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Introduction

Current therapeutic approaches for ischemic stroke mainly focus on the re-canalization of occluded blood vessels in the brain; however, reperfusion following thrombolytic therapy induces a cascade of pathological events, including an inflammatory response and blood-brain barrier (BBB) breakdown (1,2). BBB injury is the key event that leads to vascular edema and hemorrhage transformation, two high-risk factors for intracranial hypertension and brain death (1,3,4). Thus, preventing BBB breakdown is a promising strategy to halt the progression of ischemic stroke.

In Traditional Chinese Medicine, Ligusticum wallichii Franchat. (Chuan Xiong) is widely used in the treatment of cardiovascular and neurovascular diseases. Ligustrazine (Fig. 1), also known as 2,3,5,6-tetramethylpyrozine (TMP), is one of the most important active ingredients of Chuan Xiong (5). The compound can penetrate BBB and can be found throughout the brain. Chuan Xiong and TMP are widely applied by Chinese herbalists to treat ischemic stroke (6). Several animal studies have demonstrated that TMP is capable of reducing the infarct volume, neurological score and brain edema in the model of permanent and temporal cerebral ischemia injuries (7–11). TMP also reduces cellular inflammatory responses that follow brain ischemia (11) and enhances neurogenesis and spatial cognition during ischemic injury (8). The neuroprotective effect of TMP has been confirmed by multiple in vitro studies (12,13). TMP has been shown to have neuroprotective effects against Parkinson's disease (14), neuropathic pain (15) and spinal cord ischemia (16).

Figure 1. Chemical structure of ligustrazine (2,3,5,6-tetramethylpyrazine).

With regard to the mechanism underlying the TMP-induced neuroprotection, TMP has been reported to possess diverse pharmacological properties, including free radical scavenging and the inhibition of cytokine production and release (17–19). The inflammatory response and free radical production during cerebral ischemia and reperfusion are the main causes of BBB breakdown. Thus, it is possible that TMP could protect the BBB during cerebral ischemia and reperfusion, due to its association with free radical scavenging and inflammatory inhibition. In the present study, a classical brain ischemia and reperfusion model was utilized to investigate the neuroprotective and BBB-protective role of TMP. The relevant mechanisms involved in the effects of TMP were additionally investigated.

Materials and methods

Ethics statement

Male adult Sprague Dawley rats (250–270 g) were obtained from the Animal Center of the Guangzhou University of Chinese Medicine (Guangzhou, China). The present study was conducted in strict accordance with the international ethical guidelines and the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health. All animal experiments were approved by the Institutional Animal Care and Use Committee of Guangzhou University of Chinese Medicine. Every effort was made to minimize the number and suffering of the animals used.

Middle cerebral artery occlusion (MCAO) and drug treatment

MCAO was selected as the model for cerebral ischemia/reperfusion (20). Briefly, anesthesia was induced in the rats through the inhalation of 5% isoflurane and maintained with 2% isoflurane in a mixture of 70% N2O and 30% O2. Following vessel isolation, a 3-0 monofilament nylon suture (Johnson & Johnson, New Brunswick, NJ, USA) was inserted into the external carotid artery and then threaded forward into the internal carotid artery and anterior cerebral artery to occlude the MCA. Following the surgery, the rats were transferred to an intensive care incubator where the temperature was maintained at 37°C until the animals regained consciousness. The suture was removed following a 1.5-h occlusion period, in order to induce reperfusion. TMP (20 mg/kg; Forever Biotech, Shanghai, China) (Fig. 1) or a saline vehicle was intraperitoneally injected into the rats 15 min before suture insertion. Following 22.5 h of reperfusion, each rat brain was isolated and subjected to infarct volume, BBB permeability and brain edema measurement, as well as protein preparation for western blotting and the determination of matrix metalloproteinase (MMP) activity (Fig. 2).

Figure 2. Outline of the experimental design showing the time scale of drug administration and cerebral ischemia and reperfusion. Rats were administered 20 mg/kg 2,3,5,6-tetramethylpyrazine (or saline vehicle) 15 min before the induction of ischemia. After 1.5 h of ischemia plus 22.5 h of reperfusion, the rat brains were isolated and subjected to infarct volume, brain edema, MMP activity and tight junction impairment evaluations. BBB, blood-brain barrier; MMP, matrix metalloproteinase.

Measurement of infarct volume, water content and neurological score

Following reperfusion, each rat brain was isolated and sliced into 5 coronal sections (2-mm), which were stained with 2,3,5-triphenyltetrazolium chloride (Sigma, St. Louis, MO, USA). The evaluation of the infarct area was accomplished by calculating the hemispheric lesion area with ImageJ-1.38x software (National Institutes of Health, Bethesda, MD, USA). The relative infarct volume percentage (RIVP) was calculated using the following formula: RIVP = IVA - TA × 100% (IVA, total infracted area of the 5 coronal sections; TA, total area of the 5 sections).

Neurological evaluation was performed at 3 h after occlusion and scored as follows: 0, no neurological deficit; 1, difficulty in fully extending the left forepaw; 2, unable to extend the left forepaw; 3, mild circling to the left; 4, severe circling to the left; and 5, falling to the left.

In order for the brain water content to be determined, the isolated brain samples were dried in an oven at 110°C for 24 h, and the water content of these samples was subsequently evaluated using the following formula: Water content (%) = [wet weight - dry weight] - wet weight × 100.

BBB permeability evaluation

BBB permeability was evaluated by measuring the Evans blue dye extravasation in accordance with a previously described method with certain modifications (21). Briefly, 2% Evans blue was injected intravenously (2 ml/kg, Sigma) 0.5 h before sacrifice; transcardial saline perfusion was then performed for the removal of all intravascular dye from the vessels, until the drainage was colorless. The ipsilateral hemisphere was then removed and incubated for 24 h in N,N′-dimethyl formamide (Sigma) in a water bath at 60°C, and a spectrophometer (Analytik Jena AG, Jena, Germany) was used to measure the Evans blue content in the supernatants at a wavelength of 632 nm. Rats were euthanized after 22.5 h of reperfusion, by an intraperitoneal injection of pentobarbitone (200 mg/ml).

In situ zymography

The kit used for the analysis of the gelatinolytic activities of MMP-2 and -9 in cryosections from brain tissues by in situ zymography was the EnzChek® collagenase kit (Invitrogen Life Technologies, Carlsbad, CA, USA); the manufacturer's instructions were followed. Brain coronal cryosections were incubated in a reaction buffer, which contained 40 µg/ml fluorescein isothiocyanate-labeled dye-quenched (DQ)-gelatin, at 37°C for 2 h. Gelatinases cleaved the DQ-gelatin to yield the peptides and form fluorescence, which was representative of the net gelatinolytic activity. A fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany) was used to examine the fluorescent imaging.

Western blot analysis

Denatured protein samples were resolved using SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Subsequent to blocking, the membrane was incubated overnight at 4°C, with rabbit polyclonal anti-MMP-9 (#3852; 1:500; Cell Signaling Technology, Inc., Boston, MA, USA) and anti-occludin (#71-1500; 1:1,000; Invitrogen Life Technologies), and mouse monoclonal anti-claudin-5 (#4C3C2; 1:500; Invitrogen Life Technologies) and anti-β-actin (#sc-47778; 1:2,000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) primary antibodies. The membrane was then incubated with goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:2,000; Santa Cruz Biotechnology Inc.). ECL Advance™ western blotting detection reagents (GE Healthcare, Little Chalfont, UK) were used to perform immunodetection.

Statistical analysis

Data are expressed as the mean ± standard deviation. For two-group experiments, comparisons were made using an unpaired Student's t-test. SPSS 16.0 statistical software (SPSS, Inc., Chicago, IL, USA) was used for the statistical analysis. P<0.05 was considered to indicate a statistically significant difference.

Results

Reduced infarct volume, neurological score and brain edema in a rat model of cerebral ischemia/reperfusion injury following TMP treatment

The first experiment was performed to investigate the neuroprotective effect of TMP during cerebral ischemia and reperfusion. As shown in Fig. 3A and B, the brains subjected to ischemia/reperfusion injury exhibited a maximal infarct volume of 31.2±4.14%, whereas following treatment with TMP that volume dropped to 18.9±3.75% (P<0.01). TMP was also found to reduce the neurological score and brain edema in brains with ischemia/reperfusion injury (P<0.05, Fig. 3C and D). These results reveal that TMP plays a neuroprotective role against cerebral ischemia/reperfusion injury.

Figure 3. TMP reduces the infarct volume, neurological score and brain edema during cerebral ischemia and reperfusion in rats. (A) The infarct volume of each treatment group was assayed by 2,3,5-triphenyltetrazolium chloride staining. (B) The relative infarct volume percentage was calculated with ImageJ-1.38x software (National Institutes of Health, Bethesda, MD, USA) and presented as a bar graph. (C) The neurological score of each rat was determined and recorded. (D) The water content was calculated according to the difference between the wet and dry weight, and presented as a bar graph. Data are expressed as the mean ± standard deviation, n=8. **P<0.01, *P<0.05. TMP, 2,3,5,6-tetramethylpyrazine.

Decreased BBB permeability during rat cerebral ischemia and reperfusion following TMP treatment

BBB leakage is the characteristic feature of cerebral ischemia/reperfusion injury, contributing to the formation of brain edema and hemorrhage. Based on the effect of the TMP-induced reduction in brain edema in rats with cerebral ischemia/reperfusion injuries, the next experiment was performed to examine the effect of TMP on BBB integrity. The results demonstrated that TMP significantly reduced the BBB permeability (from 9.49±1.97 to 4.82±1.14 µg/g brain) (P<0.01, Fig. 4A). The impairment of tight junctions plays the key role in BBB opening, and it was observed that the expression of occludin and claudin-5, two tight junction proteins, was significantly elevated in the ischemia/reperfusion-injured brains in the TMP treatment group compared with that in the vehicle group (P<0.05, Fig. 4B and C). These data indicate that TMP can significantly reduce BBB permeability during cerebral ischemia/reperfusion injury.

Figure 4. TMP decreases BBB permeability and tight junction impairment in brains with cerebral ischemia/reperfusion injury. (A) BBB permeability in each group was evaluated by measuring the Evans blue extravasation. (B) Western blot analysis was used to determine the expression of occludin and claudin-5 in the protein samples of the brains isolated from the TMP- or saline-treated rats. (C) The blots of occludin and claudin-5 were quantified with ImageJ-1.38x software (National Institutes of Health, Bethesda, MD, USA) and presented in the bar graph. Data are expressed as the mean ± standard deviation, n=6. **P<0.01, *P<0.05. TMP, 2,3,5,6-tetramethylpyrazine; BBB, blood-brain barrier.

Decreased MMP activity during rat cerebral ischemia and reperfusion following TMP treatment

MMPs are a group of zinc-dependent proteinases responsible for the matrix and tight junction degradation (22). MMP activation and the secondary impairment of tight junction proteins are the main causes of BBB leakage. In order to study the relevant mechanisms involved in BBB protection, the MMP activities in brain sections and the MMP-9 expression were observed. Fig. 5A and B show that TMP treatment reduced the protein level of MMP-9. Correspondingly, TMP markedly reduced the elevated MMP activity in the brains of rats subjected to MCAO, as shown by in situ zymography with a fluorescence-conjugated substrate (Fig. 5C). These results suggest that TMP decreased the MMP activity during rat cerebral ischemia and reperfusion.

Figure 5. TMP reduces MMP-9 expression and MMP activity in the brain. (A) The MMP-9 expression level in the brains of TMP- or saline-treated rats was determined by western blotting. (B) Quantitative data of the western blot bands of MMP-9 expression. The average band intensity of MMP-9 was quantified with ImageJ-1.38x software (National Institutes of Health, Bethesda, MD, USA) and presented as a bar graph. (C) MMP activity in cryosections in each group was determined in situ, using a fluorescence-conjugated substrate. Data are expressed as the mean ± standard deviation, n=6. *P<0.05. TMP, 2,3,5,6-tetramethylpyrazine; MMP, matrix metalloproteinase.

Discussion

The present study investigated the effects of TMP at a neurovascular level and found that i) TMP simultaneously reduced the infarct volume, neurological score, brain edema and BBB breakdown in a rat model of cerebral ischemia and reperfusion, a finding that is consistent with previously reported findings; ii) TMP reduced BBB permeability in the brain of rats with ischemia/reperfusion damage; iii) activated MMPs and impaired tight junctions in ischemic brains were significantly attenuated by TMP treatment.

During cerebral ischemia and reperfusion, toxic substances can readily pass from the blood to the brain due to the increased permeability of the BBB induced by reperfusion insults; this results in an enlarged infarct volume. Thus, maintaining BBB integrity is a critical therapeutic strategy for the prevention of cerebral ischemia/reperfusion injury. The most notable finding of the present study is that TMP can protect the BBB integrity during cerebral ischemia and reperfusion.

The BBB is composed of extracellular matrix, microvascular endothelial cells and astrocytic endfeet. The primary responsibility of the BBB is the strict regulation of trans-BBB permeability. In this regard, the endothelial tight junctions of the capillary are the main mediators, restricting the transfer of vascular-derived substances (23). The most important membrane-associated tight junction proteins include occludins, claudins and junctional adhesion molecules, which are connected to the cytoskeleton by the zonula occludens (24,25). Elevated MMP activity and tight junctional impairment are typical events associated with the BBB dysfunction during cerebral ischemia/reperfusion. The in situ zymography assay and the western blot analysis found that TMP treatment reduced MMP activity, decreased MMP-9 expression and attenuated the loss of the tight junction proteins occludin and claudin-5. These results further confirmed that TMP was capable of protecting the BBB from increased permeability during cerebral ischemia and reperfusion.

In conclusion, this study is the first, to the best of our knowledge, to demonstrate that TMP can preserve the BBB integrity in this model of ischemic stroke in rats. The mechanisms underlying this protection could be associated with the regulation of MMPs and tight junctions. The findings of the present study could be applied in a therapeutic or preventive strategy for patients that have or are at high risk of suffering a stroke.

Acknowledgements

This study was supported by the National Natural Science of Foundation of China (no. 81072947), the Guangdong Natural Science Foundation (no. 8152800007000001) and the Collaborative Innovation Center for Key Technology of the Wudang Genuine Medicines Industry in Hubei Province (no. 2011JH-2103CXTT08).

References