A validated set of neural gene reporter mice and chemical tracers tools for mapping knee innervating neurons

Joint pain is an increasing concern for our aging population, as current therapies to slow joint disease progression or reduce pain are largely ineffective and often carry significant health and dependency risks. Age and joint disease induce changes to all tissues that make up the joint, including the dense neural network that innervates the joint. Several studies have correlated some joint innervation changes in joint diseases such as osteoarthritis or rheumatoid arthritis, but little is known about their respective functional consequences. In general, knee innervating neurons are classified as either non/peptidergic nociceptive or sympathetic neurons. How subtypes of these neurons affect individual pain experience remains relatively uncharacterized. The few studies typically focused on a single neural subtype due to the limited availability of validated tools to study joint innervation1-5. To better understand the relationship between aging, joint disease, and pain, systematic characterization of pain nociceptors and other neural subtypes that regulate joint homeostasis and pain signaling is urgently needed. The objective of this study was to establish a validated molecular and genetic toolbox for accurate mapping of the neuroarchitecture in the murine knee. We screened a panel of genetic reporter mice, consisting of Cre and Flp recombinase driver mouse lines that activate recombinase responsive reporter alleles in either peripheral nociceptors or post-ganglionic sympathetic neurons, for their specificity and accuracy in labeling their respective neural subtype in the dorsal root ganglion and knee joint. In addition, we compared the performance of a series of conventional retrograde tracers for effective tracking of primary afferent sensory and post-ganglionic sympathetic neurons from the knee joint. The validated molecular and genetic tools identified in this study will facilitate the creation of comprehensive joint innervation maps in physiological and pathological contexts, setting the stage for identifying the cellular and molecular changes responsible for mediating joint pain, a necessary goal for improved therapeutic interventions.