Protective effect of ultrashortwave versus radix salviae miltiorrhizae on brains of rats with cerebral ischemia-reperfusion injury

BACKGROUND： HOW to control the effect of oxygen-derived free radicals on development of cerebral injury and cerebral edema is a key factor for treating cerebral ischemia-reperfusion injury. OBJECTIVE： To observe and compare the protective effects, synergistic action and mechanisms of ultrashortwave （USW） and radix salviae miltiorrhizae （RSM） on the focal cerebral ischemia-reperfusion injuries in rats. DESIGN： Randomized controlled animal study SEI-FING： Department of Rehabilitation Medicine, First Hospital affiliated to China Medical University MATERIALS： A total of 160 healthy Wistar rats of both genders and aged 18-20 weeks weighing 250-300 g of clean grade were selected in this study. 5 mL/ampoule RSM injection fluid was produced by the First Pharmaceutical Corporation of Shanghai （batch number： 011019, 0.01 mug）. The USW therapeutic device was produced by Shanghai Electronic Device Factory with the frequency of 40.68 MHz and the maximal export power of 40 W. The first channel of power after modulation was 11 W. METHODS： The experiment was carried out in the Rehabilitation Medicine Department of the First Hospital affiliated to China Medical University from May 2002 to January 2003. Focal ischemia-reperfusion model was established in rats by reversible right middle cerebral artery occlusion with filament. Right cerebral ischemia was for 2 hours and then with 24 hours reperfusion. The scores of neurological deficits were evaluated by 0 to 4 scales. After surgery, 64 successful rats models were divided into four groups according to digital table： control group, USW group, RSM group and RSM ＋ USW group with 16 cases in each group. Rats in control group were intraperitoneally injected with the same volume of saline （0.1 mL/g）; rats in USW group were given small dosage of USW on head for 10 minutes at 6 hours after reperfusion; rats in RSM group were intraperitoneally injected with 0.01 mL/g RSM solution at 30 minutes before reperfusion; rats in RSM ＋ USW group were intraperitoneally injected with 0.01 mL/g RSM parenteral solution at 30 minutes before reperfusion and given small dosage of USW on head for 10 minutes once at 6 hours after reperfusion; sixteen rats in sham operation group did not receive any treatment. All 80 rats were taken brains at 24 hours after reperfusion to measure wet and dry weights to calculate water content： Cerebral water content （%） = （1-dry/wet weight） × 100%. Superoxide dismutase （SOD） activity was measured by hydroxylamine method and malondialdehyde （MDA） content was measured by TBA photometric method. MAIN OUTCOME MEASURES ： Cerebral water content, SOD activity and MDA content RESULTS： All 160 rats except 80 failing in modeling were involved in the final analysis. （① The cerebral water content of left hemisphere made no significant difference （P 〉 0.05）. The cerebral water content of right hemisphere in the control group and the three treatment groups was obviously higher than that of the sham operation group [（81.26±0.77）%, （79.74±0.68）%, （79.76±0.81）%, （79.61±0.79）%, （77.43±0.61）%, P 〈 0.05]. The cerebral water content of right hemisphere in the three treatment groups was obviously lower than that of the control group （P〈 0.05）. There was no significant difference among the three treatment groups （P 〉 0.05）. ② Compared with the control group, SOD activity （right） of the control group decreased obviously （P 〈 0.05）, while MDA content increased obviously （P 〈 0.05）. SOD activity in the three therapeutic groups increased obviously, while MDA content decreased obviously （P 〈 0.05）; there was no significant difference among the three treatment groups （P 〉 0.05）. CONCLUSION： ① USW and RSM therapy have neuroprotective effects against focal cerebral ischemia-reperfusion injuries by means of decreasing cerebral water content and MDA and increasing the activity of SOD. ② Synergistic action was not observed between these two therapeutic methods.

Effects of acupuncture on iron metabolism in the hippocampus of the rats with cerebral ischemia-reperfusion injury based on the imbalance of iron homeostasis

Objective:This study aimed to observe the effects of acupuncture on iron metabolism in the hippocampus of rats with cerebral ischemia-reperfusion injury(CIRI)and to explore the mechanism of acupuncture on neural repairinCIRIrats.Methods:After general feeding for 3 days,16 of 48 healthy Sprague-Dawley rats were selected according to a random number table to form a sham-occlusion group,and the models were prepared in the remaining rats.After successful modeling,the rats were randomized into model and acupuncture groups,with 16 rats in each group.The CIRI models were prepared using the modified Zea Longa suture-occlusion method.In the acupuncture group,the rats were fixed and acupuncture was delivered at“Shuigou"(GV26),“Dazhui"(GV14),and“Baihui"(GV20),and the needles were manipulated once every 15 min.In the sham-occlusion and model groups,rats were fixed without acupuncture.The intervention was performed for 30 min in each group,once every 12 h,seven times.The degree of neurological impairment was assessed by using the modified Garcia score.Cerebral blood flow(CBF)was monitored using a laser speckle blood flow imaging system.The cerebral infarction area ratio was measured using triphenyltetrazolium chloride staining.Ferric ion aggregation in brain tissue was noted using Prussian blue staining.The ferric ion and glutathione(GSH)contents of the hippocampal tissue on the ischemic side were detected using ferric ion and GSH kits.The protein expression of transferrin receptor 1(TFR1),iron regulatory protein 2(IRP2),and ferroportin(FPN)was detected via western blotting.Results:(1)Before intervention,compared with the sham-occlusion group,the Garcia neurological score was significantly reduced in the model and acupuncture groups(both P<0.01).After the intervention,compared with the sham-occlusion group,the Garcia neurological score in the model group decreased significantly(P<0.01),and the score in the acupuncture group was elevated compared with the model group(P<0.o5).Compared to before the intervention,the Garcia neurological score increased after the intervention in the acupuncture group(P<0.05).(2)Compared with the sham-occlusion group,in the model group,CBF was reduced,the infarct area was significantly enlarged,ferric ion aggregation increased in the hippocampus,the content of ferric ion increased,and that of GSH decreased significantly(both P<0.01),while the protein expression of TFR1 and IRP2 was upregulated and that of FPN was downregulated(both P<0.05).When compared with the model group,in the acupuncture group,CBF was increased,the infarct area was reduced(P<0.01),the ferric ion aggregation was reduced in the hippocampus,the content of ferric ion decreased,and that of GSH increased significantly(P<0.05,P<0.01);the protein expression of TFR1 and IRP2 was downregulated and that of FPN was upregulated(both P<0.05).Conclusion:Acupuncture can alleviate CiRI,which may be related to the regulation of iron homeostasisassociated transferrin in the hippocampus,attenuation of ferric ion aggregation in hippocampal tissue,reduction of lipid peroxidation,and inhibition of ferroptosis.

Experimental Study on the Protection of Agrimony Extracts from Different Extracting Methods against Cerebral Ischemia-Reperfusion Injury

Objective To study the protective effect of agrimony extracts from different extracting methods on cerebral ischemia-reperfusion injury in rats, in order to optimize the extraction scheme of agrimony.Methods Male rats were randomly assigned into seven groups: 1. Sham-operated group, 2. Untreated MCAO group (MCAO), 3. Petroleum ether extract of Agrimonia pilosa treated MCAO group (PEA), 4. Ethyl acetate extract of Agrimonia pilosa treated MCAO group (EAEA), 5. Ethanol extract of Agrimonia pilosa treated MCAO group (EEA), 6. Water extract of Agrimonia pilosa treated MCAO group (WEA), 7. Nimodipine treated MCAO group (NP). Intragastrical drug administration (i.g) was performed at 0 and 6 hours after MCAO.Neurological function tests were performed after reperfusion for 24 hours, then the brain was removed for the evaluations of the cerebral infarction volume (percentage of total brain volume) by immunohistochemistry,histological changes (hematoxylin-eosin staining), Na+/K+-ATPase, Ca2+-ATPase (modified method of Svoboda and Mosinger), mRNA expression of Tumor suppressor gene (P53) and hot shock protein (HSP70)(quantitative real-time PCR).Results The neurological function of MCAO group had significantly higher scores than the sham group (P<0.01). The WEA group showed a significantly lower neurological score than the MCAO group (P<0.05),indicating the protective effect of WEA on neurological deficits. The mean infarction volumes of WEA (13.5±6.6%, F=4.75, P<0.01), EEA (19.90±6.90%, F=5.23, P<0.01), PEA (20.40±5.30%, F=4.68,P<0.01) and EAEA (22.50±10.50%, F=6.25, P<0.05) group were all significantly smaller than that of MCAO group (29.40±6.50%). HE staining demonstrated that, compared to the treated groups, the infarcted cerebral tissue of MCAO group had more swelling neural cells, lighter stained nucleus, fewer and irregularly distributed neurons. The activity of Na+/K+-ATPase and Ca2+-ATPase reduced in the MCAO group (3.67±0.48 U/mg,1.28±0.26 U/mg, respectively), and were significantly higher in WEA group (7.56±0.85 U/mg, F=12.65,P=0.010; 3.59±0.22 U/mg, F=8.32, P=0.041, respectively). The MCAO group showed significantly elevated P53 and HSP70 mRNA expressions compared to the sham group (P<0.01, P<0.05). P53 mRNA expressions in Agrimony extracts treated groups were significantly lower than that of the MCAO group (all P<0.01), with the WEA group showing the greatest difference from MCAO group. The HSP70 mRNA level of the treated groups were not significantly different from that of the MCAO group.Conclusions Treatment using water extracts of agrimony can promote the best functional and metabolic recovery for rat model of cerebral ischemia-reperfusion injury, which maybe relate with the upregulation of energy metabolism in nerve cells after MCAO.

葛根素预处理对大鼠脑缺血再灌注保护作用的初步研究

目的:探讨葛根素预处理是否对大鼠脑缺血再灌注具有保护作用。方法:在6个不同时间点对已进行脑缺血再灌注损伤的大鼠腹腔注射葛根素注射液,分别测量6个时间点的大鼠脑组织血流量及抗氧化酶含量。结果:葛根素治疗组在缺血再灌注损伤后,脑血流量降低,腹腔注射葛根素注射液4h后增加,然后降低。葛根素治疗组的抗氧化酶活性比缺血再灌注组增加明显,丙二醛含量显著下降。结论:葛根素可能通过增加脑组织抗氧化酶活性和脑血流量来对抗脑缺血再灌注损伤。