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Modulating microglia and macrophage functions to promote stroke recovery

调节小胶质细胞和巨噬细胞功能促进中风恢复

基本信息

批准号：
10612811
负责人：
Yejie Shi
金额：
$ 39.75万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-05-01 至 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10612811
关键词：
Acute Affect Brain Brain Infarction Brain Injuries Cells Cerebral Ischemia Cerebrum Clinical Clinical Treatment Coculture Techniques Cognitive deficits Cues Equilibrium Foundations Functional disorder Future Genes Genetic Guidelines Homeostasis Hour Immune Immune response In Vitro Infiltration Inflammation Inflammatory Injury Interferons Invaded Ischemia Ischemic Stroke Knock-out Lentivirus Macrophage Mediator Microglia Middle Cerebral Artery Occlusion Modeling Molecular Mus Nature Nervous System Physiology Neurons Outcome Peripheral Phenotype Phosphorylation Pilot Projects Quality of life Recovery Recovery of Function Rehabilitation therapy Resolution Role STAT1 protein Signal Induction Signal Transduction Solid Stroke Tamoxifen Testing Therapeutic Therapeutic Effect Time Titrations Transgenic Organisms Treatment Protocols Work aged arginase brain remodeling brain repair brain tissue clinical translation clinically relevant effective therapy fludarabine gene repair gray matter immune function improved in vitro testing in vivo in vivo evaluation inhibitor intraperitoneal knock-down long term recovery monocyte neuroinflammation neurological recovery neuron loss neuroprotection neurorestoration new therapeutic target novel overexpression pharmacologic post stroke preservation prevent primary endpoint repaired response sex small hairpin RNA stroke outcome stroke recovery stroke therapy stroke victims therapeutic evaluation transcription factor white matter young adult

项目摘要

Emerging evidence implicates a pivotal role of cerebral inflammation in the pathophysiology of ischemic stroke. Microglia and macrophages (Mi/MΦ) are important mediators of post-stroke neuroinflammation and assume diverse functional states in response to specific microenvironmental signals, thereby regulating inflammation, injury progression, and brain repair. The key molecular switches and networks that determine the overall functional state of Mi/MΦ after stroke are poorly understood. Identification of these signaling mechanisms may reveal novel therapeutic targets to improve long-term stroke outcomes by boosting beneficial Mi/MΦ functions and harnessing the power of restorative neuroinflammation. Signal transducer and activator of transcription 1 (STAT1) is a transcription factor that is potently activated in canonical interferon signaling and defined as an important mediator of macrophage M1 polarization. Despite clear implication in regulating immune responses, the role of STAT1 in ischemic stroke has been studied solely for its contribution to acute neuronal death during the first 24 hours. To date, how STAT1 controls Mi/MΦ function under the temporally evolving long-term recovery period, the primary endpoint of clinical stroke, is unknown. Our pilot studies show for the first time that: 1) STAT1 is activated (phosphorylated) primarily in Mi/MΦ at the subacute stage (1-5 days) after transient focal cerebral ischemia (tFCI). 2) Mi/MΦ-specific knockout (mKO) of STAT1 does not reduce acute brain infarct yet improves long-term outcomes after tFCI in mice of both sexes. 3) Uniquely, STAT1 mKO not only downregulates proinflammatory genes in post-tFCI Mi/MΦ, but also elevates a panel of pro-repair genes, including Arginase 1, a key protective and pro-repair factor. 4) Intraperitoneal administration of fludarabine, a brain-penetrant and selective STAT1 inhibitor, upregulates Arginase 1 in Mi/MΦ, alleviates the brain invasion of peripheral immune cells, and improves both short-term and long-term outcomes after tFCI. Given these observations, we propose three specific aims to test the novel central hypothesis that genetic deletion or pharmacological inhibition of STAT1 improves long-term outcomes after ischemic stroke by promoting inflammation-resolving and pro-repair microglial/macrophage responses. Aim 1: Test if inducible deletion of STAT1 selectively in Mi/MΦ improves long-term stroke outcomes. Mice with tamoxifen-inducible STAT1 knockout in Mi/MΦ will be subjected to tFCI induced by 1-h middle cerebral artery occlusion (MCAO). Outcomes will be assessed for 5 weeks after tFCI. Aim 2: Test if STAT1 knockout alleviates proinflammatory Mi/MΦ responses but promotes the beneficial pro-resolving/pro-repair activities of Mi/MΦ via Arginase 1 signaling, using the in vivo tFCI model and in vitro primary cultures and co-cultures. We will also test if Mi/MΦ-specific overexpression of Arginase 1 boosts pro-repair Mi/MΦ responses and neurological recovery after tFCI. Aim 3: Test the therapeutic potential of the selective STAT1 inhibitor fludarabine in long-term (5 weeks) stroke outcomes in young adult and aged mice (20 months old) of both sexes following STAIR guidelines.

新出现的证据表明脑炎症在缺血性卒中的病理生理学中起着关键作用。小胶质细胞和巨噬细胞（Mi/MΦ）是脑卒中后神经炎症的重要介质，响应特定微环境信号的不同功能状态，从而调节炎症，损伤进展和大脑修复关键的分子开关和网络，决定了整体脑卒中后Mi/MΦ的功能状态知之甚少。识别这些信号机制可能揭示了新的治疗靶点，通过增强有益的Mi/MΦ功能来改善长期卒中结局并利用恢复性神经炎症的力量。信号转导子和转录激活子1（STAT 1）是一种转录因子，其在细胞内被有效激活，典型的干扰素信号传导，并被定义为巨噬细胞M1极化的重要介质。尽管 STAT 1在调节免疫应答中的明确意义，仅研究了其在缺血性卒中中的作用在最初的24小时内导致急性神经元死亡。迄今为止，STAT 1如何控制Mi/MΦ功能在随时间演变的长期恢复期内，临床卒中的主要终点未知。我们的初步研究首次表明：1）STAT 1主要在Mi/MΦ中激活（磷酸化），短暂局灶性脑缺血（tFCI）后的亚急性期（1-5天）。2)Mi/MΦ特异性敲除（mKO）在两种性别的小鼠中，STAT 1并没有减少急性脑梗死，但改善了tFCI后的长期结局。第三章独特的是，STAT 1 mKO不仅下调了tFCI后Mi/MΦ中的促炎基因，而且还上调了tFCI后Mi/MΦ中的促炎基因。一组促修复基因，包括精氨酸酶1，一种关键的保护和促修复因子。4)腹膜内给予氟达拉滨，一种脑渗透剂和选择性STAT 1抑制剂，上调Mi/MΦ中的精氨酸酶1，减少外周免疫细胞的脑侵袭，改善短期和长期结果在tFCI之后。鉴于这些观察结果，我们提出了三个具体目标来检验新的中心假设， STAT 1基因缺失或药物抑制可改善缺血性卒中后的长期预后通过促进炎症消退和促修复小胶质细胞/巨噬细胞反应。目的1：测试Mi/MΦ中选择性STAT 1的可诱导缺失是否改善长期卒中结局。小鼠 Mi/MΦ中他莫昔芬诱导的STAT 1敲除将经受由1-h大脑中动脉诱导的tFCI 闭塞（MCAO）。将在tFCI后5周评估结局。目标2：测试STAT 1敲除是否减轻促炎性Mi/MΦ反应，但通过以下途径促进Mi/MΦ的有益促消退/促修复活性：精氨酸酶1信号传导，使用体内tFCI模型和体外原代培养物和共培养物。我们还将测试如果Mi/MΦ特异性精氨酸酶1过表达促进促修复Mi/MΦ反应和神经恢复在tFCI之后。目的3：测试选择性STAT 1抑制剂氟达拉滨的长期（5周）治疗潜力根据STAIR指南，年轻成年小鼠和老年小鼠（20个月大）的中风结果。