IBS-like Functional Chronic Sensitization Of Visceral Pain And Role Of Ionotropic Glutamate Receptor In It

Chronic functional visceral pain can become the consuming focus of a patient's life. These pains may be onerous to the treating clinician, particularly in the absence of an identifiable etiology. Irritable bowel syndrome (IBS) is one of common reasons of chronic functional visceral pain. So that it has been necessary to explore the pathogenesis and effective treatment of IBS.Al-Chaer's model first reported in 2000 is an avail animal model to study pathogenesis of IBS and functional chronic visceral pain. However, we found that neonatal rats were liable to death when we repeated Al-Chaer's model. Thus it is really necessary to improve Al-Chaer's model and to increase modeling success rates in order to supply a stable and reliable model to studying IBS.In rats, afferent information from colon is conveyed to the thoracolumbar (TL) and lumbosacral (LS) segments of the spinal cord via the lumbar hypogastric and pelvic nerves, respectively. Our group (U.S.) found that the chronic visceral hypersensitivity in IBS rats was related to central sensitivity of spinal cord. But it is little known whether it is related to hypersensitivity of primary afferent fibers. If this suppose is correct , is the characteristic of TL dorsal roots (DRs) the same as or different from that of LS DRs? If these questions are solved, it is helpful to discover peripheral mechanisms of the chronic visceral hypersensitivity in IBS model and distinguish the roles of primary afferent fibers between TL and LS in this process.It was reported that visceral sensitisation produced by colonic inflammation is mediated by spinal NMDA and non-NMDA receptors. It was also reported that NMDA receptor NR2B subunit played an important role in pathogenesis of somatic pain. However, little is know about the roles of NMDA or non-NMDA glutamate receptors in chronic visceral hypersensitivity of IBS rats. Based on the above questions, we improved Al-Chaer's model and increased modeling success rates to provide a stable and reliable model for the study of IBS. With this model, we explored hypersensitivity of TL & LS primary afferent and LS neurons. Then we explored the effect of NMDA and non-NMDA receptors in IBS like functional chronic visceral hypersensitivity. It will be helpful in understanding the pathogenesis of IBS and functional chronic visceral pain. It will also supply a novel avail strategy and target to clinical prevention and medicine treatment of IBS.1 The characteristics and assessment of IBS model of functional chronic visceral pain1.1 The improvement of Al-Chare's modelModeling according to Al-Chare's report, the survival rate of neonatal rats of 4 wk old was less than 30%. Later we modified the method, i.e., decreasing the frequency of 60 mmHg noxious colorectal distension (CRD). The model rats received CRD once daily on post neonatal (PN) days 8,10,12. The survival rate of the neonatal rats increased to near 90% at 4wk. This indicates the improved method elevate the achievement ratio of modeling. This model rats were called CI rats in short.After the rats grew up to 8 weeks old, we examined whether CI rats have chronic visceral hypersensitivity by abdominal withdrawal reflect (AWR) and spike response of external oblique muscle of abdomen (EOMA) to graded CRD.1.2 Increased AWR score and decreased pain threshold in CI ratsThe behavior change to graded CRD (20 ,40 ,60 ,80 mmHg) was compared between awake control and CI rats. CI rats manifested obvious pain reaction under 20 mmHg CRD , which average AWR score (20 mmHg) was even higher than those of the control rats (40 mmHg). The AWR scores of CI rats were significantly higher than those of the control rats under graded CRD, especially under 20 mmHg and 40 mmHg CRD. The pain threshold of CI rats was significantly lower than that of the control rats. This indicated that CI rats had chronic visceral hypersensitivity. AWR scores and pain threshold were compared between control and CI rats of 6th wk and 12th wk. Compared to control rats, AWR scores (20-60 mmHg) significantly increased, but the pain thresholds significantly decreased in 6 wk CI rats. However, there was no significant difference between the AWR scores of CI rats (6th wk and 12th wk old). This indicates that 6 wk CI rats had visceral hypersensitivity.1.3 Increased discharge of abdominal electromyograms and decreased myoelectricity response threshold in adult CI ratsThe myoelectricity response to graded CRD (20 ,40 ,60 ,80mmHg) was compared between awake control and CI rats. Compared to the control rats, the myoelectricity responses to 20-80 mmHg CRD were significantly increased and the average myoelectricity response threshold was significantly decreased in CI rats. The results further confirmed that CI rats had chronic visceral hypersensitivity.1.4 Somatic hypersensitivity in adult CI ratsTo identify whether there may exist somatic hypersensitivity in CI rats, we compared responses of footplate skin of two hind limbs to hot stimulus between awake control and CI adult rats. The latent periods of two hind limb skin to hot stimulus were significantly shorter in CI rats than those in control rats. Note that there existed somatic hypersensitivity in CI rats.1.5 Comparison of body weight between control and CI ratsThe body weight was observed and compared between control and CI peer rats from 4 to 12 wks old. The results showed that there was no significant difference between two groups . This indicated neonatal CRD had no significant influence on body weight in CI rats.1.6 Comparison of diarrhea and constipation between control and CI ratsThe diarrhea and constipation were observed at the same day in control and CI adult rats. The result indicates that the incidence of these signs was significant higher in CI rats than that in control rat.1.7 The colorectal pathological change in CI rats The local colorectal histological change was observed in control and CI rats. No apparent pathological change was observed by naked eyes and microscope in control and CI rats.2 The enhanced responses of primary afferent fibers to CRD in CI rats2.1 Increased number of TL and LS afferent fibers activated by CRD in CI ratsThe number of primary afferents activated by CRD was significantly higher in CI rats than that in control rats whether recording was made at LS or TL. The total number of response of primary afferents to graded CRD at LS levels was significantly higher than that at TL levels in control rats. But there was no significant difference between LS and TL in CI rats.2.2 Spontaneous background activity of TL and LS afferent fibers in CI ratsThe average spontaneous background activity (SBA) of LS dorsal roots (DRs) was significantly higher in CI rats than that in control rats. There was no significant difference between SBA of TL DRs in control and CI rats. In control rats, the average SBA of TL DRs was significantly higher than that of LS DRs. However, in CI rats, there was no significant difference between the TL and LS DRs. This indicated that neonatal CRD has significant effect on SBA of LS DR, but no effect on SBA of TL DR.2.3 Excited threshold of TL and LS DRs in CI ratsThe mean excited threshold of LS DRs to CRD in CI rats was significantly lower than that in control rats. The threshold of response of TL DRs to CRD in CI rats was not significantly different from that in control rats. In control rats, there was no significant difference between the two levels. However, in CI rats, the excited threshold of LS DRs to CRD was significantly lower than that of TL DRs. This indicats that excited threshold of LS DRs to CRD is changed in CI rats.2.4 Responses of TL and LS DRs to CRD in CI rats2.4.1 Increased responses of LS DRs to graded CRD in CI rats.The responses of LS DRs to the same CRD were compared between control and CI rats. The average responses of afferent units to all intensities of CRD in CI rats were significantly higher than those in control rats. This indicates that sensitization of both high and low threshold primary afferent fibers in CI rats.2.4.2 Increased responses of TL DRs to 40-80 mmHg CRD in CI rats.The responses of TL DRs to the same intensity of CRD were compared between control and CI rats. Show that the average responses of TL DRs to CRD (40-80 mmHg) were significantly higher in CI rats than those in control rats This indicates that sensitization of TL DRs of high threshold in CI rats.2.4.3 Increased responses of LS DRs to graded CRD in CI rats.Interlevel comparisons between LS and TL DRs responses showed that: in control rats, only the average response of LS DRs to 80 mmHg CRD was significantly higher than that of TL DRs. However, in CI rats, the average responses to graded CRD (20-80 mmHg) in LS DRs were significantly higher than those in TL DRs. This indicates that responses to noxious and innocuous stimulation of LS DR were significantly higher than those of TL DR in CI rats. However, only response to nociceptive stimulus of LS DRs was significantly higher than that of TL DRs in control rats.3 Increased responses of LS neurons to both visceral and somatic stimulus in CI rats.3.1 Increased responses of LS neurons to graded CRD in CI rats. The responses of LS dorsal horn neurons to CRD were compared between CI and control rats. The average responses of LS neurons to 20～80 mmHg CRD in CI rats were higher than those of control rats. This indicates the responses of LS dorsal horn neurons to noxious and innocuous CRD are increased in CI rats.3.2 Increased responses of LS neurons to corresponding somatic stimulation in CI rats.The responses of LS dorsal horn neurons to all kinds of somatic stimulation were compared berween CI and control rats. Responses of LS neurons to noxious and innocuous somatic stimulation were significantly higher in CI rats than those in control rats. This indicates CI rats have chronic visceral hypersensitivity and corresponding somatic hypersensitivity.4 Role of ionotropic glutamate receptors in chronic visceral hypersensitivity 4.1 Morphology evidence for the effect of NMDA receptor on IBS-like functional chronic visceral pain.4.1.1 Increased expression of NMDA receptor NR2B subunit in DRG of CI ratsThe expression of NMDA receptor NR2B subunit in DRG was compared between control and CI rats by immunohistochemistry method. Image analysis showed that average optical density which represents the expression of NMDA receptor NR2B subunit in DRG was significantly increased in CI rats than that in control rats. This indicates that NMDA-NR2B may play a role in the periphery mechanism of IBS like functional chronic visceral pain sensitivity.4.1.2 Increased expression of NMDA receptor NR2B subunit in spinal visceral related neurons of CI rats.Expression of NMDA receptor NR2B subunit in TL & LS neurons was compared between control and CI rats by immunohistochemistry method. Image analysis shows that average optical which represents the expression of NMDA receptor NR2B subunit in TL and LS neurons was significantly higher in CI rats than those in control rats. This indicates that NMDA-NR2B may play a role in the spinal mechanism of IBS like functional chronic visceral pain.4.2 The effects of NMDA receptor on IBS like functional chronic visceral hypersensitivity.4.2.1 AP-7(i.p.) significantly inhibited the response of LS DRs to CRD in CI rats.Responses of LS DRs to 20～80 mmHg CRD were compared between control and CI rats before and after systemic administration of AP-7 (0.5 mg/kg,i.p.). AP-7 significantly inhibited responses of LS DRs to 20～80 mmHg CRD in control and CI rats, but the percent inhibition of the responses of LS DR to CRD caused by AP-7 was significantly higher in CI rats than those in control rats. This indicates NMDA receptors may mediate IBS like functional chronic visceral hypersensitivity.4.2.2 Spinal AP-7 significantly inhibited responses of LS neurons to CRD in CI rats.4.2.2.1 Percentage of LS neurons inhibited by spinal AP-7 Spinal AP-7 significantly inhibited the percentage of LS neurons responded to CRD in CI rats than that in control rats at the same dosage. Note that there could be a more active function of spinal NMDA receptor in CI rats.4.2.2.2 Spinal AP-7 significantly inhibited average responses of LS neurons to CRD in CI rats.The effect of spinal AP-7 on the average responses of LS neurons to CRD was compared between control and CI rats. In CI rats, AP-7 (0.01 mmol/L) significantly attenuated the average responses to graded CRD in a dose-dependant manner, indicating NMDA receptor play a role in functional chronic visceral sensitivity.The above results indicate spinal NMDA receptor may partly mediate IBS-like functional chronic visceral hypersensitivity.4.3 Effect of non-NMDA receptors on IBS like functional chronic visceral hypersensitivity.4.3.1 Percentage of LS neurons inhibited by spinal CNQXSpinal CNQX obviously inhibited the percentage of LS neurons responded to CRD in CI rats than that in control rats at the same dosage. Note that there could be a more active function of spinal non-NMDA receptor in CI rats.4.3.2 Spinal AP-7 significantly inhibited average responses of LS neurons to CRD in CI rats.Effect of spinal CNQX on average responses of LS neurons to CRD was compared between control and CI rats. In CI rats, CNQX (2μmol/L) significantly attenuated the average responses of LS neurons to graded CRD. At the same time, responses of LS neurons to noxious and innocuous CRD were decreased in a dose-dependant manner by CNQX (2～10μmol/L) in CI rats. Note that non-NMDA receptor may partly mediates IBS like functional chronic visceral hypersensitivity.This study indicates:1. Improved method of modeling leads to a higher achievement ratio of modeling. Colon irritation in neonatal rats can cause long-term visceral hypersensitivity accompanied with diarrhea , constipation and referred pain, despite the lack of inflammation signs in the colon. This indicates that CI model can be used for study on IBS like functional chronic visceral pain.2. IBS like functional chronic visceral pain is associated with periphery afferent nerve sensitization. TL DRs may have greater effect on colon chronic pain sensitivity than on acute noxious colon input, but LS DRs may have equal effect on acute and chronic visceral pain sensitivity.3. Neonatal colon irritation leads to LS neuron sensitization to CRD stimulation in adult rat, i.e., IBS like functional chronic visceral pain rat has spinal dorsal horn hypersensitivity.4. Expression of NMDA receptor NR2B subunit was increased in DRG and spinal visceral related neurons of CI rats, which provides morphology base for that NR2B subunit may involve in peripheral and spinal sensitizing mechanism in IBS rats. Spinal NMDA and non-NMDA receptor may involve in IBS like functional chronic visceral hypersensitivity.