1Universitätsklinikum Düsseldorf Institut für Biochemie und Molekularbiologie II Düsseldorf, Deutschland; 2Universitätsklinikum Düsseldorf Klinik für Anästhesiologie Düsseldorf, Deutschland; 3Universitätsklinikum Düsseldorf Institut für Molekulare Kardiologie Düsseldorf, Deutschland
Background. Histological staining with 2,3,5-triphenyltetrazolium chloride (TTC) is the most frequently used tool to delineate viable tissue in the ischemic heart. Although relatively simple and straightforward, the methodology is still technically challenging, as the quality of TTC images is often poor in contrast, resulting in ambiguous borderline definition. To overcome these limitations, we have scrutinized each step involved in the staining protocol and elaborated a refined approach that permits precise determination of infarct size (IS) in the mouse heart.
Methods. Myocardial infarction (MI) in mice was created by transiently occluding the left artery descending (LAD, 50 min). Sequential in-vivo determinations of IS were performed first by late-gadolinium enhancement (LGE) and manganese-enhanced magnetic resonance imaging (MEMRI) and ex-vivo by TTC staining. For whole heart staining, the heart was infused with freshly prepared TTC solution (1%) thrice into the coronary arteries via an aortic cannula every 5 min and further immersed in TTC solution overnight at 4°C. The heart sample was then embedded in OCT and sliced transversally at thickness of 50 µm with intervals of 100 µm from the apex to the base part of the heart. The slices were fixed in Zamboni solution, in which picric acid further labeled the necrotic cardiomyocytes (necCM) beige. As the formazan intensity correlates to the number of functional mitochondria seen mostly in viable cardiomyocytes (viaCM), the crimson precipitates readily identify the viaCM apart from the necCM with dramatically sharp boundary.
As area-at-risk (AAR) is an important parameter and usually calculated as the area distal to remote myocardium (RM), we employed Coomassie Brilliant blue R250 (CBB) as a feasible pigment for stable RM determination. CBB is a water-soluble dye and, after interacting with proteins, forms insoluble precipitates that warrant persistent staining without smearing in the slicing and fixation processes. In the heart after TTC infusion and LAD re-ligation, sequential infusion of CBB (1%, 300 µl) markedly stained the RM area blue, whereas the area downstream the occlusion, i.e., AAR, remained unstained with clearly distinguishable borderline. With the aid of computerized digital planimetry, the area of IS/AAR and surviving CM (surCM) were automatically contoured and calculated.
Methods. Myocardial infarction (MI) in mice was created by transiently occluding the left artery descending (LAD, 50 min). Sequential in-vivo determinations of IS were performed first by late-gadolinium enhancement (LGE) and manganese-enhanced magnetic resonance imaging (MEMRI) and ex-vivo by TTC staining. For whole heart staining, the heart was infused with freshly prepared TTC solution (1%) thrice into the coronary arteries via an aortic cannula every 5 min and further immersed in TTC solution overnight at 4°C. The heart sample was then embedded in OCT and sliced transversally at thickness of 50 µm with intervals of 100 µm from the apex to the base part of the heart. The slices were fixed in Zamboni solution, in which picric acid further labeled the necrotic cardiomyocytes (necCM) beige. As the formazan intensity correlates to the number of functional mitochondria seen mostly in viable cardiomyocytes (viaCM), the crimson precipitates readily identify the viaCM apart from the necCM with dramatically sharp boundary.
As area-at-risk (AAR) is an important parameter and usually calculated as the area distal to remote myocardium (RM), we employed Coomassie Brilliant blue R250 (CBB) as a feasible pigment for stable RM determination. CBB is a water-soluble dye and, after interacting with proteins, forms insoluble precipitates that warrant persistent staining without smearing in the slicing and fixation processes. In the heart after TTC infusion and LAD re-ligation, sequential infusion of CBB (1%, 300 µl) markedly stained the RM area blue, whereas the area downstream the occlusion, i.e., AAR, remained unstained with clearly distinguishable borderline. With the aid of computerized digital planimetry, the area of IS/AAR and surviving CM (surCM) were automatically contoured and calculated.
Planimetric IS in each slice was quantified as area percentage of the necCM in relation to the entire left ventricle and volumetric IS detection was performed using 3D projection by multi-planimetric reconstruction.
Results. We found that, in the same animal, the IS percentage derived by microscopic TTC analysis (38.4 ± 4.2%) was fairly identical to the IS values of in-vivo measurements derived by LGE (35.9 ± 4.1%) and MEMRI (35.1 ± 3.9%), proposing microscopic TTC image as a simple and reliable measure for IS detection. Compared to previously reported data, the percentage of surCM in the present experiment was unexpectedly modest, suggesting conventional TTC analysis potentially overestimates the true extent of surCM.
Results. We found that, in the same animal, the IS percentage derived by microscopic TTC analysis (38.4 ± 4.2%) was fairly identical to the IS values of in-vivo measurements derived by LGE (35.9 ± 4.1%) and MEMRI (35.1 ± 3.9%), proposing microscopic TTC image as a simple and reliable measure for IS detection. Compared to previously reported data, the percentage of surCM in the present experiment was unexpectedly modest, suggesting conventional TTC analysis potentially overestimates the true extent of surCM.
In summary, we report for the first time an important technical advance of TTC staining that consistently yields reliable and unequivocal detection of IS/AAR in the infarcted heart with microscopic accuracy. This approach is an extremely useful tool with broad applications and serves as an improvement over the conventional macroscopic TTC staining in cardiovascular research.