Tracking labeled stem cells in TBI model by cellular MRI

实验性脑损伤的病理表现包括急性炎症反应、血脑屏障破坏、出血、脱髓鞘、轴索横断、慢性轴索神经元丢失和胶质细胞增生。干细胞治疗是一种潜在的治疗方法，既可以作为替代疗法，也可以通过旁分泌作用释放生长因子和抗炎细胞因子来治疗颅脑损伤。在啮齿动物模型上进行的脑创伤的实验研究一直很有限，通常在实验性损伤后24至72小时内只注射一次细胞。在急性炎症和水肿性损伤阶段，最大限度地提高功能恢复和移植存活率的最佳细胞输送时间和剂量尚不清楚。我们通过系列磁共振成像评价了控制性皮质撞击(CCI)诱发大鼠脑内脑损伤的自然过程。在雌性Wistar大鼠左侧运动皮质(Bregma+1外侧2 mm)进行CCI，撞击深度为2 mm，撞击时间为50ms，速度为5m/s。在7特斯拉时进行MRI检查。在CCI后不同时间点分别获得图像分辨率为117×117×500m(第2、9、30、58天)和行为(第1、14、28、56天)的T2w和T2*w图像。我们观察到，尽管CCI损伤环境相同，但CCI诱导的损伤在第2、9和30天的外观和体积是高度不同的。第2天CCI侧至对侧皮质体积的变化百分比与随后第9天和第30天的检查之间几乎没有相关性(CCI皮质/对侧皮质体积的平均变化百分比：第2天=2611%；第9天=-214%；第30天=-1014%)。在CCI后第2天至第9天，45%的大鼠发生了CCI损伤内的出血转化。MRI和病理学显示，使用相同的撞击参数和技术，皮质丢失、胶质增生、轴突丢失和囊性形成存在显著差异。尽管CCI损伤的差异可能归因于技术上的差异，但相似损伤在第2天和第30天之间的差异表明了CCI大鼠模型固有的生物学变异性。第2天至第9天出血演变的可能性也引发了人们对先前使用磁性标记细胞追踪其向CCI病变输送的解释的担忧，因为出血在MRI和普鲁士蓝染色上看起来是相同的。根据这项研究，磁共振损伤或皮质体积可以作为实验性脑外伤新疗法的结果指标，然而，需要在22-受试者之间进行随机平行组试验设计，才能在治疗后检测到阿尔法0.05和功率=0.8的皮质体积差异10%到20%的能力。在30天时，病变体积或皮质差异百分比与行为测量之间没有相关性。我们开发了一种图像分析方法，用于将MRI扩散张量成像(DTI)所见与免疫组织学染色相融合。在模型中，分数各向异性(FA)的增加被认为与卒中或脑外伤后轴突的重组和再生长有关，但这种变化的组织学证据似乎很薄弱。大鼠CCI后2个月取脑组织切片，进行GFAP(星形胶质细胞)、SMI31和SMI32(神经丝)、MAP2(微管)和MBP(髓鞘)染色。基于组织学的傅立叶分析捕捉到了DTI测量的微观各向异性和取向。在CCI损伤的白质(WM)中，FA减少，并与轴突和髓鞘损伤一致，尽管星形胶质细胞也具有高度的各向异性。在灰质，FA增加，这与一致的星形胶质细胞增生相一致，在较小程度上与树突组织一致。这些结果表明，导致正常脑各向异性的微观细胞和亚细胞成分(轴突和髓鞘)不一定是造成损伤脑各向异性的唯一因素。星形胶质细胞的重塑和迁移也与DTI测量的各向异性一致。这种分析方法应该有助于提供微观各向异性和DTI派生的各向异性之间的定量和具体关系。

The pathology of traumatic brain injury in experimental models includes acute inflammatory reaction, blood brain barrier disruption, hemorrhage, demyelination, axonal transection and chronically with axonal neuronal loss and gliosis. Stem cell (SC) therapy is a potential treatment either as replacement therapy or via paracrine effect with release of growth factors and anti-inflammatory cytokines for TBI injury. Experimental studies in rodent models of TBI have been limited and usually a single dose of cells is administered within 24 to 72 hours after experimental injury. The optimal timing and dose of cell delivery to maximize functional recovery and transplantation survival during the acute inflammatory and edematous phase of damage is unknown. We evaluated the natural history of control cortical impact (CCI) to induce TBI in the rat brain by serial MRI. CCI was performed in left motor cortex (Bregma +1 lateral 2 mm) with impact time 50 msec at depth of 2mm and velocity 5 meters/seconds in female Wistar rats. MRI was performed at 7 Tesla. T2w and T2*w images with image resolution of 117x117x500m (days 2,9,30 and 58) and behavior (days 1,14, 28 and 56) were obtained at various time points after CCI. We observed that the appearance and volume of CCI-induced lesions at days 2, 9, and 30 was highly variable despite identical CCI injury settings. There was little correlation between the percent change of CCI side to contralateral side cortical volumes on days 2 to subsequent exams on 9, and 30 (mean percent change CCI cortical/contralateral cortex volume: Day 2=2611%; Day 9 = -214%; Day 30 = -1014%). Hemorrhagic conversion within the CCI lesion occurred in 45% of rats between days 2 and 9 post CCI. MRI and pathology demonstrate significant variation in cortical loss, gliosis, axonal loss and cyst formation using identical impact parameters and techniques. Although the variation in CCI lesions may be attributed to differences in technique, the divergence of similar lesions between days 2 and 30 demonstrates the inherent biological variability of the CCI rat model. The possibility of hemorrhagic evolution between days 2 and 9 also raised concerns about previous interpretation of using magnetically labeled cells to track their delivery to CCI lesions without obtaining a pre-stem cell infusion MRI, as hemorrhage appears identical on MRI and Prussian blue staining. Based on this study, MRI lesion or cortical volumes could be used as an outcome measure for novel therapies of experimental TBI, however the ability to detect 10 to 20% difference in cortical volume with alpha 0.05 and power =0.8 as a result of treatment, would require between 22-64 subjects would be needed to see significant differences in a randomized parallel group trial design. There was no correlation of lesion volume or percent cortical differences and behavioral measures at 30 days. We have developed an image analysis method for fusing MRI diffusion tensor imaging (DTI) findings to immunohistological stains. In models, increased Fractional Anisotropy (FA) has been argued to be associated with axonal reorganization and re-growth following stroke or TBI, but the histological evidence for such changes appeared tenuous. Histological sections from rats at 2 months post CCI were stained for GFAP (astrocytes), SMI31 & SMI32 (neurofilaments), MAP2 (microtubules), and MBP (myelin). The histological-based Fourier analysis captured the microscopic anisotropy and orientation as measured by DTI. In the CCI injured white matter (WM), FA decreased and was consistent with axonal and myelin injury, although astrocytes also had high anisotropy. In gray matter, FA increased and this was consistent with coherent astrogliosis and to a lesser extent, dendrite organization. These results demonstrate the microscopic cellular and subcellular elements that contribute to anisotropy in normal brain (axons and myelin) are not necessarily the only contributors to anisotropy in the injured brain. Astrocyte remodeling and migration also appears consistent with the DTI-measured anisotropy. This analytical method should help provide a quantitative and specific relationship between the microscopic anisotropy and DTI-derived anisotropy.