Prism 5.0 program

Statistical analysis was done by using Graph Pad Prism 5.0® software (Graph Pad Software Inc. USA). The values of the above study parameters were statistically evaluated for verification and confirmation of conditions such as adhesiveness and integration of the cellulosic membrane urethral wall, compared with silicon tape.
Parametric continuous variables (height of urothelium and urethral wall) were compared using the t test. For non-parametric (density of blood vessels) the Mann-Whitney test was applied. Scores (adhesiveness and integration, collagen deposition and answers inflammatory) were compared using the chi-square Pearson test. Statistical significance was set at p≤0.05. The statistical tests were performed using the GraphPad Prism 5.0 Program® (GraphPad Software Inc., USA).

Cerebrospinal fluid (CSF) samples were collected from the cisterna magna at ZT13-ZT15. Since repeated CSF collection might alter the physiological state of the animals, CSF was obtained right before the sacrifice. Collected CSF samples were centrifuged at 2000× g for 10 min and stored at −80 °C until further analysis. The samples were thawed and run in at least triplicate. The melatonin levels were quantified using commercially available enzyme-linked immunosorbent assay kits (Cloud-Clone Corp., Houston, TX, USA). Immunoassay was performed using the Fluorescence Multi-Detection Reader (BIOTEK, Winooski, VT, USA) at an absorbance of 450 nm. The concentration of melatonin was quantified using the GraphPad PRISM 5.0 program (GraphPad Software, La Jolla, CA, USA). A nonlinear regression analysis was used to derive an equation to predict the concentration of the unknown samples.

The HT29 and Caco2 cells were transfected in six‐well plates before being seeded overnight in 96‐well plates (Corning) at a density of 3  × 10³ cells per well. Irinotecan (Sigma) was dissolved in dimethyl sulfoxide in a concentration of 100 mM and stored at −80°C. The cells were incubated with gradient concentration of Irinotecan for 48 h. Cell viability was tested using CCK8 method (Bimake) in accordance with the manufacturer's protocol. For each well, 10 µl CCK8 was diluted in 90 µl medium. After incubation at 37°C for 1 h, the absorbance was examined at 450 nm using a spectrophotometer (Bio‐Rad Laboratories, Inc.). GraphPad Prism 5.0 program (GraphPad Software, Inc.) was used to construct the cell growth inhibition curve and calculate the IC₅₀ value.