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 othe...