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Evaluation of alternative proximal gastric vagotomy techniques after a 9-month interval in a rat model
Evaluation of alternative proximal gastric vagotomy techniques after a 9-month interval in a rat model Todd J. Neuberger, MD, Catherine M. Wittgen, MD, Thomas A. Schneider, II, MD Charles H. Andrus, MD, W. Michael Panneton, PhD, Donald L. Kaminski, MD st. Louis, Missouri Proximal gastric vagotomy (PGV) is an accepted operation for patients with ulcers that are refractory to medical management. Results comparable to those of standard, operative PGV have previously been demonstrated using endoscopic chemoneurolytic injection or laparoscopic laser seromyotomy in a porcine model. In this study, we evaluated several PGV techniques in regard to long-term effects on acid secretion, ulcer prophylaxis, and permanent vagal denervation in a rat model. Trans-mucosal injection of chemoneurolytic agents (cobaltous chloride, benzalkonium chloride, and phenol) and seromyotomy by C02 laser were performed. After 9 months, all rats received sub-serosal gastric injections of horseradish peroxidase (HRP) during laparotomy. Twenty-four hours later, an ulcerogenic dose of pentagastrin was administered sub-cutaneously. Three days after administration of HRP (to allow time for retrograde axonal transport and labeling of cells of the dorsal vagal nucleus with HRP), necropsy was performed. The pre-pyloric gastric mucosa was inspected for ulcerogenic changes, and a Congo red solution was applied to the gastric mucosa to map the acid-secreting areas. All PGV methods significantly diminished pentagastrin-induced ulceration when compared to sham controls. Benzalkonium chloride chemoneurolytic and laser methods were most effective for decreasing the size of acid-secreting areas. A reduced number of HRP-stained cells in the dorsal vagal nucleus indicated permanent denervation of vagal-gastric connections by operative and laser techniques. (Gastrointest Endosc 1994j40:316-20.)
In an attempt to develop minimally invasive techniques of proximal gastric vagotomy (PGV), we have previously evaluated the use of laser and chemoneurolytic agents in short-term porcine! and rat2, 3 models. These studies have demonstrated that on a short-
Received April 1, 1993. For revision June 15, 1993. Accepted November 3, 1993. From the Departments of Surgery and Anatomy & Neurobiology, St. Louis University School of Medicine and John Cochran VA Medical Center, St. Louis, Missouri. Research supported in part by a Merit Grant through the Research Division of the Department of Veterans Affairs. Reprint requests: Charles H. Andrus, MD, Associate Professor of Surgery, Department of Surgery, 3635 Vista Avenue at Grand Blvd., P.O. Box 15250, St. Louis, MO 63110-0250. 37/1/53117
316
term basis, results with both laser and chemoneurolytic agents are comparable to results with operative PGV in regard to the inhibition of pentagastrin-stimulated gastric acid secretion as documented by Congo red staining. The purpose of this study was to evaluate the long-term effectiveness of alternative PGV techniques in achieving permanent parietal cell denervation. MATERIALS AND METHODS
Seven groups of 300- to 400-g male Sprague-Dawley rats were studied to determine the long-term efficacy of alternative PGV techniques in achieving the effects of parietal cell denervation: (1) sham operative control (Sham), (2) PGV operative control (Op PGV), (3) trans-mucosal injection of 0.1 % benzalkonium chloride (BZC), (4) trans-mucosal injection of 0.75% cobaltous chloride (CoCI 2), (5) transGASTROINTESTINAL ENDOSCOPY
mucosal injection of 5% phenol (Phenol), (6) anterior CO 2 laser seromyotomywith posterior truncal vagotomy (APTV), and (7) anterior and posterior CO 2 laser seromyotomy (A&P). All procedures were performed under general anesthesia induced by intra-peritoneal injection of sodium pentobarbital (35 to 50 mg/kg). Each procedure was performed after a 24-hour fast through a mid-line celiotomy incision. In the Sham group the stomach was mobilized and then restored to its anatomic position before the abdomen was closed. In the Op PGV group, the neurovascular bundles from the vagal trunks to the serosa of the anterior and posterior portions of the lesser curvature of the stomach were ligated and divided between the gastroesophageal junction and the nerves of Latarjet (approximately 3 mm proximal to the pylorus). Chemoneurolytic injections were performed with one of three agents: 0.75% cobaltous chloride, 0.1 % benzalkonium chloride, or 5 % phenol (Sigma Chemical, St. Louis, MoV After mid-line celiotomy and mobilization of the stomach, a gastrostomy was performed on the greater curvature. Approximately 0.1 mL of one of the chemoneurolytic solutions was injected trans-mucosally with a 25-gauge needle so that wheals were raised at multiple sites between the gastroesophageal junction and the nerves of Latarjet along the anterior and posterior aspects of the stomach. A total of 1 to 1.5 mL was injected. Two CO 2 laser PGV techniques were employed. 3 Rats subjected to the first technique (APTV) received 3 W of defocused CO 2 laser (Lasersonics, Milpitas, Cal.) to the serosa of the anterior stomach in the same distribution as described for the trans-mucosal injection groups; the vagal neurovascular trunk was divided as in the Op PG V group. Rats in the other laser seromyotomy group (A&P) received 3 W of defocused CO 2 laser energy to both the anterior and posterior serosal surfaces of the stomach. After each initial procedure, the animals were returned to a regular diet and maintained in a homeostatic environment for 9 months. The rats were observed daily for the development of any complications. Weights were recorded biweekly during this period. Seventy-two hours before necropsy, six animals from each group underwent a second mid -line celiotomy using the same technique with general anesthesia. With the stomach mobilized, 40 to 50 JIL (1 mg/50 JIL) of a wheat-germ agglutinin horseradish peroxidase (HRP, Sigma Chemical) diluted with a green vital dye (Fast Green FCS dye, Sigma Chemical) was injected into the anterior and posterior sub-serosal walls in equally divided doses until the entire gastric serosa was stained. This was done to retrograde label surviving dorsal vagal neurons innervating the stomach. The puncture sites were sealed with cyano-acrylate glue. Before closure, the abdomen was irrigated with 20 to 25 mL of normal saline solution to remove any gross spillage of HRP. In order to assess the possible prophylactic benefit of each of these procedures, a subcutaneous injection of a previously determined ulcerogenic dose of pentagastrin3 (120 Jig/kg) (Peptavlon, Ayerst Laboratories, New York, N.Y.) was administered to each rat 48 hours before necropsy. At necropsy the long-term effects of each procedure were assessed by three separate methods: visual inspection of the gastric mucosa, Congo red (Eastman Kodak, Rochester, VOLUME 40, NO.3, 1994
N.Y.) staining of the pentagastrin-stimulated gastric mucosa, and HRP staining of intact motor neurons of the vagal nucleus. At necropsy, general anesthesia was induced with intra-peritoneal sodium pentobarbital. After celiotomy through the previous incision, the mobilized stomach was opened along the entire greater curvature; the cut edges were transfixed with 3-0 sutures placed at the margins and retracted to allow adequate exposure of the gastric mucosa. The mucosa of the stomach and proximal duodenum was carefully inspected and then graded according to the following criteria: 0 = no mucosal changes; 1 = superficial mucosal inflammation; 2 = ulceration with evidence of bleeding; 3 = ulceration with perforation. At the time of anesthesia each rat also received a physiologic pentagastrin dose (6 Jig/kg) in order to stimulate acid secretion. After 15 minutes, with the stomach opened and properly displayed, the mucosa was sprayed with 0.5% NaHC0 3 to neutralize existing acid. The mucosa was then blotted dry before being sprayed with a solution of Congo red (100 mg Congo red/250 mL 0.5% NaHC0 3). Any area of mucosa with a pH of less than 3 was stained black by the Congo red. Photographic slides were taken after 5 minutes utilizing Ektachrome (ASA 100) film (Eastman Kodak). After development, 8 X ll-in prints were produced on a laser copier (Canon color copier). The data were quantitated by a blinded observer using a computer-driven digitized area calculation program (Sigmascan Software, Jandel Scientific, Corte Madera, Cal.). The total mucosal area was mapped first, after which the black-stained areas were mapped. The percentage of acid-secreting mucosa was then calculated as the ratio of stained gastric mucosal area to total gastric mucosal area multiplied by 100. After the photographs were completed, a median sternotomy was performed. The right atrium was incised for venous outflow, and the left ventricle was punctured for serial perfusion with a chilled saline solution, a fixative solution (2 % paraformaldehyde and 1.25% glutaraldehyde in 0.1 mol/L phosphate buffer solution at pH 7.4), and fixative solutions with 5 % sucrose buffer and 10 % sucrose buffer as previously reported. 3 Each fixative solution was administered under positive pressure through the vasculature via the left ventricle and aorta until the right atrial effluent was the same as the affluent fixative. Several minutes were required for each perfusion step. Brains were sectioned at 50-Jim intervals using a sliding microtome. The tetramethylbenzidine technique of Mesulam 4 was used for staining the sections. Under light microscopy at a magnification of 100, every other section was inspected by a neuropathologist in a blinded manner; each labeled neuron with at least one dendrite visible on an individual section was counted. The final tabulation was multiplied by two to correct for having counted only half of the sections. Only rats that had completed the 9-month growth period and had their gastric mucosa successfully stained with Congo red were included in the final analysis. Using an analysis of variance (ANOVA), all data obtained were compared at a 95% confidence level. RESULTS
With the exception of the BZC group, which had 10 animals, all groups included 8 animals at the start of 317
Table 2. Dorsal vagal neurons counted in rats undergoing alternative PGV techniques
Table 1. Weight change in control and experimental groups Group
N
Sham OpPGV BZC CoCl 2 Phenol A&P APTV
7 7 10 8 8 4 8
Avg wt change/wk (g ± SD) 3.81 3.26 5.40 5.86 5.05 3.79 4.36
± ± ± ± ± ± ±
0.77 1.13 1.54*,t 1.90*,t,:j: 2.20* 1.57 0.84
Total wt gain (g ± SD) 142.3 126.2 171.2 162.5 150.2 144.8 161.3
± ± ± ± ± ± ±
24.5 44.5 22.2* 37.8 51.4 67.2 43.1
Sham, Sham operative control; Op PGV, operative proximal gastric vagotomy; BZC, trans-mucosal injection of 0.1 % benzalkonium chloride; CoCI], trans-mucosal injection of 0.75% cobaltous chloride; Phenol, trans-mucosal injection of 5% phenol; A&P, anterior and posterior seromyotomy performed by CO 2 laser blanching of the anterior and posterior gastric serosal surfaces; APTV, anterior seromyotomy performed by CO 2 laser blanching of the anterior gastric serosal surface and posterior truncal vagotomy. *Significant at a 95% confidence level when compared with the Op PGV group. tSignificant at a 95% confidence level when compared with the Sham group. :j:Significant at a 95% confidence level when compared with the A&P C02 laser group.
the study. Two of the control animals, one each from the Sham group and the Op PG V group, expired before sacrifice; no causes of death were determined at autopsy. No deaths occurred in the chemoneurolytic or APTV laser groups. Four premature deaths in the A&P laser group were attributed to pneumonia at autopsy. The six animals that had expired before planned necropsy were excluded from the final Congo red, HRP, and ulcer analysis. During the 9 months after the initial procedure, a net weight gain was noted in all groups (Table 1). The BZC and CoCl2 chemoneurolytic groups demonstrated average weight gains per week that were significantly greater than that in either the Sham or Op PGV control groups. In order to minimize cost but still obtain statistically significant information for comparison between groups, six animals from each group were chosen for histochemical labeling of the vagal nerves. Because of technical difficulties in the perfusion and fixation of the brain stems, not all microscopic sections were suitable for review. Results are based on those sections that could be evaluated (Table 2). At the time of necropsy, the gastric and duodenal mucosa was visually inspected and graded according to the arbitrary, prospective ulcer scale described above (Table 3). As determined previously,3 a pentagastrin dose 20 times (120 ~g/kg) the normal physiologic dose (6 ~g/kg) injected sub-cutaneously 3 days before necropsy stimulated mucosal erythema and ulceration in the pre-pyloric region of the stomach in the major318
Group
N
Sham OpPGV BZC CoCl 2 Phenol A&P APTV
6 6 4 3 4 1 4
Labelled dorsal vagal neurons 1060 76 734 684 335
± ± ± ± ±
536 103t 201 *,:j:,§ 100*,§ 267t
8t 79 ± 102t
Sham, Sham operative control; Op PGV, operative proximal gastric vagotomy; BZC, trans-mucosal injection of 0.1 % benzalkonium chloride; COCi2, trans-mucosal injection of 0.75% cobaltous chloride; Phenol, trans-mucosal injection of 5% phenol; A&P, anterior and posterior seromyotomy performed by CO 2 laser blanching of the anterior and posterior gastric serosal surfaces; APTV, anterior seromyotomy performed by CO 2 laser blanching of the anterior gastric serosal surface and posterior truncal vagotomy. *Significant at a 95% confidence level when compared with the Op PGV group. tSignificant at a 95% confidence level when compared with the Sham group. :j:Significant at a 95% confidence level when compared with the A&P C02 laser group. §Significant at a 95% confidence level when compared with the APT V C02 laser group.
ity of Sham animals. Significant ulcer prophylaxis was observed in all the PGV groups when compared with the Sham control group. However, the ulcer prophylaxis score in the Phenol chemoneurolytic group was significantly poorer than in the Op PGV group. As has been previously described,1-3 a Congo red staining technique was used to quantitate the acidsecretory ability of the gastric mucosa. The area of mucosal pH decrease was significantly diminished in the BZC, A&P, and APTV groups when compared with that in the Sham controls (Table 3). The physical intactness of the vagal-gastric connections was evaluated by retrograde HRP staining techniques. 3,4 As would be predicted, the mean number of neurons of the dorsal vagal nucleus stained was greatest in the Sham group (Table 2). Significantly reduced neuronal staining was observed in the Op PGV, Phenol, A&P, and APTV groups. The BZC and CoCh chemoneurolytic groups showed intermediate numbers of stained neurons, which were significantly greater than those of the "mechanical" PG V (Op PG V, A&P, and APTV) groups. DISCUSSION
With the recent expansion of indications for operative laparoscopic surgery, parietal cell vagotomy is now being evaluated as a possible routinely practiced procedure. 5-? In attempts to determine the completeness of parietal cell vagotomies, we have previously utilized intra-operative Congo red staining techniques in a GASTROINTESTINAL ENDOSCOPY
porcine model 1 and in humans. 8 In rat 2, 3 and porcine 1 studies, we have demonstrated chemoneurolytic injection PGV techniques to be equivalent to operative laparoscopic and CO 2 laser laparoscopic PGV techniques on a short-term basis. However, the long-term effectiveness of those various chemoneurolytic techniques has never been documented in rats, pigs, or humans. Throughout this 9-month study, no gross adverse outcomes could be attributed to any of the PGV techniques. The consistent weight gain in all groups confirmed that long-term side effects of these alternative PGV techniques were minimal (Table 1). Animals undergoing the mechanical procedures (Op PGV, A&P, and APTV) demonstrated less of an average weight gain per week overall than the chemoneurolytic groups, which may reflect the increased stomach distension and presumably decreased gastric emptying (gastroparesis) that has been observed previously in these groups.3 As we demonstrated earlier in short-term rat studies,3 histamine-like stimulation of duodenal ulcers with cysteamine (2-amino-ethanethiol hydrochloride)9 was not prevented by any PGV techniques. In these same studies, we demonstrated that a pharmacologic dose of pentagastrin (120 ~g/kg) caused reproducible pre-pyloric gastric mucosal irritation and ulceration in control animals but was inhibited by PGV techniques. Thus, we concluded that because pentagastrin stimulates the gastrin receptor on the parietal cell, which shares a common parietal intra-cellular biochemical pathway with the vagal (acetylcholine) stimulus,lO the ulcerogenic stimulation of a pharmacologic dose of pentagastrin can be reproducibly inhibited by vagotomy in the short-term rat model. In this present study, we confirmed the long-term effectiveness of ulcer prophylaxis by several PGV techniques (Op PGV, chemoneurolytic, and laser) (Table 3). In this long-term study, the reduction in the pentagastrin-stimulated acid-secretion area of the gastric mucosa was quantitated using a modified Congo red test, as previously described for the short-term rat mode1. 2,3 A significant decrease in acid secretion was demonstrated in the laser PGV groups (A&P and APTV) and in the benzalkonium chemoneurolytic group (BZC) (Table 3). Although a significant difference in acid secretion from control was demonstrated for these groups, the effect of all PGV techniques collectively for acid reduction was not as impressive as that seen in the short-term rat studies. 2, 3 In the longterm setting, the variability in innervation may reflect incompleteness or non-permanence of denervation by the various PGV techniques, re-innervation phenomenon, tachyphylaxis of the treated parietal cells, or some other mechanism. It is important to note that the greatest reduction occurred in the A&P group. This VOLUME 40, NO.3, 1994
Table 3. Results of alternative PGV techniques for ulcer prophylaxis and inhibition of acid secretion Group
N
Ulcer score
Sham OpPGV BZC CoCl 2 Phenol A&P APTV
7 7 10 8 8 4 8
2.1 0.3 0.7 1.0 1.1 0.5 0.5
± ± ± ± ± ± ±
1.2 0.5t 0.5t 0.8t 0.6*,t 0.6t 0.5t
Acid-secretion area by Congo red test (%)
17.5 11.0 8.8 15.1 14.5 2.7 7.4
± ± ± ± ± ± ±
8.6 6.2 6.5t 10.0t 10.0t 2.2t 5.1 t
Sham, Sham operative control; Op PGV, operative proximal gastric vagotomy; BZC, trans-mucosal injection of 0.1 % benzalkonium chloride; CoCI2, trans-mucosal injection of 0.75% cobaltous chloride; Phenol, trans-mucosal injection of 5% phenol; A&P, anterior and posterior seromyotomy performed by C02 laser blanching of the anterior and posterior gastric serosal surfaces; APTV, anterior seromyotomy performed by CO 2 laser blanching of the anterior gastric serosal surface and posterior truncal vagotomy. *Significant at a 95% confidence level when compared with the Op PGV group. tSignificant at a 95% confidence level when compared with the Sham group. :j:Significant at a 95% confidence level when compared with the A&P CO 2 laser group.
was the one group in which both the anterior and posterior serosal surfaces of the stomach were subjected to a significant char, and it may be the group in which the greatest permanent cellular injury or denervation was obtained. Long-lasting anatomic vagal disconnection was confirmed for those PGV techniques employing mechanical methods of vagal denervation (Op PGV, A&P, APTV) (Table 2). Although HRP staining was diminished with the chemoneurolytic techniques, only the Phenol group demonstrated significantly reduced neuronal staining when compared with the Sham group. This may reflect the non-specific cellular injury (chemical burn) of phenol, which results in mechanical vagal disconnection. Although diminished neuronal staining was observed in the other two chemoneurolytic groups (BZC and CoCI2), it was not significantly different from that in the Sham group, suggesting that these techniques produce either quantitatively less mechanical vagal disconnection or more selective denervation or parietal cell inhibition than do the other PGV techniques studied. What can be concluded from these initial long-term rat PGV studies? None of the PGV methods seems to inhibit normal animal growth, and all are significantly effective, to various degrees, for ulcer prophylaxis and inhibition of acid secretion. Chemoneurolytic techniques may promote ulcer prophylaxis at a different site along the vagal-parietal cell axis, than do the mechanical methods (Op PGV, A&P, APTV), and their effects may be more specific and selective or limited 319
and transient. Studies regarding techniques of administration, solution concentrations, and drug dosage are required to perfect current methods of chemoneurolytic suppression of gastric acid secretion. Further investigation of the exact mechanism of action of the chemoneurolytic agents with regards to the vagal-parietal cell axis and at the cellular level seem indicated. ACKNOWLEDGMENT
We wish to thank the Department of Veterans Affairs for its continued support of this project. We also wish to thank Thomas Curry, Animal Care Technician, St. Louis University.
REFERENCES 1. Schneider TA, Wittgen CM, Andrus CR, Kaminski DL. Com-
parison of minimally invasive methods of parietal cell vagotomy in a porcine model. Surgery 1992;112:649-55. 2. Schneider TA, Fitzgerald SD, LaRegina MC, Andrus CR, Kaminski DL. Evaluation of proximal gastric vagotomy by cobaltous chloride (CoCh) chemoneurolysis in a rat model. American College of Surgeons Surgical Forum 1991;42:177-9.
320
3. Wittgen CM, Schneider TA, Fitzgerald SD, et al. Proximal gastric vagotomy by minimally invasive methods in an acute rat model. Surg Endosc 1993;7:319-34. 4. Mesulam MM. Tetramethylbenzidine for horseradish peroxidase neurochemistry: a noncarcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents. J Ristochem Cytochem 1978;26:106-7. 5. Katkhouda N, Mouiel J. A new technique of surgical treatment of chronic duodenal ulcer without laparotomy by video celioscopy. Am J Surg 1991;161:361-4. 6. Shapiro S, Gordon L, Dayhkovsky L, Grundfest W, Chandra M. Development of laparoscopic anterior seromyotomy and right posterior truncal vagotomy for ulcer prophylaxis. J Laparoendosc Surg 1991;1:45-9. 7. Lirici MM, Buess G, Becker RD. The laparoscopic approach to modified Taylor's procedure in the treatment of chronic duodenal ulcer: an improved technique. Surgical Laparoscopy & Endoscopy 1992;2:199-204. 8. Schneider TA, Andrus CR. The efficacy of endoscopic Congo red confirmation of completeness of proximal gastric vagotomy: an essential procedure. Surg Endosc 1992;6:16-7. 9. Robert A, Nezamis JE, Lancaster C, Badalamenti IN. Cysteamine induced duodenal ulcers: a new model to test anti-ulcer agents. Digestion 1974;11:199-214. 10. Radke R, Stach W, Weiss R. Innervation of the gastric wall related to acid secretion: a light and electron microscopy study on rats, rabbits and guinea pigs. Acta Biologica at Medica Germanica 1980;39:687-96.
GASTROINTESTINAL ENDOSCOPY
In an attempt to develop minimally invasive techniques of proximal gastric vagotomy (PGV), we have previously evaluated the use of laser and chemoneurolytic agents in short-term porcine! and rat2, 3 models. These studies have demonstrated that on a short-
Received April 1, 1993. For revision June 15, 1993. Accepted November 3, 1993. From the Departments of Surgery and Anatomy & Neurobiology, St. Louis University School of Medicine and John Cochran VA Medical Center, St. Louis, Missouri. Research supported in part by a Merit Grant through the Research Division of the Department of Veterans Affairs. Reprint requests: Charles H. Andrus, MD, Associate Professor of Surgery, Department of Surgery, 3635 Vista Avenue at Grand Blvd., P.O. Box 15250, St. Louis, MO 63110-0250. 37/1/53117
316
term basis, results with both laser and chemoneurolytic agents are comparable to results with operative PGV in regard to the inhibition of pentagastrin-stimulated gastric acid secretion as documented by Congo red staining. The purpose of this study was to evaluate the long-term effectiveness of alternative PGV techniques in achieving permanent parietal cell denervation. MATERIALS AND METHODS
Seven groups of 300- to 400-g male Sprague-Dawley rats were studied to determine the long-term efficacy of alternative PGV techniques in achieving the effects of parietal cell denervation: (1) sham operative control (Sham), (2) PGV operative control (Op PGV), (3) trans-mucosal injection of 0.1 % benzalkonium chloride (BZC), (4) trans-mucosal injection of 0.75% cobaltous chloride (CoCI 2), (5) transGASTROINTESTINAL ENDOSCOPY
mucosal injection of 5% phenol (Phenol), (6) anterior CO 2 laser seromyotomywith posterior truncal vagotomy (APTV), and (7) anterior and posterior CO 2 laser seromyotomy (A&P). All procedures were performed under general anesthesia induced by intra-peritoneal injection of sodium pentobarbital (35 to 50 mg/kg). Each procedure was performed after a 24-hour fast through a mid-line celiotomy incision. In the Sham group the stomach was mobilized and then restored to its anatomic position before the abdomen was closed. In the Op PGV group, the neurovascular bundles from the vagal trunks to the serosa of the anterior and posterior portions of the lesser curvature of the stomach were ligated and divided between the gastroesophageal junction and the nerves of Latarjet (approximately 3 mm proximal to the pylorus). Chemoneurolytic injections were performed with one of three agents: 0.75% cobaltous chloride, 0.1 % benzalkonium chloride, or 5 % phenol (Sigma Chemical, St. Louis, MoV After mid-line celiotomy and mobilization of the stomach, a gastrostomy was performed on the greater curvature. Approximately 0.1 mL of one of the chemoneurolytic solutions was injected trans-mucosally with a 25-gauge needle so that wheals were raised at multiple sites between the gastroesophageal junction and the nerves of Latarjet along the anterior and posterior aspects of the stomach. A total of 1 to 1.5 mL was injected. Two CO 2 laser PGV techniques were employed. 3 Rats subjected to the first technique (APTV) received 3 W of defocused CO 2 laser (Lasersonics, Milpitas, Cal.) to the serosa of the anterior stomach in the same distribution as described for the trans-mucosal injection groups; the vagal neurovascular trunk was divided as in the Op PG V group. Rats in the other laser seromyotomy group (A&P) received 3 W of defocused CO 2 laser energy to both the anterior and posterior serosal surfaces of the stomach. After each initial procedure, the animals were returned to a regular diet and maintained in a homeostatic environment for 9 months. The rats were observed daily for the development of any complications. Weights were recorded biweekly during this period. Seventy-two hours before necropsy, six animals from each group underwent a second mid -line celiotomy using the same technique with general anesthesia. With the stomach mobilized, 40 to 50 JIL (1 mg/50 JIL) of a wheat-germ agglutinin horseradish peroxidase (HRP, Sigma Chemical) diluted with a green vital dye (Fast Green FCS dye, Sigma Chemical) was injected into the anterior and posterior sub-serosal walls in equally divided doses until the entire gastric serosa was stained. This was done to retrograde label surviving dorsal vagal neurons innervating the stomach. The puncture sites were sealed with cyano-acrylate glue. Before closure, the abdomen was irrigated with 20 to 25 mL of normal saline solution to remove any gross spillage of HRP. In order to assess the possible prophylactic benefit of each of these procedures, a subcutaneous injection of a previously determined ulcerogenic dose of pentagastrin3 (120 Jig/kg) (Peptavlon, Ayerst Laboratories, New York, N.Y.) was administered to each rat 48 hours before necropsy. At necropsy the long-term effects of each procedure were assessed by three separate methods: visual inspection of the gastric mucosa, Congo red (Eastman Kodak, Rochester, VOLUME 40, NO.3, 1994
N.Y.) staining of the pentagastrin-stimulated gastric mucosa, and HRP staining of intact motor neurons of the vagal nucleus. At necropsy, general anesthesia was induced with intra-peritoneal sodium pentobarbital. After celiotomy through the previous incision, the mobilized stomach was opened along the entire greater curvature; the cut edges were transfixed with 3-0 sutures placed at the margins and retracted to allow adequate exposure of the gastric mucosa. The mucosa of the stomach and proximal duodenum was carefully inspected and then graded according to the following criteria: 0 = no mucosal changes; 1 = superficial mucosal inflammation; 2 = ulceration with evidence of bleeding; 3 = ulceration with perforation. At the time of anesthesia each rat also received a physiologic pentagastrin dose (6 Jig/kg) in order to stimulate acid secretion. After 15 minutes, with the stomach opened and properly displayed, the mucosa was sprayed with 0.5% NaHC0 3 to neutralize existing acid. The mucosa was then blotted dry before being sprayed with a solution of Congo red (100 mg Congo red/250 mL 0.5% NaHC0 3). Any area of mucosa with a pH of less than 3 was stained black by the Congo red. Photographic slides were taken after 5 minutes utilizing Ektachrome (ASA 100) film (Eastman Kodak). After development, 8 X ll-in prints were produced on a laser copier (Canon color copier). The data were quantitated by a blinded observer using a computer-driven digitized area calculation program (Sigmascan Software, Jandel Scientific, Corte Madera, Cal.). The total mucosal area was mapped first, after which the black-stained areas were mapped. The percentage of acid-secreting mucosa was then calculated as the ratio of stained gastric mucosal area to total gastric mucosal area multiplied by 100. After the photographs were completed, a median sternotomy was performed. The right atrium was incised for venous outflow, and the left ventricle was punctured for serial perfusion with a chilled saline solution, a fixative solution (2 % paraformaldehyde and 1.25% glutaraldehyde in 0.1 mol/L phosphate buffer solution at pH 7.4), and fixative solutions with 5 % sucrose buffer and 10 % sucrose buffer as previously reported. 3 Each fixative solution was administered under positive pressure through the vasculature via the left ventricle and aorta until the right atrial effluent was the same as the affluent fixative. Several minutes were required for each perfusion step. Brains were sectioned at 50-Jim intervals using a sliding microtome. The tetramethylbenzidine technique of Mesulam 4 was used for staining the sections. Under light microscopy at a magnification of 100, every other section was inspected by a neuropathologist in a blinded manner; each labeled neuron with at least one dendrite visible on an individual section was counted. The final tabulation was multiplied by two to correct for having counted only half of the sections. Only rats that had completed the 9-month growth period and had their gastric mucosa successfully stained with Congo red were included in the final analysis. Using an analysis of variance (ANOVA), all data obtained were compared at a 95% confidence level. RESULTS
With the exception of the BZC group, which had 10 animals, all groups included 8 animals at the start of 317
Table 2. Dorsal vagal neurons counted in rats undergoing alternative PGV techniques
Table 1. Weight change in control and experimental groups Group
N
Sham OpPGV BZC CoCl 2 Phenol A&P APTV
7 7 10 8 8 4 8
Avg wt change/wk (g ± SD) 3.81 3.26 5.40 5.86 5.05 3.79 4.36
± ± ± ± ± ± ±
0.77 1.13 1.54*,t 1.90*,t,:j: 2.20* 1.57 0.84
Total wt gain (g ± SD) 142.3 126.2 171.2 162.5 150.2 144.8 161.3
± ± ± ± ± ± ±
24.5 44.5 22.2* 37.8 51.4 67.2 43.1
Sham, Sham operative control; Op PGV, operative proximal gastric vagotomy; BZC, trans-mucosal injection of 0.1 % benzalkonium chloride; CoCI], trans-mucosal injection of 0.75% cobaltous chloride; Phenol, trans-mucosal injection of 5% phenol; A&P, anterior and posterior seromyotomy performed by CO 2 laser blanching of the anterior and posterior gastric serosal surfaces; APTV, anterior seromyotomy performed by CO 2 laser blanching of the anterior gastric serosal surface and posterior truncal vagotomy. *Significant at a 95% confidence level when compared with the Op PGV group. tSignificant at a 95% confidence level when compared with the Sham group. :j:Significant at a 95% confidence level when compared with the A&P C02 laser group.
the study. Two of the control animals, one each from the Sham group and the Op PG V group, expired before sacrifice; no causes of death were determined at autopsy. No deaths occurred in the chemoneurolytic or APTV laser groups. Four premature deaths in the A&P laser group were attributed to pneumonia at autopsy. The six animals that had expired before planned necropsy were excluded from the final Congo red, HRP, and ulcer analysis. During the 9 months after the initial procedure, a net weight gain was noted in all groups (Table 1). The BZC and CoCl2 chemoneurolytic groups demonstrated average weight gains per week that were significantly greater than that in either the Sham or Op PGV control groups. In order to minimize cost but still obtain statistically significant information for comparison between groups, six animals from each group were chosen for histochemical labeling of the vagal nerves. Because of technical difficulties in the perfusion and fixation of the brain stems, not all microscopic sections were suitable for review. Results are based on those sections that could be evaluated (Table 2). At the time of necropsy, the gastric and duodenal mucosa was visually inspected and graded according to the arbitrary, prospective ulcer scale described above (Table 3). As determined previously,3 a pentagastrin dose 20 times (120 ~g/kg) the normal physiologic dose (6 ~g/kg) injected sub-cutaneously 3 days before necropsy stimulated mucosal erythema and ulceration in the pre-pyloric region of the stomach in the major318
Group
N
Sham OpPGV BZC CoCl 2 Phenol A&P APTV
6 6 4 3 4 1 4
Labelled dorsal vagal neurons 1060 76 734 684 335
± ± ± ± ±
536 103t 201 *,:j:,§ 100*,§ 267t
8t 79 ± 102t
Sham, Sham operative control; Op PGV, operative proximal gastric vagotomy; BZC, trans-mucosal injection of 0.1 % benzalkonium chloride; COCi2, trans-mucosal injection of 0.75% cobaltous chloride; Phenol, trans-mucosal injection of 5% phenol; A&P, anterior and posterior seromyotomy performed by CO 2 laser blanching of the anterior and posterior gastric serosal surfaces; APTV, anterior seromyotomy performed by CO 2 laser blanching of the anterior gastric serosal surface and posterior truncal vagotomy. *Significant at a 95% confidence level when compared with the Op PGV group. tSignificant at a 95% confidence level when compared with the Sham group. :j:Significant at a 95% confidence level when compared with the A&P C02 laser group. §Significant at a 95% confidence level when compared with the APT V C02 laser group.
ity of Sham animals. Significant ulcer prophylaxis was observed in all the PGV groups when compared with the Sham control group. However, the ulcer prophylaxis score in the Phenol chemoneurolytic group was significantly poorer than in the Op PGV group. As has been previously described,1-3 a Congo red staining technique was used to quantitate the acidsecretory ability of the gastric mucosa. The area of mucosal pH decrease was significantly diminished in the BZC, A&P, and APTV groups when compared with that in the Sham controls (Table 3). The physical intactness of the vagal-gastric connections was evaluated by retrograde HRP staining techniques. 3,4 As would be predicted, the mean number of neurons of the dorsal vagal nucleus stained was greatest in the Sham group (Table 2). Significantly reduced neuronal staining was observed in the Op PGV, Phenol, A&P, and APTV groups. The BZC and CoCh chemoneurolytic groups showed intermediate numbers of stained neurons, which were significantly greater than those of the "mechanical" PG V (Op PG V, A&P, and APTV) groups. DISCUSSION
With the recent expansion of indications for operative laparoscopic surgery, parietal cell vagotomy is now being evaluated as a possible routinely practiced procedure. 5-? In attempts to determine the completeness of parietal cell vagotomies, we have previously utilized intra-operative Congo red staining techniques in a GASTROINTESTINAL ENDOSCOPY
porcine model 1 and in humans. 8 In rat 2, 3 and porcine 1 studies, we have demonstrated chemoneurolytic injection PGV techniques to be equivalent to operative laparoscopic and CO 2 laser laparoscopic PGV techniques on a short-term basis. However, the long-term effectiveness of those various chemoneurolytic techniques has never been documented in rats, pigs, or humans. Throughout this 9-month study, no gross adverse outcomes could be attributed to any of the PGV techniques. The consistent weight gain in all groups confirmed that long-term side effects of these alternative PGV techniques were minimal (Table 1). Animals undergoing the mechanical procedures (Op PGV, A&P, and APTV) demonstrated less of an average weight gain per week overall than the chemoneurolytic groups, which may reflect the increased stomach distension and presumably decreased gastric emptying (gastroparesis) that has been observed previously in these groups.3 As we demonstrated earlier in short-term rat studies,3 histamine-like stimulation of duodenal ulcers with cysteamine (2-amino-ethanethiol hydrochloride)9 was not prevented by any PGV techniques. In these same studies, we demonstrated that a pharmacologic dose of pentagastrin (120 ~g/kg) caused reproducible pre-pyloric gastric mucosal irritation and ulceration in control animals but was inhibited by PGV techniques. Thus, we concluded that because pentagastrin stimulates the gastrin receptor on the parietal cell, which shares a common parietal intra-cellular biochemical pathway with the vagal (acetylcholine) stimulus,lO the ulcerogenic stimulation of a pharmacologic dose of pentagastrin can be reproducibly inhibited by vagotomy in the short-term rat model. In this present study, we confirmed the long-term effectiveness of ulcer prophylaxis by several PGV techniques (Op PGV, chemoneurolytic, and laser) (Table 3). In this long-term study, the reduction in the pentagastrin-stimulated acid-secretion area of the gastric mucosa was quantitated using a modified Congo red test, as previously described for the short-term rat mode1. 2,3 A significant decrease in acid secretion was demonstrated in the laser PGV groups (A&P and APTV) and in the benzalkonium chemoneurolytic group (BZC) (Table 3). Although a significant difference in acid secretion from control was demonstrated for these groups, the effect of all PGV techniques collectively for acid reduction was not as impressive as that seen in the short-term rat studies. 2, 3 In the longterm setting, the variability in innervation may reflect incompleteness or non-permanence of denervation by the various PGV techniques, re-innervation phenomenon, tachyphylaxis of the treated parietal cells, or some other mechanism. It is important to note that the greatest reduction occurred in the A&P group. This VOLUME 40, NO.3, 1994
Table 3. Results of alternative PGV techniques for ulcer prophylaxis and inhibition of acid secretion Group
N
Ulcer score
Sham OpPGV BZC CoCl 2 Phenol A&P APTV
7 7 10 8 8 4 8
2.1 0.3 0.7 1.0 1.1 0.5 0.5
± ± ± ± ± ± ±
1.2 0.5t 0.5t 0.8t 0.6*,t 0.6t 0.5t
Acid-secretion area by Congo red test (%)
17.5 11.0 8.8 15.1 14.5 2.7 7.4
± ± ± ± ± ± ±
8.6 6.2 6.5t 10.0t 10.0t 2.2t 5.1 t
Sham, Sham operative control; Op PGV, operative proximal gastric vagotomy; BZC, trans-mucosal injection of 0.1 % benzalkonium chloride; CoCI2, trans-mucosal injection of 0.75% cobaltous chloride; Phenol, trans-mucosal injection of 5% phenol; A&P, anterior and posterior seromyotomy performed by C02 laser blanching of the anterior and posterior gastric serosal surfaces; APTV, anterior seromyotomy performed by CO 2 laser blanching of the anterior gastric serosal surface and posterior truncal vagotomy. *Significant at a 95% confidence level when compared with the Op PGV group. tSignificant at a 95% confidence level when compared with the Sham group. :j:Significant at a 95% confidence level when compared with the A&P CO 2 laser group.
was the one group in which both the anterior and posterior serosal surfaces of the stomach were subjected to a significant char, and it may be the group in which the greatest permanent cellular injury or denervation was obtained. Long-lasting anatomic vagal disconnection was confirmed for those PGV techniques employing mechanical methods of vagal denervation (Op PGV, A&P, APTV) (Table 2). Although HRP staining was diminished with the chemoneurolytic techniques, only the Phenol group demonstrated significantly reduced neuronal staining when compared with the Sham group. This may reflect the non-specific cellular injury (chemical burn) of phenol, which results in mechanical vagal disconnection. Although diminished neuronal staining was observed in the other two chemoneurolytic groups (BZC and CoCI2), it was not significantly different from that in the Sham group, suggesting that these techniques produce either quantitatively less mechanical vagal disconnection or more selective denervation or parietal cell inhibition than do the other PGV techniques studied. What can be concluded from these initial long-term rat PGV studies? None of the PGV methods seems to inhibit normal animal growth, and all are significantly effective, to various degrees, for ulcer prophylaxis and inhibition of acid secretion. Chemoneurolytic techniques may promote ulcer prophylaxis at a different site along the vagal-parietal cell axis, than do the mechanical methods (Op PGV, A&P, APTV), and their effects may be more specific and selective or limited 319
and transient. Studies regarding techniques of administration, solution concentrations, and drug dosage are required to perfect current methods of chemoneurolytic suppression of gastric acid secretion. Further investigation of the exact mechanism of action of the chemoneurolytic agents with regards to the vagal-parietal cell axis and at the cellular level seem indicated. ACKNOWLEDGMENT
We wish to thank the Department of Veterans Affairs for its continued support of this project. We also wish to thank Thomas Curry, Animal Care Technician, St. Louis University.
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