Spinal Afferent Innervation of the Rat Stomach: Anterograde Tracing

Abstract

Nociceptive afferents from spinal dorsal root ganglia (DRG) and vagal nodose ganglia innervate the stomach and send nociceptive signals centrally to the brain and locally to the enteric nervous system. In our other two posters (Mistareehi et al. 2022, Nguyen et al. 2022), nociceptive afferents are detected with SP and CGRP markers. To specifically study spinal afferent innervation of the stomach, we injected tracer Dextran Biotin (DB) into the left DRG (T7‐T11; 1.5 µl/each ganglion). 14 days after tracer injection, rats were perfused through the left ventricle with 40℃ PBS (1M phosphate‐buffered saline, pH = 7.4). Tissues were then fixed by perfusion with 4% paraformaldehyde in 0.1 M PBS (4℃, pH 7.4). After the perfusion, the distal esophagus and the proximal duodenum were transected, and the stomach was removed. Next, the ventral and dorsal stomach walls were separated by an incision along the lesser and greater curvatures, thus yielding two flat‐mounts per animal. The external muscle wall of the stomach was then isolated as a whole mount by removing the gastric mucosa and submucosa. Ventral stomach flat‐mounts were examined using the Neurolucida system. We found that Tracer DB‐labeled axons formed serval categories of the terminal structures. The simple type: long varicose axons without branching; The branching type: axons with multiple branches running in parallel with either circular or longitudinal muscles; The complex type: single axons with multiple branches running in various directions in both circular and longitudinal muscle layers; The ganglionic type:axons innervating myenteric ganglia; The blood vessel type: axons innervating blood vessels. Our work will provide a foundation for specific labeling of spinal afferent innervation of the stomach. In the future, we will use tracer injection of DRG and nociceptive markers (e.g., SP, CGRP, TRPV1) to examine topographical innervation of spinal nociceptive afferents in the stomach. The first two authors contributed equally to this work.