| IntrodnctionAtherosclerosis-induced coronary artery disease(CAD)is the leading cause of morbidity and mortality in Western societies and increases at an alarming speed in developing countries,despite continued advances in the treatment of CAD such as PTCA (Percutaneous Transluminal Coronary Angioplasty),CABG(Coronary Artery Bypass Graft)and intracoronary stent placement.Except for surgical interventions,an effective therapeutic method for treatment has been lacking.An increasing number of patients are not suitable for conventional revascularization strategies because of diffuse small vessel disease,multi-vessel disease,long lesions,occluded vessels without collateral recanalization,or other comorbidities such as diabetes mellitus and obesity.Therapeutic angiogenesis induced by angiogenic growth factors may provide an alternative approach to the treatment of myocardial ischemia for these patients.Therapeutic angiogenesis is a process that seeks to stimulate collaterogenesis and to improve myocardial perfusion and function by delivery of proangiogenic factors to the ischemic myocardium. Numerous experimental and clinical studies have evaluated therapeutic angiogenesis as a treatment for ischemic heart disease. Despite the promising results of therapeutic angiogenesis in preclinical studies,clinical evaluation of these individual proangiogenic molecules has produced unfulfilled promises since this idea has been proposed for more than 30 years.After more than a decade of clinical practice with different single proangiogenic factors for the treatment of ischemic disorders,almost all large randomized,double-blinded,and placebo-controlled human trials have proven to be non-beneficial.Pharmaceutical companies that sponsor clinical trials often choose an angiogenic agent based on the intellectual properties owned by the company and the possible easy approval by the Food and Drug Administration(FDA).Most previous preclinical and clinical studies on the development of proangiogenic therapies for treating ischemic myocardium have been based on mono-therapeutic approaches.In addition,most studies have focused on evaluation of the therapeutic efficacy of vascular endothelial cell growth factor(VEGF)-A,which primarily targets the endothelial cell compartment.The establishment of stable and functional blood vessel networks,however,is a complex process that requires several angiogenic factors targeted different cell populations in the vasculature.The reason for this discrepancy is unclear,and in part that current angiogenic therapies in clinical trials are based on a single angiogenic factor delivered, which is insufficient to initiate the entire cascade of events leading to a mature,functional and stable vascular network.Neovascularization in the ischemic adult heart is a combination of several processes including angiogenesis,arteriogenesis and potentially vasculogenesis.Angiogenesis is defined as the sprouting of new blood vessels from pre-existing vasculature and consists of distinct stages including cell migration,proliferation and differentiation into capillaries and blood vessels.Arteriogenesis refers to the process of maturation or de novo growth of collateral conduits and produce vessels capable of carrying significant blood flow.These vessels are of a sufficient diameter to be visualized with angiography.The angiogenic factors,VEGF and FGF-2,are perhaps the most extensively studied angiogenic growth factors,which has been used in clinical trials.VEGF exerts its effects on endothelial cells that include enhanced survival,increased permeability,enhanced migration and proliferation,all of which contribute to angiogenesis. VEGF is also named VPF(vascular permeability factor)due to its high permeability of vessel.Like VEGF,FGF-2 also stimulates endothelial cell proliferation and migration,which plays a critical role in triggering an initial robust angiogenic response.Although the release of the signal peptide-less FGF-2 from its synthetic cells remains enigmatic,this growth factor plays a crucial role extracellularly in the regulation of the growth of its target cells including endothelial cells.Especially important,administration of FGF-2 in vivo selectively stimulates angiogenesis but not other tissue growth.However,endothelia cells channels,induced by single angiogenic factor,are naked,leaky,and fragile,which are easily ruptured and bleed.Insufficient perfusion eventually leads to vessel regression.In contrast to FGF-2 and VEGF,PDGF-BB acts mainly on vascular mural cells including pericytes and vascular smooth muscle cells(VSMCs).PDGF-BB interacts with both homodimeric and heterodimeric PDGFR-αand PDGFR-βcomplexes,especially PDGFR-β,which plays an essential role in stabilization of blood vessels via the recruitment of pericytes and VSMCs onto the nascent vasculature.For improvement of perfusion of high-oxygenated blood in ischemic tissues,it is essential to re-establish functional arterial vascular networks,which should remain stable for long term.The clinical failures with this attractive approach have raised several unresolved fundamental issues regarding the basic mechanisms of neovasculorazation.These include the underlying mechanisms of angiogenesis versus arteriogenesis,choice of proangiogenic agents, monotherapy versus combinatorial therapy,selection of optimal combination and drug release systems,evaluation standard for functional arterial networks and appropriate animal models for preclinical evaluation.Based on these conditions,we propose a hypothesis as following: the interplay and synergy between various angiogenic and arteriogenic factors for promoting arteriogenesis is specifically limited to certain combinations,and that each factor has its own specific partners.The optimal combination of angiogenic and arteriogenic factors could induce stable arterial networks and improve collateral growth and functional recovery.The purpose of this study was,based on the selection of optimal combination of angiogenic and arteriogenic factors in mouse cornea, to explore therapeutic evaluation of the combination in a pig myocardial infarction model.1.Methods1.1 Selection of optimal combination of angiogenic and arteriogenic factorsThe avascular feature of the corneal tissue made it an ideal model for assessing vascular network formation.A micropellet(0.35×0.35 mm)of sucrose aluminum sulfate coated with hydron polymer type NCC containing 160 ng of VEGF,PDGF-AA,-AB,-BB,or 80 ng of FGF-2,or 160 ng PDGF-AA/80 ng FGF-2,or 160 ng PDGF-AB/80 ng FGF-2,or 160 ng PDGF-BB/40 ng FGF-2,or 160 ng PDGF-BB/160 ng VEGF was surgically implanted into a micropocket in the mouse cornea(one pellet/eye,one eye implanted/mouse).The corneal neovascularization was examined and quantified on day 5 after pellet implantation.Vascularization areas were calculated by measuring vessel lengths and clock-hours(the circumferential area of neovascularization if the eye is considered as a clock).For vascular stability analysis,the corneal neovascularization pattern/profile was examined on days 5,12,24,and 70 after pellet implantation.Finally, we thus chose an optimal combination for the therapeutic evaluation and further studies in a pig model of myocardial infarction.1.2 Pig myocardial infarction model and growth factor implantation40 Chinese experimental mini-pigs weighing between 25-30 kg/each were used for the myocardial infarction model(China Agricultural University,Beijing).All animal care and experimental protocols complied with the Animal Management Rules of the Ministry of Health of the People's Republic of China(document No 55,2001)and the guidelines for the Care and Use of Laboratory Animals of Qi Lu Hospital,Shandong University,China.Animals were randomly divided into four groups(n=10 pigs/group):(A)PBS, (B)FGF-2(5μg),(C)PDGF-BB(10μg),and(D)FGF-2 and PDGF-BB(5μg and 10μg,respectively).Before all experimental procedures,all animals were anesthetized by intramuscular administration of ketamine hydrochloride(20 mg/kg,Heng Rui Medicine Co.LTD.Jiang Su, China)together with intravenous injection of sodium pentobarbital (30 mg/kg,Beijing Chemical Reagent Company,Beijing,China)and were mechanically ventilated with a volume respirator(Newport E100m Ventilator,Newport Medical Instruments,CA,USA).Median sternotomy was performed to expose the heart followed by incision of the pericardium.Acute myocardial infarction was produced by ligation of the LAD coronary artery distal to its third diagonal branch using a 7.0-prolene suture.Selective left and right angiography, performed through a standard procedure,was made to confirm complete occlusion of the LAD and to assess baseline levels of collaterals to assess the collateral index.A slow-releasing pellet of sucrose aluminum sulfate coated with hydron polymer(0.2×0.2 cm)containing PBS,5μg FGF-2,10μg PDGF-BB,or 5μg FGF-2 and 10μg PDGF-BB was attached to an aseptic application(0.5×0.5 cm),which was sutured(7.0-prolene) onto the border zone adjacent to the mid and distal LAD.1.3 Coronary arteriographySix and fourteen weeks after treatments,repeated,selective angiography was performed.Under general anesthesia,the animals received a contrast agent,meglucamine diatrizoate,through a standard femoral puncture using the digital subtraction angiography (CGO-2100,Wandong Medical Equipment Co.LTD,China).The angiographic index was assessed using a standard protocol based on Rentrop's grading scales from 0 to 3:0,none;1,filling of side branches of the LAD;2,partial filling of the LAD main artery via collateral channels;3,complete filling of the LAD.1.4 Regional myocardial blood flowFor determination of regional myocardial blood flow at different time points,colored microspheres(10±2μm diameter;E-Z Trac,Los Angeles,CA)labeled with red,yellow and green were used prior to treatment,and at the end of week 6 and 14.5×106 microspheres were injected into the left ventricle by a 5F pigtail catheter(Cordis,USA)with 10 ml of saline.After completing the injection,the catheter was flushed with 10 ml of saline.An arterial Reference Blood Sample was collected for each colored microsphere injection.Starting 10 seconds before injection,reference blood samples were withdrawn by a withdrawal pump at a constant rate of 10 ml/min for a period of 90 seconds.Recovery of microspheres from tissue and blood was performed according to the manufacturer's instruction.Blood flow values are calculated from the following equation:Qm=(Cm×Qr)/Cr.Where Qm is the myocardial blood flow per gram(ml/min/g),Cm is the microsphere count per gram of tissue, Qr is the withdrawal rate of the reference blood sample(ml/min),and Cr is the microsphere count in the reference blood sample.1.5 EchocardiographyTwo-dimensional echocardiography was used to measure global and regional left ventricular function in all animals with open chest following angiographic assessment before and 14 weeks after treatment using an ultrasound scanner(SONOS 7500,Philips Medical Systems Inc.,Andorver,MA).Analyses of LVEF and systolic wall thickening(WT%)were performed to determine the global function of the left ventricle and the regional function of the myocardium, respectively.LVEF was determined from the apical two-chamber and four-chamber views using a modified Simpson's algorithm. Regional wall thickness was measured at end-diastole(the peak of R wave of the ECG)and end-systole(the end of T wave of the ECG) individually on two-dimensional echocardiograms.Left ventricular systolic wall thickening(WT%)was calculated as: WT%=(SWT-DWT)/DWT×100%.1.6 ImmunohistochemistryThe treated areas of the left ventricle myocardium in the LAD territory were fixed with paraformaldehyde or were immediately frozen in liquid nitrogen.To determine vessel density, paraffin-embedded 5μm sections were incubated with a rabbit anti-von Willebrand factor(vWF)antibody or a mouse anti-alpha smooth muscle actin(αSMA)antibody,followed by incubation with secondary antibodies labeled with horseradishperoxidase.Ischemic myocardial sections were also used for detection of vWF/αSMA double positivity using immunofluorescent analysis.A maturation index(percentage of smooth muscle cell positive vessels vs.total vessel numbers)was calculated.1.7 Data analysisData are presented as the mean±standard errors of the mean and were analyzed with SPSS 11.5 software.A 2×2 factorial analysis of variance was used to examine the interaction between FGF-2 and PDGF-BB.Continuous variables were compared by using repeated measures analysis of variance(RM ANOVA)followed by the LSD or Dunnutt T3 corrected post hoc analysis for multiple comparison procedure.Nonparametric variables were compared between groups by using two-sided Kruskal-Wallis(multiple group comparison)and Mann-Whitney(two group comparisons)tests.Collateral index was compared within groups before and after treatment using Wilcoxon test.All reported P values were two-tailed,and a P value＜0.05 was considered statistically significant. 2.Results2.1 Selection of optimal combination of angiogenic and arteriogenic factors in mouse corneaCombination of PDGF-AA/VEGF,PDGF-AB/VEGF or PDGFBB/VEGF didn't result in synergistic angiogenesis. PDGF-AA/FGF-2,PDGF-AB/FGF-2,or PDGF-BB/FGF-2 combinations synergistically induced angiogenesis as compared with single growth factor-induced angiogenesis.At day 70 after implantation,PDGF-AA/FGF-2-induced vessels were almost completely regressed,suggesting that this combination is unable to stabilize the newly formed vasculature.Interestingly,both PDGF-AB/FGF-2- and PDGF-BB/FGF-2-induced corneal vascular networks remained stable at this and later time points.These findings demonstrate that only PDGF-AB/FGF-2 or PDGFBB/FGF-2,but not PDGF-AA/FGF-2,are able to stabilize the newly formed vasculature although all three combinations promote angiogenic synergisms.We thus chose a combination of PDGF-BB/FGF-2 for the therapeutic evaluation and further studies.Forty animals received surgery and five animals died in the initial operation,of which two animals died of anesthesia accident and three animals died of ventricular fibrillation during ligation of the left anterior descending(LAD)coronary artery to make myocardial infarction model.One animal(PBS control)died of anesthesia accident in the mid-study.Thus,the remaining thirty-four animals(PBS,n=7;FGF-2,n=10;PDGF-BB,n=8;FGF-2+PDGF-BB, n=9)completed the entire study.No abnormal adverse events were noted during the entire course of these experiments in those animals that survived to the 14-week end point.2.2 Angiographic analysis of collateral formation Analysis of the collateral index demonstrated a difference among the four groups at week 6(Kruskal-Wallis test,P=0.026), whereas baseline collateral index in all groups was similar.Further multiple comparisons showed that PDGF-BB/FGF-2 significantly induced myocardial collateral growth as compared with various individual FGF-2 or PDGF-BB or PBS -treated myocardium(P=0.037, P=0.021 and P=0.002,respectively).These newly established collaterals formed vascular networks proximally and distally from the ligation site.Compared with the baseline,collateral index had increased significantly in the FGF-2 group and in PDGF-BB/FGF-2 groups at week 6(P=0.018 and P=0.001,respectively).Despite a trend toward increase in collateral index in the PDGF-BB group compared with the baseline,the difference did not reach statistical significance(P=0.058).To study the stability of these newly established collateral networks,coronary angiography was again performed on week 14 after treatment.Remarkably,the PDGF-BB/FGF-2-induced collaterals had developed into well-established coronary arterials, indicating the re-established collaterals remained stable.2.3 Assessment of regional myocardial blood flow(MBF)The myocardial blood flow was assessed by a colored microsphere method.Prior to treatment,the basal levels of myocardial blood flow in the LAD territory were indistinguishable among all groups.At week 6,the PDGF-BB/FGF-2 together-treated group exhibited remarkably higher MBF than either factor alone-treated myocardium(P＜0.05).The high level of MBF persisted for long term in the PDGF-BB/FGF-2-treated group and no reduction of MBF was observed at the end point of the experiment(14 weeks). Statistical analysis of these data using factorial analysis of variance showed that MBF in the PDGF-BB/FGF-2 together-treated group was markedly greater than the sum effects obtained from two factors alone-treated myocardium(F=7.317,P=0.011,at week 6;and F=4.930,and P=0.034,at week 14).2.4 Echocardiographic analysis of global and regional myocardial functionEchocardiography was performed to monitor global and regional myocardial function.Measurement of left ventricular ejection fraction(LVEF),a sfandard parameter to monitor global myocardial contractile function,analysis showed that the basal cardiac dysfunction was indistinguishable among all groups before treatment. At week 14 after treatment,a significant improvement of LVEF was observed in the PDGF-BB/FGF-2-treated group as compared with the buffer-treated group,however,there were no significant differences between the combination treatment group and groups treated with either FGF-2(P=0.10)or PDGF-BB(P=0.082)alone.Neither single growth factor-treated group showed significant improvements of LVEF.To assess regional wall motion,left ventricular systolic wall thickening(WT%)was calculated.There were also no significant differences in the baseline regional function among groups.Similar to LVEF results,PFGF-BB- and FGF-2-single treated groups did not show any significant improvement in WT%as compared with buffer-controls.In contrast,the PDGF-BB/FGF-2-treated group significantly improved the WT%as compared with either buffer- or single factor-treated groups(P＜0.01).2.5 Vessel density and maturation indexCollateral micro-vessel formation was further examined by measuring the number of capillaries and arterioles by von Willebrand factor(vWF)andα-smooth muscle actin(α-SMA) immunohistochemistry in light microscopic sections taken from ischemic myocardium.Myocardial capillary density in the border zone in FGF-2 and the combination group was significantly higher compared with PBS and PDGF-BB group(P＜0.05).No apparent difference was observed between the FGF-2 and the combination group.Arteriolar density was significantly higher in growth factors treated animals than in PBS controls.Consistent with the coronary angiographic analysis,high numbers of arterial vessels were detected in the PDGF-BB/FGF-2-treated myocardium at week 14 post-treatment as compared with those of single factor-treated or control samples.In addition,for assessing the maturity and stability of myocardial newly vessels,a maturation index defined as proportion of vessels surrounded by smooth muscle cells was determined by vWF andα-SMA double-labeled immunofluorescence.At week 14 after treatment,α-SMA positive vessels in the PFGF-BB/FGF-2 together-treated group was significantly higher than those detected in the PFGF-BB,FGF-2,or buffer alone-treated myocardium(P＜0.05).2.6 Upregulation of FGFR-1,PDGFR-αand PDGFR-βThe proangiogenic factors exert their cellular effects by binding to and activating protein tyrosine kinase receptors expressed on target cells.FGFR-1,PDGFR-αand PDGFR-βwere examined by immunohistochemistry.FGFR-1,PDGFR-αand PDGFR-βexpression significantly higher in the PFGF-BB/FGF-2 group than others.To further identify whether VEGF may in part involve in vascular stability by activating flk,we also examined the expression of VEGF mRNA and protein by realtime PCR and immunohistochemistry,respectively.There were no significant differences at both mRNA and protein level in VEGF and flk among groups.Conclusion(1)A protein-based angiotherapeutic approach,based on dual delivery of arteriogenic and angiogenic factors,significantly improves myocardial collateral growth,blood perfusion,and cardiac function in a pig ischemic myocardial model.(2)A possible mechanism of angiogenic synergism involves upregulation of the expression of FGFR-1,PDGFR-αand PDGFR-βin angiogenesis,and the crosstalk between PDGFR and FGFR. INTRODUCTIONExperimental studies have demonstrated that left ventricular systolic wall thickening stems mainly from the contraction of the subendocardial myocardial fibers with the inner half layer of the ventricular wall contributing to about two-thirds and the outer half layer contributing to about one-thirds of systolic wall thickening. Therefore,the subendocardium sustains a higher systolic stress and demands more oxygen consumption and blood supply than the subepicardium.Thus,the subendocardium is more susceptible to ischemia than the subepicardium.The transmural gradient of myocardial perfusion is of negligible significance in physiological conditions but becomes extremely important in the presence of epicardial coronary artery stenosis because an accurate assessment of the transmural perfusion gradient may significantly improve the sensitivity and specificity of diagnostic techniques in the early detection of coronary artery disease.Therefore,an ideal imaging technique for detecting coronary artery disease should be able to measure subendocardial and subepicardial myocardial perfusion separately.Most conventional imaging modalities,however,can only assess myocardial perfusion across the entire ventricular wall rather than in different layers due to the lack of a high spatial resolution, and thus,the clinical value of these techniques in detecting myocardial ischemia is rather limited.Recently real-time myocardial contrast echocardiography has shown the potential to delineate transmural distribution of myocardial perfusion but the lack of an ideal myocardial contrast has significantly hindered development of this technique.Strain and strain rate imaging is a new modality of echocardiographic techniques designed for measuring the regional myocardial deformation or the velocity of deformation.With a high spatial resolution and frame rate as well as digital processing capability of these techniques,it has been possible to measure the transmural gradient of myocardial strain and strain rate in real time. As myocardial longitudinal shortening plays an important role in cardiac contraction,and longitudinal myocardial fibers contract earlier and are likely more vulnerable to ischemia than circular fibers, measurement of the transmural gradient of the longitudinal myocardial strain and strain rate may provide important information on the transmural distribution of myocardial contraction in physiological conditions and myocardial ischemia.It is still unclear whether the transmural systolic strain and strain rate could predict the transmural myocardial blood flow at different perfusion levels. Therefore,we propose the hypothesis that the transmural gradient of the longitudinal myocardial contraction can be assessed by strain and strain rate imaging,which can in turn be used to predict the transmural gradient of myocardial blood flow(MBF)measured by colored microspheres in the normal,ischemic and infarct segments in a pig model with acute myocardial infarction. OBJECTIVE:To study the contraction patterns in subendocardium and subepicardium across the normal,ischemic and infarct segments by SRI and to explore the relationship between the regional myocardial blood flow and strain and strain rate in the subendocardium and subepicardium at different perfusion levels.1.METHODS1.1 Animal preparationTwelve Chinese experimental mini-pigs(males,25-30kg,China Agricultural University,Beijing)were included in this study.All animal care and experimental protocols complied with the Animal Management Rules of the Ministry of Health of the People's Republic of China(document No 55,2001)and the guidelines for the Care and Use of Laboratory Animals of Shandong University Qilu Hospital, China.General anesthesia was achieved in all pigs by intramuscular injection of ketamine hydrochloride(20 mg/kg,Heng Rui Medicine Co.LTD.Jiang Su,China)together with intravenous injection of sodium pentobarbital(30 mg/kg,Beijing Chemical Reagent Company, Beijing,China).Pigs were placed in the supine position,intubated and mechanically ventilated with a volume respirator(Newport E100m Ventilator,Newport Medical Instruments,Inc,Newport Beach, CA)at a rate of 16-20 breaths per minute.All animals underwent continuous electrocardiographic(ECG)monitoring throughout the experiment.The heart was exposed through a median sternotomy and supported by a pericardial cradle.A 6F Judkins right catheter was introduced via the femoral artery for the left coronary angiography. Distal to the second diagonal branch,the left anterior descending (LAD)coronary artery was completely occluded for 60 minutes with a 7.0-prolene suture.Successful construction of an acute myocardial infarction model was confirmed by left coronary angiography and by the presence of typical ST segment elevation on an electrocardiogram.1.2 Strain and Strain Rate ImagingEchocardiographic imaging was performed 1 hour after LAD ligation with a Vivid 7TMultrasound system(Vivid 7,GE Vingmed, Horten,Norway)with a M3S transducer(1.5 to 4.0MHZ).Strain and strain rate imaging were derived from the apical four-chamber view with the interventricular septum as parallel as possible to the direction of the ultrasound beam.A high frame rate(＞100 frames/s) was used to obtain as much data as possible in a given period of time and a latex bag filled with degassed saline served as an acoustic interface between the epicardium and the transducer.A cine-loop comprising least 6 cardiac cycles was stored digitally on a magneto optic disk for later offline analysis.The ventricular septum was divided into three segments based on their two-dimensional images:the dramatically thinned and akinetic or dyskinetic region,usually localized in the distal septum in the present model,was defined as an infarct segment.The hypokinetic region which was close to the infarct segment and usually located in the middle septum was defined as an ischemic segment.The basal septum,whose blood supply stems from the right coronary artery, was defined as a normal segment.A myocardial length of 4 mm was used for strain analysis and two distinct and parallel sample volumes with an elliptical shape and a size of 5 mm×1 mm were placed in the subendocardial and subepicardial layers in the middle of the normal, ischemic and infarct segments,respectively.The strain curves from both subendocardial and subepicardial layers were simultaneously displayed in the normal,ischemic and infarct segments and the peak systolic strain from the subendocardium(Sp-endo)and subepicardium(Sp-epi)was measured from the two curves.The transmural gradient of myocardial strain was calculated as the ratio of Sp-endo/ Sp-epi(SpoEER)in the three segments,respectively. Similarly,The strain rate curves from both subendocardial and subepicardial layers were simultaneously displayed in the normal, ischemic and infarct segments,respectively and the peak systolic strain rate from the subendocardium(SRp-endo)and subepicardium (SRp-epi)was measured from the two curves.The transmural gradient of myocardial strain rate was calculated as the endocardial to epicardial ratio of strain(SRp-EER),i.e.,SRp-endo/SRp-epi in the three segments.Three cardiac cycles were measured and the values averaged.To ensure sample volumes to be kept in the same desired position,mild manual readjustment of the position of sample volumes during systole was necessary.1.3 Regional Myocardial Blood Flow MeasurementRegional MBF was measured by injection of 5×106 colored microspheres(10±2μm in diameter,E-Z Trac,Los Angeles,CA) into the left ventricle by a 5F pigtail catheter(Cordis,USA)which was followed by a flash injection of 10 ml saline.Starting 10 seconds before each colored microspheres injection,an arterial reference blood sample was withdrawn by a withdrawal pump at a constant rate of 10 ml/min for a period of 90 seconds.After the animal was euthanized,the cardiac sections were cut in accordance with the echocardiaographic apical four chamber views and the transmural tissue slices from normal,ischemic and infarct zones in the ventricular septum according to sample volume positions were derived,each of which was divided into subendocardial and subepicardial layers and weighed individually.Microspheres in the myocardial tissue and blood sample were recovered according to the manufacturer's instruction.MBF values in the subendocardial (MBF-endo)and subepicardial(MBF-epi)layers were calculated separately from the following equation:Qm=(Cm×Qr)/Cr,where Qm is the myocardial blood flow per gram(ml/min/g),Cm is the microspheres count per gram of tissue,Qr is the withdrawal rate of the reference blood sample(ml/min),and Cr is the microspheres count in the reference blood sample.The transmural gradient of myocardial perfusion(MBF-EER)was calculated as the ratio of MBF-endo/MBF-epi in the three segments.1.4 Inter-observer and intra-observer variabilityIn order to assess the reproducibility of strain and strain rate measurement,Sp and SRp in the subendocardium and subepicardium in all animals were measured in the ischemic segment.The inter-observer variability was calculated from the repeated measurements performed by two independent observers and the intra-observer variability was calculated from the repeated measurements performed by one observer who measured twice one week apart.Variability was expressed as the percentage of the absolute difference between two measurements divided by the mean value of the two measurements.1.5 Statistical AnalysisData analysis was performed using SPSS 11.5 statistical software (SPSS Inc.,Chicago,USA).Values were expressed as mean±SD. Comparisons of MBF,Sp and SRp among all segments were performed by one-way ANOVA,and LSD test was used to assess the difference between multiple comparisons.Paired student's t-test was used to analyze the differences between parameters measured from the subendocardium and subepicardium in the same segment. Correlations between MBF and Sp or SRp from all three segments were performed by linear regression analysis.P value＜0.05 was considered statistically significant.2.RESULTS2.1 Strain and Strain Rate Imaging MeasurementsTwo pigs died of ventricular fibrillation after LAD occlusion and 10 pigs entered the final data analysis.The peak systolic strain in both subendocardium(Sp-endo)and subepicardium(Sp-epi) decreased progressively from the normal to the ischemic and the infarct segments and there were significant differences among the three segments(all p＜0.001).Sp-endo was significantly higher than Sp-epi in the normal segment(p=0.001)whereas significantly lower than Sp-epi in the ischemic segment(p=0.002).On the other hand, there was no significant difference between Sp-endo and Sp-epi in the infarct segment(p＞0.05).Consequently,Sp-EER calculated was the highest in the normal segment and the lowest in the ischemic segment and there was a significant difference in Sp-EER between normal and ischemic segments(p＜0.001)and between normal and infarct segments(p=0.013).Moreover,Sp-EER was significantly lower in the ischemic than in the infarct segment(p=0.004).Peak systolic strain rate showed similar changes as peak systolic strain and exhibited a progressive decline from the normal to the ischemic and the infarct segments.(all p＜0.001).SR-endo was significantly higher than SR-epi in the normal segment(p=0.007)but significantly lower than SR-epi in the ischemic segment(p=0.006).In contrast,there was no significant difference between SR-endo and SR-epi in the infarct segment.Compared with the normal segment, SR-EER was significantly reduced in the ischemic(p＜0.001)and infarct segments(p=0.028).Also SR-EER was significantly lower in the ischemic than in the infarct segment(p=0.001).2.2 Regional Myocardial Blood FlowThe regional MBF was similar in the subendocardium and the subepicardium in the normal segment with a calculated MBF-EER of 1.03±0.07.Compared with MBF in the normal segment,MBF in both subendocardium and subepicardium in the ischemic segment decreased significantly(both P＜0.001)with a more remarkable decline in MBF-endo(p?... |
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