Qiangli Tianma Duzhong capsule (TMDZ), a Chinese herbal drug, is clinically used to improve functional outcome in patients with ischemic stroke in China. This study was conducted to establish whether postischemic long-term treatment with TMDZ could reduce the loss of injured hemisphere and confer the improvements of neurological outcome in chronic survival of rats with 2 h middle cerebral artery occlusion (MCAO)/reperfusion brain injury and its primary mechanisms. We found that TMDZ (44.5, 89, or 178 mg/kg), administered per os 6 h after the onset of ischemia and for 28 consecutive days, significantly improved the behavior deficits, beginning on day 7, and further improved later. TMDZ treatment also markedly reduced the tissue loss of the injured hemisphere and improved histopathology. In the meantime, TMDZ treatment could improve hemorrheology and inhibit platelet aggregation. These results provide the first evidence that post-ischemic long-term treatment with TMDZ confers the improvements of neurological outcome and the loss of injured hemisphere in an animal ischemic stroke model, and its mechanisms might be associated with the improvements of hemorrheology and the inhibition of platelet aggregation.
Cerebral ischemia or stroke, one of the leading causes of death and long-term disability in aged populations, often results in irreversible brain damage and subsequent loss of neuronal function. The medications commonly used for stroke can be divided into four groups: thrombolytic agents, antiplatelet agents, anticoagulants, and neuroprotective agents [
In recent years, much attention has been paid to traditional herbal medicines [
Sprague Dawley (SD) male rats (280–310 g) were purchased from the Center for Experimental Animals, Soochow University (certificate no. 20020008, Grade II). They were housed four per cage in a standard animal room with a 12 h light/dark cycle and given free access to food and water. NIH guidelines for the care and use of laboratory animals were followed in all animal procedures.
Qiangli Tianma Duzhong capsule (TMDZ) was provided by ChunKe Guiyang Pharmaceutical R & D Co., Ltd. TMDZ 44.5, 89, or 178 mg/kg (dissolved indistilled water) or vehicle (distilled water) was administrated per os 6 h after the onset of ischemia and consecutive 28 days after ischemia. Sham-operated or ischemic-reperfusion (IR) control animals received vehicle (distilled water) per os.
Rats (
Neurological deficits were examined at 2 h ischemia and 4 h reperfusion using a 5-point scale adapted and modified from Zhang et al. [
Cylinder Tests were performed 1, 3, 7, 14, 21, and 28 days after ischemia reperfusion. Briefly, animals were placed into a plexiglass cylinder, and their behavior was observed for forelimb use asymmetry during vertical movements along the wall of the cylinder. The final score was calculated as (nonimpaired forelimb movement − impaired forelimb movement)/(nonimpaired forelimb movement + impaired forelimb movement + both movements), as previously described in the rat [
Corner tests were performed 1, 3, 7, 14, 21, and 28 days after ischemia reperfusion. Briefly, in the home cage, an animal was placed between the two angled boards. When entering deep into the corner, both sides of the vibrissae are stimulated together. The animal then rears forward and upward then turns back to face the open end. Twenty trials were performed for each rat, and the percentage of right turns versus left turns was calculated. Only turns involving full rearing along either boards were recorded [
Rats were anesthetized with isoflurane, were placed in an animal holder/MRI probe apparatus, and were positioned inside the magnet. The animal’s head was held in place inside the imaging coil. All MRI measurements were performed using a 7 Teslar, 18 cm bore superconducting magnet (Oxford Magnet Technologies) interfaced to a UNITYINOVA console (Oxford Instruments, UK, and Varian Inc., Palo Alto, CA, USA). T2-weighted images (T2WI) were obtained from a 1.0 mm thick coronal section with a 0.5 mm gap using a 30 mm × 30 mm field of view, TR = 3000 ms, TE = 37 ms, and b value = 0 and reconstructed using a
Blood samples were taken from the abdominal aorta of rats and were mixed with Heparin Li for blood viscosity and plasma viscosity, 3.2% citric acid for FBG, EDTA
Rat platelet suspensions were prepared as previously described [
Statistical analysis was carried out by one-way analysis of variance (ANOVA) followed by a post hoc Tukey test. Differences were considered significant when
Compared to the rats in ischemia-reperfusion control (IR control), TMDZ treatment significantly increased body weight from day 7 (the second week), which persisted throughout the 4-week survival period (Figure
Post-ischemic long-term treatment with TMDZ increases the body weight after ischemic stroke. Animals underwent 2 h MCAO and reperfusion and received either an oral administration of TMDZ or vehicle (distilled water) at 2 h MCAO, 4 h reperfusion, and every day later for 28 days, and the body weight was measured every week. Compared to rats in ischemia-reperfusion control (IR Control), TMDZ treatment significantly increased body weight from day 7 (the second week), which persisted throughout the additional 3-week survival period. Values shown are mean ± S.D.,
To reduce errors associated with processing tissue for histological analysis, the residual brain volume is presented as the percentage of ipsilateral (right) hemisphere volume of the contralateral (left) hemisphere volume (indirect volume calculation). The residual brain volumes (the integrated right hemisphere volume) after a 4-week stroke were reduced compared with the sham-operated hemisphere or the contralateral hemisphere in both ischemia-reperfusion control and TMDZ-treated groups. However, compared with the residual brain volume in ischemia-reperfusion control rats, TMDZ treatment significantly increased the residual brain volumes after consecutive administration for 4 weeks (Figure
Post-ischemic long-term treatment with TMDZ reduces brain tissue loss after ischemic stroke. Animals underwent 2 h MCAO and reperfusion and received either an oral administration of TMDZ or vehicle (distilled water) at 2 h MCAO, 4 h reperfusion, and every day later for 28 days. The representative images of the whole brain and the sliced brain (a). The volume of right hemisphere (injured hemisphere) was indicated as the percentage of ipsilateral hemisphere volume of the contralateral hemisphere volume (indirect volume calculation) (b). Values shown are mean ± S.D.,
Post-ischemic long-term treatment with TMDZ improves the histopathological changes after ischemic stroke. Animals underwent 2 h MCAO and reperfusion and received either an oral administration of TMDZ or vehicle (distilled water) at 2 h MCAO, 4 h reperfusion, and every day later for 28 days, and the HE staining was measured 28 days after stroke. Computer generated MosaiX processed images (Carl Zeiss MicroImaging, Inc, Thornwood, NY, USA) of HE paraffin-embedded brain sections at coronal level (bregma
In addition, the ischemic lesion size was estimated using an in vivo MRI. T2WIs were obtained 28 days after TMDZ or vehicle consecutive treatment. Coronal forebrain sections were obtained at the level of caudate-putamen complex (Figure
Post-ischemic long-term treatment with TMDZ reduces ischemic lesion with MRI. Animals underwent 2 h MCAO and reperfusion and received either an oral administration of TMDZ or vehicle (distilled water) at 2 h MCAO, 4 h reperfusion, and every day later for 28 days. T2-weighted images were obtained from experimental animals 28 days after MCAO and TMDZ were administrated.
These results suggest that post-ischemic long-term treatment with TMDZ reduces brain damage and brain tissue loss after stroke.
Neurological score was normal in all animals before MCAO (score, 0). High-grade contralateral deficits (score, 2–5, Table
Neurological scores after stroke. Neurological function was measured by 5-point test after 2 h MCAO and 4 h reperfusion. High-grade contralateral deficits were presented at 2 h MCAO and 4 h reperfusion in all rats, and there was no significant difference among the groups.
Group | Ischemia/reperfusion | Tianma Duzhong capsule | ||
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Control | 44.5 mg/kg | 89 mg/kg | 178 mg/kg | |
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Post-ischemic long-term treatment with TMDZ improves neurological outcome after ischemic stroke. Animals underwent 2 h MCAO and reperfusion and received either an oral administration of TMDZ or vehicle (distilled water) at 2 h MCAO, 4 h reperfusion, and every day later for 28 days followed by neurological testing at 1, 3, 7, 14, 21, and 28 days. (a) The corner test demonstrated preferential turning to the right in animals that had undergone right MCAO/reperfusion and vehicle administration after stroke. Deficits persisted up to 28 days after stroke. Animals that received TMDZ, however, showed reduction of deficits over time. (b) The cylinder test demonstrated preferential right forearm placement in animals that had undergone right MCAO/reperfusion and vehicle administration after stroke. Deficits persisted up to 28 days after stroke. Animals that received TMDZ, however, showed reduction of deficits over time. Values shown are mean ± S.D.,
TMDZ did not change any of the hemorrheology parameters after normal rats were administrated with TMDZ for 4 weeks (Table
Hemorrheology parameters in normal rats. Animals received an oral administration of TMDZ or vehicle (distilled water) for 28 days, and then the blood was obtained, and hemorrheology parameters were detected. Values shown are mean ± SD,
Parameters | Control | Tianma Duzhong capsule | ||
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44.5 mg/kg | 89 mg/kg | 178 mg/kg | ||
WBV: low shear (mPas) ∣ 10 s−1 |
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WBV: moderate shear (mPas) ∣ 60 s−1 |
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WBV: high shear (mPas) ∣ 150 s−1 |
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PV |
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WBRV: low shear |
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WBRV: moderate shear |
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WBRV: high shear |
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Fb |
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HCT (%) |
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ESR(MM/H) |
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ESRK |
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RBCAI |
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RBCRI |
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RBCDI |
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RBCEI |
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WBV: whole blood viscosity; WBRV: whole blood reduced viscosity; PV: Plasma viscosity; Fb: fibrinogen; HCT: hematocrit; ESR: erythrocyte sedimentation rate; ESRK: erythrocyte sedimentation rate equation K value; RBCAI: red blood cell aggregation index; RBCRI: red blood cell rigidity index; RBCDI: red blood cell deformation index; RBCEI: red blood cell electrophoresis index.
Hemorrheology parameters in stroke rats. Animals underwent 2 h MCAO and reperfusion and received either an oral administration of TMDZ or vehicle (distilled water) at 2 h MCAO, 4 h reperfusion, and every day later for 28 days, and then the blood was obtained, and hemorrheology parameters were detected. Values shown are mean ± S.D.,
Parameters | Sham | Ischemia/reperfusion control | Tianma Duzhong capsule | ||
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44.5 mg/kg | 89 mg/kg | 178 mg/kg | |||
WBV: low shear (mPas) ∣ 10 s−1 |
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WBV: moderate shear (mPas) ∣ 60 s−1 |
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WBV: high shear (mPas) ∣ 150 s−1 |
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PV |
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WBRV: low shear |
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WBRV: moderate shear |
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WBRV: high shear |
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Fb |
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HCT (%) |
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ESR(MM/H) |
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ESRK |
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RBCAI |
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RBCRI |
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RBCDI |
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RBCEI |
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TMDZ significantly decreased the in vitro ADP- or thrombin-induced platelet aggregation after normal rats were administrated with TMDZ for 4 weeks (Figure
Long-term treatment with TMDZ inhibits ADP- or thrombin-induced platelet aggregation in normal rats. Animals received an oral administration of TMDZ or vehicle (distilled water) for 28 days, and then the blood was obtained, and the platelet aggregation induced by ADP or thrombin was detected. (a) and (c): representative curve of platelet aggregation induced by ADP (a) or thrombin (c). (b) and (d): quantitative analysis of changes in the platelet aggregation from 10 independent experiments of (a) and (c), respectively. Values shown are mean ± S.D.,
Long-term treatment with TMDZ inhibits ADP- or thrombin-induced platelet aggregation in ischemic stroke rats. Animals underwent 2 h MCAO and reperfusion and received either an oral administration of TMDZ or vehicle (distilled water) at 2 h MCAO, 4 h reperfusion and every day later for 28 days, and then the blood was obtained and the platelet aggregation induced by ADP or thrombin was detected. (a) and (c): representative curve of platelet aggregation induced by ADP (a) or thrombin (c). (b) and (d): quantitative analysis of changes in the platelet aggregation from 10 independent experiments of (a) and (c), respectively. Values shown are mean ± S.D.,
Ischemic hypoxic brain injury often causes irreversible brain damage and permanent behavioral performance deficits. A well-controlled animal model of MCAO in rats, which produces consistent cortical and subcortical infarcts, closely resembles the large hemispheric infarcts resulting from proximal MCAO in patients [
At present, the remarkable effect of TMDZ is not largely understood. An elevated blood viscosity value has been demonstrated in patient after both acute cerebral ischemia (24 h after the onset of stroke) and remote cerebral ischemic episode (3–6 months after the onset of stroke) although there are some differences in hemorheological parameters between patient with acute cerebral ischemia and patient with remote cerebral ischemic episode [
The cascade of events leading to neuronal injury and death in ischemia includes the release of cytokines, free radicals, and platelet activation [
In summary, the present results firstly established that post-ischemic long-term treatment with TMDZ confers the improvements of neurological outcome and the loss of injured hemisphere in animal ischemic stroke models, and its mechanisms might be associated with the improvements of hemorrheology and the inhibition of platelet aggregation. These results are very encouraging for the wide application of TMDZ in patients with ischemic stroke. Whether post-ischemic long-term treatment with TMDZ produces neuroregeneration in the treatment of ischemic stroke remains to be further investigated in the near future.
Li-Zhi Hong and Wei-wei Gu contributed equally to this work.
This work was supported by Grants from The National Natural Science Foundation of China (30973510 and 81171104), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, and a project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).