title: A new method for the intravital microscopic quantification of hepatic sinusoidal perfusion failure using the dye bisbenzamide H33342.
abstract: 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.
authors.affiliation: Institute for Clinical & Experimental Surgery, University of Saarland, Homburg, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Vollmar
authors.forename: M
authors.initials: M
authors.lastname: Rücker
authors.forename: M D
authors.initials: MD
authors.lastname: Menger
mesh_terms: D000818:Animals; D001562:Benzimidazoles; D005069:Evaluation Studies as Topic; D005456:Fluorescent Dyes; D008099:Liver / Q000098:blood supply* / Q000293:injuries; D008856:Microscopy, Fluorescence / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000209:etiology
chemical_list: D001562:Benzimidazoles; D005456:Fluorescent Dyes; C017807:bisbenzimide ethoxide trihydrochloride