In our study, a ligation of bilateral CCA surgery [44] was adopted to prepare rats as CH animal model [45]. A second surgery, ORIF [46], requiring general anaesthesia, was operated 30 days later so that the effects of anaesthetics on cognitive function of these CH rats could be assessed.
Animals
Male Wistar rats, 16-18 months of age and 450-570 g 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 housing environment was maintained at a temperature of 20-22℃ and a humidity of 45%~65% under a 12 h light/dark cycle. All animal experiments were carried out according to the Guide for the Care and Use of Laboratory Animals [47] and were approved by the Institutional Animal Care and Use Committee of Tianjin Medical University. Rats were housed individually per cage 3 days before ligation of the CCA and fasted 12 h before surgery a normally supply of drinking water. After surgery, rats were also housed individually per cage for recovery.
Ligation of the CCA
Rats were first anaesthetized with intraperitoneal (i.p.) injection of 10% thiobutabarbital (100 ml/kg). After disappearance of body motion and the righting reflex, the rat was fixed on the operation platform. The surgical field was maintained sterile throughout the entire procedure. 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 the 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 surgery, a heating lamp was used to help maintain the body temperature of anaesthetized rats at 37 ± 0.5°C [44].
Anaesthesia and ORIF surgery
During ORIF surgery, rats were administered isoflurane via inhalation or propofol through tail vein injection. For the induction phase of anaesthesia, the rat was 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 rat’s righting reflex disappeared, the chamber was replaced by a mask. Each rat was then assigned to one of the following 5 groups (n=32/group) and administered the respective anaesthesia as maintenance: (1) Group C: local administration of anaesthesia 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; and (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 surgery.
ORIF surgical model: Under different modes of general anaesthesia, the rats underwent an open tibial fracture of the left hind paw with intramedullary fixation. Supplemental analgesia was provided using less than 1 ml buprenorphine (0.3 mg/kg in saline) administered intraperitoneally [46]. Surgery was carried out via aseptic techniques. The left hind paw of the rat was shaved and disinfected with iodine tincture. After the skin was incised, a 0.38 mm pin was inserted into the intramedullary canal. Once the tibia was internally fixated, the bone was fractured at the middiaphysis (tibial, midshaft) using surgical pliers. The skin was sutured with 8/0 Prolene sutures. In Group C, only the skin was incised and sutured. During surgery, a heating lamp was used to help maintain the body temperature of the anaesthetized rats at 37 ± 0.5°C. Postintervention rats were moved to heated pads for recovery and then returned to their home cage supplied with sufficient food and water. For post-procedural pain relief, the rats were administered buprenorphine (0.05 mg/kg, subcutaneous) twice daily for 3 days [48].
Fear conditioning test
The FC test was utilized to evaluate cognitive function [38]. The FC test consisted of a training phase at 24 h prior to ORIF surgery 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, a 20 s 70-dB tone (conditional stimulus) was delivered, followed by an interval of 25 s. After the interval, an 0.70 mA electrical foot shock was delivered to the rat for 2 s (unconditional stimulus). After six pairs of conditional-unconditional stimuli, the rats learned the association and had 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 placement of the next 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 behaviour (without any movements) was analysed by using the ANY-Maze Video Tracking System (Stoelting, Illinois, USA). The percentage of time spent exhibiting freezing behaviour was calculated using the formula of 100*f/5 min, where f was the total of freezing time within 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 [38]. Thus, the results in this experiment were used to assess hippocampus-dependent memory.
Nissl staining
On days 1 and 7 after ORIF, rats (n=8/group) were first anaesthetized with 10% thiobutabarbital (100 ml/kg, i.p.). Rats were perfused with saline before the heart stopped, followed by perfusion with 4% paraformaldehyde solution. Then, the brain was taken out and fixed in 4% paraformaldehyde for 24 h. Coronal slices (3.0-mm thick) from each brain containing the dorsal hippocampus and the medial dorsal prefrontal cortex were dehydrated and embedded in paraffin. A series of 10-μm-thick coronal sections was obtained from each slice, and the sections were stained with cresyl violet [49]. For each brain, five sections at the dorsal hippocampus located at coordinates -3.14 from the bregma to -4.52 from bregma were analysed for Ammon's horn pyramidal cell counts [50]. Sections were examined by an observer who was blinded to the experimental conditions under light microscopy at a magnification of 200x. The number of surviving neurons in a 30,000 μm2 area of the CA1 was counted in each section. Only pyramidal neurons showing normal morphology with distinct cytoplasmic and nuclear outlines and a visible nucleolus were counted. Analysis of the data was performed by using Image Pro Plus 6.0 software (Media Cybernetics Co., USA).
Western blotting
On days 1 and 7 after ORIF, rats (n=8/group) were sacrificed with sodium pentobarbital (240 mg/ml, Department of Pharmacy, Tianjin Medical University General Hospital, i.p., 800 mg/kg) [51]. After ensuring that the heart of the rat had stopped, the brain was removed, and the hippocampal tissue was separated. The 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 supernatant was determined using a bicinchoninic acid (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, the membranes were blocked by 5% skim milk Tris-buffered saline containing 0.1% Tween (TBST) buffer for 90 min and washed with TBST buffer for 5 min. The membranes were incubated with the following 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), and anti-β-actin (1:10,000, Proteintech, Wuhan, China) overnight at 4℃. After washing with TBST 5 times (each for 5 min), the 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. The membranes were again washed 5 times (each for 5 min) 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 the expression of anti-pan-cadherin (1:2,000, Sigma) and anti-β-actin (1:10,000, Proteintech).
Statistical analysis
The data were analysed using SPSS 20.0 software (IBM Corp., Armonk, NY, USA). Data are presented as the mean ± standard deviation (SD). Behavioural data were tested using a two-way analysis of variance (ANOVA) with repeated measures. Other data were analysed using a one-way ANOVA with Tukey post hoc comparisons. P < 0.05 was the criterion for statistical significance.