| ObjectiveCardiac Shock Wave Therapy(CSWT)is a new technology for severe coronary heart disease.Clinical studies have shown the data of angiogenesis,alleviation of inflammation and oxidative stress inhibition in ischemic areas after CSWT,the improvement of myocardial perfusion,the amelioration of left ventricular function were also reported.The apoptosis caused by the cardiac ischemia plays an important role in myocardial cell reduction and heart dysfunction.According to our initial studies,we demonstrated that shock wave(SW)could effectively attenuate apoptosis of H9c2 cells in condition of ischemia and hypoxia.Based on the pre-cytological study,we used the rat model of acute myocardial infarction(AMI)by coronary artery ligation to verify the effects of CSWT on cardiomyopathy apoptosis and the study confirmed that CSWT could improve cardiomyopathy mitochondrial membrane integrity and attenuate cytochrome C release after AMI and CSWT up-regulate the expression of Bcl-2 and down regulate the expression of Bax and Cleaved-Caspase-3.JNK-dependent signaling pathway might be inhibited by CSWT through anti-appototic effects.Which will protect the myocardial cells.At the same time,safety was proved by tissue,cytology and molecular level.We will study the angiogenesis of CSWT on the as model of AMI sequentially and use transmission electron microscopy(TEM)to evaluate the possible damage to myocardium ultrastructures of CSWT treatment for the first time.The study is helpfully to provide a novel idea for the treatment of end-stage ischemic heart disease patients.MethodsAt first,based on the methods of references,we practiced to establish the rat model of AMI by left anterior descending coronary artery ligation.And we evaluate the model of AMI rat assessed by electrocardiogram and echocardiogram and pathological method.Secondly,2-month old of male Sprague-Dawley(SD)rats were used for the AMI model in the study of angiogenesis by CSWT on rat heart by ligation of the left anterior descending coronary artery.All rats were enrolled into four groups,including sham group(n=6),SHAM+SW group(n=6),AMI group(n=6)and AMI+SW group(n=6)randomly.Rats in AMI+SW group and SW group were treated with SW at day 1,3 and 5 after random grouping(energy:0.1mJ/mm2,shock wave times:200shots,frequency:4Hz.All the rats were murdered after 14 days after enrollment.Heart tissue was observed by hematoxylin and eosin staining.Angiogenesis were analysed by CD31,CD34,vWF and SMA immumohistochemical staining.For quantitative histology,digitalized images were made with an inverted microscope at 400 magnification and quantification was performed with ImagePro Plus6.0 analysis software.At last,our paper concentrated on the safety study of CSWT on rat Heart assessed by transmission electron microscopy,2-month old of male SD rats were enrolled randomly into normal control(NC,N=6)group and normal control with shock wave group(NC+SW,N=6).NC+SW group was treated with SW at day 1,3 and 5 after random grouping(energy:0.1mJ/mm2,shock wave times:800shots,frequency:4Hz.)Electrocardiogram,blood pressure(BP)and heart rate(HR)was accounted for before and after SW.And blood samples were harvested.All rats were killed after 8 days of random grouping and heart tissue were observed histologically by hematoxylin and eosin staining and Masson staining.The heart samples were respected by transmission electron microscopy to evaluate the changes in myocardial tissue ultrastructure.And semi-quantitative analysis of tissue ultrastructure damage was evaluated by the scores of myocardial ultrastructure ruin.ResultsAMI rate model was successfully established with the survival rate of 72.2%,mortality 27.8%and the success rate of survival model was 85.7%.In the study of angiogenesis by CSWT on rat heart,Hematoxylin and Eosin staining revealed that the degeneration,necrosis and infiltration were present in the border region of AMI group compared with the sham group.Similar changes were seen in AMI+SW group.Different ways of immumohistochemical staining were utilized to confirm the angiogenesis.CD31 immumohistochemical staining showed micro vascular density were 9.92±2.13/mm2 vs 10.02±1.94/mm2(P>0.05)in SHAM and SHAM+SW groups respectively;micro vascular density in border region were 22.69±11.46/mm2 和51.22±21.17/mm2(P<0.0001)in AMI and AMI+SW groups respectively.CD34 immumohistochemical staining showed micro vascular density were 15.12±1.54/mm2 vs 16.26±2.01/mm2(P>0.05)in SHAM and SHAM+SW groups respectively;micro vascular density in border region were 49.15±14.17/mm2 and 86.91±25.69/mm2(P<0.0001)in AMI and AMI+SW groups respectively.vMF immumohistochemical staining showed micro vascular density were 3.31±1.57/mm2 and 3.72±1.13/mm2(P>0.05)in SHAM and SHAM+SW groups respectively;micro vascular density in border region were 14.59±5.35/mm2 and 23.07±6.74/mm2(P<0.0001)in AMI and AMI+SW groups respectively.SMA immumohistochemical staining showed micro vascular density were 3.09±1.15/mm2 and 2.99±1.53/mm2(P>0.05)in SHAM and SHAM+SW groups respectively;micro vascular density in border region were 11.77±6.47/mm2 and 45.48±15.62/mm2(P<0.0001)in AMI and AMI+SW groups respectively.Microvascular density in border regions of AMI rates increased significantly after CSWT treatment,which was verified by four different immumohistochemical stainings.From safety study of CSWT on rat heart,we found there was no obvious anamorphosis.Necrosis,or infiltration were observed histologically by Hematoxylin and eosin staining in both groups.No collagen fibrosis staining was observed in the NC or NC+SW group;therefore.CSWT did not cause an inflammatory response and fibrosis in the myocardial tissue by Masson staining in both groups.Assessing the ultrastructures after CSWT by myocardial ultrastructure damage scoring system,the average scores of myocardial ultrastructure damage in the NC and NC+SW groups was 1.39±0.982 and 2.42±1.009,respectively(P=0.103).No abnormal changes were noted on ECG,BP,HR before and after SW treatment.Differences were not statistically significant.CSWT did not cause an increase in serum TNI levels(p=0.08).Conclusions1.Microvascular density in border regions of AMI rats increased significantly after CSWT treatment.2.CSWT did not cause an inflammatory response and fibrosis in the myocardial tissue.3.CSWT did not cause microstructure changes,but ultrastructure changes were seen in both groups.There were no significant difference between two groups.4.CSWT with appropriated protocol is a safe treatment for the adult rat. |
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