The goal of our proposed Small Project is: 1) to develop a more efficient, accessible, and accurate 3D structural analysis workflow using virtual reality (VR) and the Neurodata Without Borders (NWB) data standard, and 2) to implement this software/hardware ecosystem into our current SCI/D research program in neuropathic pain and spasticity. By developing this 3D-VR Structural Analysis Software Ecosystem (3D-VR SASE), we will powerfully expand our Center's experimental reach and capacity for translational studies in the future. We will carry out experiments in two Specific Aims to address the goal of this Project. In Specific Aim 1, we will establish a VR-based 3D structural analysis software environment (3D-VR SASE) that will expand our ability to investigate the dynamic changes in abnormal dendritic spine plasticity associated SCI/D. Our VA Center already operates imaging systems that have allowed us to perform a multitude of anatomical studies of the injured CNS. However, our project trajectory has now severely outpaced the capacity of these systems, and no commercial tools for our needs are available or in-development. Thus, to expand the capabilities of our current hardware, we will develop a faster, more accurate reconstruction/visualization hardware/software ecosystem with the established NWB data standard solution in our Center. In Specific Aim 2, we will validate the 3D-VR SASE platform. We demonstrate its utility using publicly available neuronal datasets and perform a study of dendritic spines using spinal cord tissue of transgenic reporter mice. We will perform two experimental studies with the analysis platform. First, we will reconstruct neuronal image stacks from the DIADEM challenge, which is a public neuroscience competition designed to encourage the development of algorithmic methods for improved neuron morphological tracing. The DIADEM challenge provides high-quality light-microscopy images with established specifications that allow us to score our VR traces using metrics of accuracy against a “gold standard” trace. Second, we will perform a retrospective study of dendritic spines in wide-dynamic range (WDR) dorsal horn neurons in post-mortem spinal tissue collected from our previous study. WDR neurons have been of interest because they project fibers in the ascending spinothalamic tract, a major pain pathway to the thalamic VPL nociceptive processing region. Additionally, dendritic spine dysgenesis in WDR neurons has been associated with neuropathic pain. In summary, this project will develop the 3D-VR SASE workflow that increases analytical speed, 3D visualization and neuroanatomical reconstruction, and implements the NWB data standard. The flexibility and broad utility of the 3D-VR SASE platform will facilitate our current SCI/D research program as well as the study of neuroanatomical pathologies in other chronic diseases that are also prevalent among US Veterans, e.g., Alzheimer's disease, PTSD, MS, TBI. Overall, we expect t...