Male C57BL/6J mice (n=104), aged 12–14 weeks, weighing 20–25 g (Vital River Laboratory Animal Technology Co., Ltd., Beijing, China), were used for all experiments. The animals were housed under controlled conditions (21±2°C; 50±10% humidity, 12:12 h light:dark cycle) with access to food and water ad libitum. All mice were allowed to adapt to the environment for 7 days prior to beginning the experiments. All experimental protocols were approved by the animal ethics committee of Capital Medical University (Beijing, China), and were in accordance with the guidelines for animal experiments of the local Animal Care and Use Committee.

Based on preliminary time course experiments (15–17), to investigate the effects of orthopedic surgery on the activation of mTOR, β-amyloid accumulation and tau phosphorylation, the present study examined the changes of relevant proteins. The animals were divided into three groups (n=8/group). Normal, untreated animals were used as a control group. In the surgery group, the mice underwent orthopedic surgery of left hindpaw under isoflurane anesthesia and analgesia. In the sham surgery group, mice received the same anesthesia and analgesia as the surgery group. The mice in each group received contextual fear conditioning (CFC) to estimate the learning and memory abilities of the mice, following which the animals were sacrificed by cervical dislocation, and hippocampal tissue samples were obtained for further experiments. Animals were anesthetized prior to cervical dislocation through a single intraperitoneal injection of chloral hydrate (10%; 0.3 ml/100 g).

The experiments described above showed the activation of mTOR signaling with β-amyloid accumulation and tau phosphorylation in response to surgical stimulation. Therefore, subsequent experiments were performed to examine the effects of pretreatment with rapamycin, an inhibitor of mTOR, on postoperative cognitive function, and to determine the levels of Aβ_1-42 and tau phosphorylation. This was performed to confirm whether the hyperactivation of mTOR signaling was involved in cognitive defects following surgery via the upstream regulation of Aβ_1-42 and the phosphorylation of tau. To investigate the effects, the mice were divided into four groups (n=8/group): Ctrl group (mice received injections of vehicle without surgery), Ctrl+rapa group (mice received rapamycin pretreatment without surgery), Sur group (mice received orthopedic surgery with injections of vehicle) and Sur+rapa group (mice received orthopedic surgery with rapamycin pretreatment). The animals received CFC 1 day following surgery, and the mice were then sacrificed by cervical dislocation for western blot and enzyme-linked immunosorbent assays.

Anesthesia was prepared using a procedure described by Degos et al (18). In brief, the animals were placed in a sealed plastic box and anesthesia was induced with 5% isoflurane mixed with air. The anesthesia was maintained with 1.2–1.5% isoflurane, which was delivered through a nose cone to the mouse for 15 min. The gas concentrations and respiratory rate were continuously monitored using a multi-function monitor (Datex-Ohmeda, Helsinki, Finland). In the surgery group, an open tibial fracture of the left hind paw with intramedullary fixation was performed in aseptic conditions under general anesthesia with isoflurane (19,20). Buprenorphine was used to provide supplemental analgesia (0.1 mg/kg administered subcutaneously). The surgical aspect of the left hindpaw was sterilized with povidone-iodine, and a median incision on the surgical region was made. Following incision, a 0.38 mm pin was inserted in the intramedullary canal, the periosteum was stripped and the wound was irrigated. Finally, the skin of the wound was closed with 5–0 nylon sutures and covered with antibiotic ointment. Following surgery, the mice were moved back to their original cage for recovery with a sufficient supply of food and water. Throughout the entire anesthesia and surgical procedures, all vital signs were monitored, and blood gas analysis was performed following anesthesia and surgery. For inhibition of the mTOR signal, rapamycin was used to reduce the activity of mTORC1. The dose of rapamycin used was selected based on previous studies (21,22). The mice received intraperitoneal injections of 0.5 mg/kg/day rapamycin for 3 days prior to undergoing orthopedic surgery, with the final dose administered 2 h prior to surgery. In the negative control group, animals received an intraperitoneal injection of 0.5 mg/kg/day rapamycin for 3 days without surgery.

The animals were transported to the laboratory at least 2 h prior to CFC training. For CFC, a clear plexiglas chamber was placed in a soundproof box and a camera (Meidi Electronic Co., Ltd., Shenzhen, China) was fixed to the top of the box to capture videos of each animal during CFC training using ANY-maze software (Stoelting Co., Wood Dale, IL, USA). Foot shocks were delivered through a grid floor, which comprised 28 stainless steel bars. Prior to and following each session, the chambers were cleaned using pine solution.

Training was performed 24 h prior to surgery. The animals were allowed free movement in the chambers for 5 min prior to training, following which they received three tone (2,000 Hz; 90 dB)-electric shock (0.85 mA for 2 sec) pairings, which were separated by 60 sec. Following fear establishment, the fear response of the animals was measured based on freezing times. The percentage of freezing time was used to reflect the hippocampal-dependent memory. Following the different treatments, the mice were returned to the training environment to assess the contextual fear memory. During CFC assessment test, each mouse was placed once again into the chamber for three 3 min without tone or shock. Freezing time was measured by two observers blinded to the group assignments.

For western blot analysis and the enzyme-linked immunosorbent assay, the mice were sacrificed under deep anesthesia. The hippocampus was removed and stored at −80°C.

The hippocampal tissues were homogenized in RIPA buffer containing protease and phosphatase inhibitors, and then centrifuged at 4°C at 12,000 g for 25 min. The concentration of protein in the supernatants was determined using a Bradford protein assay kit (Beyotime Institute of Biotechnology Co., Ltd., Shanghai, China). Equal quantities of the protein samples (30 μg per sample) were denatured at 100°C for 5 min, and were then separated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred electrophoretically onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membranes were blocked using 5% skim milk-Tris-buffered saline (TBS) buffer for 60 min and then incubated with the following primary antibodies: Rabbit polyclonal anti-mTOR (1:1,000; cat. no. 2972; Cell Signaling Technology, Inc., Boston, MA, USA), rabbit polyclonal anti-phosphorylated (phospho)-mTOR (Ser2448; 1:1,000; cat. no. 2971; Cell Signaling Technology, Inc.), rabbit polyclonal anti-P70S6K (1:1,000; cat. no. 9202; Cell Signaling Technology, Inc.), rabbit polyclonal anti-phospho-P70S6K (Thr389; 1:1,000; cat. no. 9205; Cell Signaling Technology, Inc.), rabbit poly-clonal anti-Tau (1:500; cat. no. YT4554; Immunoway, Newark, DE, USA) and rabbit polyclonal anti-phospho-Tau (Ser396; 1:500; cat. no. YP0263; Immunoway) overnight at 4°C. Following three washes (10 min each) in TBS with Tween solution, the membranes were incubated with horseradish peroxidase conjugated goat anti-rabbit IgG (1:2,000; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) secondary antibodies at room temperature for 1 h. The bands were treated with an enhanced chemiluminescence detection kit (EMD Millipore), and the intensity of each band was quantified by densitometric analysis. The relative expression levels of protein were normalized by GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.).

The hippocampal tissue samples were weighed and sonicated in phosphate-buffered saline with 50 mM protease inhibitor cocktail, followed by centrifugation at 20,000 g 4°C for 10 min. The supernatant was collected to quantify the protein concentration in the samples using a BCA protein assay kit (cat. no. 23225; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Equal quantities of protein sample (50 μg) were used for the measurement of Aβ_1-42 (Wuxi Donglin Sci & Tech Development Co., Ltd., Jiangsu, China) using an enzyme-linked immunosorbent assay, according to the manufacturer's protocol. The intensity of the color was measured at a wavelength of 450 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

All data were analyzed using SPSS statistical software, version 18.0 (IBM SPSS, Armonk, NY, USA). The data were expressed as the mean ± standard deviation, and statistical analysis was performed using one-way analysis of variance followed by Newman-Keuls post-hoc test where appropriate. P<0.05 was considered to indicate a statistically significant difference.

To elucidate the effect of surgery on cognitive function, CFC assessment was performed to assess learning and memory function. Compared with the control group and sham group, animals in the surgery group presented with significantly reduced freezing time percentages on day 1 (42.4±9.8, vs. 65.9±13.8%, respectively; P<0.05) and day 3 (48.95±9.97, vs. 73.88±13.9%, respectively; P<0.05). No significant difference in freezing time was found between the sham group and control group (Fig. 1).

To determine the effects of surgical trauma on mTOR/p70S6K signaling, the present study detected the levels of mTOR and p70S6K in the homogenates of the hippocampal tissue using western blot analysis (Fig. 2). Although there were no significant differences in the levels of total mTOR or p70S6K (Fig. 2D and E), the levels of phospho-mTOR at Ser2448 and phospho-p70S6K at Thr396 were significantly increased on day 1 and day 3 in the hippocampal tissues of the surgery group (P<0.05; n=8), as shown in Fig. 2B and C. No significant differences were found in the levels of phospho-mTOR at Ser2448 or phospho-p70S6K at Thr396 between the sham surgery group and the control group (Fig. 2B and C). These data indicated that surgery elevated the activation of mTOR signaling on day 1 and day 3. However, the effect of surgery on mTOR signaling was reversed on day 7. Surgical trauma induced the upregulation of mTOR/p70S6K signaling, which suggested that the activation of mTOR signaling may be involved in the development of postoperative dysfunction.

To investigate the effect of orthopedic surgery on the production of β-amyloid, the present study measured the level of Aβ_1-42. Surgery increased the production of Aβ_1-42 at 24 h (2.88±0.34, vs. 1.93±0.19 pg/ml) and 3 days post-surgery (3.0±0.32, vs. 1.86±0.20 pg/ml; P<0.05; n=8; Fig. 3). No significant differences were observed between the sham surgery group and control group (P>0.05; n=8).

Tau protein hyperphosphorylation is associated with the upregulation of mTOR/p70S6K signaling in the development of AD. The present study also investigated whether tau phosphorylation occurred following surgery accompanying the increased mTOR activity. The results showed that orthopedic surgery upregulated the levels of phosphorylated protein at Ser396 on day 1 and day 3 post-surgery, compared with the sham surgery and control group (P<0.05; n=8; Fig. 4B). However, surgery did not alter the expression of total tau protein (Fig. 4C).

As mTOR/p70S6K signaling is hyperactive under the condition of traumatic stimulation and rapamycin is a well known mTOR inhibitor, the present study investigated the effect of rapamycin administration on mTOR/p70S6K signaling. The data showed that rapamycin treatment significantly reduced the levels of phospho-mTOR at Ser2448 and phospho-p70S6K at Thr396 induced by surgical trauma (P<0.05; n=8; Fig. 5B and C). However, rapamycin had no effect on the total levels of mTOR or p70S6K (Fig. 5D and E). Rapamycin treatment also reduced the levels of phospho-mTOR at Ser2448 and phospho-p70S6K at Thr396 in the normal control mice (P<0.05; n=8; Fig. 5B and C).

In order to investigate whether the hyperactivation of mTOR/p70S6K signaling was involved in the accumulation of β-amyloid and hyperphosphorylation of tau protein, the present study examined the effect of rapamycin on the levels of Aβ_1-42, total tau and phospho-tau. It was found that rapamycin significantly attenuated the production of Aβ_1-42 (2.32±0.18 pg/ml), compared with the surgery group (2.99±0.27 pg/ml), as shown in Fig. 6 (P<0.01; n=8) and attenuated the level of hyperphosphorylated tau at Ser396, (P<0.05; n=8; Fig. 7A and B) which were triggered by surgical injury. However, rapamycin had no effect on the total level of tau protein (Fig. 7A and C). Rapamycin did not affect the levels of Aβ_1-42, total tau or phospho-tau in the normal control mice (Figs. 6 and 7).

The present study subsequently investigated whether the increase in mTOR/p70S6K signaling contributed to the cognitive dysfunction caused by surgery. The effect of rapamycin administration on learning and memory function was determined using a CFC assessment, which was also used to measure hippocampal-dependent learning and memory. It was found that surgical injury significantly reduced the percentage freezing time (P<0.05; n=8), however, rapamycin pretreatment significantly compromised the decreased freezing time caused by surgery (60.0±8.1% in Sur+rapa group, vs. 43.4±8.0% in the Sur group), as shown in Fig. 8 (P<0.05; n=8). Rapamycin treatment had no significant effect in the normal mice (P>0.05; n=8). These data suggested that rapamycin treatment rescued the impairment of hippocampal-dependent memory induced by surgery.