Animal management

Adult male C57BL/6 mice (2 and 18 months old) were provided by the Experimental Animal Center of Nanjing Medical University, Nanjing, China. All experimental protocols and procedures were approved by the Nanjing Medical University Animal Care and Use Committee, in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

C57BL/6 mice (2 months old and 18 months old) were grouped as follows: young control, young surgery, aged control, aged surgery, aged surgery + IPA, aged surgery + DEX, aged surgery + Lac., young + PA, young surgery + PA, young surgery + youth FMT, and young surgery + aged FMT groups, aged surgery + youth FMT, and aged surgery + aged FMT groups. These experiments on the different groups of mice were not conducted simultaneously. After establishing suitable control groups, these animals underwent at least 6 independent experiments.

Except for the young and aged groups, mice in all other groups underwent laparotomy. Briefly, these mice which had an operation were anesthetized with pentobarbitone (30 mg/kg), had a 2-cm wound made in the abdominal cavity, and the abdominal viscera was exposed for 5 min. Then, the peritoneal muscle and skin layers were sutured. The procedures, including anesthesia and surgery, were used as the overall conditions to induce PND; therefore, the mice in the young and aged groups did not receive any treatment.

For IPA administration, mice were supplemented daily with 100 mg/kg body weight IPA (Sigma-Aldrich, St. Louis, MO, USA) in CMC-Na (vehicle, 0.1%) orally for 4 weeks before surgery. For Lactobacillus acidophilus (Lac.) administration, mice were supplemented daily with 0.1 ml/10 g body weight Lac. (10⁹ CFU/ml) orally for 4 weeks before surgery. For dexamethasone (DEX) (ST1254, beyotime, China) administration, mice were supplemented daily with 1 mg/kg body weigh DEX in CMC-Na (vehicle, 0.1%) orally for 4 weeks before surgery. For PA administration, mice were supplemented daily with 50 mg/kg PA (Shanghai Yuanye Biotechnology Co., Ltd, Shanghai, China) in CMC-Na (vehicle, 0.1%) orally. The FMT groups were aged surgical mice that received fecal microbiota transplantation from young or aged mice.

Mice that received FMT receive antibiotic-laced drinking water (vancomycin 0.5 g/L + ampicillin 1 g/L) to suppress their own gut microbes for 7 days before surgery, and received FMT once a day for seven consecutive days after surgery. Specifically, the feces of the mice in the young and old groups (naïve mice) were collected at 7:00 am every day, mixed with the mice feces in the same group into a pre-deoxygenated sterile centrifuge tube, and added to sterile saline-PBS at a ratio of 100 mg:1 mL to fully suspend the feces. They were then left at room temperature until the supernatant appeared (approximately 10 min), and the supernatant was administered to recipient mice (0.1 mL/10 g) by gavage. All mice underwent behavioral tests after 7 days, including the MWM, FCT, and NORT. On the 20th postoperative day, the mice were sacrificed after collection of feces, blood, colon, and intact brain tissue was obtained. In some groups, intestinal blood flow was measured with a Doppler instrument before the mice sacrificed.