Interaction of GCSF with the Endocannabinoid System in Promoting Brain Repair

GCSF 与内源性大麻素系统的相互作用促进大脑修复

• 批准号: 10292953
• 负责人: Shijie Song
• 金额: --
• 依托单位: JAMES A. HALEY VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-10-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10292953
• 关键词: 2-arachidonylglycerol; Agonist; Amides; Animals; Anti-Inflammatory Agents; Apoptosis; Astrocytosis; Basic Science; Behavioral; Blood Cells; Bone Marrow; Bone Marrow Transplantation; Brain; Brain Injuries; Brain region; Brain-Derived Neurotrophic Factor; CB2 receptor antagonist; CNR1 gene; CNR2 gene; CSF3 gene; Cannabinoids; Cell Differentiation process; Cells; Clinic; Colony-Stimulating Factor Therapy; Contralateral; Corpus striatum structure; Disease; Endocannabinoids; Ethanolamines; Fatty Acids; Fostering; Goals; Granulocyte Colony-Stimulating Factor; Hippocampus (Brain); Human; IL3 Gene; Injury; Knockout Mice; Lesion; Leukocytes; Ligands; Mediating; Messenger RNA; Microglia; Mus; Neuraxis; Neurodegenerative Disorders; Neurons; Performance; Phenotype; Population; Process; Proteins; Protocols documentation; Radial; Recovery; Recovery of Function; Research; Role; Site; Stroke; System; Testing; Therapeutic Agents; Time; Transgenic Organisms; Traumatic Brain Injury; Traumatic Brain Injury recovery; Veterans; anandamide; antagonist; arm; bone circulation; brain repair; chemokine; controlled cortical impact; cytokine; design; endogenous cannabinoid system; frontal lobe; glial cell-line derived neurotrophic factor; improved; inhibitor; military veteran; monocyte; mouse model; neurogenesis; neuroinflammation; neurotrophic factor; programs; receptor; receptor expression; recruit; regenerative; relating to nervous system; repaired; response; stem; stem cells; translational goal; treatment guidelines; water maze

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.

背景：G-CSF 已被确定为 TBI 的潜在治疗剂。 损伤后脑脊液很快就会导致骨髓来源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。 这些细胞又分化为小胶质细胞，进而产生许多神经营养因子和细胞因子。 大量研究表明 TBI 会影响修复和再生过程。 内源性大麻素系统 (eCB)，改变 eCB 受体 CB1 和 CB2 的表达，以及 内源性大麻素 anandamide（N-花生四烯酰乙醇胺；AEA）和 2-AG（2- 此外，大麻素药物的施用已被证明可以增强。 由抗炎细胞因子介导的 TBI 恢复以及与之平行的再生过程 该研究计划的具体目标是测试以下假设：a) G-CSF 与内源性大麻素系统 (eCS) 相互作用，促进大脑修复，b) 阻断 CB1 和/或 CB2 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CB1和CB2及其天然配体表达的影响 TBI 后的大脑（皮层、纹状体和海马体）因变量：CB1 和 CB2 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、 小胶质细胞增多症、星形细胞增多症、神经炎症、神经营养因子（BDNF、GDNF）水平的变化； 目标 2a：确定大脑内源性大麻素系统是否受到刺激。 a) 脂肪酸酰胺 (FAAH) 抑制剂以增加 AEA 和 2AG 的水平，或 b) 施用 选择性 CB1 和 CB2 受体拮抗剂将增强（或减弱）G-的已知有益作用 CSF 对大脑修复和恢复的影响与目标 1 相同，但有附加内容。 径向臂水迷宫 (RAWM) 中的性能恢复参数 目标 2b：确定 CB1-R 或 CB2-R 负责增强 TBI 恢复的程度，CB1-R 和 将使用 Aim1 中详述的方案研究 CB2-R 敲除小鼠：CB1 和 CB2受体表达（mRNA和蛋白），皮质、纹状体和海马中AEA、2AG的水平； 这 3 个大脑区域的小胶质细胞增生和星形细胞增多的程度，海马的 BDNF 和 GDNF 水平； RAWM 中的神经发生和性能恢复 目标 3：确定 CB2 受体的作用。 在动员 BMDC 介导 G-CSF 的有益作用时，我们将施用 CB2 拮抗剂（或利用 CB2-R 敲除小鼠）对用 G-CSF 或 BMDC 追踪治疗的动物进行治疗。 将利用从转基因“绿色小鼠”进行骨髓移植的嵌合小鼠。 变量：共表达 GFP 和标记物的循环白细胞总数的百分比 单核细胞，定量估计病变和对侧 GFP+ 细胞的分布 半球，分化为小胶质细胞表型（M1、M2）的 GFP+ 细胞比例，概况 细胞因子/趋化因子、海马神经发生和 RAWM 中的表现 目标 4：效果。 使用 G-CSF 延迟治疗（“严重”TBI 后 1、2、4 周）以促进行为恢复和修复介导 由 eCB 决定的因变量：与目标 1 相同，附加参数为 RAWM 表现的恢复对大脑修复领域的影响：TBI 的后果， 中风和神经退行性疾病在退伍军人群体中非常普遍，因此有必要。 TBI 后 G-CSF 治疗的最佳时机。 或没有本项目确定的大麻素激动剂或拮抗剂，将有助于 制定治疗脑损伤患者的指南。