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.