Bifunctional staining for ex vivo determination of area at risk in rabbits with reperfused myocardial infarction

AIM: To develop a method for studying myocardial area at risk(AAR) in ischemic heart disease in correlation with cardiac magnetic resonance imaging(c MRI). METHODS: Nine rabbits were anesthetized, intubated and subjected to occlusion and reperfusion of the left circumflex coronary artery(LCx) to induce myocardial infarction(MI). ECG-triggered c MRI with delayed en-hancement was performed at 3.0 T. After euthanasia, the heart was excised with the LCx re-ligated. Bifunctional staining was performed by perfusing the aorta with a homemade red-iodized-oil(RIO) dye. The heart was then agar-embedded for ex vivo magnetic resonance imaging and sliced into 3 mm-sections. The AAR was defined by RIO-staining and digital radiography(DR). The perfusion density rate(PDR) was derived from DR for the AAR and normal myocardium. The MI was measured by in vivo delayed enhancement(i DE) and ex vivo delayed enhancement(e DE) c MRI. The AAR and MI were compared to validate the bifunctional straining for cardiac imaging research. Linear regression with Bland-Altman agreement, one way-ANOVA with Bonferroni's multiple comparison, and paired t tests were applied for statistics.RESULTS: All rabbits tolerated well the surgical procedure and subsequent c MRI sessions. The openchest occlusion and close-chest reperfusion of the LCx, double suture method and bifunctional staining were successfully applied in all animals. The percentage MI volumes globally(n = 6) and by slice(n = 25) were 36.59% ± 13.68% and 32.88% ± 12.38% on i DE, and 35.41% ± 12.25% and 32.40% ± 12.34% on e DE. There were no significant differences for MI determination with excellent linear regression correspondence(r global = 0.89; r slice = 0.9) between i DE and e DE. The percentage AAR volumes globally(n = 6) and by slice(n = 25) were 44.82% ± 15.18% and 40.04% ± 13.64% with RIO-staining, and 44.74% ± 15.98% and 40.48% ± 13.26% by DR showing high correlation in linear regression analysis(r global = 0.99; r slice = 1.0). The mean differences of the two AAR measurements on BlandAltman were almost zero, indicating RIO-staining and DR were essentially equivalent or inter-replaceable. The AAR was significantly larger than MI both globally and slice-by-slice(P < 0.01). After correction with the background and the blank heart without bifunctional staining(n = 3), the PDR for the AAR and normal myocardium was 32% ± 15% and 35.5% ± 35%, respectively,which is significantly different(P < 0.001), suggesting that blood perfusion to the AAR probably by collateral circulation was only less than 10% of that in the normal myocardium.CONCLUSION: The myocardial area at risk in ischemic heart disease could be accurately determined postmortem by this novel bifunctional staining, which may substantially contribute to translational cardiac imaging research.

AIM： To develop a method for studying myocardial area at risk （AAR） in ischemic heart disease in correlation with cardiac magnetic resonance imaging （cMRI）. METHODS： Nine rabbits were anesthetized, intubated and subjected to occlusion and reperfusion of the left circumfex coronary artery （LCx） to induce myocardial infarction （MI）. ECG-triggered cMRI with delayed enhancement was performed at 3.0 T. After euthanasia, the heart was excised with the LCx re-ligated. Bifunc-tional staining was performed by perfusing the aorta with a homemade red-iodized-oil （RIO） dye. The heart was then agar-embedded for ex vivo magnetic reso-nance imaging and sliced into 3 mm-sections. The AAR was defined by RIO-staining and digital radiography （DR）. The perfusion density rate （PDR） was derived from DR for the AAR and normal myocardium. The MI was measured by in vivo delayed enhancement （iDE） and ex vivo delayed enhancement （eDE） cMRI. The AAR and MI were compared to validate the bifunctional straining for cardiac imaging research. Linear regres-sion with Bland-Altman agreement, one way-ANOVA with Bonferroni’s multiple comparison, and paired t tests were applied for statistics.RESULTS： All rabbits tolerated well the surgical pro-cedure and subsequent cMRI sessions. The open-chest occlusion and close-chest reperfusion of the LCx, double suture method and bifunctional staining were successfully applied in all animals. The percentage MI volumes globally （n = 6） and by slice （n = 25） were 36.59% ± 13.68% and 32.88% ± 12.38% on iDE, and 35.41% ± 12.25% and 32.40% ± 12.34% on eDE. There were no signifcant differences for MI determina-tion with excellent linear regression correspondence （ rglobal = 0.89; r slice = 0.9） between iDE and eDE. The percentage AAR volumes globally （ n = 6） and by slice （ n = 25） were 44.82% ± 15.18% and 40.04% ± 13.64% with RIO-staining, and 44.74% ± 15.98% and 40.48% ± 13.26% by DR showing high correlation in linear re-gression analysis （ rglobal = 0.99; r slice = 1.0）. The mean differences of the two AAR measurements on Bland-Altman were almost zero, indicating RIO-staining and DR were essentially equivalent or inter-replaceable. TheAAR was signifcantly larger than MI both globally and slice-by-slice （P 〈 0.01）. After correction with the back-ground and the blank heart without bifunctional stain-ing （n = 3）, the PDR for the AAR and normal myocar-dium was 32% ± 15% and 35.5% ± 35%, respectively,which is signifcantly different （ P 〈 0.001）, suggesting that blood perfusion to the AAR probably by collateral circulation was only less than 10% of that in the nor-mal myocardium.CONCLUSION： The myocardial area at risk in ischemic heart disease could be accurately determined post-mortem by this novel bifunctional staining, which may substantially contribute to translational cardiac imaging research.