Mechanisms Of Modulating Effect Of Anterior Cingulate Cortex On Visceral Hyperalgesic Behaviors In The Chronic Pancreatitic Rat

Background & Objectives:Chronic pancreatitis(CP)is a progressive inflammatory disease,in which pancreatic exocrine substance is destroyed and replaced by fibrous tissue.The main manifestation is postprandial abdominal pain in the upper abdomen(sometimes radiates to the back)and is often accompanied with nausea and vomiting.Abdominal pain,is the most prominent symptom and major clinical challenge in CP.It is the main reason for hospitalization in up to 90% of patients,and also a huge problem faced by clinicians when they treat CP.Although the study of CP-induced abdominal pain has undergone a transition from pancreatic anatomical mechanisms to neurobiological mechanisms,it has not yet fully revealed the neural pathway of visceral nociceptive information in CP and its regulation mechanism in central nervous system.It has been well-documented that part of the pancreatic sensory afferent fibers is transmitted through the spinal nerves of the bilateral T4~L4 segments,and is usually transmitted through the spinal dorsal horn into the brain regionslike insula and the somatosensory cente by spinal thalamic tract(STT).Another part of the afferent fibers is transmitted via the bilateral vagus nerve and projected into the nucleus tractus solitarii(NTS).The projection fibers of the NTS were transmitted through the parabrachial nucleus to the emotionslimbic system and the advanced cognitive center.NTS is an important relay nucleus for visceral sensation,yet how it relates to pain-related cortex and its functional regulation remains elusive.In recent years,anterior cingular cortex(ACC)has received more and more attention in terms of cognition,emotions and pain.Some studies have observed the reorganization of the structure and function of ACC in CP patients and animal models with electroencephalography(EEG)and functional magnetic resonance(fMRI).In addition,CP patients still have residual pain after resection of the pancreas,suggesting that central sensitization of ACC plays a key role in maintenance of visceral pain caused by CP.However,how the CP nociceptive information is transmitted to ACC via the NTS,whether there are direct or indirect fiber connections,and how ACC participates in regulating the mechanism of CP visceral hypersensitivity remains unclear.Therefore,we established trinitrobenzene sulfonic acid(TNBS)-induced CP visceral pain model in rats.By combining neural tracing,molecular biology,electrophysiology,and behavioral methods,we tend to 1)explore direct neural projections from NTS to ACC;2)examine the plasticity and synaptic transmission of pyramidal neurons within the ACC and the morphological and functional changes of glutamate receptors in CP visceral pain;3)explore the role of ACC in the CP visceral hyperalgesia by combining pharmacology,optogenetics and chemical genetics.Methods:(1)CP model was established by injecting trinitrobenzene sulfonic acid(TNBS)into the pancreaticobiliary tract of SD rats.H&E staining was used to observe the pathological changes of the pancreas.(2)The von Frey filaments,open-field test,elevated maze test,rotarod test and forced swimming test were used to observe the behavioral changes and to determine the phasic behavioral changes.(3)The rAAV-CaMKIIα-EYFR-WPRE-pA was injected into the NTS,and the fiber connection within the brain was observed.Immunohistochemical staining was used to detect the expression of FOS within ACC and NTS after operation.(4)Med64 multi-site field potential Methods were operate to examine the changes of multi-site LTP in ACC.Spontaneous excitatory postsynaptic currents(sEPSCs)and paired pulse facilitation(PPF)were recorded by whole-cell patch-clamp technique in pyramidal neurons of ACC V layer.(5)The expression of vesicular glutamate transporter 1(VGluT1)in ACC was observed by both immunohistochemical double staining and Western blot Western blot was used to detect the expression of glutamate N-methyl-D-aspartate(NMDA)receptor and α-amino-3-hydroxy-5Methyl-4-isoxazolepropionic acid(AMPA)receptor and phosphorylation level.Immuno-electron microscopy was used to observe the distance relationship between AMPA receptor subtype GluR1 and postsynaptic membrane.(6)Bilateral lesion of ACC in rats by using Kainic acid(KA)and the mechanical visceral pain was detected.In pharmacological experiments,bilateral catheter were placed within the ACC one week ahead of the establishment of the CP model.Three weeks later,NMDA receptor antagonist DL-2-amino-phosphoric acid(DL-AP5)and AMPA receptor antagonist6-cyano-7-nitro-quinoxaline-2,3-dione(CNQX)were microinjected into bilateral ACC through cannula.The visceral pain induced by mechanical stimulation and behavioral changes were detected one hour later.(7)Optogenetic regulation was used by injecting the rAAV-CaMKIIα-ChR2-mCherry virus into bilateral ACC of CP rats and sham rats.All animals were allowed one week of recovery and then optical fibers were implanted above the ACC.Three weeks later,mechanical visceral pain and open field experiments were detected with 473 nm blue laser.(8)Using chemical genetics method,the rAAV-CaMKIIα-hM4Di-mCherry was injected into the bilateral ACC of CP / sham rats.Three weeks later,CNO was given intraperitoneally.After 1 hour,the visceral pain response induced by mechanical stimulation and the open field test were performed.Results:(1)Preparation of rat CP model and morphological changes of pancreas.Compared with sham rats,CP rats showed slower increase of body weight.Pathological changes like the reduction of pancreatic lobular acini,atrophy,interlobular septal thickening,edema and lymphocyte infiltration were also observed in CP rats(2)Pain behavior of CP model rats and its influence on high brain activity.7-28 days after the surgery,the abdominal withdraw threshold(AWT)of CP rats decreased significantly.The total active distance and the percentage of central moving distance in open field test decreased.The results of elevated plus-maze test showed that the total crossings and the percentage of time in open arms significantly decreased.In forced swimming test,CP rats did not move for an extended period of time.The results of rotarod test showed that there was no significant difference in drop latency compared with control group.(3)Morphological evidence of CP pain information ascending to ACC via NTS.3-4 weeks after virus injection into NTS,the sections were observed directly.The results showed that the terminal of the NTS projecting neurons could be seen in ACC.At different time points,neurons expressing FOS could be found in NTS and ACC of CP rats.Significant differences could be observed in the expression of FOS in ACC between layer II-III and V-VI.The changes of FOS immunoreactivity(FOS-ir)neurons in NTS and ACC was consistent,and there was a positive correlation between them.(4)Plasticity of synaptic transmission in pyramidal neurons of ACC in rat model of CP.Med64 multi-channel field potential recording demonstrated that the amplitude of LTP in CP rats decreased significantly,and the number and distribution of LTP-producing channels decreased.Whole-cell patch-clamp recording revealed that the frequency and amplitude of sEPSCs increased in V layer of ACC in CP rats,and the amplitude of PPF in pyramidal neurons in CP rats decreased.AMPA receptor and NMDA receptor-mediated excitatory postsynaptic currents were enhanced,and the excitability of ACC pyramidal neurons in CP rats was observed in current clamp mode.(5)Plasticity of glutamate receptor expression and distribution in ACC of CP rats.Immunohistochemistry and Western blot confirmed that VGluT1 expression was up-regulated in ACC of CP rats.Western blot analysis of membrane proteins and plasma proteins showed that CP rat membrane protein Glu R1,p GluR1,NR2 B,p NR2 B were upregulated,while the expression of plasma protein GluR1,pGluR1,pNR2 B remained unchanged and the expression of NR2 B decreased.Immunoelectron microscopy results showed that GluR1 positive marker particles in ACC of rats were mainly located in postsynapses.In ACC of CP rats,the distance of GluR1 positive particles to the postsynaptic membrane decreased,indicating that they had a tendency to move towards the active zone of the synapse.(6)Effects on the visceral pain and high brain function in rats with bilateral lesions of ACC or glutamate receptor antagonists administration in CP rats.Bilateral lesions of ACC by KA could significantly improve AWT of CP rats.Compared with control groups,total distance and the percentage of central distance of activity decreased significantly in CP rats in open field experiments.ACC-application of AMAP receptor antagonist DL-AP5 and NMDA receptor antagonist CNQX could significantly increase the CP rat’s AWT.In the open field test,the percentage of central distance increased significantly.(7)Effects of activation and inhibition of pyramidal neurons activity in ACC of CP rat on visceral pain behavior and advanced brain function by optogenetic and pharmacogenetic approaches.Photoactivation of ACC pyramidal neurons did not affect AWT in CP rats.In the open field test,there was no statistical difference in total and central distance traveled neither between CP rats and control group nor between that of CP rats before and after stimulation.Administration of CNO increased the AWT and motor exacerbation in CP rats by activating genetically engineered muscarinic receptor in ACC cells,but had no effect on control group.Conclusions:(1)NTS neurons sent direct neural projections to ACC.TNBS-induced chronic pancreatitis caused behavioral changes.FOS-ir neurons increased in NTS and ACC in CP rats which were both time-phasic.The general locomotor ability decreased in CP rats which might be related with anxiety-like emotions.(2)In the state of CP visceral pain,the excitatory synaptic transmission in ACC was enhanced and neural plasticity was observed in CP state,which was partially related to the release of glutamate and its receptor expression.(3)In the state of CP visceral pain,activating pyramidal neurons in ACC could not aggravate the visceral pain and negative emotions of CP rats;Inhibition of pyramidal neurons in ACC could effectively relieve the visceral pain and negative emotions of CP rats.The above results suggest that ACC may be a new therapeutic target for visceral hyperalgesia of chronic pancreatitis.