Objective: Enteric nervous system dysfunction is linked to digestive and neurological disorders. Hirschsprung's disease (HSCR) is characterized by the loss of enteric neuron cells (ENCs) in the distal colon. Embryonic enteric neural crest cell (ENCC) migration defects contribute to HSCR development in some infants, but postnatal factors that regulate ENC fate are undetermined. We sought to establish how postnatal changes contribute to HSCR by profiling the colonic microenvironment of HSCR infants. Design: In this study, we recruited infants with HSCR, infants with anorectal malformations but normal ENC development (CT), and an group of age-matched healthy control subjects. Laboratory findings and clinical manifestations were recorded. Single-cell and spatial transcriptome sequencing were applied to colonic tissues from a sub-cohort of CT and HSCR infants. Patient specimens, a mouse model of neonatal ischemic enterocolitis, and Sox10 knockdown mouse (Sox10WT/MUT) were used to reveal the factors that leads to ENC loss in HSCR infants. Results: We discover that intestinal ischemia promotes CLDN1+ hypertrophic nerve trunk formation and ENC death. Mechanistically, ischemia leads to defective nitric oxide (NO) signaling in ENCs, which aggravates mitochondria damage and caspase-mediated apoptosis that can be ameliorated by a NO donor drug. Conclusion: We show that ischemia contributes to postnatal ENC loss in HSCR infants and suggest that NO donor drugs may alleviate ischemia-related ENC death.

The authors have declared no competing interest.

This project was jointly supported by the National Natural Science Foundation of China (82125015 to Yuxia.Z., 81970450 to Yan.Z., 82100582 to W.L.), China Postdoctoral Science Foundation (2021TQ0083 to W.L.), Science and Technology Planning Project of Guangdong Province (2019B020227001 to H.X.), Guangdong Basic and Applied Basic Research Foundation (2021A1515220146 to Yan.Z.), and Guangzhou Basic Research Plan City School (Institute) Enterprise Joint Funding Project (SL2024A03J01 to Yan.Z.)

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Medical Ethics Committee of Guangzhou Women and Children's Medical center gave ethical approval for this work

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All data produced in the present study are available upon reasonable request to the authors