A total of 138 adult male Sprague-Dawley rats (2.5 months old, 350–400 g) were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China) and this was approved by the Institutional Experimental Animal Care and Use Committee of Nanjing Medical University (Nanjing, China). All animals were kept in a standard and comfortable laboratory environment at ~25°C and a relative humidity of 70%, with a 12 h light/dark cycle and free access to food and water for 10 days prior to the experiments. The 138 rats were randomly assigned to one of three experimental groups: The control group (n=18), the sham-surgery group (n=60) and the DAVF group (n=60). Rats in the DAVF group were subjected to intracranial VH, rats in the sham-surgery group were subjected to a similar procedure but without intracranial VH and rats in the control group did not undergo any surgery. A total of 18 rats from the sham-surgery and DAVF groups at each time point (1, 4 and 7 days following surgery) and 18 control rats were used for western blotting, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and analysis of brain water content. The remaining 6 rats from the sham-surgery and DAVF groups were sacrificed at day 1 post surgery and used for immunofluorescence staining for Nrf2. The present study was approved by the Ethics Committee of Nanjing Medical University (Nanjing, China).

A DAVF rat model was produced according to a method described previously by Shin et al (24), which was partly modified. Briefly, rats were anesthetized by intraperitoneal injection of 50 mg/kg pentobarbital sodium (CAS57-33-0; Haling Biological Technology, Shanghai, China). An incision was made via the front middle cervix and then the left common carotid artery (CCA) and the left external jugular vein (EJV) were separated and exposed. Subsequently, the CCA and the EJV were crosscut following clamping using temporary aneurysm clips. An end-to-end anastomosis was performed between the near-end of CCA and the encephalic end of EJV using 11-0 medical sutures. The residual end of the CCA and EJV were cauterized by bipolar coagulation. Subsequently, the draining vein of the transverse sinus was separated, exposed and cauterized. The skull was removed over the sagittal sinus and the wall of the sinus was incised and filled with surgical hemostatic material to form thrombosis of the sagittal sinus. Surgery incisions were sutured using 6-0 nylon sutures. The sham group received cervical medial, post-aurem and frontal incision and suture without the induction of VH. Rats in the control group did not undergo surgery.

The brain tissue at the coronal level 4 mm on either side to the occluded sinus was collected, homogenized and lysed in radioimmunoprecipitation assay buffer [1% NP40, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM EGTA, 1 mM EDTA, 1 mM Na₃VO₄, 0.5 mM dithiothreitol, 20 mM NaF, 1 nM phenylmethylsulfonyl fluoride and protease inhibitor cocktail in PBS (pH 7.4)]. To extract nuclear and cytoplasmic proteins, a nuclear and cytoplasmic protein extraction kit (Beyotime Institute of Biotechnology, Nantong, China) was used following the manufacturer's protocol. Protein concentration was determined using a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA) following the manufacturer's protocol. Equal amounts of total protein (50 µg) were loaded per lane, separated using 8% SDS-PAGE and transferred to PVDF membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 3% skimmed milk for 2 h at room temperature. Subsequently, membranes were incubated with primary antibodies against Nrf2 (cat. no. ab137550; 1:500), NQO-1 (cat. no. ab34173; 1:1,000), HO-1 (cat. no. ab13243; 1:1,000) and histone H3 (cat. no. ab1791; 1:1,000), all purchased from Abcam (Cambridge, MA, USA), and β-actin (cat. no. sc-130657; 1:2,000) purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) at 4°C overnight. Membranes were then washed three times with washing buffer (tris-buffered saline containing 0.05% Tween-20) and incubated with goat anti-rabbit horseradish peroxidase (HRP)-conjugated IgG (cat. no. 7074S; 1:2,000; Cell Signaling Technology, Inc., Danvers, MA, USA) at room temperature for 2 h. Membranes were washed using washing buffer for 15 min, visualized using immobilon Western chemiluminescent HRP substrate (EMD Millipore) and exposed to X-ray film (Fuji Hyperfilm, Tokyo, Japan). Quantification of band density was performed using the UN-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA).

To obtain the brain tissues, animals were deeply anesthetized with pentobarbital sodium and the cerebral cortex was harvested, immediately frozen in liquid nitrogen and stored at −80°C until use. Total RNA was isolated from brain tissues at the coronal level 4 mm either side of the occluded sinus using TRIzol reagent (Takara Biotechnology Co., Ltd., Dalian, China) following the manufacturer's protocol. The quantity and purity of the extracted total RNA were determined at the optical densities of 260 and 280 nm using a NanoDrop^™ 2000c (Thermo Fisher Scientific, Inc.). In order to avoid RNA degradation, part of the RNA was reverse transcribed to cDNA immediately using a PrimeScript™ RT reagent kit (Takara Biotechnology Co., Ltd.). qPCR was performed using a previously described method (25). The qPCR reaction mixture was prepared according to the SYBR^® Premix Ex Taq™ kit protocol (Takara Biotechnology Co., Ltd.), which contained diluted cDNA, SYBR-Green I Nucleic Acid Stain, 0.2 µM of each gene-specific primer and nuclease-free water to a final volume of 25 µl. The primer sequences used in the present study were as follows: Nrf2 forward, 5′-TCAGCGACGGAAAGAGTATGA-3′ and reverse, 5′-CCACTGGTTTCTGACTGGATGT-3′; NQO1 forward, 5′-ATGGTCGGCAGAAGAGC-3′ and reverse, 5′-GGAAATGATGGGATTGAAGT-3′; HO-1 forward, 5′-TCTCCGATGGGTCCTTACACTC-3′ and reverse, 5′-GGCATAAAGCCCTACAGCAACT-3′; and β-actin forward, 5′-CTGAATGGCCCAGGTCTGAG-3′ and reverse, 5′-AAGTCAGTGTACAGGCCAGC-3′. β-actin was selected as an endogenous reference ‘housekeeping’ gene. Relative changes in target mRNA expression following surgery was determined using the 2^−∆∆Cq method (26).

For immunofluorescence, consecutive coronal sections were cut at 4-µm intervals from the hippocampus and the cortex near the sagittal sinus thrombosis in the DAVF and the sham groups. Following routine deparaffinization and 2 h blocking in 10% normal goat serum (Wuhan Boster Biological Technology, Ltd., Wuhan, China) in PBS at room temperature, sections were incubated overnight with primary antibodies for Nrf2 (cat. no. ab137550; 1:100, Abcam) at 4°C. On the second day, the slides were washed three times with PBS and incubated with Alexa Fluor 594^®-conjugated Goat anti-Rabbit IgG secondary antibodies (cat. no. A-11037; 1:200; Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Subsequently, slides were washed three times with PBS, counterstained with DAPI for 2 min at room temperature and covered with mounting media. Fluorescence was observed using an inverted microscope (Olympus IX71; Olympus Corporation, Tokyo, Japan) and analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

The change of water content in the brains of rats subjected to DAVF was detected using a previously reported method (27). Briefly, the rat brain was removed and placed a cooling brain matrix for 24 h at 1, 4 and 7 days after surgery. Following separation and removal of the brainstem and cerebellum, ipsilateral brain hemisphere tissue was reserved and weighed up as wet weight (ww). Subsequently, the brain was kept in a drying oven at 80°C for 3 days to dehydrate and weighed up to obtain the dry weight (dw). At last, the brain water content was calculated using the following formula: [(ww-dw)/ww] ×100%.

At least three separate experiments were performed for each measurement and the data were expressed as the mean ± standard deviation. All analyses were performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Data were analyzed by one-way analysis of variance followed by Tukey's post-hoc test and P<0.05 was considered to represent a statistically significant difference.

All rats in the DAVF group survived following surgery. To determine the secondary brain edema induced by DAVF, brain water content was measured 1, 4 and 7 days after surgery. As presented in Fig. 1, no significant difference was detected in brain water content between the sham-surgery group and the control group at each time point (1, 4 and 7 days). However, there was a significant aggravation of cerebral edema 1 day post-surgery in the DAVF group compared with the sham-surgery group at the same time point and compared with the control group (P<0.05). Additionally, 4 days following surgery, the brain water content in the DAVF group decreased but remained significantly higher than in the sham-surgery and control groups (P<0.05). Brain water content in the DAVF group returned to normal levels 7 days post surgery.

The expression of Nrf2 in the cortex and hippocampus of rat brain was detected by immunofluorescence 1 day following surgery. As depicted in Fig. 2, in the cortex of the rat brain, only a small number of Nrf2-stained cells were observed in the sham-surgery group, whereas a larger number of Nrf2-stained cells were observed in the DAVF group. Similar results were observed in the hippocampus (Fig. 2).

Nrf2 translocation is the primary mechanism by which the Nrf2-ARE signaling pathway is activated; thus the expression of Nrf2 in the nucleus was determined using western blot analysis. As presented in Fig. 3A and B, the results demonstrated that there was no significant difference in the expression of nuclear Nrf2 at each time point (1, 4 and 7 days) between the sham-surgery and the control groups. However, following DAVF surgery, the expression of nuclear Nrf2 significantly increased and reached a peak 1 day following surgery (P<0.001). It began to gradually decrease but remained significantly higher 4 days after surgery compared with the control group (P<0.01). Nuclear Nrf2 expression returned to normal 7 days post-surgery. The expression of cytoplasmic Nrf2 was also measured (Fig. 3C and D) and the results were consistent with those for nuclear Nrf2. Subsequently, the expression of the Nrf2 downstream target proteins HO-1 and NQO1 was also measured. It was demonstrated that HO-1 and NQO1 expression was significantly upregulated 1 and 4 days following DAVF surgery compared with the sham-surgery and control groups (P<0.05; Fig. 3E and F).

The aforementioned results indicate that DAVF activates the Nrf2/ARE signaling pathway by increasing the expression of Nrf2 protein in the nucleus and cytoplasm. To determine whether DAVF surgery altered the expression of Nrf2/ARE mRNA, RT-qPCR was performed. As demonstrated in Fig. 4A, no significant differences in Nrf2 mRNA expression were observed between the sham-surgery and control groups at each time point (1, 4 and 7 days). However, the expression of Nrf2 mRNA significantly increased 1 day following DAVF surgery compared with the control and sham-surgery groups (P<0.01). Nrf2 mRNA expression decreased gradually 4 days post-surgery but remained significantly higher than in the control and sham-surgery groups (P<0.05). It then returned to baseline 7 days after surgery. Similar results were obtained regarding the expression of HO-1 and NQO1 mRNA (Fig. 4B and C).