Materials and methods

All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. This study was designed, conducted and reported in accordance with the ARRIVE Guidelines 2.0. After approval by the Institutional Animal Care and Use Committee (IACUC) of Konkuk University (approval number: KU24046, KU26028), all experiments were conducted at the Konkuk University Laboratory Animal Research Center in accordance with IACUC guidelines.

Animal preparation

Eighteen-month-old male Sprague–Dawley rats were purchased from Koatech (Pyeongtaek, Korea). The rats were housed in cages with free access to food and water. The room was maintained on a standard 12-h light–dark cycle (lights on at 7:00 and lights off at 19:00) at a temperature of 25°C. The rats were acclimated to the experimental conditions for 7 days before the study and fed a standard diet ad libitum, with free access to water.

Behaviour test

Schedule and apparatus

Behaviour test was performed just as the previous studies (Cho et al., 2024a, 2024b). After acclimation, the Y-maze test was performed to evaluate cognitive function on the day before general anaesthesia and 2 days after general anaesthesia. The Y-maze consisted of three arms (A, B, and C). Each arm was 50 cm long, 25 cm high, and 10 cm wide, and was connected to the other arms at an angle of 120°.

Testing protocol

Before the start of the test, all arms were wiped with 70% alcohol, and arm C was blocked with a board at the center of the equilateral triangle to prevent access to arm C. The rat was placed at the end of arm A and allowed to adapt to the apparatus for 15 min, moving freely between arms A and B. After this 15-min adaptation period, the rat was returned to its cage and rested for 1 h. After the 1-h rest, all arms were wiped again with 70% alcohol, the board blocking arm C was removed to allow free access to all arms, and the rat was placed again at the end of arm A. Movement was recorded with a video camera for 5 min.

Quantification and exclusion criteria

The total number of entries into each arm and spontaneous alternations were recorded, and the alternation ratio was calculated using the formula [(number of spontaneous alternations)/(total arm entries − 2)] × 100% (Cho et al., 2024a, 2024b; Li et al., 2020). Rats showing an alternation ratio of < 40% on the day before general anaesthesia were regarded as having pre-existing cognitive impairment and were excluded from the study (Kim et al., 2023b).

Animal grouping and general anaesthesia

Animal grouping and drug administration

Grouping and general anaesthesia were performed just as the previous studies (Cho et al., 2024a, 2024b; Piao et al., 2022; Seo et al., 2023). The animals were randomly allocated to the Control group or the Ulinastatin group. The Control group received 1 mL normal saline and the Ulinastatin group received 1 mL ulinastatin (50,000 U; Ulistin®, Hanlim Pharm., Korea), administered intraperitoneally.

General anaesthesia and mechanical ventilation

After administration, anaesthesia was induced with intraperitoneal ketamine 100 mg/kg (Yuhan, Korea) and xylazine 10 mg/kg (Sigma-Aldrich, USA). A heating pad was placed on the surgical platform to maintain the body temperature at approximately 37°C during anaesthesia. The rat was secured in the supine position and fastened on the surgical platform for endotracheal intubation using a 16 G catheter (45 mm; Dukwoo Medical, Korea). Correct placement of the catheter was confirmed by symmetrical chest expansion. The catheter was connected to a ventilator (Harvard Apparatus, USA). The ventilator settings were as follows: fraction of inspired oxygen, 0.5; inspired flow rate, 150 mL/min; tidal volume, 6 mL/kg; respiratory rate, 50 breaths/min; inspiration:expiration ratio, 1:1; and positive end-expiratory pressure, 5 cmH2O. Anaesthesia was maintained with isoflurane 1.5 vol% for 2 h, and the ventilator settings were maintained throughout. After 2 h of isoflurane maintenance, the isoflurane vaporiser was stopped. Mechanical ventilation was continued until full recovery of spontaneous ventilation was confirmed. After confirming recovery of spontaneous ventilation, the endotracheal catheter was removed and the rat was transferred to its cage.

Microbiome analysis using lactobacillus viable count

Fecal sample collection and storage

Dysbiosis was evaluated using Lactobacillus viable counts. Faecal samples were collected to assess the gut microbiome on the day before general anaesthesia and 2 days after general anaesthesia. Samples were placed in 1-mL microtubes and stored at −20°C.

Serial dilution and plating

For analysis, the stored samples were thawed. After thawing, 1 g of faeces was mixed with 9 mL normal saline in a 15-mL conical tube and thoroughly homogenised. The mixture was then serially diluted from 102 to 107 in conical tubes containing 0.9% normal saline. After dilution, 100 μL of each dilution was spread onto de Man–Rogosa–Sharpe agar plates (Microgen, Germany) using a spreader (SPL, Korea).

Incubation and colony quantification

The plates were placed in a HEPA class 100 CO2 incubator (Thermo, USA) at 37°C with 5% CO2 and cultured for 24 h. After incubation, the plates were examined and colonies were counted. The viable count was expressed as log10 colony-forming units (CFU) per gram of faeces.

Tissue collection

Samples were obtained in the following order: blood, brain, and gut.

Animal sacrifice under anaesthesia

After the Y-maze test 2 days after general anaesthesia, the rat was anaesthetised with isoflurane 5 vol% with oxygen 0.3 L/min and nitrous oxide 0.7 L/min in an induction chamber (Cho et al., 2024a, 2024b). After confirmation of adequate anaesthesia, the animal was sacrificed.

Blood sample processing

The abdomen was opened to expose the abdominal aorta. A blood sample was obtained from the abdominal aorta using a 1-mL syringe and stored in an ethylenediaminetetraacetic acid tube (BD Vacutainer®, USA). The tube was then centrifuged at 1500 × g for 20 min at 4°C to separate the plasma. The separated plasma was transferred to a 5-mL conical tube and stored at −20°C.

Brain tissue isolation and preparation

After blood collection, 1× phosphate-buffered saline (PBS) was perfused through the abdominal aorta until complete exsanguination was achieved and the liver colour changed from red to pale. Using scissors and forceps, the skull was opened and the brain was removed. The brain was divided into two hemispheres. The right hemisphere was prepared to evaluate short-chain fatty acids (SCFAs) by enzyme-linked immunosorbent assay (ELISA) and nuclear factor erythroid 2-related factor 2 (Nrf2) expression by Western blot. It was transferred to an Eppendorf® tube (Eppendorf, Germany) and stored at −20°C. The left hemisphere was prepared for assessment of Nrf2 expression by immunohistochemical staining and stored in a 15-mL conical tube containing 4% paraformaldehyde (PFA) (BIOSESANG, Korea) at 4°C for immunofluorescence analysis. This approach was adopted to avoid potential interference arising from repeated processing of the same tissue and to enable complementary analyses from the same animal, consistent with our previous studies (Cho et al., 2024a, 2024b). Because all interventions in this study were systemic rather than hemisphere-specific, systematic differences between the left and right hemispheres were not expected.

Gut tissue isolation and preparation

After brain collection, the colon was removed from the gut. It was opened longitudinally and divided evenly into two parts. One portion was prepared for analysis of SCFAs by ELISA and Nrf2 expression by Western blot, transferred to an Eppendorf® tube, and stored at −20°C. The other portion was prepared for assessment of Nrf2 expression by immunohistochemical staining and stored in a 15-mL conical tube containing 4% PFA at 4°C for immunofluorescence staining.

SCFAs from gut, blood, and brain

The stored colon samples were thawed at room temperature and homogenised in 1× PBS (100 mg/mL). The homogenised tissue was transferred to a microtube and centrifuged at 12,000 × g for 15 min at 4°C (Cho et al., 2024a, 2024b). After centrifugation, the supernatant was collected into a microtube and analysed using an ELISA kit (MyBioSource, Canada). A microplate reader, reflecting combined SCFA levels rather than individual metabolites (acetate, propionate and butyrate), was used to measure SCFA concentrations.

The stored plasma was thawed at room temperature, transferred from the conical tube to a microtube, and analysed using the same method as for the colon samples. The stored right hemisphere was also analysed using the same procedure.

Immunofluorescence staining and Western blot for Nrf2 in gut and brain

Paraffin-embedded colon and brain sections were subjected to immunofluorescence staining. Sections were incubated with an anti-Nrf2 primary antibody (Invitrogen, USA) followed by an Alexa Fluor 488-conjugated anti-rabbit secondary antibody (Invitrogen, USA), and nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole). Fluorescence images were acquired using a fluorescence microscope (Olympus, Japan). Detailed procedures are described in the Supplementary Materials.

Proteins were extracted from colon and brain tissues, and protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo, USA). Equal amounts of protein were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride membranes, and incubated with an anti-Nrf2 primary antibody followed by a horseradish peroxidase-conjugated goat anti-rabbit IgG (H + L) secondary antibody (GenDEPOT, USA). Protein bands were visualized using enhanced chemiluminescence and the iBright CL1000 imaging system (Thermo, USA). Detailed procedures are described in the Supplementary Materials.