2.4. Liver Histology

Routine histology was used to produce tissue sections for histological analyses of the rat livers. For semi-quantitative assessment of histopathological alterations to the rat liver, we adopted and extended methods previously described [14].

In our proposed model, a semi-quantitative and quantitative approach was used. Pathological changes were classified into six reaction patterns: steatosis, glycogen storage, atrophy, apoptosis, necrosis, and nuclear alterations. Histopathological alterations of the liver tissue were given one of two assessment values: (1) focal appearance-minimal pathological importance (easily reversible lesions) (2) diffuse appearance-moderate pathological importance (reversible lesions if the stressor is neutralized), and in the case of steatosis, microvesicular (1) and macrovesicular (2). Secondly, in order to obtain a more detailed semi-quantitative analysis, we introduced a second score (s) for each alteration, ranging from 1–10, depending on the degree of histopathological alteration: 1–3 = control, 4–7 = moderate, 8–10 = severe. For example, a particular tissue could show severe diffuse apoptosis and, as such, be assigned as a = 2 and s = 9. Finally, these two values were multiplied to obtain score values (see equation below). The arithmetic mean of these products (mean scored value) represents the degree of damage to a liver tissue after feeding with the experimental diets, following the calculation principle. Nuclear alterations were calculated by assessing ten high-power microscopic fields (×400) in ten random and non-overlapping fields for each specimen.