Histopathology
The general condition of each mammary tissue of mice was assessed by clinical scoring. The clinical score ranked from 1 to 5, with higher scores corresponding to greater degrees of tissue damage. Specifically, 1 indicated no damage, 2 indicated slight redness, 3 indicated redness and slight bleeding, 4 indicated redness and bleeding, and 5 indicated redness and obvious bleeding.
The mammary tissues were fixed in 4% cell tissue fixative (Solarbio Life Science, Beijing, China) immediately after the mice were sacrificed. Tissues were embedded in paraffin and sliced into 5-μm slices. To evaluate histological changes, tissues were stained with hematoxylin-eosin and observed under a microscope. The semi-quantitative scoring was performed using a histological score based on the following criteria. The histological score ranged from 1 to 5, and higher scores corresponded to greater degrees of tissue damage. Specifically, 1 indicated the absence of histological features (e.g., necrosis, neutrophils, and lymphocytes), 2 indicated minimal histological features (i.e., individual neutrophils), 3 indicated mild histological features (i.e., a small amount of neutrophils), 4 indicated moderate histological characteristics (i.e., many neutrophils and slight damage to glandular structure), and 5 indicated severe histological characteristics (i.e., a large number of neutrophils and severe damage to glandular structure). Both clinical and histological scoring was made by experienced veterinary pathologists who were blinded to treatments.