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The mouse eye is used to model central nervous system development, pathology, angiogenesis, tumorigenesis, and regenerative therapies. To facilitate the analysis of these processes, we developed an optimized tissue clearing and depigmentation protocol, termed InVision, that permits whole-eye fluorescent marker tissue imaging. We validated this method for the analysis of normal and degenerative retinal architecture, transgenic fluorescent reporter expression, immunostaining and three-dimensional volumetric (3DV) analysis of retinoblastoma and angiogenesis. We also used this method to characterize material transfer (MT), a recently described phenomenon of horizontal protein exchange that occurs between transplanted and recipient photoreceptors. 3D spatial distribution analysis of MT in transplanted retinas suggests that MT of cytoplasmic GFP between photoreceptors is mediated by short-range, proximity-dependent cellular interactions. The InVision protocol will allow investigators working across multiple cell biological disciplines to generate novel insights into the local cellular networks involved in cell biological processes in the eye.
To repair neural circuitry following spinal cord injury (SCI), neural stem cell (NSC) transplantation has held a primary focus; however, stochastic outcomes generate challenges driven in part by NSC differentiation and tumor formation. The recent ability to generate regionally specific neurons and their support cells now allows consideration of directed therapeutic approaches with pre-differentiated and networked spinal neural cells. Here, we form encapsulated, transplantable neuronal networks of regionally matched cervical spinal motor neurons, interneurons, and oligodendrocyte progenitor cells derived through trunk-biased neuromesodermal progenitors. We direct neurite formation in alginate-based neural ribbons to generate electrically active, synaptically connected networks, characterized by electrophysiology and calcium imaging before transplantation into rodent models of contused SCI for evaluation at 10-day and 6-week timepoints. The in vivo analyses demonstrate viability and retention of interconnected synaptic networks that readily integrate with the host parenchyma to advance goals of transplantable neural circuitry for SCI treatment.
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