Statistics

Sample size determination from a pilot study

Because there is no report for the expression of Lactobacillus at the occurrence of POCD, the pilot study was conducted to determine sample size. On the hypothesis, the expression of Lactobacillus in the faeces 2 days after general anaesthesia, as the primary outcome, and SCFA concentrations and Nrf2 expression in the gut and brain as the secondary outcomes, respectively were determined. In the pilot study, which included 3 rats in the Control group and 3 in the Ulinastatin group, faecal Lactobacillus expression 2 days after general anaesthesia was 4.17 ± 1.94 log10 CFU in the Control group and 10.00 ± 6.29 log10 CFU in the Ulinastatin group. The SCFA concentration was 0.37 ± 0.40 pg/mL (gut) and 0.55 ± 0.46 pg/mL (brain) in the Control group and 8.29 ± 2.02 pg/mL (gut) and 10.99 ± 3.01 pg/mL (brain) in the Ulinastatin group. Nrf2 expression was 1.82% ± 0.46% (gut) and 1.63% ± 0.15% (brain) in the Control group and 8.05% ± 1.00% (gut) and 8.27% ± 1.37% (brain) in the Ulinastatin group. Based on the results of the pilot study, sample size determination was performed, using G*Power version 3.1.9.7 (Heinrich Heine University Düsseldorf, Düsseldorf, Germany). The effect size for the primary outcome was estimated as Cohen's d = 1.25. A total sample size of 30 for the primary outcome, as well as 4 samples each for SCFAs (gut and brain) and 6 samples each for Nrf2 (gut and brain) as secondary outcomes, was calculated to achieve a power of 0.9 with an α value of 0.05.

Statistical analysis

Statistical analyses and graphical presentations were performed, using GraphPad Prism software version 8.0.1.244 (GraphPad Software, La Jolla, CA, USA). For outcomes measured before and after general anaesthesia, including the Y-maze alternation ratio and faecal Lactobacillus count, a two-way repeated-measures analysis of variance (ANOVA) with time and group as factors was used to assess the time × group interaction. Planned comparisons were performed to compare values before and after general anaesthesia within each group using paired two-tailed t-tests and to compare values between the Control and the Ulinastatin groups at each time point using unpaired two-tailed t-tests. For outcomes measured once after general anaesthesia, including SCFA concentrations in the gut, blood and brain, and Nrf2 expression in the gut and brain between-group comparisons were performed using unpaired two-tailed t-tests. For all unpaired two-tailed t-tests, Welch's correction was applied when the assumption of equal variances was not met. Data are presented as mean ± standard deviation and p values < 0.05 were considered statistically significant.