Spinal cord Ca2+ imaging reveals glial-driven central sensitization in post-traumatic osteoarthritis

Abstract

Central sensitization may be defined behaviourally, cellularly, or molecularly; yet these can all vary depending on the model and duration. Current electrophysiological approaches are time and labour intensive. Here, we developed a Ca2+ imaging and analysis pipeline (CuMIN) that implements semi-automated detection and analysis of cellular Ca2+ activity in rodent spinal cord slices, from which distinct signatures were defined for various acute and chronic pain models. Spinal cord slices from male mice were isolated after inducing pathological pain in a variety of well-established surgical or pharmacological approaches, incubated in a cell-permeant Ca2+ indicator, and imaged with epifluorescence microscopy. Intensity and temporal features of spontaneous and glutamate-evoked Ca2+ events were processed by linear discriminant analysis to map unique clusters of activity for each pain model. The resulting activity map of spinal dorsal horn activity is substantially different in the surgical model of chronic pain induced by post-traumatic osteoarthritis (PTOA), which lacks clear mechanistic evidence of central sensitization. Specifically, the PTOA Ca2+ activity signature overlapped with chemotherapy-induced neuropathy and neuropathic pain models, both of which are associated with gliosis-induced central sensitization. We confirmed gliosis in the PTOA model by immunostaining IBA1 and GFAP and observed analgesic effects of intrathecal carbenoxolone, Gap27, and minocycline that targeted glial activity. These findings validate CuMIN as a sensitive and specific approach for defining the basic cellular signatures of spinal central sensitization, with the utility of identifying potential therapeutic targets and serving as a translational platform for novel drug discovery across various acute and chronic pain models.