Involvement Of Spinal NMDA Receptor In IBS Chronic Visceral Pain And Acupuncture Alleviating Chronic Visceral Hypersensitivity

Internal organs are sensitive to physiological or nocuous stimulus,which is called visceral hyperalgesia.And its mechanism is complex.Previous study was confined to gastrointestinal local affection such as activated enterochromaffin cells,mast cells and so on.However,the formation of visceral sensation is a complex process.The abnormality of viceral sensorius path and cerebral sophisticated center can affect visceral sensation, including some neurotransmitters and their receptors.Irritable bowel syndrome(IBS) is clinically a common disease characterized most commonly by abdominal pain or discomfort,which belongs to chronic visceral pain. Alouthgh no conspicuous structural affection on local intestines was found in IBS patients,some research indicated that people who suffered from IBS had a colon,or large intestine,that was particularly sensitive,which was also the base of chronic viseral hypersensitivity.Recent studies of IBS demonstrated that increased levels of sensitivity of visceral afferent projection to spinal cord play a role in IBS's formation.Numerous studies indicated abnormal excitement of visceral sense projection neuron and endaxoneuron in spinal dorsal horn and lateral horn is one of central sensitization mechanism,with some neurotransmitters or neuromodulators such as glutamate involved.Many sufferers of IBS seek treatment using acupuncture,one of the important parts of Traditional Chinese Medicine.Evidences have already indicated that acupuncture is effective for treating various pains including viscral pain.Al-Chaer reported a model of chronic visceral hypersensitivity in adult rats,which can also simulate clinical IBS symptoms.In our previous study,we examined the effect of electro-acupuncture(EA) on chronic visceral pain in rats using an IBS model(a chronic visceral hypersensitivity model) and found that EA could significantly alleviate visceral hyperalgesia.However, how dose acupuncture work that still needs to be further explained. In the study,spinal N-methyl-D-aspartate receptors-1(NMDAR1,an NMDA subtype receptor of glutamata) was investigated to obtain the evidences of possible involvement of the receptors in acupuncture's effect on chronic visceral hyperalgesia in rats.Ⅰ.Content of the study1.Animal modelTo produce IBS model rats,daily mechanical colon distention which was repeated twice a day at a 30 min interval was performed on male Sprague-Dawley neonatal rats beginning 8-21 days after their birth according to the scale of Al-Chaer et al.The model was assessed by observing both the behavioral responses such as body weight,diarrhea, abdominal withdrawal reflex(AWR).2.Experiments were performed to observe pain threshold of IBS rats before & after intrathecal injection(i.t.) of MK-801(a non-competitive antagonist of the NMDA receptor).3.The expression of NMDA R1 in the spinal cord was investigated by western blot analysis,RT-PCR in IBS rats.1) Western blotting was used to detect the expression of NMDA R1 at protein level.2) RT-PCR was used to detect the expression of NMDA R1 at mRNA level.4.The expression of NMDA R1 in the spinal cord was investigated by western blot analysis and RT-PCR in IBS rats under EA treatment.1) Western blotting was used to detect the expression of NMDA R1 before & after single EA stimulus.2) RT-PCR was used to detect the expression of NMDA R1 before & after single EA stimulus.Ⅱ.Results of the study1.Assessment of IBS model 1.1 production of IBS modelAccording to Al-Chaer's report,we produced the IBS model rats successfully. Further,we improved the method of model production and made the IBS model more stable with long term lasting visceral pain and more apparent sign of diarrhea.1.2 Behavioral test for assessing visceral pain sensationBehavioral responses to graded strengths ofcolorectal distention(CRD)(20,40,60, and 80 mmHg) were assessed by observing the abdominal withdrawal reflex(AWR). Semi-quantitative scores were used for judgment of the responses to CRD stimulus.AWR scores were assigned according to the scale of Al-Chaer et al.And the averaged responsive value was taken for analysis.There is a significantly enhanced response to CRD stimulation in IBS rats,but no such effects in normal rats stimulated by graded CRD stimulation at above strengths.1.3 Comparison of diarrhea signs between IBS rats and normal onesThere is a significant increase on incidence of diarrhea in IBS rats compared with normal rats.1.4 Comparison of body weight between IBS rats and normal onesThere were no significant differences on body weight among the above group of rats at the same time.2.NMDA receptor plays a role in chronic visceral pain of IBS rat2.1 The effect intrathecal injection of MK-801 on AWR scores in IBS ratThe rats were treated with intrathecal injection of MK-801,3 days after which the location was assessed and then experiment was conducted one day later.Before injection of MK-801 in each group,AWR scores were detected to obtain the base value.Rats were divided into 2 groups(n=8 in each group):normal group(normal rats) and IBS model group(IBS rats).Both groups were treated with MK-801 at doses of 0.01μg,0.1μg and 1μg.The IBS rats which were treated with MK-801 at dose of 0.1μg and 1μg produced a remarkable reduction of the enhanced responses to CRD stimulation,but not in 0.01μg group.That indicates intrathecal injection of MK-801 could depress pain threshold in a dose-dependent manner.2.2 The expression of spinal NMDA R1 mRNA in IBS ratRT-PCR was used to detect the expression of NR1 at mRNA level in the spinal cord. Rats were divided into 2 groups(n = 8 in each group):normal group(normal rats) and IBS model group(IBS rats).In contrast to normal rats,there was significant increase of NR1 mRNA expression in model rats.2.3 The expression of spinal NMDA R1 protein in IBS ratWestern blotting was used to detect the expression of NR1 at protein level in the spinal cord.Rats were divided into 2 groups(n = 8 in each group):normal group(normal rats) and IBS model group(IBS rats).In contrast to normal rats,there was significant increase of NR1 expression in model rats.3.Relief of visceral pain in IBS rats by EA treatmentAWR assessment was performed during 20-90 min after EA treatment.Rats were divided into 4 groups(n = 8 in each group):normal group(normal rats),IBS model group (IBS rats),EA group(IBS rats treated with EA) and sham EA group(IBS rats treated with EA).In the EA group,EA produced a partial but significant reduction of the enhanced responses to CRD stimulation,but no analgesic effects by sham EA on AWR scores induced by graded CRD stimulation at above strengths.It shows that single EA treatment could alleviate visceral pain within a short time period.4.NMDA receptor plays a role in EA relieving visceral pain in IBS rat4.1 The expression of spinal NMDA R1 mRNA in IBS rat treated with single EART-PCR was used to detect the expression of NR1 at mRNA level in the spinal cord. Rats were divided into 4 groups(n = 8 in each group):normal group(normal rats),IBS model group(IBS rats),EA group(IBS rats treated with single EA) and sham EA group (IBS rats treated with sham EA).There was significant reduction of NR1 mRNA expression in EA group rats but not in other groups.4.2 The expression of spinal NMDA R1 protein in IBS rat treated with single EAWestern blotting was used to detect the expression of NR1 at protein level in the spinal cord.Rats were divided into 4 groups(n = 8 in each group):normal group(normal rats),IBS model group(IBS rats),EA group(IBS rats treated with single EA) and sham EA group(IBS rats treated with sham EA).There was significant reduction of NR1 expression in EA group rats but not in other groups.Ⅲ.Conclusion of the study1,The IBS model rats in this study can simulate clinical IBS symptoms such as conspicuous abdominal pain and diarrhea in humanbeings.It is an experimental model of chronic visceral hypersensitivity.We improved the method of IBS model production,made the model more stable and increased the survival rate of model animals.2,The increased expression of NMDA R1 receptors in the spinal cord of IBS model rats indicates that the excitatory amino acid system may be involved in IBS chronic visceral pain.3,EA treatment can alleviate chronic visceral hypersensitivity effectively in the IBS model rats.4,The above effect of EA treatment may be induced by a decrease in the expression of NMDA receptors in the spinal cord of IBS model rats.