| 【Background】Visceral sensation generated from internal organs. Noxious visceralstimuli contributes to economic burdens associated with lost workdays and productivity.Visceral pain accompanied with internal organs disease has a considerable negative impacton lives with psychological distress and contribute significantly to a large segment ofhealthcare resource consumption. Nevertheless, our current understanding of visceral painmechanisms falls further behind that of somatic pain, possibly because of greatercomplications associated with accessing visceral structures with adequate visceral stimuliin research models. It has been shown that afferent fibers innervating viscera project to theCNS through autonomic sympathetic and parasympathetic nerves, a dual sensory innervations. Some spinal afferents travel along hypogastric, lumbar colonic andsplanchnic nerves to terminate in thoracolumbar regions as part of sympathetic innervation,traversing both prevertebral and paravertebral ganglia en route to the spinal cord. Vagaland pelvic afferents respectively terminate in the brainstem and lumbosacral cord andcontribute to parasympathetic innervations. However, there still lacks detailedmorphological evidence for the pelvic visceral nociceptive transmission and modulationpathway in central nervous system. Furthermore, the central process of visceralinformation and emotional responses has not been revealed yet. Therefore research intothe mechanisms mediating visceral nociception and the role of higher centers inmodulating excitability through changes in biphasic descending modulation can provide abetter understanding of the differences between visceral neurotransmission and the morethoroughly explored signaling mechanisms of somatic stimuli. Ultimately, appreciatingthese contrasts and similarities between the development and maintenance of somatic andvisceral pain states and the means by which central excitability occurs in visceral disorders,in its own right, is also crucial for providing a better understanding of therapeutictreatments for visceral pain syndromes.【Aims】 To investigate the central mechanisms of pelvic visceral noxiousinformation transmission and modulation through acute and chronic animal visceral painmodels.【Methods】⑴Establishment of acute colitis model. The number of visceral painbehavior was calculated every15min during the180min observation window according;⑵Quantification of FOS expression in spinal cord through immunohistochemicalstaining;⑶Quantification of NK1R internalization in spinal cord throughimmunofluorescence staining;⑷Observation of visceral pain behavior after intrathecalinjection of NK1R inhibitor L732138;⑸After injection of retrograde tracer Fluoro-Gold(FG) into the right LPB, colorectal distension was introduced to induce the visceralnociceptive stimuli. The coexistence of retrograde FG and neuronal activation marker FOSwithin the DCN neurons was used to identified DCN-LPB projection neurons that receiveand transmit colorectal derived visceral nociceptive information;⑹Wheat-germ agglutinin (WGA) was injected into the pelvic nerve (convey visceral nociceptiveinformation);⑺Biotionylated dextran amine (BDA) was injected into sciatic nerve(convey somatic nociceptive information) nerves;⑻After injection of retrograde tracerFG (or HRP) into the right LPB, anterograde tracer WGA and BDA were injected into thepelvic and sciatic nerves, respectively. Confocal microscope observation of thedistribution of WGA-immunoreactive (WGA-ir) terminals, BDA-immunoreactive(BDA-ir) terminals and FG-immunoreactive (FG-ir) neurons in DCN. Electronmicroscope observation of the WGA-ir axons, BDA-ir axons and HRP-immunoreactive(HRP-ir) neurons in DCN;⑼Establishment of zymosan induced chronic colitis model.The number of visceral pain behavior was calculated in ten minutes;⑽The spontaneousresponse and locomotor activity of model animals were tested by open field (OF);⑾Theanxiety-like behaviors were tested by in the elevated plus maze (EPM) and light/dark box(LDB);'The counts of FOS immunoreactive (FOS-ir) neurons in the brain regionswere represented as the average number of neurons per section;⑿NB001or gabapentinwas intraperitoneal injected into ZIC mice45min prior to behavior test on day1st,7th,14thand28th;'Western Blot detection of GluA1expression in anterior cingulate cortex(ACC) was performed on ZIC mice;'Paired pulse facilitation (PPF), miniatureexcitatory post synaptic currents (mEPSC) and input-output curve were recorded by wholecell patch recording on ACC of ZIC mice;'Late phase long term potentiation (L-LTP)were recorded by Med64multi-electrode array systems on ACC of ZIC mice.【Results】⑴After formalin instillation, animals showed a two-phase visceral painresponse. The first phase reached a peak at15min following injection and a second PSpeak occurred90min after injection;⑵FOS was expressed in many neurons in the L6and S1spinal cord after formalin instillation. FOS-ir neurons were primarily distributed inthe DCN, with a few cells also observed in the IML and the medial aspects of laminae Iand II of SDH. The number of FOS-ir neurons increased significantly at15min andreached the maximum at30min;⑶After formalin instillation, NK1R endocytosis wasgreatly enhanced in the DCN neurons, with clumped endosomes distributed in thecytoplasm and dendrites;⑷The intrathecal injection of L732138significantly inhibited the pain behavior, but the effect was not persistent. It was also observed that L732138regulated the NK1R internalization effect. And L732138was involved in the regulation ofFOS expression;⑸After FG microinjected into PBN, the FG retrogradely labeledneurons were mainly found in the DCN and expressed FOS protein under the condition ofacute colitis;⑹After FG injected into PBN, WGA injected into pelvic nerve and BDAinjected into sciatic nerve, WGA or BDA labeled primary afferent fibers (PAFs) wereobserved from the L6to S1spinal cord;⑺Under confocal microscope, both the visceral(WGA-ir) and somatic (BDA-ir) PAFs converged onto a same DCN neuron whichprojected to the LPB (FG labeled neurons). Under electron microscope, WGA-ir andBDA-ir boutons formed both asymmetrical and symmetrical synapses with the HRP-irDCN neurons.⑻After three consecutive days transanally instilled with zymosan, ZICmice developed long-lasting abdominal visceral pain;⑼When test in OF, the vertical,ambulatory, stereotypic, jump counts and travel distance decreased significantly ondifferent stage of ZIC mice;⑽ZIC mice spent less time in the open arms of the EPMand the light compartment of the LDB apparatus;⑾FOS highly expressed in theanterior cingulate cortex (ACC), prefrontal cortex (PFC), insula cortex (IC), amygdale(Amy), periaqueductal gray (PAG), parabrachial nucleus (PBN) and solitary nucleus (Sol)of ZIC mice;⑿Intraperitoneal injection of NB001, an adenylyl cyclase1inhibitor,could relieve the increased spontaneous pain but had no significant effect on behavioralanxiety on ZIC mice;'Intraperitoneal injection of gabapentin, which originallydeveloped to treat epilepsy, could reduce both ZIC related long term spontaneous pain andbehavioral anxiety;'Expression of GluA1in ACC was upregulated after ZICcompared with control group and last a long term;'Chronic visceral noxious stimuli ofZIC caused PPF, mEPSC and I-V curve changed in ACC;'ZIC caused reduced L-LTPin ACC.【Conclusions】（1）In the present study, colon formalin instillation induced a two-phase visceralpain response that consisted of an acute phase (before30min) followed by a tonic phase(75-105min)。 （2）Colon formalin instillation induces strong FOS expression and significant NK1Rinternalization in the DCN. These data highly suggest that NK1R internalization correlateswith formalin-induced pain behavior. Intrathecal pretreatment with the NK1R antagonistL732138attenuates NK1R internalization, FOS expression and the nociceptive responses,but the effect could not last a long time.（3）Both visceral (via pelvic nerve) and somatic (via sciatic nerve) primary afferentfibers (PAFs) make close appositions (potential convergence) onto the same DCN neuronthat projects to the LPB. The results have suggested that neurons within DCN might be theconverging loci for pelvic visceral nociceptive and lower limbs somatic non-nociceptiveinputs, which provide morphological evidences to clinical referred pain.（4）Serious visceral pain and anxiety could be induced in animals with ZIC, whichmay be closely related to the activation of cortical areas.（5）For translational medicine point of view, NB001relieved the visceral pain buthas no significant effect on behavioral anxiety on ZIC mice, whereas gabapentin reducesboth ZIC related visceral pain and anxiety.（6）Chronic visceral pain causes development of cortical plasticity in ACC. Thiscortical plasticity related to pain process and emotional responses. Our findings suggestthe important role of ACC neuron in visceral pain and pain related negative emotion. Theresults also produce a possible modulation target in visceral pain therapy. |
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