Distribution and Morphology of Spinal Afferent Innervation of the Gastric Muscular and Submucosal Layers of Rats

Chronic pain affects 25 million Americans, with 2 million developing opioid addiction due to inadequate treatment options. Visceral chronic pain, particularly abdominal pain, is a significant clinical issue; however, the neural mechanisms driving this pain are poorly understood. Our research focuses on the spinal afferent innervation of the stomach, a key pathway for visceral pain transmission. Although many studies have explored vagal afferents, the role of spinal afferents remains under-investigated, primarily due to limitations in visualization and tracing techniques. Previously, our lab determined and characterized the spinal afferent innervation in the stomach muscular wall, identifying muscle, ganglionic, and mixed-type endings. Building on this work, we conducted a study to characterize spinal afferent nerves in the submucosa of the Sprague Dawley rat stomach (male, 3-5 months). Using anterograde tracing techniques, we injected dextran-biotin into the left dorsal root ganglia (T7-T11) to label spinal afferent axons and terminals in the muscle and submucosa of the stomach. The stomach muscle and submucosa were separated from the mucosal layer, and they were further separated to produce two flat mounts of muscular and mucosal layers. Tracer-labeled spinal axons were traced using the Neurolucida tracing software, leading to detailed distribution maps of spinal afferent innervation. High-resolution photomicrographs of spinal afferent axon morphology were captured using a Zeiss M2 imager. The results were: 1) Spinal afferents were found in all regions of the stomach muscle and submucosa (corpus, fundus, antrum, cardia). 2) In the muscular layer, spinal afferent axons target specific structures, with varicose contacts with longitudinal and circular muscles, myenteric ganglia, and blood vessels. 3) In the submucosal layer, spinal afferent formed varicose axons traveling in connective tissues andalong blood vessels. 4) Spinal afferent innervation of the flat-mounts of the whole muscular and submucosal layers were mapped and intergrated into a 3D stomach scaffold. These findings bridge a gap in our understanding of spinal afferent distribution and morphology in the muscle and submucosa and provide an anatomical foundation for future functional studies and pathological remodeling. Supported by NIH 1 U01 HEAL/SPARC NS113867-01(ZC), NIH R15HL137143-01A1(ZC). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.