Male adult Sprague-Dawley rats (n=144), aged ~3 months and weighing 220-250 g, were included in present study. All efforts were made to mini-mize the number of animals used in the experiments. The rats were fed standard chow and water, and were housed under standard experimental conditions (temperature, 20-25°C; humidity, 50-70%) under a 12-h light/dark cycle. All rats were fasted with access to water overnight prior to the experiments. The rat model of hypercapnia/hypoxemia was established as described in our previous study (6). To minimize suffering and distress, the rats were anesthetized with 2% pentobarbital sodium (30 mg/kg by intraperitoneal injection) followed by mechanical ventilation. The tidal volume (9 ml/kg body weight), respiratory rate (45 breaths/min), and inspiratory:expiratory ratio (1:1) were fixed. The rats were randomly divided into four groups according to different concentrations of O₂ and CO₂ as follows: Sham group (exposed to air), hypercapnia group (exposed to 5% CO₂), hypoxemia group (exposed to 16% O₂), and hypercapnia + hypoxemia group (HH group; exposed to 16% O₂ mixed with 5% CO₂). These concentrations (16% O₂ and 5% CO₂) were used to maintain the PO₂ of arterial blood at ~60 mmHg and the pH at 7.20-7.25. The right femoral artery was cannulated to collect arterial blood samples. The PO₂, PCO₂ and pH of the arterial blood samples were immediately measured by a Blood Gas/Electrolyte Analyzer (Model 5700; Werfen Corporation).

The rats that were used for western blotting and immu-nofluorescence staining were not subjected to invasive manipulation apart from ventilation. All animals were ventilated for 3 h, after which time they were euthanized by intraperitoneal injection of pentobarbital sodium (150 mg/kg).

At 3 h after ventilation, 2% Evans blue (EB) solution (4 ml/kg) was injected through the caudal vein. After 1 h, the rats were perfused transcardially with normal saline to remove the intravascular dye, and 4% paraformaldehyde was used to perfuse the brain. The brains were harvested and incubated in formamide (1 ml/100 mg) at 60°C for 24 h. The supernatant was separated after centrifugation at 12,000 x g at 4°C for 20 min. The optical density (OD) values were measured at 620 nm using a spectrophotometer (Multiskan FC IVD; Thermo Fisher Scientific, Inc.).

Whole blood was donated by 4 healthy male volunteers (mean age ± standard deviation, 35±9.4 years). None of the volunteers had a history of cancer, hematological disorders, infections, autoimmune diseases, transplantation, or use of immunosuppressive drugs. The whole blood was cultured as reported previously (21). Briefly, 2 ml of whole blood were collected from each volunteer. Subsequently, the whole-blood samples were mixed with 18 ml RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., cat. no. 8118329), which was supplemented with 10% human serum (Gemini Bio-Products; cat. no. H91S001) and 1% penicillin-streptomycin solution (Aladdin Biochemical Technology Co., Ltd.; cat. no. P113150). The blood samples were randomly divided into four groups as follows: Control, high concentration of CO₂ (HC), hypoxia, and hypoxia + + HC groups. The control group was exposed to 5% CO₂ + 20% O₂. The HC group was exposed to 10% CO₂ + 20% O₂. The hypoxia group was exposed to 5% CO₂ + 0.2% O₂. The hypoxia + HC group was exposed to 10% CO₂ + 0.2% O₂. These concentrations (0.2% O₂ and 10% CO₂) were used to maintain the PO₂ of the medium at ~60 mmHg and the pH at 7.20-7.25. The PO₂, PCO₂ and pH of the medium were measured by a Blood Gas/Electrolyte Analyzer (Model 5700; Werfen Corporation).

The levels of IL-1β were evaluated using ELISA kits (applied to human blood cultures, cat. no. ab100562; applied to rat blood, cat. no. ab100768; Abcam) following the manufacturers' instructions. Briefly, the samples and standards were added to the plate wells coated by IL-1β antibodies labeled with horseradish peroxidase (HRP). Subsequently, a TMB substrate solution was pipetted to the wells. Then, stop buffer was added, and the OD was measured spectrophotometrically at a wavelength of 450 nm. The concentration of IL-1β in the samples was determined by comparing the OD of the samples to the standard curve.

Primary cultures of RBECs were prepared as previously described (22–24). Briefly, the gray matter was isolated from the brain of 3-week-old Sprague-Dawley rats. The gray matter was minced into tiny particles, digested with DMEM containing collagenase type 2 (1 mg/ml), gentamycin (50 μg/ml), and 300 μl DNase (15 μg/ml) at 37°C for 1.5 h, then neutralized with bovine serum albumin (Calbiochem-Novabiochem Corp.), and finally centrifuged at 1,000 x g at 4°C for 20 min. The cells were further digested in DMEM containing collagenase-dispase (1 mg/ml) and DNase (6.7 μg/ml) for 1 h at 37°C. Microvessel endothelial cell clusters were separated on a 33% continuous Percoll gradient, then collected and washed twice with DMEM before plating into collagen type IV‑ and fibronectin‑coated dishes. RBECs were cultured in DMEM/F12 supplemented with 10% plasma-derived serum, heparin (100 μg/ml), basic fibroblast growth factor (1.5 ng/ml), transferrin (5 μg/ml), insulin (5 μg/ml), sodium selenite (5 ng/ml), puromycin (4 μg/ml) and gentamycin (50 μg/ml) (RBEC medium I) at 37°C in a humidified incubator with 5% CO₂/95% air for 2 days. On the following day, RBECs were cultured in RBEC medium II (RBEC medium I without puromycin). The medium was changed every other day from the 4th day onwards. The RBECs were randomly divided into four groups as follows: Control, IL-1β, IL-1β + IL-1 receptor antagonist (IL-1Ra), and IL-1Ra groups. The control group was treated with 0.01 M PBS. The IL-1β group was treated with IL-1β (40 ng/ml; MedChemExpress; cat. no. HY-P7028). The IL-1β + IL-1Ra group was treated with IL-1β (40 ng/ml) and IL-1Ra (40 ng/ml; MedChemExpress; cat. no. HY-P7029). The IL-1Ra group was treated with IL-1Ra (40 ng/ml) (25).

Total proteins from the hippocampal tissue samples and RBECs (n=4 for each group) were extracted using a Total Protein Extraction kit (BestBio; cat. no. BB-3101-100T). Equal amounts (40 μg) of proteins from each sample were separated in a 10% SDS-PAGE gel and transferred to PVDF membranes (EMD Millipore), which were then blocked with 5% non-fat milk for 1 h at room temperature. Subsequently, the membranes were incubated overnight at 4°C with primary antibodies against the following target proteins: IL-1R1 (1:1,000, Abcam; cat. no. ab106278), ZO-1 (1:1,000; Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. SL258826), occludin (1:1,000; Abcam; cat. no. Ab216327), claudin-5 (1:1,000; Abbkine Scientific Co., Ltd.; cat. no. Abp50990), t‑IRAK‑1 (1:1,000; Abcam; cat. no. ab238) and p-IRAK-1 (1:1,000; Abcam; cat. no. ab218130). On the following day, HRP-labeled goat anti-rabbit antibody (1:3,000; Cell Signaling Technology, Inc.; cat. no. 7074S) was added and the membranes were incubated for 2 h at 4°C. The immunoblots were visualized using a chemiluminescence kit (Bioworld Technology, Inc.; cat. no. AC36131), and detected by an imaging densitometer (ImageQuant LAS 500, GE Healthcare Bio-Sciences AB). The gray value was quantified using FluorChem 8900 software (version 4.0.1, Alpha Innotech Corporation). β-actin was used as the control. The relative density was calculated through dividing the gray value of β-actin by that of the target protein. Both the target protein and β-actin were from the same lane of the same membrane.

In vivo, the rats were anesthetized with 2% pentobarbital sodium (30 mg/kg by intraperitoneal injection) and transcardially perfused with saline and 4% paraformaldehyde at 3 h after ventilation. The brains were harvested and post‑fixed overnight at 4°C in 4% paraformaldehyde. Subsequently, the tissue samples were dehydrated in graded sucrose and cut into 10-μm sections. The sections were blocked in 5% normal donkey serum (Abbkine Co., Ltd. cat. no. BMS0140) for 0.5 h at room temperature. In vitro, the coverslips with adherent RBECs were fixed with 4% paraformaldehyde for 20 min at room temperature at 24 h after treatment. The coverslips were blocked in 5% normal donkey serum for 0.5 h at room temperature sequentially.

Subsequently, the sections/coverslips were incubated overnight at 4°C with the following primary antibodies: IL‑1R1 (1:100, Abcam; cat. no. ab106278), ZO-1 (1:100; Invitrogen; Thermo Fisher Scientific Inc.; cat. no. SL258826), occludin (1:100; Abcam; cat. no. Ab216327), claudin-5 (1:100; Abbkine Scientific Co., Ltd.; cat. no. Abp50990), and CD31 (1:100; Abcam; cat. no. Ab24590). The sections/coverslips were washed on the following day, the secondary antibodies Alexa Fluor^® 549 goat anti-rabbit IgG (H + L) (1:100; Invitrogen; Thermo Fisher Scientific Inc.; cat. no. ATRJN1301) and Alexa Fluor^® 488 goat anti-mouse IgG (1:100; Invitrogen; Thermo Fisher Scientific Inc.; cat. no. ATRMR2301) were added, and the sections/coverslips were incubated for 1 h at room temperature. Finally, the sections/coverslips were mounted using fluorescent mounting medium with DAPI (Sigma-Aldrich; Merck KGaA; cat. no. SLBW4468) and examined using a fluorescence microscope (DP73, Olympus Corporation). At a magnification of x400. Four fields of view per section/coverslip were examined in each group.

The statistical analysis was performed using SPSS v.19.0 (IBM Corp.). All values are expressed as mean ± standard deviation. Student's t-test was used to analyze the data of two-group univariate-factor measurements. One-way analysis of variance (ANOVA) was used to analyze the data of 3 or more group univariate-factor measurements followed by Tukey's post hoc test. Repeated measures ANOVA was used to analyze the repeated measurement data. Factorial ANOVA was used for the interaction effects. When an interaction was examined, simple effects analyses were evaluated. P<0.05 was considered to indicate statistically significant differences.

The PO₂ levels in the arterial blood were maintained at ~60 mmHg in the HH and hypoxemia groups, and were significantly decreased compared with those in the Sham group (HH vs. Sham: P<0.01; hypoxemia vs. Sham: P<0.01) and the hypercapnia group (HH vs. hypercapnia: P<0.01; hypoxemia vs. hypercapnia: P<0.01). The PO₂ levels of the HH group were not significantly different when compared with those of the hypoxemia group (P>0.05; Fig. 1A-a). The PCO₂ levels in the arterial blood were maintained at 60-70 mmHg in the HH and hypercapnia groups, and were significantly higher compared with those in the Sham group (HH vs. Sham: P<0.01; hypercapnia vs. Sham: P<0.01) and the hypoxemia group (HH vs. hypoxemia: P<0.01; hypercapnia vs. hypoxemia: P<0.01). There was no significant difference between the HH and hypercapnia groups in the PCO₂ levels (P>0.05; Fig. 1A-b). The pH of the arterial blood was maintained at 7.20-7.25 in the HH and hypercapnia groups, and was significantly lower compared with that in the Sham group (HH vs. Sham: P<0.01; hypercapnia vs. Sham: P<0.01) and the hypoxemia group (HH vs. hypoxemia: P<0.01; hypercapnia vs. hypoxemia: P<0.01). There was no significant difference in the pH levels between the HH and hypercapnia groups (P>0.05; Fig. 1A-c).

The PO₂ levels were maintained at ~60 mmHg in the hypoxia + HC and hypoxia groups, and were significantly decreased compared with those in the control group (hypoxia + HC vs. control: P<0.01; hypoxia vs. control: P<0.01) and the HC group (hypoxia + HC vs. HC: P<0.01; hypoxia vs. HC: P<0.01). The PO₂ levels of the hypoxia + HC group were not significantly different when compared with those of the hypoxia group (P>0.05; Fig. 1B-a). The PCO₂ levels were maintained at ~80 mmHg in the hypoxia + HC and HC groups, and were significantly higher compared with those in the control group (hypoxia + HC vs. control: P<0.01; HC vs. control: P<0.01) and the hypoxia group (hypoxia + HC vs. hypoxia: P<0.01; HC vs. hypoxia: P<0.01). There was no significant difference in the PCO₂ levels between the hypoxia + HC and HC groups (P>0.05; Fig. 1B-b). The pH was maintained at 7.20-7.25 in the hypoxia + HC and HC groups, and was significantly decreased compared with that in the control group (hypoxia + HC vs. control: P<0.01; HC vs. control: P<0.01) and the hypoxia group (hypoxia + HC vs. hypoxia: P<0.01; HC vs. hypoxia: P<0.01). There was no significant difference in the pH levels between the hypoxia + HC and HC groups (P>0.05; Fig. 1B-c).

An interaction effect was observed between hypoxia treatment and hypercapnia treatment (P<0.01; Fig. 2A). Simple effects analyses found increased IL-1β expression in the hypoxemia group (P<0.01), but not in the hypercapnia group (P>0.05) compared with the Sham group. The HH group exhibited the highest expression levels of IL-1β when compared with the hypoxemia group (P<0.01) and the hypercapnia group (P<0.01; Fig. 2B).

An interaction effect was observed between 10% CO₂ treatment and 0.2% O₂ treatment (P<0.01; Fig. 2C). Simple effects analyses found increased IL-1β expression in the hypoxia group (P<0.01), but not in the HC group (P>0.05) compared with the control group. The hypoxia + HC group exhibited the highest expression levels of IL-1β compared with the hypoxia group (P<0.01) and the HC group (P<0.01; Fig. 2D).

An interaction effect was observed between hypoxia treatment and hypercapnia treatment (P<0.05; Fig. 3B). Simple effects analyses found increased IL-1R1 expression in the hypoxemia group (P<0.05), but not in the hypercapnia group (P>0.05) when compared with the Sham group. The expression levels of IL-1R1 were the highest in the HH group compared with those in the hypoxemia group (P<0.01) and the hypercapnia group (P<0.01; Fig. 3C). Double immunofluorescence was used to examine IL-1R1 expression in cerebrovascular endothelial cells. Enhanced IL‑1R1 immunofluorescence was observed in the hypoxemia group, but not in the hypercapnia group compared with the Sham group. The HH group exhibited the most intense IL-1R1 fluorescence compared with the hypoxemia and hypercapnia groups (Fig. 3D).

IL-1R1 expression was increased in the IL-1β group compared with that in the control group (P<0.01; Fig. 4B). Double immunofluorescence was used to examine IL-1R1 expression in RBECs. Enhanced IL‑1R1 immunofluorescence was observed in the IL-1β group compared with that in the Sham group (Fig. 4C). Increased p-IRAK-1 expression was observed in the IL-1β group compared with that in the control group (P<0.01). The protein expression of p‑IRAK‑1 was significantly suppressed with IL-1Ra pretreatment (P<0.05; Fig. 4E).

Significant interaction effects were observed between hypoxia treatment and hypercapnia treatment (ZO-1: P<0.05; occludin: P<0.05; and claudin-5: P<0.05; Fig. 5B-D). Simple effects analyses found decreased expression of tight junctional proteins in the hypoxemia group (ZO-1: P<0.01; occludin: P<0.01; and claudin-5: P<0.05), but not in the hypercapnia group (ZO-1: P>0.05; occludin: P>0.05; and claudin-5: P>0.05) compared with the Sham group. The HH group exhibited the lowest expression levels of tight junctional proteins in comparison with the hypoxemia group (ZO-1: P<0.01; occludin: P<0.05; and claudin-5: P<0.05) and the hypercapnia group (ZO-1: P<0.01; occludin: P<0.01; and claudin-5: P<0.01; Fig. 5E). Double immunofluorescence was used to examine tight junctional protein expression in cerebrovascular endothelial cells (Figs. S1-S3). Reduced ZO-1, occludin and claudin-5 immunofluorescence was observed in the hypoxemia group, but not in the hypercapnia group, when compared with the Sham group. The HH group exhibited the weakest ZO-1, occludin and claudin‑5 fluorescence compared with the hypoxemia and hypercapnia groups.

The IL-1β group exhibited decreased tight junctional protein expression compared with the control group (ZO-1: P<0.01; occludin: P<0.01; and claudin-5: P<0.01). The tight junctional protein expression was significantly upregulated with IL-1Ra pretreatment (ZO-1: P<0.01; occludin: P<0.01; and claudin-5: P<0.05; Fig. 6). Double immunofluorescence was used to examine tight junctional protein expression in RBECs (Figs. S4-S6). Reduced ZO-1, occludin and claudin‑5 immunofluorescence was observed in the IL-1β group compared with that in the control group. Of note, ZO‑1, occludin and claudin‑5 fluorescence was enhanced with IL-1Ra pretreatment in RBECs.

An interaction effect was observed between hypoxia treatment and hypercapnia treatment (P<0.01; Fig. 7A). Simple effects analyses found increased extravasation of EB in the hypoxemia group (P<0.01), but not in the hypercapnia group (P>0.05) compared with the Sham group. The extravasation of EB was the most prominent in the HH group compared with the hypoxemia group (P<0.01) and the hypercapnia group (P<0.01; Fig. 7B).