The goals of this proposal are to establish a method to repetitively image injured spinal axons in live mice with two-photon microscopy over an extended period of time and to start to explore this methodology to study spinal axon regeneration following various manipulations, including growth stimulation and disrupting axon growth inhibitors.
The capacity to regenerate axons after traumatic injury in the adult mammalian central nervous system (CNS) is rather limited. Injured axons attempt to regenerate, but this attempt generally fails in the CNS. The failure of CNS axon regeneration underlies the permanent functional deficits and paralysis in spinal cord injury patients. Understanding the molecular mechanisms of CNS axon regeneration failure will significantly enhance our ability to design rational therapeutic intervention for spinal cord injury, white matter stroke, and certain neurodegenerative disorders. Several hypotheses have been intensively investigated to explain the failure of CNS axon regeneration, including a lack of intrinsic growth potential in the CNS neurons, a lack of growth promoting factors or tissue bridges, and the presence of axon growth inhibitors that actively block axon regeneration.
Despite rapid progress in this field in the past decade, it remains difficult to convincingly demonstrate axon regeneration after various cellular and molecular interventions in animal models of spinal cord injury. Conventional methods entail tract tracing and immunohistochemistry in end-point analyses to assess axonal response to injury. A major problem in such analyses is the difficulty in distinguishing regenerated axons from axons that have been spared by incomplete lesion. The ability to follow axonal response in real time and at various time intervals after injury would be highly desirable, as it would allow for unequivocal identification of regenerating axons and for temporal dynamics of axonal response to injury to be revealed.
We have experimented with in vivo imaging of genetically labeled sensory axons in live mice with two-photon microscopy. Here we present preliminary data to prove feasibility of this approach and propose to develop this technology to monitor axonal response to injury for up to 8 weeks after injury. We then propose to validate this technology as a novel method to analyze axon regeneration by examining the effect of two manipulations that are known to promote sensory axon regeneration: cAMP elevation and a conditioning peripheral nerve lesion. Finally, we propose to apply this technology to examine the role of Nogo and NgR, which have been hypothesized to be a major myelin-derived axon growth inhibitor and receptor respectively, and to determine whether disrupting such inhibitory molecules have a synergistic effect with growth stimulation.
Together, such analyses will reveal axonal dynamics after injury that have not been possible with conventional methods following various genetic, pharmacological, and surgical manipulations, and will provide a proof of principle for future studies of spinal axon regeneration. Furthermore, our in vivo imaging experiments involve laser ablation of single axons that will minimize scar formation, which would allow us to separate the contribution of myelin inhibitors from that of glial scar to CNS axon regeneration failure.
