【作者】 崔可密；

【导师】 吴根诚； 李为民；

【作者基本信息】 复旦大学 ， 中西医结合基础， 2005， 博士

【摘要】 慢性疼痛是现代医学发展和人类健康、保健所面临的严重问题之一,其中内脏疾患引起的慢性疼痛是临床上常见的疾病症候。内脏痛的发生部位深且定位模糊,同时伴有牵涉痛和植物神经反射等复杂的临床表现和特点。因此,人们对于内脏痛的认识远滞后于对躯体痛的了解,也使得慢性内脏痛的治疗缺乏特异性措施。 众所周知,由于临床研究的局限性,动物实验研究成为研究内脏痛的重要手段。但是导致内脏痛研究滞后的一个重要方面就是缺乏较为理想的模拟人类慢性内脏痛的动物模型。2000年Al-Chaer等用大鼠成功地制作了一种新的质量较高的慢性内脏痛敏模型（IBS大鼠模型）。该模型的出现,为深入探讨慢性内脏痛的中枢机制提供了一个新的契机。 研究表明,在内脏感觉敏感性增高状态下,机体对非伤害性刺激和伤害性刺激的处理,无论在外周还是在中枢水平都发生了改变。其中,中枢敏化的作用是研究热点之一,它是内脏痛发生发展过程中的关键性因素。有研究提示,如果抑制中枢敏化,则能有效地缓解慢性内脏痛。中枢敏化的形成涉及多种神经递质、受体系统,包括兴奋性氨基酸受体（EAA-R）、速激肽受体（NK-R）、5-羟色胺（5-HT）受体、α₂肾上腺素能受体、阿片受体等。其中兴奋性氨基酸递质受体系统在其中的作用引人注目。研究提示NMDA受体和non-NMDA受体均参与急性内脏伤害性感受,但究竟在慢性内脏痛疾病长期的病理演变过程中发挥了什么作用,这些与其在躯体痛中的作用有何不同和联系等,均值得进一步研究。 国内外很多临床实践证实针刺对慢性内脏痛有着良好的治疗效果。但以往研究所利用的动物模型尚不理想。新的慢性内脏痛模型是否适用于针刺研究?如何利用该模型深化慢性内脏痛以及针刺治疗慢性内脏痛的机制探讨?对这些问题的解答,将为临床针刺治疗慢性内脏痛提供科学依据和理论基础,推动内脏痛的基础研究,促进针刺疗法不断走向世界。 因此本项研究采用新的慢性内脏痛敏模型,并应用疼痛行为学观察、电生理细胞外记录和组织学技术对其进行评价;在该动物模型上,观察针刺对慢性内脏痛敏的治疗作用和作用规律,包括单次电针的即刻效应和多次电针的累加效应和不同针刺次数的治疗效应比较等;进而采用电生理细胞外记录等方法探讨脊髓兴奋性氨基酸递质受体系统在慢性内脏痛敏和针刺治疗慢性内脏痛敏中的作用。更多还原

【Abstract】 Chronic pain originated from viscera is one of the most common symptoms in clinic and also is one of serious problems to be faced with in modern medicine development and human health care. Visceral pain occurs deeply and has complex clinical characteristics of diffuse and poorly localized, referred pain, autonomous nerve reflex and etc. Thus the understanding of viscera pain is far lagged behind that of somatic pain and less specific therapies of chronic visceral pain are valid.It is well known animal experiment is a very important method in the research of visceral pain owing to the limit of clinical study. While absence of ideal animal models mimicing human chronic visceral pain is a key aspect for the draggling of visceral pain. A new rat model of chronic visceral hyperalgesia was invented by Al-Chaer and his colleague in 2000, which provided a new approach for further exploration of the central mechanism of chronic visceral pain.It is reported that altered response to non-noxious and noxious stimulus happened in both peripheral and central levels under the state of increased visceral sensitivity. Central sensitization is one of hot spots in the study of visceral pain and it is a pivotal factor in the occurrence and progress of visceral pain. It is proved that chronic visceral pain could be effectively relieved if central sensitization was inhibited. Many systems of neurotransmitter and their receptors are involved in the forming and maintenance of central sensitization including systems of excitory amino acid receptor （EAA-R）, tachykinin receptor （NK-R）, 5-hydroxytryptamine （5-HT） receptor, α₂ adrenergic receptor, opiod receptor, etc. The role of EAA-R system in central sensitization of somatic pain is conspicuous and there is evidence shows that NMDA receptor and non-NMDA receptor take part in nociception of acute visceral pain, but what is the function of EAA-R system in the pathological development of chronic visceral pain and the relation and difference among them need further investigation.It has been proved by domestic and overseas practice in clinic that chronic visceral pain could be relieved by acupuncture treatment. But no ideal animal models were used in experimental study. Whether the new animal model could serve acupuncture research or not? How to make further exploration about the mechanism of chronic visceral pain and acupuncture analgesia? To answer the questions is to provide scientific evidence and academic theory of acupunctureagainst chronic visceral pain, to promote base research in visceral pain and also to advance acupuncture therapy to be more widely used in the world.New chronic visceral pain animal model will exert and behavioral pain reflex observation, electrophysiological extracellular recording and histological techniques will be used in the study. Then the therapeutic effect and the rule of the effect including instant effect of single treatment, cumulative effect of repetitive treatments and comparison of different effects of different treatments times will be observed. Finally the role of spinal EAA-R system in chronic visceral pain and therapeutic effect by acupuncture will be investigated by methods including electrophysiological extracellular recording.Results:1 Fabrication and evaluation of IBS rat model of chronic visceral hyperalgesia1.1 Fabrication of IBS rat modelMale neonatal SD rats （8th-21st postnatal） were given colorectal distention stimulation daily by using self-made balloons with reference to the method of Al-Chaer’s. Evaluations were made when they became adults.1.2 Increased abdominal withdrawal reflex （AWR） score in IBS adult ratsSemi-quantitative score was given by observing AWR to graded colorectal distention （CRD） when the rats were awake. The results indicated that AWR of IBS group was significantly increased to graded CRD compared with that of normal rats. IBS rats manifested the sign of visceral hyperalgeisa.1.3 Increased discharge of abdominal electromyograms （AEMGs） in IBS ratsThe changes of AEMGs in response to graded CRD stimulation was recorded when the rats were under mild and stable anesthesia. The results indicated that AEMGs of IBS rats was abnormal increased in different intensities of CRD stimulation compared with that of normal rats, which reaffirm the occurrence of visceral hyperalgesia in IBS rats.1.4 Increased discharge of spinal wide dynamic range （WDR） neuron in IBS ratsThe changes of discharge of spinal WDR neuron in response to graded CRD stimulation was recorded when the rats were under anesthesia. The results indicated that discharge of WDR neuron was significantly increased in different intensities of CRD stimulation compared with that of normal rats, which suggested that theexcitability of spinal neuron in IBS rats was obviously enhanced.1.5 Comparison of AWR score in IBS rats of 8th and 12th weeks oldThe changes of AWR score to graded CRD stimulation in IBS rats of 8th week old and 12th weeks old was observed to decide the persistence of visceral hyperalgesia. The results indicated that no significant difference between AWR in 8th IBS rats and that of 12th IBS rats, which suggested that visceral hyperalgesia could at least last when the rats were 12 weeks old.1.6 Comparison of diarrhea sign in IBS and normal adult ratsThe diarrhea sing was observed at the same day in adult IBS and normal rats. The result indicated that the incidence of diarrhea sign in IBS rats was distinctly higher to that of normal rats, which showed that the symptom of diarrhea in IBS rats was evident.1.7 The comparison of body weight in adult IBS and normal ratsThe body weight of adult IBS and normal rat （7th week old） was compared. The results indicated that no significant difference between body weight in adult IBS and normal rats, which implied that model fabrication itself had no influence on body weight.1.8 Stoll examination in adult IBS ratsThe stool of adult IBS and normal rats was examined and no abnormal changed in adult IBS rat was showed compared with that of adult normal rats.1.9 Histological examination of colorectal tissue in IBS ratsLocal histological changes were observed in adults IBS and normal rats. The results indicated that no abnormal infiltration of neutrophil and interstitial edema were observed by naked eye and microscope in adult IBS and normal rat.Summary:Obvious signs of visceral hyperalgesia were observed at peripheral and central levels by different methods in well-made adult IBS rats. The symptom of diarrhea was evident in IBS rat and the animal model belonged to functional chronic visceral hyperalgesia model. The behavior of IBS rats was stable and could last for long time. A new ideal animal model was provided for the research in the treatment of chronic visceral pain by acupuncture.2 Electro-acupuncture （EA） relived visceral hyperalgesia in IBS rats2.1 Single EA treatment decreased AWR score in IBS ratsThe instant effect （20-90 min after EA treatment stopped） of single EA on AWR score in IBS rats was observed. The rats were divided into three groups, model group （model）, model plus EA treatment group （model + EA） and model plus sham EA treatment group （model + sham EA）. The results indicated that AWR in response to graded CRD stimulation （20, 40, 60, 80 mmHg） of model + EA was significantly decreased compared with that in model group, while no change in group of model + sham EA. It suggested that single E A treatment could remarkably relieve visceral hyperalgesia in short time after EA stopped.2.2 Single EA treatment inhibited AEMGs discharge in IBS ratsThe instant effect （20-90 min after EA treatment stopped） of single EA on AEMGs discharge in IBS rats was observed. The rats were divided into three groups, model group （model）, model plus EA treatment group （model + EA） and model plus sham EA treatment group （model + sham EA）. The results indicated that discharge of AEMGs in response to graded CRD stimulation （20,40, 60, 80 mmHg） of model + EA was significantly inhibited compared with that in model group, while no change in group of model + sham EA. It again suggested that single EA treatment could remarkably suppress visceral hyperalgesia in short time after EA stopped.2.3 The effect of repeated EA treatments on AWR in IBS ratsThe effect of repeated EA treatments on AWR in IBS rats was observed. Repeated EA treatments were applied for 30 min every other day and lasted for 13 days. Consecutive assessments of AWR scores were conducted on the day when EA was not applied for 22 days. The results indicated that the effect of EA began 2 days after EA treatment （after one EA application）, gradually increased to its maximum by the 8th day （4 EA applications） and lasted 5 days after the termination of EA treatment under different intensities of evoked CRD stimulations. Simultaneously, the effect of repeated sham EA treatments （needles were inserted into the acupoints similar to those in EA treatment but kept stilly without electrical stimulation） on AWR in IBS rats was observed. The results showed that no effect on AWR in IBS rats by sham EA treatments under different intensities of CRD stimulations. 2.4 The effect of repeated EA treatments on AEMGs in IBS ratsThe effect of repeated EA treatments on AEMGs in IBS rats was observed. Repeated EA treatments were applied for 30 min every other day and lasted for 13 days. Consecutive recording of AEMGs was conducted on the day when EA was notapplied for 22 days. The results indicated that the effect of EA began 4 days after EA treatment （after two EA application）, gradually increased to its maximum by the 8th day （4 EA applications） and lasted 5 days after the termination of EA treatment under different intensities of evoked CRD stimulations. Simultaneously, the effect of repeated sham EA treatments on AEMGs in IBS rats was observed. The results showed that no effect on discharge of AEMGs in IBS rats by sham EA treatments under different intensities of CRD stimulations.2.5 The effect of different times of EA treatments on AWR in IBS ratsThe effect of different times of EA treatments （1, 3, 5 and 7 EA treatments respectively） on AWR in IBS rats under the same CRD stimulus （60 mmHg） was observed. Repeated EA treatments were applied every other day and a consecutive assessment of AWR was conducted on the day when EA was not applied. Two to four extented AWR assessments were conducted after EA shopped. The results showed one time EA treatment could decrease AWR score to one day after EA shopped, three to five EA treatments could decrease AWR score to 3 days after EA shopped and 7 times of EA treatments could decrease AWR score to 5 days after EA shopped. It suggested that more EA treatments could prolong the post-effect after EA shopped.2.6 The effect of different times of EA treatments on AEMG in IBS ratsThe effect of different times of EA treatments （1, 3, 5 and 7 EA treatments respectively） on AEMGs in IBS rats under the same CRD stimulus （60 mmHg） was observed. Repeated EA treatments were applied every other day and consecutive recordings of AEMGs were conducted on the day when EA was not applied. Two to four prolonged AEMG recordings were conducted after EA shopped. The results showed one time EA treatment had no effect on discharge of AEMG, three to five EA treatments could inhibit AEMGs discharge to 3 days after EA shopped and 7 times of EA treatments could inhibit AEMGs discharge to 5 days after EA shopped. It again suggested that more EA treatments could prolong the post-effect after EA shopped.SummarySingle EA treatment could significantly relieve chronic visceral hyperalgesia. Cumulative effect of EA treatments was observed in the study, which began 2-4 days after EA treatment （after one or two EA applications）, gradually increased toits maximum by the 8th day （4 EA applications） and lasted some days after the termination of EA treatment. The time of persistence of post-effect of repeated EA treatments was prolonged as more EA treatments applied. The results showed instant and cumulative effects of acupuncture on chronic visceral hyperalgesia and the therapeutic effects were lengthened as times of treatments increased.3 The role of spinal EAA-R system in visceral hyperalgesia and the therapeuticeffect of EA in IBS rats3.1 The effect of intrathecal injection of MK-801 on evoked discharge of spinal WDR neuron in IBS ratsThe effect of intrathecal injection of different dose of specific antagonist of NMDA receptor （MK-801） on evoked discharge of spinal WDR neuron in IBS rats was observed. Test for confirmation of cannula location was conducted 3 days after intrathecal cannulatation and experiments were done the next day. The base of evoked discharge of WDR neuron was recording first before injection. The rats were divide into four groups, low, medium and high dose of MK-801 (0.01 （ig, 0.1 jj.g and 1 ug respectively） and vehicle group. The results indicated that no effect on WDR neuron discharge in vehicle and low dose of MK-801 groups, injection of 0.1 ng MK-801 could significantly decrease WDR discharge 5 min after injection and the effect could last for 15 min, injection of 1.0 ug MK-801 could remarkably decrease WDR discharge and the effect could last for 45 min. The results suggested the i.t MK-801 could dose-dependently inhibit the evoked discharge of spinal WDR neuron to noxious stimulus.3.2 The effect of intrathecal injection of CNQX on evoked discharge of spinal WDR neuron in IBS ratsThe effect of intrathecal injection of specific antagonist of non-NMDA receptor （CNQX） on evoked discharge of spinal WDR neuron in IBS rats was observed. Test for confirmation of cannula location was conducted 3 days after intrathecal cannulatation and experiments were done the next day. The base of evoked discharge of WDR neuron was recording first before injection and then high dose of CNQX （100 ng） and vehicle were injected to investigate the effect on evoked WDR neuron discharge. The results indicated that no effect on WDR neuron discharge in vehicle and high dose of CNQX groups, which suggested i.t CNQX （100 ng） have no effect on the evoked discharge of spinal WDR neuron to noxiousstimulus.3.3 The effect of EA on evoked discharge of spinal WDR neuron in IBS ratsThe effect of EA on evoked discharge of spinal WDR neuron in IBS rats was observed. Base discharge of evoked WDR neuron to noxious stimulus was firstly recorded and then the changes of evoked WDR neuron discharge were recorded during （30 min） and after EA treatment （240 min）. The results indicated that the discharge of evoked WDR neuron was decreased 5 min after EA began, reached its maximum inhibition 20-30 min after EA treatment, gradually increased after EA stopped and no difference to base response to noxious stimulus 60 min after EA stopped. The results suggested that EA could significantly inhibited the evoked WDR neuron discharge to noxious stimulation and the maximum effect of EA was 20-30 min after EA treatment started.3.4 The effect of combination of EA treatment and intrathecal injection of MK-801 on evoked discharge of spinal WDR neuron in IBS ratsThe effect of combination of intrathecal injection of low dose of MK-801 and EA treatment on evoked discharge of spinal WDR neuron in IBS rats was observed. The rats were divided into four groups, EA group, MK-801 group （0.01 jxg）, EA + MK-801 group and vehicle group. MK-801 was i.t. injected when EA began. The results showed no effect on WDR neuron discharge in vehicle and MK-801 groups while obviously suppressive effects in EA and EA + MK-801 groups, but no significant difference between EA and EA+ MK-801 groups. It suggested that low dose of MK-801 could not reinforce the inhibition of discharge of WDR neuron in IBS rats by acupuncture.SummaryThe results in the part indicated that spinal EAA-R system was involved in the chronic visceral hyperalgesia and NMDA receptor might mainly contributed to the maintenance of visceral hyperalgesia. EA could significantly inhibit the excitability of spinal WDR neurons, which maybe one possible mechanism of acupuncture against chronic visceral hyperalgesia.ConclusionsI. The well-made IBS model prettily mimiced the symptoms of clinical disease of IBS mainly including evident and stable visceral hyperalgesia, so it was an ideal更多还原