Animals
Male Sprague–Dawley rats of 3 months (young, n = 120) and 20 months (aged, n = 120) were purchased from Chengdu da shuo biotechnology co., LTD. All animal procedures were performed under the standard conditions in Beijing Chao-yang Hospital of Capital Medical University, and all experiments conformed animal care protocols of the institution. Young or aged rats were divided into five groups respectively, according to table of random numbers. Decapitation was used for rat euthanasia.
Anesthesia
All rats were housed individually at a constant room temperature (20–22 °C) and humidity (50–60%) with a 12-h light/dark cycle. They were freely fed with a standard diet and tap water. To induce anesthesia, propofol of 100 mg/kg were injected intraperitoneally. Anesthesia status was kept with propofol at a rate of 50 mg/kg/h through caudal vein for 3 hours. In control group, rats underwent the same procedure, but propofol was replaced by an equivalent volume of saline.
Rats undergoing propofol anesthesia with no respond to the tail pinch test were chosen for the following study. They were placed in a temperature-controlled incubator (28 °C) until the end of anesthesia. Arterial blood pressure and blood gases of propofol-treated rats were continuously measured and were kept in the normal range during anesthesia. All rats were then housed before further experiments.
Behavioral experiment
Shuttle box was used for learning and memory measurement. Training for rats started since 1, 3, 7 or 14d after the end of anesthesia between 8AM and 12AM and continued for 7 days. Protocols were controlled through computer. The latency of escaping response (LER), active avoidance reaction (AAR), and passive avoidance reaction (PAR) were examined and compared on the 7th day after training.
F-actin content and spines
Rats were sacrificed and brains were removed after training and testing. Brains were fixed by 4% formalin for 12 hours, and were then put into 20–30% sucrose for gradient dehydration. The dehydrated brains were kept in 30% sucrose with sodium azide or were sectioned into slices of 10-lm thickness for further experiments through a microtome (CM1860; Leica, Nussloch, Germany).
Slices were incubated with a rabbit antibody against postsynaptic density protein 95 (PSD-95, ab18258, Abcam, USA) or a rabbit anti rat antibody against microtubule associated protein-3 (MAP-2, ab32454 abcam, USA ) at 4℃ for 40 hours. After that, slices were washed with 1% phosphate buffered saline (PBS; HyClone) for three times, and were incubated with Alexa Fluor 488-labeled goat anti-rabbit IgG(1:500)for 15 min at room temperature. For F-actin observation, a mouse anti rat F-actin-NH3 (ab205, abcam, USA) and a secondary antibody of Alexa Fluor594-labeled goat anti-mouse IgG༈1:500༉were used. All slices were washed by PBS for 3 times and were viewed under a laser confocal microscopy (BX53; Olympus, Tokyo, Japan).
Statistic Analysis
Protein fluorescent densities were evaluated by one-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test using SPSS version 17.0 (SPSS, Chicago, IL, USA). A P value of less than 0.05 was considered statistically significant.