All animal experiments performed in this study complied with the National Institutes of Health guidelines for the handling of laboratory animals (32) and were approved by the Ethics Committee of the Wuxi Medical College of Anhui Medical University (approval no. YXLL-2020-112). All experiments were conducted on healthy adult male C57BL/6J mice (age, 8–10 weeks; weight, 22–25 g; Anhui Medical University, Hefei, China). The mice were housed in animal care facilities under environmentally controlled temperature (25±2°C) and humidity (55±5%) with 12-h light/dark cycles, and had free access to food and water. The 60 mice were divided into four major groups (15 animals/group): Sham, TBI, TBI + DEX and TBI + DEX + Rap.

The TBI model was established in strict accordance with the Feeney weight-drop model of focal injury (33,34). Briefly, the mice were anesthetized with an intraperitoneal injection of 1% sodium pentobarbital (40 mg/kg) and then placed in a brain stereotaxic apparatus. The rectal temperature was maintained at 37±0.5°C during the operation using a heating pad. Then, a burr hole was made in the left hemisphere at the following coordinates: 0.2 mm posterior, 1 mm lateral and 2.2 mm below the horizontal plane of the bregma. The bone flap was removed to expose the dura mater. The dura was placed under a weight-drop device with an impact sensor. A metal (weight 240 g, tip diameter 3 mm) was dropped from 1 cm above the dura onto the dura mater through a catheter. Then, the scalp was closed and the mice were removed from the apparatus. Finally, the hole was covered with medical bone wax. The animals in the Sham group received similar surgical procedures but without weight-drop impact. At 72 h following TBI, the mice were sacrificed with 100 mg/kg sodium pentobarbital via i.p. injection. Mortality was confirmed by observing respiration and by using the corneal reflection method.

DEX was purchased from Jiangsu Hengrui Medicine Co., Ltd., dissolved in 0.9% sterile saline and administered i.p. at a dose of 20 µg/kg 2 h after recovery (12) (Fig. 1A). A dose of 2 mg/kg rapamycin (Selleck Chemicals; dissolved in 2% DMSO) was administered i.p. 30 min before TBI induction in the TBI + rapamycin + DEX group.

The severity of brain injury was evaluated by determining neurological function 72 h after TBI using a previously described neurological grading system (15). The neurological scores of the animals in each group were evaluated by an independent observer. The scoring system consisted of motor, sensory, reflex and balance tests. The neurological scores (mNSS scores) ranged from 0 to 18 points and were calculated by adding the scores together; all mice in each group underwent a behavioral assessment and a higher score represented worse neurological function. All mouse behavior scores were recorded by the same independent observer who was blinded to the study groups.

The severity of brain edema was evaluated by measuring the brain water content using the standard wet-dry method, as previously reported (18,31,35). The mice were sacrificed 72 h after TBI and the entire brain was harvested and separated into the ipsilateral and contralateral cortices, ipsilateral and contralateral basal ganglia and cerebellum (wet weight). Then, brain specimens from each group were dehydrated at 105°C for 24 h to acquire the dry weight. The percentage of brain water content was equal to (wet weight-dry weight)/wet weight ×100%.

Evans blue extravasation was performed as previously described (36). Briefly, mice were anesthetized by 1% sodium pentobarbital (50 mg/kg) injection 72 h after TBI. Evans blue dye (2%, 5 ml/kg; Sigma-Aldrich; Merck KGaA) was injected into the left femoral vein over 2 min and circulated for 60 min. Then, the mice were sacrificed with 100 mg/kg sodium pentobarbital via i.p. injection and with phosphate-buffered saline (PBS) intracardial perfusion. Mortality was identified by observing respiration and by using the corneal reflection method. The brains were removed and quickly divided into the left and right cerebral hemispheres, weighed, homogenized in saline and centrifuged at 15,000 × g for 30 min at room temperature. Subsequently, the resultant supernatant was added with an equal volume of trichloroacetic acid, incubated overnight at 4°C and centrifuged at 15,000 × g for 30 min at room temperature. Next, the resultant supernatant was collected and spectrophotometrically quantified at 610 nm for Evans blue dye.

IL-1β (cat. no. ab197742; Abcam), IL-6 (cat. no. ab222503; Abcam), TNF-α (cat. no. ab208348; Abcam) and NF-κB (cat. no. ab176663; Abcam) levels were measured using ELISAs according to the manufacturer's instructions.

The nonfluorescent diacetylated 2′,7′-dichlorofluorescein (DCF-DA) probe (Sigma-Aldrich; Merck KGaA), which becomes highly fluorescent upon oxidation, was used to evaluate intracellular ROS production according to the manufacturer's instructions.

Malondialdehyde (37) levels were detected with a lipid peroxidation (37) assay kit (Ex/Em 532/553 nm, cat. no. ab118970; Abcam) according to the manufacturer's instructions.

A TUNEL assay was conducted to assess neuronal death in the hippocampus. The TUNEL reaction mixture (50 µl) was added to each sample and the slides were incubated in a humidified chamber for 60 min at 37°C in the dark. The slides were then incubated with DAPI (0.1 mg/ml) for 5 min at room temperature in the dark to stain the nuclei, followed by imaging with a fluorescence microscope. The procedure was performed with a TUNEL staining kit according to the manufacturer's instructions (cat. no. TUN11684817, Roche Diagnostics GmbH). A negative control (without the TUNEL reaction mixture) was used. The apoptotic index (%) was calculated as the ratio of the number of TUNEL-positive cells/total number of cells ×100. The cell count was confirmed in four randomly selected high-power fields and the data obtained from each field were averaged (magnification, ×400).

Western blot analyses were performed as previously described (18). Briefly, cerebral cortex samples were collected, homogenized and total protein was extracted using RIPA buffer (CoWin Biosciences). A BCA Protein Assay kit (Beyotime Institute of Biotechnology) was used to measure protein concentrations with the bicinchoninic acid method. Total protein (30 µg) was separated via 12% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% nonfat milk at room temperature for 1 h. The membranes were then incubated with the following primary antibodies overnight at 4°C: Rabbit anti-βactin (1:1,000; rabbit polyclonal; cat. no. ab8227), rabbit anti-caspase-3 (1:2,000; rabbit polyclonal; cat. no. ab184787), rabbit anti-Nrf2 (1:1,000; rabbit polyclonal; cat. no. ab31163), rabbit anti-heme oxygenase (HO)-1 (1:1,000; rabbit polyclonal; cat. no. ab13243), rabbit anti-Beclin-1 (1 µg/ml, rabbit monoclonal; cat. no. ab62557) and rabbit anti-LC-3B (1 µg/ml, rabbit monoclonal; cat. no. ab48394; all from Abcam). After washing the membranes with TBST (0.5% Tween-20) three times at room temperature for 20 min, HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies (1:2,000; cat. no. 7074s; Cell Signaling Technology, Inc.) were applied and the membranes were incubated with the secondary antibodies at room temperature for 1.5 h. The protein bands were detected using a Bio-Rad imaging system (Bio-Rad Laboratories, Inc.) and quantified with ImageJ software (version 1.52; National Institutes of Health).

The data are reported as the means ± standard error of mean. SPSS v14.0 (SPSS, Inc.) and GraphPad Prism 6 (GraphPad Software, Inc.) software were used for the statistical analyses. Student's t-test (unpaired) was used if two groups were compared and one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test was used if two independent variables were compared. For nonnormally distributed data and/or data with a nonhomogeneous variance, the Kruskal-Wallis test followed by Dunn's post hoc test was used. P<0.05 was considered to indicate a statistically significant difference.

The modified neurological severity score (mNSS) was calculated to evaluate neurological deficits and the brain water content determined using the wet-dry method at 72 h after TBI to evaluate brain damage and clarify the neuroprotective effect of DEX on TBI. TBI significantly increased the neurological scores and DEX administration significantly improved neurological function (Fig. 1B). Similar results were obtained for brain water content, which were increased significantly after TBI and was alleviated by DEX treatment (P<0.05; Fig. 1C).

Neuronal apoptosis and BBB permeability are the main factors that lead to EBI following TBI. Therefore, a TUNEL assay was used to evaluate the level of cell death in TBI mice treated with and without DEX at 72 h after model construction. The expression levels of apoptosis-related proteins were detected using Western blotting. Evans blue extravasation was analyzed to clarify BBB permeability. The results revealed more hippocampal neuronal death following TBI and DEX decreased neuronal apoptosis (Fig. 2A). The western blot results also indicated that DEX reduced the expression levels of the apoptosis-related protein caspase-3 (Fig. 2B-D). Compared with the sham and TBI groups, DEX significantly decreased the extent of damage to the blood-brain barrier (Fig. 2E). Based on these results, DEX exerted neuroprotective effects following TBI.

As previous studies have identified a vital role for neuroinflammation in EBI following TBI, increased neuroinflammation aggravates EBI (12,20,22,24). The inflammatory complex induces the secretion of pro-inflammatory cytokines, including IL-1β, IL-6 and TNF-α and the subsequent activation of pro-inflammatory signaling through NF-κB to initiate pyroptosis. Therefore, the hippocampal levels of IL-1β, IL-6, TNF-α and NF-κB were measured using ELISAs. The levels of the pro-inflammatory cytokines were increased significantly following TBI, while their levels decreased significantly following DEX treatment (Fig. 3A-D). Hence, these results suggested that DEX exhibited potent anti-inflammatory activity against TBI-induced neuroinflammation.

To clarify whether autophagy serves an important role in TBI and the regulatory effect of DEX on autophagy, the levels of the autophagy-related proteins Beclin-1 and LC3 in each group were detected using western blotting (Fig. 4A). The levels of both Beclin-1 and LC3 were increased following TBI but decreased significantly following DEX treatment (Fig. 4B). LC3 is a biomarker for autophagy activation and LC3 was chosen to mark autophagic cells. Immunofluorescence staining showed few LC3-positive neurons in the hippocampus of the Sham group, but were widespread in the hippocampus following TBI induction. However, the number of LC3-positive neurons decreased following DEX administration (Fig. 4C).

Rapamycin is a specific activator of autophagy (18). Mice were pretreated with rapamycin before the induction of TBI to investigate the relationship between autophagy and the neuroprotective effect of DEX. Pretreatment with rapamycin dramatically enhanced neurological deficits (Fig 5A), aggravated brain edema (Fig 5B) and BBB permeability (Fig 5C) and reversed the neuroprotective effect of DEX. Additionally, the TUNEL assay also showed that rapamycin also significantly increased neuronal apoptosis in the injured hippocampus compared with the TBI + DEX group (Fig 5D). Levels of autophagy-related proteins and apoptosis-related proteins were detected using western blotting (Fig. 5E). DEX significantly decreased the levels of caspase 3, Beclin-1 and LC3 (Fig. 5F) but these changes were partially blocked by rapamycin administration. Thus, rapamycin activated autophagy, abolished the anti-autophagy effects of DEX and reversed the neuroprotective effects of DEX on TBI.

AKT/mTOR is a core signaling pathway of cell autophagy. According to previous studies, activation of the AKT/mTOR signaling pathway is partially dependent on ROS production (38,39). The present study explored whether autophagy inhibition by ROS occurred through the ROS/Nrf2 signaling pathway following DEX treatment. ROS levels were detected with a DCF-DA probe and the degree of membrane lipid peroxidation evaluated by measuring the MDA content. The levels of ROS and MDA were significantly increased following TBI but decreased following DEX treatment (Fig. 6A and B). The levels of the Nrf2 and HO-1 protein were detected by performing western blotting to investigate neuronal autophagy (Fig. 6C). The levels of Nrf2 and HO-1 were decreased significantly in the TBI group and increased following DEX administration (Fig. 6D and E). Thus, these results showed that DEX may inhibit TBI-induced autophagy by regulating the ROS/Nrf2 signaling pathway.