Ethical approval for all experiments were granted by the 900th Hospital Ethics Committee (Fuzhou, China, 2020/50) and conducted under strict supervision. Adult male Sprague-Dawley rats, ranging between 280 and 320 g, were purchased from the Animal Experimental Center of 900th Hospital. All animals were housed at room humidity > 50% and temperature (24–26 °C) with free access to water and food.
Experimental model and grouping
All rats were randomized into several different groups as follows: a sham group, a CVO group, an ASDH group, and an ASDH+CVO group (CVO operating 6 h after establishment of the ASDH model). After modeling, the groups were then divided into three subgroups: a 1-day group (n=24), a 3-day group (n=6), and a 7-day group (n=6). Six rats in each group were used for neurological evaluation and the measurement of brain water content; six rats in the one-day group were sacrificed for histological studies. The remaining rats were used for immunofluorescence detection and Western blot studies. Another 24 rats with the ASDH+CVO model were used for pharmacological treatment. The ASDH rat model was made using the modified Miller method[18, 19]. First, anaesthesia was induced using 4% isoflurane, and then maintained with 2% isoflurane delivered by a small-animal anaesthetic machine. Every rat was fixed in the stereotactic frame. A cranial hole 2 mm in diameter was drilled 4 mm to the right of the sagittal suture and 4 mm posterior to the coronal suture. Using an operating microscope, the dura was incised. Then, nonheparinized venous blood (0.4 ml) was injected into the subdural space by a blunt Olive Tip 8-gauge needle, the hole was sealed, and the scalp was sutured. The injection procedure lasted over 7 min. In the sham group, holes were drilled in the sham group, but nothing was injected. The CVO rat model was established by ligating the retro-orbital venous plexus and petrosquamosal fissure venous plexus where the intracranial venous sinus is interconnected supraorbitally with the branches of facial vein and the superficial temporal vein, and returns to the external jugular venous system. To validate the model, we monitored cortical cerebral blood flow (CBF) using laser speckle imaging as described previously.
XPro1595(Xencor Inc., Monrovia, United States) was administered intravenously once, at a dose of 10 mg/kg, 30 minutes after ASDH+CVO model (n=12). Saline was used as the vehicle. another group rats were injected with simple saline as a control.
Assessment of neurological injury
Neurological injury was assessed using modified neurological severity scores (mNSSs), which consist of evaluating the motor, sensory, and reflex examinations of rats. Neurological injury was recorded when when the corresponding reflex was lost or a task was completed unsuccessfully. The mNSS test was graded on a scale of 0-18, in which a score of 0 indicated normal performance and a total score of 18 points indicated severe neurological deficits, 1-6 indicated mild injury, 7-12 indicated mean-moderate injury, and 13-18 indicated severe injury. Neurological deficits were measured 1, 3, and 7 days after the experiment by investigators who were blinded to the group information.
Measurement of brain water content
Brain water content was measured using the wet weight-dry weight method. After assessment of neurological injury, the rats were sacrificed by giving a lethal overdose of anesthesia, and the brains were removed immediately. the cerebral cortex was isolated at the edge of the bone (2 mm around craniotomy; 200 ± 20 mg), excess blood and cerebrospinal fluid were removed with the help of filter paper. A digital scale was used to measure wet weight; samples were then placed in an oven to dry for 24h at 100°C. Dry weight was measured again for each sample. Brain water content was calculated according to the formula: Brain water content (%) = (total wet weight of brain–dry weight of brain)/total wet weight of brain × 100%.
Evans blue staining of the brain
Measuring the extravasation of Evans blue was used to investigate BBB permeability. Evans blue was intravenously injected 2 h before the rats were sacrificed 1 day after modelling. Rats were transcardially perfused with PBS immediately after sacrifice. The brains were quickly removed and homogenized in a solution with 1 mL 50% trichloroacetic acid. Then, the samples were centrifuged. The absorption of the supernatant was calculated via a spectrophotometer at a wavelength of 620 nm. The quantity of Evans blue was measured according to a standard curve and expressed as micrograms of Evans blue/g of brain tissue.
Transmission electron microscopy
Sodium pentobarbital (1%) was intraperitoneally injected to anaesthetize the rats deeply one day after modelling. Cardiac perfusion was performed with 4% paraformaldehyde, and then the brain tissue was carefully separated and fixed in a combination of 1.5% paraformaldehyde and 3% glutaraldehyde for 24 h, then followed by 1% osmic acid for 2 h. The samples were embedded conventionally and sliced for uranyl acetate and lead citrate staining. Then, the sections were observed by a transmission electron microscope. The vascular lumen and endothelial cells were localized at a low magnification, and TJs were observed at a high magnification.
Cortical tissues under the compress of subdural hematoma were embedded in paraffin after formaldehyde-fixed and cut into 4μm thick sections. Slices went through xylene dewaxing, a graded series of alcohol, and a citric acid buffer protocol for antigen retrieval. For immunofluorescence, the sections were incubated overnight at 4 °C with antibodies against ADAM17(1:200, Abcam, Cambridge, UK), ionized calcium-binding adapter molecule (Iba-1) (1:200; Santa Cruz, CA, USA), glial fibrillary acidic protein (GFAP) (1:200, Abcam). After washing, the sections were then incubated with secondary goat anti-rabbit IgG antibodies (Alexa Fluor 488, 1:200, Invitrogen, NY, USA) for 1 h at room temperature. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The positive cells in five selected ROIs were calculated under a microscope at 400 magnification by a pathologist who was blinded to the experimental groups.
Enzyme-linked immunosorbent assay (ELISA)
Inflammatory factors of brain tissue were measured using ELISA kits for TNF-α, IL-1β, IL-6, IL-10, and HMGB1 (Boster Biotech, Wuhan, China). According to the manufacturer’s instructions, samples and standards were sequentially incubated with respective monoclonal antibodies, biotinylated anti-rat antibody, and then horseradish peroxidase. The detected OD values were transformed into a concentration value.
Western blotting analysis
Brain tissues were rapidly dissected in ice-cold phosphate-buffered saline, and western blotting was performed as described previously. We extracted proteins with radioimmunoprecipitation assay (RIPA) lysis buffer. 30μg of the proteins were loaded on a gel and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to polyvinylidene difluoride membranes and probed with primary antibodies against ADAM17, MMP9(1:500, Abcam), ZO-1(40-2200, Thermo Fisher Scientific, USA), Occludin (33-1500, Thermo Fisher Scientific), Iba-1, phosphorylated (p)-IκB, and NF-κB p65 (1:200, Cell Signaling Technology), followed by incubation with appropriate horseradish peroxidase-conjugated IgG (1:5000, Abcam) secondary antibodies. Chemiluminescent signals were acquired using the Millipore ECL Western Blotting Detection System (Millipore, Billerica, MA, USA). Expression levels were normalized against β-actin (1:5000, Abcam).
The statistical analyses were processed via SPSS 20.0 statistical software (IBM SPSS, Inc., Chicago, USA). The quantitative data were presented as the mean ± standard deviation. Different groups were compared by one-way analysis of variance (ANOVA), and Student-Newman-Keuls tests were used for post hoc multiple comparisons. Values with a p < 0.05 were considered statistically significant.