Animals. Pathogen-free, 20-month-old Sprague-Dawley rats weighing 500-550 g were purchased from the Medical Experimental Animal Center of Guangdong Province, China (certificate no. SCXK‑Yue‑2015‑0002). Rats were fed a normal diet and kept at a room temperature of 23±1 ℃ in air with 70±10% relative humidity before experiments. The experimental protocol was approved by the Animal Care and Use Committee at Lihuili Eastern Hospital of Ningbo Medical Center.
Reagents. The autophagy inducer rapamycin (RAP; catalog no. v900930) and autophagy inhibitor 3-methyladenine (3-MA; catalog no. M9281) were purchased from Sigma-Aldrich. TRIzol, primers, reverse transcription and a polymerase chain reaction (PCR) kit were obtained from Takara for real time-quantitative PCR. Primary antibodies against the following proteins were obtained from Cell Signaling Technology: ATG5 (catalog no. 12994), LC3B (3868s), Beclin1 (3495), SQSTM1/p62 (39749), and GAPDH (4970). Horseradish peroxidase-conjugated mouse anti-rabbit secondary antibody (7074) was also purchased from Cell Signaling Technology. Primary antibody against Cys C (catalog no. ab109508) was purchased from Abcam. A Morris water maze was purchased from Beijing Shuolinyuan Science and Technology Company. Tans-Blot® Turbo omnipotent protein transfer system, Mini PROTEIN®Tetracell system and ChemiDoc MP system were both obtained from Bio-Rad biotechnology Co., Ltd, USA. LB940 Functional Enzyme Marker was purchased from Guangzhou Nebula Scientific Instruments Co., Ltd and Mx3000P Fluorescent PCR Amplifier was a product of Agilent Technologies Company (USA).
Animal group allocations and splenectomy. This animal model of splenectomy-induced POCD was established as described [15, 16]. Rats were randomized into the follow four groups (10 per group). Control (CON) animals did not undergo any surgery and received a daily intraperitoneal injection of saline. Surgery (SUR) animals underwent splenectomy as described below and received a daily intraperitoneal injection of saline. RAP animals underwent splenectomy, followed by a daily intraperitonal injection of rapamycin (1.0 mg/kg). The 3-MA group underwent splenectomy, followed by a daily intraperitonal injection of 3-MA (3.0 mg/kg). The injection volume was the same in all four groups. Intraperitoneal injections began in all groups on postoperative day 3 and continued for four weeks.
For splenectomy, rats were anesthetized intraperitoneally with 80 mg/kg ketamine and 60 mg/kg sodium pentobarbital. During experiments, anesthesia was maintained by injecting one‑third of the initial dose of ketamine every 45 min. Rats were placed in a supine position on an adjustable warming pad and superclean bench. A transverse incision 2.0 cm long was made from the lower edge of the left rib in order to open the abdominal cavity, then the spleen was excised. After confirmation of no hemorrhage, the abdominal cavity was stitched and the wound was disinfected with iodophor.
Collection of samples. After the four-week treatment period, learning and memory were assessed in a subset of the 10 animals in each group using a Morris water maze. Tests were conducted on postoperative day 1, 3, 5 and 7. Rats were sacrificed by cervical dislocation and hippocampus tissue was collected.
Morris water maze. Animals' ability to navigate and explore space was assessed on postoperative day 1, 3, 5, and 7. Tests were conducted four times per day. The platform was placed in the center of the southwest quadrant. Rats facing the water maze wall were randomly positioned at one of four starting positions (southeast, northeast, southwest and northwest) and allowed to swim around the maze until finding the platform. If the rats did not find the platform within 2 min, we assisted them and recorded the latency as 120 s. Rats were allowed to rest on the platform for 15 s, and training was repeated at 30-s intervals. Then the time for rats to swim to the platform (escape latency) was recorded using an automatic camera and motion recorder. At 24 h after the last training, the platform was removed to perform space exploration experiments. Rats were placed in the Morris water maze in the northwest quadrant, and the time required to cross the original platform was recorded. Animals who failed to cross the platform within 2 min were recorded as having a crossing time of 120 s.
Assessment of inflammatory response. Hippocampus tissue was minced using Mayo-Noble scissors and homogenized using a tissue homogenizer. The homogenate was dissolved using Phosphate buffer saline and centrifuged. The supernatant was assayed for inflammatory factors interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α using commercial enzyme-linked immunosorbent assays (ThermoFisher Scientific).
Analysis of mRNA levels using RT-qPCR. Total RNA was extracted from hippocampus tissues using TRIzol reagent. Single-stranded cDNAs encoding ATG5, Beclin1, p62 and Cys C were synthesized and then amplified by PCR using specific primers (Table 1). Levels of target mRNAs were quantitated relative to the level of mRNA encoding glyceraldehyde 3‑phosphate dehydrogenase (GAPDH).
Analysis of protein expression by Western blotting. Total protein was isolated from hippocampal tissue using ice‑cold RIPA lysis buffer with protease inhibitors (Beyotime Institute of Biotechnology, Haimen, China), and protein concentration was determined using the BCA Protein Assay Kit (Pierce). Equal amounts of protein were fractionated by sodium dodecyl sulfate‑polyacrylamide gel electrophoresis and transferred onto polyvinylidene ﬂuoride membranes. Membranes were blocked and then incubated overnight with primary antibodies against ATG5, LC3B, Beclin1, SQSTM1/p62, Cys C and GADPH. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibody to allow chemiluminescent visualization using the ChemiDoc MP system (Bio‑Rad).
Statistical analysis. Data were analyzed using SPSS 22.0 (IBM, Chicago, IL, USA) and reported as mean ± SD. Inter‑group differences were assessed for significance using one-way ANOVA, and pair-wise comparisons were assessed using the LSD and t tests. A two-tailed P < 0.05 was defined as statistically significant.