Authors :
Presenting Author: Yan Guo, MS – Ningxia Key Laboratory of Cerebrocranial Disease, Incubation Base of National Key Laboratory, Ningxia Medical University, Yinchuan 750004, China
Jiali Zhao, MS – Ningxia Key Laboratory of Cerebrocranial Disease, Incubation Base of National Key Laboratory, Ningxia Medical University, Yinchuan 750004, China
Yan Zhang, MS – Ningxia Medical University
Jiaqi Wang, BS – Ningxia Key Laboratory of Cerebrocranial Disease, Incubation Base of National Key Laboratory, Ningxia Medical University, Yinchuan 750004, China
Yanan Gong, MS – Ningxia Key Laboratory of Cerebrocranial Disease, Incubation Base of National Key Laboratory, Ningxia Medical University, Yinchuan 750004, China
Jinhai Gu, PhD – Ningxia Key Laboratory of Cerebrocranial Disease, Incubation Base of National Key Laboratory, Ningxia Medical University, Yinchuan 750004, China
Yuanyuan Qiang, PhD – Ningxia Key Laboratory of Cerebrocranial Disease, Incubation Base of National Key Laboratory, Ningxia Medical University, Yinchuan 750004, China
Lifei Xiao, PhD – Department of Anesthesiology, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People’s Hospital, Shenzhen Institute of Translational Medicine, Shenzhen, 518000, China
Yangyang Sun, PhD – Ningxia Key Laboratory of Cerebrocranial Disease, Incubation Base of National Key Laboratory, Ningxia Medical University, Yinchuan 750004, China
Kang Ma, PhD – School of Basic Medicine, Qingdao University, 308 Ningxia Road, Qingdao 266071, China
Yujun Wen, PhD – Ningxia Key Laboratory of Cerebrocranial Disease, Incubation Base of National Key Laboratory, Ningxia Medical University, Yinchuan 750004, China
Rui Zhang, PhD – Ningxia Key Laboratory of Cerebrocranial Disease, Incubation Base of National Key Laboratory, Ningxia Medical University, Yinchuan 750004, China
Rationale:
Sudden unexpected death in epilepsy (SUDEP) is a leading cause of epilepsy-related mortality, with patients facing a significantly elevated risk compared to the general population. Seizure-induced respiratory arrest (S-IRA) has been identified as a key contributor. Activation of dorsal raphe nucleus (DRN) serotonergic (5-HT) neurons mitigates seizure-related respiratory suppression. Anatomically, DRN 5-HT neurons densely project to higher-order cortical regions, including the insular cortex (IC), which also participates in respiratory regulation. However, whether the DRN→IC 5-HTergic projection contributes to SUDEP remains unproven. This study aimed to investigate the role of this projection in a heat-induced SUDEP mouse model.Methods:
The study consisted of two parts. (1) Model construction: A heat-induced SUDEP model was established using Scn1a⁺/⁻ mice on the B6/129SF1 background during their peak spontaneous death period (P24–P28). Behavioral seizure scoring, cortical EEG, respiratory parameters, and c-Fos expression in DRN and IC neurons were recorded. (2) Projection manipulation: Four groups were included: Scn1a⁺/⁺-hM3Dq-DCZ, Scn1a⁺/⁻-hM3Dq-Saline, Scn1a⁺/⁻-hM3Dq-DCZ, and Scn1a⁺/⁻-hM3Dq-DCZ-Ondansetron (a 5-HT₃ receptor antagonist used to block circuit activation). AAV-TPH2-DIO-hM3D(Gq)-mCherry and AAV-retro-CAG-CRE-EGFP were injected into the DRN and anterior IC to selectively modulate the DRN→IC 5-HTergic projection and assess its protective effect. Results:
(1) SUDEP model establishment: Scn1a⁺/⁻ mice exhibited significantly lower body temperatures at first seizure, shorter latencies to first seizure and SUDEP, higher seizure scores, increased seizure frequency and duration, longer Racine stage 3–5 seizures, and more frequent high-grade seizures. EEG showed significantly elevated amplitude post-heat induction. Respiratory parameters, including peak inspiratory flow and tidal volume, were reduced. Kaplan–Meier survival curves showed 0% survival in Scn1a⁺/⁻ mice vs. 100% in Scn1a⁺/⁺. c-Fos-positive neurons were significantly reduced in DRN and IC. (2) Projection activation improved outcomes: Scn1a⁺/⁻-hM3Dq-DCZ mice exhibited a rightward shift in the survival curve, with survival increasing from 12.5% to 50%. This group also showed a significant reduction in seizure frequency and EEG amplitude, along with increased tidal volume and minute ventilation. In contrast, Scn1a⁺/⁻-hM3Dq-DCZ-Ondansetron mice displayed a leftward-shifted survival curve, decreased survival (to 12.5%), elevated seizure frequency and EEG amplitude, and respiratory suppression comparable to the Saline group.Conclusions:
Activation of the 5-HTergic projection from the DRN to the IC significantly improves survival, reduces seizure burden, and alleviates respiratory suppression in Scn1a⁺/⁻ mice subjected to heat-induced SUDEP. This projection plays a key protective role in the pathophysiology of SUDEP.
Funding:
Supported by National Natural Science Foundation of China (82301646); Natural Science Foundation of Ningxia (2023AAC02032); Open Research Project of Ningxia Key Laboratory for Cranial and Brain Diseases (LNZR202305).