High-resolution visualization of oxygen distribution in the liver in vivo

Abstract

Microcirculatory failure after stress events results in mismatch in oxygen supply and demand. Determination of tissue oxygen distribution in vivo may help elucidate mechanisms of injury, but present methods have limited resolution. Male Sprague-Dawley rats were anesthetized, prepared for intravital microscopy, and received intravenously the oxygen-sensitive fluorescent dye Tris(1,10-phenanthroline)ruthenium(II) chloride hydrate [Ru(phen)3(2+)]. An impaired hepatic oxygen distribution was induced by either phenylephrine or hemorrhage. Intensity of Ru(phen)3(2+) fluorescence was compared with NADH autofluorescence indicating changes in the mitochondrial redox potential. Ethanol was injected to affect the NADH-to-NAD+ ratio without altering the P(O2). Infusion of Ru(phen)3(2+) resulted in a heterogeneous fluorescence under baseline conditions reflecting the physiological acinar P(O2) distribution. A decrease in oxygen supply due to phenylephrine or hemorrhage was paralleled by an increase in Ru(phen)3(2+) and NADH fluorescence reflecting an impaired mitochondrial redox state. Ethanol did not alter Ru(phen)3(2+) fluorescence but increased NADH fluorescence indicating independence of P(O2) and redox state imaging. Intravenous administration of Ru(phen)3(2+) for intravital videomicroscopy represents a new method to visualize the hepatic tissue P(O2). Combined with NADH autofluorescence, it provides additional information regarding the tissue redox state.

Rolipram Improves Renal Perfusion and Function during Sepsis in the Mouse

“…Data are mean 6 S.E.M., n = 4-8 mice/group. NADPH autofluorescence can be quantified during IVVM and is considered a marker of cellular stress (Paxian et al, 2004;Wunder et al, 2005;Wu and Mayeux, 2007). CLP increased renal tubular NADPH autofluorescence at 18 hours after CLP compared with Sham (447 6 56 units/mm 2 for CLP 1 Vehicle versus 250 6 21 units/mm 2 for Sham 1 Vehicle, n 5 5, P , 0.05).…”

“…The RNS peroxynitrite preferentially oxidizes DHR-123 to fluorescent rhodamine that is visualized at 535-nm excitation and 590-nm emission (Gomes et al, 2006). Autofluorescence of NADPH can be detected at 365-nm excitation and 420-nm emission, and can be used as a marker of cellular redox stress (Paxian et al, 2004;Wunder et al, 2005;Wu et al, 2007a;Wang et al, 2012). Still images exposed for 500 milliseconds were captured from the same fields of view used to determine capillary perfusion.…”

Evidence for the Role of Reactive Nitrogen Species in Polymicrobial Sepsis-Induced Renal Peritubular Capillary Dysfunction and Tubular Injury

“…One RNS, peroxynitrite (ONOO Ϫ ), is formed from the chemical reaction between NO and superoxide (26) and can damage not only membranes but also nitrate protein tyrosine and change the activities of a number of important enzymes (27,28). Moreover, decreased capillary perfusion can lead to parenchymal hypoxia (29) and superoxide generation to fuel RNS synthesis (30). Therefore, it is likely that increased NO generation (as a result of iNOS induction) in the setting of a pro-oxidant, hypoxic microenvironment caused by decreased perfusion, exacerbates renal injury (31).…”

Optimizing normoxic conditions in liver devices using enhanced gel matrices

“…As previously described (McClelland and Coger, 2000;Paxian et al, 2004) Dichlorotris ruthenium(II) hydrate (Ru 2þ ), 98% light sensitive oxygen quenching dye particles (#34371-4, Aldrich, Milwaukee, WI) is effective for visualizing oxygen transport in fluorescent light (ex: 465-495 nm, em: 577-632 nm). For all O 2 transport visualization experiments in the current study, the Ru 2þ dye was first added into 0.1 M NaOH to produce 0.02 mM dye solution.…”

“…A decreased O 2 tension is paralleled by decreases in the concentration ratio of nicotinamide adenine dinucleotide (NAD) compared to its reduced form (NADH). As such, the autofluorescence of NADH has been widely used as an indicator of cellular metabolic status to elucidate the states of both hepatic tissue and cells under hypoxic conditions (Chandel and Budinger, 1997;Croce et al, 2004;Obi-Tabot and Hanrahan, 1993;Paxian et al, 2004).…”