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Jarrod P Meadows1,2, James A Tumlin3, Mark G Clemens4, Iain H McKillop1,4, Susan Evans1,21Department of Surgery; 2FH “Sammy” Ross Jr Trauma Center, Carolinas Medical Center, Charlotte, NC, USA; 3Department of Medicine, Division of Nephrology, University of Tennessee College of Medicine at Chattanooga, Chattanooga, TN, USA; 4Department of Biology, University of North Carolina at Charlotte, Charlotte, NC, USABackground: Neutrophils are implicated in initiating perioperative inflammation and postoperative outcome, particularly in patients with acute renal failure. The aim of the current study was to assess the role of neutrophil function in patients following cardiovascular surgery with mild preoperative renal dysfunction (PRD).Materials and methods: Patients scheduled for cardiovascular operations were screened for renal function, and divided into those with glomerular filtration rate (GFR) < 60 mL/minute (PRD, n = 9), and GFR > 60 mL/minute (normal controls; [C] n = 7). Neutrophils were isolated from plasma preoperatively, and 2, 24, and 48 hours postoperatively. Neutrophil activity (respiratory burst; RB) and apoptosis were then determined.Results: In the whole population, neutrophil apoptosis decreased from 67% preoperatively, to 42% postoperatively, with no significant difference between the groups (C = 64.8%–41.2%, PRD = 68.2%–42.6%). RB increased by 70% postoperatively in the whole population, with no significant difference between the groups (C = 68.1%, PRD = 72.9%, P = 0.86).Conclusion: Collectively these data demonstrate that, in addition to decreasing neutrophil apoptosis, cardiovascular surgery leads to increased neutrophil RB, thus contributing to postoperative systemic oxidative stress. This can lead to clinically significant complications and tissue damage. Our data also suggest that differences in postoperative inflammation between patients with and without PRD, do not exist, at least in the setting of mild preoperative renal insufficiency.Keywords: neutrophil, renal function, apoptosis, respiratory burst, glomerular filtration rate

Ischemia-reperfusion injury is a critical liver condition during hepatic transplantation, trauma, or shock. An ischemic deprivation of antioxidants and energy characterizes liver injury in such cases. In the face of increased reactive oxygen production, hepatocytes are vulnerable to the reperfusion driving ROS generation and multiple cell-death mechanisms. In this study, we investigate the importance of hydrogen sulfide as part of the liver's antioxidant pool and the therapeutic potency of the hydrogen sulfide donors sodium sulfide (Na 2 S, fast releasing) and sodium thiosulfate (STS, Na 2 S 2 O 3 , slow releasing). The mitoprotection and toxicity of STS and Na 2 S were investigated on isolated mitochondria and a liver perfusion oxidative stress model by adding text-butyl hydroperoxide and hydrogen sulfide donors. The respiratory capacity of mitochondria, hepatocellular released LDH, glutathione, and lipid-peroxide levels were quantified. In addition, wild-type and cystathionine-γ-lyase knockout mice were subjected to warm selective ischemia-reperfusion injury by clamping the main inflow for 1 h followed by reperfusion of 1 or 24 h. A subset of animals was treated with STS shortly before reperfusion. Glutathione, plasma ALT, and lipid-peroxide levels were investigated alongside mitochondrial changes in structure (electron microscopy) and function (intravital microscopy). Liver tissue necrosis quantified 24 h after reperfusion indicates the net effects of the treatment on the organ. STS refuels and protects the endogenous antioxidant pool during liver ischemia-reperfusion injury. In addition, STS-mediated ROS scavenging significantly reduced lipid peroxidation and mitochondrial damage, resulting in better molecular and histopathological preservation of the liver tissue architecture. STS prevents tissue damage in liver ischemia-reperfusion injury by increasing the liver's antioxidant pool, thereby protecting mitochondrial integrity., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Liver cryopreservation has the potential to enable indefinite organ banking. This study investigated vitrification-the ice-free cryopreservation of livers in a glass-like state-as a promising alternative to conventional cryopreservation, which uniformly fails due to damage from ice formation or cracking. Our unique "nanowarming" technology, which involves perfusing biospecimens with cryoprotective agents (CPAs) and silica-coated iron oxide nanoparticles (sIONPs) and then, after vitrification, exciting the nanoparticles via radiofrequency waves, enables rewarming of vitrified specimens fast enough to avoid ice formation and uniformly enough to prevent cracking from thermal stresses, thereby addressing the two main failures of conventional cryopreservation. This study demonstrates the ability to load rat livers with both CPA and sIONPs by vascular perfusion, cool them rapidly to an ice-free vitrified state, and rapidly and homogenously rewarm them. While there was some elevation of liver enzymes (Alanine Aminotransferase) and impaired indocyanine green (ICG) excretion, the nanowarmed livers were viable, maintained normal tissue architecture, had preserved vascular endothelium, and demonstrated hepatocyte and organ-level function, including production of bile and hepatocyte uptake of ICG during normothermic reperfusion. These findings suggest that cryopreservation of whole livers via vitrification and nanowarming has the potential to achieve organ banking for transplant and other biomedical applications., (© 2022. The Author(s) under exclusive licence to Biomedical Engineering Society.)

Competing Interests: The authors report no conflicts of interest.

Cells have evolved extensive signaling mechanisms to maintain redox homeostasis. While basal levels of oxidants are critical for normal signaling, a tipping point is reached when the level of oxidant species exceed cellular antioxidant capabilities. Myriad pathological conditions are characterized by elevated oxidative stress, which can cause alterations in cellular operations and damage to cellular components including nucleic acids. Maintenance of nuclear chromatin are critically important for host survival and eukaryotic organisms possess an elaborately orchestrated response to initiate repair of such DNA damage. Recent evidence indicates links between the cellular antioxidant response, the DNA damage response (DDR), and the epigenetic status of the cell under conditions of elevated oxidative stress. In this emerging model, the cellular response to excessive oxidants may include redox sensors that regulate both the DDR and an orchestrated change to the epigenome in a tightly controlled program that both protects and regulates the nuclear genome. Herein we use sepsis as a model of an inflammatory pathophysiological condition that results in elevated oxidative stress, upregulation of the DDR, and epigenetic reprogramming of hematopoietic stem cells (HSCs) to discuss new evidence for interplay between the antioxidant response, the DNA damage response, and epigenetic status.

Sepsis is a life-threatening clinical syndrome defined as a deregulated host response to infection associated with organ dysfunction. Mechanisms underlying the pathophysiology of septic liver dysfunction are incompletely understood. Among others, the iron containing tetrapyrrole heme inflicts hepatic damage when released into the circulation during systemic inflammation and sepsis. Accordingly, hemolysis and decreased concentrations of heme-scavenging proteins coincide with an unfavorable outcome of critically ill patients. As the liver is a key organ in heme metabolism and host response to infection, we investigated the impact of labile heme on sinusoidal microcirculation and hepatocellular integrity. We here provide experimental evidence that heme increases portal pressure via a mechanism that involves hepatic stellate cell-mediated sinusoidal constriction, a hallmark of microcirculatory failure under stress conditions. Moreover, heme exerts direct cytotoxicity in vitro and aggravates tissue damage in a model of polymicrobial sepsis. Heme binding by albumin, a low-affinity but high-capacity heme scavenger, attenuates heme-mediated vasoconstriction in vivo and prevents heme-mediated cytotoxicity in vitro. We demonstrate that fractions of serum albumin-bound labile heme are increased in septic patients. We propose that heme scavenging might be used therapeutically to maintain hepatic microcirculation and organ function in sepsis., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

We sought to determine if the baseline hepatic levels of miR-122, miR-29b, Claudin, Occludin, Protein Kinase R (PKR) or PKR activator (PRKRA) were correlated with HCV RNA or stage of fibrosis in patients with chronic hepatitis C (CHC). A total of 25 CHC patients (genotype 1) who were treatment naive at the time of sample collection enrolled in this study. By multivariate analysis, CLDN RNA was found as the single independent factor positively correlated with HCV RNA levels (p=0.003), while hepatic miR-29b levels was found as the single independent factor for predicting advanced stage of fibrosis (p=0.028). Conclusion: Our results highlight miR-29b and CLDN as novel predictors of advanced stage of liver fibrosis and baseline HCV RNA in CHC., Competing Interests: Conflict of Interest None declared.

Background: Isochoric freezing systems enable ice-free preservation of biological matter at subfreezing temperatures under the increased hydrostatic pressure., Objective: To examine the effects of pressure and exposure period on rat hearts preserved in an isochoric chamber., Materials and Methods: Rat hearts were preserved in the UW solution in isochoric chambers at temperatures from -2°C to -8°C and pressure from the atmospheric level to 78 MPa for up to eight hours, with and without the addition of glycerol. Hearts were evaluated via Langendorff perfusion and HE histology., Results: Hearts were compromised quickly as pressure increased, suggesting an acute time-pressure sensitivity. With the addition of 1 M glycerol, which reduces the pressure experienced at a given temperature, the survival time at -4°C was doubled., Conclusion: The enhanced hydrostatic pressure encountered during isochoric preservation yields time-dependent negative effects on the heart, which can potentially be alleviated by the addition of a cryoprotectant.

Isochoric (constant volume) preservation at subfreezing temperatures is being investigated as a novel method for preserving cells and organs. This study is a first initial effort to evaluate the efficacy of this method for heart preservation, and to provide a preliminary outline of appropriate preservation parameters. To establish a baseline for further studies, rat hearts were preserved in a University of Wisconsin (UW) intracellular solution for one hour under isochoric conditions at: 0 °C (atmospheric pressure - 0.1 MPa), - 4 °C (41 MPa), - 6 °C (60 MPa) and - 8 °C (78 MPa). The viability of the heart was evaluated using Langendorff perfusion and histological examination. The physiological performance of hearts preserved at - 4 °C (41 MPa) was comparable to that of a heart preserved on ice at atmospheric pressure, with no statistically significant difference in histological injury score. However, hearts preserved at -4 °C displayed substantially reduced interstitial edema compared to hearts preserved by conventional hypothermic preservation in UW on ice at atmospheric pressure, suggesting significant protection from increased vascular permeability following preservation. Hearts preserved at - 6 °C (60 MPa) suffered injury from cellular swelling and extensive edema, and at - 8 °C (78 MPa) hearts experienced significant morphological disruption. To the best of our knowledge, this is the first publication showing that a mammalian organ can survive low subfreezing temperatures without the use of a cryoprotective additive. Lowering the preservation temperature reduces metabolism and improves preservation quality, and these results suggest that improvements in preservation are possible at subzero temperatures with low to moderate pressures observed at -4 °C. Notably, tissue damage was observed at lower temperatures (-6 °C or below) accompanying further elevation of pressure associated with isochoric preservation that may prove detrimental. Therefore, subfreezing temperature isochoric preservation protocols should optimize, a combination of temperature and pressure that will minimize the negative effects of elevated pressure while retaining the beneficial effect of lower temperatures and reduced metabolism., (Copyright © 2018. Published by Elsevier Inc.)

Simultaneous assessment of excretory liver and kidney function is still an unmet need in experimental stress models as well as in critical care. The aim of the study was to characterize two polymethine-dyes potentially suitable for this purpose in vivo. Plasma disappearance rate and elimination measurements of simultaneously injected fluorescent dyes DY-780 (hepato-biliary elimination) and DY-654(renal elimination) were conducted using catheter techniques and intravital microscopy in animals subjected to different organ injuries, i.e. polymicrobial sepsis by peritoneal contamination and infection, ischemia-reperfusion-injury and glycerol-induced acute kidney-injury. DY-780 and DY-654 showed organ specific and determined elimination routes in both healthy and diseased animals. They can be measured simultaneously using near-infrared imaging and spectrophotometry. Plasma-disappearance rates of DY-780 and DY-654 are superior to conventional biomarkers in indicating hepatic or kidney dysfunction in different animal models. Greatest impact on liver function was found in animals with polymicrobial sepsis whereas glomerular damage due to glycerol-induced kidney-injury had strongest impact on DY-654 elimination. We therefore conclude that hepatic elimination and renal filtration can be assessed in rodents measuring plasma-disappearance rates of both dyes. Further, assessment of organ dysfunction by polymethine dyes correlates with, but outperforms conventional biomarkers regarding sensitivity and the option of spatial resolution if biophotonic strategies are applied. Polymethine-dye clearance thereby allows sensitive point-of-care assessment of both organ functions simultaneously.

Mitochondria contribute to redox and calcium balance, and apoptosis thus regulating cellular fate. In the present study, mitochondrial staining applying a novel dye, V07-07059, was performed in human embryonic kidney cells, a human vascular endothelial cell line and primary human mononuclear cells. The new fluorescent mega Stokes dye (peak excitation: 488 nm, peak emission: 554 nm) showed superior fluorescent properties and stability. V07-07059 stains mitochondria dependent on their membrane potential and is safe to use in vitro and in vivo. Unlike other dyes applied in this context (e.g. Tetramethylrhodamine methyl ester), V07-07059 only marginally inhibits mitochondrial respiration and function. V07-07059 enables real time imaging of mitochondrial trafficking and remodeling. Prolonged staining with V07-07059 demonstrated the dyes suitability as a novel probe to track cells. In comparison to the widely used standard for cell proliferation and tracking studies 5(6)-diacetate N-succinimidyl ester, V07-07059 proved superior regarding toxicity and photostability., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

It has been shown that microcirculation is hypersensitized to endothelin1 (ET-1) following endotoxin (lipopolysaccharide [LPS]) treatment leading to an increased vasopressor response. This may be related in part to decreased activation of endothelial nitric oxide synthase (eNOS) by ET-1. eNOS can also be uncoupled to produce superoxide (O2). This aberrant eNOS activity could further contribute to the hyperconstriction and injury caused by ET-1 following LPS. We therefore tested whether LPS affects ROS production by vascular endothelial cells and whether and how this effect is altered by ET-1. Human umbilical vein endothelial cells (HUVEC) or human microvascular endothelial cells (HMEC) were subjected to a 6-h treatment with LPS (250 ng/mL) or LPS and sepiapterin (100 μM) followed by a 30-min treatment with 100 μM L-Iminoethyl Ornithine (L-NIO) an irreversible eNOS inhibitor and 30-min treatment with ET-1 (10 nM). Conversion of [H]L-arginine to [H]L-citrulline was used to measure eNOS activity. Superoxide dismutase (SOD) inhibitable reduction of Cytochrome-C, dihydro carboxy fluorescein (DCF), and Mitosox was used to estimate ROS. LT-SDS PAGE was used to assess the degree of monomerization of the eNOS homodimer. Stimulation of HUVECs with ET-1 significantly increased NO synthesis by 1.4-fold (P < 0.05). ET-1 stimulation of LPS-treated HUVECs failed to increase NO production. Western blot for eNOS protein showed no change in eNOS protein levels. LPS alone resulted in an insignificant increase in ROS production as measured by cytochrome C that was increased 4.6-fold by ET-1 stimulation (P < 0.05). L-NIO significantly decreased ET-1-induced ROS production (P < 0.05). Sepiapterin significantly decreased ROS production in both; unstimulated and ET-1-stimulated LPS-treated groups, but did not restore NO production. DCF experiments confirmed intracellular ROS while Mitosox suggested a non-mitochondrial source. ET-1 treatment following a chronic LPS stress significantly monomerized the eNOS homodimer that was inhibited by sepiapterin loading. The two concomitant phenomena of decreased NO production and increased ROS formation seem to be multifactorial in nature with ROS production dependent upon pterin availability.

Background: The aim of extracorporeal albumin dialysis (ECAD) is to reduce endogenous toxins accumulating in liver failure. To date, ECAD is conducted mainly with the Molecular Adsorbents Recirculating System (MARS). However, single-pass albumin dialysis (SPAD) has been proposed as an alternative. The aim of this study was to compare the two devices with a prospective, single-centre, non-inferiority crossover study design with particular focus on reduction of bilirubin levels (primary endpoint) and influence on paraclinical and clinical parameters (secondary endpoints) associated with liver failure., Methods: Patients presenting with liver failure were screened for eligibility and after inclusion were randomly assigned to be started on either conventional MARS or SPAD (with 4% albumin and a dialysis flow rate of 700 ml/h). Statistical analyses were based on a linear mixed-effects model., Results: Sixty-nine crossover cycles of ECAD in 32 patients were completed. Both systems significantly reduced plasma bilirubin levels to a similar extent (MARS: median -68 μmol/L, interquartile range [IQR] -107.5 to -33.5, p = 0.001; SPAD: -59 μmol/L, -84.5 to +36.5, p = 0.001). However, bile acids (MARS: -39 μmol/L, -105.6 to -8.3, p < 0.001; SPAD: -9 μmol/L, -36.9 to +11.4, p = 0.131), creatinine (MARS: -24 μmol/L, -46.5 to -8.0, p < 0.001; SPAD: -2 μmol/L, -9.0 to +7.0/L, p = 0.314) and urea (MARS: -0.9 mmol/L, -1.93 to -0.10, p = 0.024; SPAD: -0.1 mmol/L, -1.0 to +0.68, p = 0.523) were reduced and albumin-binding capacity was increased (MARS: +10%, -0.8 to +20.9%, p < 0.001; SPAD: +7%, -7.5 to +15.5%, p = 0.137) only by MARS. Cytokine levels of interleukin (IL)-6 and IL-8 and hepatic encephalopathy were altered by neither MARS nor SPAD., Conclusions: Both procedures were safe for temporary extracorporeal liver support. While in clinical practice routinely assessed plasma bilirubin levels were reduced by both systems, only MARS affected other paraclinical parameters (i.e., serum bile acids, albumin-binding capacity, and creatinine and urea levels). Caution should be taken with regard to metabolic derangements and electrolyte disturbances, particularly in SPAD using regional citrate anti-coagulation., Trial Registration: German Clinical Trials Register ( www.drks.de) DRKS00000371. Registered 8 April 2010.

Background & Aims: Analysis in silico suggests that occludin (OCLN), a key receptor for HCV, is a candidate target of miR-122; the most abundant hepatic micro RNA. We aimed to determine if miR-122 can decrease HCV entry through binding to the 3' UTR of OCLN mRNA., Design: Huh7.5 cells were cotransfected with luciferase construct containing 3' UTR of OCLN (pLuc-OCLN) and with selected miRNAs (0-50 nM) and luciferase activity was measured. Huh7.5 cells were also infected by viral particles containing lenti-miR122 genome or control virus. After 48 h, the cells were infected with HCV pseudo-particles (HCVpp) and VSV pseudo-particles (VSVpp). After 72 h of infection, luciferase activity was measured and HCVpp activity was normalized to VSVpp activity., Results: miR-122 binds to the 3'-UTR of OCLN and down-regulates its expression; cotransfection of miR-122 mimic with pLuc-OCLN resulted in a significant decrease in luciferase activity [by 55% (P < 0.01)], while a non-specific miRNA and a mutant miR-122 did not have any effect. miR-122 mimic significantly down-regulated [by 80% (P < 0.01)] OCLN protein in Huh7.5 cells. Accordingly, patients with chronic hepatitis C and higher levels of hepatic miR-122 have lower hepatic expression of OCLN. Immuno-fluorescence imaging showed a decrease in colocalization of OCLN and CLDN following miR-122 over-expression in HCV infected cells. Huh7.5 cells transiently expressing Lenti-miR122 system showed 42% (P < 0.01) decrease in HCV entry., Conclusion: This study uncovers a novel antiviral effect of miR-122 on human liver cells and shows that over-expression of miR-122 can decrease HCV entry into hepatocytes through down-regulation of OCLN., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Efficient delivery of short interfering RNAs reflects a prerequisite for the development of RNA interference therapeutics. Here, we describe highly specific nanoparticles, based on near infrared fluorescent polymethine dye-derived targeting moieties coupled to biodegradable polymers. The fluorescent dye, even when coupled to a nanoparticle, mimics a ligand for hepatic parenchymal uptake transporters resulting in hepatobiliary clearance of approximately 95% of the dye within 45 min. Body distribution, hepatocyte uptake and excretion into bile of the dye itself, or dye-coupled nanoparticles can be tracked by intravital microscopy or even non-invasively by multispectral optoacoustic tomography. Efficacy of delivery is demonstrated in vivo using 3-hydroxy-3-methyl-glutaryl-CoA reductase siRNA as an active payload resulting in a reduction of plasma cholesterol levels if siRNA was formulated into dye-functionalised nanoparticles. This suggests that organ-selective uptake of a near infrared dye can be efficiently transferred to theranostic nanoparticles allowing novel possibilities for personalised silencing of disease-associated genes.

The purpose of this study is to understand the role of rho-kinase (ROCK-2) in the regulation of liver microcirculation after inflammatory stress. Endothelin-1 (ET-1)-induced nitric oxide (NO) is essential in the regulation of blood flow in hepatic sinusoids. Lipopolysaccharide (LPS) inhibits this ET-1-induced NO production and disrupts liver microcirculation; however, the exact molecular mechanism is unknown. Liver sinusoidal endothelial cells were isolated, pretreated with 10 ng/mL LPS for 6 h, and treated with 10 μM Y27632 (ROCK-2 inhibitor) for 30 min and 10 nM ET-1 for 30 min. Lipopolysaccharide induced RhoA membrane translocation that was attenuated by methyl-β-cyclodextrin (cholesterol sequester) or targeted mutation of caveolin-1. Lipopolysaccharide increased ROCK-2 expressions (+60%) and ROCK-2 activity (+36%). Endothelin-1 increased endothelial NO synthase (eNOS) activity (+70%), but LPS inhibited this ET-1-mediated eNOS response. Treatment with Y27632 restored ET-1-mediated eNOS activity (+61%) and stimulated NO production in the perinuclear region after LPS pretreatment. This treatment reduced cofilin-Ser3 phosphorylation (-73%), increased vasodilator-stimulated phosphoprotein-Ser239 phosphorylation (+88%), and stimulated globular actin/eNOS association. Lipopolysaccharide induces Rho/ROCKs signaling pathway to disrupt the ET-1-mediated eNOS activation in liver sinusoidal endothelial cells. Rho-kinase ROCK-2 inhibition restores ET-1-mediated NO production after the LPS pretreatment, in part, through an increase in actin depolymerization.

Hydrogen sulfide (H₂S) affects vascular resistance; however, its effect on the hepatic microcirculation has not been investigated. Hepatic sinusoidal perfusion is dysregulated during sepsis, contributing to liver injury. Therefore, the present study determined the effect of H₂S on the hepatic microcirculation and the contribution of endogenous H₂S to hepatic microcirculatory dysfunction in an endotoxin model of sepsis. Portal infusion of H₂S increased portal pressure in vivo (6.8 ± 0.2 mmHg before H₂S vs. 8.6 ± 0.8 mmHg peak during H₂S infusion, P < 0.05). Using intravital microscopy, we observed decreased sinusoidal diameter (6.2 ± 0.27 μm before H₂S vs. 5.7 ± 0.3 μm after H₂S, P < 0.05) and increased sinusoidal heterogeneity during H₂S infusion (P < 0.05) and net constriction. Since hepatic H₂S levels are elevated during sepsis, we used the cystathionine γ lyase inhibitor DL-propargylglycine (PAG) to determine the contribution of H₂S to the hypersensitization of the sinusoid to the vasoconstrictor effect of endothelin-1 (ET-1). PAG treatment significantly attenuated the sinusoidal sensitization to ET-1 in endotoxin-treated animals. ET-1 infusion increased portal pressure to 175% of baseline in endotoxemic animals, which was reduced to 143% following PAG treatment (P < 0.05). PAG abrogated the increase in sinusoidal constriction after ET-1 infusion in LPS-treated rats (30.9% reduction in LPS rats vs. 11.6% in PAG/LPS rats, P < 0.05). Moreover, PAG treatment significantly attenuated the increase in NADH fluorescence following ET-1 exposure during endotoxemia (61 grayscale units LPS vs. 21 units in PAG/LPS, P < 0.05), suggesting an improvement in hepatic oxygen availability. This study is the first to demonstrate a vasoconstrictor action of H₂S on the hepatic sinusoid and provides a possible mechanism for the protective effect of PAG treatment during sepsis.

Despite being protective in many disease states, hydrogen sulfide (H(2)S) contributes to organ injury in sepsis. Like the other gasotransmitters, nitric oxide and carbon monoxide, H(2)S is a modulator of the microcirculation. Because microcirculatory dysfunction is a main cause of organ injury during sepsis, the present study was designed to test the effect of H(2)S on microvascular dysfunction in isolated perfused livers. In most microcirculatory beds, endotoxin activates the endothelium, resulting in hyporesponsiveness to catecholamines and a derangement in blood flow distribution. We demonstrate that H(2)S treatment attenuates the increase in portal pressure during infusion of the α1 adrenergic agonist, phenylephrine (PE) (P < 0.01). Hydrogen sulfide almost completely negated the increase in portal pressure in livers isolated from endotoxemic rats. Treatment with an inhibitor of endogenous H(2)S, DL-propargylglycine (PAG), reversed lipopolysaccharide-induced hyporesponsiveness to PE. Because hepatic microcirculatory dysfunction is associated with excessive sinusoidal vasoconstriction and not dilation, we investigated whether H(2)S affects endothelin 1 (ET-1)-induced vasoconstriction in isolated livers. Contrary to PE treatment, H(2)S did not affect the increase in portal pressure during infusion of ET-1, nor did it attenuate the hypersensitization of the liver to ET-1 during endotoxemia. Hepatic resistance in control rats was increased by PAG treatment during ET-1 infusion, but this increase was not exacerbated during endotoxemia. We monitored hepatic O(2) consumption to assess the effect of vascular changes on oxygen consumption following ET-1 treatment. Low-dose ET-1 infusion caused an increase in hepatic O(2)consumption, whereas low-dose ET-1 infusion decreased O(2) consumption in endotoxemic livers. Interestingly, whereas we observed no effect of PAG on the vascular response to ET-1 infusion during endotoxemia, PAG treatment did maintain O(2), suggesting a more complex effect of H(2)S inhibition. In summary, the discrepancies between the hepatic response to PE and ET-1 suggest that H(2)S differentially contributes to microcirculatory dysfunction in the systemic and hepatic microcirculations. We propose that this is due to H(2)S exerting a differential vasoactive function on presinusoidal and sinusoidal sites within the liver. Moreover, our findings suggest that H(2)S may contribute to the progression of sepsis by contributing to microvascular failure.

Activated protein C (aPC) promotes fibrinolysis while inhibiting coagulation and inflammation. In septic patients, aPC levels are depleted, and aPC treatment has emerged as a therapeutic option. To better understand the mechanism(s) by which aPC improves survival in sepsis, we sought to determine the effect of aPC treatment on hepatic vasoactive gene and protein expression, leading to changes in hepatic vascular responsiveness in a septic animal model. Under anesthesia, rats underwent sham or cecal ligation and puncture followed by aPC treatment (1 mg/kg, twice daily, i.v.). Treatment with aPC significantly decreased hepatic endothelin 1 (ET-1)/ET A receptor mRNA and protein expression. To determine the effect of aPC on hepatic microvasculature, ET-1-induced changes in liver microcirculation were assessed by intravital microscopy. This approach demonstrated aPC significantly improved hepatic perfusion index in the animals that underwent cecal ligation and puncture in the absence of significant changes in portal venous pressure. Furthermore, although aPC did not affect ET-1-dependent sinusoidal vasoconstriction, aPC induced hepatoprotective effects via enhanced red blood cell velocity. Collectively, these data demonstrate aPC ameliorates ET-1-dependent changes in hepatic microcirculation and improves hepatic function in the setting of sepsis.

The liver is likely exposed to high levels of hydrogen sulfide (H2S) from endogenous hepatic synthesis and exogenous sources from the gastrointestinal tract. Little is known about the consequence of H2S exposure on the liver or hepatic regulation of H2S levels. We hypothesized that the liver has a high capacity to metabolize H2S and that H2S oxidation is decreased during sepsis, a condition in which hepatic O2 is limited and H2S synthesis is increased. Using a nonrecirculating isolated and perfused liver system, we demonstrated rapid hepatic H2S metabolism up to an infusion concentration of 200' μM H2S. Hydrogen sulfide metabolism was associated with an increase in O2 consumption from a baseline 96.7 ± 7.6 μmol O2/min/kg to 109 ± 7.4 μmol O2/min/kg at an infusion concentration of 150 μM H2S (P < 0.001). Removal of O2 from the perfusate decreased H2S clearance from a maximal 97% to only 23%. Livers isolated from rats subjected to cecal ligation and puncture (CLP) did not differ significantly from control livers in their capacity to metabolize H2S, suggesting that H2S oxidation remains a priority during sepsis. To test whether H2S induces O2 consumption in vivo, intravital microscopy was utilized to monitor the oxygen content in the hepatic microenvironment. Infusion of H2S increased the NADH/NAD+ ratio (645 gray-scale-unit increase, P = 0.035) and decreased hepatic O2 availability visualized with Ru(Phen)3(2+) (439 gray-scale-unit increase, P = 0.040). We conclude that the liver has a high hepatic capacity for H2S metabolism. Moreover, H2S oxidation consumes available oxygen and may exacerbate the tissue hypoxia associated with sepsis.

Leukocyte motion represents an important component in the innate immune response to infection. Intravital microscopy is a powerful tool as it enables in vivo imaging of leukocyte motion. Under inflammatory conditions, leukocytes may exhibit various motion behaviors, such as flowing, rolling, and adhering. With many leukocytes moving at a wide range of speeds, collisions occur. These collisions result in abrupt changes in the motion and appearance of leukocytes. Manual analysis is tedious, error prone,time consuming, and could introduce technician-related bias. Automatic tracking is also challenging due to the noise inherent in in vivo images and abrupt changes in motion and appearance due to collision. This paper presents a method to automatically track multiple cells undergoing collisions by modeling the appearance and motion for each collision state and testing collision hypotheses of possible transitions between states. The tracking results are demonstrated using in vivo intravital microscopy image sequences.We demonstrate that 1)71% of colliding cells are correctly tracked; (2) the improvement of the proposed method is enhanced when the duration of collision increases; and (3) given good detection results, the proposed method can correctly track 88% of colliding cells. The method minimizes the tracking failures under collisions and, therefore, allows more robust analysis in the study of leukocyte behaviors responding to inflammatory conditions.