In our study, a ligation of bilateral CCA (common carotid arteries) surgery was adopted to prepare rats as CH animal model . A second surgery, open reduction and internal fixation (ORIF), requiring general anesthesia, was operated so that the effects of anesthetics on cognitive function of these CH rats could be assessed.
Male Wistar rats, 16-18 months of age, 450-570 in weight, were purchased from the Academy of Military Medical Science of the Chinese People's Liberation Army, and housed in groups of six per cage with ad libitum access to food and water. The environment temperature was 20-22℃ and humidity was 45%~65% with a 12 h light/dark cycle. All animal experiments were carried out according to the Guide for the Care and Use of Laboratory Animals  and approved by the Institutional Animal Care and Use Committee of Tianjin Medical University. Rats were housed alone per cage 3 days before the ligation of CCA and fasted 12 h before the surgery with normally supplied drinking water. After surgeries, rats were also housed alone per cage for recovery.
Ligation of CCA
The rats were first anaesthetized with intraperitoneal (i.p.) injection of 10% thiobutabarbital (100 ml/kg). After the body motion reaction and righting reflex disappeared, rat was fixed on the operation platform. Throughout the whole procedure, the surgical field was maintained sterile. The skin of the rat’s neck was shaved and disinfected with iodine tincture. A median incision of approximately 2-3 cm was made in the neck. The muscles and surrounding tissues were separated to expose CCA. The CCA and a blunt end syringe needle (0.45 mm in diameter, 1 cm in length) were ligated tightly at the proximal side 1.5 cm from the bifurcation of the internal and external carotid arteries. The slipknot was firmly fixed and the needle was carefully removed. The wound was sutured and disinfected. During the surgery, a heating lamp was used to help maintaining the body temperature of anesthetized rats at 37 ± 0.5°C. 
Anesthesia and surgery of ORIF
During the ORIF surgery, rats were administered isoflurane inhalation or propofol through the tail vein. For the induction phase of anesthesia, rats were placed in a transparent chamber (W 25 cm × D 15 cm × H 10 cm) connected to a vaporizer and anaesthetized with 5% isoflurane and 40% oxygen. When the rats’ righting reflex disappeared, the chamber was replaced by a mask. Then all the rats were separated into 5 groups (n=32/group) and given different anesthesia maintenance methods. (1) Group C: local infiltration anesthesia with 2% Lidocaine and inhalation with air containing 40% oxygen via the mask for 3 h. (2) Group I: inhalation with air containing 40% oxygen and 1.9% isoflurane for 3 h. (3) Group P: venous transfusion with 40 mg·kg-1·h-1 propofol and inhalation with air containing 40% oxygen via the mask for 3 h. (4) Group IP1: venous transfusion with 20 mg·kg-1·h-1 propofol and inhalation with air containing 40% oxygen and 1% isoflurane for 3 h. (5) Group IP2: venous transfusion with 10 mg·kg-1·h-1 propofol and inhalation with air containing 40% oxygen and 1.4% isoflurane for 3 h. The concentration of isoflurane was detected continuously by a gas monitor (Puritan-Bennett; Tewksbury, MA, USA) during the process of surgery.
ORIF surgical model: under different general anesthesia, rats received an open tibial fracture on the left hind paw with an intramedullary fixation. Supplemental analgesia was provided by buprenorphine (0.3 mg/kg in saline) intraperitoneally in less than 1 ml. The surgery was carried out with aseptic techniques. The left hind paw of surgical rats was shaved and disinfected with iodine tincture. After the skin was incised, an insertion of a 0.38 mm pin was performed in the intramedullary canal. Once the tibia was internally fixated, the bone was fractured in the middiaphysis (tibial, midshaft) using surgical pliers. The skin was sutured with 8/0 Prolene sutures. Only skin incisions and sutures were performed on rats in group C. During the surgery, a heating lamp was used to help maintaining the body temperature of anesthetized rats at 37 ± 0.5°C. Postintervention rats were moved to heated pads for recovery and then delivered back to their own cage, where food and water were sufficient. For post-procedural pain relief, the rats were administered buprenorphine (0.05 mg/kg, subcutaneous) twice daily for 3 days.
Fear conditioning test
The fear conditioning (FC) test was carried on to estimate cognitive function. The FC test consisted of a training phase at 24 h prior to ORIF operation and an evaluation phase on days 1 and 7 after ORIF when hippocampal-dependent memory was assessed.
During the training phase, rats were placed in a chamber (Ugo Basile, Italy) and allowed to adapt to the environment for 120 s. After adaption, they were stimulated by a 20 s 70-dB tone (conditional stimulus). Then there was an interval of 25 s. After the interval, rats were stimulated by a 0.70 mA electrical foot-shock for 2 s (unconditional stimulus). After six pairs of conditional-unconditional stimuli, the rats learned the association and established long-term memory. The pairs of conditional-unconditional stimuli were separated by 60 s inter-training intervals. Each training chamber was cleaned with 95% ethyl alcohol before the placement of a rat and was illuminated only with a 10 W bulb in a dark experimental room.
During the evaluation phase, rats were placed again in the training chamber for 5 min without tone and foot shock. Each animal’s freezing behavior (without any movements) was scored by the ANY-maze video tracking system (Stoelting, Illinois, USA). The percentage of time in freezing behavior was calculated using the formula of 100*f/5min, where f was the total of freezing time in the 5 min. Freezing time measured during exposure to the known context, or after a conditional stimulus in the known context, reflects hippocampal-dependent memory whereas assessment during delivery of the conditional stimulus (tone) assesses hippocampal-independent memory. Thus the results in this experiment were used to assess hippocampus-dependent memory.
On days 1 and 7 after ORIF, rats (n=8/group) were sacrificed with sodium pentobarbital (240mg/ml, Department of Pharmacy, Tianjin Medical University General Hospital, i.p., 800 mg/kg). After ensuring that the heart of rat stopped, the brain was taken out. Paraffined coronal hippocampal slices (10 μm in thickness) were prepared for Nissl staining. All slices were heated at 60 ℃ for 2 h in an incubator in order to melt the paraffin. Then, slices were dewaxed in Xylene for 5-10 min, three times. Slices were bathed in anhydrous ethanol for 5 min, 90% ethanol for 2 min, 70% ethanol for 2 min, distilled water for 2 min, and cresyl violet for 10 min, successively.
Then, slices were washed with distilled water twice (each for 2 min) and dehydrated in 95% ethanol twice (each for 5 min). To assess hippocampal injury, five continuous random fields (200 in magnification) in the pyramidal layer of CA1 region (8 slides/group) was selected and the number of survival neurons was counted under light microscopy (Leica, DMIRB) in a blinded manner according to the method described by Tian. The analysis of data was performed through Image Pro Plus 6.0 software (Media Cybernetics Co., USA).
On days 1 and 7 after ORIF, rats (n=8/group) were sacrificed with sodium pentobarbital (240mg/ml, Department of Pharmacy, Tianjin Medical University General Hospital, i.p., 800 mg/kg). After ensuring that the heart of rat stopped, the brain was taken out and hippocampus tissue was separated. Hippocampus was homogenized in RIPA solution (Biomart, Beijing, China) buffer and then centrifuged at 4℃ at 12000r/min for 10 min (Sigma 3-30KS, Sigma Laboratory Centrifuges, Germany). The quantity of protein in the supernatants was determined using a BCA protein assay kit (Beyotime Biotechnology, Beijing, China). Equal amounts of protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. Then membranes were blocked by 5% skim milk Tris-buffered saline containing 0.1% Tween (TBST) buffer for 90 min and then washed by TBST buffer for 5 min. The membranes were incubated with primary antibodies: anti-GABAAR α1 (1:1,000, Abcam, Cambridge, UK), anti-BiP (1:1,000; Abcam), anti-pan-cadherin (1:2,000, Sigma, St. Louis, MO, USA), anti-β-actin (1:10,000, Proteintech, Wuhan, China) overnight at 4℃. After washing with TBST 5 times (each for 5 min), membranes were incubated with a secondary polyclonal antibody conjugated to horseradish peroxidase, anti-rabbit immunoglobulin G (IgG) (1:5000, KPL, Gaithersburg, MD), and anti-mouse IgG (1:5000, KPL) at room temperature for 1 h. Then the membranes were washed 5 times (each for 5 min) again and treated with an enhanced chemiluminescence detection kit (EMD Millipore, Billerica, MA, USA). The intensity of each band was quantified by densitometry using a gel image analysis software (Image Pro Plus, Media Cybernetics, USA). Relative expression was normalized to that of anti-pan-cadherin (1:2,000, Sigma) and anti-β-actin (1:10,000, Proteintech).
The data were analyzed using SPSS 20.0 software (IBM Corp., Armonk, NY, USA). Data were presented as mean ± standard deviation (SD). Behavioral data were tested using a two-way analysis of variance (ANOVA) with repeated measures. Other data were analyzed using a one-way ANOVA with Tukey post hoc comparisons. P < 0.05 was the criterion for statistical significance.