Cumulative Effect Of Electro-Acupuncture On Visceral Pain And Spinal P38MAPK And C-Fos Expression In Goats With Visceral Hypersensitivity

Visceral hypersensitivity(VH)is considered as a significant characteristic of gastrointestinal(GI)disorders in humans and animals.It commonly refers to the development of either hyperalgesia,an enhanced response to a nociceptive stimulus and/or allodynia,a painful response to an innocuous stimulus with altered bowel movement and increased mucosal secretion.Visceral pain usually acompanies VH.VH or visceral pain has a considerable negative impact on daily lives,causes a huge economic burden,and creates tremendous pressure on healthcare systems worldwide.The small intestine is more prone to visceral inflammatory pain and hypersensitivity than other segments of the gut because of its greater length and enhanced peristaltic movement.The small intestine is the focus of common clinical presentations,and VH remains a difficult entity to treat because its etiopathogenesis is not completely understood.Most studies have investigated VH utilizing a colitis model.However,such model is not appropriate for studying the exact mechanism by which small intestinal inflammation(such as ileitis)induces VH because its location,duration,and central regulating mechanism are different.An animal small intestinal inflammation model is needed to study the mechanism underlying VH or pain and to evaluate the efficacy of its treatment.Increasing evidences suggest p38 MAPK play significant roles in mediating the generation and maintenance of inflammation and pain.However,it is not known whether the spinal p38 MAPK activation contributes for the development of VH.The current existing clinical pain management mainly includes the combination of agents from several drug classes such as opioids,antidepressants,antispasmodics/anticonvulsant agents,or nonsteroidal anti-inflammatory drugs.The long-term uses of these agents have prominent adverse effects including inadequate pain relief,addiction,tolerance,fatigue,and gastrointestinal and cardiovascular toxicity.It is imperative to explore a safe and effective anti-inflammatory and analgesic treatment with low-risk complementary therapy.Acupuncture,the ancient Chinese art,has been used for 3000 years for the treatment of various pain conditions.Now,this technique is popular throughout the world because it is characterized by the high therapeutic benefits and few adverse effects,when stimulating electric current is applied to acupoints with acupuncture needles.Some studies have demonstrated that EA is effective for visceral pain and VH.Other studies have shown that it inhibits inflammatory and neuropathic pains through down regulation of p38 MAPK.The c-Fos has been considered to be valuable markers for the identification of neuronal activation and thus have been used to map functionally related neuronal pathways in the CNS in response to peripheral sensory neuronal activation.However,whether EA modifies visceral pain or VH through its regulation of p38 MAPK is not clear yet.In the present study,TNBS was used to induce ileitis,and behavioral change scores were used to evaluate visceral pain intensity in goats.Then ileitis goats were treated with repeated EA to investigate cumulative analgesic effect of EA and its effect on p38 MAPK and c-Fos expression in the spinal cord.1.Visceral pain triggered by traction on the ileocecal ligament with ileitis in goatsObjective: To quantify the intensity of visceral pain using ileocecal ligament traction on TNBS-induced ileitis goats.Methods: Thirty-six male goats,weighing 20.05±2.1 kg,were randomly allocated equally into a TNBS-group(n=18)and Saline-group(n=18).Ileitis was induced via the injection of 30 mg 2,4,6-trinitrobenzenesulfonic acid(TNBS)dissolved in 30% ethanol into the ileal wall through a laparotomy.An equal volume of normal Saline was injected into the ileal wall of the Saline goats.Behavioral responses to tractions(2,4 and 6 Newtons)on the ileocecal ligament were observed on days 3,7 and 14.Six goats from each group received a laparotomy and partial intestinal resection for ileal sample collection immediately after behavioral testing on days 3,7 and 14.Ileal histopathological changes were assessed and concentrations of myeloperoxidase,IL-1β,IL-6 and TNF-α were investigated using ELISA.Results: The TNBS-treated goats exhibited remarkably increased macroscopic scores,mast cell counts and myeloperoxidase and TNF-α concentration on days 3 and 7 compared with the Saline group and increased microscopic scores and IL-1β and IL-6 concentrations on days 3 to 14.The TNBS-treated goats exhibited behavioral changes in response to traction in the same pattern as their microscopic changes and cytokines levels.The traction force positively correlated with pain behavioral responses.Conclusion: Traction on the ileocecal ligament of goats with ileitis provoked an apparent,stable and reproducible ileum-derived pain.The current model may be helpful in evaluating the efficacy of new drugs for the management of visceral pain and in investigating its underlying mechanisms.2.Cumulative effect of electro-acupuncture on p38 MAPK and c-Fos expression in the spinal cord of goats with visceral hypersensitivityObjective To study the cumulative effects of EA applications on visceral hypersensitivity(VH)and its interference with the spinal activation of p38 MAPK and cFos in TNBS-induced ileitis goatsMethods: Thirty goats were randomly allocated into: Saline group(n =9),Sham group(n =6),TNBS group(n =9)and TNBS+EA group(n =6).The goats were laparotomized and the visceral pain model was induced by administration of 30 mg 2,4,6-trinitrobenzenesulfonic acid(TNBS)dissolved in 40% ethanol(1.2 m L)into the ileal wall.In the case of control goats,an equal volume of normal Saline instead of TNBS solution was injected in the same manner.Three goats from Saline and TNBS groups were laparotomized for partial intestinal resection for ileal sample for the assessment of inflammation at day 7.EA(with frequencies 60 Hz and intensity of 3 m A)was applied to bilateral “Zusanli”(ST 36)acupoints for 30 min in a total of six times at days 7,10,13,16,19 and 22.Visceromotor responses(VMR)to colorectal distension(CRD)stimulation at 20,40,60,80 and 100 mm Hg were measured with electromyogram(EMG)for assessing visceral hypersensitivity.At day 22,expressions of p38 and c-Fos protein and m RNA in the spinal cord of the eleventh thoracic vertibrae(T11)were detected using western blotting and RT-PCR,respectively.The 6-cm ileum of each goat was excised from 15 cm proximal to the ileocecal junction for histopathological changes at day 22.Results: VMR,and protein and m RNA expressions of spinal p38 MAPK and cFos of TNBS group were increased(P<0.01)compared with control-group.TNBS+EA group showed lower VMR to CRD stimulation at strengths of 20,40,60,80 and 100 mm Hg than TNBS group did.Repeated EA at the bilateral Zusanli(ST36)acupoints reduced the mechanical hypersensitivity induced by TNBS-injection into the ileal wall of goats.The TNBS+EA goats exhibited a remarkably decrease(P<0.05)in microscopic scores,and protein and m RNA expression of spinal p38 MAPK and c-Fos compared with the TNBS goats at day 22.Conclusion: TNBS injection into the ileum induced transmural ileitis and VH up to day 22.Effect of repeated EA applications inhibited p38 MAPK and c-Fos m RNA and protein expression in the spinal cord of ileitis-induced VH goats.These data provide evidence that EA attenuates VH probably through down-regulation of p38 MAPK and cFos in the spinal cord.