Calcitonin gene-related peptide and spinal afferents partly mediate postoperative colonic ileus in the rat

Calcitonin gene-related peptide and spinal afferents partly mediate postoperative colonic ileus in the rat Tilman T. Zittel, MD, Kent C. K. Lloyd, PhD, Ines Rothenhöfer, CM, Helen Wong, John H. Walsh, MD, and Helen E. Raybould, PhD, Los Angeles, Calif. and Tübingen, Germany

Background. Calcitonin gene-related peptide (CGRP) is a widely distributed neuropeptide contained in intrinsic and extrinsic neurons of the gastrointestinal wall that has been shown to be released by noxious stimulation, to be involved in nociception, to inhibit gastrointestinal motility, and to partly mediate postoperative gastric ileus. We hypothesized that abdominal surgery-induced release of CGRP might inhibit postoperative colonic motility and food intake. Methods. Colonic transit, stool pellet number, stool pellet weight, and food intake were measured for 48 hours after induction of postoperative ileus in rats. CGRP was immunoneutralized by preoperative injection of CGRP monoclonal antibody, or visceral afferent nerve fibers containing CGRP were functionally ablated by topical capsaicin treatment of the vagus nerves or of the celiac/superior mesenteric ganglia before abdominal surgery. Results. Abdominal surgery increased colonic transit time and decreased 24-hour cumulative stool pellet number, stool pellet weight, and food intake. CGRP immunoneutralization reversed postoperative inhibition of colonic transit, 24-hour cumulative stool pellet number, stool pellet weight, and food intake by 77%, 82%, 80%, and 52%, respectively. Whereas ablation of vagal afferent nerve fibers had no effect, spinal afferent nerve fiber ablation reversed postoperative inhibition of 24-hour cumulative stool pellet number, stool pellet weight, and food intake by 41%, 38%, and 19%, respectively. Conclusions. CGRP and spinal afferent nerve fibers partly mediate postoperative colonic ileus and inhibition of food intake in the rat. By the magnitude of reversal of postoperative ileus, CGRP seems to be an important mediator of postoperative colonic ileus. Our results for the first time show involvement of a neuropeptide and spinal afferents in the mediation of postoperative colonic ileus and postoperative inhibition of food intake in rats. (Surgery 1998;123:518-27.) From CURE/Gastroenteric Biology Center, West Los Angeles VA Medical Center, and University of California, Los Angeles, Calif., and Department of Abdominal and Transplantation Surgery, Tübingen, Germany

LAPAROTOMY AND INTRAABDOMINAL operative procedures result in inhibition of gastrointestinal motility. While gastric motility seems to recover after 1 to 2 days,1 recovery of colonic motility usually requires 3 to 5 days2,3 and occasionally even longer.1,3 In clinical practice, recovery of gastrointestinal motility is monitored by abdominal auscultation, and regular food intake is withheld until the return of bowel sounds and first appearance of flatus or defecation. Postoperative ileus has been shown to influence both the time of T. T. Zittel was the recipient of a scholarship from the Deutsche Forschungsgemeinschaft, Bonn, Germany. Accepted for publication Sept. 16, 1997. Reprint requests: Tilman T. Zittel, MD, Chirurgische Universitätsklinik, Abteilung für Allgemeinchirurgie, HoppeSeyler-Str. 3, 72076 Tübingen, Germany. Copyright © 1998 by Mosby, Inc. 0039-6060/98/$5.00 + 0 11/56/88090

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postoperative food intake and of hospital discharge,3 indicating its importance for the length of hospital stay and thus for health costs. Because of the multiplicity of chemical coding of the enteric and central nervous system,4 postoperative inhibition of gastrointestinal motility is probably a multifactorial event. In a variety of studies, several neurotransmitters have been shown to contribute to postoperative ileus.5-10 We have recently shown that calcitonin gene-related peptide (CGRP) partly mediates postoperative gastric ileus in the rat.9 CGRP is a widely distributed neuropeptide contained in intrinsic and extrinsic neurons of the gastrointestinal wall, as well as in peripheral extramural ganglia or in the central nervous system.11,12 CGRP is thought to be an important mediator of pain13,14 and to contribute to repair mechanisms in response to tissue damage.15,16 In addition, CGRP has been shown

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Table I. Effect of anesthesia and abdominal surgery on cumulative food intake (FI; gm/100 gm body weight)

Control n Body weight (gm) FI 0-24 hr FI 24-48 hr FI 0-48 hr

13 385 ± 13 7.7 ± 0.2 6.6 ± 0.3 14.3 ± 0.3

Saline 0.9%

CGRP MAb

Halothane

Abdominal surgery

7 367 ± 16 7.2 ± 0.6 6.1 ± 0.6 13. 3 ± 1.1

7 369 ± 24 8.1 ± 0.6 7.0 ± 0.5 15.1 ± 1.1

9 374 ± 15 6.8 ± 0.2* 6.2 ± 0.2 13.0 ± 0.3*

9 374 ± 12 3.7 ± 0.3* 5.7 ± 0.4 9.4 ± 0.7*

Abdominal surgery + KLH MAb

Abdominal surgery + CGRP MAb

7 312 ± 16 3.9 ± 0.8* 5.8 ± 0.5 9.6 ± 1.7*

7 368 ± 22 6.1 ± 0.9*† 7.0 ± 0.4† 13.1 ± 1.3†

*p < 0.05 vs control. †p

< 0.05 vs abdominal surgery.

Table II. Effect of halothane anesthesia in rats with capsaicin or vehicle treatment of the VNs Vehicle VN n Colonic transit (hr) Stool pellet number 0-12 hr 12-24 hr Stool pellet weight (gm) 0-12 hr 12-24 hr

Capsaicin VN

Vehicle VN + halothane

Capsaicin VN + halothane

4 8.0 ± 2.4

5 7.2 ± 1.3

4 12.0 ± 2.8

5 7.5 ± 1.7

32 ± 5 17 ± 2

39 ± 7 21 ± 3

24 ± 6 18 ± 2

30 ± 6 22 ± 4

7.3 ± 1.3 4.0 ± 0.5

9.0 ± 1.5 5.4 ± 1.0

4.6 ± 1.1 4.4 ± 0.3

6.9 ± 1.2 5.8 ± 1.7

to inhibit gastric, small intestinal, and colonic motility in vitro and in vivo.17-20 In our study, we investigated the effects of CGRP immunoneutralization on postoperative colonic ileus in rats. Because CGRP is particularly abundant in extrinsic afferent nerve fibers of the gastrointestinal tract,11 we further investigated the effects of vagal and spinal afferent nerve fiber ablation on postoperative colonic ileus. Afferent C fibers, which compose 99% of the afferent innervation of the gastrointestinal tract,16,22 can be selectively ablated by the neurotoxin capsaicin.21 By applying capsaicin topically to the vagus nerves or to the celiac/superior mesenteric ganglia (CSMG), as described previously,9,23 the afferent vagal or spinal innervation of the gastrointestinal tract can be largely ablated. In addition, postoperative food intake was measured in parallel for a variety of reasons. First, CGRP has been shown to decrease food intake,24 and recent data suggest that CGRP receptor blockade might interfere with, and possibly increase, food intake.25 Second, blockade of CGRP action has been shown to shorten postoperative gastric ileus,9,26 thereby possibly increasing postoperative food intake. Finally, colonic motility responds to food intake,27 thus possibly influencing postoperative colonic ileus. In our study, we demonstrate for the first time that

CGRP immunoneutralization and spinal afferent nerve ablation partly reverse postoperative inhibition of colonic transit, stool pellet output, and food intake, thus indicating that CGRP and spinal afferent nerve fibers partly mediate postoperative colonic ileus in the rat. MATERIAL AND METHODS Animals. Experiments were performed on adult male Sprague-Dawley rats (Harlan SpragueDawley, San Diego, Calif.) weighing 365 ± 8 gm (mean ± standard error of the mean). The rats were housed under conditions of controlled humidity, temperature (22° ± 1° C), and illumination (lights on 12 midnight to 12 midday). Institutional guidelines for the care and use of laboratory animals were followed throughout the study. Antibodies. The CGRP monoclonal antibody (MAb) and keyhole limpet hemocyanin (KLH) MAb were obtained from the laboratory of John Walsh and Helen Wong (CURE/Gastroenteric Biology Center and UCLA Digestive Diseases Center, Los Angeles, Calif.; Antibody Core National Institutes of Health Grant DK41301). The CGRP MAb has been shown to neutralize CGRP action in a variety of models, whereas KLH

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Fig. 1. Colonic transit (hr), stool pellet number/24 hr, and stool pellet weight (gm)/24 hr in control animals, after intraperitoneal injection of saline 0.9% or CGRP MAb, 2 mg in saline 0.9%, after halothane anesthesia, or after abdominal surgery with manipulation of the cecum for 5 minutes. *p < 0.001 vs control.

Fig. 2. CGRP immunoneutralization reversed postoperative inhibition of colonic transit, stool pellet number/24 hr, and stool pellet weight (gm)/24 hr by 77%, 82% and 80%, respectively. *p < 0.001 vs control; +p < 0.01 vs abdominal surgery.

MAb does not possess any specific biologic activity and served as control antibody.9,28 A total of 2 mg of antibody was dissolved in saline 0.9%, 300 µL and given intraperitoneally 2 hours before experiments. Capsaicin treatment. The vagus nerves (VNs) at the level of the neck or the CSMG were treated with either vehicle (0.1 ml of Tween 80 in olive oil)

or with capsaicin (1%; Sigma, St. Louis, Mo.) by sonicating 10 mg capsaicin in 0.1 ml 10% Tween 80. The solution was brought to 1 ml by adding 0.9 ml olive oil, as described previously.9,23 Capsaicin or vehicle treatment was performed immediately before colonic catheter implantation. Colonic catheters. Rats were equipped with a colonic cannula (polyethylene tubing, PE 90; Clay

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Adams, Parsippany, N.J.) under pentobarbital anesthesia (50 mg/kg intraperitoneally, Nembutal; Abbot Laboratories, North Chicago, Ill.). Cannulas were inserted into the ascending colon 3 cm distal to the ileocecal valve as described for the duodenum previously.23 Rats were allowed to recover for 2 weeks before experiments were started. Abdominal surgery. Rats were fasted for 18 hours with free access to water, and all experiments were performed immediately before the dark phase of the light cycle. Under halothane anesthesia (4% v/v), the cecum was exposed through a lower abdominal midline incision, wrapped in a compress soaked with warm saline, and manipulated for 5 minutes. The cecum was returned into the abdominal cavity and the wound was closed with running sutures; the whole procedure lasted 15 minutes. This model has been used repeatedly to investigate postoperative ileus in the rat.7-9,26 Colonic transit and stool pellet output. Carbamine red (250 µl; 1.2 gm in aqua destillata 100 ml; Sigma) was injected into the ascending colon through the colonic catheter, and rats were kept in wire-bottomed cages. The first appearance of carbamine red with fecal pellets and the weight and number of fecal pellets were recorded every 6 hours for 48 hours. Occasionally, the appearance of carbamine red could not be determined visually, and no colonic transit time could be noted in these experiments. Food intake. Access to food (regular rat food; Altromin) was given immediately after abdominal operation. Cumulative food intake was recorded at 24 and 48 hours and is given as food intake (gm/100 gm body weight, BW). Statistical analysis. The data are presented as the mean ± the standard error of the mean (SEM). Differences between treated groups were determined by analysis of variance followed by Fisher’s least significant differences test. A probability level of p < 0.05 was considered significant. RESULTS Experiment 1: Effect of anesthesia and abdominal surgery on colonic transit, stool pellet output, and food intake. Abdominal surgery increased colonic transit time by 61% and decreased 24-hour cumulative stool pellet number and stool pellet weight by 40% and 46%, respectively, whereas halothane anesthesia had no significant effect (Fig. 1). Abdominal surgery did not decrease 48-hour cumulative stool pellet output, the operated rats making up the deficit from the first 24 hours during the second 24 hours (data not shown).

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Fig. 3. Capsaicin treatment of the VNs had no significant effect on postoperative stool pellet number/24 hr or stool pellet weight (gm)/24 hr compared with vehicle treatment.

Halothane anesthesia and abdominal surgery decreased 24-hour cumulative food intake by 12% and 52%, and 48-hour cumulative food intake by 9% and 34%, respectively (Table I). Experiment 2: Effect of CGRP MAb treatment on postoperative colonic transit, stool pellet output, and food intake. Treatment with saline 0.9%, KLH MAb, or CGRP MAb did not influence colonic transit time, stool pellet output, or food intake (Fig. 1, Table I). Treatment with CGRP MAb before abdominal operation reversed inhibition of colonic transit by 77% and inhibition of 24-hour cumulative stool pellet number and stool pellet weight by 82% and 80%, respectively (Fig. 2). In addition, CGRP MAb treatment reversed postoperative inhibition of food intake by 52% and 62% at 24 and 48 hours, respectively (Table I). Experiment 3: Effect of capsaicin treatment of the vagus nerves on postoperative colonic transit, stool pellet output, and food intake. Capsaicin

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Table III. Effect of capsaicin and vehicle treatment of the VN on cumulative food intake (FI; gm/100 gm body weight)

n Body weight (gm) FI 0-24 hr FI 24-48 hr FI 0-48 hr

Vehicle VN

Capsaicin VN

Vehicle VN + halothane

Capsaicin VN + halothane

4 365 ± 24 6.8 ± 0.6 6.3 ± 0.4 13.0 ± 0.9

5 342 ± 15 8.0 ± 0.7 7.0 ± 0.7 15.0 ± 1.3

4 368 ± 22 6.3 ± 0.5 5.5 ± 0.3 11.8 ± 0.8

5 348 ± 19 7.5 ± 0.6 6.8 ± 0.6 14.3 ± 1.2

Vehicle VN + abdominal surgery

Capsaicin VN + abdominal surgery

4 374 ± 21 2.1 ± 0.1* 4.5 ± 0.4* 6.5 ± 0.3*

5 358 ± 19 2.9 ± 0.2* 5.0 ± 0.3* 7.9 ± 0.5*

*p < 0.05 vs vehicle VN or capsaicin VN.

Table IV. Effect of halothane anesthesia in rats with capsaicin or vehicle treatment of the CSMG

n Colonic transit (hr) Stool pellet number 0-12 hr 12-24 hr Stool pellet weight (gm) 0-12 hr 12-24 hr

Vehicle CSMG

Capsaicin CSMG

Vehicle CSMG + halothane

Capsaicin CSMG + halothane

7 9.0 ± 1.5

6 10.8 ± 1.3

7 12.0 ± 2.2

6 9.4 ± 1.3

24 ± 3 15 ± 2

23 ± 2 14 ± 2

25 ± 5 14 ± 1

26 ± 3 17 ± 3

4.9 ± 0.6 3.1 ± 0.4

4.7 ± 0.8 3.1 ± 0.7

5.5 ± 1.2 3.3 ± 0.4

5.6 ± 0.8 3.7 ± 1.0

treatment of the VN did not alter colonic transit, stool pellet number, stool pellet weight, or food intake compared with vehicle treatment (colonic transit, capsaicin VN vs vehicle VN: 7.2 ± 1.3 vs 8.0 ± 2.4 hr, not significant; stool pellet number/24 hr: 60 ± 10 vs 49 ± 7, not significant; stool pellet weight/24 hr: 14.3 ± 2.2 vs 11.3 ± 1.7 gm, not significant; food intake/24 hr: 8.0 ± 0.7 vs 6.8 ± 0.6 gm/100 gm BW, not significant). Halothane anesthesia did not reveal any differences between capsaicin and vehicle treatment of the VN as well (Table II). Capsaicin or vehicle treatment of the VNs significantly increased postoperative colonic transit time compared with control animals, with no significant difference between capsaicin and vehicle treatment (postoperative colonic transit, control animals: 14.2 ± 1.2 hr; capsaicin VN: 27.6 ± 2.7 hr, p < 0.05 vs control; vehicle VN: 24.0 ± 8.5 hr, p < 0.05 vs control). Postoperative stool pellet output and food intake were decreased after capsaicin or vehicle treatment of the VN compared with control animals, with no differences between capsaicin and vehicle treatment of the VN (Fig. 3, Table III). Experiment 4: Effect of capsaicin treatment of the celiac and superior mesenteric ganglia on postoperative colonic transit, stool pellet output, and food intake. Capsaicin treatment of the CSMG did not alter colonic transit, stool pellet number, stool

pellet weight, or food intake compared with vehicle treatment (colonic transit, capsaicin CSMG: 10.8 ± 1.3 hr, vehicle CSMG: 9.0 ± 1.5 hr, not significant; stool pellet number/24 hr, capsaicin CSMG: 37 ± 2, vehicle CSMG: 39 ± 5, not significant; stool pellet weight/24 hr, capsaicin CSMG: 7.8 ± 1.1 gm, vehicle CSMG: 7.9 ± 0.9 gm, not significant; food intake/24 hr, capsaicin CSMG: 6.2 ± 0.4 gm/100 gm BW, vehicle CSMG: 6.0 ± 0.5 gm/100 gm BW, not significant). Halothane anesthesia did not reveal any differences between capsaicin and vehicle treatment of the CSMG as well (Table IV). Postoperative colonic transit time was not affected by capsaicin or vehicle treatment of the CSMG (postoperative colonic transit, control animals: 14.2 ± 1.2 hr; capsaicin CSMG: 15.6 ± 1.6 hr; vehicle CSMG: 15.4 ± 1.9 hr, not significant). However, postoperative inhibition of stool pellet number and stool pellet was reversed by 41% and 38%, respectively, by capsaicin treatment of the CSMG compared with vehicle (Fig. 4). In addition, postoperative inhibition of food intake was reversed by 19% and 39% at 24 and 48 hours, respectively, in capsaicin compared with vehicle-treated rats (Table V). DISCUSSION In our study in awake rats, abdominal surgery

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significantly reduced colonic transit and cumulative stool pellet output by 40% to 60% in rats monitored for 24 hours after operation compared with unoperated control rats. No significant difference could be observed at 48 hours after operation for cumulative stool pellet output. The deficit from the first 24 hours was made up during the second 24 hours, suggesting gastrointestinal integrity with no permanent damage in our postoperative ileus model. Although postoperative 48-hour cumulative food intake was decreased compared with unoperated control rats, food intake was not decreased between 24 and 48 hours after the operation compared with unoperated controls, again showing recovery of the animals. Thus, our postoperative ileus model, which has been used repeatedly to induce gastric or small intestinal ileus in the rat,79,26 seems suitable to investigate postoperative colonic ileus as well. Although inhibitory effects of CGRP on rat and rabbit colon have been shown in vitro,18,19 in vivo effects of CGRP immunoneutralization on colonic motility have not been investigated. In our study, immunoneutralization of CGRP did not influence colonic transit or stool pellet output in unoperated rats, suggesting no major role for CGRP in the physiologic control of colonic motility. CGRP release and consecutive inhibition of colonic motility may occur only in response to noxious stimulation or tissue damage, as shown previously.29,30 Immunoneutralization of CGRP did not influence food intake in unoperated rats as well, although intraperitoneal injection of CGRP has been shown to decrease food intake,24 and CGRP receptor blockade partly reversed glucagon-, cholecystokinin- or bombesin-induced inhibition of food intake in rats.25 However, because we recorded food intake only at 24 and at 48 hours, our setup might not have been sensitive enough to pick up short-term changes in food intake by CGRP immunoneutralization. As expected, saline or KLH MAb injections did not alter colonic motility or food intake in control experiments, indicating that the injection procedure, saline, or unspecific control antibody did not themselves influence results. CGRP immunoneutralization reversed abdominal surgery-induced inhibition of colonic transit and 24-hour stool pellet output by approximately 80%, with no significant difference in colonic transit time and 24-hour stool pellet output in postoperative CGRP MAb-treated rats compared with unoperated control animals. These results suggest an important role for CGRP in mediating postoperative colonic ileus in rats. In addition, our results for the first time show involvement of a neuropep-

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Fig. 4. Capsaicin treatment of the CSMG reversed postoperative inhibition of stool pellet number/24 hr and stool pellet weight (gm)/24 hr by 41% and 38%, respectively, compared with vehicle treatment. *p < 0.05 vs vehicle CSMG + abdominal surgery.

tide in the mediation of postoperative colonic ileus in rats. We have recently shown that CGRP immunoneutralization reversed postoperative gastric ileus by about 45%.26 Reversal of postoperative colonic ileus by about 80% in this study would suggest an even greater contribution of CGRP in postoperative colonic ileus than in gastric ileus. Because the length of postoperative ileus is mainly influenced by recovery of colonic ileus,1-3 CGRP might be particularly important in mediating postoperative ileus. Several possibilities may explain the way in which CGRP inhibits postoperative gastrointestinal motility. CGRP might be released into circulation,31 thus inhibiting motility directly through CGRP receptors on gastrointestinal smooth muscle cells.20 Alternatively, CGRP might be released locally as part of nociceptive or intestino-intestinal inhibitory reflex pathways. CGRP is released by

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Table V. Effect of capsaicin and vehicle treatment of the CSMG on cumulative food intake (FI; gm/100 gm body weight)

n Body weight (gm) FI 0-24 hr FI 24-48 hr FI 0-48 hr

Vehicle CSMG

Capsaicin CSMG

Vehicle CSMG + halothane

Capsaicin CSMG + halothane

Vehicle CSMG + abdominal surgery

Capsaicin CSMG + abdominal surgery

7 380 ± 20 6.0 ± 0.5 6.0 ± 0.4 12.0 ± 0.9

6 361 ± 19 6.2 ± 0.4 5.8 ± 0.5 12.0 ± 0.8

7 374 ± 25 6.4 ± 0.5 5.9 ± 0.4 12.4 ± 0.9

6 369 ± 12 6.9 ± 0.7 6.2 ± 0.4 13.1 ± 1.1

7 382 ± 18 2.9 ± 0.5* 4.5 ± 0.5* 7.4 ± 1.0 *

6 388 ± 11 3.6 ± 0.2*† 5.7 ± 0.4† 9.3 ± 0.5*†

*p < 0.05 vs vehicle CSMG or capsaicin CSMG. †p

< 0.05 vs vehicle CSMG + abdominal surgery

noxious stimulation and promotes nociception.13,29 CGRP-induced nociception may activate spinal afferent nerve fibers as part of spinal reflex pathways,32 which are able to inhibit gastrointestinal motility.33,34 In addition, CGRP potentiates the release of substance P,35 which is thought to be a mediator of pain, and partly mediates postoperative ileus in the rat.10,13 Blocking CGRP action by CGRP immunoneutralization might decrease the initial painful volley of nerve impulses during operation, which may initiate postoperative ileus. Finally, CGRP might inhibit postoperative gastrointestinal motility by releasing somatostatin,36 which has been shown to inhibit acetylcholine release from myenteric neurons and gastrointestinal motility.37,38 CGRP immunoneutralization reversed postoperative inhibition of food intake by 40% at 24 hours and by 76% at 48 hours after operation, indicating improved recovery from abdominal surgery by CGRP immunoneutralization. Food intake is a complex process, involving multiple neurotransmitters, neuropeptides, and hormones.39 We have shown that abdominal surgery induces neuronal activity in the nucleus of the solitary tract,40 a brain stem center known to respond to intestinal nutrients and thought to be involved in the inhibition of food intake.41 Abdominal surgery may activate brain stem centers known to inhibit food intake via gastrointestinal afferent nerve fibers. These nerve fibers could be activated by CGRP postoperatively, as suggested by data showing inhibition of food intake by peripheral CGRP.24 CGRP immunoneutralization might have decreased afferent input to brain satiety centers postoperatively, thereby increasing postoperative food intake. Alternatively, abdominal surgery-induced inhibition of gastric motility would be expected to result in gastric distention, which is known to decrease food intake.42 CGRP immunoneutralization might have hastened recovery from postoperative gastric ileus,9,26 thus

increasing postoperative food intake. On the other hand, increased postoperative food intake might indicate not only recovery from surgical trauma but might also contribute to resolution of postoperative ileus. Food intake is known to activate colonic motor activity,27 which also has been observed in patients postoperatively.43 Increasing postoperative food intake by CGRP immunoneutralization may have stimulated colonic motility in our experiments. It is conceivable that food intake might be an important mechanism to resolve postoperative colonic ileus. We further investigated the effects of gastrointestinal afferent nerve fiber ablation on postoperative colonic motility and food intake. Topical application of capsaicin or vehicle to the cervical VNs did not influence colonic motility, stool pellet output, or food intake compared with control animals. However, both capsaicin and vehicle treatment of the cervical vagus increased postoperative colonic transit, whereas postoperative stool pellet output and food intake were decreased. Because capsaicin treatment, which selectively ablates afferent nerve fibers,21 had no specific effect, we assume that some unspecific damage might have occurred to vagal efferent nerve fibers by the topical treatment. Worsening of postoperative colonic ileus by vagal efferent nerve fiber damage would suggest that vagal efferent nerve fibers contribute to the recovery of postoperative colonic ileus, a conclusion supported by data showing that colonic motility can be stimulated by vagal efferent nerve fibers.44,45 Capsaicin treatment of the CSMG did not influence colonic motility, stool pellet output, or food intake compared with control animals. However, capsaicin treatment of the CSMG reversed postoperative inhibition of stool pellet output by about 40% and postoperative inhibition of food intake at 24 and 48 hours by 19% and 39%, respectively. Thus, capsaicin treatment of the CSMG partly reversed postoperative colonic ileus and postopera-

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tive inhibition of food intake. A large proportion of gastrointestinal extrinsic spinal afferent nerve fibers contains CGRP.11 These fibers run through the prevertebral ganglia, making contact with postganglionic sympathetic efferent nerve fibers, which, besides other functions, contribute to the regulation of gastrointestinal motility.46 The afferent innervation of the gastrointestinal tract up to the proximal colon runs in part through the CSMG,46 providing the afferent arm of intestinointestinal reflex arches able to inhibit gastrointestinal motility, if activated.47 In particular, it has been shown that afferent input from the proximal colon can inhibit distal colonic motility.33 Thus, in our postoperative ileus model, manipulating the cecum might have activated a reflex resulting in the inhibition of distal colonic motility. Ablating the afferent arm of this reflex by capsaicin treatment of the CSMG may have allowed faster recovery from postoperative colonic ileus in our experiments. Capsaicin treatment of the CSMG was not as effective as CGRP immunoneutralization for recovery from postoperative ileus in our experiments. Several reasons might have accounted for this outcome. Afferent innervation of the distal colon is not ablated by capsaicin treatment of the CSMG, because the spinal afferent innervation of the distal colon runs in part through the inferior mesenteric ganglion.46 Thus, additional inhibitory colocolonic reflexes might have been left intact, possibly mediating postoperative inhibitory input to the distal colon. Further, as already pointed out, CGRP might inhibit postoperative gastrointestinal motility directly by acting on CGRP receptors on gastrointestinal smooth muscle cells, or indirectly via the release of somatostatin.20,36 In conclusion, CGRP immunoneutralization or capsaicin treatment of the CSMG partly reversed postoperative colonic ileus in the rat. By the magnitude of reversal of postoperative ileus, CGRP seems to be an important mediator of postoperative ileus. CGRP might be released into circulation and inhibit postoperative gastrointestinal motility by acting on CGRP receptors on gastrointestinal smooth muscle cells or by releasing somatostatin. Alternatively or in parallel, locally released CGRP might stimulate spinal afferent nerve fibers, possibly running through the CSMG, thus activating gastrointestinal afferent nerve fibers as part of nociceptive or colo-colonic inhibitory reflex pathways. In addition to its effects on postoperative colonic ileus, CGRP immunoneutralization or capsaicin treatment of the CSMG partly reversed postoperative inhibition of food intake, possibly by decreas-

ing afferent input to satiety centers in the brain stem. Further studies should investigate the effects of CGRP immunoneutralization or CGRP receptor blockade on colonic ileus by recording colonic motility directly. Also, on the basis of our results, it is conceivable that postoperative inhibition of food intake contributes to postoperative ileus. Therefore, studies should address the relationship between postoperative food intake and postoperative ileus, and whether reversal of postoperative inhibition of food intake provides a mechanism to reverse postoperative ileus. REFERENCES 1. Livingston EH, Passaro EP. Postoperative ileus. Dig Dis Sci 1990;35:121-32. 2. Condon RE, Cowles VE, Schulte WJ, Frantzides CT, Mahoney JL, Sarna SK. Resolution of postoperative ileus in humans. Ann Surg 1986;203:574-80. 3. Bufo AJ, Feldman S, Daniels GA, Lieberman RC. Early postoperative feeding. Dis Colon Rectum 1994;37:1260-65. 4. Furness JB, Bornstein JC, Murphy R, Pompolo S. Roles of peptides in transmission in the enteric nervous system. Trends in Neuroscience 1992;15:66-71. 5. Dubois A, Henry D, Kopin I. Plasma catecholamines and postoperative gastric emptying and small intestinal propulsion in the rat. Gastroenterology 1975;68:466-9. 6. Sagrada A, Fargeas MJ, Bueno L. Involvement of alpha-1 and alpha-2 adrenoreceptors in the postlaparotomy intestinal motor disturbances in the rat. Gut 1987;28:955-9. 7. Barquist E, Zinner M, Rivier J, Taché Y. Abdominal surgeryinduced delayed gastric emptying in rats: role of CRF and sensory neurons. Am J Physiol 1992;262:G616-20. 8. Rivière JM, Pascaud X, Chevalier E, Le Gallou B, Junien JL. Fedotozine reverses ileus induced by surgery or peritonitis: action at peripheral κ-opioid receptors. Gastroenterology 1993;104:724-31. 9. Zittel TT, Reddy NS, Plourde V, Raybould HE. Role of spinal afferents and calcitonin gene-related peptide in the postoperative gastric ileus in anesthetized rats. Ann Surg 1994;219:79-87. 10. Espat NJ, Cheng G, Kelley MC, Vogel SB, Sninsky CA, Hocking MP. Vasoactive intestinal peptide and substance P receptor antagonists improve postoperative ileus. J Surg Res 1995;58:719-23. 11. Sternini C. Tachykinin and calcitonin gene-related peptide immunoreactivities and mRNAs in the mammalian enteric nervous system and sensory ganglia. Adv Exp Med Biol 1991;298:39-51. 12. Ishida-Yamamoto A, Atohyama M. Calcitonin gene-related peptide in the nervous tissue. Prog Neurobiol 1989;33:33586. 13. Biella G, Panara C, Pecile A, Sotgiu ML. Facilitatory role of calcitonin gene-related peptide (CGRP) on excitation induced by substance P (SP) and noxious stimuli in rat

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