Translational Research for axona regeneration by using Stroke model of minature pig

背景和目的本研究的第一个目的是在小型猪的脑回中引入一种具有最小侵入性的可重复的中风模型。本文提供了关于小型猪灰质和白色质缺血性损伤的手术技术和量化方法的信息。第二个目的是将这种新的脑卒中模型应用于干细胞移植到缺血脑内进行神经元再生的转化研究。方法16只雄性小型猪随机分为3组，分别行额颞入路眶缘截骨术（n=5）、永久性颈内动脉闭塞（n=6）和假手术（n=5）。组织学定位和磁共振（MR）成像用于描绘缺血区域。室管膜下区（SVZ）细胞分离自迷你 ...更多信息 在碱性成纤维细胞生长因子的存在下，在Iscove改良的Dulbecco培养基中培养成熟猪8天。将铁磁颗粒标记的室管膜下区细胞立体定向移植到脑卒中小型猪体内。使用磁共振成像（MRI）非侵入性地跟踪移植的细胞。为了检测宿主脑中的铁磁标记SVZ细胞，我们使用普鲁士蓝反应对矢状或冠状切片进行铁染色。结果MCAO组、ICAO组和假手术组的梗死体积分别为16.2 ±1.1、1.5±0.5和0.0±0.0 cm^3（均数±标准差）。尽管定量组织病理学测量的梗死体积小于MRI测量的梗死体积，但组织学和MRI成像体积显示出良好的相关性（r^2=0.86，p <0.0001）。使用淀粉样前体蛋白抗体（APP）的免疫组织化学染色使我们能够评估轴突损伤，MCAO中的APP总评分为43.8±3.5（平均值±SD）。移植后1周和2周MRI信号检测到移植细胞在病灶处有标记。普鲁士蓝阳性细胞也被检测到在同一病变的细胞被移植。移植的细胞可能只是在宿主大脑中归巢。我们也没有证实移植细胞和宿主脑之间的网络结构。结论本实验建立了一种简单、重复性好、创伤小的局灶性脑缺血白色和灰质损伤诱导方法。这种小型猪中风模型可用于研究脑回缺血的病理生理学，并在开始人体临床试验之前评估药物或干细胞移植的治疗效果。少

Background and Purpose The first purpose of the present set of studies was to introduce a reproducible stroke model in the gyrencephalic brain of miniature pig with minimum invasiveness. The paper provides information on both the surgical technique and the methods of quantification of ischemic damage to both grey and white matter in the miniature pig. The second purpose was to apply this new stroke model for the translational research for the neuronal regeneration by using thestem cell trasplantation into the ischemic brain. Methods Sixteen male miniature pigs were randomly divided into three groups and underwent transcranial surgery, a frontotemporal approach with orbital rim osteotomy, for : permanent middle cerebral artery occlusion (MCAO) (n=5), permanent internal carotid artery occlusion (ICAO) (n=6) and sham operation (n=5). Histological mapping and magnetic resonance (MR) imaging were used to delineate the areas of ischemia. Subventricular zone (SVZ) cells isolated from the mini … More ature pig were cultured in Iscove's modified Dulbecco's medium in the presence of basic fibroblast growth factor for 8 days. We stereotaxically transplanted subventricular zone (SVZ) cells labeled by ferromagnetic particles into stroked miniature pig. The transplanted cells were non-invasively tracked using magnetic resonance imaging (MRI). To detect ferromagnetic-labeled SVZ cells in the host brain, we stained sagittal or coronal sections for iron using Prussian blue reaction. Results The volumes of the infarction measured directly from MR images were 16.2 ±1.1, 1.5±0.5 and 0.0±0.0 cm^3 (mean±SD) in the MCAO, ICAO and sham-operation groups, respectively. The histological and MR imaging volumes showed a good correlation (r^2=0.86, p <0.0001) although the infarct volume measured by quantitative histopathology was smaller than that measured by MRI. Immunohistochemical staining using amyloid precursor protein antibody (APP) allowed us to evaluate the axonal damage, providing total APP score of 43.8±3.5 (mean±SD) in the MCAO. MRI signals detected the tranplanted cells with labeling at the lesion, and these signals were detected 1 and 2 weeks after transplantation. Prussian blue-positive cells were also detected in the same lesion where the cells were transplanted. The transplanted cells were likely to be just homing in the host brain. We have neither confirmed the construction of the network between the transplanted cells and host brain. Conclusions This paper describes a simple and reproducible method for the induction of white and grey matter damage in focal cerebral ischemia with minimum invasiveness. This miniature pig stroke model has utility for studying the pathophysiology of ischemia in gyrencephalic brain and for the assessing the therapeutic efficacy of drugs or stem cell transplants, prior to initiating human clinical trials. Less