Hypersensitivity to visceral distention has been reported

Transcription

1 GASTROENTEROLOGY 2008;134: Anterior Cingulate Cortex Modulates Visceral Pain as Measured by Visceromotor Responses in Viscerally Hypersensitive Rats ZHIJUN CAO, XIAOYIN WU, SHENGLIANG CHEN, JING FAN, RUI ZHANG, CHUNG OWYANG, and YING LI Gastroenterology Research Unit, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan Background & Aims: We have identified that the anterior cingulate cortex (ACC) neurons are responsive to colorectal distention (CRD) and shown that sensitization of ACC neurons occurs in viscerally hypersensitive rats. However, the role of the ACC in pain response has not been clearly defined. We aimed to determine if ACC neuron activation enhances visceral pain in viscerally hypersensitive rats and to identify the receptor involved in facilitation of visceral pain. Methods: The nociceptive response (visceromotor response [VMR]) to CRD was recorded in normal and viscerally hypersensitive rats induced by colonic anaphylaxis. The ACC was stimulated electrically, and ACC lesions were generated with ibotenic acid. L-glutamate, -amino-3-hydroxy-5-methyl-isoxozole propionic acid receptor antagonist cyanonitroquinoxaline dione, and N-methyl-D-aspartate receptor antagonist aminophosphonopentanoic acid were microinjected into the rostral ACC. Results: Electrical stimulation of the rostral ACC enhanced the VMR to CRD in normal rats. ACC lesions caused a decrease in the VMR in viscerally hypersensitive rats but had no effect in normal rats. ACC microinjection of 2 mmol/l glutamate increased the VMR to CRD (10 mm Hg) in viscerally hypersensitive rats, and 20 mmol/l glutamate induced a more potent VMR in viscerally hypersensitive than in normal rats. Cyanonitroquinoxaline dione did not affect the VMR in either group. Aminophosphonopentanoic acid significantly suppressed the VMR in viscerally hypersensitive rats but not in normal rats. Conclusions: The ACC plays a critical role in the modulation of visceral pain responses in viscerally hypersensitive rats. This process appears to be mediated by enhanced activities of glutamate N-methyl-D-aspartate receptors. Hypersensitivity to visceral distention has been reported in patients with irritable bowel syndrome (IBS). Magnetic resonance imaging shows that patients with IBS exhibit altered activation in regions of the brain including the anterior cingulate cortex (ACC), thalamus, insula, and prefrontal cortex. 1,2 Experiments in animals show that the ACC receives nociceptive inputs. 3 Lesions in the ACC affect animals behavioral response in the hot-plate test. 4 The ACC also has direct neural connections to autonomic effector areas (dorsal motor nucleus of the vagus, amygdala, hypothalamus, and periaqueductal gray). 3 These findings indicated that neuronal activity in the ACC may affect spinal nociceptions and the overall pain experience in experimental animals and humans. 5 Pain contains both sensory and affective dimensions. Teasing apart the mechanisms that control the neural pathways mediating pain effect and sensation is a challenge. Hence, the role of the ACC in the mediation of sensory and affective components of pain is not easily distinguished. Coffin et al investigated the somatic nociceptive flexion (RIII) reflex using visceral nociceptive signals in patients with IBS 6 ; however, the majority of previous studies of visceral hypersensitivity in humans have relied on descriptive methods of reporting visceral sensation. 2 Systematic investigations of the role of the ACC in the modulation of the sensory components of visceral pain (ie, objective reflexive responses) have not been performed. We have recently shown that viscerally hypersensitive rats exhibit enhanced ACC spontaneous activity, decreased colorectal distention (CRD) pressure threshold, and increased magnitude of ACC neuronal response to visceral stimulation. 7 However, it is not clear whether activation of the ACC is only causally involved with the perception of pain-related unpleasantness or if it is also involved with the perceived intensity of pain during noxious visceral stimulation. Rodents do not have the forebrain structures to generate the cognitive feelings of humans; the use of behavioral paradigms to assess spinal pain reflexes that do not include the assessment of cognitive perception in the conscious rat may help to identify the regulatory role of the ACC in visceral pain sensation. Abbreviations used in this paper: ACC, anterior cingulate cortex; AP5, aminophosphonopentanoic acid; AUC, area under the curve; CRD, colorectal distention; DNQX, cyanonitroquinoxaline dione; EA, egg albumin; IBS, irritable bowel syndrome; NMDA, N-methyl-D-aspartate; VMR, visceromotor response by the AGA Institute /08/$34.00 doi: /j.gastro

2 536 CAO ET AL GASTROENTEROLOGY Vol. 134, No. 2 Glutamatergic synapses are found in regions of the central nervous system related to pain transmission, plasticity, and modulation. 8 Previous studies have shown that activation of the rostral ACC may facilitate spinal nociception. 9 We designed the current study to test the hypothesis that activation of the ACC evoked by noxious stimulation is a determinant of the sensory component of visceral pain in the viscerally hypersensitive state. We also investigated the role of the glutamate receptor in ACC neurons in the mediation of the pain response to CRD in normal and viscerally hypersensitive rats. Materials and Methods All chemicals were purchased from Sigma-Aldrich (St Louis, MO). Ibotenic acid was obtained from Research Biochemicals International (Natick, MA). All protocols were approved by the University Committee on Use and Care of Animals at the University of Michigan. Experiments were performed on adult male Sprague Dawley rats ( g). For surgical preparations, rats were anesthetized with a mixture of xylazine and ketamine according to the protocol described in our previous publication. 10 Viscerally Hypersensitive Rat Model The rats were sensitized to chicken egg albumin (EA) with an intraperitoneal injection of normal saline (1 ml) containing EA (10 g) as the antigen and aluminum hydroxide (10 mg) as the adjuvant. Beginning on day 3, the antigen solution was perfused into the colon, and CRD was performed 30 minutes after instillation of EA. 7 Visceromotor response (VMR) studies were conducted 5 7 days after the induction of colonic anaphylaxis. No significant inflammatory changes were observed in the colon 7 days after colonic anaphylaxis. Hence, the EA rat model is a suitable animal model to study visceral hypersensitivity without the presence of mucosal inflammation. VMR to CRD in Control and EA Rats Measurement of visceral sensitivity in animals is mainly based on brainstem reflexes, which have been described as pseudoaffective responses. 11 Teflon-coated, 32- gauge stainless steel wires were implanted into the external oblique pelvic muscles 4 6 days before the beginning of the experimental procedures. During the experiment, the strain gauge was connected by way of a shielded cable to a chart recorder to monitor the number of abdominal muscle contractions. Graded-pressure CRD was produced by rapidly injecting saline into the colonic balloon over 1 second and maintaining the distention for 20 seconds. Pressure was regulated with a distention control device and monitored using a pressure transducer. 7 Graded-intensity stimulation trials ( mm Hg CRD) were conducted to establish stimulus response curves. Each distention trial consisted of 3 segments: a 20-second predistention baseline period, a 20-second distention period, and a 20-second post-crd termination period with a 4-minute interstimulus interval. The responses were considered stable if there was less than 20% variability between 2 consecutive trials of CRD at 60 mm Hg. Spike bursts higher than 0.3 mv were regarded as significant and therefore used to estimate the pain response. Data were presented as the number of contractions that surpassed the threshold. The results of electromyography were also quantified by calculating the area under the curve (AUC), which is the sum of all recorded data points multiplied by the sample interval (in seconds) after baseline subtraction. ACC Electrical Stimulation Using bregma as the origin and from ear bar zero, with the incisor bar set at 5.0, a Teflon-coated stainless steel wire (120- m diameter) was inserted into either the rostral ACC ( 2.7 mm) or the caudal ACC ( 0.5 mm), 0.6 mm laterally from bregma and 2.5 mm ventrally from the skull surface, and a reference electrode was implanted on the contralateral skull. Behavioral tests were performed 5 days after surgery. To identify the effective sites within the ACC, stimulations of 10, 50, and 100 A at 100 Hz were applied to the rostral and caudal ACC, according to parameters established in a previous brain stimulation study. 12 We found that high-intensity (100 A) electrical stimulation of the rostral ACC facilitated the VMR to CRD. However, high-intensity electrical stimulation of the caudal ACC did not change the VMR. Based on our pilot studies, we established the parameter for electrical stimulation of the rostral ACC to be 0.2- millisecond rectangular pulses of 10 and 100 Hz at 100 A lasting 1 second and repeated every 5 seconds, lasting 10 minutes in total. This stimulation paradigm is comparable to a previously reported stimulation paradigm designed to study spinal nociceptive transmission. 13 ACC Lesions Generated With Ibotenic Acid Axon-sparing (excitotoxic) lesions were generated in the ACC 14 to avoid causing damage to the underlying cingulum bundle, which can itself significantly impair behavior. Lesions of the ACC were generated by slowly microinfusing 0.6 L of ibotenic acid (0.5 mg/ml) over a period of 6 minutes at each of the following coordinates in the rostral ACC: (1) anterior posterior (AP) 4.2 mm from bregma, 0.7 mm lateral to midline (L), 2.5 mm ventral to brain surface; (2) AP 3.0 mm, L 0.8 mm, 2.5 mm ventral to brain surface, and in the caudal ACC: AP 0.5 mm to 0.3 mm from bregma, L 0.8 mm, 2.5 mm ventral to brain surface. Sham lesions were generated using the same coordinates. The cannulas were kept in place for 4 minutes to permit diffusion of the drug. This procedure was repeated in the opposite hemisphere. Behavioral testing was performed after a recovery period of 6 days. The extent of the lesions was determined by histologic studies, and reconstructions were made on

3 February 2008 ACC MODULATES VISCERAL PAIN 537 charts derived from the atlas of the rat brain by Paxinos and Watson. 15 Chronic ACC Cannulation ACC microinjections were performed using a method adapted from a previous study designed for thalamus cannulation. 10 This method involved accurate implantation of the bilateral guide cannulas in the direction of the rostral ACC using the following coordinates: AP 4.2 to 3.0 from bregma, DV 2.5, L 0.7. The VMR experiments were performed 6 days postoperatively. The exact location of the injections was determined by locating and marking the point of termination of the cannula track on the plates reproduced from the atlas of the rat brain. 15 Rats with cannulas inserted in the primary motor cortex (M1) using the following coordinates: AP 4.2 to 3.0 from bregma, L 2.5, DV 2.5 served as offsite control. Glutamate and Glutamate Antagonist Studies Glutamate is a major excitatory transmitter in the ACC. This agent activates only the cell bodies, not the passing fibers. After establishing a stable VMR induced by graded-pressure CRD, the following drugs were microinjected into the ACC: L-glutamate (0.2, 2.0, and 20 mmol/l; 60 nl), glutamate N-methyl-D-aspartate (NMDA) receptor blocker aminophosphonopentanoic acid (AP5; 2.0, 20, and 200 mmol/l), and -amino-3- hydroxy-5-methyl-isoxozole propionic acid/kainate receptor blocker cyanonitroquinoxaline dione (DNQX; 2.0, 20, and 200 mmol/l). A total volume of 60 nl per hemisphere was administered. Similar volumes of vehicle (saline) were administrated into the ACC as controls. The doses of AP5 and DNQX were chosen in accordance with previous studies, which showed that similar doses of glutamate receptor antagonists inhibited ACC laminar transmembrane currents (layer II/III and layer V) during noxious electrical stimulation of medial thalamus 16 and suppressed pain-related aversion and avoidance in rats. 17,18 Each rat served as its own control. Vehicle and a single drug were tested on each day. Three different doses of glutamate, AP5, or DNQX were infused into the same site at 2-hour intervals, by which time the VMR had returned to its preinjection level, as shown in the pilot study. Separate groups of rats, in which glutamate was infused into the primary motor cortex, were used and served as offsite controls. Using a similar cannula injection technique, previous studies have shown that after microinjection of [3H][3-methyl-His2]-TRH (60 nl) into the preoptic nucleus, more than 75% of the radioactivity was found within a diameter of 600 m from the injection site. 19 Postoperative care was taken as previously described. 10 Histologic Analysis After completion of the experiments, the brains were removed and fixed. The sections were stained with cresyl violet and analyzed to assess the extent of the damage caused by the lesions. Statistical Analyses Statistical comparisons of the VMR in various groups were made using one-way repeated-measures analysis of variance, followed by multiple comparisons adjusted by the Bonferroni test using baseline values as a covariate and 2 main factors (ie, distention level as the repeated factor and group as the independent factor). Results are expressed as means SEM. P.05 was considered statistically significant. Results Visceromotor Responses to CRD Both control and EA rats showed pressure-dependent increases in the VMR to CRD. These responses were Figure 1. VMR to graded distention pressures in normal control and viscerally hypersensitive rats. Under basal conditions (CRD, 0 mm Hg), there was no significant difference between normal control rats and viscerally hypersensitive rats. (A) A representative abdominal muscle electromyogram of the VMR to graded-pressure CRD recorded from the external oblique pelvic muscle in normal and viscerally hypersensitive rats. (B) Mean amplitude of the abdominal muscle contraction expressed as AUC after baseline subtraction was presented. Data were collected from 7 sham-treated control rats and 8 viscerally hypersensitive rats. Analysis of variance showed a significant effect for distention level, as well as a significant interaction between distention level and group (*P.05). Stimulus-response functions were shifted to the left in viscerally hypersensitive rats, indicating group differences in the VMR response. Values are presented as means SE.

4 538 CAO ET AL GASTROENTEROLOGY Vol. 134, No. 2 significantly enhanced in EA rats (Figure 1A). A significant VMR to the lowest distention pressure tested (20 mm Hg) in EA rats and an absence of response to the lowest distention pressure in normal rats suggest a reduced pressure threshold (ie, allodynia) in EA rats. Graded CRD pressures of 20, 40, and 60 mm Hg caused an increase in the number of muscle contractions to , 20 4, and 33 3 contractions per 5 seconds, respectively, in normal rats and to 22 3, 37 4, and 44 6 contractions per 5 seconds in EA rats. The mean amplitude of the electromyogram (AUC, in microvolts per second) is shown in Figure 1B. These results provide evidence of enhanced visceral pain responses (ie, hyperalgesia) in EA rats. ACC Electrical Stimulation Electrical stimulation of the rostral ACC at a low frequency (Figure 2) produced a mild increase in response to 20 and 40 mm Hg CRD, which did not reach significance. Compared with control, high-frequency electrical stimulation (Figure 2) of the rostral ACC caused a marked increase in muscle contraction in response to 10, 20, 40, and 60 mm Hg CRD (from 0, 1 0.5, 24 4, and 39 4 contractions per 5 seconds, respectively, after sham stimulation to 6 0.5, 38 4, 49 6, and 53 8 contractions per 5 seconds after Figure 3. Effects of ablation of the rostral ACC using ibotenic acid on the VMR to graded-pressure CRD in normal control and viscerally hypersensitive rats. Abdominal muscle electromyogram mean amplitude, expressed as the AUC, was presented. The VMR was enhanced in the EA rats (see Figure 1). ACC lesions had no effect on the VMR in normal rats. In the rostral ACC lesion/ea rats, significant inhibition of VMR compared with sham lesion/ea rats was observed, which suggests that the rostral ACC plays an important role in mediating the pain response in viscerally hypersensitive rats. Data are presented as means SEM. *P.05 compared with sham lesion/normal rats or compared with lesion/ea rats. Figure 2. Effect of electrical stimulation of the rostral ACC on the VMR in normal rats. Twelve rats were used in this study. However, results were collected from 11 rats because 1 rat lost an electrode. Electrical stimulation of the rostral ACC at 10 Hz (100 A, 0.2 milliseconds) produced a mild increase in response to 20 and 40 mm Hg CRD. These changes did not reach significance compared with sham electrical stimulation. However, activation of the ACC with an electrical stimulation of the same intensity but higher frequency (100 Hz) induced marked increases in muscle contraction compared with control, suggesting facilitation of the VMR. Data are presented as means SEM. *P.05. Analysis of variance showed a significant effect for distention level and group interaction. Post hoc comparisons of means revealed a significant difference between the group receiving electrical stimulation at 100 Hz (*P.05) and 0 or 10 Hz at all levels of CRD (one-way repeatedmeasure analysis of variance followed by the Bonferroni test). high-frequency stimulation; P.05). The stimulus-produced facilitation began to recover 5 minutes after termination of the stimulation. In contrast, stimulation of the caudal ACC at the same intensity did not produce any change in the VMR (data not shown). ACC Lesion In the pilot study, caudal ACC lesions were generated in 6 EA rats and 6 normal rats by microinjection of ibotenic acid. The caudal ACC includes portions of postgenual Brodmann areas 24a and 24b. 20 Although most damage to the caudal ACC was confined to these coordinates, lesion damage extended to AP 1.0 in 2 animals in the caudal ACC group. Lesions generated in the caudal ACC did not significantly change the VMR in either normal or EA rats (data not shown). The rostral ACC, as defined by Vogt and Peters, 20 is the area corresponding to perigenual Brodmann area 24b, portions of perigenual 24a, and caudodorsal area 32. Areas of the rostral ACC are rich in nociceptive input, as reported in the literature. 7,14 In the rats, rostral cingulate receives most afferents from mediodorsal thalamic 21 and amygdala. 22 It should be noted that the criteria used to define rostral and caudal ACC in current studies do not correspond to the terminology of ACC regions in the primates and humans. 2 Lesions were generated in the rostral ACC of 7 EA rats and 7 normal rats. One rat in the normal group died postoperatively. Histologic evaluation confirmed that lesions were successfully generated in the rostral

5 February 2008 ACC MODULATES VISCERAL PAIN 539 examination showed that neuronal loss extended from 4.2 to 2.7 mm anterior bregma, destroying perigenual Cg1 and Cg2 (Figure 4A and B). Damage to the prelimbic cortex was minimal (a few rats exhibited a small degree of neuronal loss in the most dorsal aspect of the prelimbic cortex). The infralimbic cortex, the posterior cingulate cortex, and the corpus callosum were not damaged. Microinjection of Glutamate Microinjection of L-glutamate into the rostral ACC produced differential effects on the VMR in normal compared with EA rats. Bilateral injection of 0.2 mmol/l (60 nl) L-glutamate into the rostral ACC did not change the VMR in either normal or EA rats. Although injection of a higher dose of glutamate (2.0 mmol/l) into the rostral ACC had no significant effect on the VMR to all grades of CRD pressure in normal rats (control rats: 2 0.2, 26 2, and 39 3 contractions per 5 seconds; glutamate-treated rats: 2 1, 29 2, and 41 4 contractions per 5 seconds; in response to 20, 40, and 60 mm Hg, respectively), it enhanced the VMR to CRD in EA rats. In EA rats, injection of 2.0 mmol/l glutamate into the rostral ACC increased the VMR to 10 mm Hg CRD from 0 0 (vehicle injection) to 10 2 contractions per 5 seconds (Figure 5). In response to CRD at 20, 40, and 60 mm Hg, 2.0 mmol/l glutamate produced a 46%, 31%, and 32%, respectively, increase in muscle contraction in EA rats (from 22 2, 38 4, and 50 5 contractions Figure 4. Photomicrographs of a representative coronal section through the rostral ACC show the location of the ACC lesion after injection of ibotenic acid. (A) Coronal section from a rat that received an injection of phosphate-buffered saline into the rostral ACC. (B) Coronal section after a well-placed injection of ibotenic acid into the rostral ACC. Pale areas indicate neuronal cell loss. Scale bar 250 m. ACC of 7 EA and 6 normal rats in this group. Sham lesions were generated in 6 EA and 6 normal rats. Lesions in the rostral ACC did not change the VMR in normal rats, which suggests that this region of the forebrain is not involved in the behavioral component of the visceral pain response in rats that are not viscerally hypersensitive. In contrast, lesions in the rostral ACC caused a reduction in the number of muscle contractions in the EA rats (from 26 3, 40 4, and 56 8 contractions per 5 seconds in sham lesions to 7 2, 23 3, and 40 7 contractions per 5 seconds in the lesions, in response to 20, 40, and 60 mm Hg, respectively; P.05). The mean amplitudes expressed as AUC are shown in Figure 3. These observations indicate that ablation of this region of the brain suppresses allodynia and hyperalgesia in viscerally hypersensitive rats. Microinjection of ibotenic acid into the ACC generated lesions with clearly definable borders of neuronal cell loss and gliosis, in contrast to the lesions generated with microinjection of phosphate-buffered saline. Histologic Figure 5. Original electromyograms in response to CRD in a control and a viscerally hypersensitive rat after bilateral ACC administration of L-glutamate. (A) Infusion of L-glutamate (2 mmol/l) into the ACC of a normal rat did not change the number of muscle contractions in response to CRD. (B) In the viscerally hypersensitive rat, infusion of L- glutamate (2 mmol/l) into the ACC caused greater increases in response to all CRD pressures (10, 20, 40, 60 mm Hg).

6 540 CAO ET AL GASTROENTEROLOGY Vol. 134, No. 2 of 2.0 mmol/l glutamate, injection of an even higher dose of glutamate (20.0 mmol/l) into the rostral ACC evoked allodynia and hyperalgesia in both normal and viscerally hypersensitive rats (Figure 6A). Bilateral microinjection of glutamate (2.0 mmol/l) into the primary motor cortex, a location outside the ACC, failed to alter the VMR to CRD in both normal and EA rats (Figure 6B). Microinjection of Glutamate Antagonists To determine if glutamatergic transmission in the ACC is responsible for modulating visceral pain responses in either normal or viscerally hypersensitive states, receptor antagonist studies were performed. A total of 18 normal and 21 EA rats were studied. Histologic studies confirmed the accuracy of the microinjection sites in all but 1 normal and 2 EA rats. The microinjection sites in these 3 rats were outside the ACC; therefore, the data from these 3 rats were excluded from the statistical analysis. Microinjection of either non- NMDA receptor antagonist DNQX (2, 20, 200 mmol/l) or NMDA receptor antagonist AP5 (2, 20, 200 mmol/l) into the rostral ACC did not change the VMR to gradedpressure CRD in normal rats (Figure 7). Thus, although electrical stimulation or application of high doses of glutamate in the rostral ACC caused hyperalgesia (ie, increased VMR) in both normal and viscerally hypersen- Figure 6. Effects of bilateral injection of L-glutamate into the ACC or primary motor cortex (M1) on the VMR induced by graded-pressure CRD in normal and viscerally hypersensitive rats. (A) Results were obtained from 17 normal rats and 19 viscerally hypersensitive (EA) rats. Summary of data is expressed as AUC after vehicle or glutamate injection into rostral ACC. Infusion of L-glutamate at a low dose of 2 mmol/l had no effect on the VMR to CRD in normal rats; however, it produced marked increases in the VMR to 20, 40, and 60 mm Hg CRD in EA rats. Injection of a high dose of L-glutamate (20 mmol/l) into the rostral ACC produced increases in the number of muscle contractions in both normal and EA rats. *P.05 compared with EA/vehicle. **P.05, normal/ glutamate 20 mmol/l versus normal/glutamate 2 mmol/l; EA/glutamate 20 mmol/l versus EA/glutamate 2 mmol/l. (B) Microinjection of L-glutamate into the primary motor cortex (M1) did not change the VMR to CRD in both normal and EA rats (n 4 for each group). *P.05 compared with normal/vehicle-treated rats. per 5 seconds in vehicle-treated EA rats to 32 2, 50 4, and 66 5 contractions per 5 seconds in glutamatetreated EA rats; P.05). The mean amplitudes expressed as AUC are shown in Figure 6. These observations suggest that in the viscerally hypersensitive rats, glutamate in the rostral ACC causes pain by a stimulus that does not normally provoke pain (ie, CRD pressures of 10 and 20 mm Hg) and increases the pain response to a stimulus that is normally painful (ie, CRD pressures of 40 and 60 mm Hg). In contrast to the effect evoked by an injection Figure 7. Effects of bilateral administration of the glutamate receptor antagonist on the VMR to CRD in normal and viscerally hypersensitive rats. Results were collected from 17 normal and 19 viscerally hypersensitive (EA) rats. Administration of either non-nmda receptor antagonist DNQX or NMDA receptor antagonist AP5 (2, 20, and 200 mmol/l) did not change the numbers of spontaneous muscle contractions in either normal or EA rats. Bilateral administration of either DNQX or AP5 into the ACC had no effect on the VMR to CRD in normal rats. Similar to the effect in normal rats, DNQX in the ACC of EA rats did not change the VMR to CRD. In contrast to the effect of DNQX, bilateral administration of AP5 into the rostral ACC of EA rats dose-dependently decreased the number of muscle contractions in response to graded-pressure CRD. *P.05 compared with EA/vehicle rats.

7 February 2008 ACC MODULATES VISCERAL PAIN 541 sitive rats, the failure of glutamate antagonists to affect the VMR to CRD in normal rats suggests that the ACC neuronal network is not involved in the mediation of visceral pain in normal conditions. In the EA rats, DNQX had no effect on the VMR, whereas AP5 dose-dependently decreased the VMR to CRD in EA rats. In response to graded CRD pressures of 20, 40, and 60 mm Hg, AP5 (2 mmol/l) reduced the number of muscle contractions from 24 3, 42 4, and 56 6 contractions per 5 seconds to 13 2, 30 4, and 45 3 contractions per 5 seconds, respectively, representing 46%, 30%, and 20% inhibition (P.05). This inhibitory effect appears to be dose dependent. At a dose of 20 mmol/l, AP5 produced 62%, 40%, and 30% inhibition. These inhibitory responses were significantly greater than those observed with 2 mmol/l AP5 (P.05). The mean amplitudes expressed as AUC are shown in Figure 7. A 200-mmol/L dose of AP5 did not produce additional inhibition. The microinjection sites are shown in Figure 8. These observations suggest that enhanced glutamate NMDA receptor activities in the ACC are responsible for allodynia and hyperalgesia in viscerally hypersensitive rats. Discussion The ACC is a major cortical component of the limbic loop system, and its functional relationship to emotion and pain has been well described. 2 We have shown that viscerally hypersensitive rats exhibit enhanced ACC spontaneous activity, a decreased CRD pressure threshold to activate ACC neurons, and an increase in the magnitude of the ACC response. 7 Enhanced Figure 8. Glutamate microinjection sites in the rostra ACC and the primary motor cortex (M1) of normal and viscerally hypersensitive rats. Black squares and circles indicate the ACC microinjection sites in the control and EA rats, respectively. Gray squares and circles indicate microinjection into the M1 in the control and EA rats, respectively. Cg1, cingulate cortex, area 1; IL, intralimbic cortex; M1, primary motor cortex; M2, secondary motor cortex; PrL, prelimbic cortex. NMDA receptor activation contributes to the development of lasting ACC neuronal plasticity. 23 However, the role of the enhanced ACC neuronal activation on the modulation of the sensory component of acute visceral pain has not been investigated. Visceral hypersensitivity is a key brain-gut axis abnormality observed in the majority of patients with IBS. We utilized a rat model with colonic anaphylaxis evoked by intraperitoneal injection of chicken EA. 11 Although this model has been shown by a number of investigators, 24 including ourselves, to be suitable for the study of visceral hypersensitivity, its clinical relevance to IBS is still unclear. In this model, we have observed that there was no significant mucosal inflammation in the colon 7 days after the initiation of visceral hypersensitivity (data not shown). The hypersensitivity to colonic distention, however, can be observed even up to 7 weeks following the initiation of colonic anaphylaxis and appears to be independent of mucosal inflammation. Hence, this may be a useful model to study postinflammatory conditions of visceral hyperalgesia such as postinfection IBS, which occurs in up to 20% of patients following an acute bout of gastrointestinal infection. It is well documented that the descending endogenous analgesia system, including the periaqueductal gray and the rostral ventral medulla, plays an important role in modulation of nociceptive transmission. 25 Outputs from the dorsal ACC are projected to several brainstem nuclei, including the dorsolateral periaqueductal gray, the superior colliculus, and the reticular formation. 3 The efferent projections from the ventral ACC are directed at the ventrolateral periaqueductal gray, dorsal motor nucleus of the vagus, nucleus ambiguous, and spinal structures involved in autonomic control. 3 Electrical stimulation of the ACC has been shown to elicit a variety of visceral or autonomic responses in a number of species. 26 In the rat study, electrical or chemical stimulation of the rostral ACC enhances the tail-flick reflex 9 ; this descending facilitation from the ACC relays at the rostral ventral medulla. 9 In this study, we used the behavioral paradigms of VMRs to identify the regulatory role of the ACC in visceral pain sensation. The VMR induced by CRD is a brainstem-mediated reflex contraction of the abdominal musculature. In response to stimuli that cause pain, all animals show musculoskeletal and autonomic responses, the so-called pseudoaffective reflex responses. 11 However, it is not entirely clear whether this visceromotor reflex in rodents is truly representative of actual pain experience in humans. Previous studies have shown that dorsal midcingulate cortex contains a motor component that projects to the spinal cord and regulates skeletomotor function. 27,28 It is thus possible that ACC modulates CRD-evoked VMRs via altering spinal motor neuronal activities. In this study, we show that electrical stimulation of the rostral ACC in conscious rats enhances the VMR to CRD

8 542 CAO ET AL GASTROENTEROLOGY Vol. 134, No. 2 in a frequency-dependent manner. Furthermore, we show that bilateral ACC lesion does not change the VMR in normal rats but markedly inhibits the VMR to CRD in EA rats. The reduction in the VMR after ablation of the rostral ACC suggests that neural networks in this region mediate allodynia and hyperalgesia in viscerally hypersensitive rats. Glutamate is a fast excitatory transmitter in the mammalian brain. Glutamate mediates synaptic transmission by binding to postsynaptic -amino-3-hydroxy-5-methylisoxozole propionic acid, NMDA, and kainate receptors. In most regions of the brain, synaptic responses are primarily mediated through postsynaptic -amino-3-hydroxy-5-methyl-isoxozole propionic acid and kainate receptors, because NMDA receptors are blocked by magnesium at resting membrane potential. In the ACC, NMDA receptors are highly expressed, although their physiologic function with respect to visceral pain remains unclear. In this study, we show that injection of low-dose glutamate into the ACC has no effect on the pain response in normal rats; however, in EA rats, it has a potent effect on the VMR to CRD, suggesting sensitization of glutamate receptors in ACC neurons in viscerally hypersensitive states. These behavioral findings are consistent with our previous observation showing increased firings in single ACC neurons in response to microinjection of glutamate in EA rats. 23 To determine the role of glutamatergic transmission in the modulation of visceral pain, we show that microinjection of the NMDA receptor antagonist AP5 into the ACC suppresses the CRD-induced increase in the VMR in EA rats. Hence, these data suggest visceral hyperalgesia in our rat model involves endogenous activation of descending facilitatory pathways mediated by NMDA receptors in the rostral ACC. What makes glutamatergic synapses unique is that they can sustain synaptic plastic changes that may persist for hours to days. 8 In the ACC, glutamate mediates excitatory synaptic transmission. 8 Overexpression of the NMDA receptor subunit NR2B in the forebrain significantly increases excitation of the ACC neurons in transgenic mice. 29 It is quite likely that in the viscerally hypersensitive state, NMDA receptors play an important role in the modulation of visceral pain by reinforcing glutamate sensory transmission in the ACC. Further studies are needed to explore the cellular and molecular mechanisms underlying these dynamic changes in ACC neurons in the viscerally hypersensitive state. Our results strongly suggest that cognitive and affective factors are not the exclusive elements mediated by the ACC in the pain experience; ACC neurons appear to facilitate reflexive pain responses (ie, sensation) in the viscerally hypersensitive state. We conclude that the ACC plays a critical role in the modulation of the pain reflex in viscerally hypersensitive rats. ACC sensitization characterized by enhanced glutamate NMDA receptor activation may trigger a descending modulation pathway to intensify visceral pain. References 1. Mertz H, Morgan V, Tanner G, et al. Regional cerebral activation in irritable bowel syndrome and control subjects with painful and nonpainful rectal distension. Gastroenterology 2000;118: Mayer EA, Naliboff BD, Craig AD. Neuroimaging of the brain-gut axis: from basic understanding to treatment of functional GI disorders. Gastroenterology 2006;131: Neafsey EJ, Terreberry RR, Hurley KM, et al. Anterior cingulate cortex in rodents: connections, visceral control functions, and implications for emotion. In: Vogt BA, Gabriel M, eds. Neurobiology of cingulate cortex and limbic thalamus. Boston, MA: Birkhauser, 1993: Pastoriza LN, Morrow TJ, Casey KL. Medial frontal cortex lesions selectively attenuate the hot plate response: possible nocifensive apraxia in the rat. Pain 1996;64: Calejesan AA, Kim SJ, Zhuo M. Descending facilitatory modulation of a behavioral nociceptive response by stimulation in the adult rat anterior cingulate cortex. Eur J Pain 2000;4: Coffin B, Bouhassira D, Sabate JM, et al. Alteration of the spinal modulation of nociceptive processing in patients with irritable bowel syndrome. Gut 2004;53: Gao J, Wu X, Owyang C, Li Y. Enhanced responses of the anterior cingulate cortex neurons to colonic distension in viscerally hypersensitive rats. J Physiol 2006;570: Dudek SM, Bear MF. Homosynaptic long-term depression in area CA1 of hippocampus and effects of N-methyl-D-aspartate receptor blockade. Proc Natl Acad Sci USA1992;89: Zhang L, Zhang Y, Zhao ZQ. Anterior cingulate cortex contributes to the descending facilitatory modulation of pain via dorsal reticular nucleus. Eur J Neurosci 2005;22: Li Y, Wu XY, Zhu JX, et al. Hypothalamic regulation of pancreatic secretion is mediated by central cholinergic pathway in rat. J Physiol 2003;552: Ness TJ, Gebhart GF. Visceral pain: a review of experimental studies. Pain 1990;41: Sotres-Bayon F, Torres-Lopez E, Lopez-Avila A, et al. Lesion and electrical stimulation of the ventral tegmental area modify persistent nociceptive behavior in the rat. Brain Res 2001;898: Zhuo M, Gebhart GF. Biphasic modulation of spinal nociceptive transmission from the medullary raphe nuclei in the rat. J Neurophysiol 1997;78: Johansen JP, Fields HL, Manning BH. The affective component of pain in rodents: direct evidence for a contribution of the anterior cingulate cortex. Proc Natl Acad Sci USA2001;98: Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 4th ed. New York, NY: Academic, Yang JW, Shih HC, Shyu BC. Intracortical circuits in rat anterior cingulate cortex are activated by nociceptive inputs mediated by medial thalamus. J Neurophysiol 2006;96: Lei LG, Sun S, Gao YJ, et al. NMDA receptors in the anterior cingulate cortex mediate pain-related aversion. Exp Neurol 2004; 189: Souza TM, Roesler R, Madruga M, et al. Differential effects of post-training muscimol and AP5 infusions into different regions of the cingulate cortex on retention for inhibitory avoidance in rats. Neurobiol Learn Mem 1999;72: Siren AL, Vonhof S, Feuerstein G. Hemodynamic defense response to thyrotropin-releasing hormone injected into medial preoptic nucleus in rats. Am J Physiol Regul Integr Comp Physiol 1991;261:R305 R312.

9 February 2008 ACC MODULATES VISCERAL PAIN Vogt BA, Peters A. Form and distribution of neurons in rat cingulate cortex: areas 32, 24, and 29. J Comp Neurol 1981;195: Krettek JE, Price JL. The cortical projections of the mediodorsal nucleus and adjacent thalamic nuclei in the rat. J Comp Neurol 1977;84: Sripanidkulchai K, Sripanidkulchai B, Wyass JM. The cortical projections of the basolateral amygdaloid nucleus in the rat: a retrograde fluorescent dye study. J Comp Neurol 1984;229: Gao J, Wu X, Moon KD, et al. Differential roles for NMDA and non-nmda receptors in visceral nociceptive transmission in the anterior cingulate cortex in normal and visceral hypersensitive rats (abstr). Gastroenterology 2005;128:A Nozdrachev AD, Akoev GN, Filippova LV, et al. Changes in afferent impulse activity of small intestine mesenteric nerves in response to antigen challenge. Neuroscience 1999;94: Gebhart GF. Descending modulation of pain. Neurosci Biobehav Rev 2004;27: Cechetto DF, Saper CB. Role of the cerebral cortex in autonomic function. In: Loewy AD, Spyer KM, et al. Central regulation of autonomic function. New York, NY: Oxford University Press, 1990: Vagt BA. Pain and emotion interactions in subregions of the cingulate gyrus. Nat Rev Neurosci 2005;6: Dum RP, Strick PL. The origin of corticospinal prejection from the premotor areas in the frontal lobe. J Neurosci 1991;11: Wei F, Wang GD, Kerchner GA, et al. Genetic enhancement of inflammatory pain by forebrain NR2B overexpression. Nat Neurosci 2001;4: Received May 4, Accepted November 15, Address requests for reprints to: Ying Li, MD, Division of Gastroenterology, University of Michigan, Medical Sciences Research Building I, Room 6510, 1150 West Medical Center Drive, Ann Arbor, Michigan yli@umich.edu; fax: (734) Z.C. and S.C. are currently with the Department of Gastroenterology, Renji Hospital, School of Medicine, Shanghai Jiaotong University, China. Supported by National Institute of Neurological Disorders and Stroke grant R01 NS (to Y.L.) and National Institute of Diabetes and Digestive and Kidney Diseases grants R01-DK (to Y.L.) and P30-DK (to C.O.). The authors report that no conflicts of interest exist. The authors thank Celina G. Kleer, MD (University of Michigan Medical School, Ann Arbor, MI), for examination of colon histologic sections.