PIEZO2 IS UPREGULATED IN DRG TO MEDIATE CGRP UPREGULATION, COLONIC NEUROGENIC INFLAMMATION AND HYPERSENSITIVITY IN STRESS

BACKGROUND: Stress is a strong contributor to the development and exacerbation of irritable bowel syndrome. Visceral sensation is initiated in dorsal root ganglia (DRG), manifested by neurochemical changes and modulated by environmental cues. Sympathoneuronal outflow into DRG has been documented in many pain modalities including visceral pain. Norepinephrine (NE) release into DRG by sympathetic nerve fibers can regulate the activity of DRG neurons. Piezo2 as a mechanotransducer and calcitonin gene-related peptide (CGRP) as an excitatory neurotransmitter play crucial roles in pain. CGRP release to peripheral organs contributes to neurogenic inflammation. AIMS: (1) Characterize the role of Piezo2 in CGRP production in DRG and colonic neurogenic inflammation in stress-induced colonic hypersensitivity; and (2) Measure the action of NE on Piezo2⁺ (expressing) mechanosensory neurons in mediating CGRP upregulation in DRG. METHODS: Conditional deletion of Piezo2 from nociceptors (Nav1.8⁺) was achieved by Cre-LoxP technique to generate Nav1.8-Cre^+/-;Piezo2^fl/fl (Piezo2^cKO-Nav1.8) mice. Nav1.8-Cre^+/-;Piezo2^wt/wt (Piezo2^wt-Nav1.8) mice served as wildtype control. Optogenetics was used to activate Piezo2⁺ DRG neurons. Water Avoidance (WA) stress was used to induce visceral hypersensitivity. Sympathoneuronal outflow to DRG was assessed by NE analog fluorescent false neurotransmitter (FFN 270) uptake. Immunohistochemistry and qPCR were used to assess neurochemical changes in DRG. Colonometry was used to measure colonic mechanosensing. Macroscopic visualization and H&E stain were used to assess colonic inflammation. Von Frey test was used to measure somatic mechanical pain. Intact mice and DRG explant culture were utilized. RESULTS: WA stress upregulated Piezo2 and CGRP in DRG neurons in wildtype mice, with an increase in Piezo2 and CGRP co-localization when compared to unstressed mice. Increased FFN 270 uptake in DRG was evident following WA stress. In culture, NE treatment of DRG explants resulted in an upregulation of Piezo2 and CGRP in DRG neurons. NE-induced CGRP upregulation was present in Nav1.8⁺ DRG neurons and was reduced by Piezo2^cKO-Nav1.8. Optogenetic activation of Piezo2⁺ DRG neurons increased CGRP expression measured at both the protein and transcription levels. In vivo, Piezo2^cKO-Nav1.8 attenuated WA stress-induced CGRP upregulation in DRG, colonic neurogenic inflammation and hypersensitivity, when compared to WA stress in Piezo2^wt-Nav1.8 mice. Piezo2^cKO-Nav1.8 also reduced WA stress-induced somatic mechanical pain, suggesting mechanistic generality. CONCLUSION: Stress-induced visceral hypersensitivity is accompanied by NE outflow to DRG to facilitate Piezo2-mediated CGRP upregulation in DRG neurons. Lack of Piezo2 in nociceptors blocked NE- or stress-induced CGRP upregulation, and subsequent colonic neurogenic inflammation and hypersensitivity.