Tracking labeled stem cells in TBI model by cellular MRI

创伤性脑损伤的病理变化包括急性炎症反应、血脑屏障破坏、出血、脱髓鞘、轴突横断和慢性轴突神经元丢失和胶质增生。干细胞治疗是一种潜在的治疗方法，无论是作为替代治疗还是通过旁分泌效应释放生长因子和抗炎细胞因子来治疗TBI损伤。在TBI的啮齿动物模型中的实验研究有限，并且通常在实验损伤后24至72小时内施用单剂量的细胞。在损伤的急性炎症和水肿阶段，细胞递送的最佳时机和剂量使功能恢复和移植存活率最大化尚不清楚。我们评估了自然历史的控制皮质撞击（CCI）诱导TBI在大鼠大脑中的系列MRI。在雌性Wistar大鼠的左侧运动皮质（前额叶+1外侧2mm）中进行CCI，在2mm的深度和5米/秒的速度下撞击时间为50毫秒。 在7特斯拉下进行MRI。在CCI后不同时间点获得图像分辨率为117 × 117 × 500 m的T2 w和T2 * w图像（第2、9、30和58天）和行为（第1、14、28和56天）。我们观察到，尽管CCI损伤设置相同，但CCI诱导的损伤在第2、9和30天的外观和体积是高度可变的。 第2天CCI侧与对侧皮质体积的百分比变化与随后第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百分比变化：第2天= 2611%;第9天=-214%;第30天=-1014%）。CCI后第2天至第9天，45%的大鼠发生了CCI病变内的出血性转化。MRI和病理学显示，使用相同的撞击参数和技术，皮质丢失、神经胶质增生、轴突丢失和囊肿形成存在显著差异。尽管CCI病变的变化可能归因于技术差异，但第2天和第30天之间相似病变的差异证明了CCI大鼠模型的固有生物学变异性。第2天至第9天之间出血演变的可能性也引起了对先前使用磁性标记细胞追踪其递送至CCI病变而未获得干细胞输注前MRI的解释的担忧，因为出血在MRI和普鲁士蓝染色上表现相同。基于该研究，MRI病变或皮质体积可用作实验性TBI的新疗法的结果测量，然而，在α 0.05和功效= 0.8的情况下检测皮质体积的10%至20%差异的能力作为治疗的结果，将需要22 - 64名受试者才能在随机化平行组试验设计中看到显著差异。 在30天时，病变体积或皮质差异百分比与行为测量无相关性。我们已经开发了一种图像分析方法融合磁共振扩散张量成像（DTI）的结果，免疫组织化学染色。在模型中，增加的各向异性分数（FA）被认为与中风或TBI后的轴突重组和再生长有关，但这种变化的组织学证据似乎很薄弱。在CCI后2个月，对来自大鼠的组织切片进行GFAP（星形胶质细胞）、SMI 31和SMI 32（神经丝）、MAP 2（微管）和MBP（髓磷脂）染色。基于组织学的傅立叶分析捕获了由DTI测量的微观各向异性和取向。在CCI损伤的白色物质（WM）中，FA降低，与轴突和髓鞘损伤一致，尽管星形胶质细胞也具有高各向异性。在灰质中，FA增加，这与连贯的星形胶质细胞增生和较小程度的树突组织一致。这些结果表明，在正常脑中有助于各向异性的微观细胞和亚细胞成分（轴突和髓鞘）不一定是受伤脑中各向异性的唯一贡献者。星形胶质细胞的重塑和迁移也与DTI测量的各向异性一致。这种分析方法应有助于提供一个定量和具体的微观各向异性和DTI派生的各向异性之间的关系。

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.