慢性内脏痛敏及针刺缓解内脏痛的神经生物学机制
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摘要
虽然现代医学科学有了长足进展,慢性疼痛仍然是临床医生深感棘手的一个挑战。慢性痛严重影响病人的生活质量,慢性痛的治疗疗效并不令人满意,而且费用高昂,这消耗了大量医疗和社会资源。慢性内脏痛是肠道功能紊乱疾病(如肠易激综合征,非心源性胸痛,慢性消化不良等),以及盆腔器官功能紊乱疾病(如慢性间质性膀胱炎等)的主要症状之一。肠道和盆腔器官功能紊乱疾病缺乏解剖结构、感染、代谢等指标异常,疼痛弥散无固定位点并易与内脏器官实质疾病相互混淆。
在胃肠道功能紊乱疾病之中,IBS病人所占比例最大,发病率大约为18-20%。其症状主要有：腹痛,腹胀,便秘和/或腹泻。与之相似,大约15%的女性受到慢性盆腔痛的困扰[1]。肠道和盆腔器官功能紊乱疾病主要特点是下腹痛。这里有多种原因可能导致这些功能紊乱的疾病发生,如持续精神压力、有内脏器官感染和炎症病史、遗传背景、早期的负性事件(如虐待、创伤以及疼痛等)等。
近年来对IBS发病机制的研究显示,在直肠气囊充气试验中,IBS患者以及IBS模型动物痛阈明显低于对照组,即内脏痛敏现象,而内脏痛敏可能是IBS病人慢性腹痛和不适的主要机制之一。一般认为,内脏痛敏可能与内脏传入神经和神经中枢出现了敏化(sensitization)现象有关。对动物和人的研究表明,以下三个中枢机制可能参与内脏痛敏的调制：1)脑内神经中枢对内脏感觉传导的易化作用；2)连续内脏传入信息刺激下所致脊髓感觉中枢处于敏化状态;3)来自于髓上中枢对脊髓或内脏器官感觉传输的易化调节。
脊髓是痛觉信号进入中枢后的第一级整合中枢,是痛觉信息传人的中继站,既能直接调制痛觉,又能接受脊髓上中枢的下行调节信号,是疼痛刺激调节的关键部位。大量研究显示,脊髓背角很多递质、受体、细胞因子在痛觉过敏形成的外周和中枢机制中起着关键作用,这包括5-HT、P物质、降钙素基因相关肽(CGRP),也包括NMDA受体。
延髓头端腹内侧核(RVM)是脊髓上神经中枢对内脏伤害刺激进行下行调制的共同通路。RVM对内脏痛具有双重调节作用：抑制和易化伤害输入。根据大量研究成果,Porreca等提出假设,RVM对于疼痛信息的下行调节功能紊乱,可能是疼痛感觉异常如IBS,纤维肌痛和神经性疼痛的重要原因,而RVM中谷氨酸递质及其受体,特别是NMDA受体,在内脏痛敏的调节中发挥了重要作用
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。一般认为,由脊髓上多个神经中枢,如RVM、PAG、杏仁核、扣带回等组成的下行痛觉调制系统,对脊髓背角疼痛信息的传导有着强大的抑制作用,而针刺可能是激活下行痛觉调制系统而发挥其镇痛效应的。Al-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征的治疗作用,结果发现电针能明显缓解模型大鼠慢性内脏痛敏,并发现电针对IBS大鼠慢性内脏痛敏的治疗效应,可能是通过中枢NMDA受体发挥作用的。然而目前对于针刺治疗缓解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和RVM的谷氨酸递质受体系统有着密切的联系,电针能明显缓解模型大鼠慢性内脏痛敏,并发现电针对慢性内脏痛敏的治疗效应可能是通过中枢NMDA受体发挥作用的,本研究选择了脊髓背角和RVM中c-Fos和NR1受体为研究对象,运用免疫组化方法,观察和分析IBS大鼠脊髓背角和RVM中内脏反应神经元的兴奋性在针刺治疗前后的改变,以及二者中NR1受体在针刺缓解IBS模型大鼠慢性内脏痛敏中的参与机制。
方法
1.建立IBS大鼠模型
参照Al-Chaer等报道的大鼠IBS模型和我们研究小组的改进,采用新生9天SD幼鼠分为两组,第一组通过结直肠机械扩张刺激制作IBS慢性内脏痛敏模型,持续两周；另一组仅轻柔肛周皮肤作为对照。
2.IBS模型大鼠行为学评估
饲养至6～8周后,分别通过测定模型大鼠腹部撤回反射(abdominal withdrawal reflex, AWR)和痛阈压力值(pain threshold pressure, PTP),对模型进行综合评估。
3.对IBS模型大鼠进行电针治疗并用行为学评估治疗结果
随机将IBS模型大鼠分为三组：模型组(M)、模型加电针组(EA)和模型加假电针组(SEA)。模型组不做任何治疗,电针组穴位取双侧“足三里”和“上巨虚”,连续隔日治疗四次,假电针不通电,余与电针组同。再次通过行为学评估电针治疗结果。
4.固定和冰冻切片的制备
大鼠在5%戊巴比妥麻醉下,用4%多聚甲醛约灌注30 min,取出延髓和脊髓,梯度蔗糖磷酸缓冲液脱水后,后将其制成30μm连续冰冻切片,分别置于不同标记的24孔板中。
5.观察多次电针过程后脊髓T13-L2、L6-S2节段,以及RVM中c-Fos蛋白和NRl受体表达变化规律
5.1运用免疫组化方法,观察脊髓T13-L2和L6-S2节段和RVM中c-Fos蛋白的表达,观察电针对IBS模型大鼠这些部位内脏反应神经元兴奋性的影响。
5.2运用免疫组化方法,观察脊髓L6-S2节段和RVM中NR1受体的表达,观察在多次电针后IBS模型大鼠这些部位NMDA受体表达的变化规律。
结果
1.IBS大鼠PTP值明显下降,AWR评分异常升高
与正正常对照组比较,模型组的PTP值明显下降。AWR评分结果显示,在不同强度(20、40、60、80mmHg)的CRD诱发内脏痛刺激下,模型组相比于正常对照组,AWR评分明显升高。
2.电针缓解IBS大鼠内脏痛敏
而电针组治疗后与治疗前及假电针组治疗后相比,AWR评分明显降低,PTP明显升高,而假电针组治疗后与治疗前AWR评分的差异无统计学意义。
3.IBS模型大鼠脊髓背角c-Fos和NRl受体的表达显著升高
在CRD刺激后,相对于正常对照组大鼠,IBS模型大鼠L6-S2节段浅层(the superficial laminae, SDH, laminaeⅠandⅡ)、固有层(nucleus proprius, NP, laminaeⅢandⅣ)、背角颈段(the neck of the dorsal horn, NECK, laminaeⅤandⅥ)中,c-FosI阳性神经元明显高于正常对照组,而在X层则无明显差别；而相对于正常对照组大鼠,IBS模型大鼠T13-L2节段除背角NECK层(laminae V)中c-Fos阳性神经元,明显高于高于对照组外,浅层、固有层及中央管周区的c-Fos阳性神经元数目无明显差异。NR1受体组化结果跟c-Fos结果类似,即IBS模型组L6-S2节段浅层、固有层神经元数目和IOD值明显高于正F常对照组。
4.IBS模型大鼠RVM中c-Fos和NR1受体的表达显著升高
在RVM各个亚核Gi (nucleus reticularis gigantocellularis), LPGi (nucleus lateralis paragigantocellulari), GiA (nucleus reticularis gigantocellularis pars alpha), NRM (nucleus raphe magnus)水平,IBS模型组c-Fos蛋白和NR1受体免疫阳性细胞的计数明显升高：RVM中NR1受体的IOD值与正常对照组相比明显升高。
5.电针明显降低IBS模型大鼠脊髓背角c-Fos和NR1受体的表达
在多次隔日电针治疗后,IBS模型大鼠L6-S2节段SDH、NP、NECK、X层中,c-Fos阳性神经元明显下降,而假电针则没有此效应。电针治疗后IBS大鼠T13-L2节段浅层、固有层、背角颈段及中央管周区的c-Fos阳性神经元数目无明显差异；在多次隔日电针治疗后,IBS模型大鼠L6-S2节段SDH、NP中,NR1阳性神经元数目和IOD值明显下降,NECK层中,NR1阳性神经元数目无明显差异,而IOD值明显下降,假电针则没有此效应。
6.电针明显降低IBS模型大鼠RVM中c-Fos和NRI受体的表达
IBS模型大鼠在连续四次电针治疗以后,RVM的亚核Gi, LPGi, GiA中c-Fos蛋白,以及Gi, LPGi, GiA, NRM中NR1受体免疫阳性细胞的计数明显下降;RVM整体水平NR1受体的IOD值也显著下降。但假电针组与模型组相比无明显变化。
综上所述,本研究提示：
1、PTP及AWR评分表明,IBS成年大鼠模型内脏痛敏表现明显,出现了内脏超敏和痛敏现象,较好地模拟了临床IBS病人肠道感觉过敏现象,是一个比较稳定的慢性内脏痛敏动物模型。
2、我们实验室前期研究成果表明,隔日电针4-6次,达到最大治疗效应。本研究提示,第四次电针治疗后,PTP及AWR评分显示,多次电针治疗可以明显缓解慢性内脏痛敏。
3、IBS模型大鼠L6-S2节段脊髓背角和RVM中c-Fos蛋白表达显著升高,这表巨额脊髓背角和RVM中内脏反应神经元兴奋性异常增高,可能是IBS模型大鼠内脏痛敏的发生的重要原因。
4、IBS模型大鼠脊髓背角和RVM中NR1受体表达显著升高,这表明脊髓背角和RVM中内脏反应神经元上NR1受体异常表达,可能参与了IBS模型大鼠功能性慢性内脏痛敏的维持。
5、电针可以明显抑制IBS模型大鼠L6-S2节段脊髓背角和RVM中c-Fos表达,从而使脊髓背角和RVM中内脏反应神经元异常增高兴奋性下降,这可能是针刺缓解慢性内脏痛敏的机制之一
6、电针可以明显抑制IBS模型大鼠脊髓背角和RVM中NR1受体表达,这可能是脊髓背角和RVM中内脏反应神经元异常增高兴奋性下降的重要原因,这也可能是针刺缓解慢性内脏痛敏的机制之一。
Although with the advances of medical science, many chronic pain syndromes still remain a challenge for clinicians. Suffering from chronic pain can significantly deteriorate a person's quality of life and can often lead to disability. Such chronic pain in the viscera is observed in functional bowel disorders (e.g., noncardiac chest pain, chronic idiopathic dyspepsia, functional abdominal pain, irritable bowel syndrome; IBS) and chronic pelvic pain (e.g., chronic interstitial cystitis, painful bladder syndrome) that are multifaceted problems and still poorly understood. Functional bowel disorders and chronic pelvic pain represent unexplained symptoms that have no readily identifiable infectious, anatomical, ormetabolic basis. The pain is diffusing and poorly localized and often it is confused as originating from other visceral organs.
Among all functional bowel disorders, IBS is the most common and prevalent gastrointestinal (GI) disorder (about 18-20% of the patient population), having symptoms of cramping, abdominal pain, bloating, constipation, and/or diarrhea. Similarly, chronic pelvic pain affects approximately 15% of women. Interstitial cystitis and painful bladder syndrome are the most common. All functional bowel disorder and chronic pelvic pain patients exhibit one common symptom-lower abdominal pain. There are different factors that can cause or modify these disorders, such as persistent mental and social stress, a previous episode of infection or inflammation, genetic background, and early-life adverse events (e.g., abuse, trauma, and painful experience). Presumably in all functional disorders, patients develop excessive pain to painful stimuli (e.g., hyperalgesia) and may also experience pain to a nonpainful stimulus (e.g., allodynia).
The general notion for visceral hypersensitivity is the presence of sensitization of the neural pathway (includes primary sensory afferents and spinal ascending neurons) involved in primary sensory afferents and spinal neurons to visceral stimuli and their sensitization. Both animal and human studies of IBS clearly point to a spinal mechanism, consistent with the observation that IBS patients have enhanced responses to visceral and cutaneous stimuli throughout the pain matrix of the brain (including RVM). However, on the basis of the evidence presented so far, it is not entirely clear the extent to which these enhanced responses are the result of (1) a facilitating mechanism confined within the brain, (2) a spinal sensitization maintained by tonic impulse input from the rectum and/or colon, or (3) a mechanism of descending facilitation from the brain to the spinal cord and/or gut.
Visceral hypersensitivity is the main mechanism underlying irritable bowel syndrome (IBS) patients with chronic visceral hyperalgesia, while the recurrent occurrence of visceral pain is a major cause of patients asking for medical treatment. The lack of understanding on visceral pain hindered the targeted treatment measures supposed, meanwhile the therapeutic effect of visceral pain is not satisfactory and the expense for visceral pain treatment has taken a large number of medical resources and community resources. To find effective and economical treatment methods has become an inevitable trend.
It has been already known that rostral ventromedia medulla (RVM) has a dual role in descending modulation of nociception, that is, it is able to inhibit or facilitate the nociceptive input. Exposure of this region to higher intensity of electrical stimulation or higher concentration of stimulatory neurotransmitters can induce analgesia, while low-intensity electrical stimulation or low concentration of stimulatory neurotransmitters can result in hyperalgesia. Because facilitation of pain via the RVM has been implicated in the development of central sensitization, it has been supposed therefore that disordered descending influences from the brainstem on nociceptive afferent information may underlie abnormal pain perception in both functional pain disorders, such as irritable bowel syndrome and fibromyalgia, and neuropathic pain.
The spinal cord, which is the first integration center in the transmission of pain, accepted the control signals from supraspinal centers, and plays a important role in direct modulation of pain. A large number of studies have shown that many of the spinal cord dorsal horn neurotransmitters, receptors and cytokines play a key role in the formation of peripheral hyperalgesia and central mechanisms, including 5-HT, substance P, calcitonin gene-related peptide (CGRP), and the NMDA receptor.
In east part of Asia, acupuncture as empirical medicine has long been used for patients with different kinds of diseases for thousands of years, especially in treating various types of pain. Recently, acupuncture has been focused on treatment particularly with chronic visceral pain, although a few of acupuncture trials demonstrated no efficacy or minimal superiority over placebo/control in treatment of patients or animal models. Compared with pharmacotherapy, the anti-hyperalgesia effects of acupuncture, which have a curative effect, economical and practical advantages, fewer side effects, may be beneficial to patients with chronic visceral pain.
Al-Chaer et al. developed a rat model of chronic visceral hyperalgesia by repetitive colorectal distention (CRD) during the neonatal period. This chronic visceral hyperalgesia model provides a useful animal model with which to further study the mechanisms of chronic visceral pain. It has been shown that there is an analgesic effect of electro-acupuncture (EA) on chronic visceral hyperalgesia and possibility of N-methyl-D-aspartate receptor 1 (NR1) in the central nervous system underlying the effect of EA to reduce visceral pain condition. Because the glutamate neurotransmitter system in RVM and spinal dorsal horn is closely involved in the formation and development of chronic visceral pain, and c-Fos protein is a nuclear protein product encoded by the immediate-early gene c-fos, which has been widely used as a marker of increased neural activity in the central nerval system, we selected c-Fos and NR1 in the RVM and spinal dorsal horn to investigate the specific mechanism with acupuncture relieving chronic visceral hyperalgesia in this study.
The experimental steps as follows:
1. Production of IBS rat model
Sprague-Dawley rats (9 days old) were randomly divided into 2 groups undergoing different treatments. Rats in group 1 (IBS model rats) received colon mechanical irritation, and rats in another group (normal rats or intact rats) were gently touched on the perineal area. The irritation period lasted on a daily basis between the ages of 9 and 22 days.
2. Assessment of abdominal withdraw reflex and pain threshold pressure
After a resting period of another 2～4 weeks, behavioral test of abdominal withdraw reflex (AWR) and pain threshold pressure (PTP) responded to colorectal distention (CRD) stimuli between two groups were observed.
3. Administration of electro-acupuncture treatment
the IBS model rats were randomly divided into 3 groups:IBS model group (M) without any treatment, model+EA group (EA) and model+sham EA group (SEA). Administration of EA consecutively repeated four times evcry other day at bilateral points of Zu-san-li (ST-36) and Shang-ju-xu (ST-37) in the hind limbs, while sham-EA at similar acupoints was done inserting needles without electrical stimulation. After the treatment, AWR and PTP were observed again.
4. Tissue preparation
5-8 hours after CRD stimulation, distended rats were deeply anesthetized with pentobarbital and intracardially perfused with 4% paraformaldehyde. The RVM and spinal cord subsequently allowed to equilibrate in 30% sucrose with PB. Thirty-μm transverse sections were cut on a cryostat.
5. c-Fos and NR1 immuohistochemistry
The RVM and spinal dorsal horn sections were stained for c-Fos, NR1 using strept-avidin-biotin complex (SABC) method. To measure the level of intensity of c-Fos and NR1 immunoreactivity, five slices for each rat were selected for the count of c-Fos and NR1 positive neurons. Both the integrated optical density (IOD) and the number of immunoreactive neurons of NR1 in RVM and spinal dorsal horn were counted with software image pro plus (IPP).
results:
1. PTP significantly decreased and AWR score abnormally increased in IBS rats
PTP value siginificantly decreased from 48.7±5.4 mmHg in the control group to 32.7±6.5 mmHg in the irritated groups. Parallel to the PTP change, with graded CRD stimulation, there were significant increases in AWR scores in IBS rats compared with control rats, respectively, at distention pressures of 20,40,60 and 80 mmHg.
2. Electro-acupuncture reversed abnormal PTP value and AWR score in IBS rats
After 4 consecutive EA treatments to the IBS model rats, the decreased PTP in response to CRD was reversed to the level of normal rats. The average PTP of EA group was 45.0±6.8 mmHg, significantly higher than that of the IBS group at 32.7±6.5 mmHg. There were also significant differences in AWR scores compared before and after EA treatment. AWR scores of the EA-treated rats were significantly lower than those before EA treatment (EA-B), respectively, at distention pressures of 20,40,60, and 80 mmHg. There was no significant decrease in AWR scores of sham EA(SEA) treated rats compared with those before SEA treatment (SEA-B).
3. Electro-acupuncture reduced hyper-expression of c-Fos and NR1 in IBS rats in spinal dorsal horn
After CRD stimulation, the number of immunoreactive neurons of c-Fos protein increased significantly in L6-S2 segment of spinal dorsal horn of IBS model rats compared with those of normal rats, then decreased to normal level after EA treatment in laminaes of SDH, NP, NECK. Only the c-Fos positive neurons significantly in the laminae of NECK in T13-L2 segment of spinal dorsal horn of IBS model rats. Correlated with the expression of c-Fos protein, the number of immunoreactive neurons and IOD of NR1 receptor increased significantly in L6-S2 segment, then decreased to normal level after EA treatment in laminaes of SDH, NP. Only the IOD of NR1 receptor significantly in the laminae of NECK in L6-S2 segment of spinal dorsal horn of IBS model rats. Compared with IBS model rats, there was no difference observed after sham EA treatment with c-Fos and NR1 immunostaining.
4. Electro-acupuncture reduced hyper-expression of c-Fos and NR1 in IBS rats in RVM
the number of immunoreactive neurons of c-Fos protein and NR1 increased significantly in nucleus reticularis gigantocellularis (Gi), nucleus lateralis paragigantocellulari (LPGi), nucleus reticularis gigantocellularis pars alpha (GiA), nucleus raphe magnus (NRM) with IBS model rats, both the number of immunoreactive neurons of c-Fos and NR1 significantly decreased, respectively, in the Gi, LPGi, GiA and in Gi, LPGi, GiA, NRM. The IOD also significantly increased compared with those in normal rats, then decreased to normal level after EA treatment in RVM. Compared with IBS model rats, there was no difference observed after sham EA treatment with c-Fos and NR1 immunostaining.
Conclusions
1. Behavioral tests of AWR and PTP responded to CRD stimuli demonstrated that these rats received colon mechanical irritation produce visceral allodynia and visceral hyperalgesia in their adulthood. It demonstrated that the visceral vulnerability, induced by continuous mechanical damage at early birth, could be continued until the adulthood.
2. As revealed in our previous report, repeated EA treatment gradually enhanced to its maximum within 8-12 days, AWR assessment was performed during 24 h after four EA treatments. We found that AWR score of EA treatment group was significantly decreased, and PTP value was significantly increased, while sham EA treatment didn't produce this effect.
3. This study indicated that abnormally high neuronal excitability, especially the increased NR1 receptor expression in the spinal dorsal horn, may be important reasons underlying visceral hyperalgesia in IBS model rats. It demonstrated that EA treatment can relieve chronic visceral hyperalgesia in IBS rats, and such an effect might be correlated with the modulation of abnormal neuronal excitability and NR1 expression in the spinal dorsal horn of the rat's brain by EA.
4. This study indicated that abnormally high neuronal excitability, especially the increased NR1 receptor expression in the RVM, may be important reasons underlying visceral hyperalgesia in IBS model rats. It demonstrated that EA treatment can relieve chronic visceral hyperalgesia in IBS rats, and such an effect might be correlated with the modulation of abnormal neuronal excitability and NR1 expression in the RVM of the rat's brain by EA.
在胃肠道功能紊乱疾病之中,IBS病人所占比例最大,发病率大约为18-20%。其症状主要有：腹痛,腹胀,便秘和/或腹泻。与之相似,大约15%的女性受到慢性盆腔痛的困扰[1]。肠道和盆腔器官功能紊乱疾病主要特点是下腹痛。这里有多种原因可能导致这些功能紊乱的疾病发生,如持续精神压力、有内脏器官感染和炎症病史、遗传背景、早期的负性事件(如虐待、创伤以及疼痛等)等。
近年来对IBS发病机制的研究显示,在直肠气囊充气试验中,IBS患者以及IBS模型动物痛阈明显低于对照组,即内脏痛敏现象,而内脏痛敏可能是IBS病人慢性腹痛和不适的主要机制之一。一般认为,内脏痛敏可能与内脏传入神经和神经中枢出现了敏化(sensitization)现象有关。对动物和人的研究表明,以下三个中枢机制可能参与内脏痛敏的调制：1)脑内神经中枢对内脏感觉传导的易化作用；2)连续内脏传入信息刺激下所致脊髓感觉中枢处于敏化状态;3)来自于髓上中枢对脊髓或内脏器官感觉传输的易化调节。
脊髓是痛觉信号进入中枢后的第一级整合中枢,是痛觉信息传人的中继站,既能直接调制痛觉,又能接受脊髓上中枢的下行调节信号,是疼痛刺激调节的关键部位。大量研究显示,脊髓背角很多递质、受体、细胞因子在痛觉过敏形成的外周和中枢机制中起着关键作用,这包括5-HT、P物质、降钙素基因相关肽(CGRP),也包括NMDA受体。
延髓头端腹内侧核(RVM)是脊髓上神经中枢对内脏伤害刺激进行下行调制的共同通路。RVM对内脏痛具有双重调节作用：抑制和易化伤害输入。根据大量研究成果,Porreca等提出假设,RVM对于疼痛信息的下行调节功能紊乱,可能是疼痛感觉异常如IBS,纤维肌痛和神经性疼痛的重要原因,而RVM中谷氨酸递质及其受体,特别是NMDA受体,在内脏痛敏的调节中发挥了重要作用
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。一般认为,由脊髓上多个神经中枢,如RVM、PAG、杏仁核、扣带回等组成的下行痛觉调制系统,对脊髓背角疼痛信息的传导有着强大的抑制作用,而针刺可能是激活下行痛觉调制系统而发挥其镇痛效应的。Al-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征的治疗作用,结果发现电针能明显缓解模型大鼠慢性内脏痛敏,并发现电针对IBS大鼠慢性内脏痛敏的治疗效应,可能是通过中枢NMDA受体发挥作用的。然而目前对于针刺治疗缓解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和RVM的谷氨酸递质受体系统有着密切的联系,电针能明显缓解模型大鼠慢性内脏痛敏,并发现电针对慢性内脏痛敏的治疗效应可能是通过中枢NMDA受体发挥作用的,本研究选择了脊髓背角和RVM中c-Fos和NR1受体为研究对象,运用免疫组化方法,观察和分析IBS大鼠脊髓背角和RVM中内脏反应神经元的兴奋性在针刺治疗前后的改变,以及二者中NR1受体在针刺缓解IBS模型大鼠慢性内脏痛敏中的参与机制。
方法
1.建立IBS大鼠模型
参照Al-Chaer等报道的大鼠IBS模型和我们研究小组的改进,采用新生9天SD幼鼠分为两组,第一组通过结直肠机械扩张刺激制作IBS慢性内脏痛敏模型,持续两周；另一组仅轻柔肛周皮肤作为对照。
2.IBS模型大鼠行为学评估
饲养至6～8周后,分别通过测定模型大鼠腹部撤回反射(abdominal withdrawal reflex, AWR)和痛阈压力值(pain threshold pressure, PTP),对模型进行综合评估。
3.对IBS模型大鼠进行电针治疗并用行为学评估治疗结果
随机将IBS模型大鼠分为三组：模型组(M)、模型加电针组(EA)和模型加假电针组(SEA)。模型组不做任何治疗,电针组穴位取双侧“足三里”和“上巨虚”,连续隔日治疗四次,假电针不通电,余与电针组同。再次通过行为学评估电针治疗结果。
4.固定和冰冻切片的制备
大鼠在5%戊巴比妥麻醉下,用4%多聚甲醛约灌注30 min,取出延髓和脊髓,梯度蔗糖磷酸缓冲液脱水后,后将其制成30μm连续冰冻切片,分别置于不同标记的24孔板中。
5.观察多次电针过程后脊髓T13-L2、L6-S2节段,以及RVM中c-Fos蛋白和NRl受体表达变化规律
5.1运用免疫组化方法,观察脊髓T13-L2和L6-S2节段和RVM中c-Fos蛋白的表达,观察电针对IBS模型大鼠这些部位内脏反应神经元兴奋性的影响。
5.2运用免疫组化方法,观察脊髓L6-S2节段和RVM中NR1受体的表达,观察在多次电针后IBS模型大鼠这些部位NMDA受体表达的变化规律。
结果
1.IBS大鼠PTP值明显下降,AWR评分异常升高
与正正常对照组比较,模型组的PTP值明显下降。AWR评分结果显示,在不同强度(20、40、60、80mmHg)的CRD诱发内脏痛刺激下,模型组相比于正常对照组,AWR评分明显升高。
2.电针缓解IBS大鼠内脏痛敏
而电针组治疗后与治疗前及假电针组治疗后相比,AWR评分明显降低,PTP明显升高,而假电针组治疗后与治疗前AWR评分的差异无统计学意义。
3.IBS模型大鼠脊髓背角c-Fos和NRl受体的表达显著升高
在CRD刺激后,相对于正常对照组大鼠,IBS模型大鼠L6-S2节段浅层(the superficial laminae, SDH, laminaeⅠandⅡ)、固有层(nucleus proprius, NP, laminaeⅢandⅣ)、背角颈段(the neck of the dorsal horn, NECK, laminaeⅤandⅥ)中,c-FosI阳性神经元明显高于正常对照组,而在X层则无明显差别；而相对于正常对照组大鼠,IBS模型大鼠T13-L2节段除背角NECK层(laminae V)中c-Fos阳性神经元,明显高于高于对照组外,浅层、固有层及中央管周区的c-Fos阳性神经元数目无明显差异。NR1受体组化结果跟c-Fos结果类似,即IBS模型组L6-S2节段浅层、固有层神经元数目和IOD值明显高于正F常对照组。
4.IBS模型大鼠RVM中c-Fos和NR1受体的表达显著升高
在RVM各个亚核Gi (nucleus reticularis gigantocellularis), LPGi (nucleus lateralis paragigantocellulari), GiA (nucleus reticularis gigantocellularis pars alpha), NRM (nucleus raphe magnus)水平,IBS模型组c-Fos蛋白和NR1受体免疫阳性细胞的计数明显升高：RVM中NR1受体的IOD值与正常对照组相比明显升高。
5.电针明显降低IBS模型大鼠脊髓背角c-Fos和NR1受体的表达
在多次隔日电针治疗后,IBS模型大鼠L6-S2节段SDH、NP、NECK、X层中,c-Fos阳性神经元明显下降,而假电针则没有此效应。电针治疗后IBS大鼠T13-L2节段浅层、固有层、背角颈段及中央管周区的c-Fos阳性神经元数目无明显差异；在多次隔日电针治疗后,IBS模型大鼠L6-S2节段SDH、NP中,NR1阳性神经元数目和IOD值明显下降,NECK层中,NR1阳性神经元数目无明显差异,而IOD值明显下降,假电针则没有此效应。
6.电针明显降低IBS模型大鼠RVM中c-Fos和NRI受体的表达
IBS模型大鼠在连续四次电针治疗以后,RVM的亚核Gi, LPGi, GiA中c-Fos蛋白,以及Gi, LPGi, GiA, NRM中NR1受体免疫阳性细胞的计数明显下降;RVM整体水平NR1受体的IOD值也显著下降。但假电针组与模型组相比无明显变化。
综上所述,本研究提示：
1、PTP及AWR评分表明,IBS成年大鼠模型内脏痛敏表现明显,出现了内脏超敏和痛敏现象,较好地模拟了临床IBS病人肠道感觉过敏现象,是一个比较稳定的慢性内脏痛敏动物模型。
2、我们实验室前期研究成果表明,隔日电针4-6次,达到最大治疗效应。本研究提示,第四次电针治疗后,PTP及AWR评分显示,多次电针治疗可以明显缓解慢性内脏痛敏。
3、IBS模型大鼠L6-S2节段脊髓背角和RVM中c-Fos蛋白表达显著升高,这表巨额脊髓背角和RVM中内脏反应神经元兴奋性异常增高,可能是IBS模型大鼠内脏痛敏的发生的重要原因。
4、IBS模型大鼠脊髓背角和RVM中NR1受体表达显著升高,这表明脊髓背角和RVM中内脏反应神经元上NR1受体异常表达,可能参与了IBS模型大鼠功能性慢性内脏痛敏的维持。
5、电针可以明显抑制IBS模型大鼠L6-S2节段脊髓背角和RVM中c-Fos表达,从而使脊髓背角和RVM中内脏反应神经元异常增高兴奋性下降,这可能是针刺缓解慢性内脏痛敏的机制之一
6、电针可以明显抑制IBS模型大鼠脊髓背角和RVM中NR1受体表达,这可能是脊髓背角和RVM中内脏反应神经元异常增高兴奋性下降的重要原因,这也可能是针刺缓解慢性内脏痛敏的机制之一。
Although with the advances of medical science, many chronic pain syndromes still remain a challenge for clinicians. Suffering from chronic pain can significantly deteriorate a person's quality of life and can often lead to disability. Such chronic pain in the viscera is observed in functional bowel disorders (e.g., noncardiac chest pain, chronic idiopathic dyspepsia, functional abdominal pain, irritable bowel syndrome; IBS) and chronic pelvic pain (e.g., chronic interstitial cystitis, painful bladder syndrome) that are multifaceted problems and still poorly understood. Functional bowel disorders and chronic pelvic pain represent unexplained symptoms that have no readily identifiable infectious, anatomical, ormetabolic basis. The pain is diffusing and poorly localized and often it is confused as originating from other visceral organs.
Among all functional bowel disorders, IBS is the most common and prevalent gastrointestinal (GI) disorder (about 18-20% of the patient population), having symptoms of cramping, abdominal pain, bloating, constipation, and/or diarrhea. Similarly, chronic pelvic pain affects approximately 15% of women. Interstitial cystitis and painful bladder syndrome are the most common. All functional bowel disorder and chronic pelvic pain patients exhibit one common symptom-lower abdominal pain. There are different factors that can cause or modify these disorders, such as persistent mental and social stress, a previous episode of infection or inflammation, genetic background, and early-life adverse events (e.g., abuse, trauma, and painful experience). Presumably in all functional disorders, patients develop excessive pain to painful stimuli (e.g., hyperalgesia) and may also experience pain to a nonpainful stimulus (e.g., allodynia).
The general notion for visceral hypersensitivity is the presence of sensitization of the neural pathway (includes primary sensory afferents and spinal ascending neurons) involved in primary sensory afferents and spinal neurons to visceral stimuli and their sensitization. Both animal and human studies of IBS clearly point to a spinal mechanism, consistent with the observation that IBS patients have enhanced responses to visceral and cutaneous stimuli throughout the pain matrix of the brain (including RVM). However, on the basis of the evidence presented so far, it is not entirely clear the extent to which these enhanced responses are the result of (1) a facilitating mechanism confined within the brain, (2) a spinal sensitization maintained by tonic impulse input from the rectum and/or colon, or (3) a mechanism of descending facilitation from the brain to the spinal cord and/or gut.
Visceral hypersensitivity is the main mechanism underlying irritable bowel syndrome (IBS) patients with chronic visceral hyperalgesia, while the recurrent occurrence of visceral pain is a major cause of patients asking for medical treatment. The lack of understanding on visceral pain hindered the targeted treatment measures supposed, meanwhile the therapeutic effect of visceral pain is not satisfactory and the expense for visceral pain treatment has taken a large number of medical resources and community resources. To find effective and economical treatment methods has become an inevitable trend.
It has been already known that rostral ventromedia medulla (RVM) has a dual role in descending modulation of nociception, that is, it is able to inhibit or facilitate the nociceptive input. Exposure of this region to higher intensity of electrical stimulation or higher concentration of stimulatory neurotransmitters can induce analgesia, while low-intensity electrical stimulation or low concentration of stimulatory neurotransmitters can result in hyperalgesia. Because facilitation of pain via the RVM has been implicated in the development of central sensitization, it has been supposed therefore that disordered descending influences from the brainstem on nociceptive afferent information may underlie abnormal pain perception in both functional pain disorders, such as irritable bowel syndrome and fibromyalgia, and neuropathic pain.
The spinal cord, which is the first integration center in the transmission of pain, accepted the control signals from supraspinal centers, and plays a important role in direct modulation of pain. A large number of studies have shown that many of the spinal cord dorsal horn neurotransmitters, receptors and cytokines play a key role in the formation of peripheral hyperalgesia and central mechanisms, including 5-HT, substance P, calcitonin gene-related peptide (CGRP), and the NMDA receptor.
In east part of Asia, acupuncture as empirical medicine has long been used for patients with different kinds of diseases for thousands of years, especially in treating various types of pain. Recently, acupuncture has been focused on treatment particularly with chronic visceral pain, although a few of acupuncture trials demonstrated no efficacy or minimal superiority over placebo/control in treatment of patients or animal models. Compared with pharmacotherapy, the anti-hyperalgesia effects of acupuncture, which have a curative effect, economical and practical advantages, fewer side effects, may be beneficial to patients with chronic visceral pain.
Al-Chaer et al. developed a rat model of chronic visceral hyperalgesia by repetitive colorectal distention (CRD) during the neonatal period. This chronic visceral hyperalgesia model provides a useful animal model with which to further study the mechanisms of chronic visceral pain. It has been shown that there is an analgesic effect of electro-acupuncture (EA) on chronic visceral hyperalgesia and possibility of N-methyl-D-aspartate receptor 1 (NR1) in the central nervous system underlying the effect of EA to reduce visceral pain condition. Because the glutamate neurotransmitter system in RVM and spinal dorsal horn is closely involved in the formation and development of chronic visceral pain, and c-Fos protein is a nuclear protein product encoded by the immediate-early gene c-fos, which has been widely used as a marker of increased neural activity in the central nerval system, we selected c-Fos and NR1 in the RVM and spinal dorsal horn to investigate the specific mechanism with acupuncture relieving chronic visceral hyperalgesia in this study.
The experimental steps as follows:
1. Production of IBS rat model
Sprague-Dawley rats (9 days old) were randomly divided into 2 groups undergoing different treatments. Rats in group 1 (IBS model rats) received colon mechanical irritation, and rats in another group (normal rats or intact rats) were gently touched on the perineal area. The irritation period lasted on a daily basis between the ages of 9 and 22 days.
2. Assessment of abdominal withdraw reflex and pain threshold pressure
After a resting period of another 2～4 weeks, behavioral test of abdominal withdraw reflex (AWR) and pain threshold pressure (PTP) responded to colorectal distention (CRD) stimuli between two groups were observed.
3. Administration of electro-acupuncture treatment
the IBS model rats were randomly divided into 3 groups:IBS model group (M) without any treatment, model+EA group (EA) and model+sham EA group (SEA). Administration of EA consecutively repeated four times evcry other day at bilateral points of Zu-san-li (ST-36) and Shang-ju-xu (ST-37) in the hind limbs, while sham-EA at similar acupoints was done inserting needles without electrical stimulation. After the treatment, AWR and PTP were observed again.
4. Tissue preparation
5-8 hours after CRD stimulation, distended rats were deeply anesthetized with pentobarbital and intracardially perfused with 4% paraformaldehyde. The RVM and spinal cord subsequently allowed to equilibrate in 30% sucrose with PB. Thirty-μm transverse sections were cut on a cryostat.
5. c-Fos and NR1 immuohistochemistry
The RVM and spinal dorsal horn sections were stained for c-Fos, NR1 using strept-avidin-biotin complex (SABC) method. To measure the level of intensity of c-Fos and NR1 immunoreactivity, five slices for each rat were selected for the count of c-Fos and NR1 positive neurons. Both the integrated optical density (IOD) and the number of immunoreactive neurons of NR1 in RVM and spinal dorsal horn were counted with software image pro plus (IPP).
results:
1. PTP significantly decreased and AWR score abnormally increased in IBS rats
PTP value siginificantly decreased from 48.7±5.4 mmHg in the control group to 32.7±6.5 mmHg in the irritated groups. Parallel to the PTP change, with graded CRD stimulation, there were significant increases in AWR scores in IBS rats compared with control rats, respectively, at distention pressures of 20,40,60 and 80 mmHg.
2. Electro-acupuncture reversed abnormal PTP value and AWR score in IBS rats
After 4 consecutive EA treatments to the IBS model rats, the decreased PTP in response to CRD was reversed to the level of normal rats. The average PTP of EA group was 45.0±6.8 mmHg, significantly higher than that of the IBS group at 32.7±6.5 mmHg. There were also significant differences in AWR scores compared before and after EA treatment. AWR scores of the EA-treated rats were significantly lower than those before EA treatment (EA-B), respectively, at distention pressures of 20,40,60, and 80 mmHg. There was no significant decrease in AWR scores of sham EA(SEA) treated rats compared with those before SEA treatment (SEA-B).
3. Electro-acupuncture reduced hyper-expression of c-Fos and NR1 in IBS rats in spinal dorsal horn
After CRD stimulation, the number of immunoreactive neurons of c-Fos protein increased significantly in L6-S2 segment of spinal dorsal horn of IBS model rats compared with those of normal rats, then decreased to normal level after EA treatment in laminaes of SDH, NP, NECK. Only the c-Fos positive neurons significantly in the laminae of NECK in T13-L2 segment of spinal dorsal horn of IBS model rats. Correlated with the expression of c-Fos protein, the number of immunoreactive neurons and IOD of NR1 receptor increased significantly in L6-S2 segment, then decreased to normal level after EA treatment in laminaes of SDH, NP. Only the IOD of NR1 receptor significantly in the laminae of NECK in L6-S2 segment of spinal dorsal horn of IBS model rats. Compared with IBS model rats, there was no difference observed after sham EA treatment with c-Fos and NR1 immunostaining.
4. Electro-acupuncture reduced hyper-expression of c-Fos and NR1 in IBS rats in RVM
the number of immunoreactive neurons of c-Fos protein and NR1 increased significantly in nucleus reticularis gigantocellularis (Gi), nucleus lateralis paragigantocellulari (LPGi), nucleus reticularis gigantocellularis pars alpha (GiA), nucleus raphe magnus (NRM) with IBS model rats, both the number of immunoreactive neurons of c-Fos and NR1 significantly decreased, respectively, in the Gi, LPGi, GiA and in Gi, LPGi, GiA, NRM. The IOD also significantly increased compared with those in normal rats, then decreased to normal level after EA treatment in RVM. Compared with IBS model rats, there was no difference observed after sham EA treatment with c-Fos and NR1 immunostaining.
Conclusions
1. Behavioral tests of AWR and PTP responded to CRD stimuli demonstrated that these rats received colon mechanical irritation produce visceral allodynia and visceral hyperalgesia in their adulthood. It demonstrated that the visceral vulnerability, induced by continuous mechanical damage at early birth, could be continued until the adulthood.
2. As revealed in our previous report, repeated EA treatment gradually enhanced to its maximum within 8-12 days, AWR assessment was performed during 24 h after four EA treatments. We found that AWR score of EA treatment group was significantly decreased, and PTP value was significantly increased, while sham EA treatment didn't produce this effect.
3. This study indicated that abnormally high neuronal excitability, especially the increased NR1 receptor expression in the spinal dorsal horn, may be important reasons underlying visceral hyperalgesia in IBS model rats. It demonstrated that EA treatment can relieve chronic visceral hyperalgesia in IBS rats, and such an effect might be correlated with the modulation of abnormal neuronal excitability and NR1 expression in the spinal dorsal horn of the rat's brain by EA.
4. This study indicated that abnormally high neuronal excitability, especially the increased NR1 receptor expression in the RVM, may be important reasons underlying visceral hyperalgesia in IBS model rats. It demonstrated that EA treatment can relieve chronic visceral hyperalgesia in IBS rats, and such an effect might be correlated with the modulation of abnormal neuronal excitability and NR1 expression in the RVM of the rat's brain by EA.
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