| BackgroundVentricular remodeling refers to the changes of ventricle in myocardial structure, function and phenotype in respond to myocardial injury or load pressure increase, which caused a series of changes, such as the size, shape, organization structure, wall thickness and so on. Ventricular remodeling includes cellular remodeling(abnormal changes of myocardial cell in structure, metabolism and function) and extracellular matrix(ECM) remodeling(mainly,inbalance between of collagen synthesis and degradation, resulting in the collagen excessive deposition and degradation). Ventricular remodeling mainly manifest myocardial hypertrophy,cardiac fibroblast proliferation, changes of intracellular composition and ECM collagen,which result in myocardial fibrosis.Matrix metalloproteinases(MMPs) are a group of Zn2+ dependent enzymes family, which can degrade extracellular matrix components specificity. MMPs degrading matrix component in heart, is the important driving force of substrate degradation in myocardial remodeling. MMP-9 is one of MMPs, also known as gelatinase B, which content rich in myocardium. Previous studies have demonstrated that some cardiovascular diseases always existed over-expression of MMP-9, such as,in myocardial infarction, dilated cardiomyopathy and myocarditis. By directly adjusting the level of MMP-9 or treating the diseases to decrease the level of MMP-9could reverse the ventricular remodeling. Therefore, high MMP-9 may have a close relationship to ventricular remodeling, but it remain unclear that how MMP-9to affect the ventricular remodeling. A good ventricular remodeling animal model with high MMP-9 is very important to research the mechanism, prevention and cure this kind of ventricular remodeling. However, so far, most ventricular remodeling model with high MMP-9 are some disease conditions, high MMP-9 may only be a accompany state of the diseases, rather than be a ventricular remodeling pathogenesis. In order to study the mechanism of MMP-9 in ventricular remodeling, a definite ventricular remodeling model caused by high MMP-9is necessary. In view of our previous studies have found that giving exogenous MMP-9 can cause ventricular remodeling in rats. The present study was therefore designed to investigate how to establish the ventricular remodeling model with high MMP-9 in rats by inputting exogenous MMP-9, so that provide a convenient, inexpensive tool for the study of high MMP-9ventricular remodeling.Aim To investigate how to establish the ventricular remodeling model with high MMP-9 in rats by inputting exogenous MMP-9, so that provide a convenient,inexpensive tool for the study of high MMP-9ventricular remodeling.Method1.48 of Eight weeks healthy male Wistar rats(body weight 160~200g),according to the weight were stratified random divided into 4 groups: control group(normal saline 2ml), model groups: low dose MMP-9 group(recombinant rat MMP-9, 0.7ng/body weight(g), 2ml); middle dose MMP-9 group(recombinant rat MMP-9, 1.4ng/body weight(g), 2ml); high dose MMP-9 model group(recombinant rat MMP-9, 2.1ng/body weight(g), 2ml). The control group and model group were given intraperitoneal injection, two times per week,per injection 2ml. randomized six of each group were given injection four weeks continuously(the first stage groups),others were given injection eight weeks(the second stage groups).2.Theblood pressure were measured by a noninvasive tail cuff method in the day before experiment, the 28 th day(all individual) and the 56 th day(the second stage groups only). The changes of left ventricular structure and function were measured by echocardiography after measuring blood pressure in the two stage group of experiment respectively. The cardiac structural parameters included left ventricular internal dimension at end diastole(LVIDd), left ventricular internal dimension at end systolic(LVIDs), interventricular septal thickness at end diastole(IVSd), left ventricular posterior wall thickness at end diastole(LVPWd), end-diastolic left ventricular volume(LVEDV), end-systolic left ventricular volume(LVESV) and functional parameters included left ventricular fractional shortening rate(LVFS),Cardiac Output(CO), stroke volume(SV), left ventricular ejection fraction(LVEF),ratio of mitral E peak velocity to A peak velocity(E/A), heart rate(HR).3.We measured serum levels of MMP-9by ELISA, isolated the left ventricle for the calculation of left ventricular weight index(LVWI=left ventricular weight(mg)/body weight(g)), observed the changes of the cardic myocyte and the myocardial interstitial with HE stain by light microscope, observed the distribution of myocardial interstitial collagen with VG(Van Gieson) stain, measured the collagen volume fraction(CVF) by Image Analysis, detected the levels of MMP-9in myocardial tissue by western blotting, and determine the expression of MMP-9m RNAin myocardial tissue by reverse transcription-polymerase chain reaction(RT-PCR).Result1.The general situation and the survival rateCompared with the control group rats, there were no obvious abnormal mental state, food intake, activity, weight in model group rats. All model group and control group rats are survive at the end of experiment. the survival rate is 100%.2.The baseline dataThe initial average body weight and the initial blood pressure of each group rats were not obvious difference.3.Changes of the level of MMP-9protein in myocardial tissueIn the first stage: compared with the control group,the levels of MMP-9 were significantly higher in myocardial tissue in the high dose model group(p<0.05);the levels of MMP-9 were increased in myocardial tissue in the Low, middle dose model group(p>0.05). Comparison between the model groups, the levels of MMP-9 in myocardial tissue in the high dose model group were obviously higher than thosein low dose model group(p<0.05).In the second stage: compared with the control group, the levels of MMP-9were significantly higher in myocardial tissue in the middle and high dose model group(p<0.05);the levels of MMP-9 were increased in myocardial tissue in the low dose model group(p>0.05). Comparison between the model group, the levels of MMP-9 in myocardial tissue in high dose model group were obviously higher than those in low dose model group(p<0.05).4.Changes of the level of MMP-9protein in serumIn the first stage: compared with the control group, the serous levels of MMP-9 were significantly higher in middle and high dose model groups(p<0.05);but it was mild increased in Low dose model group(p>0.05). Comparison between the model group, the serous levels of MMP-9 were higher than in high dose model group than those in low dose model group(p<0.05).In the second stage: compared with the control group,the serous levels of MMP-9 were significantly higher in all model groups(p<0.05).the serous levels of MMP-9 were higher than in high dose model group than those in low dose model group(p<0.05).5.The expression of MMP-9mRNA in myocardial tissueIn the first stage: compared with the control group, the expression of MMP-9m RNA were significantly increased in myocardial tissue in model groups except low dose model group(p<0.05).In the second stage: compared with the control group, the expression of myocardial tissue MMP-9m RNA were no significant difference in model groups(p>0.05), and the result was similar by comparison between the model group(p>0.05).6.Changes of ventricular remodeling6.1. Changes of left ventricular mass indexIn the first stage: compared with the control group, LVWI were significantly higher in the high dose model group(p<0.05); LVWI of the low and middle dose model group were mild increase(p>0.05). Comparison between the model group,LVWI of the high dose model group were obviously higher than that of low dose model group(p<0.05).In the second stage: compared with the control group, LVWI were significantly higher in the high dose model group(p<0.05);LVWI of the low and middle dose model group tend to increase(p>0.05). Comparison between the model group, LVWI in the high dose model group were obviously higher than that in low dose model group(p<0.05).6.2. Changes of structure and function of left ventricleIn the first stage: Echocardiographic left ventricular structural and functional parameters were not significant difference in the model groups compare to those in the control group(p<0.05).In the second stage: Echocardiography found, compared with the control group, the LVIDd and LVEDV were significantly larger in the middle and high dose model group(p<0.05), the LVIDd and LVEDV in the low dose model group showed a increased tendency(p>0.05).Comparison between the model group, the LVIDd and LVEDV of the high dose model group were significantly larger than those of the low dose model group(p<0.05), while other cardiac structure and function parameters were not significant difference.6.3. Changes of left ventricular myocardial histopathology1) Morphological observation(HE stain): In the first stage, compared with the control group, there were cardic myocyte arrangement disorderly, cardiomyocyte hypertrophy, part cardiac muscle fiber fracture, cytoplasm dissolved and necrosis in middle and high dose model group. But no significant change in the low dose model group.In the second stage: In all model group, we detected myocardial fiber fracture,defect, dissolved and necrosis obviously.2) Changes of myocardial interstitial collagen(VG stain): In the first stage of experiment, compared with the control group, CVF in the high dose model group were significantly lower(p<0.05), CVF in the low, middle dose model group were decreased mildly(p>0.05).And no significant difference between the model groups,(p>0.05).In the second stage, compared with the control group, CVF in middle and high dose model group were significantly lower(p<0.05). Comparison between the model group, CVF were significantly lower in the high dose model group than those in low dose model group(p<0.05).7. Changes of blood pressureIn the first stage, the systolic and diastolic blood pressure level of the high dose model group were significantly higher(p<0.05),but were only higher tendency in other model groups compared with those in control group(p>0.05).In the second stage,compared with the control group, in addition to the low dose model group, the middle and high dose model group’s systolic and diastolic blood pressure levels were significantly higher(p<0.05).Comparison between the model group, the systolic and diastolic blood pressure level in the high dose model group were significantly higher than those in low dose model group(p<0.05).8. The success rate of high MMP-9 ventricular remodeling model establishmentWith myocardial MMP-9≥mean + 2times standard deviation(SD) and LVEDV ≥mean + 2times SD and CVF ≤mean- 2times SD levels of control group as standard, the highest success rate of high MMP-9 ventricular remodeling model establishment rech 83% in high dose model group on second stage.ConclusionIt is feasible to establish high MMP-9 ventricular remodeling rat model successfully by injecting exogenous MMP-9.According to the present study,recommended optimal program is: 2.1 ng exogenous MMP-9/g(weight),intraperitoneal injection, two times per week for eight weeks. |
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