The present study was approved by the ethics committee of Fuzhou General Hospital (Fuzhou, China), and all animal experiments were performed in accordance with the institutional guidelines on the care and use of experimental animals. A total of 54 male Sprague-Dawley rats (weight, 240–260 g; age, 6 weeks old), obtained from the Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China), were used in the present study. Rats were housed in standard conditions at 26–28°C, with 10 h light/14 h dim light cycles and access to food and water ad libitum. Of the 54 rats, 36 were randomly assigned to six subgroups (n=6 rats/group) and one group was sacrificed by intraperitoneal injection of 30 mg/100 g body weight chloral hydrate at 0, 6, 12, 24, 48 and 72 h following the induction of thrombus. The remaining 18 rats were divided into three groups (n=6 rats/group): Sham, CVST, and rhs-TM. The rhs-TM group was injected with 1 mg/kg rhs-TM (dissolved in saline; Asahi Kasei Pharma Corp., Tokyo, Japan) through the tail vein 10 min prior to CVST. The CVST and the sham groups received normal saline.

In all experiment groups, rats were anesthetized with an intraperitoneal injection 30 mg/100 g body weight chloral hydrate. To induce venous sinus thrombosis, a 1.5×10 mm cranial window was made to expose the superior sagittal sinus. A strip of filter paper soaked with 40% ferric chloride was applied to the exposed cranial window for 5 min, whereas the sham group received filter paper soaked with 0.9% saline. Subsequently, the ferric chloride strip was removed and the field flushed with 0.9% saline; the removed bone strip was replaced, sealed with bone cement and the skin sutured.

Neurological evaluations were performed in a blinded manner. Rats were subjected to neurological evaluation prior to surgery and at 24 h following CVST using a score of 0–3, where 0=no observable neurological deficit (normal), 1=failure to extend left forepaw on lifting whole body by tail (mild), 2=circling to contralateral side (moderate) and 3=no spontaneous motor activity (severe) (11).

Rats were sacrificed with an intraperitoneal injection of 30 mg/100 g body weight chloral hydrate 24 h following CVST. Brains were removed, cut into 2-mm-thick coronal sections, stained with 1% TTC in phosphate-buffered saline for 20 min at 37°C and fixed in 10% paraformaldehyde solution for 10 min. The sections were analyzed with the image analysis software Image J version 1.44 (National Institutes of Health, Bethesda, MD, USA). For all 6 slices, the brain infarct volume was calculated as the product of the average slice thickness (2-mm) and the sum of the infarction area.

The water content of the brain was determined by the dry-wet weight method. The brains were removed following sacrifice by chloral hydrate administration and the cerebellum, pons and olfactory bulbs from each brain were removed. Each cerebrum was weighed to determine its wet weight. The brains were subsequently placed into an 110°C thermal oven for 24 h, and weighed to determine their dry weight. Water content was calculated using the following formula: Water content = [(wet weight - dry weight) / wet weight] × 100.

Total RNA was isolated from the penumbra regions of the infarcted hemispheres using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc. Waltham, MA, USA), according to the manufacturer's instructions. cDNA was generated with the High-Capacity cDNA RT kit (Roche Diagnostics GmbH, Mannheim, Germany). The mRNA expression levels of high mobility group box 1 (HMGB1), receptor for advanced glycation end products (RAGE), TNF-α, IL-1β, IL-6, caspase-3, B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X (Bax) were analyzed by qPCR using a SYBR Green PCR Master Mix kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) in conjunction with an ABI-Prism 7300 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Thermal cycling parameters consisted of an initial enzyme activation step at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 30 sec and amplification at 72°C for 30 sec. The concentrations of the target genes were calculated by comparing quantification cycle (Cq) values in each sample with Cq values of the internal standard curve (12). Melting curve analysis and gel electrophoresis evaluation of RT-qPCR products was routinely performed to determine the specificity of the reaction. The mRNA expressions levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels. The following primers were used: Forward, 5-CGA GTC CGT GTC TAC CAG ATT-3′ and reverse, 5′-GCGGCTGGAATGGAAACTGAA-3′ for RAGE; forward, 5′-GCTCCATAGAGACAGCGCCGGG-3′ and reverse, 5′-CCTCAGCGAGGCACAGAGTCGC-3′ for HMGB1; forward, 5′-CTCCCAGAAAAGCAAGCAAC-3′ and reverse, 5′-CGAGCAGGAATGAGAAGAGG-3′ for TNF-α; forward, 5′-CTGTGACTCGTGGGATGATG-3′ and reverse, 5′-GGGATTTTGTCGTTGCTTGT-3′ for IL-1β; forward, 5′-ACAGTGCATCATCGCTGTTC-3′ and reverse, 5′-CCGGAGAGGAGACTTCACAG-3′ for IL-6; forward, 5′-CTGGACTGCGGTATTGAG-3′ and reverse, 5′-GGAACATCGGATTTGATT-3′ for caspase-3; forward, 5′-ATACCTGGGCCACAAGTGAG-3′ and reverse, 5′-TGATTTGACCATTTGCCTGA-3′ for Bcl-2; forward, 5′-CGAGCTGATCAGACATCA-3′ and reverse, 5′-CTCAGCCCATCTTCTTCCAG-3′ for Bax; and forward, 5′-TCGGAGTCAACGGATTTGG-3′ and reverse, 5′-CATGGTGGATCATGGA-3′ for GAPDH.

Rats were sacrificed with an intraperitoneal injection of 30 mg/100 g body weight chloral hydrate and penumbra regions of the infarcted hemispheres were lysed in radioimmunoprecipitation assay buffer (EMD Millipore, Billerica, MA, USA). The concentration of the protein extracts was determined by the Bradford assay (Bradford Protein assay kit; Bio-Rad Laboratories, Inc., Hercules, CA, USA). A total of 40 µg protein was loaded onto gels, separated by 8–15% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20, and subsequently probed with primary antibodies overnight at 4°C. The following antibodies were used: Monoclonal rabbit anti-HMGB1 (cat. no. ab92310; dilution, 1:1,000; Abcam, Cambridge, MA, USA), monoclonal rabbit anti-RAGE (cat. no. ab172473; dilution, 1:500; Abcam), polyclonal rabbit anti-TNF-α (cat. no. ab6671; dilution, 1:500; Abcam), polyclonal rabbit anti-IL-1β (cat. no. ab9722; dilution, 1:500; Abcam), monoclonal mouse anti-IL-6 (cat. no. ab9324; dilution, 1:500; Abcam), polyclonal rabbit anti-caspase-3 (cat. no. ab4051; dilution, 1:500; Abcam), polyclonal rabbit anti-Bcl-2 (cat. no. ab59348; dilution, 1:500; Abcam) and polyclonal rabbit anti-Bax (cat. no. ab59348; dilution, 1:500; Abcam). The membranes were washed three times with PBS-Tween 20, before they were incubated with horseradish peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit IgG secondary antibodies (H+L; cat. nos. ab6728 and ab6721, respectively; dilution, 1:2,000; Abcam) for 1 h at room temperature. The blots were developed using the SuperSignal™ West Pico Chemiluminescent Substrate (cat. no. 34077; Invitrogen; Thermo Fisher Scientific, Inc.). Band intensities were normalized to β-actin and measured using spot densitometry analysis with Image J software (version 1.44, National Institutes of Health).

Data are expressed as the mean ± standard deviation. Statistical analyses were performed in SPSS software version 13.0 (SPSS, Inc., Chicago, IL, USA). Intergroup differences were analyzed using paired Student's t-test and multiple comparisons among various groups were conducted by analyses of variance with a post-hoc Tukey test. P<0.05 was considered to indicate a statistically significant difference.

The mRNA and protein expression levels of HMGB1-RAGE in the infarcted segments of CVST rat brains were analyzed by RT-qPCR and western blot analysis, respectively. HMGB1 mRNA expression levels were upregulated in the infarcted segments of rat brains 6 h following CVST, and RAGE mRNA expression levels were upregulated 12 h following CVST. mRNA expression levels of the two peaked at 24 h and gradually declined, although significant differences compared with the control group were observed even at 72 h following CVST (P<0.001; Fig. 1A). Western blot analysis demonstrated that HMGB1 and RAGE protein expression levels were consistent with the RT-qPCR data (Fig. 1B and C). The time point of 24 h was therefore selected for subsequent analyses.

In the sham group no neurological deficits were observed. The neurological deficit score 24 h following surgery was 1.49±0.38 in the CVST group and 1.02±0.29 in the rhs-TM group (P=0.036; Fig. 2A). This suggests that rhs-TM may protect against neurological deficits.

Brain infarctions were identified by TTC staining of the brain slices, revealing a bilateral infarction in the CVST group (Fig. 2B). The brain infarction volume was 83.3±4.6 mm³ 24 h following CVST alone, compared with 65.3±3.8 mm³ in the rhs-TM group (P<0.001; Fig. 2C). No brain lesions were observed in the sham group.

The water content of rat brains was 76.3±0.15% in the sham group, 80.3±0.34% in the CVST group, and 78.21±0.24% in the rhs-TM group. This difference between the CVST and rhs-TM groups was significant (P<0.001; Fig. 2D).

mRNA and protein expression levels of HMGB1-RAGE and their downstream effectors, TNF-α, IL-1β and IL-6 were assessed by RT-qPCR and western blot analysis, respectively. A significant downregulation of HMGB1-RAGE, TNF-α, IL-1β and IL-6 mRNA (Fig. 3) and protein (Fig. 4) expression levels was detected in infarcted segments of the brains of rats treated with rhs-TM compared with CVST alone (P<0.001). In addition, expression levels of apoptosis-associated genes and proteins (caspase-3, Bcl-2 and Bax) in infarcted segments were affected by rhs-TM treatment; however, the differences were not statistically significant when compared with the sham group (caspase-3 mRNA, P=0.052; Bcl-2 mRNA, P=0.192; Bax mRNA, P=0.077; caspase-3 protein, P=0.213; Bcl-2 protein, P=0.06; Bax protein, P=0.176).