Paraventricular Thalamus-insular Cortex Circuit Mediates Visceral Hyperalgesia Induced By Neonatal Colonic Inflammation In Mice

Irritable Bowel Syndrome(IBS)is a common functional disorder,mainly characterized by abdominal discomfort,visceral pain,and changes in bowel habit.The pathogenesis of IBS has not been fully understood,thus clinical treatments are limited and there is no cure.Visceral hyperalgesia sensitivity is widely considered to be the main manifestation of IBS whereas its complex and diverse regulating mechanisms remain poorly understood,particularly the involvements of central nervous system.Recent studies suggest that Neonatal Colonic Inflammation(NCI)has been shown to simulate the characteristics of IBS,and laboratory studies had shown that the Insular Cortex(IC)was associated with visceral pain sensitivity,and that the Paraventricular Thalamus(PVT)projected to IC and PVT was also involved in pain.Therefore,this study employs NCI mice as the research model to explore the relationship between the visceral hyperalgesia and PVT-IC circuit,in order to provide novel therapeutic targets for clinical treatment.Aims(1)To study the regulative mechanism by which IC is involved in visceral hyperalgesia in NCI mice.(2)To explore the regulative mechanism by which PVT is involved in visceral hyperalgesia in NCI mice.(3)To investigate the regulative mechanism by which PVT-IC circuit is involved in visceral hyperalgesia in NCI mice.Methods(1)On the tenth day after birth,C57BL/6J mice were divided into experimental group(NCI)and control group(CON).Mice in NCI group were given 30 μL 0.5%acetic acid in the colorectum,and mice in CON group were given 30 μL normal saline.At the age of six weeks,colorectal distension(CRD)was used to assess the visceral pain threshold to determine whether NCI model was successfully established.(2)Immunofluorescence was used to detect the c-Fos expression in the IC and PVT brain regions of mice in the NCI and CON group,and to determine the neuron types.(3)In vivo optical fiber recording technology was used to detect calcium activity of glutaminergic neurons in IC brain region of mice in NCI and CON group under the same stimulation.(4)The changes of visceral pain sensitivity in NCI and CON mice upon optogenetic manipulation of glutaminergic neurons in IC and PVT brain regions were examined by the combination of optogenetic techniques and abdominal wall electromyography recording.(5)Viral neuronal tracing technology was used to explore the projective relationship between PVT-IC,and then immunofluorescence technology was employed to confirm the types of neurons involved in this projection.(6)The mechanism of PVT to IC neural circuit involved in visceral hyperalgesia of NCI mice was explored by combining optogenetic,chemogenetic techniques and abdominal wall electromyography.Results(1)Compared with CON mice of the same age,the pain threshold of NCI mice was significantly reduced,indicating that NCI mice had visceral pain,and that the mice visceral pain model was successfully constructed.(2)Compared with CON mice of the same age,the expression of c-Fos in IC and PVT of NCI mice was significantly increased,and c-Fos was mainly expressed in glutamatergic neurons.(3)Under the same CRD stimulation,calcium ion activity in glutamatergic neurons of NCI mice was significantly increased compared with CON mice of the same age.(4)Blue light activated IC and PVT excitatory neurons to induce visceral pain sensitivity in CON mice.Yellow light inhibited IC and PVT excitatory neurons to relieve visceral pain sensitivity in NCI mice.The acute activation of glutamatergic neurons in IC brain by blue light induced visceral pain sensitivity in VGlu2-Cre normal mice.Yellow light acute inhibition of IC brain region glutamatergic neurons alleviates visceral pain sensitivity in VGlu2-Cre NCI model mice.(5)Virus tracer results showed that PVT had a direct projection to IC.The retrograde tracer AAV2/R-hSyn-EGFP was injected into IC,and the morphology of neuron body was observed in PVT,which was colocalized with CaMKII.Virus AAV2/9-EF1α-DIO-EYFP was injected into IC and virus AAV2/1-CaMKIIα-Cre was injected into PVT.EYFP-labeled neuron bodies were found in IC and colocalized with CaMKII three weeks later.(6)Blue light activated glutamatergic neurons of PVT brain in CON mice and induced visceral pain sensitivity in normal mice.Chemogenetic virus inhibited glutamatergic neurons in IC brain and reversed visceral pain sensitivity in mice.Yellow light acute inhibited glutamatergic neurons in PVT brain region and alleviated visceral pain sensitivity,and this effect was blocked by chemogenetic virus activated glutamatergic neurons in IC brain region in NCI mice.ConclusionsAbnormal activity of glutamatergic neurons in PVT brain region is involved in chronic visceral pain sensitivity induced by NCI,and its role was mainly mediated by regulating the activity of glutamatergic neurons in IC brain region.