Biomedical subjects

M G Clemens

Publications and source records attributed to M G Clemens.

At least 55 records · Page 3Linked to original sources

PubMed1994

Activated THP-1 cells depress mitochondrial respiration in Hep G2 cells infected with influenza B virus.

Influenza B virus has been aetiologically linked to Reye Syndrome (RS), but the mechanism(s) by which this pathogen could disrupt liver metabolism and produce the hepatic mitochondrial injury characteristic of the syndrome are unknown. In this study, two mechanisms by which infection of hepatocytes with influenza B virus could disrupt cellular metabolism were investigated. (1) virus-induced increase in pro-oxidant iron with subsequent iron-induced lipid peroxidation (LP) and (2) increased membrane permeability. Hep G2 cells, a well-differentiated continuous human liver cell line derived from a hepatoblastoma, were infected with allantoic-fluid derived influenza B Lee/40 virus (AFDV) at a multiplicity of infection of 10 for 24 h; productive infection was confirmed by both haemagglutination of chick erythrocytes and by plaque assay. Infection of Hep G2 cells preloaded with 59Fe-transferrin resulted in increased release of 59Fe (153 +/- 17% of controls, P < 0.03). However, the iron released did not result in increased LP (assessed by thiobarituric acid reactive substances; TBARS). To confirm that this lack of of increase in TBARS was not due to insensitivity of the cell line to pro-oxidant iron, cells were exposed to 15 microM iron ascorbate for 60 min. Production of TBARS was increased (122 +/- 4% of controls, P < 0.0003). Release of 51Cr from infected cells was also increased (128 +/- 12% of controls, P < 0.05); thus the infected cells exhibited a generalized increase in membrane permeability. However, infection did not depress mitochondrial respiration (as assessed by the formation of MTT-f3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan. To determine if the combination of viral infection and soluble products of activated macrophages would affect mitochondrial respiration, infected hepatocytes were exposed to the supernatant fluid from THP-1 cells which had previously been incubated with lipopolysaccharide at 100 ng ml-1 for 18 h. This supernate did depress the formation of MTT-f (81 +/- 5% of controls, P < 0.03). We conclude that influenza B virus does productively infect Hep G2 cells, and does increase hepatocyte membrane permeability. This effect does not impair mitochondrial respiration directly. However, infection does act in concert with soluble products of activated macrophages to depress hepatic mitochondrial respiration. Whether this interaction can be explained by virus-induced permeability changes and/or other effects of infection deserves further investigation.

Animals↗

PubMed1994

Microcirculatory failure determines lethal hepatocyte injury in ischemic/reperfused rat livers.

The contribution of microcirculatory failure to ischemia/reperfusion injury in isolated perfused rat livers was investigated using intravital epifluorescence videomicroscopy. The degree of microvascular shut-down during reperfusion was modulated by the reperfusion conditions: flow-controlled (10 ml/min), in which microcirculatory failure is minimized by maintenance of constant flow through the liver, and pressure-controlled, in which microvascular shut-down is allowed to occur. Livers underwent 60 min of ischemia, 90 min of ischemia, or no ischemia (control). Perfused sinusoids and dead hepatocytes were quantified in 10 standardized microscopic fields (9000 microns2) per liver during off-line video playback. With flow-controlled reperfusion, microvascular (sinusoid) shut-down was largely avoided; a maximum of 21% of the sinusoids failed to conduct flow. Pressure-controlled reperfusion, however, resulted in early and severe shut-down. A significant decrease of approximately 20-30% was found after 60 min of ischemia and 30 min of reperfusion, while, after 90-min ischemia and 90-min reperfusion, 90% of the sinusoids failed to conduct flow. The appearance of dead hepatocytes correlated well with the number of perfused sinusoids (r = -0.78 for flow controlled, r = -0.97 for pressure-controlled). Only an occasional dead hepatocyte was observed with control perfusion, while up to 50% stained with propidium iodide following 90-min ischemia and 90-min reperfusion under pressure-controlled conditions. These results indicate that loss of sinusoidal flow can be ameliorated by flow-controlled reperfusion; moreover, hepatocyte necrosis during reperfusion is highly dependent upon the integrity of the microcirculation.

Animals↗

PubMed1994

Hormone effects on hepatic substrate preference in sepsis.

This study addressed the effect of catecholamine stimulation on substrate utilization for gluconeogenesis, ureagenesis, and oxidation in perfused livers from septic rats. Livers were perfused with buffer containing 5 mM [14C]lactate and various concentrations of unlabeled alanine or pyruvate. Addition of alanine to lactate resulted in inhibition of gluconeogenesis and especially inhibition of gluconeogenesis from lactate. This effect was dependent upon the presence of the amino nitrogen, since the effect of pyruvate was to increase total gluconeogenesis with little effect on gluconeogenesis specifically from lactate except with phenylephrine stimulation of livers from sham-operated animals in which addition of pyruvate actually increased the rate of gluconeogenesis from lactate. Alanine itself was very poorly utilized as a gluconeogenic substrate. In contrast, the addition of alanine stimulated total oxygen consumption in both groups in the absence or presence of phenylephrine. This was the result of oxidation of added alanine in livers from sham animals, either with or without phenylephrine, and in septic animals without phenylephrine. However, in the presence of phenylephrine, the increase in total oxygen consumption was almost entirely the result of lactate oxidation. Pyruvate, on the other hand, uniformly stimulated oxygen consumption in both groups, with and without phenylephrine. Urea production was increased by alanine to a greater extent in the septic group compared to sham. However, while phenylephrine stimulated ureagenesis in the sham-operated group, it inhibited ureagenesis in the septic group. These results demonstrate that fundamental differences develop in livers from septic animals in their handling of nitrogenous and non-nitrogenous gluconeogenic substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗

PubMed1994

Effect of activation on neutrophil-induced hepatic microvascular injury in isolated rat liver.

Polymorphonuclear neutrophils (PMNs) have been implicated in microvascular injury following ischemia and reperfusion (I/R) but the relative contribution of obstruction versus toxic mediators is not well defined. Therefore, the present study was performed to determine the contribution of exogenous or endogenous activation on PMN-induced microvascular and hepatocyte injury. Rat livers were isolated and perfused at constant pressure with Krebs buffer with red cells (Hct-10%) and monitored for perfused sinusoids (PS) and dead hepatocytes (propidium iodide-stained, DH) by intravital microscopy. PMNs isolated from the peritoneum after oyster glycogen injection were added to the perfusate either without or with activation by phorbol myristate acetate (PMA, 160 nM). Unactivated PMNs stuck in the liver but had no significant effect on either perfused sinusoids (11.1 +/- .4/field, unactivated PMNs versus 11.9 +/- .5/field, the time-matched control) or dead hepatocytes (1.2 +/- .4/field, unactivated PMNs versus 1 +/- .3/field, the time-matched control). Infusion of PMA-activated PMNs resulted in significant decrease in perfused sinusoids and increase in DH (9.5 +/- .3/field for PS and 3.2 +/- .6/field for DH, respectively). In contrast, when PMNs were "activated" by infusion into a liver previously made ischemic for 30 min, DH were significantly increased after 60 min (26.2 +/- 4.5/field, I/R plus PMNs versus 12.4 +/- 2/field, I/R only) but perfused sinusoids were not different from ischemia alone. These results demonstrate that oxidatively quiescent PMNs do not cause cellular or microvascular injury in spite of microvascular accumulation. Activated PMNs damage microcirculation or hepatocytes depending on the nature of the activation.

Animals↗

PubMed1994

Hepatic intercellular communication in shock and inflammation.

The liver is well recognized as a target for injury during low flow or inflammatory states. Functionally, the result is both metabolic and host defense dysfunction. Although the liver is clearly responsive to changes in systemic levels of various mediators, it is becoming apparent that substantial changes occur within the liver that are not directly dependent on extrahepatic factors. This is the result of complex interactions among the various cell types that exist in a highly organized arrangement within the functional subunit of the liver. The purpose of this review is to summarize the structural relationships which form the basis for this system of cell-cell communication and their functional implications both in the normal liver and during both low-flow and normal-flow inflammatory states.

Animals↗

PubMed1994

Endothelin-1 induces direct constriction of hepatic sinusoids.

We studied the hepatic microvascular response to endothelin (ET) and the possible role of Ito cells (fat-storing cells) acting as pericytes in this response using isolated rat livers under high-power intravital microscopy. Livers were perfused in a modified pressure-controlled system with Krebs buffer plus rat erythrocytes (RBC, 10%), and sinusoids at the site of Ito cells were observed under a x 100 objective (total magnification x 2,533) before and during infusion of ET-1 (10(-9 M) alone, sodium nitroprusside (NP, 10(-5) M). plus ET-1, or phenylephrine (PE, 10(-7) M). Both ET-1 and PE decreased portal flow (25 and 51%) and increased inflow pressure (28 and 43%), respectively. PE had no effect on any sinusoidal parameters except that it decreased measured sinusoidal RBC velocity (P < 0.05); ET-1 decreased sinusoidal diameter by 25% and increased the calculated sinusoidal pressure gradient and resistance by 116 and 350%, respectively, but did not alter RBC velocity. NP significantly inhibited changes induced by ET-1. These results demonstrate that ET-1 induces a specific sinusoidal constriction that disrupts normal acinar flow dynamics, and the sinusoidal constriction colocalizes with Ito cells, suggesting that the constriction may be mediated at least in part by ET-1 action on Ito cells, which can be inhibited by a nitric oxide donor.

Animals↗

PubMed1994

ET-1 induced alterations of hepatic microcirculation: sinusoidal and extrasinusoidal sites of action.

Using epifluorescence microscopy, we investigated the dynamic changes in hepatic sinusoidal hemodynamics in vivo during continuous infusion of endothelin-1 (ET-1) in pentobarbital-anesthetized rats. ET-1 was infused for 20 min at rates of 2 or 10 pmol/min either systemically or into the portal vein, followed by a 90-min recovery period. In contrast to systemic application of ET-1 that did not cause a consistent hepatic microvascular effect, we observed two different patterns of microcirculatory alterations during portal application. Infusion of 2 pmol/min elicited a rapid, reversible decrease in sinusoidal diameter that was paralleled by a slight increase in red cell velocity, resulting in conservation of volumetric flow and sinusoid density. Infusion of 10 pmol/min resulted in a biphasic narrowing followed by transient increase in sinusoidal diameter and a profound and lasting decrease in red cell velocity, leading to an almost complete cessation of hepatic microvascular blood flow. These results indicate that ET-1 is a potent constrictor in the liver microcirculation in vivo and acts at both extrasinusoidal and sinusoidal sites, although the sinusoidal sites appear to be more sensitive to lower concentrations.

Animals↗

PubMed1994

Endothelin-1 as a regulator of hepatic microcirculation: sublobular distribution of effects and impact on hepatocellular secretory function.

Endothelin-1 is a potent vasoconstrictor in the portal circulation causing sinusoidal constriction as well as presinusoidal resistance changes. Using in vivo videomicroscopy we studied the sublobular differences in dose response characteristics of sinusoid constriction at 10-13 min into continuous infusion of 5, 1.0, 3.0, and 5.0 pmol of endothelin-1 (ET-1)/100 g body weight/min in pentobarbital anesthetized rats. In addition, bile flow was monitored to estimate parenchymal secretory function. ET-1 evoked a profound constrictor effect in both sublobular regions studied. However, the maximal decrease of sinusoidal width in periportal inflow region (zone 1; 1 pmol/100 g/min: 4.8 +/- .1 microns) was reached at slightly lower concentrations of the peptide than in pericentral outflow region (zone 3; 3 pmol/100 g/min: 6.2 +/- .3 microns) compared to respective baseline values (zone 1: 7.1 +/- .1 microns; zone 3: 10.2 +/- .1 microns), suggesting upstream binding and clearance of the peptide. The constrictor response in zone 1 was biphasic and at higher concentrations of the peptide (5 pmol/100 g/min: 5.5 +/- .2 microns) sinusoidal widths increased again compared to the maximal response with the 1 pmol. Secretory function as reflected by the bile flow was maintained or even slightly increased with the lower doses (.5 and 1 pmol) of ET-1 and during the first 10 min into infusion of the higher doses (3 and 5 pmol). Subsequently, an approximately 20-25% decrease in bile flow accompanied the infusion of higher doses of ET-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Maturational differences in hepatic microhemodynamics in rats.

Age-related changes in the hepatic microcirculation may contribute to the increased susceptibility of the immature liver to microvascular injury. We quantified sinusoidal and acinar diameters, sinusoidal red cell velocities (VRBC), and sinusoidal volume flows to characterize microhemodynamics of weanling and adult rat livers with and without hepatic artery (HA) ligation using intravital fluorescence videomicroscopy. Despite a 20% faster heart rate and a nearly 20% lower mean arterial and portal vein pressure in weanling rats relative to those in the adults, weanling periportal and pericentral sinusoidal velocities were approximately 30 and 25% faster, respectively, than those in adults. Furthermore, the HA was found to contribute more to maintenance of sinusoidal VRBC in the immature liver as demonstrated by a significant decrease in both periportal and pericentral VRBC following HA ligation. HA ligation had no effect on VRBC of either zone in adults. Zonal volume flow (HA intact), however, was maintained independent of age. These results suggest a lower extrasinusoidal resistance in the weanling. The 25% shorter acinar diameter that we found in weanling livers likely contributes to a lower extrasinusoidal resistance by allowing a higher ratio of inflow vessels to volume of tissue. Shorter sinusoidal pathways in weanling livers also decreases sinusoidal resistance 1.3-fold relative to that in the adult, countering the approximately 1.5 times increase in resistance due to the smaller caliber of sinusoidal vessels so that overall sinusoidal resistance is not age-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1993

Sepsis-induced alterations in phosphoenolpyruvate carboxykinase expression: the role of insulin and glucagon.

Sepsis is associated with a decrease in the intrinsic gluconeogenic capacity of hepatocytes. The mechanism underlying this depression is unknown. This study sought to investigate whether decreased expression of phosphoenolpyruvate carboxykinase (PEPCK), a rate limiting enzyme in hepatic gluconeogenesis, might contribute to the decreased gluconeogenesis in sepsis. Therefore, we determined the effects of sepsis on the steady-state level of PEPCK mRNA and on PEPCK activity. Further, levels of insulin and glucagon, which modulate PEPCK expression under normal conditions, were also measured. Rats were subjected to either cecal ligation and puncture, or sham operation. Twenty-four hr later, the steady-state level of PEPCK mRNA was determined by Northern Blot hybridization analysis, and PEPCK activity was measured by 14C incorporation into phosphoenolpyruvate. Insulin and glucagon levels were determined by radioimmunoassay, and the insulin/glucagon ratio calculated. The steady-state levels of PEPCK mRNA were significantly decreased in septic animals relative to sham-operated animals. The specific activity of PEPCK in sham-operated animals was 1.67 +/- 0.25 U/mg protein, compared to 0.93 +/- 0.18 U/mg protein in septic animals (P < 0.05). The insulin/glucagon ratio was lower in septic animals than in sham-operated controls. To investigate the specific effect of the insulin-glucagon ratios observed in septic and sham operated rats on hepatocytes under non-septic conditions, cultures of primary rat hepatocytes were used. These cells were incubated with levels of insulin and glucagon equivalent to those found following cecal ligation and puncture or sham operation. Hormonal conditions designed to mimic sepsis were associated with an increase in PEPCK expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Spatial relationship between leukocyte accumulation and microvascular injury during reperfusion following hepatic ischemia.

In order to further elucidate the possible contribution of leukocytes to microvascular injury during reperfusion following total hepatic ischemia, we studied the spatial relationship between areas of white cell accumulation and areas of microvascular damage in the rat liver in vivo. No-flow hepatic ischemia was produced for 90 min in vivo and during the ensuing reperfusion phase (I/R) leukocyte accumulation, absolute number of perfused sinusoids per unit area, and red blood cell velocity were quantitated using in vivo epi-fluorescence video microscopy. The total number of stationary leukocytes in the liver during reperfusion was found to be significantly elevated following ischemia compared to time-matched sham-operated controls. In addition, by 2 hr of reperfusion, approximately 80% of the leukocytes in the I/R group were extravascular compared to only about 50% in the controls. When leukocyte accumulation and microhemodynamics were expressed on the basis of whole liver, the increased accumulation of leukocytes was associated with decreased microvascular perfusion as indicated by decreased number of sinusoids perfused and decreased red blood cell velocity. However, when the data were analyzed on the basis of .05mm2 microscopic fields on the surface of the liver, there was no difference in leukocyte accumulation in areas with sinusoidal blood flow compared to areas that were devoid of perfused sinusoids. Moreover, in a correlation analysis of number of adherent leukocytes/microscopic field vs red blood cell velocity in perfused sinusoids in that field, only a very small negative correlation between leukocytes/field and red blood cell velocity was found (r = -.23, p < .05). These results demonstrate that at the whole organ level leukocyte accumulation appears to correlate well with microvascular damage; however, this increase in whole liver accumulation of leukocytes does not necessarily reflect accumulation at sites of overt microvascular damage. Thus, leukocyte-independent factors are likely to be of considerable quantitative importance in microvascular injury during reperfusion following hepatic ischemia.

Animals↗

PubMed1992

Effect of alpha-tocopherol on hepatic mixed function oxidases in hepatic ischemia/reperfusion.

This study was done to determine the relationship between microsomal lipid peroxidation during hepatic ischemia/reperfusion and alteration in cytochrome P-450-dependent drug metabolism. Rats were pretreated with alpha-tocopherol to inhibit lipid peroxidation or with vehicle (soybean oil) and then subjected to 60 min no-flow hepatic ischemia in vivo. Control animals were time-matched sham-ischemic animals. After 1, 5 or 24 hr of reperfusion, liver microsomes were isolated and cytochrome P-450 and mixed function oxidases were studied. In vehicle-treated ischemic rats, serum ALT levels peaked at 5 hr (5,242 +/- 682 U/L) and were significantly reduced by alpha-tocopherol pretreatment (1,854 +/- 229 U/L, p less than 0.01). Similarly, microsomal lipid peroxidation was elevated in the vehicle-treated ischemic group, but this elevation was prevented by alpha-tocopherol pretreatment. Microsomal cytochrome P-450 content and aminopyrine-N-demethylase activity were both decreased in vehicle-treated ischemic rats to 60% and 70% of sham-ischemic control levels, respectively. Although alpha-tocopherol restored cytochrome P-450 content to the level of sham-ischemic control rats, aminopyrine-N-demethylase activity remained at 76% of control with alpha-tocopherol treatment (p less than 0.01 compared with sham-ischemic control). In contrast to what was seen with cytochrome P-450 and aminopyrine-N-demethylase, aniline p-hydroxylase activity was elevated in the vehicle-treated ischemic rats compared with sham-ischemic control rats. These increases were prevented by alpha-tocopherol pretreatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine Transaminase↗

PubMed1992

Intestinal villus microcirculatory response to hemorrhage in adult and immature rats.

The response of the villus microcirculation to moderate hemorrhage was compared in adult and immature rats. As an estimate of villus blood flow, flux of red blood cells through the terminal arteriole loop at the villus tip was quantified in vivo using fluorescence epilumination videomicroscopy. Baseline red blood cell flux and mean arterial pressure (MAP) of immature rats (973 cells/s, 84 mm Hg) were significantly less than that of adults (1,435 cells/s, 131 mm Hg). Following hemorrhage of 30% of estimated blood volume, MAP of immature rats was still 24% less than that of adults; however, villus flux was not significantly different (578 cells/s immature, 640 cells/s adult; P = .63). Thus, as indicated by changes in villus flux, immature rats do not appear to be more susceptible to acute intestinal blood flow reduction induced by a single moderate hemorrhage. Immaturity of villus microvascular anatomy and of intestinal vasoregulatory mechanisms may account for the differential response.

Age Factors↗

PubMed1992

Subacinar distribution of hepatocyte membrane potential response to stimulation of gluconeogenesis.

Isolated rat livers were perfused and the membrane potentials of matched periportal and pericentral hepatocytes were determined using glass microelectrodes. O2 uptake and gluconeogenesis were increased by both phenylephrine and glucagon and the extent of the increase was not affected by the direction of perfusion. With no exogenous substrate, hepatocyte membrane potentials were approximately -27 mV. No gradients were found. Substrate produced hyperpolarization in all hepatocytes, with a small but significant gradient produced. Phenylephrine-induced hyperpolarization was higher in periportal than in pericentral hepatocytes during anterograde perfusion, but reversed during retrograde perfusion. Similar effects on membrane potential were produced by phorbol myristate acetate (PMA). Glucagon hyperpolarized homogeneously during both anterograde and retrograde perfusion with no gradients across the acinus. Octanol addition during glucagon stimulation, however, resulted in heterogeneity similar to phenylephrine or PMA. Thus when hepatocytes are stimulated by substrate or hormones, the degree of hepatocyte membrane potential heterogeneity across the acinus is highly dependent on the nature of the stimulus. We propose that the differential hormone effects on hepatocyte membrane potential may be mediated at least in part by differential modulation of cell to cell communication via gap junctions.

Animals↗

PubMed1992

Kupffer cell exacerbation of hepatocyte hypoxia/reoxygenation injury.

In order to examine the possible contribution of the Kupffer cell to the generation of hypoxia/reoxygenation injury in the liver, primary cultures of hepatocytes, either alone or in coculture with Kupffer cells, were exposed to 90 min of sublethal hypoxia followed by 120 min of reoxygenation. Prolonged incubation of cocultured hepatocytes and Kupffer cells resulted in increased release of lactic dehydrogenase (LDH) indicating cell injury even under normoxic conditions. LDH release was further increased by the presence of Kupffer cells during hypoxia/reoxygenation. To determine whether or not this effect of Kupffer cells might be the result of oxygen-derived free radical production, we assessed the efficacy of the enzymatic scavengers superoxide dismutase (SOD) + catalase in ameliorating the Kupffer cell mediated injury. SOD + catalase was effective in preventing free radical injury generated by hypoxanthine + xanthine oxidase. However, SOD + catalase did not ameliorate hepatocyte injury caused by Kupffer cells. Thus, activation of Kupffer cells may be an important factor in the genesis of liver injury, but the mediator of Kupffer cell exacerbation of hepatocyte injury appears to be a mechanism other than free radicals released into the medium. These results indicate that chemical substances from the activated Kupffer cells may cause hepatocyte damage, which cannot be blocked by SOD + catalase, and suggest that these substances at reflow may be important for the genesis of reperfusion injury in vivo.

Animals↗

PubMed1991

Interrelationship between hepatic ureagenesis and gluconeogenesis in early sepsis.

This study was performed to investigate the interrelationship between gluconeogenesis and ureagenesis during sepsis. In isolated perfused livers, gluconeogenesis was assessed using either lactate or a combination of lactate, glutamine, and alanine as substrate. Ureagenesis was assessed using either NH4Cl or glutamine plus alanine as substrate. NH4Cl stimulated urea production in livers from both septic and sham-operated control rats. Urea release was approximately 1.2 and 2.0 mg urea nitrogen.g-1.h-1 for 1 and 5 mM NH4Cl, respectively, and was equal for both groups. With amino acids as substrate, urea production was significantly greater in livers from septic animals compared with controls. Phenylephrine stimulated urea production in the sham-operated group by about twofold, whereas in the septic group urea release was slightly inhibited. Gluconeogenesis from lactate was inhibited by NH4Cl (1 and 5 mM) in both groups, with no difference between groups. In contrast to enhanced ureagenesis from amino acids in septic rats, gluconeogenesis was decreased by approximately 24% (P less than 0.5). Similarly, phenylephrine (1 microM) stimulated gluconeogenesis by 13 +/- 1 mumol.g-1.h-1 in sham-operated rats but only by 9 +/- 1 mumol.g-1.h-1 in septic rats (P less than 0.02). These results suggest that hepatic gluconeogenic and ureagenic pathways are intact in sepsis but that altered substrate preference and hormone sensitivity may result in decreased gluconeogenesis in the presence of elevated amino acid levels.

Alanine↗

PubMed1991

Conjugated desferoxamine attenuates hepatic microvascular injury following ischemia/reperfusion.

Iron-dependent oxy radicals have been implicated in reperfusion injury. Although the iron chelator desferoxamine (DFO) is beneficial, its hemodynamic effects and short vascular retention limit its use in vivo. We tested whether DFO conjugated to a high-molecular-weight starch might ameliorate in vivo hepatic microvascular injury without adverse side effects following 120 min of ischemia. Prior to reperfusion, conjugated DFO (100 mg/kg), vehicle (Veh), or saline (I/R) was administered. After 90 min of reperfusion, blood was collected for serum transaminase determination (ALT; U/liter), and fluorescein-albumin was injected to label perfused microvessels, which were quantified in frozen sections by a point-count technique. Tissue edema was estimated by wet to dry weight ratios (W/D). Reperfusion results in hepatocyte injury (rise in ALT and W/D) and a 30% loss of perfused microvessels. Intravenous administration of conjugated DFO produces no significant change in systemic hemodynamics, whereas both ALT and tissue edema were decreased by approximately 50%. Moreover, perfused microvessels were restored virtually to nonischemic control levels. Enhanced perfusion and attenuated cell injury with DFO suggest that microvascular failure and resultant cell death are mediated, at least in part, by iron-dependent mechanisms in reperfusion.

Animals↗

PubMed1990

Susceptibility of hepatic microcirculation to reperfusion injury: a comparison of adult and suckling rats.

Primary graft failure and vascular thromboses are frequent complications of liver transplantation, yet the mechanisms responsible remain unclear. Previous work from our laboratory has shown that hepatic reperfusion injury results in damage at the microvessel level. The present study was performed to determine whether an increased susceptibility of immature animals to microvascular injury during reperfusion might be a contributing factor in these complications. Suckling (35 to 50 g) or adult (250 to 400 g) rats were subjected to 30 or 60 minutes of hepatic ischemia to the left and median lobes followed by 90 minutes of reperfusion. Control animals were sham-operated, time-matched rats. At the end of reperfusion, fluorescein-labeled albumin was injected systemically to mark perfused sinusoids. Frozen sections of liver biopsies were viewed under fluorescence microscopy. The perfused sinusoid density was determined by point count analysis and expressed as the number of intersections of perfused sinusoids with 25 randomly oriented points superimposed on the sinusoid field. In sham-operated rats, at both 30 and 60 minutes, there were no differences between sucklings and adults. After 30 minutes of ischemia and 90 minutes of reperfusion, adults showed a significantly decreased density of perfused sinusoids (4.5 +/- 0.1 intersections per field) when compared with suckling rats (6.0 +/- 0.3 intersections per field, P less than .001). However, in rats subjected to 60 minutes of ischemia followed by 90 minutes of reperfusion, the microvascular injury was more severe in suckling rats (2.7 +/- 0.2 intersections per field) than in adults (4.7 +/- 0.2 intersections per field, P less than .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗