When Timing Matters: Effects of Maternal Separation and Post-Weaning High-Fat Diet on Liver Morphology in a Rodent Model

Abstract

Background: Early-life stress and dietary habits are key determinants of metabolic health. This study investigates the combined effects of maternal separation (MS) and a post-weaning high-fat diet (HFD) on liver morphology in male C57BL/6 mice. Methods: Male mice were subjected to MS during early postnatal life or kept unmanipulated (UM). After weaning, animals were assigned to either a control diet (CD) or an HFD, forming four groups: UM-CD, UM-HFD, MS-CD, and MS-HFD. Liver histology, collagen deposition, and both morphometric and stereological parameters were assessed following 16 weeks of dietary intervention. Results: MS and HFD independently altered liver structure, while the combination of both factors intensified these changes. The MS-HFD group exhibited pronounced steatosis, mixed inflammatory infiltrates, and hepatocellular ballooning, with a significantly higher NAFLD Activity Score (NAS). No significant differences were observed in liver fibrosis. Morphometric analysis revealed increased body mass in HFD-fed groups and elevated liver mass in MS-HFD. Liver volume was higher in MS-HFD, though not significantly. Liver stereology revealed reduced numerical density of hepatocytes (Nv_hep) and increased surface density (Sv_hep) in MS groups, with the most pronounced effects in MS-HFD. Conclusions: Maternal separation amplifies the hepatic alterations induced by HFD, promoting early inflammatory and steatotic changes. These findings highlight the significance of early-life stress as a factor increasing susceptibility to diet-induced liver damage.

Keywords: early-life stress; high-fat diet; liver morphology; maternal separation.

Figure 1

Schematic representation of the experimental design. PND: postnatal day, UM: unmanipulated, MS: maternal separation, CD: control diet, HFD: high-fat diet, UM-CD: unmanipulated group with control diet, UM-HFD: unmanipulated group with a high-fat diet, MS-CD: maternal separation group with control diet, MS-HFD: maternal separation group with a high-fat diet.

Figure 2

Histology of livers of C57BL/6 mice subjected to maternal separation and a post-weaning high-fat diet. UM-CD: unmanipulated group with control diet (A–D), UM-HFD: unmanipulated group with a high-fat diet (E–H), MS-CD: maternal separation group with control diet (I–L), MS-HFD: maternal separation group with a high-fat diet (M–P). CV: central vein, PA: portal area, IV: interlobular vein, IBD: interlobular bile duct, H: hepatocyte, S: sinusoid, red arrow: macrovesicular hepatic steatosis, yellow arrow: microvesicular hepatic steatosis, white arrowhead: lymphocyte-predominant leukocytes, black arrowhead: leukocytes with a predominance of neutrophils, SM: stellate macrophage, MN: macronucleus, NG: neutrophilic granule, AB: acidophilic body, LV: lipid vacuoles, HB1: grade 1 hepatocellular ballooning, HB2: grade 2 hepatocellular ballooning. H&E staining.