Intervertebral Disc Degeneration and Cross-Talk with the Nervous System

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Intervertebral disc (IVD) degeneration is one of the greatest contributors to low back pain, yet how the IVD can generate pain remains poorly understood. To date, our knowledge of “cross-talk” between degenerating IVD and sensory nerves involved in transmitting pain is limited to findings of altered protein and RNA expression in tissues of the IVD, the spinal cord or dorsal root ganglia (DRG). Recent advances in the imaging of sensory neuron activation via recording of Ca2+ sensitive fluorescent indicators, together with pre-clinical models of IVD degeneration, now enable the study of temporal and spatial changes in neuronal function and their “cross-talk” to changes in the degenerating IVD. We propose to evaluate action potential-driven Ca2+ transients and molecular changes in sensory neurons in a mouse model of injury-induced IVD degeneration. In Specific Aim 1, we will document temporal changes to pain-related behaviors and sensitivity in Thy1-GCaMP6s mice following puncture of a lumbar IVD to induce IVD degeneration. These mice carry a transgene for the calcium-sensitive fluorophore, GCaMP6s, that is expressed in sensory nerves of the DRG. We will also evaluate the presence of neuronal markers and key ion channels in innervating DRGs, and anatomic changes and nerve fiber infiltration in IVDs, to test for changes with IVD degeneration as compared to sham controls. This work will document molecular changes to IVD and DRGs for this model from 6 to 52 weeks of IVD degeneration, and test for relationships between injured IVD and the innervating lumbar DRGs as a first measure of “cross-talk.” In Specific Aim 2, we will evaluate action potential-driven Ca2+ transients in lumbar DRG neurons in the IVD degeneration model of Specific Aim 1. We will record Ca2+ transients in intact DRG of Thy1-GCaMP6s mice in vitro following electric field stimulation, and measure threshold voltage, 50% maximum (IC50), and numbers of responding DRG neurons and their nearest neighbor response. DRGs will also be tested before and after incubation with sodium channel blockers to screen for remodeling of specific ion channel function with periods of IVD degeneration. Our goal is to identify temporal and spatial changes in DRG function and “cross-talk” with changes of IVD degeneration. Finally, in Specific Aim 3, we will evaluate sensory stimuli-induced responses in the DRG of living mice using in vivo fluorescence microscopy. Working with 2-photon confocal microscopy and motion correction algorithms developed for brain imaging, we will identify the threshold response of DRG neurons in Thy1-GCaMP6s mice with and without IVD degeneration, following in vivo stimulation of brush, pinch, heat and cold. Our goal is to test for relationships between in vivo activation of lumbar DRG neurons with behavioral and sensitivity changes following onset of IVD degeneration. Completion of this study would identify functional changes to sensory neurons at sites distant to the degenerated IVD and reveal new information about IVD- nervous system “cross-talk” that may suggest novel interventions for treatment of discogenic pain.