A new method for the intravital microscopic quantification of hepatic sinusoidal perfusion failure using the dye...

In hepatic pathology, in vivo assessment of microvascular perfusion failure by intravital microscopic determination of functional sinusoidal density is a time-consuming procedure, which requires accurate identification of blood-flow conditions within each individual sinusoid. Herein we report a new method, which is easily applicable and which allows for rapid analysis of hepatic microvascular perfusion deficits by intravital fluorescence microscopy using Bisbenzamide H33342 staining of hepatocytes (excitation 330-390 nm/emission > 430 nm). To validate this method hepatic microvascular perfusion failure was induced in eight spontaneously breathing, chloralhydrate-anesthetized rats by 90-min left lobar ischemia and reperfusion. For quantitative assessment of hepatocellular bisbenzamide fluorescence intensity, gray levels were determined densitometrically, and the area of positive-stained cells was calculated automatically as percentage of the whole area of observation. Within identical acini (n = 67) area of positive hepatocellular fluorescence was correlated with functional sinusoidal density, i.e, total length of perfused sinusoids per observation area (cm/cm2), which was determined simultaneously by contrast enhancement with sodium fluorescein (450-490/ > 520 nm) using a computer-assisted image analysis system. Postischemic reperfusion was characterized by a marked heterogeneity of nutritive perfusion with a considerable number of nonperfused sinusoids. Visualization of bisbenzamide fluorescence revealed brightly stained hepatocytes in well-perfused areas, but faint hepatocellular fluorescence in areas presenting with sinusoidal perfusion failure. Linear regression analysis between area of positive hepatocellular fluorescence and functional sinusoidal density revealed a significant (P < 0.01) correlation (r2 = 0.926), indicating that automated densitometric analysis of hepatocellular bisbenzamide fluorescence represents a valid method to determine hepatic nutritive perfusion failure in vivo.