The mice were then sacrificed. Blood was sampled from the abdominal aorta, centrifuged at 4?, and plasma was stored at 20? for assays; liver tissues were collected for histological examination. Alteration in animal weight The weight of each animal was measured just before the first drug administration and after sacrifice. The alteration in animal weight was calculated from these two values. Histological examination Hepatic tissues were fixed using 4 paraformaldehyde phosphate buffered saline and embedded in paraffin wax. After deparaffinization, 5 m sections were stained with hematoxylin and eosin , and liver condition was classified according to the standard formulae published by the China Medical Association in 1995. HBsAg HBcAg expression in tissue The surface antigen of the hepatitis B virus and hepatitis B core antigen were detected using immunohistochemical staining as previously described . The stained slides were examined microscopically. Quantification of HBsAg and HBcAg positive cells was performed over several different areas of each section.
The percentage of positive cells was evaluated by counting 100 cells in three consecutive tissue sections, and square scores were marked according to the following semiquantitative criteria: 1: 25 positive cells; 2: 26 50 positive inhibitor screening cells; 3: 51 75 positive cells; 4: 75 positive cells. The intensity of the HBsAg staining within positive cells was evaluated, and the intensity scores were marked according to the following semi quantitative criteria: 1: light yellow; 2: light brown; 3: chocolate brown. The final immunohistochemical reaction score was calculated according to the formula IRS positive staining scores intensity scores. HBsAg and HBeAg quantification HBsAg and HBeAg levels in serum were determined using Enzyme Linked Immunosorbent Assay according to the manufacturer?s instructions as previously described . HBV DNA concentration determination Blood was collected via the abdominal aorta after the mice were sacrificed. After centrifugation at 8000 r min for 5 min, the serum was separated and stored at 20?.
HBV DNA content in serum was determined using realtime PCR according to the manufacturer?s instructions. Statistical analysis JAK1 inhibitor Data are presented as mean SE. Data was checked for normal distribution and equal variance. One way ANOVA test analysis was carried out by SPSS 11.0 software. Differences were considered significant at P values of less than 0.05 One mouse in group A died due to improper blood collection during the experiment at the third week. The other mice in all three groups were healthy during the experiments and their behaviour was normal.