Experimental Studies On Rat Bone Marrow Stem Cells Mobilization In Acute Myocardial Infarction

Background and objectiveMyocardial infarction remains a leading cause of morbidity and mortality in human being. Because of the increased age of the population and improved postinfarction survival rates, congestive heart failure (CHF) has become a major cardiovascular disorder increasing in prevalence and incidence. Contemporary therapy mainly includes medication ,intervention and bypass operation.These methods can improve cardiac ischemia and symptom of heart failure. However,they can not reverse necrotic cardiac muscle.On the other hand,although heart transplantation is an effective treatment choice of heart failure, organ shortage,expense and immunorejection largely limit the application of this approach. Terminally differentiated cardiomyocytes have shown some evidence of mitotic division in the adult heart, but the rate of proliferation is minute and cannot meet the demand for tissue regeneration.Bone marrow stem cells (BMSCs) possess self-renovated and differentiation potential cell colony.A lot of experiments show BMSc can differentiate myocardial cells and endothelium which may repair injured and necrosic cardiac muscle.They can form neovascular vessel which improve cardiac function.However,it is difficult to culture,expand and differentiate bone marrow stem cells in vitro.Therefore,people try to use some cytokine to mobilize BMSCs theraping ischemic heart disease.Some researches have found that BMSCs could auto-transfer to the position of cardiac necrosis after actue myocardial infarction(AMI).They could transdifferate into cardiomyocyte,endothelium and smooth muscle cells.They could regenerate myocardial tissue to repair necrostic myocardium and improve cardiac function.However, BMSCs are few at peripheral blood.Thus,BMSCs which they transfere to infracted district are fewer.These transferred BMSCs couldn't effectively repair necrotic myocardial tissue.Therefore,BMSCs mobilization could drive stem cells to peripherial blood.They could increase peripherial stem cells into the myocardial tissue to repaire necrostic myocardium.Granulocyte colony stimulating factor (G-CSF) was mighty mobilization agent so that it could increase peripherlcal stem cells from ten times to hundreds times. G-CSF may mobilizate BMSC to transfer to infarct position and may differentiate cardiomyocytes and endothelium.These cells may form angiogenesis and repaire cardiac tissue.The purpose of the study is: (1) the feasibility of mobilizating BMSCs to infarct position and differentiating cardiomyocytes.(2) to investigate whether mobilizated BMSCs could express in the infarcted myocardium. (3) to study whether mobilizated BMSCs could reduce concentration of Cardiac Troponin-I which specially reflects the degree of cardiac injury marker.(4) to assess the functional improvement in rat left ventricular after mobilizating BMSCs.Materials and methods1. Make a model of the myocardial infarction: Under general anesthesia, rats were incubated and positive pressure ventilation at 80 breaths/min was maintained. A rat heart was exposed through a 1.5-cm left lateral thoracotomy incision. Then opened the pericardium and exposed the heart. Under direct vision, the left anterior descending (LAD) was ligated with 6-0 polypropylene sutures. The incision thoracotomy, muscle and skin layers were closed with 5-0 silk sutures.2. Animal grouping: 1 hour after infarction creation, the rats were randomly divide into mobilization group(n=30) and control one(n=30).According different groups BMSCs were mobilised by daily subcutaneous injections of G-CSF at 100 n g/kg body weight for 1 day or 5 days after AMI.BrdU was injected daily by celiac injections at lOOmg/kg body weight for 7 days in the 7 days group.Troponin-I was measured at 24h and 7d.These rats were executed respectively at 24h,7d and 14d.Hearts were extracted to process immunhistochemical examinations and pathology examinations.3. Echocardiography assessment: The rats were done to observe the changes in cardiac function by echocardiography at 24 hour and two weeks respectively after myocardial infarction. A 12 MHz pediatric transducer obtained images at the papillary muscle level of left ventricle in bidimensional echocardiography. Regional fractional shortening (FS%) and Left ventricular ejection fraction (EF%) were assessed after 2 weeks of cell mobilization by echocardiography cardiac function again.4. Immunohistochemical examinations: The hearts were fixed and embedded in paraffin. Paraffin sections in 5um thickness ensured that different stains could be carried out on successive sections of tissue cut through the infarct area. One of the sections was stained with hematoxylin and eosin to depict nuclei, cytoplasm, and connective tissue; other successive sections were selected for immunostaining of BrdU and CD34+ cells.Resultsl.Cardiac Troponin-I examination:Mobilization groups were able to evidently reduce the levels of Troponin-I (ng/ml) at 24h( 10.06+0.83 vs 11.75 + 1.21) and 7d (1.37 + 0.66 vs 2.33 + 0.74) . There was significant difference between mobilization group and control group(F < 0.05).2. Immunohistochemical Examinations: Hematoxylin and eosin stain group showed that there was some cells with an immature appearance, big blue nuclei, and large nucleus-to-cytoplasm ratio in mobilization group. The mononuclear cells were aligned in mobilization group.Many scar fiber tissue, macrophages, and granulocytes were seen in the infarct region of control group.The adjacent tissue of infarction region showed that some cytoplasm were stained brown (indicating CD34+ cells),some nucleus was stained brown (indicating BrdU-stain postive cells).These cells were arranged in spindle-like structure.CD34+ stain postive cells and BrdU stain positive cells could still be seen in the adjacent region of infarction area in control group. At 14d the antigen of CD34+ became negative express between mobilization group and control group.The number of CD34+ postive cells (1.40+0.51 vs 8.30+ 1.83,P < 0.05) (cells/HP) and BrdU positive cells (1. 30 + 0.67 vs 7.90+1.37, P<0. 05) (cells/HP) was much less than mobilization group at 7d. The number of CD34+ postive cells (8.30±1.83 V5 2.90±0.87, P < 0.05) (cells/HP) in mobilization group was significant difference between 7d and 24h.3. Echocardiography assessment: 24 hour after infarction EF%(39.5±3.2 vs 39.6+3.2) and FS%(18.9+3.0 vs 19.8+2.3) were no significant difference among cell mobilization group and control group (P>0.05);but at 14 days EF% (46.4+4.3 vs 40.4 + 2.8) and FS%(25.6±2.8 vs 20.7+1.5) were significant difference between cell mobilization group and control group, (P<0.05).Conclusionsl.G-CSF could mobilizate CD34+ hematopoietic stem cells to transfer to the position of myocardial infarction.They could transdifferentiate into cardiomyocyte and endothelium.They could form angiogenesis. The mobilization BMSCs coulddecrease the levels of Troponin-I and reduce injured cardiac muscle after AMI in rats. These cells could improve cardiac function.2.The mobilizated BMSCs transdifferentiate into cardiomyocytes-like phenotype in the infarct region 24h and 7d after cell mobilization.3.The antigen express of CD34+ cells weak along with differentiating degree of cells on the membrane of hematopo i et i c stem ce 11 s (HSCs).Therefore, the antigen of CD34+ become negative express according to more immature cells. 4.BrdU can replace thymine to enter DNA during synthesization ofcells.lt could be markered cells of mitotic division.There are positive BrdU cells and CD34+ at 24h and 7d at the myocardial infarction region. BrdU cells of the mobilization groups are obviously more than the control ones.