IBS样功能性慢性内脏痛的痛觉敏化作用以及离子型谷氨酸受体的作用
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摘要
功能性慢性内脏痛不仅严重影响病人的生活,而且因其发病机制不明,给治疗带来极大的麻烦。肠易激综合征(Irritable Bowel Syndrome,IBS)为功能性慢性内脏痛的典型代表。探索IBS及功能性慢性内脏痛的发病机制,并寻找行之有效的治疗手段为目前国内外研究热点。
2000年Al-Chaer首次报道了IBS样慢性内脏痛大鼠模型,为研究IBS及功能性慢性内脏痛的发病机制提供了可借鉴的动物模型。然而,该模型制备过程中,新生鼠易死亡,成功率不高。因此,如何改进IBS样慢性内脏痛敏大鼠模型,提高模型成功率,为IBS及功能性慢性内脏痛的研究提供一个稳定、可靠的动物模型,将是十分必要的。
大鼠降结肠和直肠的传入信息可通过腹下神经和盆神经传递到脊髓胸腰和腰骶节段。本课题组(美方)其他成员研究发现,IBS大鼠的内脏痛敏与脊髓中枢敏化有关;然而它是否与原发传入神经纤维敏化有关,尚有待于研究。此外,假设上述推论成立,那么胸腰和腰骶脊髓节段原发传入神经纤维敏化的特征是否一致或有何不同?探明这些问题,将有助于揭示IBS大鼠内脏痛敏的外周敏化机制并区分胸腰和腰骶背根传入在此过程的不同作用。
有报道,结肠炎症产生的内脏痛觉过敏可由脊髓NMDA和非NMDA受体共同介导。另有报道,NMDA受体中NR2B亚单位可能在躯体疼痛发病机制中发挥重要作用。然而,NMDA与非NMDA受体在IBS样慢性功能性内脏痛敏形成中的作用,尚有待于探索。
基于以上存在的问题,本研究改良了Al-Chaer模型,提高了建模的成功率,为IBS研究提供一个稳定、可靠的动物模型。籍此研究模型,探讨IBS大鼠脊髓胸腰和腰骶节段初级传入神经纤维的敏化作用以及脊髓腰骶神经元的敏化作用;进而探索NMDA和非NMDA受体在IBS样功能性慢性内脏痛敏中的作用,以期深化理解IBS及功能性慢性内脏痛的发病机制,为IBS的药物治疗提供新的作用靶点,为IBS的临床防治提供新的策略与途径。
1 IBS样功能性慢性内脏痛模型的特点及评价
1.1 Al-Chare模型的改进
重复Al-Chare报道的方法造模,结果大鼠4 wk内的存活率不到30%。
采用本文改进的造模方法,即减少伤害性结肠刺激(CI)的次数,仅在出生后第8、10、12天,给予乳鼠60 mmHg压力的CI刺激,每天1次,共3次,大鼠4 wk内的的存活率接近90%。提示改进造模方法,提高了造模的成功率。此模型组大鼠简称为CI大鼠。
大鼠8 wk后,以结直肠扩张(CRD)引起腹壁撤退反射和腹外斜肌放电反应为指标,了解CI模型大鼠是否出现慢性内脏痛敏。
1.2 CI大鼠腹壁撤退反射评分显著升高,痛反应阈显著降低。
比较对照组与CI模型组成年大鼠在清醒状态下对不同强度(20、40、60、80 mmHg)的CRD刺激所产生的行为学变化。CI组大鼠在20 mmHg CRD刺激下即表现出明显的疼痛反应,其平均腹壁撤退反射(AWR)评分甚至超过对照组大鼠40 mmHg时的平均评分。CI大鼠在各个压力CRD下AWR评分均显著高于对照大鼠,尤其是在20与40mmHg较低压力刺激下评分增高更为显著。CI组大鼠痛反应阈较对照组显著降低。提示CI大鼠存在慢性内脏痛觉敏化现象。
比较6 wk与12 wk CI模型组大鼠AWR评分及痛反应阈。虽然与同龄对照大鼠相比,20～60 mmHg CRD刺激下6 wk CI大鼠的AWR评分显著增高,痛反应阈明显降低;但不同周龄CI大鼠AWR评分之间无显著性差异。提示CI幼年大鼠即存在内脏痛觉敏化
1.3 CI大鼠腹外斜肌肌电显著升高,肌电反应阈显著降低。
比较对照与CI大鼠在清醒状态下对不同强度(20、40、60、80 mmHg)CRD刺激所引起的肌电反应。CI大鼠对20～80 mmHg各等级CRD刺激的肌电反应均较对照组显著增高,平均肌电反应阈显著降低。上述结果进一步证实了CI大鼠存在内脏痛觉敏化。
1.4 CI大鼠躯体感觉敏感性增加
为明确CI大鼠是否存在躯体感觉敏化,比较对照与CI成年大鼠在清醒状态下,双后肢足底皮肤对热刺激的敏感性差异。CI大鼠双后肢皮肤对热刺激反应的潜伏期较对照组显著缩短,提示CI大鼠相应的躯体皮肤对热刺激的敏感性增加。
1.5 CI大鼠与对照大鼠体重的比较
观察比较4～12周龄对照与CI大鼠在同一时期生长发育过程中体重的变化。结果表明,两组大鼠体重之间无显著性差异,提示造模本身对CI大鼠体重无明显影响。
1.6 CI大鼠与对照大鼠腹泻与便秘体征的比较
观察比较对照与CI大鼠生长发育过程中腹泻、便秘等体征的改变。对照大鼠无明显体征出现;但60 % 8 wk CI大鼠有腹泻或便秘体征。
1.7 CI大鼠结直肠局部组织学的病理变化
比较对照和CI大鼠远端降结肠和直肠的局部组织学的病理改变。对照与CI大鼠经过肉眼观察和显微镜检查,均未见明显病理改变。
2 CI大鼠原发传入神经纤维对结直肠扩张反应显著增强
2.1 CI大鼠结直肠扩张激活的胸腰和腰骶段传入神经纤维数目增加
CI大鼠可以被CRD激活的传入神经纤维数量不论是在胸腰,还是在腰骶水平,均比对照大鼠显著增多。对照大鼠在腰骶水平记录到对CRD有反应的传入神经纤维数目比在胸腰水平记录到的显著增多,而CI大鼠被CRD激活的胸腰和腰骶段传入神经纤维的数量之间差别无显著性。
2.2 CI大鼠胸腰和腰骶段传入神经纤维电紧张电位的改变
与对照大鼠相比,CI大鼠腰骶段(LS)传入神经纤维(DR)电紧张电位(BKG)显著增高;而CI大鼠胸腰段(TL)DR电紧张电位无显著改变。对照大鼠TL DR(n=14)的平均电紧张电位显著高于LS DR(n=29);而CI大鼠,两者之间则无显著性差异。提示造模对LS DR的电紧张电位有显著影响,而对TL DR则无显著影响。
2.3 CI大鼠胸腰和腰骶段传入神经纤维兴奋阈的改变
与对照大鼠相比,CI大鼠LS DR对CRD刺激的兴奋阈显著降低。CI大鼠TL DR(n=126)对CRD的兴奋阈与对照大鼠TL DR (n=14)之间差异无显著性。对照大鼠TL DR与LS DR兴奋阈之间差异无显著性,而CI大鼠LS DR的兴奋阈显著低于TL DR,提示CI大鼠腰骶背根传入神经纤维兴奋阈改变较显著。
2.4 CI大鼠胸腰和腰骶段传入神经纤维对CRD反应的改变
2.4.1 CI大鼠腰骶传入神经纤维对各等级CRD反应显著增强比较对照与CI大鼠LS DR对相同强度CRD刺激的反应差异。CI大鼠LS DR对各个等级强度CRD刺激的平均反应强度均显著高于对照大鼠,提示CI大鼠腰骶高、低阈值的初级传入神经纤维均敏化。
2.4.2 CI大鼠胸腰传入神经纤维对较高强度CRD反应显著增强
比较对照与CI大鼠TL DR在相同强度CRD刺激下的反应。CI大鼠脊髓背根对40～80 mmHg CRD的平均反应较对照大鼠显著增高,提示CI大鼠胸腰高阈值的传入神经纤维敏化。
2.4.3 CI大鼠腰骶传入神经纤维对各等级CRD反应显著高于胸腰传入
比较TL和LS DR对CRD的反应。对照大鼠LS DR只对80 mmHg的CRD的平均反应显著强于TL DR;而CI大鼠LS DR对20～80 mmHg各个等级的CRD的平均反应都显著高于TL DR。提示CI大鼠LS DR对伤害与非伤害刺激反应的强度均显著高于TL DR,而对照大鼠LS DR仅对伤害性刺激反应的强度显著高于TL DR。
3 CI大鼠脊髓神经元对内脏与躯体刺激的反应增强
3.1 CI大鼠脊髓腰骶神经元对结直肠扩张刺激的反应增强比较对照与CI大鼠LS脊髓背角神经元对CRD刺激的反应。CI大鼠LS神经元对20～80 mmHg各个压力CRD的平均反应均显著高于对照大鼠。提示CI大鼠LS脊髓背角神经元对伤害与非伤害结直肠扩张刺激的反应显著增强。
3.2 CI大鼠脊髓腰骶神经元对相应躯体刺激的反应增强
比较对照与CI大鼠LS神经元对伤害与非伤害性各种不同躯体刺激的反应。与对照大鼠相比,CI大鼠LS神经元对伤害与非伤害性各种不同躯体刺激的反应均显著增强。提示CI大鼠除了出现慢性内脏痛敏,还伴随相应躯体感觉敏化。
4谷氨酸受体在CI模型大鼠慢性内脏痛敏中的作用
4.1 NMDA受体涉及IBS样功能性慢性内脏痛的形态学证据
4.1.1 CI大鼠脊髓背根神经节NMDA受体NR2B亚单位表达增强采用免疫组织化学方法,比较对照与CI大鼠背根神经节NMDA受体NR2B亚单位的表达。图像分析结果,CI大鼠NR2B表达的平均光密度较对照组显著升高。提示CI大鼠背根神经节NMDA受体NR2B亚单位可能涉及IBS样功能性慢性内脏痛的外周敏化机制。
4.1.2 CI大鼠脊髓内脏相关神经元NMDA受体NR2B亚单位表达增强
采用免疫组织化学方法,比较对照与CI大鼠脊髓背角神经元NMDA受体NR2B亚单位的表达。图像分析结果,CI大鼠胸腰和腰骶段NR2B亚单位表达的平均光密度均显著高于对照大鼠。提示,CI大鼠脊髓NMDA受体NR2B亚单位可能涉及IBS样功能性慢性内脏痛的脊髓敏化机制。
4.2 NMDA受体在IBS样功能性慢性内脏痛敏中的作用
4.2.1腹腔注射AP-7显著抑制CI大鼠腰骶传入神经纤维对CRD刺激的反应
本研究选用中等剂量AP-7 (0.5 mg/kg,i.p.),比较对照和CI大鼠给药前后LS DR对20～80 mmHg CRD的反应。结果显示,NMDA受体拮抗剂AP-7显著抑制对照和CI大鼠LS DR对20～80 mmHg CRD的反应),CI大鼠AP-7的抑制百分率显著高于对照大鼠。提示体内NMDA受体可能介导IBS样功能性慢性内脏痛敏。
4.2.2脊髓局部给予AP-7显著抑制CI大鼠腰骶神经元对CRD刺激的反应
4.2.2.1脊髓局部给予AP-7抑制CI大鼠腰骶神经元对CRD反应的百分数
比较脊髓局部给予相同剂量AP-7在对照与CI大鼠之间对腰骶神经元产生效应的百分数。结果表明,相同剂量AP-7对CI组大鼠神经元产生效应的百分数较对照组大。
4.2.2.2脊髓局部给予AP-7显著抑制CI大鼠腰骶神经元对CRD的反应
比较脊髓局部给予不同浓度AP-7抑制对照与CI大鼠腰骶神经元对CRD刺激反应的程度。结果显示,与对照组大鼠相比,0.01 mmol AP-7则能剂量依赖性显著减弱CI组大鼠LS神经元对各压力等级CRD的平均反应。以上结果提示,脊髓NMDA受体参与介导IBS样功能性慢性内脏痛敏。
4.3脊髓非NMDA受体在IBS样功能性慢性内脏痛敏中的作用
4.3.1脊髓局部给予CNQX抑制CI大鼠腰骶神经元对CRD反应的百分数
比较脊髓局部给予相同剂量CNQX在对照与CI大鼠之间对腰骶神经元产生效应的百分数。与对照大鼠相比,相同剂量CNQX对CI大鼠神经元产生效应的百分数较大。
4.3.2脊髓局部给予CNQX抑制CI大鼠腰骶神经元对CRD的反应
比较脊髓局部给予不同浓度CNQX抑制对照与CI大鼠腰骶神经元对CRD刺激反应的程度。结果,与对照大鼠相比,2μmol CNQX就显著降低CI大鼠腰骶神经元对所有等级CRD的平均反应,同时2～10μmol CNQX呈剂量依赖性显著减弱神经元对伤害性和非伤害性CRD的平均反应。以上结果提示,脊髓非NMDA受体参与介导IBS样功能性慢性内脏痛敏。
综上所述,本工作提示:
1.改进后的CI模型制作方法可提高建模的成功率。CI模型大鼠慢性内脏痛敏显著,且伴有腹泻、便秘症状与牵涉痛,其结直肠局部组织未见明显的病理改变。提示本CI模型可做为IBS样功能性慢性内脏痛的研究模型。
2.IBS样功能性慢性内脏痛大鼠存在外周传入神经纤维敏化。与胸腰背根在结肠急性伤害性传入的作用相比,其在结肠慢性痛觉敏化过程中可能发挥了更大作用;而腰骶背根传入在急性与慢性痛觉敏化过程中可能发挥了同等重要的作用。
3.新生期的结肠伤害性刺激敏化了成年大鼠腰骶神经元对结直肠扩张刺激的反应,即IBS样功能性慢性内脏痛大鼠存在脊髓背角神经元敏化。
4. CI大鼠背根神经节和脊髓内脏相关神经元NMDA受体NR2B亚单位表达增强,这为NR2B亚单位可能涉及功能性慢性内脏痛的外周和脊髓敏化机制提供形态学基础。脊髓NMDA受体和非NMDA受体参与介导功能性慢性内脏痛敏作用。此外,似乎还提示功能性慢性内脏痛时,脊髓与内脏痛觉相关的离子型谷氨酸受体功能可能更多地被激活。
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 pain
1.1 The improvement of Al-Chare’s model
Modeling 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 rats
The 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 rats
The 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 rats
To 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 rats
The 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 rats
The 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 rats
2.1 Increased number of TL and LS afferent fibers activated by CRD in CI rats
The 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 rats
The 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 rats
The 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 rats
2.4.1 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 rats
The 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 CNQX
Spinal 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.
2000年Al-Chaer首次报道了IBS样慢性内脏痛大鼠模型,为研究IBS及功能性慢性内脏痛的发病机制提供了可借鉴的动物模型。然而,该模型制备过程中,新生鼠易死亡,成功率不高。因此,如何改进IBS样慢性内脏痛敏大鼠模型,提高模型成功率,为IBS及功能性慢性内脏痛的研究提供一个稳定、可靠的动物模型,将是十分必要的。
大鼠降结肠和直肠的传入信息可通过腹下神经和盆神经传递到脊髓胸腰和腰骶节段。本课题组(美方)其他成员研究发现,IBS大鼠的内脏痛敏与脊髓中枢敏化有关;然而它是否与原发传入神经纤维敏化有关,尚有待于研究。此外,假设上述推论成立,那么胸腰和腰骶脊髓节段原发传入神经纤维敏化的特征是否一致或有何不同?探明这些问题,将有助于揭示IBS大鼠内脏痛敏的外周敏化机制并区分胸腰和腰骶背根传入在此过程的不同作用。
有报道,结肠炎症产生的内脏痛觉过敏可由脊髓NMDA和非NMDA受体共同介导。另有报道,NMDA受体中NR2B亚单位可能在躯体疼痛发病机制中发挥重要作用。然而,NMDA与非NMDA受体在IBS样慢性功能性内脏痛敏形成中的作用,尚有待于探索。
基于以上存在的问题,本研究改良了Al-Chaer模型,提高了建模的成功率,为IBS研究提供一个稳定、可靠的动物模型。籍此研究模型,探讨IBS大鼠脊髓胸腰和腰骶节段初级传入神经纤维的敏化作用以及脊髓腰骶神经元的敏化作用;进而探索NMDA和非NMDA受体在IBS样功能性慢性内脏痛敏中的作用,以期深化理解IBS及功能性慢性内脏痛的发病机制,为IBS的药物治疗提供新的作用靶点,为IBS的临床防治提供新的策略与途径。
1 IBS样功能性慢性内脏痛模型的特点及评价
1.1 Al-Chare模型的改进
重复Al-Chare报道的方法造模,结果大鼠4 wk内的存活率不到30%。
采用本文改进的造模方法,即减少伤害性结肠刺激(CI)的次数,仅在出生后第8、10、12天,给予乳鼠60 mmHg压力的CI刺激,每天1次,共3次,大鼠4 wk内的的存活率接近90%。提示改进造模方法,提高了造模的成功率。此模型组大鼠简称为CI大鼠。
大鼠8 wk后,以结直肠扩张(CRD)引起腹壁撤退反射和腹外斜肌放电反应为指标,了解CI模型大鼠是否出现慢性内脏痛敏。
1.2 CI大鼠腹壁撤退反射评分显著升高,痛反应阈显著降低。
比较对照组与CI模型组成年大鼠在清醒状态下对不同强度(20、40、60、80 mmHg)的CRD刺激所产生的行为学变化。CI组大鼠在20 mmHg CRD刺激下即表现出明显的疼痛反应,其平均腹壁撤退反射(AWR)评分甚至超过对照组大鼠40 mmHg时的平均评分。CI大鼠在各个压力CRD下AWR评分均显著高于对照大鼠,尤其是在20与40mmHg较低压力刺激下评分增高更为显著。CI组大鼠痛反应阈较对照组显著降低。提示CI大鼠存在慢性内脏痛觉敏化现象。
比较6 wk与12 wk CI模型组大鼠AWR评分及痛反应阈。虽然与同龄对照大鼠相比,20～60 mmHg CRD刺激下6 wk CI大鼠的AWR评分显著增高,痛反应阈明显降低;但不同周龄CI大鼠AWR评分之间无显著性差异。提示CI幼年大鼠即存在内脏痛觉敏化
1.3 CI大鼠腹外斜肌肌电显著升高,肌电反应阈显著降低。
比较对照与CI大鼠在清醒状态下对不同强度(20、40、60、80 mmHg)CRD刺激所引起的肌电反应。CI大鼠对20～80 mmHg各等级CRD刺激的肌电反应均较对照组显著增高,平均肌电反应阈显著降低。上述结果进一步证实了CI大鼠存在内脏痛觉敏化。
1.4 CI大鼠躯体感觉敏感性增加
为明确CI大鼠是否存在躯体感觉敏化,比较对照与CI成年大鼠在清醒状态下,双后肢足底皮肤对热刺激的敏感性差异。CI大鼠双后肢皮肤对热刺激反应的潜伏期较对照组显著缩短,提示CI大鼠相应的躯体皮肤对热刺激的敏感性增加。
1.5 CI大鼠与对照大鼠体重的比较
观察比较4～12周龄对照与CI大鼠在同一时期生长发育过程中体重的变化。结果表明,两组大鼠体重之间无显著性差异,提示造模本身对CI大鼠体重无明显影响。
1.6 CI大鼠与对照大鼠腹泻与便秘体征的比较
观察比较对照与CI大鼠生长发育过程中腹泻、便秘等体征的改变。对照大鼠无明显体征出现;但60 % 8 wk CI大鼠有腹泻或便秘体征。
1.7 CI大鼠结直肠局部组织学的病理变化
比较对照和CI大鼠远端降结肠和直肠的局部组织学的病理改变。对照与CI大鼠经过肉眼观察和显微镜检查,均未见明显病理改变。
2 CI大鼠原发传入神经纤维对结直肠扩张反应显著增强
2.1 CI大鼠结直肠扩张激活的胸腰和腰骶段传入神经纤维数目增加
CI大鼠可以被CRD激活的传入神经纤维数量不论是在胸腰,还是在腰骶水平,均比对照大鼠显著增多。对照大鼠在腰骶水平记录到对CRD有反应的传入神经纤维数目比在胸腰水平记录到的显著增多,而CI大鼠被CRD激活的胸腰和腰骶段传入神经纤维的数量之间差别无显著性。
2.2 CI大鼠胸腰和腰骶段传入神经纤维电紧张电位的改变
与对照大鼠相比,CI大鼠腰骶段(LS)传入神经纤维(DR)电紧张电位(BKG)显著增高;而CI大鼠胸腰段(TL)DR电紧张电位无显著改变。对照大鼠TL DR(n=14)的平均电紧张电位显著高于LS DR(n=29);而CI大鼠,两者之间则无显著性差异。提示造模对LS DR的电紧张电位有显著影响,而对TL DR则无显著影响。
2.3 CI大鼠胸腰和腰骶段传入神经纤维兴奋阈的改变
与对照大鼠相比,CI大鼠LS DR对CRD刺激的兴奋阈显著降低。CI大鼠TL DR(n=126)对CRD的兴奋阈与对照大鼠TL DR (n=14)之间差异无显著性。对照大鼠TL DR与LS DR兴奋阈之间差异无显著性,而CI大鼠LS DR的兴奋阈显著低于TL DR,提示CI大鼠腰骶背根传入神经纤维兴奋阈改变较显著。
2.4 CI大鼠胸腰和腰骶段传入神经纤维对CRD反应的改变
2.4.1 CI大鼠腰骶传入神经纤维对各等级CRD反应显著增强比较对照与CI大鼠LS DR对相同强度CRD刺激的反应差异。CI大鼠LS DR对各个等级强度CRD刺激的平均反应强度均显著高于对照大鼠,提示CI大鼠腰骶高、低阈值的初级传入神经纤维均敏化。
2.4.2 CI大鼠胸腰传入神经纤维对较高强度CRD反应显著增强
比较对照与CI大鼠TL DR在相同强度CRD刺激下的反应。CI大鼠脊髓背根对40～80 mmHg CRD的平均反应较对照大鼠显著增高,提示CI大鼠胸腰高阈值的传入神经纤维敏化。
2.4.3 CI大鼠腰骶传入神经纤维对各等级CRD反应显著高于胸腰传入
比较TL和LS DR对CRD的反应。对照大鼠LS DR只对80 mmHg的CRD的平均反应显著强于TL DR;而CI大鼠LS DR对20～80 mmHg各个等级的CRD的平均反应都显著高于TL DR。提示CI大鼠LS DR对伤害与非伤害刺激反应的强度均显著高于TL DR,而对照大鼠LS DR仅对伤害性刺激反应的强度显著高于TL DR。
3 CI大鼠脊髓神经元对内脏与躯体刺激的反应增强
3.1 CI大鼠脊髓腰骶神经元对结直肠扩张刺激的反应增强比较对照与CI大鼠LS脊髓背角神经元对CRD刺激的反应。CI大鼠LS神经元对20～80 mmHg各个压力CRD的平均反应均显著高于对照大鼠。提示CI大鼠LS脊髓背角神经元对伤害与非伤害结直肠扩张刺激的反应显著增强。
3.2 CI大鼠脊髓腰骶神经元对相应躯体刺激的反应增强
比较对照与CI大鼠LS神经元对伤害与非伤害性各种不同躯体刺激的反应。与对照大鼠相比,CI大鼠LS神经元对伤害与非伤害性各种不同躯体刺激的反应均显著增强。提示CI大鼠除了出现慢性内脏痛敏,还伴随相应躯体感觉敏化。
4谷氨酸受体在CI模型大鼠慢性内脏痛敏中的作用
4.1 NMDA受体涉及IBS样功能性慢性内脏痛的形态学证据
4.1.1 CI大鼠脊髓背根神经节NMDA受体NR2B亚单位表达增强采用免疫组织化学方法,比较对照与CI大鼠背根神经节NMDA受体NR2B亚单位的表达。图像分析结果,CI大鼠NR2B表达的平均光密度较对照组显著升高。提示CI大鼠背根神经节NMDA受体NR2B亚单位可能涉及IBS样功能性慢性内脏痛的外周敏化机制。
4.1.2 CI大鼠脊髓内脏相关神经元NMDA受体NR2B亚单位表达增强
采用免疫组织化学方法,比较对照与CI大鼠脊髓背角神经元NMDA受体NR2B亚单位的表达。图像分析结果,CI大鼠胸腰和腰骶段NR2B亚单位表达的平均光密度均显著高于对照大鼠。提示,CI大鼠脊髓NMDA受体NR2B亚单位可能涉及IBS样功能性慢性内脏痛的脊髓敏化机制。
4.2 NMDA受体在IBS样功能性慢性内脏痛敏中的作用
4.2.1腹腔注射AP-7显著抑制CI大鼠腰骶传入神经纤维对CRD刺激的反应
本研究选用中等剂量AP-7 (0.5 mg/kg,i.p.),比较对照和CI大鼠给药前后LS DR对20～80 mmHg CRD的反应。结果显示,NMDA受体拮抗剂AP-7显著抑制对照和CI大鼠LS DR对20～80 mmHg CRD的反应),CI大鼠AP-7的抑制百分率显著高于对照大鼠。提示体内NMDA受体可能介导IBS样功能性慢性内脏痛敏。
4.2.2脊髓局部给予AP-7显著抑制CI大鼠腰骶神经元对CRD刺激的反应
4.2.2.1脊髓局部给予AP-7抑制CI大鼠腰骶神经元对CRD反应的百分数
比较脊髓局部给予相同剂量AP-7在对照与CI大鼠之间对腰骶神经元产生效应的百分数。结果表明,相同剂量AP-7对CI组大鼠神经元产生效应的百分数较对照组大。
4.2.2.2脊髓局部给予AP-7显著抑制CI大鼠腰骶神经元对CRD的反应
比较脊髓局部给予不同浓度AP-7抑制对照与CI大鼠腰骶神经元对CRD刺激反应的程度。结果显示,与对照组大鼠相比,0.01 mmol AP-7则能剂量依赖性显著减弱CI组大鼠LS神经元对各压力等级CRD的平均反应。以上结果提示,脊髓NMDA受体参与介导IBS样功能性慢性内脏痛敏。
4.3脊髓非NMDA受体在IBS样功能性慢性内脏痛敏中的作用
4.3.1脊髓局部给予CNQX抑制CI大鼠腰骶神经元对CRD反应的百分数
比较脊髓局部给予相同剂量CNQX在对照与CI大鼠之间对腰骶神经元产生效应的百分数。与对照大鼠相比,相同剂量CNQX对CI大鼠神经元产生效应的百分数较大。
4.3.2脊髓局部给予CNQX抑制CI大鼠腰骶神经元对CRD的反应
比较脊髓局部给予不同浓度CNQX抑制对照与CI大鼠腰骶神经元对CRD刺激反应的程度。结果,与对照大鼠相比,2μmol CNQX就显著降低CI大鼠腰骶神经元对所有等级CRD的平均反应,同时2～10μmol CNQX呈剂量依赖性显著减弱神经元对伤害性和非伤害性CRD的平均反应。以上结果提示,脊髓非NMDA受体参与介导IBS样功能性慢性内脏痛敏。
综上所述,本工作提示:
1.改进后的CI模型制作方法可提高建模的成功率。CI模型大鼠慢性内脏痛敏显著,且伴有腹泻、便秘症状与牵涉痛,其结直肠局部组织未见明显的病理改变。提示本CI模型可做为IBS样功能性慢性内脏痛的研究模型。
2.IBS样功能性慢性内脏痛大鼠存在外周传入神经纤维敏化。与胸腰背根在结肠急性伤害性传入的作用相比,其在结肠慢性痛觉敏化过程中可能发挥了更大作用;而腰骶背根传入在急性与慢性痛觉敏化过程中可能发挥了同等重要的作用。
3.新生期的结肠伤害性刺激敏化了成年大鼠腰骶神经元对结直肠扩张刺激的反应,即IBS样功能性慢性内脏痛大鼠存在脊髓背角神经元敏化。
4. CI大鼠背根神经节和脊髓内脏相关神经元NMDA受体NR2B亚单位表达增强,这为NR2B亚单位可能涉及功能性慢性内脏痛的外周和脊髓敏化机制提供形态学基础。脊髓NMDA受体和非NMDA受体参与介导功能性慢性内脏痛敏作用。此外,似乎还提示功能性慢性内脏痛时,脊髓与内脏痛觉相关的离子型谷氨酸受体功能可能更多地被激活。
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 pain
1.1 The improvement of Al-Chare’s model
Modeling 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 rats
The 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 rats
The 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 rats
To 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 rats
The 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 rats
The 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 rats
2.1 Increased number of TL and LS afferent fibers activated by CRD in CI rats
The 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 rats
The 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 rats
The 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 rats
2.4.1 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 rats
The 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 CNQX
Spinal 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.
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