The Effects And Mechanisms Of Nav1.9 Channels On The Intestinal Pain Sense And Motility In Mice

Background:The Nav1.9 channel is a voltage-gated sodium channel that regulates the excitability of some neurons.Nav1.9 channels are highly expressed in small diameter neurons in dorsal root ganglia.These neurons are the primary neurons of pain conduction in the trunk and extremities.Theαsubunit of the human Nav1.9 channel is encoded by the SCN11A gene.Pathogenic variants in the SCN11A gene can cause three genetic disorders,namely familial episodic pain,congenital insensitive to pain,and genetic small fiber neuropathy.Nav1.9 channels are also highly expressed in intrinsic primary afferent neurons and intestinofugal afferent neurons of the enteric nervous system,and may be involved in intestinal motility regulation.Scn11a^-/-mice exhibit enhanced colonic migrating motor complex.Visceral hyperalgesia and gastrointestinal dysmotility are important pathogenesis of irritable bowel syndrome.At present,the role and mechanism of Nav1.9 channel in intestinal hyperalgesia and dysmotility are still not completely clear.Therefore,this study constructed gene editing mice carrying gain-of-function variation of the Scn11a gene.Then we tested whether the model mice had intestinal hyperalgesia and dysmotility,and analyzed the underlying mechanisms.Methods:1.A pedigree with familial episodic pain was collected for genetic testing.The p.R222S mutation was detected in the SCN11A gene.And the p.R222S mutation was recombined into the homologous allele locus of the Scn11a gene in C57BL/6 mice using CRISPR/Cas9 gene editing technology.The Scn11a ^R222Sknock-in mice were constructed and the recombinant region was verified by Sanger sequencing.2.The alterations of somatic pain thresholds in Scn11a ^R222Sknock-in mice were detected using the Hargreaves and formalin tests.The changes of visceral（mainly refers to the bowel）pain sensitivity were tested using the abdominal wall withdrawal reflex,acetic acid writhing and formalin enema-induced visceral pain tests.3.We isolated and cultured dorsal root ganglion neurons innervating the distal colon of model mice.Then whole-cell patch clamp was used for electrophysiological analysis of small diameter neurons.4.The intestinal motility of the model mice was detected by in vivo carbon powder propelling test and in vitro intestinal segments mechanical contraction frequency measurement experiment.5.The levels of norepinephrine,substance P and vasoactive intestinal peptide in the intestinal tissue were detected by enzyme-linked immunosorbent assay.Depression scores were calculated using the tail suspension and forced swimming tests.Results:1.The gene-edited mice were constructed in this study,which carried the homologous allele p.R222S mutation of human SCN11A gene.2.The homozygous mutant knock-in mice(Scn11a ^R222S/R222Smice)had a shorter response time to thermal pain stimulation in the Hargreaves test,and a longer duration of pain behaviors in the formalin test.Scn11a ^R222S/R222Smice showed a lower pressure threshold for pain behaviors in the abdominal withdrawal reflex test,an increased number of writhing times in the acetic acid writhing test,and a prolonged duration of pain behaviors in the formalin enema-induced visceral pain test.3.The small diameter dorsal root ganglion neurons from the sixth lumbar vertebrae to the first sacral vertebrae of Scn11a ^R222S/R222Smice showed hyperexcitability.4.In Scn11a ^R222S/R222Smice,the intestinal carbon powder propelling rate was decreased,and the contraction frequency of isolated intestinal segment was decreased.5.The levels of substance P and vasoactive intestinal peptide in the small intestine of Scn11a ^R222S/R222Smice were significantly decreased,while the levels of norepinephrine were slightly increased.Depression scores in Scn11a ^R222S/R222Smice were not significantly different from those of wild type mice.Conclusions:1.Scn11a ^R222Sknock-in mice were successfully constructed in this study.Scn11a ^R222S/R222Smice partially simulated somatic hyperalgesia symptoms of patients with familial episodic pain.2.The gain-of-function changes of Nav1.9 channels cause intestinal hyperalgesia in Scn11a ^R222S/R222Smice.The small diameter dorsal root ganglion neurons that innervate the gut are hyperexcitability,which lead to intestinal hyperalgesia.3.The gain-of-function alterations of Nav1.9 channels result in intestinal motility inhibition in Scn11a ^R222S/R222Smice.Intestinofugal afferent neurons of Scn11a ^R222S/R222Smice may be hyperexcitability,which abnormally activates the entero-enteric inhibitory reflex.Then sympathetic nerves of prevertebral ganglia release more norepinephrine,which inhibits intestinal motility.In addition,primary afferent neurons in the enteric nervous system may be hyperexcitability,which abnormally activates the peristaltic reflex.That disrupts the pressure gradient required for peristalsis,which inhibits intestinal motility.