Histological grading evaluation of non-alcoholic fatty liver disease after bariatric surgery: a retrospective and longitudinal observational cohort study

Figure 1. Patients enrolled in the observational study. Flowchart showing the patients who were excluded from the study. Obese patients operated by the Department of Surgery of the University of Campinas from 2008 to 2019. The exclusion criteria were based in age (<18 e > 80 years-old), BMI less than 30 kg/m2 at the time of surgery, lack of information in medical records and pre-existing liver diseases. RYGB: Roux-en-Y Gastric Bypass. BMI: body mass index.

Figure 2. Histological evaluation of non-alcoholic fatty liver disease in the obese patients who underwent bariatric surgery – Retrospective cross-sectional analysis. Liver biopsies were performed during the bariatric surgery in the Clinical Hospital by the Surgery Department of the University of Campinasfrom 2008 to 2019. Number of patients included in the study = 895. (A) Classification of the NAFLD according to the histopathological changes. (B) Percentage of patients that were classified in each histological category: no liver damage, steatosis, steatohepatitis and cirrhosis. (C–F) Representative images of Masson’s Trichrome staining showing no histological change in the liver (C), hepatocytes displaying vacuoles related to triglyceride accumulation (macrogoticular steatosis) (D), hepatocellular ballooning (Mallory hyaline), macrogoticular steatosis and perisinusoidal fibrosis in acinar zone 3 (E), steatohepatitis with distortion of lobular architecture, collagen fibers highlighted in blue delineate hepatocytes forming septa that join portal structures and lobular center, in nodular transformation (cirrhosis) (F). 10× objective lenses were used as indicated in each image.

Figure 3. Triglycerides and cholesterol levels at the time of bariatric surgery (T1) and after (T2) the procedure. * p ≤ 0.05 T1 vs. T2. Mann Whitney test and Dunn’s multiple comparisons test.

Figure 4. Evaluation of non-alcoholic fatty liver disease at the time of (T1) and after (T2) bariatric surgery. Liver biopsies were performed during the bariatric surgery and also during the second abdominal procedure which the patients were submitted in their follow-up (median of 22 months between surgeries). (A) Classification of the NAFLD according to histopathological changes of patients before (T1) and after (T2) bariatric surgery (n = 30). (B) Percentage of T1 and T2 patients that were classified in no liver damage (No NAFLD), non-alcoholic steatosis (NAFL), non-alcoholic steatohepatitis (NASH) and cirrhosis according to histopathological changes. (C) Fibrosis staging of T1 and T2 patients according to histopathological changes.

Figure 5. Histopathological analysis of liver biopsies at the time (T1) and after (T2) bariatric surgery showed NAFLD regression after the procedure. A-C. Representative images of hematoxylin and eosin (H&E) and Masson’s Trichrome staining of the T1 and T2 groups. Collagen fibers stained by the Masson’s Trichrome are shown in blue. (A) NAFLD regression (*p = 0.0152, n = 7). (B) NAFLD maintainance (n = 2). (C) Development of liver disease after bariatric surgery (n = 3). 4×, 10× and 40× objective lenses were used as indicated in each image. (D–F) Quantitative analysis of Masson’s Trichrome staining in T1 and T2 of patients who presented NAFLD in T1 and/or T2.

Figure 6. Immunostaining of liver biopsies at the time (T1) and after (T2) bariatric surgery showed Kupffer cell infiltration and myofibroblast formation. Representative images of the immunohistochemical analysis of EGF-like molecule containing mucin-like hormone receptor 1 (EMR1) and alpha-smooth muscle actin (α-SMA) were performed on paraffin-embedded slides from the T1 and T2 groups. Positive cells for immunostaining are shown in brown. 4×, 10× and 40× objective lenses were used as indicated in each image.