The Institutional Animal Care and Use Committee of Hebei Medical University (Shijiazhuang, China) approved all experiments, which were performed according to the guidelines of the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publications no. 80–23, revised 1978; NIH, Bethesda, MD, USA). All efforts were made to minimize the number of animals used and their suffering. A total of 75 male Sprague Dawley rats, weighing 280–320 g (6–8 weeks), were supplied from the Experimental Animal Center of Hebei Medical University (Shijiazhuang, Hebei, China). All animals were housed in plastic boxes at a temperature of 22–24°C, 50% humidity and were provided food and water ad libitum under a 12-h reversed light-dark cycle.

The TBI model was produced using a modified weight-drop device (9). Following 10% chloral hydrate anesthesia (3 ml/kg), a midline longitudinal incision was performed to expose the skull between bregma and lambda suture lines. A steel disk (diameter, 10 mm; thickness, 3 mm) was adhered to the skull using dental acrylic. Animals were moved onto a foam mattress underneath a weight-drop device where a weight of 450 g fell freely through a vertical tube from 1.5 m onto the steel disk. Sham-operated animals underwent the same surgical procedure without weight-drop impact. Rats were placed on heat pads (37°C) for 2–4 h to maintain normal body temperature during the recovery period.

All rats were randomly arranged into 3 groups as follows: Sham group (n=25); TBI group (n=25); and TBI + quercetin group (Que; n=25). Quercetin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany; dissolved in 0.9% saline solution) was administered intraperitoneally at a dose of 50 mg/kg at 30 min, 12 and 24 h following the TBI insult.

In addition, 15 rats (5/group) underwent behavioral testing. All investigations were blinded and the animal groupings were revealed only at the end of the behavioral and histological analyses.

Brain water content was determined at day 1, 3 and 5 following TBI (45 rats, 15/group). In order to reduce the use of animal population, at 5 days after TBI, 15 rat brains were taken from rats which had completed the behavioral experiments. Rat brains were separated and weighed immediately with a chemical balance to obtain the wet weight (WW). Following drying in a desiccating oven for 24 h at 100°C, dry tissues were weighed again to obtain the constant dry weight (DW). The percentage of water in the tissues was calculated according to the following formula: brain water (%)=[(WW-DW)/WW] ×100.

The neurobehavioral status of the rats was evaluated at day 1, 3 and 5 after TBI using a set of 10 tasks, collectively termed the Neurological Severity Score (NSS) (10), which test reflexes, alertness, coordination and motor abilities. A point is awarded for failure to perform a particular task; therefore, a score of 10 reflects maximal impairment, whereas a healthy rat scores 0. Post-injury, NSS was evaluated at day 1, 3 and 5. Each animal was assessed by an observer who was blinded to the treatment group of the animal. The difference between the initial NSS (performed at day 1) and that at any subsequent time point was calculated for each rat, and this value (ΔNSS) reflects the spontaneous or treatment-induced recovery of motor function.

At 24 h post-TBI, 15 rats (5/group) were anesthetized as described above, and perfused intracardially with isotonic sodium chloride solution, followed by 4% (w/v) paraformaldehyde in 0.1M sodium phosphate buffer (pH=7.4). The brains were removed and fixed for 48 h in 4% (w/v) paraformaldehyde at 22–24°C. Following fixation, brains were embedded in paraffin, and sliced into 6 µm coronal sections at the level of the bregma and stained with hematoxylin (20 min) and eosin (3 sec) at 22–24°C. The staining was visualized by light microscopy at ×400 magnification (Olympus Corporation, Tokyo, Japan). The surviving and dying neurons per mm² cortex were quantified (the nuclei of dead cells were shrunk and thickened).

As for HE staining, the brain tissues were fixed, embedded and cut into 6 µm slices. Sections were deparaffinized with xylene and rehydrated at 60°C with graded ethanol (100, 95, 90, 80 and 70%). Endogenous peroxidase activity was blocked using 3% hydrogen peroxide for 30 min at room temperature, followed by 5% normal goat serum (AR0009; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 1 h to block non-specific protein interactions. The sections were subsequently incubated overnight at 4°C with an anti-activated caspase3 antibody (1:500; AB2302; Abcam, Cambridge, UK). Following three washes with PBS, the slides were incubated with a biotinylated goat anti-mouse horseradish peroxidase conjugated secondary antibody (1:100, BA1051; Wuhan Boster Biological Technology Ltd.) for 2 h at room temperature. The sections were washed with PBS again, and incubated with the kit-provided horseradish peroxidase (HRP)-streptavidin for 30 min at room temperature. The peroxidase reaction was visualized using 0.05% diaminobenzidine + 0.01% hydrogen peroxide. Immunohistochemical procedures were performed in accordance with the manufacturer's protocols. The positive cells were visualized by a microscope at ×100 magnification.

The rats were deeply anesthetized as described above 24 h following TBI. The cortical region of the rat brain was rapidly isolated. The segments were immediately stored at −80°C for further analysis. Total protein samples were extracted from brain tissues using whole cell lysis buffer (WD2072; Beyotime Institute of Biotechnology, Shanghai, China) a bicinchoninic acid protein assay kit (P10310; Beyotime Institute of Biotechnology) was used to determine the protein concentration of each sample. The homogenate was heated to 100°C for 10 min and centrifuged again at 15,294 × g for 1 min at 22–24°C. Equal amounts (80 µg) of protein were subjected to Tris-HCl SDS-PAGE on 8 and 12% gels (Bio-Rad Laboratories, Inc., Hercules, CA, USA) for 30 min at 70 V and 60 min at 120 V. Following electrophoresis, the proteins were transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA) at 300 mA for immunoblotting. Following blocking with 5% skimmed milk for 2 h at room temperature, membranes were incubated overnight at 4°C with the following primary antibodies: Rabbit anti-activated Caspase3 (AB2302; 1:1,000; Abcam), rabbit anti-Akt (AB81283; 1:1,000; Abcam), rabbit anti-p-Akt (AB38449; 1:1,000; Abcam), rabbit anti-ERK1/2 (AB17942; 1:1,000; Abcam), rabbit anti-p-ERK1/2 (AB214362; 1:1,000; Abcam), and rabbit anti-β-actin (AB227387; 1:5,000; Abcam). Following three washes in TBS-Tween 20, membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (BL003A; 1:5,000; EMD Millipore) for 2 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence kit (Beyotime Institute of Biotechnology). Band density was quantified via detection with a DNR Micro Chemi chemiluminescence gel imaging system (DNR Bio-Imaging Systems Ltd., Neve Yamin, Israel). Each band density was normalized to the density of β-actin.

SPSS software version 16.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. A statistical evaluation of the data was performed using a one-way analysis of variance, followed by post hoc comparisons using the least significant difference or Kruskal-Wallis method. All experimental data are expressed as the mean ± standard error of the mean, and P<0.05 was considered to indicate a statistically significant difference.

Following injury, brain edema leads to an elevation in intracranial pressure, reducing cerebral perfusion pressure and brain oxygenation. Edema is associated with the resultant pathology following TBI (11). In order to evaluate the effects of quercetin on brain edema, the wet-dry weight method was used in the present study to evaluate brain edema at day 1, 3 and 5 after TBI. As presented in Fig. 1, cerebral water content was significantly increased at day 1, 3 and 5 after TBI compared with the sham group (P<0.01). However, treatment with quercetin attenuated this increased compared with the TBI model group (P<0.05).

Motor deficit recovery was expressed as ΔNSS in present study. Alterations in the functional recovery of rats at day 1, 3 and 5 are depicted in Fig. 2. It was observed that rats exhibited marked motor deficits following TBI. Post-injury administration of quercetin significantly improved the motor function between day 1 and 5 after trauma compared with the TBI group (P<0.05).

Cortical regions of brains were collected and neuronal survival was assessed at 24 h via H&E staining. As presented in Fig. 3, the nuclei of normal neurons were round and stained pale, whereas nuclei of dying neurons were pyknotic and darkly stained following TBI. The survival rate of neurons in the quercetin-treated group was significantly improved compared with that of the TBI group (P<0.05).

In order to assess the effect of quercetin on neuronal apoptosis following TBI, immunohistochemical and western blot analyses were used to assess alterations in caspase3 expression, respectively. As depicted in Fig. 4A, representative photomicrographs exhibited a high density of caspase3-positive cells in the TBI group compared with the sham group at 24 h. However, the expression of activated caspase3-positive cells notably decreased in the quercetin treatment group. In addition, western blot analysis revealed that, compared with the sham group, the protein expression levels of activated caspase3 increased significantly in the TBI group at 24 h (P<0.01), and the levels of caspase3 exhibited a significant downregulation at the same time point following treatment with quercetin (P<0.05; Fig. 4B and C).

Western blot analysis was performed to investigate the expression of Akt and p-Akt at 24 h after TBI in the 3 groups. As presented in Fig. 5, the level of p-Akt was increased following TBI compared with that in the sham group (P<0.05). Additionally, administration of quercetin produced a significant elevation of p-Akt (P<0.01). No significant difference in total Akt protein expression was observed among the 3 groups.

Western blot analysis was performed to investigate the expression of ERK1/2 and p-ERK1/2 at 24 h after TBI in the 3 groups. As presented in Fig. 6, the level of p-ERK1/2 was increased significantly post-TBI, compared with the sham group (P<0.01). However, the administration of quercetin produced a significant attenuation of p-ERK1/2 levels compared with the TBI group (P<0.01). No significant difference in total ERK1/2 protein expression was observed among the 3 groups.