Effect Of Electro-acupuncture On Expression Levels Of Glutamate Transporter EAAT2 And NMDAR-2B In Spinal Cord In Goats With Chronic Ileitis

Inflammatory bowel diseases(IBDs)are common chronic inflammatory disorders of the gastrointestinal tract in human and animals.The major symptoms are visceral hypersensitivity(VH,an enhanced visceral pain with altered bowel movement,increased mucosal secretion,weight loss,malnutrition,fever and lack of appetite).VH or visceral pain is an important characteristic of IBDs,but VH in ruminants is rarely reported.Most studies have investigated VH utilizing a colitis model.However,such model is not appropriate for studying the exact mechanism by which ileitis induces VH because its location,duration,microbiota and central regulating mechanism are different.There is an urgent need to establish a small intestinal inflammation model to study the mechanism underlying visceral pain or VH and to appreciate efficacy of pharmacological and nonpharmacological treatments.Pain sensitization or VH is a big challenge for clinicians and become a very thorny work in veterinary practices,which includes pharmacological and non-pharmacological treatment modalities.Sometimes pharmacological treatment is unsatisfactory with profound side effects such as immune suppression,intestinal hemorrhage,mental retardation,addiction.Electro-acupuncture(EA)is a non-pharmacological treatment which is generally safe and effective when applied appropriately by the trained practitioner.EA is relatively simple to operate and its parameters can be easily controlled,so it is more appropriate for clinical application.EA analgesia involves a variety of neural and hormonal factors in peripheral and central nervous system.Research on the central modulating mechanism of EA analgesia is becoming an interesting topic.Accumulating evidence shows that EA can modulate central descending pathway to release neurotransmitters(serotonin,norepinephrine and glutamate)or neuromodulators(opioids)to suppress nociception.There is little concern about effect of EA on Glutamate Nmethyl-D-aspartate(NMDA)receptors and Glutamate transporters(the only vehicle for removal of glutamate from the extracellular fluid)in central nervous system in chronic pain.Studies show that activation of NMDA receptors NR2 B subunit contributes to pain sensitization after tissue and nerve injury,in the spinal cord.Inhibition of NMDAR-2B or enhancement of glutamate transporter(excitatory amino acid transporter;EAAT2)has been shown to attenuate somatic and neuropathic pains and VH in different animal pain models.However,it is not clear whether EA modifies VH through reducing the NMDAR-2B activation and enhancing the glutamate transporters EAAT2 level in the spinal cord.In the present study,TNBS was injected in ileal wall to induce ileitis,and visceromotor response(VMR)to colorectal distension(CRD)was used to evaluate the intensity of VH in goats.Then ileitis goats received repeated EA therapy to investigate cumulative analgesic effects and its influence on NMDAR-2B and glutamate transporters EAAT2 in the spinal cord.1.A model for studying ileitis-induced visceral hypersensitivity in goatsObjective: To establish a novel model for chemically inducible ileitis accompanied with VH in goat.Procedures: In the present study,60 healthy goats of either sex were equally divided into a 2,4,6-trinitrobenzenesulfonic acid(TNBS)group and Saline group.A volume of 1.2 ml of TNBS-ethanol solution(30 mg TNBS in 40% ethanol)or an equal volume of isotonic saline was injected into the wall of the terminal ileum through laparotomy.Six goats were taken from each group at days 3,7,14,21 and 28,respectively,for sampling.The severity of the resultant ileitis was determined according to macro-and microscopic pathologic scores and the levels of myeloperoxidase,interleukin-1?,interleukin-6 and tumor necrosis factor-?,and VH was evaluated with VMR to CRD.VMRs were assessed with a continuous ramp distention mode with six second for each pressure(20,40,60,80 and 100 mm Hg).Results: Compared to the saline group,the TNBS-treated goats showed apparent transmural pathological changes and a significant increase(P < 0.05)in macroscopic and microscopic changes score,and levels of myeloperoxidase,interleukin-1?,interleukin-6 and tumor necrosis factor-? in the ileum and VMR to colorectal distension.The goats exhibited apparent ileitis at days 3 to 21,and VH at days 7 to 28 following TNBS treatment.Conclusion: This experiment successfully established a reproducible ileitis and VH with administration of TNBS-ethanol solution in the ileal wall of goats.This model is useful for studying the pathogenesis of the IBD and the mechanism underlying VH,and for evaluating the efficacy of new therapeutic regimens.2.Effect of Electro-acupuncture on expression levels of Glutamate transporter EAAT2 and NMDAR-2B receptor in spinal cord after TNBS-induced ileitis in goatsObjective: This study was designed to evaluate the effect of repeated administration of EA on VH and on the expression of NMDAR-2B and glutamate transporter EAAT2 in a goat ileitis model with VH.Procedures: Twenty four animals were randomly divided into four equal groups i.e.salinel group,TNBS group,TNBS+EA group and sham EA group.A 6-cm incision was performed on the right flank abdomen,and the distal part of the ileum was exteriorized.For TNBS,TNBS+EA and Sham groups,1.2 m L of TNBS solution(30mg TNBS in 40% of ethanol)was injected into the wall of the ileum through five points,approximately 15 cm proximal to the ileoceacal junction,with a 30 gauge needle.In case of Saline group,the same volume of saline was injected in the same manner as in other groups.Then administration of EA at acupoints of zusanli(ST36)bilaterally in the hind limbs for 30 min with frequencies 60 Hz and intensity of 3 m A at day 7,10,13,16,19 and 22 after TNBS treatment.In Sham group,needle will insert in the same acupoints for 30 minutes without receiving electrical stimulation at the same days.All the experimental goats were immobilized with the same restraint method.Electromyography(EMG)was used to record VMR to CRD of goats.All animals from each group were euthanized at day 22 immediately after CRD test.Following euthanasia,the spinal cord of the eleventh thoracic vertibrae(T11)was removed very carefully and divided into two parts.The anterior part was taken and stored in 10% formalin for immunohistochemistry,and the posterior part was weighed,frozen in liquid nitrogen and transferred to store in-80 oC for RT-PCR and western blotting analysis.The ileum was removed from 15 cm proximal to the ileocecal junction for histological examination.Results: The results showed that repeated administration of EA attenuated(P < 0.05)VH in TNBS-treated goats.Such an effect was not seen in Sham group.Quantitative PCR,and western blotting showed an increase(P < 0.01)in NMDAR-2B expression and a decrease(P < 0.01)in EAAT2 expression at both m RNA and protein levels in the spinal cord of TNBS group compared with saline goats.EA at Zusanli acu-points(ST36)increased(P < 0.01)EAAT2 expression and attenuated(P < 0.01)NMDAR-2B expression compared with the TNBS group.However,there was no changed expression of these substances observed in the sham-EA group.Conclusion: EA can effectively attenuate VH through upregulation of EAAT2 and downregulation of NMDAR-2B in the spinal cord of TNBS-ileitis goats.The present findings suggest the potential of EA as a therapy to relieve chronic visceral pain and VH in patients with gastrointestinal disorders.