The histological evaluation is presented in Fig. 4. After 16 weeks, hepatic steatosis was present in NASH, HFD and HFr-fed rats as confirmed by corresponding Oil Red O stains. No pathological changes were observed in livers of Control-fed rats. Steatosis was phenotypically distinguishable between NASH-, HFD- and HFr-fed rats. While zonal distribution was comparable (originating in zone 1, periportally), steatosis in HFD-rats was almost exclusively found to be of the microvesicular type; in HFr-fed rats almost exclusively macrovesicular; while the NASH-fed rats represented an intermediate between the two, with both macro- and microvesicular steatosis. Furthermore, steatosis in the NASH-group at week 16 was much more pronounced, involving not only zone 1, but occasionally also zones 2 and 3 and in some animals, only few areas displayed normal liver architecture. Inflammatory hepatic infiltration was identified in both NASH-, HFD- and HFr-fed rats. In all three groups, inflammation was characterized by being of primarily mononuclear composition with occasional neutrophilic involvement. However in HFr-fed rats, inflammatory cells appeared to center around lipid-loaded hepatocytes and form structures resembling lipogranulomas, whereas inflammatory infiltrates observed in HFD-fed rats were randomly scattered and not consistently associated with steatosis. As with the steatosis, inflammatory changes in the NASH-group were more pronounced, as exemplified by larger and more disseminated infiltrates. Accordingly, hepatic macrophage numbers were two- to threefold increased in the NASH-group compared to Control- and HFD-fed rats (p < 0.001 and p < 0.01, Fig. 5a, b). Hepatic deposition levels of collagen was higher in NASH-animals than in other groups, albeit only significantly increased compared to HFD (p < 0.001, Fig. 5c, d).Fig. 4Histological evaluation of liver sections from Control-, NASH-, HFD- and HFr-fed rats. Row 1: representative H&E-stains of normal liver from a Control-fed rat, and hepatic steatosis in b NASH-fed rat, c HFD-fed rat and d HFr-fed rat. Hepatic steatosis in HFD-fed rats was almost exclusively found to be microvesicular (c); in HFr-fed rats almost exclusively macrovesicular (d); while steatosis in NASH-fed rats represented an intermediate between the two, with both macro- and microvesicular steatosis (b). Row 2: higher magnification of representative H&E stained liver sections. e Liver morphology appeared normal in Control-fed rats, whereas inflammatory infiltrates were observed in livers from f NASH-fed rats, g HFD-fed rats and h HFr-fed rats Row 3: Oil Red O stains of liver sections from j NASH-fed rats, k HFD-fed rats and l HFr-fed rats confirmed hepatic steatosis observed in b–d. i Oil Red O staining of liver from Control-fed rat Fig. 5Macrophage infiltration and collagen deposition. A significant increase in macrophage infiltration was seen in NASH-fed rats, compared to Control- and HFD.fed rats (a) and collagen deposition in NASH-fed rats trended towards being increased, although only significantly when compared to HFD-rats (b). Representative CD68- (c) and Picro Weigert-stains (d) of liver from NASH-fed rat. Statistical significance: **p < 0.01; ***p < 0.001. Results in a and c are shown as mean ± SEM