2.1 Animals

Male Sprague-Dawley rats (weight, 280-300 g) were obtained from the Animal Center of Fujian Medical University, Quanzhou, China. The animals were maintained in controlled temperature (37.0 ± 0.5°C) conditions on a 12 hours/12 hours light/dark cycle. All rats were maintained on an ad libitum diet for 1 week before the experiment. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Fujian Medical University and were in accordance with the guide of the Care and Use of Laboratory Animals by the National Institutes of Health.

2.2 TBI model

The rats were placed in a stereotaxic frame after being anaesthetized by exposure to isoflurane. A 2 cm midline scalp incision was made to expose the skull. Then, a 6 mm hole was made over the left parietal cortex, and the centre of the hole was 2.5 mm lateral to the midline on the mid-coronal plane. The dura was not torn during the operation. Then, a 40 g weight was released onto the dura from 25 cm along a stainless steel string. The scalp wound was sutured, and the animals were placed back in their cages. Sham animals received the same procedures except that they were not injured by releasing the weight.

2.3 Experimental design

Part 1: A total of 33 rats were used in this experiment. Three rats died after the operation and were excluded. A total of 24 rats were divided into four groups post-TBI, and rats in three groups were killed at 6, 12, 24, 48 and 72 hours after TBI (n = 6 each). Then, six rats in each group were subjected to Western blot analysis at peak SIRT1 expression (n = 6 each).

Part 2: Rats (112 rats were used, and 22 rats died) were divided into five groups (n = 18 each) to determine the role of SIRT1 after TBI: Sham group; TBI group; TBI + vehicle group; TBI + activator 3 (A3) group and TBI + sirtinol group. All animals were killed 24 hours post-TBI after neurological function measurement. The rats were evaluated for neurological function and cerebral oedema, and they were subjected to Western blotting and a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay.

Part 3: Seventy-two rats (101 rats were used, and 11 rats died) were placed in the following groups (n = 18 each) to explore the relationship between SIRT1 and neuroinflammation: Sham group; TBI group; TBI + vehicle group; TBI + activator 3 (A3) group and TBI + sirtinol group. The animals were killed 24 hours post-TBI for Western blot and TUNEL assay.

2.4 Drug administration

All drugs were dissolved in a vehicle (0.9% saline + 3% dimethyl sulphoxide) (Sigma, St. Louis, MO, USA). Sirtinol (2 mmol/L, 30 μL/kg) or vehicle was injected intraperitoneally 2 hours before TBI. A3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was diluted to 5 mg/kg. The rats were intraperitoneally injected with A3 30 minutes after TBI. The drug dose was confirmed in a previous study.¹⁷

2.5 Western blot analysis

Protein extracts were separated by 10% or 15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% non-fat milk for 2 hours, and then they were incubated overnight at 4°C with SIRT1 (1:200, Santa Cruz Biotechnology), Bcl-2 (1:200, Santa Cruz Biotechnology), Cleaved caspase-3 (1:1000, Cell Signaling Technology, Danvers, MA, USA), Bax (1:200, Santa Cruz Biotechnology), caspase-3 (1:400, Cell Signaling Technology), ac-NF-кB (1:400, Cell Signaling Technology), IL-6 (1:200, Santa Cruz Biotechnology), TNF-α (1:200, Santa Cruz Biotechnology) and β-actin (1:5000, Bioworld Technology, St. Louis Park, MN, USA) in blocking buffer. After three washes with TBST for 15 minutes each, the membranes were subsequently incubated in secondary antibody conjugated with horseradish peroxidase (HRP) (1:1000, Bioworld Technology) at room temperature for 2 hours. The protein bands were exposed to the Tanon-5200 Chemiluminescent Imaging System, and strip grey levels were quantified by software (version 4.62; Bio-Rad Laboratories).

2.6 Immunohistochemical staining

Brain tissue samples were cut to 5 μm after fixation in formalin for 24 hours. The sections were incubated overnight at 4°C with primary antibody against SIRT1 (1:50, Santa Cruz Biotechnology). After three 15 minutes washes in PBS, the sections were incubated with HRP-conjugated IgG (1:500; Bioworld Technology) for 60 minutes. After three washes for 30 minutes each, counterstaining was performed with diaminobenzidine and haematoxylin. Control tissue was not exposed to the primary antibody.

2.7 TUNEL analysis

Apoptotic cells were determined using a TUNEL detection kit (Roche Inc, Indianapolis, IN, USA). The sections were incubated at 37°C with TUNEL reaction fluid for 60 minutes. The slides were washed three times with PBS for 45 minutes, followed by blocking with 10% goat serum in 0.1 mol/L Tris for 15 minutes. The slides were covered with slide glass and mounting medium after three washes. DNA was fixed with streptavidin-HRP peroxidase (1:40 dilution) and dyed with DAB Chromogen. The apoptotic cells were atrophied with a crimped brown nucleus according to the microscopic analysis (ZEISS HB050 inverted microscope; Carl Zeiss, Thornwood, NY, USA). The positive cells were monitored and analysed by two observers blinded to the experiment. TUNEL-positive cells from each sample were selected for quantification. The average percentage of the four aspects was regarded as the data for analysis.

2.8 Brain water content

Animals were anaesthetized as described above. The brain stem and cerebellum were removed, and the left cerebral hemisphere was separated. The wet weight (ww) of fresh tissue was determined after removing the brain. The hemispheres were parched at 80°C for 72 hours, and dry weight (dw) was measured in succession. The percentage of brain water content was calculated by the following formula: ((ww – dw)/ww).

2.9 Neurobehavioural evaluation

Neurological deficit was evaluated 24 hours and 3 days after TBI using the beam-walking test. The investigators performed seven different tasks to test motor function, balance and alertness. One point was given for failing on all tasks; 0 = minimum deficit and 7 = maximum deficit. The severity of injury was defined by the initial grade and evaluated 1 hour after TBI, and it is a reliable predictor of the final outcome. The behavioural test was carried out by two independent investigators who had no prior knowledge of the experimental groups.

2.10 Statistical analysis

All statistical analyses were performed with SPSS 17.0 software (SPSS Inc, Chicago, IL, USA). Data are expressed as mean with standard deviation, and the differences were analysed using analysis of variance and Tukey's test. A P-value <.05 was considered significant.

3.1 Expression of SIRT1 in the brain cortex after TBI

Western blot was performed to evaluate the time course of SIRT1 expression after TBI. The SIRT1 level peaked at 24 hours and remained high until 48 hours following TBI (Figure 1). Significant variation was detected among the Sham, 24 hours and 48 hours groups.

3.2 Effects of sirtinol on SIRT1 expression and neuroinflammation under TBI conditions

As shown in Figure 2A, the expression of SIRT1 increased significantly after TBI compared with that in the sham group. On the contrary, sirtinol administration significantly reduced the level of SIRT1. Similarly, immunohistochemical staining (Figure 2B) revealed elevation of the percentage of SIRT1-positive neurons in the cerebral cortex after TBI, which decreased after sirtinol administration.

To investigate the effect of sirtinol on SIRT1 expression, the levels of NF-кB, IL-6 and TNF-α were evaluated by Western blot (Figure 2B,C). Compared with the sham group, the levels of NF-кB, IL-6 and TNF-α were significantly increased after TBI. After sirtinol treatment, the NF-кB, IL-6 and TNF-α expression levels were further promoted compared with those in the TBI + vehicle group.

3.3 Effects of sirtinol on brain oedema and neurological function

The brain water content was significantly increased after TBI (Figure 3C). Further, sirtinol restrained SIRT1 and further raised the water content of the cerebral cortex compared with that in the TBI + vehicle group.

After 24 hours of TBI, the scores in the TBI and TBI + vehicle groups were lower than that in the Sham group. After sirtinol administration, the neurological score was significantly promoted than that in the TBI + vehicle group (Figure 3C). The sirtinol-treated group showed neurological deficits compared with the TBI + vehicle group.

3.4 Effects of sirtinol on neuronal apoptosis

TUNEL-positive cells were detected, a few positive neurons in the rat brain, in the Sham group (Figure 3A,B). There were much more TUNEL-positive neurons in the TBI and TBI + vehicle groups. However, the percentage of TUNEL-positive neurons was raised after sirtinol treatment.

Compared with the TBI + vehicle group, sirtinol administration significantly elevated cleaved caspase-3 and Bax, and deduced the level of Bcl-2. In addition, the percentage of caspase-3-positive neurons was significantly increased after sirtinol administration compared with that in the TBI + vehicle group (Figure 4).

3.5 Effects of A3 on the expression of SIRT 1, neuronal apoptosis, brain water content and neurological function

As shown in Figure 5, Western blot and immunohistochemical staining revealed that A3 treatment significantly increased the level of SIRT1 compared with that in the TBI + vehicle group. Western blot presented that A3 administration could impair the Bax level and raise the level of Bcl-2 (Figure 6). Further, neuronal apoptosis was reduced following A3 administration (Figure 6A). Results of brain oedema and neurological function showed that administration of A3 could markedly alleviate the damage of neurologic behaviour and brain oedema compared with that in the TBI + vehicle group (Figure 6D,G).