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Hepatic fibrosis is characterized by excess type I collagen deposition and exacerbated inflammatory response. Naltrexone, an opioid receptor antagonist used for treating alcohol abuse, attenuates hepatocellular injury in fibrotic animal models, which can be accompanied by deleterious side effects. Additionally, opioid neurotransmission is upregulated in patients with inflammatory liver disease. Several derivatives of Naltrexone, Nalmefene (Nal) and JKB-119, exert immunomodulatory activity; however, unlike Nal, JKB-119 does not show significant opioid receptor antagonism. To delineate the potential hepatoprotective effects of these compounds, we investigated if JKB-119 and Nal could modulate activation of hepatic stellate cells (HSCs), primary effector cells that secrete type I collagen and inflammatory mediators during liver injury. Our results demonstrated that Nal or JKB-119 treatment decreased smooth muscle α-actin, a marker of HSC activation, mRNA and protein expression. Despite decreased collagen mRNA expression, both compounds increased intracellular collagen protein expression; however, inhibition of collagen secretion was observed. To address a possible mechanism for suppressed collagen secretion or retention of intracellular collagen, endoplasmic (ER) protein expression and matrix metalloproteinase (MMP) activity were examined. While no change in ER protein expression (Grp78, PDI, Hsp47) was observed, MMP13 mRNA expression was dramatically increased. In an acute LPS inflammatory injury animal model, JKB-119 treatment decreased liver injury (ALT), plasma TNFα and PMN liver infiltration. Overall, these results suggest that JKB-119 can directly inhibit HSC activation attributed to anti-inflammatory activity and may, therefore, attenuate inflammation associated with HSC activation and liver disease., (Copyright © 2011 Elsevier Inc. All rights reserved.)

IL-27 is a heterodimeric cytokine composed of p28 and Epstein Barr virus induced gene 3 (Ebi3) protein subunits. In the present study, we questioned whether murine gammaherpesvirus 68 (HV-68) could induce expression of Ebi3, p28, and IL-27 in this mouse model of an EBV-like infection. Cultured macrophages and dendritic cells exposed to HV-68 upregulated p28 mRNA expression and increased secretion of the p28 and IL-27 (p28+Ebi3) proteins. B220(+) and CD11b(+) cells also upregulated p28 mRNA expression following in vivo infection with this virus. Surprisingly, no significant increases in p28 or IL-27 protein production were observed in vivo during the acute or mononucleosis phases of the disease. The possibility that HV-68-induced upregulation of p28 mRNA expression primed cells for IL-27 secretion was suggested by the ability of a TLR4 agonist to augment cytokine production. When cultured macrophages and dendritic cells were exposed to virus plus a suboptimal dose of LPS, increased levels of p28 protein expression were observed. More importantly, when latently infected mice were challenged with a sublethal dose of LPS, augmented p28 and IL-27 protein production occurred. Using a model of sepsis, mice latently infected with HV-68 had exaggerated p28 protein production when compared to mice that were singularly infected or subjected to cecal ligation and puncture. Taken together, these studies define expression of HV-68 induced IL-27, and suggest that mice latently infected with this gammaherpesvirus will have exaggerated responses when confronted with other stimuli capable of inducing this member of the IL-12 family of cytokines., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Dysfunction of hepatic microcirculation during inflammatory stress conditions is associated with overexpression of caveolin 1 (Cav-1) in sinusoidal endothelial cells. Because Cav-1 binds and inhibits eNOS, it was suggested that Cav-1 overexpression inhibits endothelin 1 (ET-1)-mediated eNOS activation after endotoxemia in the liver; however, a causal link between stress-mediated suppression of eNOS and Cav-1 overexpression has not been fully established. We hypothesize that genetic knockout of Cav-1 reverses the LPS-suppressed ET-1-mediated eNOS activation. In this report, liver sinusoidal endothelial cells (LSECs) from wild-type (WT) and Cav-1 knockout (KO) mice were isolated, pretreated with 100 ng/mL LPS for 6 h, and treated with 10 nmol ET-1 for 30 min. Data showed that LPS increased Cav-1 protein expression (+88%; P < 0.05) and inhibited ET-1-mediated eNOS activation and NO production in WT LSECs. Genetic deletion of Cav-1 increased basal eNOS activity (0.40 in KO vs. 0.15 fmol/min per well in WT; +262%; P < 0.05) and reversed LPS inhibition of ET-1-stimulated eNOS activity (+25.7%; P < 0.05) by increasing eNOS-Ser1177 (+40.3%; P < 0.05) and decreasing eNOS-threonine-495 (-8.8%; P < 0.05) phosphorylation. The reversal of LPS inhibition resulted in an increase in ET-1-induced eNOS translocation to the plasma membrane and an augmentation of NO production in the perinuclear region and plasma membrane of Cav-1 KO LSECs. These results showed that genetic knockout of Cav-1 increased basal eNOS activity and at least partially restored ET-1-mediated eNOS translocation and NO production in LSECs after LPS treatment. In conclusion, Cav-1 overexpression is a requirement for decreased eNOS activity in LSECs after endotoxemia.

Background: Sepsis remains a serious complication after trauma. Although hepatic microvascular dysfunction occurs during trauma and sepsis, the mechanism responsible is unclear. Since Kupffer cells can provide the signals that regulate the hepatic response in inflammation and contribute to multiple organ failure, this study investigated the role of Kupffer cells in the imbalance of the expression of vasoactive and inflammatory mediators during trauma and sepsis., Materials and Methods: The Kupffer cells were inactivated by gadolinium chloride (GdCl(3), 7.5 mg/kg body weight, i.v.) 1 and 2 d before surgery. The animals then underwent femur fracture (FFx) followed 48 h later by cecal ligation and puncture (CLP). After 24 h, blood was obtained to determine the alanine aminotransferase (ALT) activity. Liver samples were also taken for RT-PCR analysis of the mRNA for genes of interest., Results: Serum ALT levels increased in FFx and CLP. This increase was potentiated by FFx + CLP and was attenuated by GdCl(3). The mRNA expression levels in the FFx showed no change in the ET(A), ET(B), iNOS, and HO-1, and showed a slight increase of 2.6-fold, 2.2-fold, and 2.8-fold for ET-1, eNOS, and TNF-alpha, respectively. The ET-1 mRNA expression level increased after CLP and FFx + CLP. The ET(A) mRNA level showed no change, whereas the ET(B) transcripts increased after CLP. This increase was potentiated after FFx + CLP, and was attenuated by GdCl(3). After CLP alone, iNOS, eNOS, and TNF-alpha mRNA expression levels were increased 20.3-fold, 5.8-fold, and 11.9-fold, respectively. This increase was potentiated after FFx + CLP, and was attenuated by GdCl(3). HO-1 mRNA expression significantly increased after FFx + CLP, and this increase was attenuated by GdCl(3)., Conclusions: Mild trauma alone causes little change in the expression of vasoactive mediators while sequential injury potentiates the imbalanced gene expression induced by sepsis. Kupffer cells might be essential for hepatic microvascular dysfunction after sequential stress.

Endothelin-1 (ET-1) plays a key role in the regulation of endothelial nitric oxide synthase (eNOS) activation in liver sinusoidal endothelial cells (LSECs). In the presence of endotoxin, an increase in caveolin-1 (Cav-1) expression impairs ET-1/eNOS signaling; however, the molecular mechanism is unknown. The objective of this study was to investigate the molecular mechanism of Cav-1 in the regulation of LPS suppression of ET-1-mediated eNOS activation in LSECs by examining the effect of caveolae disruption using methyl-beta-cyclodextrin (CD) and filipin. Treatment with 5 mM CD for 30 min increased eNOS activity (+255%, P < 0.05). A dose (0.25 microg/ml) of filipin for 30 min produced a similar effect (+111%, P < 0.05). CD induced the perinuclear localization of Cav-1 and eNOS and stimulated NO production in the same region. Readdition of 0.5 mM cholesterol to saturate CD reversed these effects. Both the combined treatment with CD and ET-1 (CD + ET-1) and with filipin and ET-1 stimulated eNOS activity; however, pretreatment with endotoxin (LPS) abrogated these effects. Following LPS pretreatment, CD + ET-1 failed to stimulate eNOS activity (+51%, P > 0.05), which contributed to the reduced levels of eNOS-Ser1177 phosphorylation and eNOS-Thr495 dephosphorylation, the LPS/CD-induced overexpression and translocation of Cav-1 in the perinuclear region, and the increased perinuclear colocalization of eNOS with Cav-1. These results supported the hypothesis that Cav-1 mediates the action of endotoxin in suppressing ET-1-mediated eNOS activation and demonstrated that the manipulation of caveolae produces significant effects on ET-1-mediated eNOS activity in LSECs.

Background: Recurrent ankle injury occurs in 70% of individuals experiencing a lateral ankle sprain. The cause of this high level of recurrence is currently unknown. Researchers have begun to investigate sensorimotor deficits as one possible cause with inconclusive and often conflicting results. The purpose of this study was to further the understanding of the role of sensorimotor deficits in the chronically unstable ankle by establishing which specific measures best distinguish between chronically unstable and healthy ankles., Methods: Twenty-two participants with chronic ankle instability and 21 healthy matched controls volunteered. Twenty-five variables were measured within four sensorimotor constructs: joint kinesthesia (isokinetic dynamometer), static balance (force plate), dynamic balance (Star Excursion Balance Test) and motoneuron pool excitability (electromyography)., Findings: The above variables were evaluated using a discriminant function analysis [Wilks'Lambda=0.536 chi(2)(7, N=43)=22.118, P=0.002; canonical correlation=0.681]. The variables found to be significant were then used to assess group discrimination. This study revealed that seven separate variables from the static balance (anterior/posterior and medial/lateral displacement and velocity) and motoneuron pool excitability constructs (single-legged recurrent inhibition and single- and double-legged paired reflex depression) accurately classified over 86% of participants with unstable ankles., Interpretation: These results suggest that a multivariate approach may be necessary to understand the role of sensorimotor function in chronic ankle instability, and to the development of appropriate rehabilitation and prevention programs. Out of the four overall constructs, only two were needed to accurately classify the participants into two groups. This indicates that static balance and motoneuron pool excitability may be more clinically important in treatment and rehabilitation of chronic ankle instability than functional balance or joint kinesthesia.

Objective: To further understanding of the role that segmental spinal reflexes play in chronic ankle instability (CAI)., Design: A 2 x 2 repeated-measures case-control factorial design. The independent variables were ankle group with 2 levels (healthy, CAI) and stance with 2 levels (single, double legged)., Setting: University research laboratory., Participants: Twenty-two participants with CAI and 21 matched healthy controls volunteered., Interventions: Not applicable., Main Outcome Measures: The dependent variables were 2 measures of motoneuron pool excitability: paired reflex depression (PRD) and recurrent inhibition., Results: A 2 x 2 repeated-measures multivariate analysis of variance revealed a significant interaction between group and stance on the linear combination of PRD and recurrent inhibition variables (Wilks lambda=.808, F(2,40)=4.77, P=.014). Follow-up univariate F tests revealed an interaction between group and stance on the PRD (F(1,41)=9.74, P=.003). Follow-up dependent t tests revealed a significant difference between single- and double-legged PRD in the healthy participants (t(20)=-3.76, P=.001) with no difference in CAI participants (t(21)=-0.44, P=.67). Finally, there was a significant difference in recurrent inhibition between healthy (mean, 83.66) and CAI (mean, 90.27) (P=.004)., Conclusions: This study revealed that, compared with healthy participants, CAI participants were less able to modulate PRD when going from a double- to a single-legged stance. Additionally, CAI participants showed higher overall levels of recurrent inhibition when compared with healthy matched controls.

Previous studies report S-adenosyl-L-methionine (SAMe) can exert hepatoprotective effects. At present, the role of SAMe in affecting the activation and/or proliferation of hepatic stellate cells (HSCs) during alcohol-induced fibrotic disease progression is poorly understood. In the human disease state, chronic ethanol intake increases hepatic exposure to LPS and magnifies the hepatic insult leading to fibrosis and cirrhosis. In this study, we developed a "2-hit" ethanol-LPS fibrotic liver rat model with which to investigate the effects of SAMe as a hepatic antifibrotic treatment. Male rats were maintained on liquid diets containing either ethanol or isocalorically matched controls for 8 weeks. Animals received ethanol alone (E), ethanol concomitant with twice weekly LPS injections (EL), or ethanol, LPS, and daily SAMe injections. When using this model, SAMe-treated animals demonstrated significantly decreased fibrosis, oxidative stress, steatosis, and improved liver function versus the EL group. In addition, the EL group showed increased HSC activation, an effect that was abrogated by the addition of SAMe. Analysis of the transforming growth factor-beta (TGF-beta) signaling pathways demonstrated increased hepatic TGF-beta and Smad3 messenger RNA expression in the E and EL groups, which was inhibited in the presence of SAMe. Conversely, SAMe led to increased Smad7 (an inhibitor of TGF-beta signaling) messenger RNA expression. These data demonstrate chronic ethanol feeding combined with LPS induces liver fibrosis, and the addition of SAMe significantly reduces hepatic injury and fibrosis through inhibition of oxidative stress and HSC activation.

Background and Aim: We previously reported that hypothermic machine perfusion (HMP) for liver preservation is feasible, but hepatic microcirculatory dysfunction and significant liver damage remain major obstacles in its application when the preservation is extended to 24 h. The underlying injury mechanism is not well understood. The present study sought to investigate the role of thromboxane A(2) (TXA(2)) in the pathogenesis of liver injury after prolonged HMP., Methods: Livers isolated from Sprague-Dawley rats were subjected to continuous machine perfusion with University of Wisconsin (UW) solution at a flow rate of 0.4 mL/min/g liver at 4 degrees C for 24 h. A specific TXA(2) synthase inhibitor, OKY-046 (OKY), was added to UW solution during the preservation period and to the Krebs-Henseleit buffer during reperfusion. The performance of the livers after preservation was evaluated using an isolated liver perfusion system with Krebs-Henseleit buffer at a flow rate of 15 mL/min at 37 degrees C for 30 min., Results: Prolonged HMP induced a significant release of TXA(2) into the portal circulation as indicated by markedly increased levels of TXB(2) in the perfusate during reperfusion (at 30 min, 1447.4 +/- 163.6 pg/mL vs 50.91 +/- 6.7 pg/mL for control). Inhibition of TXA(2) synthesis with OKY significantly decreased releases of TXA(2) (69.8 +/- 13.4 pg/mL) concomitant with reduced lactate dehydrogenase (LDH) releases (at 30 min, HMP + OKY: 144.9 +/- 27.9 U/L; HMP: 369.3 +/- 68.5 U/L; simple cold storage or SCS: 884.4 +/- 80.3 U/L), decreased liver wet/dry weight ratio (HMP + OKY vs SCS and HMP: 3.6 +/- 0.3 vs 4.4 +/- 0.1 and 3.9 +/- 0.2, respectively) and increased hyaluronic acid uptake (at 30 min, HMP + OKY vs SCS, HMP: 33.1 +/- 2.9% vs 13.9 +/- 3.6%, 18.6 +/- 2.4%, respectively). Liver histology also showed significant improvement in tissue edema and hepatocellular necrosis with OKY compared with HMP without OKY., Conclusion: The results demonstrate that TXA(2) is involved in the development of hepatocellular injury induced by HMP, and inhibition of TXA(2) synthesis during preservation and reperfusion protects liver hepatocytes and sinusoidal endothelial cells from injuries caused by prolonged HMP.

Endotoxemia produces hepatic vascular dysregulation resulting from inhibition of endothelin (ET)-stimulated NO production. Mechanisms include overexpression of caveolin-1 (Cav-1) and altered phosphorylation of endothelial nitric oxide (NO) synthase (NOS; eNOS) in sinusoidal endothelial cells. Since ischemia-reperfusion (I/R) also causes vascular dysregulation, we tested whether the mechanisms are the same. Rats were exposed to either mild (30 min) or moderate (60 min) hepatic ischemia in vivo followed by reperfusion (6 h). Livers were harvested and prepared into precision-cut liver slices for in vitro analysis of NOS activity and regulation. Both I/R injuries significantly abrogated both the ET-1 (1 microM) and the ET(B) receptor agonist (IRL-1620, 0.5 microM)-mediated stimulation of NOS activity. 30 min I/R resulted in overexpression of Cav-1 and loss of ET-stimulated phosphorylation of Ser1177 on eNOS, consistent with an inflammatory response. Sixty-minute I/R also resulted in loss of ET-stimulated Ser1177 phosphorylation, but Cav-1 expression was not altered. Moreover, expression of ET(B) receptors was significantly decreased. This suggests that the failure of ET to activate eNOS following 60-min I/R is associated with decreased protein expression consistent with ischemic injury. Thus hepatic vascular dysregulation following I/R is mediated by inflammatory mechanisms with mild I/R whereas ischemic mechanisms dominate following more severe I/R stress.

For in vitro liver replacement devices, such as packed bed bioreactors, to maintain the essential functions of the liver, they must at least successfully support hepatocytes, the parenchymal cell of the liver. In vivo, the liver is a major consumer of oxygen. Hence it is unsurprising that the limited transport distance of oxygen (O(2)) governs the dimensions of the cellular space of engineered devices. Because cellular space capacity directly affects the device's performance, O(2) transport is a critical issue in the scale up of bioreactor designs. In the current investigation, the microporosity of the extracellular matrix (ECM) has been modified to further improve O(2) transport in packed bed devices beyond that previously reported in the literature. These improvements to the O(2) enhancement technique enabled O(2) transport distances of 481.7 +/- 12.5 microm to be achieved under acellular conditions; and distances of 418.1 +/- 6.0 microm to be attained in the presence of 1 million hepatocytes. Both values are significantly greater than the 170 microm baseline attained when 10(6) hepatocytes are packed within normal non-enhanced ECM gels. The study's results also illustrate that the O(2) enhancement technique has the added benefit of preventing regions of severe hypoxia and hyperoxia from developing within the cellular space. As such, enhanced ECM gels enable packed hepatocytes to maintain better hepatocellular metabolic status than is possible with normal non-enhanced gels., (Copyright 2007 Wiley Periodicals, Inc.)

Hypothermic machine perfusion (HMP) has the potential to improve recovery and preservation of Donation after Cardiac Death (DCD) livers, including uncontrolled DCD livers. However, current perfusion solutions lack the needed substrates to improve energy recovery and minimize hepatic injury, if warm ischemic time (WIT) is extended. This proof-of-concept study tested the hypothesis that the University of Wisconsin (UW) solution supplemented with anaplerotic substrates, calcium chloride, thromboxane A2 inhibitor, and antioxidants could improve HMP preservation and minimize reperfusion injury of warm ischemic livers. Preflushed rat livers subjected to 60 min WIT were preserved for 5 h with standard UW or supplemented UW (SUW) solution. Post preservation hepatic functions and viability were assessed during isolated perfusion with Krebs-Henseleit solution. Livers preserved with SUW showed significantly (p < .001) improved recovery of tissue ATP levels (micromol/g liver), 2.06 +/- 0.10 (mean +/- SE), as compared to the UW group, 0.70 +/- 0.10, and the level was 80% of that of fresh control livers (2.60 +/- 0.13). At the end of 1 h of rewarming, lactate dehydrogenase (U/L) in the perfusate was significantly (p < .05) lower in the SUW group (429 +/- 58) as compared to ischemia-reperfusion (IR) (781 +/- 12) and the UW group (1151 +/- 83). Bile production (microg/min/g liver) was significantly (p < .05) higher in the SUW group (280 +/- 13) as compared to the IR (224 +/- 24) and the UW group (114 +/- 14). The tissue edema formation assessed by tissue wet-dry ratio was significantly (p < .05) higher in UW group. Histology showed well-preserved hepatic structure in the SUW group. In conclusion, this study suggests that HMP with SUW solution has the potential to restore and preserve livers with extended WIT.

Intravital microscopy has been used to visualize the microcirculation by imaging fluorescent labeled red blood cells (RBCs). Traditionally, microcirculation has been modeled by computing the mean velocity of a few, randomly selected, manually tracked RBCs. However, this protocol is tedious, time consuming, and subjective with technician related bias. We present a new method for analyzing the microcirculation by modeling the RBC motion through automatic tracking. The tracking of RBCs is challenging as in each image, as many as 200 cells move through a complex network of vessels at a wide range of speeds while deforming in shape. To reliably detect RBCs traveling at a wide range of speeds, a window of temporal template matching is applied. Then, cells appearing in successive frames are corresponded based on the motion behavior constraints in terms of the direction, magnitude, and path. The performance evaluation against a ground truth indicates the detection accuracy up to 84% TP at 6% FP and a correspondence accuracy of 89%. We include an in-depth discussion on comparison of the microcirculation based on motion modeling from the proposed automated method against a mean velocity from manual analysis protocol in terms of precision, objectivity, and sensitivity.

Improvement of O(2) supply in bioartificial liver devices remains a critical issue in maintaining hepatocyte viability and functions. Therefore, the current study investigates whether enhanced oxygen (O(2)) transport through collagen extracellular matrix (ECM) can produce a more stable antioxidant defense in different O(2) tensions during prolonged incubation times. Total glutathione concentration of cultured hepatocytes in enhanced ECM was significantly higher than in normal ECM under the lowest O(2) tension phase (2.60mm of thickness from O(2) source), and was also significantly increased in 0.52 mm transport distance of hypoxia as compared to normoxic conditions. Catalase and glutathione reductase activities for hepatocytes within enhanced ECM were also significantly preserved relative to their values for the normal collagen ECM. Specifically, the enhanced ECM produced higher activities at a further transport distance (1.56 mm) from the O(2) source at the 24 h time-point, and remained higher up to the 96 h incubation time. In contrast, the glutathione peroxidase activities in both collagen ECM systems were similar. Hepatocyte viability in the enhanced ECM system was also consistently greater than that for normal ECM. These results suggest that the O(2) enhanced collagen ECM preserves the antioxidant defense system as compared to normal collagen ECM, ostensibly via increased micropathways for O(2) transport to the hepatocytes.

Background and Aim: Partial portal vein ligation (PPVL) is a commonly used procedure to induce prehepatic portal hypertension in animal models. The aim of this study was to test the hypothesis that the hepatic arterial flow becomes the primary source feeding the sinusoids in the liver after PPVL., Methods: Sprague-Dawley rats underwent either sham operation or partial portal vein ligation (PPVL). The number of vessels in the liver at 2 weeks postoperatively was determined by factor VIII immunolocalization and the gene expression of angiogenic factors was assessed by RT-PCR. The total hepatic arterial supply to the liver was measured using the fluorescent microsphere injection technique. To further test the hypothesis, two additional groups of rats underwent hepatic artery ligation (HAL) or PPVL plus HAL (PPHAL). The integrity of hepatic microcirculation was then evaluated in all four groups by intravital microscopy., Results: At 2 weeks after operation, the number of vessels detected by factor VIII staining was significantly higher in PPVL compared to sham. Densitometric analysis of RT-PCR bands revealed a significant increase of vascular endothelial growth factor gene expression in PPVL compared to sham. Arterial flow to the liver measured by fluorescent microspheres was increased by 190% in PPVL compared to sham. When all four groups were compared, no prominent histological abnormality was observed in sham, HAL, and PPVL groups; however, PPHAL livers showed focal necrosis and inflammatory cell infiltration around the portal triads. Additionally, only the PPHAL livers showed a decreased sinusoidal diameter and significantly lower perfusion index (PPHAL 42.9+/-6.1; sham 85.7+/-7.0, PPVL 80.2+/-6.5, HAL 70.9+/-4.5)., Conclusions: These results suggest that the hepatic artery flow becomes the primary source for the blood supply of sinusoids and the compensatory change in the hepatic arterial system plays a critical role in maintaining microcirculatory perfusion following the restriction of the portal vein flow by PPVL.

Objective: The vasoconstrictor endothelin-1 can induce vasomodulators release like nitric oxide in the liver. Here the authors explored whether endothelin-1 can stimulate endothelial and Kupffer cells release of other vasomodulators under normal and stress conditions., Methods: Cells were cultured for 24 h and treated with H2O2 (25 microM) for 6 h and subsequently with endothelin-1 (10 nM) for 10 min. Eicosanoid release was assessed in the media by enzyme immunoassay., Results: Endothelin-1 mediated cPLA2 phosphorylation and increased prostaglandin (PG) I2, PGE2 and thromboxane A2 (TXA2) release in endothelial cells while it only increased TXA2 in Kupffer cells. H2O2 significantly increased PGI2, PGE2 and TXA2 in endothelial cells through an upregulation of cyclooxygenase-2, thromboxane synthase A2, and phosphorylation of cPLA2. Endothelin-1-induced PGI2, PGE2, and TXA2 release in endothelial cells were inhibited by H2O2 correlating with the absence of further cPLA2 phosphorylation. In Kupffer cells, H2O2 only increased TXA2 synthesis and further endothelin-1 stimulation of TXA2 was possible through a higher increase in cPLA2., Conclusion: These results indicate that under normal conditions endothelial cells play a pivotal role in liver microcirculation regulation. Oxidative stress not only disrupts the basal balance of vasomodulators in the liver but also affects endothelin-1-induced effects in both Kupffer cells and endothelial cells.

Endothelin-1 (ET-1) has been shown to regulate the expression of various genes in addition to its vasoconstrictor role in the liver. Elevated levels of ET-1 during cirrhosis play an important role in the observed microcirculatory dysfunction; however, its role as a transcription regulator remains unclear. This study aimed to determine the role of ET-1 in the hepatic gene expression of vasomediators after cirrhosis in response to LPS. Cirrhosis was induced by bile duct ligation (BDL) for 1 or 3 weeks in male Sprague-Dawley rats. Following 1 or 3 weeks of BDL or sham operation (sham), rats received an intravenous (i.v.) injection of bosentan, a dual-selective ETA/B receptor antagonist (30 mg/kg bw) or saline, and an intraperitoneal (i.p.) injection of LPS (1 mg/kg bw). Plasma alanine aminotransferase (ALT) levels were significantly elevated in 1- and 3-week BDL animals. Six hours following LPS, the elevated ALT levels were markedly exacerbated in 3-week BDL animals, which were significantly ameliorated with bosentan treatment. LPS resulted in increased ET-1, inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX)-2 mRNA expressions in both sham and BDL rats. Bosentan significantly inhibited the up-regulations of ET-1, iNOS, and COX-2 mRNA. Our data strongly suggest that ET-1 plays an important role in up-regulating the expression of iNOS, COX-2, and ET-1 itself in hepatic tissue following LPS challenge, which may contribute to the observed hepatocellular injury during endotoxemia in cirrhosis. Thus, due to significant increases in ET-1 levels during cirrhosis, ET-1 receptor blockade may prove to be of great therapeutic value in the treatment of cirrhotic patients exposed to secondary injuries such as endotoxemia.

During endotoxemia, liver microcirculation disruption is characterized by a hypersensitivity to the constrictor effects of endothelin 1 (ET-1). The shift of ET-1-mediated effects toward vasoconstriction may result from depressed ET-1-mediated vasodilation through decreased ET-1-induced nitric oxide (NO) production. We have previously shown that lipopolysaccharide (LPS) pretreatment abrogates ET-1-induced endothelial nitric oxide synthase (eNOS) translocation, but its effects on eNOS activation are yet to be determined. Our aim was to assess the effects of LPS on ET-1-mediated eNOS activation in hepatic sinusoidal endothelial cells (SECs) and to investigate the molecular mechanisms involved. SECs were treated with LPS (100 ng/mL) for 6 hours followed by 30 minutes ET-1 (10 nmol/L) stimulation. LPS significantly inhibited ET-1-mediated eNOS activation. This inhibition was associated with upregulation of Caveolin-1 (CAV-1) and a shift in ET-1-mediated eNOS phosphorylation from an activation (Ser1177) to an inhibition (Thr495). LPS treatment has been shown to induce ET-1 expression and secretion from endothelial cells. We therefore investigated the role of endogenous ET-1 in the inhibition of ET-1 activation of eNOS after LPS. Antagonizing ET-1 effects and blocking its activation in LPS pretreated SECs decreased the LPS-induced overexpression of CAV-1 as well as the inhibition of ET-1-induced NOS activity. Furthermore, 6 hours of ET-1 treatment exerted the same effects on eNOS activity, phosphorylation, and CAV-1 expression as LPS treatment. In conclusion, LPS-induced suppression of ET-1-mediated eNOS activation is ET-1 dependent and suggest a pivotal role of CAV-1 in eNOS induction inhibition under stress.

Angiogenic factors including endothelin-1 (ET-1) play a key role in the progression of breast metastases to bone. We investigated the impact of ET-1 on the development of bone metastases in an immunocompetent murine skin-fold chamber model. Murine mammary carcinoma 4T1 was injected in a skin-fold chamber implanted on CB6 mice along with bone explants. Furthermore, mice were treated with or without a dual selective antagonist of both ET-1 receptors. The progression of the vascularization within the chamber was monitored over time by intravital microscopy (IVM). The tumor growth and the development of bone metastases were assessed by cytokeratin-19 gene expression and histological studies. Results indicate that this new model associated with IVM allows for the continuous monitoring of the change in vascularization associated with the development of bone metastases. Additionally, treatment with an antagonist of both ET-1 receptors was associated with the presence of significantly less vessels near the tumor mass compared to control mice. These changes were correlated with smaller tumor masses and reduced bone invasion (P < 0.05). Thus, in an immunocompetent murine model of breast carcinoma metastases to bone, our data support the hypothesis that vascularization plays a role in tumor development and progression and that ET-1 specifically modulates the angiogenesis associated with breast metastases to the bone.

Oxidative stress may mediate vascular disruption associated with a loss of endothelial nitric oxide synthase (eNOS) activity and a hypersensitivity to the constrictor effects of endothelin-1 (ET-1). We hypothesize that this is due, in part, to uncoupling of ET(B) receptors from eNOS activation. Thus, we tested whether oxidative stress (OS) affects liver vascular relaxation by reducing basal and ET-1-induced NO production. Primary sinusoidal endothelial cell cultures were pretreated with H(2)O(2) (25 microM) for 1 or 6 h before a 10-min ET-1 stimulation. OS resulted in a significant basal and ET-1-induced decrease in NO production. Acute OS increased the monomeric form of the inhibitory protein caveolin-1 (1.2 +/- 0.05 vs 0.9 +/- 0.02, p < 0.01) and increased the eNOS-caveolin association as determined by coimmunoprecipitation (1.24 +/- 0.04 vs 0.97 +/- 0.04, p < 0.05). ET-1 stimulation further exacerbated these effects. Subacute OS inhibited ET-1-induced eNOS phosphorylation of serine 1177 (activation residue) (1 +/- 0.07 vs 1.6 +/- 0.04, p < 0.05) and dephosphorylation of the inhibitory residue threonine 495 (1.5 +/- 0.08 vs 0.7 +/- 0.02, p < 0.01). Additionally subacute OS resulted in dissociation of eNOS from ET(B) (0.8 +/- 0.09 vs 1.2 +/- 0.06, p < 0.05). Our findings indicate that acute and subacute oxidative stress result in the inhibition of induced nitric oxide synthase activity through distinct mechanisms dependent on caveolin-1 inhibition, ET(B) dissociation, and eNOS phosphorylation.

In vivo studies have shown that chronic alcohol consumption sensitizes the liver to endotoxemic shock, leading to liver microcirculation disruption. In the present study, we investigated the molecular mechanisms involved, focusing on endothelial nitric oxide synthase (eNOS) activity and regulation, which represents one of the major vasodilatory pathways. Male Sprague-Dawley rats were fed an alcohol liquid diet or a control isocaloric diet for 5 weeks. Priming effects of ethanol were studied in a model with or without a 24-h LPS treatment (1 mg/kg body weight). At the end of the diet, liver tissue was harvested for western blot, reverse transcriptase-PCR, histological analysis, and immunostaining and blood for serum alanine aminotransferase analysis. Chronic ethanol and LPS alone induced a mild hepatitis and infiltration, respectively. Combined, LPS and chronic ethanol feeding showed a synergistic effect on the liver, leading to extensive steatohepatitis with extensive focal necrosis associated with significantly higher levels of serum ALT. Chronic ethanol and LPS significantly inhibited eNOS activity, but exerted their effects through different mechanisms. Caveolin-1, an eNOS inhibitory protein, was upregulated after LPS and chronic alcohol consumption. Additionally, chronic alcohol consumption down-regulated endothelin B receptor, eNOS protein levels, and eNOS phosphorylation. In conclusion, chronic ethanol consumption and LPS share a similar pathophysiology and both lead to the impairment of eNOS activity, but through distinct molecular mechanisms. The presence of focal necrosis in a mild stress model could provide a good animal study to investigate the advanced stages of alcoholic liver diseases.

Trauma and subsequent sepsis lead to hepatic microcirculation disruption through various molecular mechanisms in which endothelin-1 (ET-1) plays a pivotal role. These stresses are thought to alter hepatic perfusion, heterogeneously leading to a mismatch of oxygen supply and demand. We hypothesize that mild remote stresses prime the liver to sequential sepsis through direct effects on the hepatic lobular flow distribution. We also propose to investigate the extent and the localization of the stress-induced microcirculation disruption. Sprague-Dawley rats were randomly divided into four experimental groups: sham, femur fracture (FFX), cecal ligation and puncture (CLP), and sequential stress (SS). Hepatic intravital microscopy was performed for in vivo assessment of the liver microcirculation flow distribution under baseline and after ET-1 infusion. Red blood cell motion distribution was used to quantify intralobular and interlobular heterogeneity of perfusion (HoP). Intralobular HoP, which reflects lobular regulation sites, was significantly increased in the FFX and CLP groups, but was not changed or decreased in the SS group compared with control. ET-1 infusion exerted opposite effects depending on the pathological condition, further increasing the difference between groups. SS induced decrease in intralobular HoP, contrasted with a significant increase in interlobular HoP, suggesting multiple disruption sites. Our data suggest that increased intralobular HoP may be indicative of a compensatory response to moderate stress; its decrease under sequential stress conditions corresponds with a total breakdown of hepatic lobular flow regulation. This may be another instance of the rich variability characteristic of normal physiology that "decomplexifies" under critical decompensated conditions.

Objective: The objectives of this study were to develop a model for studying endothelin-1-mediated eNOS regulation in cultured sinusoidal endothelial cells and determine the effect of endothelin-1 and endotoxin (LPS) on eNOS localization., Methods: Changes in caveolin-1, calmodulin, and eNOS expression were determined by western blot and densitometric analysis. Endothelin receptor expression and localization and the intracellular localization of eNOS and caveolin-1 were assessed by confocal microscopy., Results: Sinusoidal endothelial cells expressed caveolin-1 and calmodulin, and expression was altered in cultured and passaged cells. eNOS expression decreased significantly in 24-h cultured cells, with expression dropping below the level of detection in passaged cells. Both endothelin A and endothelin B receptors were expressed on the cell surface after 24 h in culture. In 24-h cultured cells, caveolin-1 was localized in the perinuclear region and cell membrane, while eNOS was predominantly localized in the perinuclear region, where it co-localized with caveolin-1. Endothelin-1 stimulated eNOS translocation to the cell membrane. Pretreatment with LPS markedly inhibited the endothelin-1-mediated eNOS translocation., Conclusions: These studies demonstrate an LPS-mediated uncoupling of endothelin receptor activation and eNOS translocation. This functional uncoupling may, in part, account for the hyperconstrictive effects of endothelin-1 during inflammatory conditions.

Stress alters hepatic perfusion heterogeneously leading to mismatch of oxygen supply and demand. Endothelin-1 (ET-1) has been linked to stress-mediated microcirculation disruption; however, perfusion heterogeneity is difficult to quantify. Therefore, we developed an image analysis based automated methodology for quantification of heterogeneity (Ht) of perfusion from hepatic intravital microscopy images (HIVM). In vivo assessment of the liver microcirculation flow distribution, before and after ET-1 infusion, was performed in male Sprague-Dawley rats using HIVM combined with FITC-labeled RBC infusion. Our method aimed to analyze parallel/spatial and series/temporal intralobular Ht of perfusion as well as interlobular Ht of perfusion and was based on automated RBC detection, tracking, and motion analysis. Compared with manual analysis, this method detected 40 times more RBC without increasing false positives (<0.5%). At baseline, parallel/spatial Ht was higher than series/temporal Ht of perfusion which suggested higher hepatic stellate cells asynchrony between sinusoids than within the same sinusoid. Following ET-1 infusion, parallel/spatial Ht transiently increased contrasting with a decrease in series/temporal Ht. In conclusion, we proposed a HIVM analysis methodology that allows assessment of Ht of perfusion within and between hepatic lobules and improved our understanding of ET-1 mediated effects on the hepatic microcirculation.

Background: Macrophages undergo maladaptive alterations after trauma. In this study, we assessed the role of Kupffer cells in hepatic microcirculatory response to endothelin-1 (ET-1) after femur fracture (FFx) and cecal ligation and puncture (CLP)., Methods: Sprague-Dawley rats (200-300 g) underwent sham, FFx, CLP, or FFx + CLP. To ablate Kupffer cells, group 1 animals were treated with gadolinium chloride, and group 2 animals received saline. Hepatic microcirculation was assessed by intravital microscopy. Liver mitochondrial redox state and tissue oxygen (tPo2) were determined by NADH and ruthenium fluorescence, respectively. Liver damage was estimated by alanine aminotransferase levels. Differences were assessed using analysis of variance followed by Student-Newman-Keuls post hoc test., Results: After 10 minutes of ET-1, CLP and FFx + CLP caused significant reduction in hepatic perfusion index (2.5-fold and 5-fold vs. sham, p < 0.05, respectively), redox state (36% and 45% vs. sham, p < 0.01, respectively), tPo2 (10% and 12% vs. sham, p < 0.05, respectively), and more liver damage compared with sham and FFx-treated animals. Kupffer cell depletion restored microcirculation, redox state, and tPo2 and abrogated hepatocellular damage., Conclusion: Kupffer cells contribute directly to hepatic microcirculatory dysfunction and liver injury after inflammatory stress. Furthermore, Kupffer cell depletion ameliorates the microcirculatory perturbations of trauma and sepsis. Thus, modulation of Kupffer cell response may prove beneficial.

Background: Prostaglandins, synthesized by cyclooxygenase (COX), play an important role in the pathophysiology of inflammation. Severe injuries result in immunosuppression, mediated, in part, by maladaptive changes in macrophages. Herein, we assessed Kupffer cell-mediated cyclooxygenase-2 (COX-2) expression on liver function and damage after trauma and sepsis., Materials and Methods: To ablate Kupffer cells, Sprague Dawley rats were treated with gadolinium chloride (GdCl3) 48 and 24 h before experimentation. Animals then underwent femur fracture (FFx) followed 48 h later by cecal ligation and puncture (CLP). Controls received sham operations. After 24 h, liver samples were obtained, and mRNA and protein expression were determined by PCR, Western blot, and immunohistochemistry. Indocyanine-Green (ICG) clearance and plasma alanine aminotransferase (ALT) levels were determined to assess liver function and damage, respectively. One-way analysis of variance (ANOVA) with Student-Newman-Keuls test was used to assess statistical significance., Results: After CLP alone, FFx+CLP, and GdCl3+FFx+CLP, clearance of ICG decreased. Plasma ALT levels increased in parallel with severity of injury. Kupffer cell depletion attenuated the increased ALT levels after FFx+CLP. Femur fracture alone did not alter COX-2 protein compared with sham. By contrast, COX-2 protein increased after CLP and was potentiated by sequential stress. Again, Kupffer cell depletion abrogated the increase in COX-2 after sequential stress. Immunohistochemical data confirmed COX-2 positive cells to be Kupffer cells., Conclusions: In this study, sequential stress increased hepatic COX-2 protein. Depletion of Kupffer cells reduced COX-2 and attenuated hepatocellular injuries. Our data suggest that Kupffer cell-dependent pathways may contribute to the inflammatory response leading to increased mortality after sequential stress.

We examined the role of thromboxane A2 (TXA2) in LPS-induced hyperresponsiveness of hepatic portal circulation to endothelins (ETs) and whether Kupffer cells are the primary source of TXA2 release in response to ET-1 in endotoxemia. After 6 h of LPS (1 mg/kg body wt ip) or saline (control), liver was isolated and perfused with recirculating Krebs-Henseleit bicarbonate buffer at a constant flow rate (100 ml.min(-1).kg body wt(-1)). ET-1 (10 pmol/min) was infused for 10 min. Portal pressure (PP) was continuously monitored during perfusion. Perfusate was sampled for enzyme immunoassay of thromboxane B2 (TXB2; the stable metabolite of TXA2) and lactate dehydrogenase (LDH) assay. ET-1 infusion resulted in a significantly greater increase of PP in the LPS group than in controls. Both TXA2 synthase inhibitor furegrelate (Fureg) and TXA2 receptor antagonist SQ-29548 (SQ) substantially blocked enhanced increase of PP in the LPS group (4.9 +/- 0.4 vs. 3.6 +/- 0.5 vs. 2.6 +/- 0.6 mmHg for LPS alone, LPS + Fureg, and LPS + SQ, respectively; P < 0.05) while having no significant effect on controls. GdCl3 for inhibition of Kupffer cells had similar effects (4.9 +/- 0.4 mmHg vs. 2.9 +/- 0.4 mmHg for LPS alone and GdCl3 + LPS, respectively; P < 0.05). In addition, the attenuated PP after ET-1 was found concomitantly with significantly decreased releases of TXB2 and LDH in LPS rats treated with Fureg, SQ, and GdCl3 (886.6 +/- 73.4 vs. 110.8 +/- 0.8 vs. 114.8 +/- 54.7 vs. 135.2 +/- 45.2 pg/ml, respectively; P < 0.05). After 6 h of LPS, Kupffer cells in isolated cell preparations released a significant amount of TXA2 in response to ET-1. These results clearly indicate that hyperresponsiveness of hepatic portal circulation to ET-1 in endotoxemia is mediated at least in part by TXA2-induced receptor activation, and Kupffer cells are likely the primary source of increased TXA2 release.

This study was conducted to investigate the effect of starch in the preservation solution during hypothermic machine perfusion (HMP) on endothelial cell and hepatocyte functions in an isolated perfused rat liver model. Livers isolated from male Sprague-Dawley rats were perfused with the University of Wisconsin (UW) solution (HMP + starch group); modified UW solution (starch omitted) (HMP - starch group) at 0.4 mL/min per g liver; or simply stored in the UW solution (SCS group) at 4 degrees C for 24 hours. Following preservation, livers from HMP + starch, HMP - starch, SCS, and control group (without preservation) were perfused with Krebs-Henseleit Buffer solution at 37 degrees C for 30 minutes. Samples were taken every 10 minutes during 30-minute warm perfusion to assess hepatocyte and endothelial cell function and damage. After 24 hours of hypothermic preservation and 30 minutes rewarming, livers in the HMP + starch group displayed significantly lower lactate dehydrogenase levels and higher bile production. Endothelial cell function was also improved as indicated by hyaluronic acid uptake and shorter transient time for albumin observed in a multiple indicator dilution study. Liver wet and dry ratio and histological findings confirmed reduced edema formation in the tissue of the HMP + starch group livers compared with that of the HMP - starch and SCS group livers. These results suggest that HMP with the UW solution containing starch improve endothelial cell function and induce less hepatocellular damage following 24-hour preservation compared to SCS and HMP with the starch-free UW solution. These results also suggest that oncotic support may be an important component in preserving hepatic microcirculation in HMP.

Hypothermic machine perfusion (HMP) has a potential to relieve the current donor liver crisis by providing an improved and extended preservation method. This study examined the effect of HMP on hepatocellular functions, using a prototype liver transporter capable of preserving livers for 24 hours. Livers obtained from adult farm pigs (28 to 32 kg body weight) were divided into three groups: fresh control, HMP, and simple cold storage (n = 4 each). A 4-hour normothermic reperfusion of livers was conducted to assess hepato-metabolic and cellular functions. The hepatic transport function, as indicated by canalicular excretion of indocyanine green, was improved in the HMP group than in the SCS group. The overall tissue viability, as indicated by oxygen consumption levels, was notably improved in HMP and control livers as compared to the SCS group. Higher bile production in both the preserved groups as compared to the fresh control livers could be a result of biliary edema and leakage of plasma into the canaliculus. The hepato-cellular injury, measured by ALT, release was significantly greater in the SCS group as compared to the HMP and control groups. These findings suggest that HMP could be a better method to preserve hepatic function and overall tissue viability as compared to SCS. Improved hepatic functions are indirect indicators of superior microcirculation and sinusoidal endothelial cell functions. Further studies in progress will evaluate these functions to confirm the significance of these observations.

This study addresses the microvascular mechanisms by which a remote, mild stress such as blunt trauma sensitizes the liver to injury. Rats received closed femur fracture (FFx), and 24 h later livers were isolated and perfused at a similar starting flow rate for assessment of vascular response to endothelin-1 (ET-1). Sinusoidal volumetric flow (QS), red blood cell velocity (VRBC), and sinusoidal diameter (Ds) were determined by intravital microscopy. Baseline portal resistance in livers from FFx rats was not changed. The FFx group showed a lower baseline VRBC (322.9 +/- 26.4 and 207.3 +/- 17.2 microm/s in sham and FFx,) and QS (28.4 +/- 4.2 and 17.6 +/- 2.1 pL/s in sham and FFx, P < 0.05). ET-1 caused a decrease in the VRBC in sham but no change after FFx. In contrast, Ds was unchanged by ET-1 in sham but decreased in FFx (10.3 +/- 0.4 to 10.7 +/- 0.5 vs. 10.6 +/- 0.4 to 9.0 +/- 0.4 microm at 10 min in sham and FFx groups, P < 0.05). The overall result of these changes was a greater decrease in sinusoidal flow in FFx compared with sham. There was no significant change in mRNA for ET-1, endothelin A (ETA) receptor, or iNOS (inducible nitric oxide synthase) in FFx compared with sham. However, endothelin B (ETB) receptor mRNA and eNOS (endothelial nitric oxide synthase) mRNA were increased in the FFx group (ETB, 54.81 +/- 8.08 in sham vs. 83.28 +/- 8.19 in FFx; eNOS, 56.11 +/- 2.53 in sham vs. 83.31 +/- 5.51 in FFx; P < 0.05) while the levels of these proteins remained unchanged. Caveolin-1 (cav-1) protein levels were elevated in FFx, and coimmunoprecipitation with both ETB and eNOS showed increased associations with these proteins, suggesting a possible inactivation of eNOS. The eNOS activity was also blunted in FFx animals in the presence of increased cav-1 expression. Taken together, these results demonstrate that remote trauma sensitizes the liver to the sinusoidal constrictor effect of ET-1. We propose that this hyperresponsiveness occurs as a result of uncoupling of the ETB receptor from eNOS activity mediated by interaction of eNOS and possibly the ETB receptor with increased caveolin-1. This vascular sensitization that occurs after FFx may contribute to the exacerbation of injury during subsequent stresses.

Objective: Endothelins and their receptors play a crucial role in regulating liver microcirculation in pathophysiological conditions. The authors investigated the functional significance of the coupling of ET(B) receptors and eNOS in maintaining regional perfusion and tissue oxygenation in the normal liver., Methods: The effect of endothelin-1 or the ET(B) agonist IRL1620 on oxygen consumption was determined in isolated perfused liver and isolated hepatocytes. Oxygen delivery to the liver tissue was determined in vivo. Following eNOS or iNOS blockade, either ET-1 or IRL1620 was infused via the portal vein. Hepatic tissue oxygenation, redox state, and microcirculation were investigated by intravital microscopy. Injury was estimated by serum LDH., Results: Although IRL1620 and endothelin-1 increased oxygen consumption in isolated hepatocytes, in intact liver, endothelin decreased oxygen consumption while IRL1620 produced no change. In vivo, ET(B) stimulation modestly altered hepatic tissue P(O(2)), redox potential, and microcirculation. eNOS inhibition and ET(B) activation dramatically reduced microcirculatory blood flow, oxygen supply, and increased LDH release. Inhibition of iNOS resulted in small but not significant changes in these parameters. Concomitant ET(A)/ET(B) receptor activation increased microcirculatory failure and decreased tissue oxygen even without NOS inhibition. In contrast, hepatocellular injury was significantly increased following eNOS inhibition., Conclusions: Coupling between ET(B) receptor stimulation and eNOS activation decreases sinusoidal constriction and plays a functionally important role in maintaining microcirculation and tissue oxygenation in the normal liver.

The effects of glucagon on the subacinar distribution of hepatic transmembrane potentials were studied in the perfused fasted rat liver. The livers were perfused with a Krebs-Henseleit buffer, and membrane potentials of matched periportal and pericentral hepatocytes were determined using glass microelectrodes. Lactate- and pyruvate-induced glucose production and O2 uptake were potentiated by 10(-8) M glucagon. Twenty-five micromoles 8-bromoadenosine 3',5'cyclic monophosphate (8-BrcAMP) exhibited stimulatory effects similar, in terms of glucose production and O2 uptake to those of glucagon. Octanol (0.1 and 0.5 mM) had no effect on glucose production but reversibly increased O2 uptake by 16% to 30% over all experiments. Under basal conditions (no exogenous substrate) hepatocyte membrane potentials averaged approximately -27 mV, and no gradients were found between periportal and pericentral hepatocytes. Addition of lactate and pyruvate produced hyperpolarization in all hepatocytes. However, there was a small but statistically significant gradient produced across the hepatic acinus in membrane potential, i.e., the hyperpolarization was higher in the periportal region compared with the pericentral region. Glucagon and 8-BrcAMP induced marked hyperpolarization in periportal and pericentral hepatocytes with no gradients across the acinus. Although no changes were found under basal and lactate plus pyruvate, 0.5 mM octanol induced heterogeneity of membrane potential during glucagon and 8-BrcAMP stimulation. Our findings suggest that glucagon-induced homogeneity of membrane potential may be mediated by increased gap junctional coupling. In addition, cAMP may be responsible for the increase in the intercellular communication during glucagon stimulation.

Background: Although hypothermic machine perfusion (HMP) preservation has been shown to improve organ function and to expand the organ donor pool, problems still exist with the current HMP technology for liver preservation. The present study was conducted to investigate endothelial and hepatocellular functions following extended HMP (> r =24 hr) in rat liver model., Methods: Following 24-hour hypothermic HMP with University of Wisconsin (UW) solution or 24-hour simple cold storage (SCS), livers were reperfused with Krebs-Henseleit buffer solution at 37 degree C for 30 minutes. Hepatocyte damage and function were assessed by measuring lactate dehydrogenase (LDH) activity, bile production, and indocyanine green (ICG) extraction. Sinusoidal endothelial cell (SEC) function and permeability were determined by hyaluronic acid (HA) uptake and multiple indicator dilution (MID) method, respectively., Results: After 24-hour hypothermic preservation, HMP livers showed lower released LDH levels, higher bile flow rate, and greater hepatic ICG uptake compared with SCS livers. However, LDH levels became significantly higher in HMP than in SCS after 30 minutes of warm perfusion. The increased enzyme levels were accompanied by a significant increase in endothelial permeability to albumin and a decrease in hyaluronic acid uptake in HMP compared to SCS. Liver wet/dry weight ratio confirmed a greater edema in HMP livers than SCS livers., Conclusion: These results suggest that 24-hour hypothermic HMP may help preservation of hepatocyte function, but endothelial cell dysfunction during the cold preservation may play a key role in hepatocyte dysfunction and parenchymal cell death upon reperfusion.

Liver transplantation is often the only effective treatment for end stage liver diseases resulting from cirrhosis, hepatitis, progressive jaundice, and biliary atresia. Hypothermic machine perfusion (HMP) preservation may enhance donor pool by extending preservation time and reclaiming marginal donor livers including those from non-heart beating donors (NHBD), as demonstrated in the kidney. However, current HMP protocols have not been successful in improving extended preservation of livers and the major cause of preservation injury remains unknown. An intravital microscopy study was conducted to understand the flow dynamics of sinusoidal perfusion during 24h HMP with cold modified University of Wisconsin (UW) solution. Fluorescein isothiocynate (FITC) labeled albumin was utilized to visualize microvascular space and FITC labeled red blood cells (RBCs) were used to visualize flow dynamics during HMP. A heterogeneous flow pattern with regions of red cell stasis was observed after 24-h HMP. To examine the cause of red cell stasis, intravital and confocal microscopy studies of endothelial cells (ECs) structure labeled with DiI acetylated low-density lipoprotein (DiI acLDL) were conducted. These studies suggest that morphological changes in EC structures occurred during 24h HMP, which may cause obstruction to the sinusoidal flow. Histological findings confirm these results. As a result, heterogeneous flow pattern, red cell stasis, and edema occur, which may lead to the failure of these tissues following extended HMP.

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.

Background: Non-heart-beating donor (NHBD) livers are an untapped source with the potential to provide relief to the current donor shortage problem. Hypothermic machine perfusion (MP) has the potential to reclaim and preserve these marginal donor organs., Methods: This study compared 5-day survival in a rat NHBD liver transplantation model with simple cold storage (SCS) and MP-preserved tissues that had experienced 30 min of warm ischemia followed by a 5-hr preservation period with the University of Wisconsin solution. Total release of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) were determined at major time points. Bilirubin levels and histology were examined after 5-day survival., Results: Six of seven control livers and five of six MP livers survived, whereas SCS tissues had survival in zero of seven. The results showed that MP livers had reduced release of LDH and ALT after 5 hr of storage, 5.07+/-1.42 and 2.02+/-0.69 U (mean+/-SE), respectively, compared with SCS, 15.54+/-0.81 and 3.41.3+/-0.73 U, respectively. Bilirubin values after 5-day survival of MP livers (1.17+/-0.49 mg/dL) were comparable to controls (0.91+/-0.36 mg/dL). Histology confirms that SCS displayed increased necrosis and MP tissue showed regions of near normal hepatic structure., Conclusions: These results suggest that MP for 5 hr improves survival and reduces cellular damage of liver tissue that has experienced 30 min of warm ischemia when compared with SCS tissues. Further studies need to be conducted, but this study suggests that MP preservation has the potential to reclaim and preserve NHBD liver tissues.

Hemorrhagic shock is associated with decreased systemic oxygen delivery, but also with impaired microvascular perfusion, which can result in diminished local oxygen availability even in the presence of adequate cardiac output after resuscitation. Beside surgical interventions to control blood loss, transfusion of stored packed red blood cells represents the current standard of care in the management of severe hemorrhagic shock. Because stored red blood cells are less deformable and show a higher O2 affinity that affects the O2 off-load to tissues, perfluorocarbon-based artificial oxygen carriers might improve local O2 delivery under these conditions. To test this, rats were subjected to hemorrhagic shock (1 h, mean arterial pressure [MAP] 30-35 mmHg) and were resuscitated with fresh whole blood, pentastarch, stored red blood cells, perflubron emulsion (2.7 and 5.4 g/kg body weight) together with pentastarch, or stored red blood cells together with 2.7 g/kg perflubron emulsion. Hepatic microcirculation, tissue oxygenation, and mitochondrial redox state were investigated by intravital microscopy. In addition, hepatocellular function and liver enzyme release were determined. After hemorrhagic shock and resuscitation with perflubron emulsion, volumetric sinusoidal blood flow was significantly increased compared with resuscitation with stored red blood cells. Furthermore, resuscitation with perflubron emulsion resulted in higher hepatic tissue PO2 and normalized mitochondrial redox potential, which was accompanied by lessened hepatocellular injury as well as improved liver function. These results indicate that, in this model of hemorrhagic shock, asanguineous fluid resuscitation with addition of perflubron emulsion is superior to stored blood or pentastarch alone with respect to increased local O2 availability on the cellular level. This effect is primarily due to improved restoration of hepatic microcirculatory integrity.