Subtypes of Enteric Neurons in Hirschsprung Disease: A Comparison of Fully Ganglionic and Transitional Zone.

BACKGROUND: Hirschsprung disease (HD) is characterized by aganglionosis of the distal bowel and an adjacent transitional zone with structural abnormalities of the enteric nervous system (ENS). While previous studies have focused primarily on neuronal density, it remains unclear whether the neurochemical composition of enteric neurons differs between transitional and fully ganglionic bowel. This study therefore aimed to characterize neuronal subtype composition in these regions of HD specimens. METHODS: Colonic specimens resected during pullthrough surgery from pediatric HD patients were analyzed. Chromogenic immunohistochemistry and multiplex fluorescent immunohistochemistry (HuC/D, nNOS, ChAT) were performed and analyzed across myenteric and submucosal plexuses. Digital image analysis (QuPath) enabled automated cell detection and subtype classification. Cell counts were normalized per segment length for density analysis. Neuronal subtype composition was analyzed by paired testing and complementary Bayesian Dirichlet-multinomial regression modeling. KEY RESULTS: HuC/D-, nNOS-, and ChAT-positive neurons were identified in both transitional and fully ganglionic bowel, with only minor differences in neuronal density. Paired analysis and Bayesian Dirichlet-multinomial modeling of neuronal subtype proportions indicated that the relative composition of major neurochemical neuronal subtypes was largely preserved between transitional and fully ganglionic bowel. Although expected differences in subtype distribution were observed between enteric plexuses, no consistent zone-specific alterations were identified. CONCLUSIONS & INFERENCES: Neurochemical subtype composition of enteric neurons is largely preserved between transitional and fully ganglionic bowel in HD. Within the limitations of this study, we did not detect major differences in neuronal subtype composition between transitional and fully ganglionic bowel that could explain persistent postoperative bowel dysfunction.