Remodeling of the residual gastric mucosa after roux-en-y gastric bypass or vertical sleeve gastrectomy in diet-induced obese rats

Abstract

Whereas the remodeling of intestinal mucosa after bariatric surgeries has been the matter of numerous studies to our knowledge, very few reported on the remodeling of the residual gastric mucosa. In this study, we analyzed remodeling of gastric mucosa after Roux-en-Y gastric bypass (RYGB) and vertical sleeve gastrectomy (VSG) in rats. Diet-induced obese rats were subjected to RYGB, VSG or sham surgical procedures. All animals were assessed for food intake, body-weight, fasting blood, metabolites and hormones profiling, as well as insulin and glucose tolerance tests before and up to 5 weeks post-surgery. Remodeling of gastric tissues was analyzed by routine histology and immunohistochemistry studies, and qRT-PCR analyses of ghrelin and gastrin mRNA levels. In obese rats with impaired glucose tolerance, VSG and RYGB caused substantial weight loss and rats greatly improved their oral glucose tolerance. The remaining gastric mucosa after VSG and gastric pouch (GP) after RYGB revealed a hyperplasia of the mucous neck cells that displayed a strong immunoreactivity for parietal cell H+/K+-ATPase. Ghrelin mRNA levels were reduced by 2-fold in remaining fundic mucosa after VSG and 10-fold in GP after RYGB. In the antrum, gastrin mRNA levels were reduced after VSG in line with the reduced number of gastrin positive cells. This study reports novel and important observations dealing with the remaining gastric mucosa after RYGB and VSG. The data demonstrate, for the first time, a hyperplasia of the mucous neck cells, a transit cell population of the stomach bearing differentiating capacities into zymogenic and peptic cells.

Fig 1. Surgical procedures of Vertical Sleeve Gastrectomy (VSG) and Roux-en-Y Gastric Bypass (RYGB) in rats.

(A) Photography and (B) contrast radiography of a rat stomach showing forestomach (rumen), fundus and antrum. In both surgeries, first step is the resection of the rumen (C). In VSG: vertical resection of the fundus (D, E) resulted in a reduction of the gastric volume visible by opacification with gastrograffine (F). In RYGB: reconstructed gastric pouch directly anastomosed to the jejunum (G) and verification of the gastro-jejunal anastomosis (alimentary limb) after opacification (H). In RYGB: jejuno-jejunal anastomosis limb (I) and verification of the gastro-jejunal anastomosis after opacification (J). (K) Example of a fistula, the main cause of the post-operative mortality. Photography of the necropsy of one rat showing a fistula (star) in the remaining stomach. Insert: radiography imaging after opacification with gastrograffine of the gastrointestinal tract showing a fistula (star).

Fig 2. Time-course of body weight after VSG and RYGB in HFD obese rats.

(A) VSG- and (B) RYGB-induced weight loss in HFD obese rats and in the corresponding sham-operated rats. Black boxes correspond to the period of post-operative care and liquid diet consumption before the animals had free access to solid ND. Results are expressed as percent of loss of body weight over preoperative weight. Two-Way ANOVA was used to compare body-weight curves.

Fig 3. RYGB and VSG improve oral glucose tolerance.

(A-B) Fasting glucose levels of HFD rats before (HFD Preop) and 5 weeks after VSG (A) or RYGB (B) in comparison to ND fed rats (ND). (C) Comparative time-dependent blood glucose levels after an oral load of 1g/kg BW glucose and (D) the corresponding calculated area under curves (AUC) in HFD-fed rats before (HFD Preop) and 5 weeks after VSG or RYGB (n = 12 for HFD, n = 8 for VSG or RYGB). ** P<0.01 and ***P<0.001 vs. HFD Preop. (E-H) Plasma levels of metabolic hormones in HFD-fed rats before surgery (pre-operative) and 5 weeks after VSG or RYGB surgery (E) Glucagon, (F) insulin, (G) leptin and, (H) C-peptide levels. n = 12 pre-operative, n = 6 VSG and n = 6 RYGB.* P<0.05; ** P<0.01 vs. HFD Preop: NS: Not significantly different between RYGB and VSG.

Fig 4. Histology of gastric fundic mucosa in sham-, VSG- and RYGB- operated rats.

Panel A. Post-mortem representative macroscopic photomicrographs of stomach of sham-, VSG- or RYGB-operated rats. (top) normal stomach showing fundus (1) and antrum (2); (middle) residual stomach after VSG with fundus (1), antrum (2), duodenum (3) and, pylorus (arrow); (bottom) excluded stomach in RYGB and gastric pouch (1) directly anastomosed to the jejunum (4). Panel B: representative Hematoxylin and Eosin staining (H&E) of fundic mucosa. Note the hyperplasia of mucous neck cells (black arrow heads) after VSG and RYGB. Panel C: fundic mucosa sections stained with periodic acid Schiff (PAS)/Alcian blue (AB). Mucous neck cells are PAS/AB positive (black arrows). Panel D: immunostaining of Ki67-proliferating cells (brown nuclei) in formalin-fixed fundic mucosa section from sham (top), remaining stomach after VSG (middle), and GP after RYGB (bottom).

Fig 5. Immunostaining of parietal cell H⁺/K⁺-ATPase in fundic mucosa.

(A) Overview of a representative immunostaining of parietal cell H+/K⁺-ATPase along the formalin-fixed gastric pouch anastomosed to jejunum in the alimentary limb after RYGB. Comparative immunostaining of parietal cell H+/K⁺-ATPase in fundic mucosa from sham (B), residual stomach after VSG (C), and GP after RYGB (D). Note the enlarge parietal cells (blue stars) with dense H⁺/K⁺ATPase immunoreactivity as compared to sham. (E) Percent of H⁺/K⁺-ATPase positive cells per fundic mucosa area (μm²). Values are shown as mean ± SEM n = 5 for sham and n = 6 for VSG and RYGB. **P<0.01 and ***P<0.001 vs. sham.

Fig 6. Levels of ghrelin and gastrin mRNA in remodeled stomach after VSG and RYGB.

(A) Insert: scheme with location of fundus and antrum in normal stomach, residual stomach after VSG and gastric pouch (GP) or excluded stomach after RYGB. (A) Quantification of ghrelin mRNA in mucosa scrapings of residual fundic mucosa after VSG, fundic mucosa in gastric pouch (GP) after RYGB and antral mucosa of the excluded stomach after RYGB in HFD-obese operated rats in comparison to fundic and antral mucosa from sham-operated ND or HFD rats. (B) Quantification of gastrin mRNA in mucosa scrapping from antrum of sham-operated ND and HFD rats, antrum of residual stomach after VSG, and antrum of excluded stomach after RYGB. L19 was used as reference. n = 5 rats per group. (C) Representative photomicrographs of immunfluorescence of gastrin-positive cells in antral mucosa of sham- and VSG-operated HFD rats. (D) Comparative number of gastrin positive cells per antral mucosa area (μm²) showing significant decrease in G cell number. Values are means ± SEM for n = 4 in each group.