 3.9% (p = 0.002).As can be seen, there is virtually no difference between any of the treatment groups, with all of them showing significant improvement over the control group 2 weeks after ICH.The mAb 1281, BrdU, Synaptophysin and TUJ1 histochemical staining data suggest that there was a significant increase in the positive-staining cells in the region of ICH for all treatment groups compared with the controls. Specifically, for TUJ1, and synaptophysin labeling, the area of positive-staining cells was significantly increased in treated animals compared with controls. Labeling of mAb 1281 was seen in the treated animals in the region of the ICH, verifying that the injected hBMSCs did reach the site of injured brain preferentially compared with the contralateral hemisphere. Staining for BrdU was significantly increased in the boundary zone around the ICH, implying localized new cell formation in the rats treated with hBMSCs compared with control animals. Control immunostaining, which omitted the primary antibodies, did not show positive-staining cells. ConclusionsIntravenous injection of hBMSCs at doses of 1, 3 and 6 million cells 1 day after experimental ICH improves neurological function and is associated with a significant reduction in local tissue loss.By 14 days posttreatment, the injected human cells are found in high concentrations at the site of the hemorrhage. There were significant increases in immature neurons, neuronal migration, synaptogenesis, and new cell formation in the striatum and SVZ near the site of the ICH in the animals receiving the hBMSCs.This improvement in the treated animals is associated with reduced tissue loss and increased local presence of the hBMSCs, mitotic activity, immature neurons, synaptogenesis, and neuronal migration. BackgroundSpontaneous IVH is an infrequent but severe complication of hemorrhage stroke. Morality rate has been reported to be as high as 42.6 to 83.3%, morality rate of surgery 35.7 to 100%. When IVH is large enough to impede normal CSF circulation, acute obstructive hydrocephalus can occur in the subacute and chronic stages of IVH, communicating hydrocephalus may develop if fibrosis of the basal leptomeninges occurs or reabsorption of CSF becomes impaired from fibrosis of the arachnoid villus. Although there have been some medical or surgical therapies for IVH, none of them are encouraging. Among the frequently used are: ①external ventricular drainage (EVD) combined with urokinase; ② hematoma evacuation of craniotomy with bone flap or bony opening;③evacuation and drainage of intraventricular hematoma with stereotactic operation. EVD combined with urokinase offers a simple operation technique with less injury on cerebral cortex, and no further injury on the deep nucleus mass. Patients thus have a quick recovery after operation. It is especially fit for patients older in age or with multiple organ dysfunctions. It also can be applied to treatment for intraventricular hemorrhage in pregnancy. However, secondary infection and rebleeding etc may happen due to less accuracy in setting catheters, longer drainage time, catheter easier to get blocked and poorer effect of pressure reduction. Craniotomy with bone flap can evacuate hematoma deep in the brain and intraventricular hematoma, with a complete reduction of pressure. On the other hand, however, this operation technique may cause too much traction to cortex, and is likely to aggravate injury to deep nucleus mass. Craniotomy with small bony opening simplifies the operation, and can evacuate and remove hematoma with pincers under direct vision. No active hemorrhage may occur if there is no forced evacuation of blood-clots on the perisporium and bottom of hematoma cavity. However, it does not help minimize operative injury since it is difficult to accurately locate the hematoma. Evacuation and drainage of intraventricular hematoma with stereotactic operation place great emphasis on protection for the cortex and deep nucleus mass. Pressure reduction does not have a good effect sometimes since the operation is not conducted under direct vision and rebleeding rate might be 4 to 10% with more difficulty in evacuating hard blood clots; Injury of negative pressure to brain tissue cannot be avoided completely when aspiration is conducted. The purpose of this study was to probe a method of minimally invasive treatment for intraventricular hemorrhage, i.e. a neuroendoscopic approach to IVH. Methods1 Patients Forty-two patients with IVH were treated surgically in our department between December 2002 and February 2004. A CT scan was performed on all patients preoperatively. ICH hematoma volume was measured on the head CT scan with the use of the ABC/2 method, in which A is the greatest diameter on the largest hemorrhage slice, B is the diameter perpendicular to A, and C is the approximate number of axial slices with hemorrhage multiplied by the slice thickness. Angiography was obtained when intracerebral aneurysms or arteriovenous malformations (AVM) were suspected.All patients were screened and enrolled if an IVH with or without ICH had occurred within 48 hours before admission and was diagnosed by clinical and brain CT criteria, and ICH volume is <30ml. In addition, when intracerebral aneurysms or arteriovenous malformations were suspected, they had to be excluded by appropriate diagnostic studies. The patients presenting with IVH resulting from cerebellar and brainstem hemorrhage were excluded from this study. This study is prospective and randomized. All patients were divided into external ventricular drainage (EVD) group and neuroendoscope (NE) group randomly, underwent external ventricular drainage and neuroendoscopic operation respectively.2 Operation In NE group, the neuroendoscopic operation was performed with patients in supine or latericumbent position within 48 hours from onset under general anesthesia.For patients presenting with spontaneous primary or secondary IVH (ICH volume <20ml), operative access was precoronal or postcoronal by a 30 mm burr hole. The incision was 3-4 cm long. The dura was opened in a cross. A variable-angled (0° or30°) rigid neuroendoscope with an outer diameter of 6 mm was introduced into monolateral or bilateral ventricle.If there is less intraventricular hematoma, with bigger clearance leftover inside ventricle, and with hematoma partially liquefied, the neuroendoscope was used alone and the procedure performed through the endoscopic channels (EN, endoscopic neurosurgery). Aspiration was alternated with irrigation with normal saline and was promptly stopped when the whitish color of the ventricular walls appeared.If there is large amount of intraventricular hematoma, with smaller clearance leftover inside ventricle and with hematoma adhered to the wall of ventricle, microneurosurgical techniques and the endoscope were used in combination, with part of the operation being carried out with the assistance of the endoscope (NEAMN, neuroendoscopic assisted microneurosurgery). Suction tube is inserted close by the endoscope to evacuate hematoma by means of better lighting ,clear and enlarged visual field provided by the endoscope. In case of bleeding, bipole coagulator can be used for hemostasis.Once the chorodial plexus and Monro foramen were identified, the instrument was advanced into the third ventricle to remove hematoma.Finally, the endoscope was flexed toward the frontal or occipital horn and the trigonus to evacuate these sections from the clots. Leave the endoscope inside ventricle for 5-10minutes and withdraw the endoscope after making sure there is no bleeding. At the end of the procedure, monolateral or bilateral ventriculostomy was performed in all patients by placing an intraventricular catheter (IVC) for both ICP monitoring and drainage (with a constant gradient of 15 mm Hg).Within 24 hours and 1 week after surgery, a CT scan was obtained respectively.For patients without any significant remaining intraventricular hematoma, the external ventricular diversion was kept open for 2 to 3 days. On pressure stabilization, it was removed. If findings show that the remaining IVH volume is>10ml, the patient should receive injection of urokinase (UK) into the ventricle. The UK preparation made in Tianjin Biochemical pharmaceutical factory was a sterile, lyophilized preparation intended for intraventricular injection .A vial of UK contains 250,000IUas powder, which is reconstituted with sterile normal saline to yield a solution that contains 25,000IU of UK per milliliter. Patients received a dose that ranged from 25,000IU to 30,000IU of UK. After injection, the IVC was closed for 1 hour to prevent drainage of UK away from the clot and to allow adequate time for drug-clot interaction. After 1 hour of closure, the IVC was reopened with an appropriate drainage gradient. The UK was administered every 8 hours, for about one week until the IVC was removed based on the patient tolerance to IVC closure for 24 hours (i.e., no sustained ICP elevation > 15 mm Hg).For patients presenting with spontaneous secondary intraventricular hemorrhage (ICH volume >20ml), the operative approach was selected based on the projection of intracerebral hematoma on skull surface by means of CT scan image. Remove intracerebral hematoma and by its breaking into ventricular route remove intraventricular hematoma according to the method described above. Postoperative treatment was the same as that described above.In EVD group, traditional external ventricle drainage with dissolving hematoma with urokinase was performed with patients in supine position within 48 hours from onset under local anesthesia. Conventional puncture into lateral ventricle was adopted from forehead. Postoperative treatment was the same as that described above. 3 Therapeutic Evaluation, Follow-Up and Statistical analysisAfter 2 months of treatment, grading was concluded based on Glasgow outcome scale (GOS) put forward by Jennett and Bond in 1975.Excellent (5 points): fully independent with no residual disability. Good (4 points): independent with moderate disability. Fare (3 points): significant disabilities requiring assistance in most daily life activities. Poor (2 points): persistent vegetative state. Dead (1 points): death.Clinical follow-up was at 2 months (GOS).Chi-square test and Fisher"s exact test of probabilities were applied to evaluate the GOS score. ResultsIn Neuroendoscope group, CT scan 24 hours after surgery showed that, almost compete removal (> 90%) of intracerebral or /and intraventricular hematoma was achieved in 15 cases, < 90% removal in 7 cases. There was not intracranial infection and rebleeding after surgery in all cases.In EVD group, CT scan 24 hours after surgery showed that, partial removal (< 60%) of intraventricular hematoma was achieved in 3 cases, almost no removal in 17 cases. There was no rebleeding in all cases. Intracranial infection after surgery was observed in 2 cases.All patients were followed up for two months and evaluated at two months from surgery according to GOS. The follow-up results were listed in Table 1.The result was dead in 2 cases of neuroendoscope group (died of pneumonia and acute myocardial infarction respectively), and in 2 cases of EVD group (died of pneumonia and pneumonia with stress ulcer respectively). Patients in EVD group, compared with neuroendoscope group, showed poor recovery after two months of surgery. The excellence and goodness rate (ratio between number of excellent and good patients and number of all patients in this group) in EVD group is < in Neuroendoscope group, and the difference between two groups is statistically significant (x~2 =4.752, P<0.05).The difference in mortality rate between two groups was not statistically significant (x~2 =0.010, P>0.05).Conclusions Neuroendoscopic neurosurgery for intraventricular hemorrhage offers better surgical treatment because it is characterized by visualized manipulation, effective hemorrhage evacuation and excellent postoperative outcomes.
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