Drugs and Reagents
The TLR4 antagonist TAK-242 (MedChemExpress). HMGB1 inhibitor ethyl pyruvate (Sigma-Aldrich; Merck KGaA). Anti-glial fibrillary acidic protein (cat. no. M00213-7; Boster Biological Technology). Anti-Claudin-5 (cat. no. 34-1600; Thermo Fisher Scientific, Inc.). FITC conjugated anti-Rabbit IgG (cat. no. BM2012; Boster Biological Technology). Anti-HMGB1 (cat. no. ab18256; Abcam). Anti-TLR4 (cat. no. ab13556; Abcam). Anti-MMP-9 (cat. no. ab58803; Abcam). Anti-Occludin (cat. no. ab222691; Abcam). Anti-Claudin-5 (cat. no. ab15106; Abcam). Anti-Zo-1 (cat. no. ab216880; Abcam). Horseradish peroxidase (HRP)-conjugated goat anti-rat IgG (cat. no. ab97057; Abcam). DAPI (Boster Biological Technology). In Situ Cell Apoptosis Detection Kit Ⅲ，FITC (cat. no. MK1023; Boster Biological Technology).
RRD is comprised of two components, Rehmannia and Rhubarb (Traditional Chinese Medicine Pharmacy, Shuguang Hospital Affiliated to Shanghai University of TCM, Shanghai, China). To obtain the RRD, Rehmannia and Rhubarb (dose ratio, 3:1) were diluted in distilled water at a 1:10 ratio and incubated for 12 h before the whole filtered suspensions from every decoction for 1 h of three times were collected and centrifuged at 2,000 x g for 20 min at room temperature. Dehydrated alcohol was then added until the concentration of the alcohol reached 75% (v/v). The liquid was stirred overnight, which was evaporated to a final concentration of 2 g/ml (w/v) and the precipitate was discarded. Finally, the liquid was autoclaved and stored at -20˚C until further use.
Animals and treatments
Male spontaneous hypertensive rats (Beijing vitalriver laboratory animal technology co., LTD.; certificate: SCXK (Beijing) 2016-0006), 3-4 months old，weighing 280-320 g and systolic pressure ≤180-200 mmHg as determined using the tail set method, were used for all experiments(n=182). All experimental procedures were conducted in accordance with the guidelines for animal experiments of Shanghai University of Traditional Chinese Medicine and approved by the university’s committee on animal experimentation (ethical code, PZSHUTCM170721001).
The animal study was divided into two steps. The first step aimed to investigate neuroprotective effects of RRD on the BBB following acute ICH, where the rats were divided into the following three groups: i) Sham group (Sham); ii) ICH group; and iii) RRD group. After confirming the neuroprotective effect of RRD, further experiments were conducted to verify if the neuroprotective effects of RRD on the BBB following acute ICH was mediated via the HMGB1/TLR4/MMP9 signaling pathway. Rats were randomly divided into the following 7 groups: i) Sham group (Sham); ii) ICH group; iii) RRD group (RRD); iv) TAK-242 group; v) TAK-242 + RRD group; vi) EP group; and vii) EP + RRD group. The administration of RRD was given at a dosage of 18.51 g/kg/d by intragastric administration for 3 days after ICH induction based on observations from previous studies. Rats in the TAK-242 group were administered with TAK-242 by intraperitoneal injection at 3 mg/kg  whilst those in the EP group were treated with an intraperitoneal injection of 80 mg/kg EP, once daily for 3 days after ICH induction .
ICH was induced by the autologous blood injection method. Briefly, rats were deprived of food except water before 12h of ICH modeling to prevent peritonitis. And on the following day, they were first anesthetized with 10% chloral hydrate (350 mg/kg) by intraperitoneal injection and then placed in a stereotaxic frame (David Kopf Instruments). When the rats were monitored to lose pain reflex meant deep anesthesia, a 26-gauge needle was then inserted through a burr hole into the caudate nucleus (3.0 mm lateral to the midline, 0.2 mm anterior to bregma, 6.0 mm in depth below the skull). A total of 100μl autologous whole blood taken from the tail vein was injected at a rate of 10μl/min . Following the completion of the injection, the needle was withdrawn slowly after standing 10 mins, paraffin was placed around the burr hole and the skin incision was closed with sutures. For rats in the sham group, only drilling was performed.
Neurobehavioral function evaluation
Neurological function deficit were evaluated using the Longa five-point scale detailed as follows : i) 0, no neurological deficit; ii) 1, a slight neurologic deficit (unable to stretch the left forepaw fully); iii) 2, a moderate neurological deficit (hemiplegia or circling to the left); iv) 3, a severe neurological deficit (unable to stand or roll), and iv) 4, a critical neurological deficit (no spontaneous locomotor activity and disturbance of consciousness). On day 3 after operation, rats scoring 1-3 were selected for further study, while rats scoring 4 were were so severely impaired in neurological function that they were unable to continue the next stage of the experiment.
Measurement of BBB structure
Transmission electron microscopy
On day 3 after operation, the rats were anesthetized with 10% chloral hydrate (350 mg/kg) by intraperitoneal injection. When the rats were monitored to lose pain reflex meant deep anesthesia, following which they were and perfused with saline that was pre-cooled to 4˚C through the left ventricle until clear. The rat brain was then rapidly removed and placed on an ice box. The tissue around the hematoma of ICH was harvested quickly and soaked in a pre-cooled 2.5% glutaraldehyde specimen bottle, which was then cut to the size of 1 mm3 for full pre-fixation. After 1% osmium acid fixation and dehydration using an ascending alcohol gradient, the tissues were embedded in epoxy resin to make ultrathin sections. The sections were then mounted onto copper grids, stained with uranyl acetate and lead citrate and then observed under a transmission electron microscope (FEI Tecnai G2 Spirit TEM; Thermo Fisher Scientific, Inc.) to observe the ultrastructural changes of the BBB around the hematoma. Three rats were selected from each group for this examination.
Immunofluorescence staining for Glial fibrillary acidic protein(GFAP) and Cladudin-5 was performed to observe the gross structure of the BBB. On day 3 after operation, the rats were anesthetized with 10% chloral hydrate (350 mg/kg), following which they were perfused with normal saline through the left ventricle with until clear and then with 250 ml 4% paraformaldehyde. The brain was quickly removed and soaked in 4% paraformaldehyde for >24 h. All brain tissues were embedded in paraffin and then sliced into 5-μm thick sections. The primary antibodies of GFAP(diluted 1:100; Boster Biological Technology) and Cladudin-5(diluted 1:100; Thermo Fisher Scientific, Inc.) were used to incubate the sections overnight at 4˚C after permeabilization with 0.3 % Triton X-100 for 25 min. The next day, sections were incubated with the fluorescent-labeled secondary antibody FITC-conjugated anti-Rabbit IgG (1:100; Boster Biological Technology) at 37˚C for 1 h, followed by DAPI for 30 min after washing in PBS. The sections were then imaged by fluorescent microscopy, following which fluorescence intensity was determined using Image J 1.52a(National Institutes of health). Three rats were selected from each group for this experiment.
Measurement of BBB function
Evans blue assay
On day 3 after operation, the rats were first anesthetized with 10% chloral hydrate (350 mg/kg) before 2% EB dye (5 ml/kg) was injected into the blood circulation through the tail vein. After 2 h, rats were perfused with pre-cooled saline through the left ventricle until clear. The rat brain was then rapidly removed and weighed. Each sample was homogenized using 50% trichloroacetic acid and centrifuged at 15,000 x g for 30 min at 4˚C to obtain the supernatant, following which anhydrous ethanol was then added and incubated overnight at 4˚C. The samples were again centrifuged at 15,000 x g for 30 min at 4˚C and the optical density value was measured using a fluorescence spectrophotometer (excitation wavelength, 620 nm; emission wavelength, 680 nm) to calculate the sample EB concentration. EB extravasation was calculated according to the measured concentration and was defined as the amount of EB (μg) per g wet-weight brain tissue. (standard curve, y=0.23+6.58x10-3x, R2=0.984). Three rats were selected from each group for this experiment.
Brain water content
On day 3 after operation, the rats were anesthetized with 10% chloral hydrate (350 mg/kg) and perfused with pre-cooled 4˚C saline through the left ventricle until clear. The wet brain was then quickly removed and weighed using an electronic analytical balance. After drying in an electrothermostat for >24 h at 100˚C overnight, the dry brain was weighed again. Brain water content (%) was calculated using the following formula: (Wet weight - dry weight)/wet weight x100%. Five rats were selected from each group or this experiment.
Rats were anesthetized with 10% chloral hydrate (350 mg/kg) 3 days after operation and placed on the rat coil (channel 4) to perform MRI (Philips Achieva 1.5T; Philips Healthcare) using the following parameters: i) T1, TR/TE (repetition time/ echo time) =550/15; ii) T2, TR/TE=4000/112; iii) SL (slice thickness), 2; and iv) FOV (field of view), 100. During MRI, a series of imaging data were obtained. Areas of hyperintensity and the mixed regions of hyperintensity-hypointensity were measured from the MRI images and used to calculate brain edema volume using DicomGov220.127.116.11(Shenyang Link Imaging Technology Co., Ltd). Three rats were selected from each group for this experiment.
On day 3 after operation, rats were anesthetized with 10% chloral hydrate (350 mg/kg) and perfused with saline through the left ventricle until clear, followed by 250 ml 4 % paraformaldehyde. The brain was then quickly removed and soaked in 4% paraformaldehyde for >24 h. All brain tissues were embedded in paraffin and then sliced into 5-μm thick sections. After deparaffinization and rehydration, the slides were stained with cresyl violet (Nissl Staining Solution, Beyotime Institute of Biotechnology) for 10 min at 37-50˚C. The stained slides were then dehydrated by incubation with 100% alcohol twice at 5 min each followed by incubation with xylene twice at 5 min each. After the sections were mounted using a permanent mounting medium, the numbers of Nissl body in the tissue around the hematoma were counted per image (magnification, x400). Three rats were selected from each group for this experiment.
Apoptotic cells in the tissue around the hematoma were observed by TUNEL staining according to manufacturer’s protocol (In Situ Cell Apoptosis Detection Kit Ⅲ, FITC). After deparaffinization and rehydration of the coronal sections, the slides were labeled with the TUNEL reaction mix and converter-peroxidase. Following washing with TBS, the slides were incubated with streptavidin-biotin complex-FITC to induce a chromogenic reaction, where apoptotic nuclei were stained brown. The number of TUNEL-positive cells were counted in each image using a fluorescence microscope (magnification, x400). Three rats were selected from each group for this experiment.
Western blotting. On day 3 after operation, the rats were anesthetized with 10% chloral hydrate (350 mg/kg) and perfused with saline through the left ventricle until clear. The rat brain tissues were then quickly removed, flash-frozen in liquid nitrogen and then preserved at -80˚C. In total, 20 mg brain tissues around the hematoma were homogenized and lysed separately in ice-cold RIPA lysis buffer (Beyotime Institute of Biotechnology), which were then centrifuged for 20 min at 14,000 x g at 4˚C. The supernatants were collected and protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Protein samples (20 μg per lane) were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The following primary antibodies were used for western blotting: Anti-HMGB1 antibody (1:1,000), anti-TLR4 antibody (1:1000), anti-MMP-9 (1:1,000), anti-Claudin-5 antibody (1:1,000), anti-Occludin antibody (1:1,000) and anti-ZO-1 antibody (1:500). The membranes were then incubated with the primary antibodies with gentle agitation at 4˚C overnight. After washing with TBS-T, the membranes were incubated with the HRP-conjugated secondary antibody (Abcam 1:2,000) for 2 h. The relative density of the blot bands was determined using Image J 1.52a (National Institutes of Health). Three rats were selected from each group for this experiment.
All data were presented as the mean ± S.E.M. The neurobehavioral deficit scores were used Repeated Measures of General Linear Model, whilst all other data were analyzed using after one-way ANOVA. The differences among multiple groups were compared by LSD as a post hoc tests. SPSS 21.0 (IBM Corp.) software was used for all statistical analyzes. P<0.05 was considered to indicate a statistically significant difference.