Interaction of GCSF with the Endocannabinoid System in Promoting Brain Repair

背景：G-CSF已被确定为TBI的潜在治疗剂。G的管理- 损伤后不久的CSF导致骨髓源性细胞增殖并释放到循环中 （BMDC）。血液中的单核细胞被募集到大脑中的病变部位， 分化成小胶质细胞。这些又产生了许多神经营养因子和细胞因子 参与修复和再生过程。许多研究表明，TBI影响 内源性大麻素系统（eCB），改变eCB受体CB 1和CB 2的表达，以及 改变内源性大麻素anandamide（N-花生四烯酸-乙醇胺; AEA）和2-AG（2- 花生四烯酰甘油）。此外，已显示给予大麻素剂可增强 由抗炎细胞因子介导的TBI恢复和与这些细胞因子平行的再生过程 由G-CSF触发本研究计划的具体目的是为了测试假设a）， G-CSF与内源性大麻素系统（eCS）相互作用以促进脑修复，和B）阻断内源性大麻素系统（eCS）的作用。 CB 1和/或CB 2受体将减弱或增强大脑对TBI的修复反应。目标1： 研究G-CSF对小鼠CB 1、CB 2及其天然配体表达的影响 脑（皮质、纹状体和海马）。因变量：CB 1和CB 2受体 表达（mRNA和蛋白质），eCB配体AEA和2-AG的水平，细胞凋亡的程度， 小胶质细胞增生、星形细胞增多、神经炎症、神经营养因子（BDNF、GDNF）水平; 海马神经发生目的2a：确定用内源性大麻素刺激大脑内源性大麻素系统是否有效。 a）脂肪酸酰胺（FAAH）的抑制剂以增加AEA和2AG的水平，或B）施用 选择性CB 1和CB 2受体拮抗剂将增强（或减弱）G- CSF对脑修复和恢复的影响。因变量与目标1相同，但增加了 在径向臂水迷宫（RAWM）中恢复性能的参数。目标2b：确定 CB 1-R或CB 2-R负责增强从TBI的恢复的程度，CB 1-R和 将使用Aim 1中详述的方案研究CB 2-R敲除小鼠。因变量：CB 1和 皮质、纹状体和海马CB 2受体mRNA和蛋白表达、AEA、2AG水平; 这3个脑区域中的小胶质细胞增生和星形细胞增生的程度; BDNF和GDNF、海马 神经发生和RAWM中的性能恢复。目的3：确定CB 2受体的作用 在动员BMDC介导G-CSF的有益作用中，我们将给予CB 2 拮抗剂（或利用CB 2-R敲除小鼠）对用G-CSF或媒介物处理的动物的作用。追踪BMDC 将利用从转基因“绿色小鼠”移植骨髓的嵌合小鼠。依赖 变量：共表达GFP和标志物的总循环白色血细胞的百分比 单核细胞，定量估计GFP+细胞在损伤和对侧中的分布 半球，分化成小胶质细胞表型（M1，M2）的GFP+细胞比例， 细胞因子/趋化因子、海马神经发生和RAWM中的表现。目标4：影响 G-CSF延迟治疗（“严重”TBI后1、2、4周）对行为恢复和修复介导的 eCB的。因变量：与目标1相同，但增加了速率参数 在RAWM中恢复性能。对脑修复领域的影响：TBI的后果， 中风和神经变性疾病在退伍军人群体中非常普遍。有必要 有效和安全的治疗方法。TBI后G-CSF治疗的最佳时机， 或没有大麻素激动剂或拮抗剂，如本项目所确定的，将有助于 为遭受脑损伤的人制定治疗指南。

Background: G-CSF has been identified as a potential therapeutic agent for TBI. Administration of G- CSF soon after injury results in proliferation and release into the circulation of bone marrow-derived cells (BMDC). Monocytes from blood are recruited into the brain to the site of the lesion where they differentiate into microglia. These, in turn generate a number of neurotrophic factors and cytokines involved in repair and regenerative processes. Numerous studies indicate that TBI impacts the endogenous cannabinoid system (eCBs), altering expression of eCB receptors CB1 and CB2, and changing levels of the endocannabinoids anandamide (N-arachidonoyl-ethanolamine; AEA) and 2-AG (2- arachidonoylglycerol). Moreover, administration of cannabinoid agents have been shown to enhance recovery from TBI mediated by anti-inflammatory cytokines and regenerative processes that parallel those triggered by G-CSF. Specific Aims of this research program are designed to test the hypotheses a) that G-CSF interacts with the endocannabinoid system (eCS) to promote brain repair and b) that blockade of CB1 and/or CB2 receptors will diminish or enhance the brain’s repair response to TBI. Aim 1: To investigate the effects of G-CSF on the expression of CB1 and CB2 and their natural ligands in mouse brain (cortex, striatum and hippocampus) following TBI. Dependent variables: CB1 and CB2 receptor expression (mRNA and protein), levels of the eCB ligands AEA and 2-AG, extent of apoptosis, microgliosis, astrocytosis, neuro-inflammation, levels of neurotrophic factors (BDNF, GDNF); changes in hippocampal neurogenesis. Aim 2a: To determine if stimulation of the brain endocannabinoid system with a) an inhibitor of fatty acid amide (FAAH) to increase levels of AEA and 2AG, or b) administration of selective CB1 and CB2 receptor antagonists will potentiate (or diminish) the known beneficial effects of G- CSF on brain repair and recovery. Dependent variables are the same as in Aim 1, with the additional parameter of recovery of performance in the radial arm water maze (RAWM). Aim 2b: To determine the extent to which the CB1-R or the CB2-R is responsible for enhanced recovery from TBI, the CB1-R and CB2-R knockout mice will be studied using the protocol detailed in Aim1. Dependent variables: CB1 and CB2 receptor expression (mRNA and protein), levels of AEA, 2AG in cortex, striatum and hippocampus; extent of microgliosis and astrocytosis in these 3 brain regions; levels of BDNF and GDNF, hippocampal neurogenesis and recovery of performance in RAWM. Aim 3: To determine the role of the CB2 receptor in the mobilization of BMDC in mediating the beneficial effects of G-CSF, we will administer a CB2 antagonist (or utilize CB2-R knockout mice) to animals treated with G-CSF or vehicle. Tracking of BMDC will utilize chimeric mice that have had bone marrow transplants from transgenic “green mice”. Dependent variables: percentage of total circulating white blood cells that co-express GFP and markers of monocytes, quantitative estimates of the distribution GFP+ cells in the lesioned and contralateral hemisphere, proportion of GFP+ cells that differentiate into microglial phenotypes (M1, M2), profile of cytokines/chemokines, hippocampal neurogenesis, and performance in RAWM. Aim 4: Effects of delayed treatment (1, 2, 4 wks after “severe” TBI) with G-CSF on behavioral recovery and repair mediated by eCBs. Dependent variables: are the same as in Aim 1, with the additional parameter of rate of recovery of performance in RAWM. Impact on the Field of Brain Repair: The consequences of TBI, stroke and neurodegenerative diseases are highly prevalent in the veteran population. There is a need for effective, and safe therapies for these disorders. The optimal timing of G-CSF therapy after TBI, with or without cannabinoid agonists or antagonists as determined from this project, will be helpful in developing guidelines for treatment of humans who have suffered brain injury.