We examined the effects of hypoxia and reoxygenation in isolated, perfused rat livers. Hypoxia induced by a low rate of perfusion led to near anoxia confined to centrilobular regions of the liver Iobule. Periportal regions remained normoxic. Within 15 min, anoxic centrilobular hepatocytes developed surface blebs that projected into sinusoids through endothelial fenestrations. Periportal hepatocytes were unaffected. Both scanning and transmission electron microscopy suggested that blebs developed by transformation of preexisting microvilli. Upon reoxygenation by restoration of a high rate of perfusion, blebs disappeared. Other changes included marked shrinkage of hepatocytes, enlargement of sinusoids, and dilation of sinusoidal fenestrations. There was also an abrupt increase in the release of lactate dehydrogenase and protein after reoxygenation, and cytoplasmic fragments corresponding in size and shape to blebs were recovered by filtration of the effluent perfusate.We also studied phalloidin and cytochalasin D, agents that disrupt the cytoskeleton. Both substances at micromolar concentrations caused rapid and profound alterations of cell surface topography.We conclude that hepatic tissue is quite vulnerable to hypoxic injury. The morphological expression of hypoxic injury seems mediated by changes in the cortical cytoskeleton. Reoxygenation causes disappearance of blebs and paradoxically causes disruption of cellular volume control and release of blebs as cytoplasmic fragments. Such cytoplasmic shedding provides a mechanism for selective release of hepatic enzymes by injured liver tissue.

Hypoxia was produced in isolated, hemoglobin-free, perfused rat liver by reducing the flow rate of oxygen-carrying fluid entering the organ. The procedure caused anoxia in centrilobular regions. In these anoxic areas, structural derangements developed rapidly, characterized by bleb-like protrusions of hepatocyte plasma membrane through fenestrations in the sinusoidal endothelium. Periportal tissue remained normoxic and was completely spared. Cellular injury resulting from localized anoxia may play an important role in the pathogenesis of centrilobular liver disease.

Pyridine nucleotide fluorescence made from the surface of the hemoglobin-free perfused rat liver was measured continuously by using a "micro-light guide" placed on selected periportal and pericentral regions of the liver lobule. From the portal oxygen tension at which pyridine nucleotide reduction first occurred in pericentral regions, the oxygen gradient across the liver lobule was estimated in livers from rats treated chronically with ethanol or sucrose. Chronic treatment with ethanol increased the average lobular oxygen gradient from 275 to 400 torr (1 torr = 133 Pa), primarily due to the increase in the oxygen gradient in pericentral regions. Ethanol treatment also increased hepatic oxygen uptake significantly, from 110 to 144 (Iumol/g)/hr. Treatment with the antithyroid drug 6-propyl-2-thiouracil reversed the effect of ethanol on 02 uptake and on the lobular oxygen gradient. The oxygen gradients measured with the micro-light guide were confirmed by direct measurement oftissue oxygen tensions in periportal and pericentral areas by using an oxygen electrode. These data are consistent with the hypothesis that chronic treatment with ethanol causes the pericentral region of the liver lobule to become susceptible to hypoxic cellular injury. This may be responsible, at least in part, for the localized hepatotoxic effects of ethanol.Ethanol-induced liver damage is often confined to the pericentral region of the liver lobule (1-3). Israel et al. (3) have postulated that ethanol-induced pericentral necrosis results from an accentuated gradient of decreasing oxygen tension from the portal to the central venous end of the sinusoid leading to pericentral hypoxia. This hypothesis is based on the observations that ethanol treatment increased the rate of oxygen uptake in liver slices (4-6) and perfused liver (7,8). In support of this postulate, Israel et aL (3) demonstrated that, after brief exposure to hypoxia, pericentral necrosis was greater in ethanol-treated rats than in controls. Treatment ofrats with the antithyroid drug 6-propyl-2-thiouracil abolished the ethanol-induced increase in liver respiration and diminished the tissue damage produced by hypoxia (3).We have used a "micro-light guide" fluorometric technique (9-12) to test this hypothesis directly. This technique allows continuous measurement of pyridine nucleotide fluorescence from the periportal or pericentral regions of the perfused liver. Chance and co-workers (13, 14) have shown that tissue pyridine nucleotide fluorescence can be used to monitor intracellular oxygen tension indirectly because pyridine nucleotide fluorescence increases when tissue oxygen tension falls below the level (<1 torr; 1 torr = 133 Pa) necessary to satisfy the oxygen requirement of mitochondrial cytochrome oxidase. By measuring the oxygen tension in the influent perfusate at which pyridine nucleotide fluorescence begins to increase in pericentral regions, it is possible to estimate the oxygen gradient across the liver lobule. The results of the present study indicate that ...

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Three-way light guides containing one or more strands of 25-micron or 80-micron diameter optical fibers in each channel have been constructed and used to measure the NADH fluorescence and UV reflectance from mitochondrial suspensions, the perfused, hemoglobin-free rat liver, and the perfused beating interventricular septum of the rabbit. The optical changes measured with these so-called micro-light guides, which have channels containing one or several strands of optical fibers less than 100 micron, are comparable in magnitude with those measured using much larger conventional light guides. The effect of light scattering on the fluorescence channel has been determined and an empirical equation for correcting the fluorescence channel for light scattering has been obtained for mitochondrial suspensions. A mathematical equation characterizing the optical behavior of a two-way micro-light guide has been derived and has been shown to account satisfactorily for reflectance and fluorescence measurements of a mat surface in air.