Evans blue staining of cardiomyocytes induced by myocardial contrast echocardiography in rats: evidence for necrosis instead of apoptosis.

High mechanical index (MI) echocardiography with contrast agent has been shown to induce Evans blue staining of cardiomyocytes, seen 1 d after exposure, in addition to contraction band necrosis, seen immediately after exposure. This research examined the roles of necrosis vs. apoptosis in these bioeffects. Myocardial contrast echocardiography at high MI with 1:4 electrocardiogram triggering was performed in anesthetized rats at 1.5 MHz. Histologically observable cell injury was accumulated by infusing a high dose of 50 microL/kg ultrasound contrast media via tail vein for 5 min at the start of 10 min of scanning. Evans blue dye or propidium iodide was injected as an indicator of cardiomyocyte plasma membrane integrity. Histologic sections were stained using the terminal dUTP nick-end labeling (TUNEL) method for labeling nuclei with DNA degradation (e.g., apoptosis). Evans blue fluorescent cells were counted on frozen sections or on hematoxylin-stained and TUNEL-labeled paraffin sections. In addition, transmission electron microscopy was used to assess potential apoptotic nuclei. Hypercontraction and propidium iodide staining were observed immediately after imaging exposure. Although TUNEL-positive cells were evident after 4 h, these also had indications of contraction band necrosis, and features of apoptosis were not confirmed by electron microscopy. Inflammatory cell infiltration was evident after 24 h. A second, more subtle injury was recognized by Evans blue staining, with minimal inflammatory cell infiltration at the morphologically intact stained cells after 24 h. Apoptosis was not detected by the TUNEL method in the cardiomyocytes stained with Evans blue at 24 h. However, Evans blue-stained cell numbers declined after 48 h, with continued inflammatory cell infiltration. The initial insult for Evans blue-stained cardiomyocytes apparently induced partial permeability of the plasma membrane, which led to gradual degeneration (but not apoptosis) and necrosis after 24 to 48 h.