Osteoarthritis Progression And Sensory Pathway Alterations

The overarching aim of this project has been to characterize the sensory innervation of the knee, in order to explain its role in painful knee osteoarthritis (OA). We have identified two fundamental processes underlying OA pain: (1) mechanical sensitization and (2) anatomical neuroplasticity of joint nociceptors. In the course of experimental OA, joint nociceptors become sensitized to mechanical stimuli, manifest as knee hyperalgesia (early disease) and weightbearing asymmetry (late). Concurrently, remodeling of joint innervation occurs, with sprouting of nociceptors into the synovium and the subchondral bone (SCB). Using transcriptomic data, we have generated compelling evidence that a specific subset of nociceptors, nonpeptidergic NP1, is absent from healthy joints but sprouts into the SCB of OA joints and mediates weightbearing pain. This NP1 subset is molecularly defined by druggable receptors, including the Mas related G-protein coupled receptor d (Mrgprd). MrgprD+ fibers have been shown to be mechanosensitive and hyperexcitable. Our pilot data indicate that Mrgprd knock out mice are protected from weightbearing deficits and sprouting after DMM. We aim to test the hypothesis that NP1 fibers represent a subset of nociceptors that can be targeted for OA pain. In Aim 1, we will establish the pattern of innervation by NP1 fibers in murine and human knees. We will document their spatio-temporal neuroplasticity in murine knees of both sexes (PMX, primary OA), and validate findings in human knee tissues from postmortem donors and total knee arthroplasty. We will also profile NP1 neurons in murine and human DRGs. In Aim 2, we will examine the effects of agents that can reverse the hyperexcitability and sprouting of NP1 neurons, focusing on Mrgprd inverse agonists and other drug candidates. Aim 2A will examine effects on excitability, using (1) electrophysiological recordings from optogenetically identified Mrgprd+ neurons, (2) excitation of Mrgprd- GCaMP6 neurons driven by knee manipulation, and (3) OA pain. Aim 2B will investigate the effects of these agents on sprouting of murine Mrgprd-eGFP or human iPSC-derived neurons, using microchips to assess axon growth. We will examine whether agents that reduce the excitability of these neurons also impact their sprouting. Aim 3 will examine the consequences of chronic inhibition of Mrgprd signaling using Mrgprd ko mice. We will perform PMX or sham surgery in mice and monitor them for pain-related behaviors, DRG molecular profiles, joint innervation, and joint integrity. We will also use Mrgprd ko x Pirt-GCaMP3 mice to perform in vivo Ca2+ imaging of DRG in order to assess the functional responses to mechanical stimuli as a result of decreased sprouting.