Effects of acupuncture plus mild hypothermia on apoptosis-related factors in rats with cerebral ischemia-reperfusion

Objective

To investigate the effect of acupuncture plus mild hypothermia on neurological function impairment score, cerebral infarct size and apoptosis-related factors in cerebral ischemia reperfusion injury (CIRI) rats.

Methods

Sixty healthy male Sprague-Dawley (SD) rats were routinely reared for 1 week. Ten rats were randomly selected as the sham operation group and 10 rats as the blank control group, while the remaining 40 rats were subjected to preparing the middle cerebral artery occlusion (MCAO) model by modified filament occlusion method. The 40 MCAO rats were further randomly divided into a model group, an acupuncture group, a mild hypothermia group and an acupuncture plus mild hypothermia group, with 10 rats in each group. Rats in the sham operation group, the blank control group and the model group did not accept treatment except binding; rats in the acupuncture group received acupuncture treatment; rats in the mild hypothermia group received mild hypothermia treatment; rats in the acupuncture plus mild hypothermia group received acupuncture and mild hypothermia treatment. 72 h after the treatment, neurological function impairment score was performed; the infarct area ratio was determined by 2,3,5-tripheyl tetrazolium chloride (TTC) staining; apoptosis of brain cells was observed by TUNEL method; the expressions of Bcl-2, Bax and Caspase-3 were detected by immunohistochemistry.

Results

Compared with the blank control group and the sham operation group, the neurological function impairment score, cerebral infarct area ratio, apoptosis, and the expressions of Bax and Caspase-3 in the model group were significantly increased, while the expression of Bcl-2 was significantly decreased, and there were significant between-group differences (all P<0.05). After the treatment, there were statistically significant differences among the treatment groups in the neurological function impairment score, cerebral infarct area ratio and apoptosis in the ischemic side of rats, as well as the expressions of Bcl-2, Bax and Caspase-3 (all P<0.05), and from the figures, tables and statistical analysis, it was found that a better tendency in the acupuncture plus mild hypothermia group than the acupuncture group or mild hypothermia group.

Conclusion

Acupuncture plus mild hypothermia can protect the brain cells by improving neurological function impairment, decreasing cerebral infarct area ratio, reducing the number of apoptotic cells in the ischemic area and regulating the expressions of apoptosis related proteins to inhibit apoptosis.

     

Protective effect of hydroalcoholic extract of Mimusops elengi Linn. flowers against middle cerebral artery occlusion induced brain injury in rats

Ethnopharmacological relevance

In the traditional Indian and Thai system of medicine, Mimusops elengi Linn., flower is used as brain tonic and to calm anxiety and panic attacks.

Aim of the study

The present study was designed to investigate the neuroprotective effect of hydroalcoholic extract of Mimusops elengi (ME) against cerebral ischemic reperfusion injury in rats.

Materials and methods

Male rats were pretreated with ME (100 and 200 mg/kg) for seven days and focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) method. After 60 min of MCAO and 24 h of reperfusion, a battery of behavioral tests assessed the extent of neurological deficits. Infarct volume and brain edema were measured in TTC stained brain sections and the extent of blood brain barrier (BBB) disruption was observed by Evan's blue extravasation. Oxidative and nitrative stress parameters were estimated in the brain homogenates. Further, simultaneous quantification of five polyphenolic biomarkers were done using HPLC.

Results

Pretreatment with ME at doses of 100 and 200 mg/kg significantly improved the neurobehavioral alterations and reduced the infarct volume, edema and extent of BBB disruption induced by ischemia reperfusion injury. It also prevented the alteration in the antioxidant status and reduced the nitrite levels when compared to ischemic animals. Further, HPLC studies revealed that ME contains five bioactive polyphenolic compounds.

Conclusions

These results clearly indicate the neuroprotective effect of ME against stroke like injury. The observed protective effect might be attributed to the polyphenolic compounds and their antioxidant and anti-inflammatory property.     

Protective effect of Sheng-Nao-Kang decoction on focal cerebral ischemia-reperfusion injury in rats

Ethnopharmacological relevance

Sheng-Nao-Kang decoction (SNK), a modified traditional Chinese medicine (TCM), has been used clinically for the treatment of acute and chronic cerebrovascular related diseases. To evaluate the protective effect of SNK on focal cerebral ischemia-reperfusion (I/R) injury in rats and investigate the underlying mechanisms.

Materials and methods

Focal cerebral I/R injury in rats was induced by middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion for 24 h. Adult male Sprague-Dawley (SD) rats were randomly divided into six kinds of groups: Sham group; I/R group; SNK-treated groups at doses of 0.7 g/kg, 1.4 g/kg and 2.8 g/kg; and nimodipine (NMP)-treated group. The recoveries of neurological function in rats were estimated by neurological defect scoring and 2,3,5-triphenyltetrazolium chloride (TTC) staining after 24 h reperfusion. Various biochemical indexes in serum were assayed by colorimetry, including malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), inducible nitric oxide synthase (iNOS) and total nitric oxide synthase (TNOS). Histological structures of the brain in rats were observed by hematoxylin and eosin (H&E) staining. Immunohistochemistry was performed to detect the caspase-3 protein content in rats.

Results

SNK administration significantly reduced the neurological defect scores and lessened the cerebral infarction volume. The treatment of SNK lowered MDA content, up-regulated SOD and GSH-Px levels, down-regulated iNOS and TNOS levels in serum. Furthermore, histological examination indicated that dense neuropil and largely surviving neurons were seen in SNK-treated rats. SNK administration restrained the expression of caspase-3 positive protein significantly.

Conclusion

The results suggest that SNK demonstrates a strong and ameliorative effect on cerebral I/R damage in rats. The protective mechanisms of SNK are associated with its properties of anti-apoptosis and anti-oxidation as well as regulation of iNOS and TNOS.     

Effects of acupuncture on the number of associated protein phosphorylation in brain tissues of MCAO rats based on protein microarray technique

Objective

To investigate the effects of acupuncture on the number of associated phosphorylated proteins in brain tissues of middle cerebral artery occlusion (MCAO) rats, based on the protein microarray technique.

Methods

The MCAO model was prepared according to the modified occlusion method using occlusion lines. Forty healthy Sprague-Dawley (SD) rats were randomly divided into 4 groups using the lottery method: a sham operation group, a model group, a control point group and an acupoint group, with 10 rats in each group. Rats in the sham operation group and the model group only received binding without acupuncture. Rats in the acupoint group received acupuncture at Dazhui (GV 14), Baihui (GV 20) and Shuigou (GV 25); rats in the control point group received acupuncture at non-acupoint control points. The needle was twisted once for 1 min after insertion and another time in the middle of the 30 min needle retaining. Acupuncture was conducted once every 12 h for 6 consecutive times. At the end of the experiment, the neurological impairment score was collected, and cells of the ischemic brain tissues were extracted. The protein phosphorylation of the related signaling was detected using the 720 phosphorylated antibody microarray technique, and the differentially expressed proteins between groups were screened.

Results

The neurological impairment scores after 72 h of treatment: compared with the sham operation group, the scores of the model group, the control point group and the acupoint group were significantly increased (P<0.01); compared with the model group, the scores of the acupoint group and the control point group were significantly decreased (P<0.01, P<0.05); the score of the acupoint group was better than that of the control point group (P<0.05). The results of the protein microarray: compared with the sham operation group, 48 proteins showed up-regulated phosphorylation (≥1.5 times) in the model group and the down-regulated was 28; compared with the model group, 35 proteins showed up-regulated phosphorylation in the control point group, and the down-regulated was 24. There were 29 proteins showing up-regulated phosphorylation in the acupoint group and the down-regulated was 51. The numbers of proteins involved in the function and signal transduction pathways were also different.

Conclusion

Acupuncture at Dazhui (GV 14), Baihui (GV 20) and Shuigou (GV 25) can effectively repair brain injury. The ischemic injury of brain tissue may be caused by imbalance of a variety of proteins, and acupuncture can promote brain tissue repair by multi-functional and multi-channel regulation of the protein disorders.

     

Neuroprotective effect of Artemisia absinthium L. on focal ischemia and reperfusion-induced cerebral injury

Ethnopharmacological relevance

Artemisia absinthium L. has long been used as traditional herbal medicine in China, Europe and Pakistan for the treatment of gastric pain, cardiac stimulation, to improve memory and for the restoration of declining mental function.

Aim of the study

The present study was designed to investigate the potential protective effects of Artemisia absinthium on cerebral oxidative stress and damage as well as behavioral disturbances induced by cerebral ischemia and reperfusion injury in rats.

Materials and methods

Focal ischemia and reperfusion were induced by middle cerebral artery occlusion (MCAO) for 90 min, followed by 24 h reperfusion. MCAO led to significant rise in infarct size and lipid peroxidation, and depletion in glutathione content, superoxide dismutase and catalase activity in brain. Further, behavioral deficits like motor incoordination and impairment of short-term memory were also significantly impaired by MCAO as compared with sham group.

Results

The brain oxidative stress and damage, and behavioral deficits were significantly attenuated by pre-treatment with the methanol extract of Artemisia absinthium (100 mg/kg and 200 mg/kg, p.o.).

Conclusion

These findings suggested that Artemisia absinthium is neuroprotective and may prove to be useful adjunct in the treatment of stroke.     

Preconditioning with the traditional Chinese medicine Huang-Lian-Jie-Du-Tang initiates HIF-1α-dependent neuroprotection against cerebral ischemia in rats

Ethnopharmacological relevance

Huang-Lian-Jie-Du-Tang (HLJDT) is a classical heat-clearing and detoxicating formula of traditional Chinese medicine that is widely used to treat stroke. The present study was designed to investigate the effects of HLJDT preconditioning on neurons under oxygen and glucose deprivation (OGD) and rats subjected to middle cerebral artery occlusion (MCAO).

Materials and methods

A stroke model of rats was obtained through MCAO. Following HLJDT preconditioning, the cerebral infarction volume, cerebral water content, and neurological deficient score were determined. Cerebral cortical neurons cultured in vitro were preconditioned with HLJDT and then subjected to OGD treatment. The release of lactate dehydrogenase (LDH) from neurons was detected. The levels of hypoxia-inducible factor-1α (HIF-1α) and PI3K/Akt signaling were analyzed by western blotting, and the levels of erythropoietin (EPO) and vascular endothelial growth factor (VEGF) in the supernatant of the neurons and the plasma of MCAO rats were measured through a radioimmunological assay. The apoptosis and proliferation of neurons were analyzed by immunohistochemistry.

Results

HLJDT preconditioning significantly reduced the cerebral infarction volume and cerebral water content and ameliorated the neurological deficient score of MCAO rats. In addition, HLJDT preconditioning protected neurons against OGD. Increased HIF-1α, EPO, and VEGF levels and the activation of PI3K/Akt signaling were observed as a result of HLJDT preconditioning. Furthermore, HLJDT preconditioning was found to inhibit ischemia-induced neuron apoptosis and to promote neuron proliferation under conditions of ischemia/reperfusion.

Conclusion

Both rats and neurons subjected to HLJDT preconditioning were able to resist ischemia/reperfusion or hypoxia injury through the inhibition of apoptosis and the enhancement of proliferation, and these effects were primarily dependent on the activation of the PI3K/Akt signaling pathway and HIF-1α.     

Effect of electroacupuncture on expressions of VEGF and CD31 in MCAO model rats

Objective

To investigate the effect of electroacupuncture (EA) at Ganshu (BL 18) and Shenshu (BL 23) on vascular endothelial growth factor (VEGF) and platelet endothelial cell adhesion molecule-1 (PECAM-1)/CD31 around the cerebral infarction focus in middle cerebral artery occlusion (MCAO) rats and the possible mechanism, thus to provide a new strategy for the treatment of cerebral ischemic stroke by acupuncture.

Methods

A total of 180 healthy male Sprague-Dawley (SD) rats were randomly divided into a sham operation group, a model group, an acupoint group and a non-acupoint group, 45 rats in each group. MCAO model was established using the modified line-embolus method in all rats except for those in the sham operation group; rats in the acupoint group were treated with EA at Ganshu (BL 18) and Shenshu (BL 23); rats in the non-acupoint group were treated with EA at the control points; rats in other 2 groups were only subjected to bundling without treatment. Ten rats in each group were randomly selected on the 3rd day, the 14th day and the 21st day after acupuncture stimulation to test the neurological function impairment. The expression levels of CD31 and VEGF were also detected.

Results

Compared with the model group and non-acupoint group, the neurological function score of the acupoint group was decreased at each time point, and the differences were statistically significant (P<0.05, P<0.01). The expressions of VEGF and CD31 in each group were the lowest on the 3rd day, reached the peak on the 14th day and still remained at high level on the 21st day. And the differences among groups were statistically significant both on the 14th day and the 21st day (P<0.05, P<0.01). Compared with the model group and the non-acupoint group, the expressions of VEGF and CD31 in the acupoint group were increased, and the differences were statistically significant (all P<0.05).

Conclusion

EA at Ganshu (BL 18) and Shenshu (BL 23) can significantly improve the neurological function score of MCAO model rats, and shows protective effect on cerebral ischemia. The protective mechanism may be related to the up-regulation of CD31 and VEGF expression around the cerebral infarction focus in the MCAO model rats and induction of angiogenesis.

     

Neuroprotective effect of calycosin on cerebral ischemia and reperfusion injury in rats

Ethnopharmacological relevance

Radix Astragali has been commonly used as traditional herbal medicine in China for reinforcing vital energy, strengthening superficial resistance and promoting the discharge of pus and the growth of new tissue.

Aim of the study

The present study was to investigate the neuroprotective effect of calycosin isolated from the roots of Radix Astragali on cerebral ischemic/reperfusion injury.

Materials and methods

After 24 h of reperfusion following ischemia for 2 h induced by middle cerebral artery occlusion (MCAO), Sprague-Dawley rats were intragastrically administered different doses of calycosin (7.5, 15, 30 mg/kg, respectively). Neurological deficit, infarct volume, histopathology changes and some oxidative stress markers were evaluated after 24 h of reperfusion.

Results

Treatment with calycosin significantly ameliorated neurologic deficit and infarct volume after cerebral ischemia reperfusion. Calycosin also reduced the content of malondialdehyde (MDA), protein carbonyl and reactive oxygen species (ROS), and up-regulated the activities of superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-Px) in a dose-dependent manner. Moreover, calycosin can also inhibit the expression of 4-Hydroxy-2-nonenal (4-HNE).

Conclusion

These results suggest that calycosin has a neuroprotective effect against cerebral ischemia/reperfusion injury. The mechanism might be attributed to its antioxidant effects.     

Effects of different frequencies of electro-acupuncture at shuigou (GV 26) on recovery of motor function in rats with focal cerebral ischemic injury

Objective

To investigate the effects of different frequencies of electro-acupuncture at Shuigou (GV 26) on the latent period and wave amplitude of motor evoked potentials (MEPs) in rats with focal cerebral infarction.

Methods

Fifty healthy male Wistar rats were randomly divided into five groups: controls, model, 2 Hz Shuigou, 50 Hz Shuigou and 100 Hz Shuigou. There were 10 rats in each group. Using a modification of a technique for middle cerebral artery occlusion, focal cerebral ischemic injury was induced in all rats except those in the control group. The rats in the control group received no treatment. After behavioral deficit had been evaluated using the Zausinger 6-point neurological function score, the rats in the Shuigou groups underwent acupuncture and continuous wave stimulation at a frequency of 2 Hz, 50 Hz or 100 Hz (intensity 1 mA) for 10 min twice daily for 3 days. The control and model groups underwent no intervention. Zausinger 6-point neurological function score and MEPs were measured 72 h after the start of treatment.

Results

The neurological function scores of the three Shuigou groups were significantly higher than those of the model group (P<0.05). There was no significant difference between sides in the latency and amplitude of MEPs in the model group (P>0.05). The latency on the affected side in the model group was significantly longer than that in the control group (P<0.05) and the amplitude on affected side was significantly reduced (P<0.01). After 3 days of electro-acupuncture, the latency on the affected side in the 2 Hz Shuigou group was significantly shortened (P<0.05) and the amplitude was significantly increased (P<0.05) compared with the model group.

Conclusion

Low frequency electro-acupuncture at Shuigou (GV 26) can promote recovery of motor function after focal cerebral ischemic injury in rats.     

Therapeutic effect of acupuncture and massage for shoulder-hand syndrome in hemiplegia patients: a clinical two-center randomized controlled trial

Objective

To evaluate the therapeutic effects of acupuncture and massage for shoulder-hand syndrome in hemiplegia patients.

Methods

One hundred and twenty hemiplegia patients with stage I shoulder-hand syndrome were randomly divided into a group treated with standardized electric acupuncture and massage, and a group treated with rehabilitation therapy for 6 weeks. The primary indices evaluated were pain on passive movement of the shoulder using the numeric pain rating scale (NPRS), and the number of patients with shoulder-hand syndrome at Steinbrocker stage II or III after treatment. The secondary indices were Fugl-Meyer evaluation of functional movement of the upper limb and hand using the modified rankin scale (MRS).

Results

At post-treatment evaluation and a 12-week follow-up visit, NPRS score, number of patients with stage II or III shoulder-hand syndrome, and MRS score were all improved in the acupuncture-massage group compared with the rehabilitation group (P<0.05). On Fugl-Meyer evaluation, functional movement of the upper limb was also improved in the acupuncture-massage group compared with the rehabilitation group (P<0.05).

Conclusion

Standardized acupuncture-massage therapy may have curative effects on shoulder-hand syndrome in hemiplegia patients.     

Neuroprotective activity of Matricaria recutita Linn against global model of ischemia in rats

Ethnopharmacological relevance

Traditionally, the whole plant is used for various diseases, including neuronal disorders.

Aim of the study

To evaluate the neuroprotective effect of Matricaria recutita L. against global cerebral ischemia/reperfusion (I/R) injury-induced oxidative stress in rats.

Materials and methods

Neuroprotective activity was carried out by global cerebral ischemia on Sprague–Dawley rats by bilateral carotid artery (BCA) occlusion for 30 min followed by 60 min reperfusion. The antioxidant enzymatic and non-enzymatic levels were estimated along with cerebral infarction area and histopathological studies.

Results

The Matricaria recutita L. methanolic extract showed dose-dependent neuroprotective activity by significant decrease in lipid peroxidation (LPO) and increase in the superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and total thiol levels in extract treated groups as compared to ischemia/reperfusion group. Cerebral infarction area was significantly reduced in extract treated groups as compared to ischemia/reperfusion group.

Conclusion

The methanolic extract of Matricaria recutita L. showed potent neuroprotective activity against global cerebral ischemia/reperfusion injury-induced oxidative stress in rats.     

Protective effect of Danhong injection on cerebral ischemia–reperfusion injury in rats

Ethnopharmacological relevance

Danhong injection (DH), a Chinese medical product, is used extensively for the treatment of cerebrovascular diseases such as acutely cerebral infarction in clinic.

Aim of the study

To explore the protective effect and the relevant mechanisms of DH on cerebral ischemia–reperfusion (I/R) injury.

Materials and methods

Cerebral I/R injury was induced through four-vessel occlusion (4-VO) or middle cerebral artery occlusion (MCAO). Adult male SD rats were randomly divided into six kinds of groups: normal control group, sham-operated group, I/R injury group, DH-treated groups at doses of 0.5 ml/kg, 1.0 ml/kg and 2.0 ml/kg. The effects of DH on murine neurological deficits and cerebral infarct volume, 6-keto-prostagladin F_1α (6-keto-PGF_1α) level, malondialdehyde (MDA) level and superoxide dismutase (SOD) activity in brain tissue, as well as the activities of plasma tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) after I/R were evaluated. Moreover, the expressions of Bcl-2 and Bax protein were detected by immunohistochemistry.

Results

There was no significant difference between the control group and the sham-operated group based on the measurement indicators. Compared with the vehicle-treated group, rats treated with DH showed dose dependent reductions in brain infarction size, and improvement of neurological outcome. The level of 6-keto-PGF_1α and the activities of SOD and plasma t-PA were enhanced significantly, whereas the level of MDA and the activity of plasma PAI were declined significantly. The immunohistochemical staining results also revealed that the expression of Bcl-2 protein was up-regulated and that of Bax protein was down-regulated when exposed to DH.

Conclusion

DH demonstrates a strong ameliorative effect on cerebral I/R damage in rats by its anticoagulant, antithrombotic, antifibrinolytic and antioxidant activities. Furthermore, suppressing apoptosis through regulating Bcl-2 and Bax protein expressions should be another potential mechanism by which DH exerts its neuroprotective function.     

Effect of acupuncture anesthesia on acne vulgaris of pricking-bloodletting cupping: a single-blind randomized clinical trail

Objective

To evaluate the effect on acne vulgaris of pricking-bloodletting cupping at Dazhui (GV 14) under acupuncture anesthesia, and establish whether providing anesthesia to the treatment area by manipulating Hegu (LI 4) and Quchi (LI 11) might have an additional therapeutic benefit.

Methods

Thirty-eight patients were recruited and randomized into a control group and an intervention group with a single-blind (observer-blind) method. The control group was treated by pricking-bloodletting cupping at Dazhui (GV 14) and the studied group by pricking-bloodletting cupping at Dazhui (GV 14) under acupuncture anesthesia at Hegu (LI 4) and Quchi (LI 11). Both groups were treated twice weekly for 6 weeks. The analgesic and therapeutic effects of acupuncture were evaluated on a visual analog scale (VAS) and global acne grading system (GAGS), respectively.

Results

There were differences in the VAS scores of pain on pricking and in the pricked area, and the duration of pain between the groups. After 12 treatments, there was a significant reduction in GAGS scores from baseline in both groups, but there was no significant difference between the groups.

Conclusion

Acupuncture anesthesia at Hegu (LI 4) and Quchi (LI 11) is an effective means of alleviating the pain of pricking-bloodletting cupping and reducing the duration of pain in the treatment area. Pricking-bloodletting cupping at Dazhui (GV 14) improves the skin lesions of patients with moderate acne vulgaris, but acupuncture anesthesia does not appear to have an additional therapeutic effect.     

An-Gong-Niu-Huang Wan protects against cerebral ischemia induced apoptosis in rats: Up-regulation of Bcl-2 and down-regulation of Bax and caspase-3

Ethnopharmacological relevance

The An-Gong-Niu-Huang Wan (AGNH), a Chinese traditional medicine, has been used for treatment of cerebral diseases for centuries in China and other Asian countries, and is approved by the State Food and Drug Administration of China for the treatment of stroke. The aim of present study is to test the neuroprotective effects of AGNH on cerebral ischemia in rats and to explore the underlying mechanisms.

Materials and methods

75 Male Sprague-Dawley rats were randomly divided into 5 groups: sham, ischemia–reperfusion (I/R), and I/R plus 0.065 g/kg/d AGNH, 0.125 g/kg/d AGNH and 0.25 g/kg/d AGNH. Cerebral ischemia was induced by 1.5 h of middle cerebral artery occlusion (MCAO). Neurological functional deficits were evaluated according to Zea longa?s score, cerebral infarct area was measured by tetrazolium staining. Cell injury and apoptosis were assessed by Nissl staining and DNA fragmentation assay. The expression of Bax, Bcl-2 and caspase-3 were analyzed by Western blot.

Results

Rats subjected to MCAO exhibited worsened neurological score, infarct area, cell damage and apoptosis. These were all attenuated by AGNH (0.125 and 0.25 g/kg/d). Moreover, AGNH reversed cerebral ischemia induced decreases in Bcl-2 expression and increases in Bax and caspase-3 expression.

Conclusions

These results suggest that AGNH exerts neuroprotective effects, and the neuroprotection is likely to relate to depressed Bax/Bcl-2 ratio and caspase-3 level, leading to inhibition of apoptotic cell death.     

血塞通与阿司匹林联合用药对大鼠脑缺血再灌注损伤的保护作用

Objective:To investigate the protective effects of the combination of Xuesaitong(XST)and aspirin on cerebral ischemia and reperfusion injury(CIRI)in rats,and further explore the underlying mechanisms.Methods:A total of 150 male Sprague-Dawley(SD)rats were randomly divided into five groups with 30rats in each group:sham group,middle cerebral artery occlusion/reperfusion(MCAO/R)model group,XST group,aspirin group,and XST+aspirin group.Rats were pretreated with XST,aspirin,or XST+aspirin for7 d.One hour after the last administration,a model of CIRI was induced by MCAO/R.Neurological deficits were assessed using Longa’s five-point scale.Cerebral edema was detected by the measurement of brain water content.The volume of cerebral infarction was determined by 2,3,5-triphenyltetrazolium chloride(TTC)staining.The activities of superoxide dismutase(SOD),catalase(CAT),and glutathione peroxidase(GSH-Px),as well as levels of malonaldehyde(MDA)were detected by commercial kits.Enzyme-linked immunosorbent assay(ELISA)was used to determine the levels of interleukin-1(),interleukin-4(IL-4),interleukin-6(IL-6),interleukin-10(IL-10),tumor necrosis factor-alphamonocyte chemotactic protein 1(MCP-1),and kynurenine in serum,cerebral cortex,and hippocampus of MCAO/R rats.The protein expression of nuclear factor erythroid 2-related factor(Nrf2),heme oxygenase-1(HO-1),I-kappa B alpha(IκBα),and nuclear factor kappa B(B)/p65 in the cortex were analyzed by western blotting.Results:Treatment of XST,aspirin,and XST+aspirin significantly alleviated the neurological deficits,cerebral edema,and cerebral infarct volume induced by MCAO/R.Treatment of XST,aspirin,and XST+aspirin also reduced MDA,,MCP-1,and kynurenine levels,and increased SOD,CAT,GSH-Px,IL-4,and IL-10 levels in serum,cerebral cortex,and hippocampus of MCAO/R rats.Furthermore,treatment of XST,aspirin,and XST+aspirin decreased the expression of nuclearB/p65 and increased the expression of IκBα,nuclear Nrf2,and HO-1.Importantly,the combination of XST and aspirin enhanced the protective effects of XST or aspirin treatment alone on CIRI in rats.Conclusion:The combination of XST and aspirin significantly inhibited oxidative stress and inflammation in serum,cerebral cortex,and hippocampus of MCAO/R rats.The combination of XST and aspirin exerted more protective effects than XST or aspirin treatment alone.The combination of XST and aspirin might provide the synergistic therapeutic effects on CIRI,and deserve further clinical investigation.     

Evaluation of the Effect of Metoclopramide on Acupuncture and Electro-acupuncture Analgesia – A Blind and Controlled Study in Rats

Background

Metoclopramide is an antagonist that is selective of dopamine DA₂-receptors. In high doses it appears to have antagonist properties also towards DA₁ receptors. It has been demonstrated that the dopaminergic system is closely correlated to opioid actions; in particular it has been observed that the destruction of DA₂ receptors in rats increases analgesia by μ and k₁ agonists. The role of dopamine on acupuncture-induced analgesia is not fully defined; the data available is scarce and contrasting.

Aim

The aim of the present study was to verify whether the administration of metoclopramide potentiates the analgesic efficacy of manual and electro-acupuncture in the rats.

Methods

In this study we used 140 Sprangue-Dawley rats weighing between 180–220 g, divided into 7 groups of 20 on the basis of type of treatment. Control group 1: no manipulation; control group 2: subjected to all the manipulations without treatment (acupuncture and/or metoclopramide); group 3: treated with metoclopramide; group 4: treated with acupuncture; group 5: treated with metoclopramide and acupuncture; group 6: treated with metoclopramide and electro-acupuncture; group 7: treated with electro-acupuncture. The pain threshold was measured using the plantar test.

Results

Throughout the experiment there were no significant variations in the pain thresholds of either of the two control groups. Rats treated with metoclopramide showed an increased pain threshold. Both groups treated with acupuncture and electro-acupuncture showed an increased pain thresholds in the plantar test. Pre-treatment with metoclopramide increased the analgesic effect of the stimulation.

Conclusion

It can be demonstrated that metoclopramide possesses analgesic properties and can potentiate the effect of acupuncture and electro-acupuncture.     

Effect of acupuncture plus mild hypothermia on MAPK/ERK pathway of brain tissues in rats with cerebral ischemia-reperfusion injury

Objective

To observe the protective effect of acupuncture plus mild hypothermia on brain tissues in rats with cerebral ischemia-reperfusion injury (CIRI), and the influence on protein expression levels of phosphorylated Raf-1, MEK-2 and ERK1/2 in the mitogen-activated protein kinase (MAPK)/extracellular regulated protein kinases (ERK) pathway, and to explore the mechanism of acupuncture plus mild hypothermia therapy for the ischemic stroke.

Methods

Ninety Sprague-Dawley (SD) rats were randomly divided into a blank control group, a sham operation group, a model group, an acupuncture group, a mild hypothermia group and an acupuncture plus mild hypothermia group, 15 rats in each group. Except the rats in the blank control group, the remaining rats were used to prepare the middle cerebral artery occlusion (MCAO) models according to the modified occlusion method using lines, while only the occlusion lines were inserted without blocking the brain arteries of rats in the sham operation group. When the vital signs of rats were stable, rats in the blank control group did not receive any intervention; rats in the sham operation group and the model group received fastening without treatment; rats in the acupuncture group, the mild hypothermia group, and the acupuncture plus mild hypothermia group were treated with the corresponding therapeutic methods. 72 h later, observed neurologic injury score, evaluated infarction area ratio by 2,3,5-tripheyl tetrazolium chloride (TTC) staining, determined apoptosis by TUNEL assay, and measured the phosphorylated Raf-1, MEK-2 and ERK1/2 protein expression levels in rat ischemic hippocampal tissues by Western blot assay.

Results

Compared with the blank control group and the sham operation group, after modeling, the neurologic injury score, infarction area ratio and apoptotic cells were increased, and phosphorylated Raf-1, MEK-2 and ERK1/2 protein expression levels were significantly increased in the model group; the differences were statistically significant (P<0.05 or P<0.01). Compared with the model group, after acupuncture or mild hypothermia therapy, neurologic injury score and infarction area ratio were decreased; apoptotic cells and phosphorylated Raf-1, MEK-2 and ERK1/2 protein expression levels were significantly decreased; the differences were statistically significant (P<0.05 or P<0.01). Compared with the acupuncture group, neurologic injury score and phosphorylated Raf-1, MEK-2 and ERK1/2 protein expression levels were decreased in the acupuncture plus mild hypothermia group; differences between the groups were statistically significant (P<0.05 or P<0.01). Compared with the mild hypothermia group, phosphorylated Raf-1, MEK-2 and ERK1/2 protein expression levels decreased in the acupuncture plus mild hypothermia group, and differences were statistically significant (P<0.01).

Conclusion

Acupuncture or mild hypothermia therapy can improve neurologic injury, reduce infarction area and apoptosis, which brought about protective effect on the brain tissues, in the MCAO model. The protective effect of acupuncture plus mild hypothermia group is the strongest. The mechanism may involve the MAPK/ERK pathway, by reducing the phosphorylated Raf-1, MEK-2 and ERK1/2 protein expression levels.

     

Calycosin-7-O-β-d-glucoside regulates nitric oxide /caveolin-1/matrix metalloproteinases pathway and protects blood–brain barrier integrity in experimental cerebral ischemia–reperfusion injury

Ethnopharmacology relevance

Astragali Radix (AR) has been used for thousands years to treat ischemic stroke. Calycosin and its glycoside form calycosin-7-O-β-d-glucoside (CG) are two representative isoflavones in Astragali Radix. However, its neurological effects and related molecular mechanisms are largely unknown. The present study aims to evaluate the neuroprotective effects of CG on blood–brain barrier (BBB) integrity of ischemic brain tissue and explore the relevant signaling mechanisms.

Material and method

Male adult Sprague-Daweley rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) plus 24 h or 14 days of reperfusion. CG (26.8 mg/kg) was intraperitoneally administered into the rats at 15 min before onset of ischemia. The neuroprotective effects of CG were evaluated by measuring infarct volume, histological damage and BBB permeability. Furthermore, the effects of CG on scavenging nitric oxide (NO), and modulating matrix metalloproteinases (MMPs) and caveolin-1 (cav-1) were investigated with in vitro cultured brain microvascular endothelial cells treated with NO donor or oxygen–glucose deprivation (OGD) and/or in vivo rat model of MCAO cerebral ischemia–reperfusion injury.

Results

CG treatment significantly reduced infarct volume, histological damage and BBB permeability in the in vivo MCAO ischemia–reperfusion rat model. CG treatment remarkably inhibited the expression and activities of MMPs, and secured the expression of cav-1 and tight junction proteins in the microvessels isolated from ischemic rat cortex. Furthermore, CG was revealed to scavenge NO, inhibit the activities of MMP-2 and MMP-9, and attenuate cell death in the in vitro cultured brain microvascular endothelial cells under OGD condition.

Conclusion

CG could protect BBB integrity in experimental cerebral ischemia–reperfusion injury via regulating NO/cav-1/MMPs pathway.     

Effect of electroacupuncture at Ganshu (BL 18) and Shenshu (BL 23) on the expression of EphB2 protein in cortex around cerebral infracted area of rat

Objective

To observe the effect of electroacupuncture (EA) on the expression of erythropoie-tin-producing hepatocyte receptor B2 (EphB2) in the cortex around the infracted area of middle cerebral artery occlusion (MCAO) rats at different timing, and to reveal the possible mechanism of acupuncture in the treatment of cerebral ischemia.

Methods

A total of 180 male Sprague-Dawley (SD) rats were randomly divided into a sham operation group, a model group, an acupoint group and a non-acupoint group, with 45 rats in each group. Rats in each group were further divided into three subgroups: postoperative 3 d, postoperative 14 d and postoperative 21 d groups, with 15 rats in each subgroup. The MCAO model was made by the modified occlusion method. The neurological function score, 2,3,5-triphenyl tetrazolium chloride (TTC) staining, immunohistochemistry assay, immunofluorescence double labeling method and Western blot were used to detect the corresponding indicators.

Results

The neurological impairment of rats was most obvious at postoperative 3 d, and then gradually improved with time, which was more significant in the acupoint group (P<0.05). The change of infarcted volume was consistent with the neurological function impairment. The number of EphB2 positive cells (EphB2⁺) around the infarcted area was decreased significantly at postoperative 3 d, and then gradually improved with time, which returned to the same level as that in the sham operation group at postoperative 21 d. The increase was most significant in the acupoint group (P<0.05), and the positive cell number was higher than that in the sham operation group (P<0.01). Western blot and immunohistochemistry results were basically consistent. Immunofluorescence displayed that EphB2⁺ and postsynaptic density-95 positive (PSD-95+) were co-expressed, after the MCAO operation, in the cortical neuron around the infracted area, and the number of co-expressing cells was increased gradually with time, which was most significant in the acupoint group (P<0.05).

Conclusion

Electroacupuncture at Ganshu (BL 18) and Shenshu (BL 23) can significantly improve the neurological function and cerebral infarcted volume ratio of MCAO rats, which may be related to the activation of EphB2 expression in cortex around the infracted area and the promotion of synaptic remodeling.