Reinforcing the Repair Response to Traumatic Brain Injury

描述(由申请人提供)： 创伤性脑损伤是一个复杂的过程，包括三个相互重叠的阶段：a)原发损伤，b)继发性损伤和c)再生反应。全身应用粒细胞集落刺激因子(G-CSF)是一种增强大脑自我修复的新方法，尤其是在第二和再生阶段。本研究计划的具体目的是检验如下假设：a)G-CSF通过促进骨髓衍生细胞(BMDC)渗透到大脑、调节神经炎症过程和分泌营养因子来间接增强脑修复；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，从而减少细胞死亡和促进神经再生。目的1确定小鼠颅脑损伤后应用G-CSF的最佳剂量和行为功能恢复的时程。未受辐射的小鼠将被用来确保G-CSF的效果不会被全身照射和骨髓移植(BMT)所混淆。在基线、伤后3天、7天和14天对小鼠的运动功能(偏向摆动；旋转杆)和行为(水迷宫)终点进行评估。次级终点将测量a)损伤体积，b)小胶质细胞增多症和星形细胞增多症的程度，以及c)脑内细胞因子的区域水平。目标2a。为了评估颅脑损伤后G-CSF调节的骨髓基质细胞树突状细胞动员的程度，将利用免疫荧光技术检测嵌合小鼠脑内绿色荧光蛋白(GFP+)骨髓基质细胞树突状细胞的表型去向和分布，以确定共表达绿色荧光蛋白的小胶质细胞、星形胶质细胞和神经元样细胞。GFP+BMDCs在伤后3、7、14天的渗入时程将通过评估伤侧和对侧的总GFP+负荷来确定。目标2b。为了确定骨髓基质细胞向脑内的渗透在多大程度上促进了脑损伤的恢复，骨髓基质细胞向中枢神经系统(CNS)的渗透将被阻断单核细胞的趋化因子信号或利用趋化因子受体CCR2基因敲除的小鼠的药物减弱或阻断。尽管骨髓间充质干细胞动员受到抑制，但恢复速度加快将支持G-CSF对神经细胞的直接作用发挥主要作用的假说。目的3.为了研究G-CSF对神经细胞的直接作用，将在神经细胞培养中评估这些细胞因子对信号转导、细胞凋亡和神经发生的分子影响。这项分析的结果将与用G-CSF或赋形剂治疗的脑损伤组织标本中信号转导和抗细胞凋亡的分子分析进行比较。方法：将产生携带GFP BMDCs的嵌合小鼠，以跟踪脑损伤后渗透到脑内的BMDCs的分布和表型命运。手术：TBI将与一名 气动控制皮质冲击(CCI)装置对小鼠。行为评估：运动不对称分析(EBST)、旋转棒试验和水迷宫(MWM)。终点：a)行为变化；b)损伤体积的变化；c)GFP+BMDC在脑内的范围、分布和表型命运；d)脑区细胞因子谱的变化；e)信号转导的变化(PKC-β)，bcl2。预期结果：G-CSF将调节BMDCs的渗透，促进行为缺陷的恢复。神经功能缺陷的改善将被证明与一系列行动有关，包括a)脑内BMDC渗透的改变；b)促进神经发生的细胞因子的分泌；c)G-CSF直接作用于神经细胞中的其受体而触发的抗凋亡信号的上调。

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.