Following injury to the central nervous system (CNS) such as a spinal cord injury, axons have very limited ability to regenerate, leading to permanent functional deficits in patients. The central goal of this proposal is to gain a better understanding of the innate axonal responses to injury and of how such responses are regulated by factors intrinsic and extrinsic to the injured neurons. In addition to conventional models of experimental spinal cord injury, the proposed study proposes to directly visualize the degenerative and regenerative responses of fluorescently labeled spinal sensory axons to axonal injury in live mice with in vivo imaging using 2-photon microscopy. In Aim 1, the applicant will examine the injury responses of individual spinal sensory axons to the genetic deletion of Nogo, a major myelin-derived inhibitor of axon growth and/or neuronal PTEN, a negative regulator of axon regeneration in the adult CNS. This will probe a potential synergistic effect of manipulating both the extrinsic inhibitory environment and neuron-intrinsic growth potential. By using the new experimental paradigm of in vivo 2-photon imaging in conjunction with laser-mediated ablation of single axons, this study will provide novel insights on the action of Nogo and PTEN, including a definitive assessment of any axon regeneration and a description of the detailed dynamics of axonal responses to injury. In Aim 2, the applicant will examine the individual and combined effects of Nogo and PTEN deletion after spinal cord injury. The findings from this study will serve to validate and extend the findings from Aim 1. These insights will be important to the development of therapeutic strategies for spinal cord and other CNS injuries. In addition, the proposed study will serve as a precedent for using in vivo 2-photon imaging to evaluate the contribution of various extrinsic and intrinsic factors in regulating CNS axon regeneration. This intensive research plan is accompanied by a comprehensive training plan so that the applicant will acquire the skills necessary in order to develop into an independent researcher in future. PUBLIC HEALTH RELEVANCE: Due to the severity, permanence of functional deficits, and the dire effect on quality of life after traumatic spinal cord injury it is of great necessity to find a therapeutic intervention to improve the lives of those who suffer from these injuries. Using experimental spinal cord injury and a new experimental paradigm of in vivo imaging, the proposed study aims to gain novel insights on the innate response of spinal axons to injury and how this response is regulated by specific intrinsic and extrinsic factors implicated in spinal cord regeneration. A better understanding of how axon regeneration is regulated will aid in the design of effective therapies for spinal cord injury, other traumatic CNS injuries and certain neurodegenerative diseases.