Peripheral nerve injuries (PNI) affects nearly one million Americans every year leaving them with permanent motor deficits such as a loss in the stretch-reflex, deficiencies in limb coordination, and excessive muscle co- contraction. These inevitable outcomes persist even when peripheral nerve regeneration is successful, suggesting that central mechanisms might be responsible for poor motor recovery. One known phenomenon that occurs after PNI, that may be in part responsible for these deficiencies, is the disappearance of the central projections of proprioceptive IA afferents. Concurrently, after PNI central microglia become activated and signal the infiltration of peripheral macrophages into the spinal cord. Both of these cells are phagocytic and surround injured IA afferent synapses, but their exact roles in synaptic remodeling remain a topic of debate. The hypothesis of this proposal is that microglia and peripheral macrophages specifically recognize the central axons and synapses of IA afferents that are injured in the peripheral nerve and are directly involved in their degradation. We will use novel transgenic mouse models to genetically distinguish, for the first time, central microglia (CX3CR1-EGFP) from infiltrating macrophages (CCR2-RFP) and study their relationships with the central terminations of IA afferents. The knowledge gained will generate insights into novel therapeutic approaches for improving patient's quality of life by preventing the reorganization of motor circuits and improving motor function recovery following regeneration after nerve injuries.
Aim 1 : Decreasing central microglia activity and blocking peripheral macrophage infiltration preserves IA afferent synapses following PNI and results in improved motor function recovery. To test if microglia and peripheral macrophages are involved in the removal of IA afferent synapses, we will block their activity by intrathecally administering minocycline or a neutralizing antibody against MCP-1, which is necessary for peripheral macrophage recruitment. Furthermore, minocycline-treated animals will be used to determine if reducing neuroinflammation improves motor outcomes after nerve regeneration. Motor function will be tested by recording electromyographic (EMG) activity from ankle flexor and extensors during treadmill locomotion.
Aim 2 : Use live cell imaging to characterize the process by which central microglia and peripheral macrophages interact with injured IA afferent central axons and synapses. Both injured and uninjured IA afferents will be labeled with fluorescent tracers for live imaging experiments in adult spinal cord slices and their interactions with genetically labelled microglia or peripheral macrophages will be investigated. We will use two-photon microscopy and time-lapse imaging to observe their relationships. The nature of these interactions will allow us to infer how IA synapses are recognized for degradation and the mechanism(s) of their removal.
Peripheral nerve injury results in the permanent removal of IA afferent synaptic terminals from motoneurons ultimately leaving individuals with persistent motor deficits such as a loss in the stretch reflex and muscle co- contraction. We plan to investigate the roles of central microglia and peripheral macrophages in the reorganization of this central motor circuit that results in persistent poor motor performance after peripheral nerves regenerate. The results from this project will not only determine the mechanisms involved in the removal of these IA afferent synapses, it will also provide potential therapeutic approaches for preventing this maladaptive plasticity from occurring.
|Schultz, Adam J; Rotterman, Travis M; Dwarakanath, Anirudh et al. (2017) VGLUT1 synapses and P-boutons on regenerating motoneurons after nerve crush. J Comp Neurol 525:2876-2889|