Chronic Visceral Hyperalgesia And Acupuncture To Alleviate The Neurobiological Mechanisms Of Visceral Pain

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 modelSprague-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 pressureAfter 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 treatmentthe 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 preparation5-8 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 immuohistochemistryThe 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 ratsPTP 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 ratsAfter 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 hornAfter 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 RVMthe 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.Conclusions1. 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.