Vagal Sparing Surgical Technique but Not Stoma Size Affects Body Weight Loss in Rodent Model of Gastric Bypass
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- Bueter, M., Löwenstein, C., Ashrafian, H. et al. OBES SURG (2010) 20: 616. doi:10.1007/s11695-010-0075-5
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The aim of this study was to evaluate whether gastric bypass with or without vagal preservation resulted in a different outcome.
Body weight, food intake and postprandial peptide YY (PYY) and glucagon-like peptide (GLP-1) levels were compared between gastric bypass (n = 55) and sham-operated rats (n = 27) in three groups. In group 1 (n = 17), the vagal nerve was not preserved, while in group 2 the vagal nerve was preserved during gastric bypass (n = 10). In group 3, gastric bypass rats (n = 28) were randomised for either one of the two techniques.
Rats in which the vagal nerve was preserved during gastric bypass showed a lower body weight (p < 0.001) and reduced food intake (p < 0.001) compared to rats in which the vagal nerve was not preserved during the gastric bypass operation. Levels of PYY and GLP-1 were significantly increased after gastric bypass compared to sham-operated controls (p < 0.05), but there was no difference between gastric bypass rats with and without vagal preservation. Differences in food intake and body weight were not related to the size of the gastro-jejunostomy in gastric bypass rats. There were no signs of malabsorption or inflammation after gastric bypass.
We propose that the vagal nerve should be preserved during the gastric bypass operation as this might play an important role for the mechanisms that induce weight loss and reduce food intake in rats. In contrast, the gastro-jejunal stoma size was found to be of minor relevance.
KeywordsGastric bypassRatsPara-oesophageal bundleVagal nerveLeft gastric vesselsWeight loss
Bariatric surgery has been proven to be the most effective treatment for severe obesity and its inherent co-morbidities resulting in significant and sustained weight loss with a proven mortality benefit [1, 2]. At present, the Roux-en-Y gastric bypass procedure (gastric bypass) provides reliable and sustainable weight loss. Given the rapid increase in gastric bypass procedures, it is important to understand the underlying mechanisms by which gastric bypass induces and sustains weight loss [3, 4]. The use of animal models for gastric bypass surgery is a valuable tool and has been shown to be a valid model to mimic human weight loss after gastric bypass [5–7]. However, there is significant variation in techniques used in humans and rodent models. The results for weight loss, food intake and mortality rates are heterogeneous [5–15].
The vagal nerve is thought to have an important role in the regulation of food intake and body weight, but only a few reports examined whether vagal preservation is effective or necessary in weight control after bariatric surgery [15–17]. Gut hormones released from enteroendocrine cells in the distal ileum like glucagon-like peptide (GLP-1) and peptide YY (PYY) can signal either through the circulation or via afferent vagal fibres .
In this study, we describe variations in the technique for gastric bypass surgery in rats in the area of the gastro-jejunostomy. Here, the para-oesophageal bundle can be found which contains the left gastric vessels and the dorsal vagal trunk that contains 4/5 of the right vagal fibres and a 1/5 of the left vagal fibres . The aim of our study was to assess whether preservation of the vagal fibres in the para-oesophageal bundle impacts on body weight and food intake after gastric bypass in rats.
Material and Methods
Male Wistar rats used were individually housed under a 12/12 h light–dark cycle and at a room temperature of 21 ± 2°C. Water and standard chow were available ad libitum, unless otherwise stated. All experiments were performed under a licence issued by the Home Office UK (PL 70-5569) or approved by the Veterinary Office of the Canton Zurich, Switzerland. Body weight and food intake were measured daily in groups 1 and 2 for a postoperative period of 60 days and in group 3 for 75 days.
All operations reported in this study were performed by one surgeon (MB). After 1 week of acclimatisation, rats were randomised to gastric bypass or sham operation. Rats were food deprived for 12 h overnight, but water was available ad libitum. Before surgery, rats were weighed, and then anaesthetised with isofluorane (4% for induction, 3% for maintenance). Preoperatively, gentamicin 8 mg/kg and carprofen 0.01 ml were administered intraperitoneally (ip) as prophylaxis for postoperative infection and pain relief. Surgery was performed on a heating pad to avoid decrease of body temperature during the procedure. Prior to a midline laparotomy, the abdomen was shaved and disinfected with surgical scrub. In the sham group, a 7 mm gastrotomy on the anterior wall of the stomach with subsequent closure (interrupted prolene 5-0 sutures) and a 7 mm jejunotomy with subsequent closure (running prolene 6-0 suture) was performed. In the gastric bypass group, the proximal jejunum was divided 15 cm distal to the pylorus to create a biliopancreatic limb. After identification of the caecum, the ileum was then followed proximally to create a common channel of 25 cm. Here, a 7 mm side-to-side jejuno-jejunostomy (running prolene 7-0 suture) between the biliopancreatic limb and the common channel was performed.
The two techniques described below in this paper relate to how the stomach was transected close to the gastro-oesophageal junction to create a small gastric pouch with no more than 3 mm of gastric mucosa left. The gastric pouch and alimentary limb was anastomosed end-to-side using a running prolene 7-0 suture. The gastric remnant was closed with interrupted prolene 5-0 sutures. The complete bypass procedure lasted approximately 60 min and the abdominal wall was closed in layers using 4-0 and 5-0 prolene sutures. Approximately 20 min before the anticipated end of general anaesthesia, all rats were injected with 0.1 ml of 0.3% buprenorphine subcutaneously to minimise postoperative discomfort. Immediately after abdominal closure, all rats were injected subcutaneously with 5 ml of normal saline to compensate for intraoperative fluid loss. After 24 h of wet diet (normal chow soaked in tap water), regular chow was offered on postoperative day 2.
The vagal fibres in the para-oesophageal bundle in the area of the gastric pouch were subjected to two different techniques. All groups were operated in chronological order. In a first group, 25 obese rats (body weight (BW) 348 ± 3.9 g) were randomised for gastric bypass (n = 17) or sham operation (n = 8). In this group the vagal fibres were not preserved in the gastric bypass rats as the para-oesophageal neurovascular bundle was completely ligated (group 1). In a subsequent group, 18 obese rats (332 ± 2.4 g) were randomised to gastric bypass (n = 10) or sham operation (n = 8). Here, the vagal fibres were preserved as the left gastric vessels were separated and selectively ligated in all gastric bypass rats (group 2). Significant differences in body weight and energy intake were observed in these two groups. As it was unclear whether these differences were related to the different techniques of vagal preservation, a third group (group 3) of 39 obese rats (471 ± 4.3 g) was randomised for gastric bypass without vagal preservation (n = 14) or gastric bypass with vagal preservation (n = 14) or sham operation (n = 11).
Animals from group 3 were fasted for 12 h from the beginning of the light cycle. At the onset of the dark cycle animals were offered 5 g of standard chow all of which was consumed within half an hour by the animals. Blood was then obtained by puncture of a sublingual vein under brief isoflurane anaesthesia from sham-operated controls, gastric bypass with and without vagal preservation (each n = 6). Blood was collected into EDTA-rinsed tubes and, immediately centrifuged at 3,000 rpm for 10 min at 4°C. The supernatant was stored at −80°C until further analysis. Concentrations of active GLP-1 and PYY were analysed using a rat endocrine lincoplex kit (RENDO-85 K, Labodia SA, Yens, Switzerland).
Measurement of Size of the Gastro-Jejunostomy
To exclude that the differences in body weight between bypass rats were due to different levels of restriction and subsequent differences in food intake, sizes of the gastro-jejunostomy were measured during necropsy in all gastric bypass rats of group 3.
Blood was obtained from all animals of group 3 by puncture of a sublingual vein under brief isoflurane anaesthesia. Blood was collected into EDTA-rinsed tubes and immediately centrifuged at 3,000 rpm for 10 min at 4°C. Plasma was stored at −80°C before analysis for C-reactive protein (Abbott, UK) to assess inflammation.
To evaluate nutrient malabsorption, faeces were collected over 24 h on postoperative days 15 and 59 from all animals in group 3. Faeces were dried in an oven and weighed; calorie content was measured using a ballistic bomb calorimeter .
All data were normally distributed and are expressed as mean ± SEM. Student’s t test for independent samples and one-way ANOVA with repeated measures and post-hoc Bonferroni test for each time point were used to test for significant differences. p < 0.05 was considered significant.
Overall surgical mortality was 13.4% (11/82). Gastric bypass-related mortality was 14.5% (8/55), while mortality after sham operation was 11.1% (3/27, p = 0.668). There was no mortality difference between bypass rats with complete ligation and with preservation of the para-oesophageal bundle. All eight bypass rats showed signs of respiratory distress along with hypersalivation and dysphagia within the first two postoperative days after the operation and were euthanized immediately after onset of symptoms. Necropsy revealed that these symptoms originated at the level of the gastro-jejunostomy where food did not pass through and was retained in the oesophagus. Whether this was due to inflammatory swelling following anastomotic leakage or due to anastomotic constriction remains unclear. The three sham-operated rats died without prior noticeable symptoms. Necropsy revealed in two cases a small bowel ileus presumably due to a volvulus after inappropriate repositioning of the viscera into the abdominal cavity at the end of the operation. In one case, a leak at the site of the gastrotomy was found.
Postprandial Plasma Levels of PYY and Active GLP-1
Size of the Gastro-Jejunostomy
There was no gastrogastric fistula in any of the gastric bypass rats of group 3. The overall size of the gastro-jejunostomy in all gastric bypass rats was 15.4 ± 0.4 mm. There was no difference in size of the anastomosis between rats in which the complete para-oesophageal bundle was ligated and rats in which the left gastric vessels were separated and selectively ligated (bypass with selective ligation, 15.2 ± 0.4 mm vs. 15.6 ± 0.7 mm, p = 0.69).
C-reactive protein levels were below 2 mg/L in all animals of group 3 indicating that there was no postsurgical infection or inflammation 28 days after surgery.
There was no increase in either fresh faecal mass (sham, 8.4 ± 0.5 g vs. bypass with vagal preservation, 7.5 ± 0.6 g vs. bypass without vagal preservation, 7.2 ± 0.6 g, p = 0.3) or faecal calorie content (sham, 3.56 ± 0.04 kcal vs. bypass with vagal preservation, 3.43 ± 0.05 kcal vs. bypass without vagal preservation, 3.65 ± 0.06 kcal, p = 0.24) in the gastric bypass animals compared to the sham-operated rats in group 3.
Our data in the rat model for gastric bypass are consistent with previous findings that gastric bypass surgery can effectively induce food intake and body weight reduction [1, 21, 22]. In this randomised study, the weight loss and food intake outcome of gastric bypass surgery was dependent on whether vagal fibres were preserved or not during the formation of the gastric pouch. Rats in which the para-oesophageal bundle including the vagal fibres was completely ligated started to regain body weight up to preoperative levels and showed no difference in average daily energy intake compared to their sham-operated counterparts. In contrast, rats in which the para-oesophageal bundle including the vagal fibres was preserved and in which the left gastric vessels were selectively ligated, maintained the reduced body weight and ate significantly less than the sham-operated controls throughout the entire study period. Gastric bypass rats had higher postprandial GLP-1 and PYY levels compared to sham-operated controls, but there were no differences in GLP-1 and PYY levels between gastric bypass rats with our without preserved vagal fibres. Furthermore, differences in food intake and body weight were not related to the size of the gastro-jejunostomy in gastric bypass rats and there were no signs of malabsorption or inflammation after gastric bypass in any of the groups.
Our data confirm previous findings that gastric bypass in rats increases postprandial levels of peptide YY and glucagon-like peptide-1, which are satiation-inducing gut hormones and hence favour an anorectic state and facilitate body weight loss [5, 21]. Both hormones are thought to activate anorectic neurons in the hypothalamic arcuate nucleus which promote weight loss [23–26]. Gut hormones released from enteroendocrine cells in the distal ileum like GLP-1 and PYY can signal either through the circulation or via afferent vagal neurons .
In this study body weight and food intake after gastric bypass were related to whether the vagal fibres within the para-oesophageal bundle were preserved or not, while there were no differences in levels of GLP-1 and PYY between these two groups. This finding highlights the potential role of the vagal nerve for mediating the inhibitory effects of gut hormones such as PYY and GLP-1 on food intake and body weight after gastric bypass surgery in rats. Our findings are consistent with previous reports showing that the ablation of the vagal–brainstem–hypothalamic pathway attenuates the inhibitory effects of PYY and GLP-1 on food intake . Vagal preservation may thus be necessary for optimum weight loss after bariatric surgery [15–17].
In contrast to our observation, Wang and Liu  described greater weight loss after gastric bypass and total vagotomy in rats. The difference was only present at 20 days after surgery, but the difference in food intake and body weight between bypass rats with or without vagal dissection was lost thereafter. Another difference to our study is that Wang and Liu  used the bypass operation to prevent obesity in rats weighing 180–200 g while we performed surgery to cause weight loss in obese rats.
Weight loss after a gastric bypass operation might also be due to nutrient malabsorption or postoperative inflammation. However, we found no evidence for an increase in either faecal mass or faecal calorie content in the gastric bypass animals with or without vagal preservation. Moreover, we did not detect any evidence of increased inflammation in animals with or without vagal ligation post-surgery.
The size of the gastric pouch and the lengths of the different limbs used in this study have been proven to effectively induce weight loss . An increasing body of evidence in humans indicates that up to certain limits the size of the gastric pouch and length of the different limbs is of less importance for the outcome of gastric bypass . In support of this observation, we demonstrated that the level of restriction measured by the size of the gastro-jejunostomy has no impact on different levels of weight loss and food intake after gastric bypass in rats.
There are two major limitations of our study. Firstly, we cannot exclude the possibility that the ligation of the para-oesophageal bundle is functionally equivalent only to a partial dissection of the vagal nerve. In addition, we did not perform a secretin test or histological analysis to collect further informations on vagal function to confirm whether the complete ligation of the para-oesophageal bundle produced a total or partial vagotomy. Secondly, it remains unclear whether our results can be translated into humans. Most bariatric surgeons usually aim to preserve the anterior and posterior vagal trunk during formation of the gastric pouch, although there is a lack of supporting data indicating that this approach has beneficial effects. Recently, the dissection of the anterior vagal trunk during pouch formation has been reported to have no effect on clinical, functional and laboratory results of a gastric bypass operation .
In conclusion, our gastric bypass technique induces reliable weight loss in rats with an acceptable mortality. We propose that vagal nerve fibres should be preserved during gastric bypass in rats. Restriction at the gastro-jejunal anastomosis does not seem to be critical for the weight loss. Although the mechanisms have not yet been fully elucidated, vagal preservation may play an important role in inducing and maintaining weight loss after gastric bypass in humans and rats.
M. B. was supported by the Deutsche Forschungsgemeinschaft (DFG). T.L. and C.L. were supported by the Swiss National Research Foundation. S.B. and C.le R. were supported by a Department of Health Clinician scientist award. Imperial College London receives support from the NIHR Biomedical Research Centre funding scheme.
The authors have no conflict of interest to disclose.