VRC: Engineered extracellular vesicles for mild TBI-induced retinal injury

创伤性脑损伤是运动损伤和车祸中导致视力丧失的主要原因。 美国，目前还没有治疗方法。我们的长期目标是发展安全、创新 对抗颅脑损伤所致视力丧失的策略。轻度颅脑损伤(MTBI)，即使对眼睛没有直接损伤，也会导致视网膜 通过小胶质细胞和Müler细胞引起的炎症造成的损害，以及由视网膜神经节细胞丢失引起的功能障碍 (RGCs)。间充质干细胞(MSC)细胞外小泡(EVS)作为一种干细胞正在迅速崛起 促进免疫调节、修复和再生的替代品。玻璃体内注射MSC EVS 啮齿动物表现出对视网膜节细胞、Müler细胞和小胶质细胞的嗜好，并触发了特定的细胞反应， 其中来自电动汽车的微小RNA(MiRNA)起着关键作用。 在这里，我们利用我们已发表的和初步的数据，证明MSC EV减弱小胶质细胞 体外激活和RGC死亡。MSC EVS的抗细胞凋亡和抗炎作用进一步增强 通过使用母体MSCs的低氧预适应为电动汽车“增压”，产生“H-电动汽车”。间充质干细胞系 基因改变以稳定地在电动汽车(即功能工程电动汽车或费用)中过表达microRNA， FICS模拟H-EVS的抗细胞凋亡和抗炎作用。这项提议针对的是中断 应用含有功能特异性miRNA的MSC-EVS减轻神经炎症的轻度脑损伤对视网膜的损伤 并伴有神经元功能障碍和细胞死亡。我们的中心假设是针对EV的特异性 关键miRNAs的表达可改善小胶质细胞和Müler细胞引起的炎症以及RGCs和RGCs的丢失 MTBI导致的继发性视觉功能障碍。 AIM 1设计了模拟H-EVS功能的EVS，以减弱小胶质细胞和Müler细胞的激活和 视网膜神经元在体外死亡。我们假设，费用增强了抗炎和抗细胞凋亡的作用 MSC-EVS和模拟H-EV。混合动力电动汽车中表达最高的两个MIR将作为费用进行比较研究 对激活的视网膜小胶质细胞，以及暴露于谷氨酸的视网膜神经节细胞和Müller细胞中的MSC EVS、H-EVS和对照组。 识别费用和H-EVS的作用机制将为精确治疗提供一条新的途径 治疗轻度颅脑损伤患者视觉功能障碍的药物。 目的2在临床相关的小鼠体内测定FECS的抗炎和抗细胞凋亡作用 MTBI模型。我们将检验这样一种假设，即进入玻璃体的费用在给予时拯救了视网膜 在mTBI之后。应用50psi的mTBI后视网膜功能、细胞凋亡、谷氨酸水平和炎症介质 对颅骨的冲击波进行定量比较，并确定H-处理组的细胞特异性效应。 电动汽车、费用、MSC电动汽车和控制。 拟议的研究有望为MSC-EVS的保护作用提供新的结果 治疗受脑外伤影响的视网膜细胞，并为其他神经退行性疾病提供新的潜在治疗方法。

Traumatic brain injury (TBI) is the leading cause of vision loss in sports injury, and automobile accidents in the United States, for which there currently is no treatment. Our long-term objective is to develop safe, innovative strategies to combat vision loss from TBI. Mild TBI (mTBI), even without direct trauma to the eye, results in retinal damage via microglia and Müller cell-driven inflammation, and dysfunction from loss of retinal ganglion cells (RGCs). Mesenchymal stem cell (MSC) extracellular vesicles (EVs) are rapidly emerging as a stem cell alternative that promote immunomodulation, repair, and regeneration. MSC EVs injected into the vitreous in rodents demonstrated tropism for RGCs, Müller cells, and microglia, and triggered specific cellular responses, with micro RNA (miRNA) from the EVs playing a key role. Here we capitalize upon our published and preliminary data demonstrating that MSC EVs attenuate microglial activation and RGC death in vitro. Anti-apoptosis and anti-inflammatory effects of MSC EVs were further boosted by “supercharging” the EVs using hypoxic preconditioning of the parent MSCs, yielding “H-EVs.” MSC cell lines were genetically altered to stably overexpress microRNAs in EVs (i.e., Functionally-Engineered EVs, or FEEs), and FEEs mimicked the anti-apoptosis and anti-inflammatory action of H-EVs. This proposal targets interrupting retinal damage from mild TBI with MSC-EVs containing function-specific miRNA to attenuate neuro-inflammation and accompanying neuronal dysfunction and cell death. Our central hypothesis is that targeted EV-specific expression of key miRNAs ameliorates microglia and Müller cell-driven inflammation, and loss of RGCs and subsequent visual dysfunction from mTBI. Aim 1 engineers EVs mimicking functionality of H-EVs to attenuate microglia and Müller cell activation and retinal neuron death in vitro. We hypothesize that FEEs enhance anti-inflammatory and anti-apoptosis effects of MSC-EVs and mimic H-EVs. Two of the highest expressed miRs in H-EVs will be studied as FEEs in comparison to MSC EVs, H-EVs, and controls in activated retinal microglia, and RGCs and Müller cells exposed to glutamate. Identifying mechanisms of action of FEEs and H-EVs will provide a novel pathway to therapeutic precision medicine for visual dysfunction in mTBI. Aim 2 determines in vivo anti-inflammatory and anti-apoptosis actions of FEEs in a clinically-relevant mouse model of mTBI. We will test the hypothesis that FEEs administered into the vitreous rescue the retina when given after mTBI. Retinal function, apoptosis, glutamate levels, and inflammatory mediators post mTBI using 50 psi blast to the cranium will be quantitatively compared, and cell-specific effects identified in groups treated with H- EVs, FEEs, MSC EVs, and controls. The proposed studies are expected to provide new results with MSC-EVs modified for protective action to treat the retinal cells affected by TBI, and provide novel potential therapy for other neurodegenerative disorders.