Results of a comparative study on Non-alcoholic fatty liver induced and microstructural fibrotic changes

Abstract

Background: In recent years, the incidence of liver diseases due to complications of non-alcoholic fatty liver disease (NAFLD) has shown a significant upward trend in Southeast Asian countries. NAFLD is a hepatic disorder characterized by lipid accumulation in the microstructure of the liver in individuals who consume little to no alcohol. It is often associated with insulin resistance and is diagnosed when steatosis affects more than 5% of hepatocytes histologically, or when the fat signal intensity on MRI exceeds 5.6%, based on fat-to-water ratio measurements. In Mongolia, histological studies using frozen liver sections with routine and special staining techniques are limited, highlighting the necessity of this study. Aim: To determine and compare the degree of steatosis and fibrosis in frozen liver tissue samples of patients with NAFLD through histological analysis. Materials and Methods: This study was conducted at the the Department of Anatomy, School of Biomedicine and Biomedical Research Institute of MNUMS in collaboration with the Second State Central Hospital. Ethical approval was obtained from the Research Ethics Committee of MNUMS (Protocol No. 2024/3-06). All procedures adhered strictly to laboratory biosafety protocols. Participants were selected among patients undergoing elective laparoscopic cholecystectomy, from whom informed consent was obtained. Based on inclusion criteria, five participants were grouped as follows: healthy control (n=1), NAFLD without fibrosis (n=2), and NAFLD with fibrosis (n=2). Liver biopsies (approx. 1 cm in size) were obtained intraoperatively, immediately deep-frozen in liquid nitrogen, and prepared for histological evaluation. Results: In patients with NAFLD compared to the healthy liver group, disruption of hepatocyte columnar architecture and mild periportal lymphocytic infiltration were observed. Oil Red O staining revealed 34–66% microand macrovesicular steatosis, corresponding to grade 2 steatosis. Masson’s trichrome staining showed no fibrotic changes in perivenular or periportal areas (Ishak grade 0/4) at this stage. However, upon progression to grade 3 steatosis, early-stage fibrosis was observed in both perivenular and periportal regions (Ishak grade 1/4). Further progression to stage 4 fibrosis was characterized by the development of connective tissue septa, although no significant changes in droplet size were observed. Conclusions: 1. Increasing stages of fibrosis are not directly influenced by the severity of hepatic steatosis in NAFLD. 2. Although the degree of steatosis increases, the absence of corresponding fibrotic changes in early stages indicates a complex progression pattern of NAFLD requiring further investigation.