脊髓NMDA受体在IBS慢性内脏痛敏及针刺对其疗效中的作用
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摘要
内脏高敏感性(visceral hyperalgesia)是指内脏对正常生理性或伤害性刺激的感觉阈值降低,反应增强,其发生机制非常复杂。以往研究多局限于胃肠道局部病变,如肠嗜铬细胞和肥大细胞的活化、肠壁内脑肠肽及其受体的表达异常等,即外周致敏机制。但内脏感觉的形成是一个复杂的过程,内脏感觉中枢传导通路和大脑高级中枢的异常,包括神经递质及其受体的表达和活性,均可影响对内脏刺激的感知。
肠易激综合征(irritable bowel syndrome,IBS)是临床上一种常见病,其主要临床表现之一是腹痛和腹部不适,属于一种慢性内脏痛敏。IBS的局部肠道虽然没有明显器质性病变,但研究表明IBS病人对阈下肠道刺激感觉过敏。近年来对IBS发病机制的研究显示,脊髓中枢对内脏感觉传入投射区域的敏感性升高是形成IBS慢性内脏痛敏的重要机制。脊髓背角和侧角的内脏感觉投射神经元和中间神经元的兴奋性异常升高是形成内脏中枢敏化的机制之一,其中一些神经递质或调质如五羟色胺递质受体系统参与发挥了作用。
针刺镇痛是祖国医学的瑰宝之一,具有疗效确切,毒副作用少,经济简单等优点。国内外大量临床和基础研究表明,针刺治疗各类疼痛包括内脏痛具有显著镇痛作用。AI-Chaer于2001年报道了一种能够模拟临床IBS的大鼠模型。我们在成功复制了该IBS模型大鼠后,观察了电针对肠易激综合症的治疗作用,结果发现电针能明显缓解模型大鼠慢性内脏痛敏。然而目前对于上述针刺治疗IBS模型大鼠慢性内脏痛敏作用的机制研究还有待于进一步的开展。
鉴于慢性内脏痛的形成和发展与脊髓的谷氨酸递质受体系统有着密切的联系,本研究选择了脊髓NR1受体为研究对象,观察和分析脊髓NR1受体在IBS慢性内脏痛敏形成中及针刺对其疗效中的作用。具体内容报告如下:
一.研究内容:
1.建立IBS大鼠模型
参照AI-Chaer等报道的大鼠IBS模型,将雄性SD新生鼠,自出生后8天起,每天给予结直肠扩张刺激(在这个过程中改进造模方法,根据老鼠的状态给予不同量的结直肠刺激),连续两周,使其在成年后形成慢性内脏痛敏状态。通过测定模型大鼠体重和腹泻情况、腹部撤回反射(abdominal withdrawal reflex,AWR),对模型进行综合评估。
2.运用神经药理学方法,观察脊髓中枢NMDA受体在IBS大鼠慢性痛敏维持中的作用
鞘内埋管,给予NMDA受体拮抗剂MK-801,观察给药前后IBS大鼠内脏痛敏行为的变化。
3.运用分子生物学方法,观察脊髓中枢部位NMDA受体在IBS大鼠慢性痛敏形成过程中的表达规律
1)运用Western Blot方法,从蛋白水平观察NR1受体的表达变化;
2)运用RT-PCR方法,从mRNA水平观察NR1受体的表达变化。
4.运用分子生物学方法,观察电针过程中脊髓中枢NMDA受体表达变化规律
1)运用Western Blot方法,从蛋白水平观察电针前后NR1受体的表达改变;
2)运用RT-PCR方法,从mRNA水平观察电针前后NR1受体的表达改变。
二.研究结果:
1.大鼠IBS慢性内脏痛敏模型的建立和评价
(1)IBS大鼠模型的建立
参照Al-Chaer等报道的大鼠IBS模型,并对报道的造模方法进行改进,制造成功的IBS大鼠内脏痛敏表现明显,腹泻体征表现突出,内脏痛持续时间长。通过造模方法的改进,大大提高了模型的存活率和稳定性。
(2)成年IBS大鼠AWR评分异常升高
成年IBS大鼠在清醒的状态下,对不同强度的结直肠扩张刺激(CRD)所产生的腹部撤回反射(AWR)进行半定量行为评分。结果表明:与正常成年大鼠相比,在各个诱发刺激强度下,IBS大鼠的AWR评分显著升高。
(3)成年IBS大鼠与正常大鼠腹泻体征比较
比较IBS大鼠(6周龄时)和同龄正常大鼠的腹泻体征的差异。结果表明,IBS大鼠伴有腹泻体征发生率与同龄正常大鼠比较具有显著性差异。该结果提示IBS大鼠具有明显的腹泻体征。
(4)成年IBS大鼠与正常大鼠体重比较
比较IBS大鼠(各个时间阶段)和同时间段正常大鼠的体重差异。结果表明,IBS大鼠(各个时间阶段)与同时间段正常大鼠体重比较无明显差异。提示造模本身对IBS大鼠体重无明显影响。
2.脊髓兴奋性氨基酸递质受体系统在IBS大鼠慢性内脏痛敏中的作用
2.1鞘内注射MK-801对IBS大鼠AWR评分的影响
本实验观察了鞘内给予不同剂量的NMDA受体特异性拮抗剂MK-801对IBS大鼠AWR评分的影响。大鼠鞘内埋管3天后进行埋管位置鉴定,4天后进行实验。给药前进行AWR评分基础值记录。实验分为两组:正常对照组(Normal)和IBS模型组(Model),两组分别给予低、中和高剂量的MK-801(分别为0.01μg、0.1μg和1μg的MK-801)。结果表明:正常对照组大鼠鞘内注射各种剂量MK-801后AWR评分与注射前相比没有明显变化;IBS模型组鞘内注射低剂量MK-801后AWR评分呈现降低的趋势,但无统计学差异;IBS模型组鞘内注射中剂量和高剂量MK-801后AWR评分与注射前相比显著降低。该结果提示,i.t.MK-801可剂量相关性地降低IBS大鼠AWR评分。
2.2 IBS模型大鼠脊髓NR1受体mRNA的表达显著升高
运用RT-PCR方法检测IBS模型大鼠脊髓NR1受体mRNA的表达变化。实验分为正常组(Normal)和IBS模型组(Model)。Trizol一步法提取总mRNA。RT-PCR结果显示,与正常组相比,模型组脊髓腰膨大NR1受体mRNA表达明显升高。
2.3 IBS模型大鼠脊髓NR1受体蛋白的表达显著升高
运用western blot方法检测IBS模型大鼠脊髓NR1受体蛋白的表达变化。实验分为:正常组(Normal)和IBS模型组(Model)。western blot结果显示,与正常组相比,模型组脊髓腰膨大NR1受体蛋白表达明显升高。
3.电针缓解IBS大鼠内脏痛敏
本实验观察了单次电针在短时间内(停针后20min)对IBS大鼠AWR的影响。实验分为四组:正常对照组(Normal),模型组(Model),模型加电针组(Model+EA)和模型加假电针组(Model+Sham EA)。结果表明:在不同强度(20,40,60,80mmHg)的内脏痛诱发刺激下,与模型组相比,模型加电针组AWR评分明显降低,而假电针组无明显改变。提示单次电针在短时间内可以明显地缓解内脏痛敏。
4.脊髓兴奋性氨基酸递质受体系统在电针缓解IBS慢性内脏痛中的作用
4.1电针降低IBS大鼠脊髓NR1受体mRNA的表达
运用RT-PCR方法检测电针对IBS模型大鼠脊髓NR1受体mRNA表达的影响。实验分为:正常组(Normal)、IBS模型组(Model)、模型加电针组(Model+EA)和模型加假电针组(Model+Sham EA)。RT-PCR结果显示,与模型组相比,模型加电针组脊髓腰膨大NR1受体mRNA表达明显降低;而模型加假电针组无明显变化。
4.2电针降低IBS大鼠脊髓NR1受体蛋白的表达
运用western blot方法检测电针对IBS模型大鼠脊髓NR1受体蛋白表达的影响。实验分为:正常组(Normal)、IBS模型组(Model)、模型加电针组(Model+EA)和模型加假电针组(Model+Sham EA)。Western blot结果显示,与模型组相比,模型加电针组脊髓腰膨大NR1受体蛋白表达明显降低,而模型加假电针组无明显变化。
三、研究结论:
1.制作成功的IBS大鼠模型能较好地模拟了临床IBS的表现:腹痛表现明显,腹泻体征表现突出,是一个比较稳定的慢性内脏痛敏动物模型。通过造模方法的改进,大大提高了模型的存活率和稳定性。
2.IBS大鼠脊髓中枢NMDA R1受体表达明显增加,提示了脊髓中枢NMDA R1受体可能参与了功能性慢性内脏痛敏的维持。
3.单次电针在短时间内(停针后20min)可以明显缓解慢性内脏痛敏。
4.电针可以明显抑制脊髓背角NMDA R1受体的表达,这可能是针刺缓解慢性内脏痛敏的机制之一。
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 study
1.Animal model
To 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 study
1.Assessment of IBS model
1.1 production of IBS model
According 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 sensation
Behavioral 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 ones
There 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 ones
There 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 rat
2.1 The effect intrathecal injection of MK-801 on AWR scores in IBS rat
The 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 rat
RT-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 rat
Western 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 treatment
AWR 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 rat
4.1 The expression of spinal NMDA R1 mRNA in IBS rat treated with single EA
RT-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 EA
Western 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 study
1、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.
肠易激综合征(irritable bowel syndrome,IBS)是临床上一种常见病,其主要临床表现之一是腹痛和腹部不适,属于一种慢性内脏痛敏。IBS的局部肠道虽然没有明显器质性病变,但研究表明IBS病人对阈下肠道刺激感觉过敏。近年来对IBS发病机制的研究显示,脊髓中枢对内脏感觉传入投射区域的敏感性升高是形成IBS慢性内脏痛敏的重要机制。脊髓背角和侧角的内脏感觉投射神经元和中间神经元的兴奋性异常升高是形成内脏中枢敏化的机制之一,其中一些神经递质或调质如五羟色胺递质受体系统参与发挥了作用。
针刺镇痛是祖国医学的瑰宝之一,具有疗效确切,毒副作用少,经济简单等优点。国内外大量临床和基础研究表明,针刺治疗各类疼痛包括内脏痛具有显著镇痛作用。AI-Chaer于2001年报道了一种能够模拟临床IBS的大鼠模型。我们在成功复制了该IBS模型大鼠后,观察了电针对肠易激综合症的治疗作用,结果发现电针能明显缓解模型大鼠慢性内脏痛敏。然而目前对于上述针刺治疗IBS模型大鼠慢性内脏痛敏作用的机制研究还有待于进一步的开展。
鉴于慢性内脏痛的形成和发展与脊髓的谷氨酸递质受体系统有着密切的联系,本研究选择了脊髓NR1受体为研究对象,观察和分析脊髓NR1受体在IBS慢性内脏痛敏形成中及针刺对其疗效中的作用。具体内容报告如下:
一.研究内容:
1.建立IBS大鼠模型
参照AI-Chaer等报道的大鼠IBS模型,将雄性SD新生鼠,自出生后8天起,每天给予结直肠扩张刺激(在这个过程中改进造模方法,根据老鼠的状态给予不同量的结直肠刺激),连续两周,使其在成年后形成慢性内脏痛敏状态。通过测定模型大鼠体重和腹泻情况、腹部撤回反射(abdominal withdrawal reflex,AWR),对模型进行综合评估。
2.运用神经药理学方法,观察脊髓中枢NMDA受体在IBS大鼠慢性痛敏维持中的作用
鞘内埋管,给予NMDA受体拮抗剂MK-801,观察给药前后IBS大鼠内脏痛敏行为的变化。
3.运用分子生物学方法,观察脊髓中枢部位NMDA受体在IBS大鼠慢性痛敏形成过程中的表达规律
1)运用Western Blot方法,从蛋白水平观察NR1受体的表达变化;
2)运用RT-PCR方法,从mRNA水平观察NR1受体的表达变化。
4.运用分子生物学方法,观察电针过程中脊髓中枢NMDA受体表达变化规律
1)运用Western Blot方法,从蛋白水平观察电针前后NR1受体的表达改变;
2)运用RT-PCR方法,从mRNA水平观察电针前后NR1受体的表达改变。
二.研究结果:
1.大鼠IBS慢性内脏痛敏模型的建立和评价
(1)IBS大鼠模型的建立
参照Al-Chaer等报道的大鼠IBS模型,并对报道的造模方法进行改进,制造成功的IBS大鼠内脏痛敏表现明显,腹泻体征表现突出,内脏痛持续时间长。通过造模方法的改进,大大提高了模型的存活率和稳定性。
(2)成年IBS大鼠AWR评分异常升高
成年IBS大鼠在清醒的状态下,对不同强度的结直肠扩张刺激(CRD)所产生的腹部撤回反射(AWR)进行半定量行为评分。结果表明:与正常成年大鼠相比,在各个诱发刺激强度下,IBS大鼠的AWR评分显著升高。
(3)成年IBS大鼠与正常大鼠腹泻体征比较
比较IBS大鼠(6周龄时)和同龄正常大鼠的腹泻体征的差异。结果表明,IBS大鼠伴有腹泻体征发生率与同龄正常大鼠比较具有显著性差异。该结果提示IBS大鼠具有明显的腹泻体征。
(4)成年IBS大鼠与正常大鼠体重比较
比较IBS大鼠(各个时间阶段)和同时间段正常大鼠的体重差异。结果表明,IBS大鼠(各个时间阶段)与同时间段正常大鼠体重比较无明显差异。提示造模本身对IBS大鼠体重无明显影响。
2.脊髓兴奋性氨基酸递质受体系统在IBS大鼠慢性内脏痛敏中的作用
2.1鞘内注射MK-801对IBS大鼠AWR评分的影响
本实验观察了鞘内给予不同剂量的NMDA受体特异性拮抗剂MK-801对IBS大鼠AWR评分的影响。大鼠鞘内埋管3天后进行埋管位置鉴定,4天后进行实验。给药前进行AWR评分基础值记录。实验分为两组:正常对照组(Normal)和IBS模型组(Model),两组分别给予低、中和高剂量的MK-801(分别为0.01μg、0.1μg和1μg的MK-801)。结果表明:正常对照组大鼠鞘内注射各种剂量MK-801后AWR评分与注射前相比没有明显变化;IBS模型组鞘内注射低剂量MK-801后AWR评分呈现降低的趋势,但无统计学差异;IBS模型组鞘内注射中剂量和高剂量MK-801后AWR评分与注射前相比显著降低。该结果提示,i.t.MK-801可剂量相关性地降低IBS大鼠AWR评分。
2.2 IBS模型大鼠脊髓NR1受体mRNA的表达显著升高
运用RT-PCR方法检测IBS模型大鼠脊髓NR1受体mRNA的表达变化。实验分为正常组(Normal)和IBS模型组(Model)。Trizol一步法提取总mRNA。RT-PCR结果显示,与正常组相比,模型组脊髓腰膨大NR1受体mRNA表达明显升高。
2.3 IBS模型大鼠脊髓NR1受体蛋白的表达显著升高
运用western blot方法检测IBS模型大鼠脊髓NR1受体蛋白的表达变化。实验分为:正常组(Normal)和IBS模型组(Model)。western blot结果显示,与正常组相比,模型组脊髓腰膨大NR1受体蛋白表达明显升高。
3.电针缓解IBS大鼠内脏痛敏
本实验观察了单次电针在短时间内(停针后20min)对IBS大鼠AWR的影响。实验分为四组:正常对照组(Normal),模型组(Model),模型加电针组(Model+EA)和模型加假电针组(Model+Sham EA)。结果表明:在不同强度(20,40,60,80mmHg)的内脏痛诱发刺激下,与模型组相比,模型加电针组AWR评分明显降低,而假电针组无明显改变。提示单次电针在短时间内可以明显地缓解内脏痛敏。
4.脊髓兴奋性氨基酸递质受体系统在电针缓解IBS慢性内脏痛中的作用
4.1电针降低IBS大鼠脊髓NR1受体mRNA的表达
运用RT-PCR方法检测电针对IBS模型大鼠脊髓NR1受体mRNA表达的影响。实验分为:正常组(Normal)、IBS模型组(Model)、模型加电针组(Model+EA)和模型加假电针组(Model+Sham EA)。RT-PCR结果显示,与模型组相比,模型加电针组脊髓腰膨大NR1受体mRNA表达明显降低;而模型加假电针组无明显变化。
4.2电针降低IBS大鼠脊髓NR1受体蛋白的表达
运用western blot方法检测电针对IBS模型大鼠脊髓NR1受体蛋白表达的影响。实验分为:正常组(Normal)、IBS模型组(Model)、模型加电针组(Model+EA)和模型加假电针组(Model+Sham EA)。Western blot结果显示,与模型组相比,模型加电针组脊髓腰膨大NR1受体蛋白表达明显降低,而模型加假电针组无明显变化。
三、研究结论:
1.制作成功的IBS大鼠模型能较好地模拟了临床IBS的表现:腹痛表现明显,腹泻体征表现突出,是一个比较稳定的慢性内脏痛敏动物模型。通过造模方法的改进,大大提高了模型的存活率和稳定性。
2.IBS大鼠脊髓中枢NMDA R1受体表达明显增加,提示了脊髓中枢NMDA R1受体可能参与了功能性慢性内脏痛敏的维持。
3.单次电针在短时间内(停针后20min)可以明显缓解慢性内脏痛敏。
4.电针可以明显抑制脊髓背角NMDA R1受体的表达,这可能是针刺缓解慢性内脏痛敏的机制之一。
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 study
1.Animal model
To 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 study
1.Assessment of IBS model
1.1 production of IBS model
According 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 sensation
Behavioral 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 ones
There 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 ones
There 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 rat
2.1 The effect intrathecal injection of MK-801 on AWR scores in IBS rat
The 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 rat
RT-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 rat
Western 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 treatment
AWR 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 rat
4.1 The expression of spinal NMDA R1 mRNA in IBS rat treated with single EA
RT-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 EA
Western 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 study
1、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.
引文
[1]Giamberardino MA,Vecchiet L.Visceral pain,referred hyperalgesia and outcome:new concepts[J].Eur J Anaesthesiol,1995,10:61-66.
[2]荣培晶,张建梁,张宏肩.内脏痛觉的病理生理研究进展.中国病理生理杂志,2004,20(3):475-480.
[3]张道麟.肠道易激综合症研究现状[J].中国肛肠病杂志,1994,3:31-33.
[4]Sengupta JN,Gebhart GF.Mechanosensitive afferent fibers in the gastrointestinal and lower urinary tracts[J].Visceral pain,Progress in pain research and management,edited by G.F.Gebhart.Seattlle:IASP press,1995,5(7):75-98.
[5]Naliboff BD,Derbyshire SW,Munakata J,et al.Cerebral activation in patients with irritable bowel syndrome and control subjects during rectosigmoid stimulation[J].Psychosom Med,2001,63:365-375.
[6]王巍峰,杨云生,孙刚,等.肠易激综合征大鼠P物质能神经通路的改变[J].世界华人消化杂志,2005,13:214-218.
[7]Sun YN,Luo JY.Effects of tegaserod on Fos,substance P and calcitonin gene-related peptide expression induced by colon inflammation in lumbarsacral spinal cord[J].World J Gastroenterol,2004,10:1830-1833.
[8]Gaudreau GA,Plourde V.Involvement of N-methyl-d-aspartate(NMDA) receptors in a rat model of visceral hypersensitivity[J].Behav Brain Res,2004,150:185-189.
[9]Willeft RP,Woolf CJ,Hobson AR,et al.The development and maintenance of human visceral pain hyperalgesia is dependent on the N-Methyl-D-Aspartate receptor[J].Gastroenterology,2004,126:683-692.
[10]Kolhekar R,Gebhart GF.Modulation of spinal visceral nociceptive transmission by NMDA receptor activation in the rat[J].J Neurophysiol,1996,75:2344-2353.
[11]AL-Chaer ED,Kawasaki M,Pasricha PJ.A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development[J].Gastroenterology,2000,119:1276-1285.
[12]Cui KM,Li WM,Gao X,et al.Electro-acupuncture relieves chronic visceral hyperalgesia in rats[J].Neuroscience Letters,2005,376(1):20-23.
[13]李为民,崔可密,吴根诚.电针治疗肠易激综合症大鼠作用观察[J].上海针灸,2006,25(3):43-47.
[14]Cervero F.Visceral pain:mechanisms of peripheral and central sensitization[J].Ann Med,1995;27:235-239.
[15]Ness TJ,Gebhart GF.Visceral pain:a review of experimental studies[J].Pain,1990,41(2):167-234.
[16]Elie D,Al-Chaer,Richard J Traub.Biological basis of visceral pain:recent developments[J].Pain,2002,96:221-225.
[17]Heady JD,Wolff H,Goodell H.Pain sensations and reactions[J].Baltimore MD:Williams &Wilkins,1952,2(3):104-116.
[18]Simone DA,Sorkin LS,Oh U,et al.Neurogenic hyperalgesia:central neural correlates in responses of spinothalamic tract neurons[J].J Neurophysiol,1991,65(1):141-142.
[19]Woolf CJ.Evidence for a central component of post-injury pain hyper-sensitivity [J].Nature,1983,306(5944):686-688.
[20]L Bueno,Fioramonti J.Visceral perception:inflammatory and non-inflammatory mediators[J].Gut,2002,51:19-23.
[21]Kolhekar R,Meller ST,Gebhart GF.Characterization of the role of spinal N-methyi-D-aspartate receptors in thermal nociception in the rat[J].Neuroscience,1993,57(2):385-395.
[22]Olivar T,Laird JM.Differential effects of N-methyl-D-aspartate receptor blockade on nociceptive somatic and visceral reflexes[J].Pain,1999,79:67-73.
[23]Ji Y,Traub RJ.Spinal NMDA receptors contribute to neuronal processing of acute noxious and nonnoxious colorectal stimulation in the rat[J].J Neurophysiol,2001,86:1783-1791.
[24]陈仁珠.肠易激综合征的流行病学和病因研究[J].华人消化杂志,1998,6(10):913-914.
[25]张荣在.肠道易激综合征研究[J].中国肛肠病杂志,1994,(3):31-33.
[26]苏皖文.机理和临床治疗方法.国外医学消化系统疾病分册[J].1993,13(2):82-84.
[27]任一鸣.乳果糖氢呼吸试验测定口-盲肠传递时间[J].新消化病学杂志,1997, 5(1):39-40.
[28]王大骏.肠易激综合征患者的胃排空.临床消化病杂志[J].1995,7(4):155-156.
[29]李兆东.促肾上腺皮质激素释放激素与胃肠道运动.国外医学内科学分册[J].1998,25(3):102-104.
[30]谢勇.肠易激综合征乙状结肠粘膜内胃肠激素及一氧化氮的变化[J].中国内镜杂志,1997,3(6):17-18.
[31]杨云生.肠易激综合征血浆及乙状结肠粘膜中CCK及SP的含量[J].新消化病杂志,1997,5(7):437-438.
[32]杨云生.血浆及乙状结肠粘膜中CCK、MOT的含量与肠易激综合征的关系[J].胃肠病学和肝病学杂志,1996,5(4):287-289.
[33]王利华.肠道感染与肠易激综合征[J].中华内科杂志,2002,41:90-93.
[34]王伟岸.精神因素对肠易激综合征患者内脏敏感性的影响[J].中华医学杂志2002,82:308-311.
[35]王巍峰,杨云生,孙刚,等.肠易激综合征大鼠P物质能神经通路的改变[J].世界华人消化杂志,2005,13:214-218.
[36]Sun YN,Luo JY.Effects of tegaserod on Fos,substance P and calcitonin gene-related peptide expression induced by colon inflammation in lumbarsacral spinal cord[J].World J Gastroenterol,2004,10:1830-1833.
[37]Kolhekar R,Gebhart GF.Modulation of spinal visceral nociceptive transmission by NMDA receptor activation in the rat[J].J Neurophysiol,1996,75:2344-2353.
[38]Gaudreau GA,Plourde V.Involvement of N-methyl-D-aspartate(NMDA) receptors in a rat model of visceral hypersensitivity[J].Brain Research,2004,150:185-189.
[39]苗明三.实验动物和动物实验技术.北京:中国中医药出版社,1997:3-7.
[40]施新猷.医学动物实验方法.北京:人民卫生出版社,1980:1-3.
[41]黄应堂.医学科学实验模型及评价方法.兰州:甘肃文化出版社,1998:1-5.
[42]杨斌,张励才,曾因明.内脏模型的研究进展[J].国外医学《麻醉学与复苏分册》,2003,24(2):101-103.
[43]Mayer EA,Gebhart GF.Basic and clinical aspects of visceral hyperalgesia[J]. Gastroenterology,1994,107:271-293.
[44]AL-Chaer ED,Traub RJ.Biological basis of visceral pain:recent developments[J].Pain,2002,96:221-225.
[45]Woolf CJ,Water ET.Common patterns of plasticity contributing to nociceptive sensitization in mammals and aplysia[J].Trends Neuroscience,1991,14:74-78.
[46]Fitzgerald M,Jennings E.The postnatal development of Spinal sensory processing [J].Proceedings of the National Academy of Sciences of the USA,1999,96:7719-7722.
[47]Andrews K,Fitzgerald M.The cutaneous withdrawal reflex in human neonates:sensitization,receptive fields,and effects of contralteral stimulation[J].Pain,1994,56:95-101.
[48]Fitzgerald M.Development of pain mechanisms[J].British Medical Bulletin,1991,47:667-675.
[49]Coutinho S,Plotsky P,Sablad M,et al.Neonatal maternal separation alters stress-induced responses to viscerosomatic nociceptive stimuli in the adult rat[J].Am J Physiol Gastrointest Liver Physiol,2002,282:G307-316.
[50]王德深.针灸基础知识.北京:科学出版社,1985:1-8.
[51]张笑平.针灸作用机理研究.合肥:安徽科学技术出版社,1983:1-1.
[52]郑关毅.针刺足三里穴治疗肠道易激综合征的临床观察[J].上海针灸杂志,1996,15(4):20-21.
[53]Zhang YQ,Ji GC,Wu GC,et al.Kynurenic acid enhances electroacupuncture analgesia in normal and carrageenan-injected rats[J].Brain Research,2003,966(2):300-307.
[54]Zhang YQ,Ji GC,Wu GC,et al.Excitatory amino acid receptor antagonists and electroacupuncture synergetically inhibit carrageenan-induced behavioral hyperalgesia and spinal fos expression in rats[J].Pain,2002,99(3):525-535.
[55]Wang SJ,Omori N,Li F,et al.Enhanced expression of phospho-Akt by electro-acupuncture in normal rat brain[J].Neurological Research,2002,24(7):719-724.
[56]Medeiros MA,Canteras NS,Suchecki D,et al.Analgesia and c-Fos expression in the periaqueductal gray induced by electroacupuncture at the Zusanli point in rats[J]. Brain Research,2003,973(2):196-204.
[57]Ting H,Liao JM,Lin CF,et al.Pressor effect on blood pressure and renal nerve activity elicited by electroacupuncture in intact and acute hemorrhage rats[J].Neuroscience Letters,2002,327(1):5-8.
[58]Chiu JH,Chung MS.Cheng HC,et al.Different central manifestations in response to electroacupuncture at analgesic and nonanalgesic acupoints in rats:a manganese-enhanced functional magnetic resonance imaging study[J].Canadian Journal of Veterinary Research,2003,67(2):94-101.
[59]张兆发,庄鼎.电针基础与临床.北京:中国科学技术出版社,1993:5-7.
[60]韩济生.针刺镇痛原理.上海:上海科技教育版社,1999:112-125.
[61]Ulett GA,Han S,Han JS.Electroacupuncture:mechanisms and clinical application [J].Biol Psychiatry,1998,44(2):129-138.
[1]W.M.Li,A.Suzuki,K.M.Cui.Responses of blood pressure and renal sympathetic nerve activity to colorectal distension in anesthetized rats.Journal of Physiological Sciences,2006,56(2):153-156.
[2]W.M.Li,A.Suzuki.Reflex inhibition of heart rate and efferent cardiac sympathetic outflow induced by colorectal distension in anesthetized rats.Journal of Physiological Sciences,2006,56(2):187-190.
[3]Giamberardino MA,Vecchiet L.Visceral pain,referred hyperalgesia and outcome:new concepts[J].Eur J Anaesthesiol,1995,10(Suppl):61-66.
[4]荣培晶,张建梁,张宏启.内脏痛觉的病理生理研究进展.中国病理生理杂志,2004,20(3):475-480.
[5]崔香淑,李在琉.胃肠感觉的神经解剖学基础.诊断学理论与实践,2006,5(1):5-7.
[6]Furness JB,Borstein JC,Kunze W AA,et al.The enteric nervous system and its extrinsicconnection[M]Yamada T,Alpers DH,Laine L.Textbook of Gastroenterology.vol 1,3rd ed,New York:Lippincott Williams and Wilkins 1999:11-34.
[7]Janig W,Koltzenburg M.Receptive properties of sacral primary afferent neurons supplying the colon[J].J Neurophysiol,1991,65(5):1067-1077.
[8]Zhang HQ,Al-Chaer ED,Willis WD.Effec of tactile inputs on thalamic responses to nociceptive colorectal distention in rat[J].J Neurophysiol.,2002,88(9):1185-1196.
[9]Rogers RC,Mc Tigue DM,Hermannn GE.Vagovagal reflex control of digestion:afferent modulation by neural and "endoneurocrine" factors[J].Am J Physiol.,1995,268(Pt 1):G1-G10.
[10]Gebhard CF,Ness TJ.Central mechanism of visceral pain[J].Can J Physiol Pharmacol,1991,69(5):627-634.
[11]张引国,田学亮,魏洁.内脏痛的研究进展.武警医学院学报,2001,10(1):85-87.
[12]Sugiura Y,Terui N,Hosoya Y.Difference in distribution of central terminals between visceral and somatic unmyelinated(C) primary afferent fibers[J].J Neurophysiol,1989,62(4):834-840.
[13]Cottrell DF,Iggo A.The responses of duodenal tension receptors in sheep to pentagastrin,cholecystokinin and some other drugs[J].J Physiol.,1984,354:477-495.
[14]El Ouazzani T,Mei N.Electrophysiologic properties and role of the vagal thermoreceptors of lower esophagus and stomach of cat[J].Gastroenterology,1982,83(5):995-1001.
[15]张建梁,张宏启.内脏痛的脊髓通路研究进展.国外医学-生理、病理科学与临床分册,2005,25(3):211-214.
[16]Cottrell DF.Mechanoreceptors of the rabbitduodenum[J].Q J Exp Physiol.,1984,69(4):677-684.
[17]李在琉.中枢神经系统与胃肠运动功能.[M]周吕,柯美云主编.神经胃肠病学与动力-基础与临床.北京:科学出版社,2005:143-170.
[18]Becker R,Gatscher S,Sure U,et al.Punctate midline myelotomy for intractable visceral pain caused by hepatobiliary or pancreatic cancer[J].J Pain Symptom Manage,2004,27(1):79-84.
[19]Lu GW,Li Q J,Meng Z,et al.Can we be aware of both visceral and somatic sensation via a single neuronal pathway? Chin Sci Bull,2002,47(23):1940-1945.
[20]Honda CN.Visceral and somatic afferent convergence onto neurons near the central in the sacral spinal cord of the cat.J Neurophysiol,1985,53(4):1059-1078.
[21]Villanueva L,Bouhassira D,Le Bars D.The medullary subnucleus reticularis dorsalis(SRD) as a key link in both the transmission and modulation of pain signals.Pain,1996,67(2-3):231-240.
[22]Ness TJ;Follett KA.The development of tolerance to intrathecal Morphine in rat models of visceral and cutaneous pain.Neuroscience letter volume,1998,22:33-36.
[23]AL-Chaer E D,KawasakiM,Pasricha P J.A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development[J].Gastroenterology,2000,119:1276-1285.
[24]Koster R,Anderson M,de Beer EJ.Acetic acid for analgesic screening.Fed Proc,1959,18:412.
[25]Schmauss C,Yaksh TL.In vivo Studies on spinal opiate receptor systems mediating antinociception.Ⅱ.Pharmacological profiles suggesting a differential association of mu,delta and kappa receptors with visceral chemical and cutaneous thermal stimuli in the rat.J Pharmacol Ther,1984,28:1-12.
[26]王宏琰,黄登凯.内脏痛的实验性研究,解剖学杂志,1995,18(3):282-287。
[27]Jian-Hua Liu,Jiangshan Li,Jie Yah.Expression of c-fos in the nucleus of the solitary tract following electroacupuncture at facial acupoints and gastric distension in rats.Neuroscience letters,2004,366(2):215-219.
[28]Huang Z,Liu N,Zhong S,Lu J,Zhang N.The role of nucleus tractus solitarii(NTS)in acupuncture inhibition of visceral-somatic reflex(VSR).Zhen ci yan jiu.1991;16(1):43-7.
[29]Gong S,Yin WP,Yin QZ.Involvement of vasopressinergic neurons of paraventricular nucleus in the electroacupuncture-induced inhibition of experimental visceral pain in rats.Sheng Li Xue Bao.1992 Oct;44(5):434-41.Chinese.
[30]Guoxi T.The action of the visceronociceptive neurons in the posterior group of thalamic nuclei:possible mechanism of acupuncture analgesia on visceral pain.Kitasato Arch Exp Med.1991 Apr;64(1):43-55.
[31]Sun M,Li Y,Zhang J,Bian J.Effects of noxious stimuli on the discharges of pain-excitation neurons and pain-inhibition neurons in the nucleus ventralis posterolalis of thalamus in the rat and a modulating action of electroacupuncture on its electric activities.Zhen ci yan jiu.1991;16(1):19-22.
[32]阎丽萍,马骋,项晓人,等.电针抑制内脏痛诱发的大鼠丘脑背内侧痛放电.中 国疼痛医学杂志,2004,10(3):177-180.
[33]Shu J,Li KY,Huang DK.The central effect of electro-acupuncture analgesia on visceral pain of rats:a study using the[3H]2-deoxyglucose method.Acupuncture&electro-therapeutics research,1994,Jun-Sep:19(2-3):107-17.
[34]Jen-Hwey Chiu.How is the motility of gastrointestinal sphincters modulated by acupmoxa? International Congress Series.Volume 1238,August 2002,Pages 141-147.
[35]Masahiro Iwa,Carmen Strickland,Yukiomi Nakade.Electroacupuncture Reduces Rectal Distension-Induced Blood Pressure Changes in Conscious Dogs.Digestive Diseases and Sciences,2005,50(7):162-167.
[36]吴红金,周雷,薛峥,等.电针抗大鼠急性炎症性内脏痛的肠神经机制.针剌研究,1999,24(2):138-142.
[37]Xiao Yu Tian,Zhao Xiang Bian.Electro-acupuncture attenuates stress-induced defecation in rats with chronic visceral hypersensitivity via serotonergic pathway.Brain research.2006,1008(1):101-108.
[38]蔡佰元,黄显奋.甲氧氯普胺加强电针镇内脏痛及其中枢机制.针刺研究,1994,19(1):66-70.
[39]曹福元,陈国斌,殷光甫,等.电针对大鼠内脏痛结肠壁、背根节和脊髓内一氧化氮合酶的影响.针刺研究,2006,31(1):15-18.
[2]荣培晶,张建梁,张宏肩.内脏痛觉的病理生理研究进展.中国病理生理杂志,2004,20(3):475-480.
[3]张道麟.肠道易激综合症研究现状[J].中国肛肠病杂志,1994,3:31-33.
[4]Sengupta JN,Gebhart GF.Mechanosensitive afferent fibers in the gastrointestinal and lower urinary tracts[J].Visceral pain,Progress in pain research and management,edited by G.F.Gebhart.Seattlle:IASP press,1995,5(7):75-98.
[5]Naliboff BD,Derbyshire SW,Munakata J,et al.Cerebral activation in patients with irritable bowel syndrome and control subjects during rectosigmoid stimulation[J].Psychosom Med,2001,63:365-375.
[6]王巍峰,杨云生,孙刚,等.肠易激综合征大鼠P物质能神经通路的改变[J].世界华人消化杂志,2005,13:214-218.
[7]Sun YN,Luo JY.Effects of tegaserod on Fos,substance P and calcitonin gene-related peptide expression induced by colon inflammation in lumbarsacral spinal cord[J].World J Gastroenterol,2004,10:1830-1833.
[8]Gaudreau GA,Plourde V.Involvement of N-methyl-d-aspartate(NMDA) receptors in a rat model of visceral hypersensitivity[J].Behav Brain Res,2004,150:185-189.
[9]Willeft RP,Woolf CJ,Hobson AR,et al.The development and maintenance of human visceral pain hyperalgesia is dependent on the N-Methyl-D-Aspartate receptor[J].Gastroenterology,2004,126:683-692.
[10]Kolhekar R,Gebhart GF.Modulation of spinal visceral nociceptive transmission by NMDA receptor activation in the rat[J].J Neurophysiol,1996,75:2344-2353.
[11]AL-Chaer ED,Kawasaki M,Pasricha PJ.A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development[J].Gastroenterology,2000,119:1276-1285.
[12]Cui KM,Li WM,Gao X,et al.Electro-acupuncture relieves chronic visceral hyperalgesia in rats[J].Neuroscience Letters,2005,376(1):20-23.
[13]李为民,崔可密,吴根诚.电针治疗肠易激综合症大鼠作用观察[J].上海针灸,2006,25(3):43-47.
[14]Cervero F.Visceral pain:mechanisms of peripheral and central sensitization[J].Ann Med,1995;27:235-239.
[15]Ness TJ,Gebhart GF.Visceral pain:a review of experimental studies[J].Pain,1990,41(2):167-234.
[16]Elie D,Al-Chaer,Richard J Traub.Biological basis of visceral pain:recent developments[J].Pain,2002,96:221-225.
[17]Heady JD,Wolff H,Goodell H.Pain sensations and reactions[J].Baltimore MD:Williams &Wilkins,1952,2(3):104-116.
[18]Simone DA,Sorkin LS,Oh U,et al.Neurogenic hyperalgesia:central neural correlates in responses of spinothalamic tract neurons[J].J Neurophysiol,1991,65(1):141-142.
[19]Woolf CJ.Evidence for a central component of post-injury pain hyper-sensitivity [J].Nature,1983,306(5944):686-688.
[20]L Bueno,Fioramonti J.Visceral perception:inflammatory and non-inflammatory mediators[J].Gut,2002,51:19-23.
[21]Kolhekar R,Meller ST,Gebhart GF.Characterization of the role of spinal N-methyi-D-aspartate receptors in thermal nociception in the rat[J].Neuroscience,1993,57(2):385-395.
[22]Olivar T,Laird JM.Differential effects of N-methyl-D-aspartate receptor blockade on nociceptive somatic and visceral reflexes[J].Pain,1999,79:67-73.
[23]Ji Y,Traub RJ.Spinal NMDA receptors contribute to neuronal processing of acute noxious and nonnoxious colorectal stimulation in the rat[J].J Neurophysiol,2001,86:1783-1791.
[24]陈仁珠.肠易激综合征的流行病学和病因研究[J].华人消化杂志,1998,6(10):913-914.
[25]张荣在.肠道易激综合征研究[J].中国肛肠病杂志,1994,(3):31-33.
[26]苏皖文.机理和临床治疗方法.国外医学消化系统疾病分册[J].1993,13(2):82-84.
[27]任一鸣.乳果糖氢呼吸试验测定口-盲肠传递时间[J].新消化病学杂志,1997, 5(1):39-40.
[28]王大骏.肠易激综合征患者的胃排空.临床消化病杂志[J].1995,7(4):155-156.
[29]李兆东.促肾上腺皮质激素释放激素与胃肠道运动.国外医学内科学分册[J].1998,25(3):102-104.
[30]谢勇.肠易激综合征乙状结肠粘膜内胃肠激素及一氧化氮的变化[J].中国内镜杂志,1997,3(6):17-18.
[31]杨云生.肠易激综合征血浆及乙状结肠粘膜中CCK及SP的含量[J].新消化病杂志,1997,5(7):437-438.
[32]杨云生.血浆及乙状结肠粘膜中CCK、MOT的含量与肠易激综合征的关系[J].胃肠病学和肝病学杂志,1996,5(4):287-289.
[33]王利华.肠道感染与肠易激综合征[J].中华内科杂志,2002,41:90-93.
[34]王伟岸.精神因素对肠易激综合征患者内脏敏感性的影响[J].中华医学杂志2002,82:308-311.
[35]王巍峰,杨云生,孙刚,等.肠易激综合征大鼠P物质能神经通路的改变[J].世界华人消化杂志,2005,13:214-218.
[36]Sun YN,Luo JY.Effects of tegaserod on Fos,substance P and calcitonin gene-related peptide expression induced by colon inflammation in lumbarsacral spinal cord[J].World J Gastroenterol,2004,10:1830-1833.
[37]Kolhekar R,Gebhart GF.Modulation of spinal visceral nociceptive transmission by NMDA receptor activation in the rat[J].J Neurophysiol,1996,75:2344-2353.
[38]Gaudreau GA,Plourde V.Involvement of N-methyl-D-aspartate(NMDA) receptors in a rat model of visceral hypersensitivity[J].Brain Research,2004,150:185-189.
[39]苗明三.实验动物和动物实验技术.北京:中国中医药出版社,1997:3-7.
[40]施新猷.医学动物实验方法.北京:人民卫生出版社,1980:1-3.
[41]黄应堂.医学科学实验模型及评价方法.兰州:甘肃文化出版社,1998:1-5.
[42]杨斌,张励才,曾因明.内脏模型的研究进展[J].国外医学《麻醉学与复苏分册》,2003,24(2):101-103.
[43]Mayer EA,Gebhart GF.Basic and clinical aspects of visceral hyperalgesia[J]. Gastroenterology,1994,107:271-293.
[44]AL-Chaer ED,Traub RJ.Biological basis of visceral pain:recent developments[J].Pain,2002,96:221-225.
[45]Woolf CJ,Water ET.Common patterns of plasticity contributing to nociceptive sensitization in mammals and aplysia[J].Trends Neuroscience,1991,14:74-78.
[46]Fitzgerald M,Jennings E.The postnatal development of Spinal sensory processing [J].Proceedings of the National Academy of Sciences of the USA,1999,96:7719-7722.
[47]Andrews K,Fitzgerald M.The cutaneous withdrawal reflex in human neonates:sensitization,receptive fields,and effects of contralteral stimulation[J].Pain,1994,56:95-101.
[48]Fitzgerald M.Development of pain mechanisms[J].British Medical Bulletin,1991,47:667-675.
[49]Coutinho S,Plotsky P,Sablad M,et al.Neonatal maternal separation alters stress-induced responses to viscerosomatic nociceptive stimuli in the adult rat[J].Am J Physiol Gastrointest Liver Physiol,2002,282:G307-316.
[50]王德深.针灸基础知识.北京:科学出版社,1985:1-8.
[51]张笑平.针灸作用机理研究.合肥:安徽科学技术出版社,1983:1-1.
[52]郑关毅.针刺足三里穴治疗肠道易激综合征的临床观察[J].上海针灸杂志,1996,15(4):20-21.
[53]Zhang YQ,Ji GC,Wu GC,et al.Kynurenic acid enhances electroacupuncture analgesia in normal and carrageenan-injected rats[J].Brain Research,2003,966(2):300-307.
[54]Zhang YQ,Ji GC,Wu GC,et al.Excitatory amino acid receptor antagonists and electroacupuncture synergetically inhibit carrageenan-induced behavioral hyperalgesia and spinal fos expression in rats[J].Pain,2002,99(3):525-535.
[55]Wang SJ,Omori N,Li F,et al.Enhanced expression of phospho-Akt by electro-acupuncture in normal rat brain[J].Neurological Research,2002,24(7):719-724.
[56]Medeiros MA,Canteras NS,Suchecki D,et al.Analgesia and c-Fos expression in the periaqueductal gray induced by electroacupuncture at the Zusanli point in rats[J]. Brain Research,2003,973(2):196-204.
[57]Ting H,Liao JM,Lin CF,et al.Pressor effect on blood pressure and renal nerve activity elicited by electroacupuncture in intact and acute hemorrhage rats[J].Neuroscience Letters,2002,327(1):5-8.
[58]Chiu JH,Chung MS.Cheng HC,et al.Different central manifestations in response to electroacupuncture at analgesic and nonanalgesic acupoints in rats:a manganese-enhanced functional magnetic resonance imaging study[J].Canadian Journal of Veterinary Research,2003,67(2):94-101.
[59]张兆发,庄鼎.电针基础与临床.北京:中国科学技术出版社,1993:5-7.
[60]韩济生.针刺镇痛原理.上海:上海科技教育版社,1999:112-125.
[61]Ulett GA,Han S,Han JS.Electroacupuncture:mechanisms and clinical application [J].Biol Psychiatry,1998,44(2):129-138.
[1]W.M.Li,A.Suzuki,K.M.Cui.Responses of blood pressure and renal sympathetic nerve activity to colorectal distension in anesthetized rats.Journal of Physiological Sciences,2006,56(2):153-156.
[2]W.M.Li,A.Suzuki.Reflex inhibition of heart rate and efferent cardiac sympathetic outflow induced by colorectal distension in anesthetized rats.Journal of Physiological Sciences,2006,56(2):187-190.
[3]Giamberardino MA,Vecchiet L.Visceral pain,referred hyperalgesia and outcome:new concepts[J].Eur J Anaesthesiol,1995,10(Suppl):61-66.
[4]荣培晶,张建梁,张宏启.内脏痛觉的病理生理研究进展.中国病理生理杂志,2004,20(3):475-480.
[5]崔香淑,李在琉.胃肠感觉的神经解剖学基础.诊断学理论与实践,2006,5(1):5-7.
[6]Furness JB,Borstein JC,Kunze W AA,et al.The enteric nervous system and its extrinsicconnection[M]Yamada T,Alpers DH,Laine L.Textbook of Gastroenterology.vol 1,3rd ed,New York:Lippincott Williams and Wilkins 1999:11-34.
[7]Janig W,Koltzenburg M.Receptive properties of sacral primary afferent neurons supplying the colon[J].J Neurophysiol,1991,65(5):1067-1077.
[8]Zhang HQ,Al-Chaer ED,Willis WD.Effec of tactile inputs on thalamic responses to nociceptive colorectal distention in rat[J].J Neurophysiol.,2002,88(9):1185-1196.
[9]Rogers RC,Mc Tigue DM,Hermannn GE.Vagovagal reflex control of digestion:afferent modulation by neural and "endoneurocrine" factors[J].Am J Physiol.,1995,268(Pt 1):G1-G10.
[10]Gebhard CF,Ness TJ.Central mechanism of visceral pain[J].Can J Physiol Pharmacol,1991,69(5):627-634.
[11]张引国,田学亮,魏洁.内脏痛的研究进展.武警医学院学报,2001,10(1):85-87.
[12]Sugiura Y,Terui N,Hosoya Y.Difference in distribution of central terminals between visceral and somatic unmyelinated(C) primary afferent fibers[J].J Neurophysiol,1989,62(4):834-840.
[13]Cottrell DF,Iggo A.The responses of duodenal tension receptors in sheep to pentagastrin,cholecystokinin and some other drugs[J].J Physiol.,1984,354:477-495.
[14]El Ouazzani T,Mei N.Electrophysiologic properties and role of the vagal thermoreceptors of lower esophagus and stomach of cat[J].Gastroenterology,1982,83(5):995-1001.
[15]张建梁,张宏启.内脏痛的脊髓通路研究进展.国外医学-生理、病理科学与临床分册,2005,25(3):211-214.
[16]Cottrell DF.Mechanoreceptors of the rabbitduodenum[J].Q J Exp Physiol.,1984,69(4):677-684.
[17]李在琉.中枢神经系统与胃肠运动功能.[M]周吕,柯美云主编.神经胃肠病学与动力-基础与临床.北京:科学出版社,2005:143-170.
[18]Becker R,Gatscher S,Sure U,et al.Punctate midline myelotomy for intractable visceral pain caused by hepatobiliary or pancreatic cancer[J].J Pain Symptom Manage,2004,27(1):79-84.
[19]Lu GW,Li Q J,Meng Z,et al.Can we be aware of both visceral and somatic sensation via a single neuronal pathway? Chin Sci Bull,2002,47(23):1940-1945.
[20]Honda CN.Visceral and somatic afferent convergence onto neurons near the central in the sacral spinal cord of the cat.J Neurophysiol,1985,53(4):1059-1078.
[21]Villanueva L,Bouhassira D,Le Bars D.The medullary subnucleus reticularis dorsalis(SRD) as a key link in both the transmission and modulation of pain signals.Pain,1996,67(2-3):231-240.
[22]Ness TJ;Follett KA.The development of tolerance to intrathecal Morphine in rat models of visceral and cutaneous pain.Neuroscience letter volume,1998,22:33-36.
[23]AL-Chaer E D,KawasakiM,Pasricha P J.A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development[J].Gastroenterology,2000,119:1276-1285.
[24]Koster R,Anderson M,de Beer EJ.Acetic acid for analgesic screening.Fed Proc,1959,18:412.
[25]Schmauss C,Yaksh TL.In vivo Studies on spinal opiate receptor systems mediating antinociception.Ⅱ.Pharmacological profiles suggesting a differential association of mu,delta and kappa receptors with visceral chemical and cutaneous thermal stimuli in the rat.J Pharmacol Ther,1984,28:1-12.
[26]王宏琰,黄登凯.内脏痛的实验性研究,解剖学杂志,1995,18(3):282-287。
[27]Jian-Hua Liu,Jiangshan Li,Jie Yah.Expression of c-fos in the nucleus of the solitary tract following electroacupuncture at facial acupoints and gastric distension in rats.Neuroscience letters,2004,366(2):215-219.
[28]Huang Z,Liu N,Zhong S,Lu J,Zhang N.The role of nucleus tractus solitarii(NTS)in acupuncture inhibition of visceral-somatic reflex(VSR).Zhen ci yan jiu.1991;16(1):43-7.
[29]Gong S,Yin WP,Yin QZ.Involvement of vasopressinergic neurons of paraventricular nucleus in the electroacupuncture-induced inhibition of experimental visceral pain in rats.Sheng Li Xue Bao.1992 Oct;44(5):434-41.Chinese.
[30]Guoxi T.The action of the visceronociceptive neurons in the posterior group of thalamic nuclei:possible mechanism of acupuncture analgesia on visceral pain.Kitasato Arch Exp Med.1991 Apr;64(1):43-55.
[31]Sun M,Li Y,Zhang J,Bian J.Effects of noxious stimuli on the discharges of pain-excitation neurons and pain-inhibition neurons in the nucleus ventralis posterolalis of thalamus in the rat and a modulating action of electroacupuncture on its electric activities.Zhen ci yan jiu.1991;16(1):19-22.
[32]阎丽萍,马骋,项晓人,等.电针抑制内脏痛诱发的大鼠丘脑背内侧痛放电.中 国疼痛医学杂志,2004,10(3):177-180.
[33]Shu J,Li KY,Huang DK.The central effect of electro-acupuncture analgesia on visceral pain of rats:a study using the[3H]2-deoxyglucose method.Acupuncture&electro-therapeutics research,1994,Jun-Sep:19(2-3):107-17.
[34]Jen-Hwey Chiu.How is the motility of gastrointestinal sphincters modulated by acupmoxa? International Congress Series.Volume 1238,August 2002,Pages 141-147.
[35]Masahiro Iwa,Carmen Strickland,Yukiomi Nakade.Electroacupuncture Reduces Rectal Distension-Induced Blood Pressure Changes in Conscious Dogs.Digestive Diseases and Sciences,2005,50(7):162-167.
[36]吴红金,周雷,薛峥,等.电针抗大鼠急性炎症性内脏痛的肠神经机制.针剌研究,1999,24(2):138-142.
[37]Xiao Yu Tian,Zhao Xiang Bian.Electro-acupuncture attenuates stress-induced defecation in rats with chronic visceral hypersensitivity via serotonergic pathway.Brain research.2006,1008(1):101-108.
[38]蔡佰元,黄显奋.甲氧氯普胺加强电针镇内脏痛及其中枢机制.针刺研究,1994,19(1):66-70.
[39]曹福元,陈国斌,殷光甫,等.电针对大鼠内脏痛结肠壁、背根节和脊髓内一氧化氮合酶的影响.针刺研究,2006,31(1):15-18.
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