Male Sprague-Dawley (SD) rats (220–260 g) were purchased from Shanghai Alac Laboratory Animal CO.LTD (Shanghai, China). License No.: SCXK (Shanghai) 2017-0005, Certificate No.: 20170005008495. All rats were fed a standard rodent diet and sterilized secondary ultrapure water ad libitum, housed at 22–25 °C, humidity 40–70% in a 12 h light-dark cycle. The animals were left to acclimatize for 7 days.
Group assignment and drug administration
60 SD rats were randomly divided into 4 groups (n = 15): Sham, MCAO, MCAO + Pel 10 mg/kg and MCAO + Pel 20 mg/kg. Pelargonidin (Chengdu Herbpurify CO., LTD, EINECS No.: 205-127-7, purity ≥ 98%, Chengdu, China) was dissolved in sterile distilled water. Rats in the experimental groups were orally administered 10 mg/kg or 20 mg/kg of pelargonidin per day, while the Sham and MCAO groups were given the same volume of normal saline daily, both for 7 days(Fig. 1). The Animal Ethics Committee of Hainan Medical University approved all the animal experimental protocols.
The rat model of focal cerebral ischemia/reperfusion injury was prepared using the suture-occluded method developed by Longa. The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (Sigma-Aldrich, CA, US, 300 mg/kg) and then fixed in the supine position. The neck was shaved and disinfected for routine skin preparation. A midline cervical incision was made to dissect the right common carotid, external and internal carotid arteries, followed by ligation of the external carotid artery and also its distal end. The proximal ends of the common carotid and internal carotid arteries were temporarily clipped. A small incision was made in the external carotid artery adjacent to the common carotid bifurcation and a silicon-coated suture inserted. The clip over the internal carotid artery was removed and the suture was gently inserted into the internal carotid artery through the external carotid artery until the origin of the middle cerebral artery was occluded; the length was typically 18–20 mm. The suture was tightened and the clip over the common carotid artery was removed, followed by cervical skin closure. After the ischemia status was maintained for 2 h, the suture was removed, allowing reperfusion of the blood supply. In the Sham group, only the internal carotid artery was dissected without any other procedure. After surgery, 100,000 units of penicillin sodium (Sigma-Aldrich, CA, US) were injected intramuscularly for 3 consecutive days to prevent infection.
After treatment, the rats were examined using MRI scanning (GE Discovery MR750W 3.0T Superconducting Magnetic Resonance Imaging System) using a 3T experimental coil (5 cm in aperture). The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (300 mg/kg) and fixed in the supine position, with the head placed through the coil centrally. A T2-weighted MRI scan (T2WI) and coronal diffusion-weighted imaging (DWI) was performed. An echo planar imaging (EPI) sequence was obtained for DWI with TR = 350 ms, TE = 50 ms, b value = 1,000 s/mm2, slice thickness = 3 mm and slice gap = 0.2 mm. The images were processed by Functool software and the largest slice of ischemic lesions was selected for analysis. ROI with an area of 2 mm² was placed in the lesion center and contralateral mirror location. The relative apparent diffusion coefficient (rADC) and index ADC (eADC) were measured. rADC and reADC, (rADC = ipsilateral ADC value/homologous contralateral ADC value, reADC = ipsilateral eADC value/homologous contralateral eADC value) were calculated.
Neurological function tests(mNSS)
On day 2 after the end of treatment, the mNSS was used to evaluate the neurological functions of MCAO rats in each group; mNSS includes motor, sensory, reflex and balance functions, with a total score of 18. The function was considered normal when the mNSS score was 0, and a higher mean mNSS score indicated a higher severity of neurological impairment . The scoring details are shown in Supplementary Table 1.
2, 3, 5-triphenyltetrazolium chloride (TTC) staining
Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (300 mg/kg), after which the thoracic cavity and right auricular appendix were opened. They were then transcardially perfused with PBS and then whole brain removed and sectioned into 5 slices. The sections were incubated in 2% TTC (Sigma-Aldrich, CA, US) at 37 °C for 30 min, fixed in 4% formaldehyde for 24 h and then photographed. Additionally, the infarct size was calculated.
Morris water maze (MWM) testing
The MWM test was performed to assess the spatial learning and memory of the rats. A round pool (120 cm diameter, 50 cm height, 30 cm depth, water temperature 22 ± 1 °C) was placed in an independent light-protected laboratory house and divided into four quadrants (E: East, S: South, W: West, N: North). Each rat was trained twice daily before the MWM at 120 s/dose for 3 days on end of treatment. On days 4, 6, 8 and 10, the place navigation test was performed. The platform was placed in any quadrant 2 cm underwater. The adjacent and opposite quadrants of the platform were selected as water entry points. The latency and times of crossing the platform were measured during a 120 s test. The assay was performed according to the instructions of the instrument supplier (SuperMaze Morris Water Maze Experimental Analysis System, Shanghai XinRuan Information Technology Co., Ltd.).
Blood was collected from the abdominal inferior vena cava. After standing at room temperature for 2 h, it was centrifuged at 3,000 rpm for 10 min at 4 °C to separate the serum. The level of TNF-α, TGF-β, IL-6, IL-10, MDA and SOD was measured as using an ELISA kit (R & D Systems, Minneapolis, US).
Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (300 mg/kg) and decapitated to facilitate removal of their brains. Brain tissues were fixed in 4% paraformaldehyde for 72 h and thereafter were embedded in paraffin and sectioned (4 µm slices). The sections were dehydrated with gradient alcohol and washed with PBS 3 times. They were blocked with 10% fetal bovine serum (Gibco Life Technologies, NY, US) for 2 h and then incubated with anti-Nrf2 antibody and anti-HO-1 antibody (1:100, Abcam, Cambridge, MA, US) at 4 °C overnight, followed by washing with PBS three times. Fluorescence (red light) conjugated secondary antibody IgG (1:200, MultiSciences, Shanghai, China) was added and incubated for 2 h at room temperature, followed by washing with PBS three times. The slides were counterstained with DAPI (Beyotime Biotechnology, Shanghai, China) for 10 min and photographed under a fluorescence microscope.
Western blot analysis
The ischemic cortex tissues were isolated and homogenized using RAPI lysate (Beyotime Biotechnology, Shanghai, China) until no obvious lump was observed. Tissues were then centrifuged at 14,000 rpm for 30 min at 4 °C to collect the supernatant containing the total protein. The protein concentration of samples was determined using a BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). The protein samples were electrophoresed using 10% SDS-PAGE after they were transferred onto PVDF membranes (Millipore, MA, US), blocked with 5% skimmed milk for 2 h, and washed three times in TBST buffered saline. Next, they were Incubated with Nrf2 and HO-1 (1:1000, Abcam, Cambridge, MA, US) monoclonal antibodies at 4 °C overnight. Subsequently, the membranes were washed 3 times with TBST buffered saline, incubated with anti-IgG antibody (1:2000, MultiSciences, Shanghai, China) at room temperature for 1 h, and washed with TBST buffered saline 3 times. The ECL Chemiluminescent kit (Beyotime Biotechnology, Shanghai, China) was used in a dark room for gel-imaging acquisition and analysis. Quantity One software was used to analyze the corresponding grayscale values of each band.
Data from 5 rats were collected separately in each experiment for statistical analysis. For each experiment, the data was expressed as the mean ± S.E.M. and was statistically analyzed using GraphPad Prism 6.0 software. Statistical differences between data were assessed using one-way ANOVA followed by the Newman-Keuls test. A P-value < 0.05 was considered to be a statistically significant finding.