Inhibitory Mechanisms of Tetramethylpyrazine in Middle Cerebral Artery Occlusion (MCAO)-Induced Focal Cerebral Ischemia in Rats

• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Materials
• MCAO-induced transient focal cerebral ischemia in rats
• Determination of the expression of iNOS protein in MCAO-insulted brains
• Determination of respiratory bursts in human neutrophils
• Determination of neutrophil migration
• Measurement of free radicals in human neutrophils by electron spin resonance spectrometry
• Statistical analysis
• Results
• Discussion
• References

Abstract

Tetramethylpyrazine (TMPZ) is an active ingredient isolated from a commonly used Chinese herb, Ligusticum wallichii Franchat, which has long been used in China for the treatment of vascular diseases. In the present study, TMPZ significantly attenuated middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia in rats. Administration of TMPZ at 10 and 20 mg/kg produced concentration-dependent reductions in infarct size compared to that of control rats. MCAO-induced focal cerebral ischemia was associated with increases in both nitrotyrosine and inducible nitric oxide synthase (iNOS) expression in ischemic regions. The expressions of nitrotyrosine and iNOS were markedly inhibited by TMPZ (20 mg/kg) treatment. Furthermore, TMPZ (100 - 250 μM) concentration-dependently inhibited respiratory bursts in human neutrophils stimulated by fMLP (800 nM) and PMA (320 nM). TMPZ (100 - 250 μM) also significantly inhibited neutrophil migration stimulated by fMLP (800 nM) and LTB₄ (160 nM). An electron spin resonance (ESR) method was used to further study the scavenging activity of TMPZ on free radicals formed in human neutrophils. TMPZ (100 and 200 μM) greatly reduced the ESR signal intensity of hydroxyl radical formation. In conclusion, we demonstrate a neuroprotective effect of TMPZ in MCAO-induced focal cerebral ischemia in vivo. TMPZ mediates at least part of the free radical-scavenging activity and inhibits neutrophil activation, resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Thus, TMPZ treatment may represent an ideal approach to lowering the risk of or improving function in ischemia-reperfusion brain injury-related disorders.

Abbreviations

ESR:electron spin resonance

fMLP:N-formyl-Met-Leu-Phe

iNOS:inducible nitric oxide synthase

LCL:lucigenin-enhanced chemiluminescence

mAb:monoclonal antibody

MCAO:middle cerebral artery occlusion

PMA:phorbol 12-myristate-13-acetate

ROS:reactive oxygen species

Introduction

Tetramethylpyrazine (TMPZ) is an active alkaloid purified from a commonly used Chinese herb named ”Chung Chong” (Ligusticum wallichii Franchat), which has been used for at least 2000 years by traditional Chinese physicians to stimulate blood circulation, relieve pain [1], and treat a variety of vascular diseases, notably ischemic stroke and pulmonary hypertension secondary to chronic obstructive pulmonary diseases (COPDs) (Fig. [1]) [1], [2]. However, there are no studies available concerning the actual concentration of TMPZ in Ligusticum wallichii Franchat. One clinical study included 156 patients with acute ischemic cerebrovascular diseases who were admitted to the hospital following a cerebrovascular stroke. TMPZ (40 - 120 mg/day) was given by intravenous infusion for 1 - 3 weeks, and about 90 % of patients treated with TMPZ showed clinical improvement regarding myodynamic changes and swallowing difficulties [1]. Wang [3] demonstrated that TMPZ could improve changes in the microcirculation of patients with acute cerebral thrombosis. Ho et al. [4] showed that TMPZ increased the survival rate of Mongolian gerbils with experimentally induced stroke. Furthermore, TMPZ has exhibited effective antiplatelet activity in both in vitro and in vivo studies [5], [6].

Ischemic brain injury often causes irreversible brain damage. The cascade of events leading to neuronal injury and death in ischemia includes the release of cytokines, the formation of free radicals, and the activation of platelets and neutrophils [7], [8]. The participation of activated platelets has been observed in brain microvessels of the ischemic microvascular bed after experimental middle cerebral artery occlusion (MCAO) [8]. Thus, platelet aggregation may play a crucial role in MCAO-induced cerebral damage. Furthermore, reperfusion of ischemic areas can exacerbate ischemic brain damage by the generation of reactive oxygen species (ROS) including superoxide anions (O₂ ^.-), hydroxyl radicals, and peroxynitrite radicals from activated neutrophils and by excessive production of nitric oxide (NO) through the induction of inducible nitric oxide synthase (iNOS) [9], [10]. Neutrophils are a potential source of ROS when activated during an inflammatory response [9]. When a tissue suffers from ischemia and reperfusion, proinflammatory cytokines produced by inflammatory cells can trigger adhesion and migration of circulating neutrophils to endothelial cells and generation of ROS [9]. It has been reported that NO immediately reacts with superoxide to form peroxynitrite (ONOO^-), which is capable of nitrating tyrosine residues of proteins and enzymes to generate nitrotyrosine leading to tissue injury [11]. Therefore, both inhibition of neutrophil activation and enhanced degradation of ROS with pharmacological agents have been found to limit the extent of brain damage following stroke-like events [12], [13].

By considering the pivotal roles of platelet aggregation, ROS, and inflammatory responses in ischemia-reprefusion-induced brain injury, the present study was designed to examine the mechanisms responsible for mediating TMPZ’s neuroprotective effects using an MCAO-reperfusion model in rats.

Materials and Methods

Materials

2,3,5-Triphenyltetrazolium (TTC), aprotinin, cremophor EL, leupeptin, lucigenin, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), N-formyl-Met-Leu-Phe (fMLP), phorbol 12-myristate-13-acetate (PMA), leukotriene B₄ (LTB₄), and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO). Ficoll-Paque plus was purchased from Amersham (Buckinghamshire, HP, UK). TMPZ purchased from Aldrich (Milwaukee, MI) was dissolved in solvent (cremophor:ethanol:normal saline, 1 : 1:4) for the in vivo studies, and dissolved in 0.5 % DMSO for the in vitro studies.

MCAO-induced transient focal cerebral ischemia in rats

Male Wistar rats (250 - 300 g) were used in this study. All animal experiments and care were performed according to the Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, DC, 1996). Before undergoing the experimental procedures, all animals were clinically normal and free of apparent infection or inflammation and showed no neurological deficits.

Animals were anesthetized with a mixture of 95 % O₂ and 5 % CO₂ gases containing 3 % isoflurane. The rectal temperature was maintained at 37 ± 0.5 °C. The right MCA was occluded as described by Longa et al. [14] and Nagasawa ad Kolgure [15]. Briefly, the right common carotid artery was exposed, and a 4 - 0 monofilament nylon thread (25 mm) coated with silicon was then inserted from the external into the internal carotid artery until the tipp occluded the origin of the MCA. After closure of the operative sites, the animals were allowed to awake from the anesthesia. During another brief period of anesthesia, the filament was gently removed after 1 h of MCAO. An observer blinded to the identity of the groups assessed neurological deficits at 1 and 24 h after reperfusion (before sacrifice) using the forelimb akinesia (also called the postural tail-hang) test, while the spontaneous rotational test was used as a criterion for evaluating the ischemic insult [16]. Animals not showing behavioral deficits at the above time points after reperfusion were excluded from the study. On the other hand, reperfusion was also ensured by an improvement in ipsilateral local blood flow to at least 60 % of the baseline following an initial sharp decrease to about 30 % of the baseline caused by MCAO as determined using a continuous laser Doppler flowmeter (Oxford Array^TM, Oxford Optronix, Oxford, UK) with a standard needle probe (pp-051).

Rats were sacrificed by decapitation after 24 h of reperfusion. Their brains were cut into 2-mm coronal slices. Each stained brain (2 % TTC) slice was drawn using a computerized image analyzer (Image-Pro plus). The calculated infarct areas were then compiled to obtain the infarct volumes (mm³) per brain. Infarct volumes were expressed as a percentage of the contralateral hemisphere volume using the formula, (the area of the intact contralateral [left] hemisphere - the area of the intact region of the ipsilateral [right] hemisphere) to compensate for edema formation in the ipsilateral hemisphere [17].

All animals were divided into four groups: (i) a sham-operated group; (ii) an MCAO (control) group; and groups treated with (iii) a solvent solution (cremophor:ethanol:normal saline, 1 : 1 : 4) or (iv) a single dose (10 or 20 mg/kg, i. p.) of TMPZ. In the group treated with the solvent or TMPZ, rats were given isovolumetric solvent or TMPZ (10 or 20 mg/kg) 20 min before MCAO.

Determination of the expression of iNOS protein in MCAO-insulted brains

The expression of iNOS in the brain was analyzed by Western blotting as described by Rodrigo et al. [18] with some modifications. In brief, MCAO-insulted and sham-operated rats were anesthetized with chloral hydrate (400 mg/kg, i. p.), and fresh brains were removed and sectioned coronally into 4 sequential parts from the frontal to the occipital lobe. The third parts of the right and left hemispheres were separately collected and stored at -70 °C. Each brain tissue sample was homogenized followed by centrifugation (10,000 g). The supernatant (50 μg protein) was subjected to SDS-PAGE and electrophoretically transferred onto PVDF membranes (0.45 μm; Hybond-P; Amersham). After incubation in blocking buffer, the blots were treated with either an anti-iNOS mAb (1 : 3000; BD Sciences) or an anti-tubulin mAb (1 : 2000; Santa Cruz Biotech) in buffer. Blots were subsequently incubated with secondary horseradish peroxidase-conjugated goat anti-mouse mAb (Amersham) for 1 h. Blots were then washed, and the immunoreactive protein was detected using film exposure with enhanced chemiluminescence detection reagents (ECL⁺ system; Amersham).

Determination of respiratory bursts in human neutrophils

Superoxide anion production by neutrophils was measured using the method of lucigenin-enhanced chemiluminescence (LCL) as described previously [19] with some modifications. Briefly, washed neutrophil suspensions (2 × 10⁶ cells/mL) were dispensed into wells of a standard scintillation microplate. Before the assay, cells were preincubated with the solvent control (0.5 % DMSO) or various concentrations of TMPZ (100, 200, and 250 μM). Then, 20-μL aliquots of lucigenin were added at a final concentration of 100 μM. The basal LCL was recorded for 1 min using a microplate luminometer (Orion®, Berthold, Germany) at 37 °C, and cells were immediately stimulated with fMLP (800 nM) or PMA (320 nM). The luminescent light was continuously recorded for 5 min. The chemiluminescent signal was represented as relative light units per second (RLU/s). The results of LCL intensity (as increments in the signal intensity) were determined by measuring basal and stimulator-induced peak values and calculating the difference between them.

Determination of neutrophil migration

The neutrophil chemotactic assay was performed using a 24-well transwell system (Corning, NY) as previously described [20]. The chemoattractant, either fMLP (800 nM), LTB₄ (160 nM), or free buffer (as the negative control) was placed in the wells of 24-well tissue culture plates. The transwell inserts (8-μm pore size) were filled with neutrophils in modified Hanks’ balanced salt solution (HBSS). Plates were incubated for 90 min at 37 °C in 5 % CO₂. The number of neutrophils that migrated into the bottom of the wells was quantitated using an inverted microscope equipped with phase-contrast objectives. Total migrating neutrophils were determined by counting the total number of cells in four randomly selected 40 × microscope fields (200 × 200 μm), and these were also captured using a digital image system (Image-Pro Plus Software 4.5.; MediaCybernetics).

Measurement of free radicals in human neutrophils by electron spin resonance spectrometry

The electron spin resonance (ESR) method used a Bruker EMX ESR spectrometer as described previously [21]. In brief, human neutrophils (1 × 10⁹/mL) were preincubated with TMPZ (100 and 200 μM) for 5 min before the addition of fMLP (800 nM). The reaction was allowed to proceed for 15 min, followed by the addition of 100 mM DMPO for the ESR study. ESR spectra were recorded on a Bruker EMX ESR spectrometer using a quartz flat cell designed for aqueous solutions. Conditions of ESR spectrometry were as follows: 20 mW power at 9.78 GHz, 1 G modulation, and 100 G scanning for 42 s, with 5 scans accumulated.

Statistical analysis

The experimental results are expressed as the means ± S.E.M. and are accompanied by the number of observations. Student’s unpaired t-test was used to determine significant differences in the study of MCAO-induced cerebral ischemia. The other experiments were assessed by the method of analysis of variance (ANOVA). If this analysis indicated significant differences among the group means, then each group was compared using the Newman-Keuls method. A P value of less than 0.05 was considered statistically significant.

Results

Animals of all groups in this study showed similar physiological values for rectal temperature, mean arterial blood pressure, plasma glucose, and hematocrit (%) before, during, and after MCAO (data not shown). Neither abnormal behavior, depression of respiratory, nor hypothermia was observed in the solvent- or TMPZ-treated groups. Cerebral infarction was examined in 2-mm-thick slices of the cerebrum of MCAO-reperfused rats through TTC staining. Fig. [2] A shows typical photographs of coronal sections of the sham-operated group (lane 1), the solvent (cremophor:ethanol:normal saline, 1 : 1 : 4)-treated (lane 2, white area), and TMPZ-treated groups (lane 3, 10 mg/kg; lane 4, 20 mg/kg) prior to the ischemic insult. Treatment with the solvent did not significantly influence the infarct size compared to the control (MCAO only) group (control, 41.4 ± 2.8 %, n = 10 vs. solvent, 37.7 ± 5.6 %, n = 7) (Fig. [2] B). Administration of TMPZ at 10 and 20 mg/kg, showed concentration-dependent reductions in infarct volume compared with the solvent group (10 mg/kg, 23.8 ± 2.9 %, n = 8; 20 mg/kg, 14.6 ± 1.3 %, n = 8) (Fig. [2] B).

Results of Western blotting of MCAO-insulted cerebral tissues are shown in Fig. [3]. iNOS, detected as a major band of approximately 135 kDa, was more pronounced in the ipsilateral hemisphere than in the contralateral hemisphere. The iNOS band showed significant increases in ischemic cerebral tissues after MCAO-reperfusion if compared to those of sham-operated rats. With administration of TMPZ (20 mg/kg), iNOS expression was markedly reduced in MCAO-reperfusion rats (Fig. [3]).

The inhibitory effect of TMPZ on neutrophil activation was evaluated by fMLP- and PMA-induced lucigenin-dependent chemiluminescence, which is an index of respiratory bursts, by determining the production of superoxide anions. When human neutrophils (2 × 10⁶ cells/mL) were treated with fMLP (800 nM), rapid generation of superoxide anions was observed with the LCL signal rising as high as 2461 ± 217 RLU/s (n = 20; data not shown). TMPZ (100 - 250 μM) markedly inhibited this increase in chemiluminescence stimulated by fMLP (Fig. [4]). At 250 μM, TMPZ abolished the LCL stimulated by fMLP about 70 % as compared with the solvent control (0.5 % DMSO). On the other hand, stimulation by PMA (320 nM) also caused a gradual generation of superoxide anions from neutrophils, with a peak of LCL as high as 728 ± 96 RLU/s (n = 15, data not shown). Similarly, the PMA-induced increase in chemiluminescence was attenuated by TMPZ (100 - 250 μM) in a concentration-dependent manner (Fig. [4]). Furthermore, the superoxide anion production induced by these two stimulators was also markedly abrogated by superoxide dismutase (200 U/mL) (data not shown).

We further investigated the effect of TMPZ on the chemotaxis of neutrophils using the transwell method. In the presence of solvent (0.5 % DMSO), human neutrophil migration increased to 58.6 ± 7.5 and 46.8 ± 6.2 cells/field when stimulated by fMLP (800 nM) and LTB₄ (160 nM), which were more than the spontaneously migrating cells at 6.2 ± 1.8 and 5.0 ± 1.2 cells/field, respectively (n = 8; data not shown). Treatment of neutrophils with TMPZ (100, 200, and 250 μM) caused significant inhibition of both fMLP- and LTB₄-induced chemotaxis in a concentration-dependent manner (Fig. [4]). At a higher concentration (250 μM) of TMPZ, the chemotactic responses stimulated by fMLP and LTB₄ were attenuated to about 63 % and 56 % of the solvent control (Fig. [4]), respectively.

The rate of free radical-scavenging activity is defined by the following equation:

inhibition rate = 1 - signal height (TMPZ)/signal height (solvent control) [21].

In this study, a typical ESR signal of the hydroxyl radical was markedly increased in activated neurophils compared with resting platelets (Figs. 5A, B). TMPZ (100 and 200 μM) concentration-dependently attenuated hydroxyl radical formation in human neutrophils (Figs. 5C, D). This observation provides direct in vitro evidence suggesting the usefulness of TMPZ’s free radical-scavenging activity.

Discussion

This study demonstrates for the first time that TMPZ possesses a neuroprotective effect if applied following MCAO-reperfusion injury. Animal models of focal cerebral ischemia, for which MCAO is usually used, reproduce the pattern of ischemic brain damage observed in many human ischemic stroke patients [22]. TMPZ can permeate the blood-brain barrier and can be enriched in the brain, especially the brainstem. The clinical dosage of TMPZ for the treatment of stroke varies from 160 to 320 mg via intravenous injection. The present MCAO study represents a more rapid and severe stroke model than that of patients with cerebrovascular diseases. In order to obtain more significant results during a short period, we used relatively higher doses of TMPZ in this animal study. Furthermore, the patients received TMPZ (40 - 120 mg/day) for 1 - 3 weeks in a clinical study for cerebrovascular diseases [1]. However, the rats was administered a single dose (10 or 20 mg/day) of TMPZ intraperitoneally in the present MCAO study. Therefore, concentrations applied in animals (10 - 20 mg/kg) are quite different from those given to patients (40 - 120 mg/day). The antiplatelet and vasodilation effects of TMPZ probably account for some of its beneficial effects in mediating the neuroprotective effect after MCAO-reperfusion injury [5].

Infiltration of neutrophils into infarct areas following cerebral ischemia-reperfusion injury plays a crucial role in the development of cerebral infarction and neuronal damage [12]. Although neutrophils produce and release a variety of toxic agents designed to kill microbes, those systems that depend on reactive products of oxygen metabolism are especially potent. These agents are produced as a consequence of respiratory bursts, which are a series of events triggered by phagocytosis or exposure to certain inflammatory mediators and featuring a dramatic increase in oxidative metabolism with direct conversion of molecular oxygen to its univalent reduction product, the superoxide anion. Subsequent reactions lead to the formation of other toxic species, including hydrogen peroxide, hydroxyl radicals, hypochlorous acid (HOCl), and singlet oxygen (¹O₂) [23].

Our results showed that TMPZ significantly inhibited both neutrophil migration and respiratory bursts by different stimulators such as fMLP, PMA, and LTB₄, suggesting that TMPZ does not act at the level of a specific ligand-receptor interaction. Both the croton oil derivative, PMA, and the chemotactic tripeptide, fMLP, can activate leukocytes, resulting in leukocyte adhesion to the endothelium, leukocyte aggregation, decreased leukocyte deformability, and release of oxidants, proteases, and lipid metabolites [24]. LTB₄, the main product of neutrophil 5-lipoxygenase, is a powerful chemoattractant for neutrophils; however, it is a weak stimulator of neutrophil respiratory bursts. In this study, the anti-migration effect of TMPZ was not due to its cytotoxic effect, because under these conditions there was no significant difference in cell viability (> 95 % in all groups) between the TMPZ-treated group and the solvent-treated group (data not shown).

TMPZ shows potent ROS-scavenging properties in cultured neurons and is almost as effective as α-tocopherol [25]. PMC (2,2,5,7,8-pentamethyl-6-hydroxychromane), in which the phytyl chain is replaced by a methyl group, is the most potent derivative of α-tocopherol regarding antioxidative activity [26]. Recently, we also demonstrated a potent neutroprotective effect of PMC on MCAO-induced focal cerebral ischemia, which is mediated at least partly by its antioxidative activity (unpublished data). Furthermore, several studies have reported that TMPZ possesses cell protective function (i. e., liver, kidney and cerebral cells) that may be mediated by ROS-scavenging activity [27], [28], [29]. Therefore, we further examined whether TMPZ has free radical-scavenging activity in human neutrophils. In this study, the superoxide anion was changed into a hydroxyl radical by the catalytic action of trace iron. Furthermore, it has also been reported that hydroxyl radicals are a secondary product of the decomposition of the DMPO-superoxide radical adduct and are also formed as a result of trace metals such as iron present in the buffer [30]. Therefore, the concentration of hydroxyl radicals was higher than that of superoxide anions in the ESR analysis. TMPZ effectively inhibited hydroxyl radical formation in vitro. Thus, the neuroprotective mechanisms of TMPZ may involve, at least partially, the inhibition of free radical formation. On the other hand, there are no data available concerning which structure of TMPZ is involved in antioxidative activity. However, based on the chemical structure of TMPZ, we suggest that pyrazine may play an important role, because the pyrazine cation can be formed by loss of an electron which can react with free radicals to form non-radical products.

In the late stages of cerebral ischemia (> 6 h), iNOS is expressed in the setting of post-ischemic inflammation. Large amounts of NO produced by this enzyme contribute to the delayed progression of damage [31]. The toxic effects of NO may be attributed to peroxynitrite, which is a reaction product of NO with superoxide. Peroxynitrite is responsible for the nitration of both free and protein-bound tyrosine residues which have been shown to disrupt cell signaling cascades [11]. Recently, Kimura et al. [32] reported that overexpression of iNOS in the rostral ventrolateral medulla (RVLM) increases blood pressure via activation of the sympathetic nervous system, which may be mediated by an increase in oxidative stress. TMPZ has been reported to abrogate the expression of iNOS in the lung and aorta, and to reduce the delayed hypotension in rats with endotoxic shock [33]. In this study, we further demonstrated that TMPZ significantly reduced the expressions of both iNOS and nitrotyrosine (see Supporting Information) in ischemic cerebral areas.

In conclusion, the most-important findings of this study suggest that the preventive effect of TMPZ on cerebral ischemic damage in MCAO-reperfusion rats is assumed to be mediated, at least partially, by inhibition of platelet aggregation, and activation of neutrophils and inflammatory responses such as iNOS expression and ROS (free radical) formation in cerebral ischemic areas. The rationale for the use of TMPZ is based on the fact that multiple deleterious processes in different cell types of organelles are initiated during ischemia-reperfusion injury which ultimately synergistically contribute towards irreversible injury; thus treatment using TMPZ may represent an ideal approach for improving function after ischemia-reperfusion brain injury.

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Joen-Rong Sheu

Graduate Institute of Medical Sciences

Taipei Medical University

No. 250 Wu-Hsing Street

Taipei 110

Taiwan

Republic of China

Phone: +886-2-2739-0450

Fax: +886-2-2739-0450

Email: sheujr@tmu.edu.tw

References

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Joen-Rong Sheu

Graduate Institute of Medical Sciences

Taipei Medical University

No. 250 Wu-Hsing Street

Taipei 110

Taiwan

Republic of China

Phone: +886-2-2739-0450

Fax: +886-2-2739-0450

Email: sheujr@tmu.edu.tw

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