SUMMARY Peripheral nerve injuries are common and debilitating conditions affecting more than one hundred thousand people annually in the United States. Prognosis for recovery of severe nerve injuries is poor, as high-grade axonotmetic and neurotmetic injuries usually do not spontaneously recover. These injuries require surgical intervention and if the nerve injury is located far from the target muscles, nerve repairs fail to provide useful function in half of patients- leading to permanent physical disabilities and enormous emotional stress. To improve clinical outcomes, a better understanding of the molecular mechanisms involved in nerve injury is critical. The traditional view that Wallerian degeneration (WD) is inevitable after nerve injury has recently been challenged with the discovery of the role of nicotinamide adenine dinucleotide (NAD) in supporting maintenance of viable axons. SARM1 with its NADase enzymatic activity, has been identified as a key gatekeeper of WD. After a severe nerve injury, SARM1 rapidly degrades NAD resulting in catastrophic structural changes to the distal axon. Blockade of this phenomenon, combined with the promise of fusogens can provide a potential mechanism for transected nerves to rapidly recover after injury. Optical imaging with its high spatial and temporal resolution is highly promising to visualize the entire process in the nerve tissue, however high scattering from the skin and other adjacent organs limit the application to only in vitro or ex vivo studies. To address these limiting issues, we developed a minimally invasive in vivo model that enables continuous imaging of a peripheral nerve injury with a high, single axon resolution. Our approach uses a flexible skin-embedded transparent optical window with the nerve surgically repositioned above the muscle layer. This modality allows daily or even hourly, longitudinal imaging of the nerve with virtually any optical reporter that can be used in living animals. When combined with fluorescent reporters and a high-resolution imaging system (i.e., two-photon imaging), this method generates a 3D view of the nerve with unprecedented resolution. In Aim 1 we will develop a double transgenic mouse reporter model (THY-1/CFP and S100/GFP) expressing different levels of SARM1 (SARM1-/-, SARM1-/+, SARM1+/+). We will then monitor differences in the degree and timing of axonal degeneration after a unilateral sciatic nerve injury, using our optical window. In Aim 2, we will synthesize a library of small activatable fluorogenic probes that mimic NAD+ to directly measure the activity of SARM1 in the distal stump. Overall, this imaging approach will provide direct visualization of the morphologic and metabolic characteristics of the distal stump degeneration in live animals. It will also establish a conceptual framework for future investigation of the fundamental processes during nerve regeneration. This approach will lead to new discoveries in the biology of these processe...