A total of 280 male C57BL/6 mice (age, 10–12 weeks; weight, 22±5 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and housed in groups of five per cage under a temperature of 23±2°C and humidity of 55±5%. They were maintained on a 12 h light/dark cycle with free access to food and water. The experimental protocols were approved by the Ethics Committee of Southern Medical University (Yancheng, China). The mice were familiarized to the housing conditions for a week prior to surgery.

The mice were randomly divided into six groups: Normal group (without any operation, n=32), sham group (with sham operation, n=64), and four TBI groups at 6 h (n=56), 1 day (n=32), 3 days (n=32), and 7 days (n=64) post-operation. Every group further was randomly divided into two equal groups: Without HBO and with HBO. According to preliminary analysis, there was no significant difference among sham groups at different time points (data not shown). Thus, the authors collected samples from mice belonging to the sham group 7 days following the sham operation.

The controlled cortical impact (CCI) of TBI was used. Briefly, mice were anesthetized with isoflurane and oxygen provided via a nose-cone mask, and then placed in a stereotactic frame. A 3 mm-diameter craniotomy was performed over the left parietal cortex adjacent to the central suture between bregma and lambda. Injury was delivered using a 2.5 mm-diameter tip with a velocity of 5.0 m/s and a depth of 1.2 mm. The temperature of mice was maintained at 37±0.5°C during the experiment. Sham-operated mice were anesthetized and only the left parietal craniotomy was made.

HBO treatment was given for 1 h at 2.0 ATA in an experimental model hyperbaric chamber of transparent acrylic plastic (701 Space Research Institute, Beijing, China). The chamber was pressurized with 100% oxygen at a rate of 2 l/min.

The mice without HBO were placed in the same chamber breathing room air. To minimize the effects of biological rhythm differences, all treatments were initiated at 2:00 pm. For repeated treatments, HBO was conducted once daily for 7 consecutive days.

The beam-balancing test was used for neurological deficit scoring, as previously reported (16). Briefly, mice (16 mice from the normal group and the TBI at 6 h, 1 and 3 days groups; 32 mice from the sham group and the TBI at 7 days group) were placed on a narrow wooden beam (6 mm wide and 120 mm in length). The status of maintaining balance was measured up to 60 sec: 0, walking easily and turning around freely; 1, maintaining a stable posture during 60 sec; 2, hugging or hooking the beam with its limb before 60 sec; and 3, falling off of the beam.

Cerebral edema (n=8) was quantified using the wet-dry method. Briefly, tissue from the ipsilateral cortex centered upon the impact site was immediately weighed (wet weight), then dehydrated at 65°C for 2 days. The sample was reweighed as dry weight. The percentage of tissue water content was calculated according to the following equation: % H₂O = [(wet weight - dry weight)/wet weight] × 100%.

The levels of IL-1β (cat. no. MLB00C) and IL-18 (cat. no. 7625) in tissues surrounding the cortical contusion site (n=8) were performed by the specific ELISA kits according to the manufacturer's instructions (R&D Systems, Inc., Minneapolis, MN, USA).

Total RNA was extracted from the ipsilateral cortex (n=6) using TRIzol^® reagent according to the manufacturer's protocol (Thermo Fisher Scientific, Inc., Waltham. MA, USA). Up to 1 µg mRNA was reversed transcribed using the PrimeScript RT Master Mix kit (Takara Bio, Inc., Otsu, Japan) with a reaction volume of 20 µl for 60 min at 42°C. The reaction was terminated by heating at 75°C for 5 min. qPCR was performed on cDNA using the ABI 9600 Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.), with a reaction volume of 10 µl. The sequences of the primers used were as follows: IL-1β (NM_008361.3) sense, 5′-TCATTGTGGCTGTGGAGAAG-3′ and antisense, 5′-AGGCCACAGGTATTTTGTCG-3′; IL-18 (NM_008360.1) sense, 5′-ACTGTACAACCGCAGTAATAC-3′ and antisense, 5′-AGTGAACATTACAGATTTATCCC-3′; NLRP-3 (NM_145827.3) sense, 5′-CCCTTGGAGACACAGGACTC-3′ and antisense, 5′-GAGGCTGCAGTTGTCTAATTCC-3′; ASC (NM_023258.4) sense, 5′-AGAGTACAGCCAGAACAGGA-3′ and antisense, 5′-GATGGAACAAAGCTGAAGAG-3′; caspase-1 (NM_009807.2) sense, 5′-TATCCAGGAGGGAATATGTG-3′ and antisense, 5′-ACAACACCACTCCTTGTTTC-3′; GAP DH (NM_008084.2) sense, 5′-AACGACCCCTTCATTGAC-3′ and antisense, 5′-TCCACGACATACTCAGCAC-3′. Amplification conditions were as follows: Initially at 95°C for 5 min, and then at 95°C for 5 sec and at 58°C for 30 sec. This loop was repeated for a total of 38 cycles. The experiment was repeated three times, and the 2^−∆∆Cq method was used for quantification of relative gene expression (17).

The homogenate from the pericontusional cortex (n=6) was centrifuged (12,000 × g, 10 min, 4°C) and the total supernatant protein was measured using a bicinchoninic acid assay kit (Pierce; Thermo Fisher Scientific, Inc.). The protein sample concentration was adjusted to 100 µg and then separated by 10% SDS-PAGE before transferring the protein to nitrocellulose membranes which were blocked (TBS containing 10% skim powder and 0.05% Tween-20) for 1 h. The membranes were incubated with primary antibodies: anti-NLRP-3 (cat. no. AG-20B-0014-C100; 1:200; AdipoGen Corporation, San Diego, CA, USA), anti-ASC (cat. no. sc-514414; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-caspase-1 (cat. no. AB1871; 1:1,000; Merck KGaA, Darmstadt, Germany), anti-IL-1β (cat. no. I3767; 1:1,000; Sigma-Aldrich; Merck KGaA) and anti-β-actin (cat. no. A5316; 1:1,000; Sigma-Aldrich; Merck KGaA) in TBS-T overnight at 4°C. Following incubation with the following horseradish peroxidase-conjugated secondary antibodies: Rabbit anti-mouse immunoglobulin (Ig) G H&L (cat. no. ab6728; 1:1,000; Abcam, Cambridge, UK), goat anti-rabbit IgG H&L (ab6721; 1:1,000; Abcam) and rabbit anti-goat IgG H&L (ab6741; 1:1,000; Abcam) for 1.5 h at room temperature, protein bands were visualized using enhanced chemiluminescence western blotting detection reagents (Pierce; Thermo Fisher Scientific, Inc.) in a gel image analysis system (Tanon Science & Technology Co., Ltd., Shanghai, China).

Mice (n=4) were anaesthetized with pentobarbital sodium (80 mg/kg), and then perfused with 40 ml saline and 40 ml 4% paraformaldehyde. Following fixing in 4% paraformaldehyde at 4°C overnight, the brains were maintained in 20% sucrose for 3 days, and in 30% sucrose for 3 days for dehydration. Coronal sections (30 µm) of the pericontusional cortex were sliced using a freezing microtome (Leica CM1950; Leica Microsystems GmbH, Wetzlar, Germany). For double immunofluorescence, the sections were incubated overnight at 4°C with the following primary antibodies: Anti-NLRP-3 (1:200; cat. no. AG-20B-0014-C100; AdipoGen Corporation) and anti-neuronal nuclei (NeuN; cat. no. MAB377, 1:1,000; EMD Millipore, Billerica, MA, USA); anti-NLRP-3 and anti-glial fibrillary acidic protein (GFAP; cat. no. G6171, 1:1,000; Sigma-Aldrich; Merck KGaA); anti-NLRP-3 and anti-ionized calcium binding adaptor molecule 1 (Iba1; cat. no. 019-19741; 1:1,000; Wako Pure Chemical Industries, Ltd., Osaka, Japan). Sections were then incubated for 1.5 h at room temperature with the following fluorescent secondary antibodies: Alexa Fluor 594-conjugated goat anti-rabbit IgG (cat. no. R37117; 1:200; Invitrogen; Thermo Fisher Scientific, Inc.) and Alexa Fluor 488-conjugated goat anti-mouse IgG (cat. no. A-11001; 1:200; Invitrogen; Thermo Fisher Scientific, Inc.). Cell nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI; cat. no. D9542; 1:500; Sigma-Aldrich; Merck KGaA) for 5 min at room temperature. Stained sections were examined under a fluorescence microscope (Nikon Optical TE2000-S; Nikon Corporation, Tokyo, Japan).

Results were presented as mean ± standard error of the mean and analyzed with one-way analysis of variance followed by post hoc Tukey's test using SPSS software (version 10.0; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To evaluate neurological functional deficits, the beam-balancing test was conducted at different time points (6 h, 1 day, 3 days and 7 days) following TBI. As presented in Fig. 1A, normal (receiving no operation) and sham (receiving sham operation) mice demonstrated no significant movement deficits regardless of HBO treatment. TBI can cause a significant impairment in beam walk performance. The score of beam balancing was highest at 6 h post-TBI and then gradually deceased during 7 days following the operation, indicating that a spontaneous recovery from motor deficits occurred (Fig. 1A). Mice treated with HBO 1 h following TBI presented a significant amelioration of motor deficits compared to mice without HBO (Fig. 1A; P<0.05). Thus, HBO was effective for alleviating TBI-induced motor deficits.

The development of posttraumatic cerebral edema following TBI exacerbates neurological deficits. As presented in Fig. 1B, TBI operation significantly increased the levels of brain water content during a 7-day observation period. Mice with HBO 1 h following TBI presented a significant reduction in water content, when compared to mice without HBO (Fig. 1B; P<0.05). Thus, HBO can protect the brain parenchyma against cerebral edema post-TBI.

Time-course ELISA experiments (Fig. 2) demonstrated that the TBI operation induced the production of IL-1β and IL-18 significantly higher in sham groups over an observation period of 6 h-7 days, when compared without normal controls. The increase in cortical IL-1β levels rapidly reached a peak at 6 h and gradually decreased to the control level over a 7-day observation period (Fig. 2A), while the levels of IL-18 were gradually increased over the 7 days of operation (Fig. 2B). HBO treatment inhibited the production of these pro-inflammatory cytokines, both IL-1β and IL-18, during the observation period. As a result, two time points of 6 h and 7 days post-TBI were chosen for further experiments.

To investigate the effects of HBO treatment on the NLRP-3 inflammasome, the authors initially examined the mRNA levels of NLRP-3 inflammasome components, including NLRP-3, ASC and caspase-1. As indicated in Fig. 3, the mRNA expression of NLRP-3 (Fig. 3A), ASC (Fig. 3B) and caspase-1 (Fig. 3C) was significantly increased in the pericontusional cortex 6 h and 7 days following TBI operation. In addition, the levels of these NLRP-3 inflammasome components at 7 d post-TBI were elevated compared with those at 6 h post-TBI. HBO treatment inhibited the increase in NLRP-3 inflammasome mRNA expression levels in TBI-operated mice brain, irrespective of whether it was 6 h or 7 days post-operation (Fig. 3).

The authors further evaluated the protein expression of NLRP-3, ASC and cleaved caspase-1 in the pericontusional cortex 6 h and 7 days following TBI operation. The expression of NLRP-3 protein was weak in the sham group and increased significantly 6 h and 7 days following TBI operation (P<0.05; Fig. 4A), which was suppressed by HBO treatment (P<0.05). Similar trends were also observed in the expression of ASC (P<0.05; Fig. 4B) and cleaved caspase-1 (P<0.05; Fig. 4C).

To clarify which cell type is primarily involved in the activation of the NLRP-3 inflammasome following TBI operation, the authors conducted double immunofluorescence labeling using NLRP-3/NeuN, NLRP-3/GFAP and NLRP-3/Iba1 in the 7 days group. As indicated in Fig. 5A, mice post-TBI had a loss of NeuN staining cells and an enhanced accumulation of NLRP-3 expression in the pericontusional cortex compared with the contralateral cortex. Furthermore, although both astrocytes and microglia were activated significantly, only microglia was accompanied with the strong expression of NLRP-3 in the pericontusional cortex. These results indicated that the activation of the NLRP-3 inflammasome was located in microglia in the cortex following TBI. Furthermore, HBO treatment significantly reduced the immunofluorescence staining of NLRP-3 in microglia (Fig. 5B).