Reinforcing the Repair Response to Traumatic Brain Injury

描述（由申请人提供）： 创伤性脑损伤（TBI）是一个复杂的过程，包括三个重叠的阶段：a）原发性损伤，B）继发性损伤和c）再生反应。粒细胞集落刺激因子（G-CSF）的全身给药代表了一种新的方法，用于加强大脑的自我修复，特别是在次级和再生阶段。本研究项目的具体目的旨在检验以下假设：a）G-CSF通过促进骨髓源性细胞（BMDC）向脑的浸润间接增强脑修复，调节神经炎症过程并分泌营养因子; B）G-CSF直接与其神经细胞受体相互作用，触发细胞内信号级联反应，减少细胞死亡并促进神经发生。目的1确定G-CSF的最佳剂量和小鼠TBI后行为障碍的恢复时间。将使用未经辐照的小鼠，以确保G-CSF的作用不受全身辐照和骨髓移植（BMT）的混淆。将在基线、TBI后3、7和14天在小鼠中评估运动功能（偏向摆动活动;旋转棒）和行为（水迷宫）终点。次要终点将测量a）病变体积，B）小胶质细胞增生和星形细胞增生的程度和c）细胞因子的脑区域水平。目标2a。为了评估由TBI触发并由G-CSF调节的BMDC动员的程度，将使用免疫荧光来确定嵌合小鼠脑中绿色荧光蛋白（GFP+）BMDC的表型命运和分布，以鉴定共表达GFP的小胶质细胞、星形胶质细胞和神经元样细胞。GFP + BMDC浸润的时程将通过在损伤后3、7和14天评估TBI同侧和对侧的总GFP+负荷来确定。目标2b。为了确定BMDC渗透到脑中导致增强的TBI恢复的程度，BMDC渗透到中枢神经系统（CNS）中将用阻断趋化因子信号传导到单核细胞的试剂或利用具有趋化因子受体CCR2敲除的小鼠来减弱或阻断。尽管BMDC动员受到抑制，但恢复增强将支持G-CSF对神经细胞的直接作用起主要作用的假设。目标3：为了研究G-CSF作用对神经细胞的直接影响，将在神经细胞培养物中评估这些细胞因子对信号转导、凋亡和神经发生的分子影响。将来自该分析的结果与从用G-CSF或媒介物处理的TBI脑解剖的组织样品中的信号转导和抗细胞凋亡的分子分析进行比较。研究方法：将产生携带GFP BMDC的嵌合小鼠，以允许追踪TBI后浸润脑的BMDC的分布和表型命运。手术：TBI将通过 电驱动的受控皮质撞击（CCI）装置对小鼠的影响。行为评估：运动不对称性（EBST）、旋转棒试验和水迷宫（MWM）分析。结束点：（a）行为改变; B）病变体积的变化; c）通过双标记程序评估脑中GFP + BMDC的范围、分布和表型命运; d）脑区域中细胞因子谱的变化; e）信号转导（PKC-β）、Bcl 2的变化。预期结果：G-CSF将调节BMDCs的浸润，并促进行为缺陷的恢复。神经功能缺损的改善将显示与以下作用的组合相关，包括a）BMDC脑浸润的变化; B）促进神经发生的细胞因子的分泌; c）由直接作用于神经细胞中其受体的G-CSF触发的抗凋亡信号传导的上调。

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) is a complex process encompassing three overlapping phases: a) primary injury, b) secondary injury and c) regenerative responses. Systemic administration of granulocyte-colony stimulating factor (G-CSF) represents a novel approach for reinforcing the brain's self-repair, especially during the secondary and regenerative phases. The Specific Aims of this research program are designed to test the hypotheses that a) G-CSF indirectly enhances brain repair by promoting infiltration of bone marrow-derived cells (BMDC) to brain, modulate neuro-inflammatory processes and secrete trophic factors; b) G-CSF directly interacts with its neural cells receptors to trigger intra-cellular signaling cascades that decrease cell death and promote neurogenesis. Aim 1 will determine the optimal dose of G-CSF and the recovery time-course of behavioral deficits after TBI in mice. Non-irradiated mice will be used to ensure the effects of G-CSF are not confounded by whole body irradiation and bone marrow transplantation (BMT). Motoric function (biased swing activity; rotarod) and behavioral (water maze) end-points will be assessed in mice at baseline, 3, 7, and 14 days after TBI. Secondary endpoints will measure a) lesion volume, b) extent of microgliosis and astrocytosis and c) brain regional levels of cytokines. Aim 2a. To assess the extent of BMDC mobilization triggered by TBI and modulated by G-CSF, the phenotypic fate and distribution of green fluorescent protein (GFP+) BMDC in chimeric mice brains will be determined using immunofluorescence to identify microglia, astrocytes, and neuron-like cells that co-express GFP. The time-course of infiltration of GFP+ BMDCs will be determined by assessing total GFP+ burden ipsilateral and contralateral to the TBI at 3, 7 and 14 days after injury. Aim 2b. To determine the extent to which BMDC penetration into brain is responsible for enhanced TBI recovery, the infiltration of BMDC into the central nervous system (CNS) will be attenuated or blocked with agents that block chemokine signaling to monocytes or utilization of mice with a knockout of the chemokine receptor CCR2. Enhanced recovery despite inhibition of BMDC mobilization will support the hypothesis that direct actions of G-CSF on neural cells play a major role. Aim 3. To investigate the direct effects of G-CSF action on neural cells, the molecular impact of these cytokines on signal transduction, apoptosis and neurogenesis will be assessed in neural cell cultures. Results from this analysis will be compared to molecular analyses of signal transduction and anti- apoptosis in tissue samples dissected from TBI brains treated with G-CSF or vehicle. Methods: Chimeric mice will be generated that harbor GFP BMDCs to permit tracking the distribution and phenotypic fate of BMDCs that infiltrate the brain after TBI. Surgery: TBI will be delivered with a pneumatically driven controlled cortical impact (CCI) device to mice. Behavioral Assessments: Analyses of motor asymmetry (EBST), rotarod test and Water Maze (MWM). Endpoints: a) changes in behavior; b) changes in lesion volume; c) extent, distribution and phenotypic fate of GFP+ BMDC in brain assessed by double-labeling procedures; d) changes in cytokine profiles in brain regions; e) changes in signal transduction (PKC-�), Bcl2. Expected Results: G-CSF will modulate BMDCs infiltration and enhance recovery of behavioral deficits. Improvement of neurologic deficits will shown to be related to a combination of actions including a) changes in brain infiltration of BMDC; b) secretion of cytokines that promote neurogenesis; c) up-regulation of anti-apoptotic signaling triggered by G-CSF acting directly on its receptor in neural cells.