The Effect of Sepsis on the Erythrocyte.

Sepsis induces a wide range of effects on the red blood cell (RBC). Some of the effects including altered metabolism and decreased 2,3-bisphosphoglycerate are preventable with appropriate treatment, whereas others, including decreased erythrocyte deformability and redistribution of membrane phospholipids, appear to be permanent, and factors in RBC clearance. Here, we review the effects of sepsis on the erythrocyte, including changes in RBC volume, metabolism and hemoglobin's affinity for oxygen, morphology, RBC deformability (an early indicator of sepsis), antioxidant status, intracellular Ca2+ homeostasis, membrane proteins, membrane phospholipid redistribution, clearance and RBC O2-dependent adenosine triphosphate efflux (an RBC hypoxia signaling mechanism involved in microvascular autoregulation). We also consider the causes of these effects by host mediated oxidant stress and bacterial virulence factors. Additionally, we consider the altered erythrocyte microenvironment due to sepsis induced microvascular dysregulation and speculate on the possible effects of RBC autoxidation. In future, a better understanding of the mechanisms involved in sepsis induced erythrocyte pathophysiology and clearance may guide improved sepsis treatments. Evidence that small molecule antioxidants protect the erythrocyte from loss of deformability, and more importantly improve septic patient outcome suggest further research in this area is warranted. While not generally considered a critical factor in sepsis, erythrocytes (and especially a smaller subpopulation) appear to be highly susceptible to sepsis induced injury, provide an early warning signal of sepsis and are a factor in the microvascular dysfunction that has been associated with organ dysfunction.

Sepsis impairs microvascular autoregulation and delays capillary response within hypoxic capillaries.

INTRODUCTION: The microcirculation supplies oxygen (O2) and nutrients to all cells with the red blood cell (RBC) acting as both a deliverer and sensor of O2. In sepsis, a proinflammatory disease with microvascular complications, small blood vessel alterations are associated with multi-organ dysfunction and poor septic patient outcome. We hypothesized that microvascular autoregulation-existing at three levels: over the entire capillary network, within a capillary and within the erythrocyte-was impaired during onset of sepsis. This study had three objectives: 1) measure capillary response time within hypoxic capillaries, 2) test the null hypothesis that RBC O2-dependent adenosine triphosphate (ATP) efflux was not altered by sepsis and 3) develop a framework of a pathophysiological model. METHODS: This was an animal study, comparing sepsis with control, set in a university laboratory. Acute hypotensive sepsis was studied using cecal ligation and perforation (CLP) with a 6-hour end-point. Rat hindlimb skeletal muscle microcirculation was imaged, and capillary RBC supply rate (SR = RBC/s), RBC hemoglobin O2 saturation (SO2) and O2 supply rate (qO2 = pLO2/s) were quantified. Arterial NOx (nitrite + nitrate) and RBC O2-dependent ATP efflux were measured using a nitric oxide (NO) analyzer and gas exchanger, respectively. RESULTS: Sepsis increased capillary stopped-flow (p = 0.001) and increased plasma lactate (p < 0.001). Increased plasma NOx (p < 0.001) was related to increased capillary RBC supply rate (p = 0.027). Analysis of 30-second SR-SO2-qO2 profiles revealed a shift towards decreased (p < 0.05) O2 supply rates in some capillaries. Moreover, we detected a three- to fourfold increase (p < 0.05) in capillary response time within hypoxic capillaries (capillary flow states where RBC SO2 < 20 %). Additionally, sepsis decreased the erythrocyte's ability to respond to hypoxic environments, as normalized RBC O2-dependent ATP efflux decreased by 62.5 % (p < 0.001). CONCLUSIONS: Sepsis impaired microvascular autoregulation at both the individual capillary and erythrocyte level, seemingly uncoupling the RBC acting as an "O2 sensor" from microvascular autoregulation. Impaired microvascular autoregulation was manifested by increased capillary stopped-flow, increased capillary response time within hypoxic capillaries, decreased capillary O2 supply rate and decreased RBC O2-dependent ATP efflux. This loss of local microvascular control was partially off-set by increased capillary RBC supply rate, which correlated with increased plasma NOx.

S-nitrosoglutathione acts as a small molecule modulator of human fibrin clot architecture.

BACKGROUND: Altered fibrin clot architecture is increasingly associated with cardiovascular diseases; yet, little is known about how fibrin networks are affected by small molecules that alter fibrinogen structure. Based on previous evidence that S-nitrosoglutathione (GSNO) alters fibrinogen secondary structure and fibrin polymerization kinetics, we hypothesized that GSNO would alter fibrin microstructure. METHODOLOGY/PRINCIPAL FINDINGS: Accordingly, we treated human platelet-poor plasma with GSNO (0.01-3.75 mM) and imaged thrombin induced fibrin networks using multiphoton microscopy. Using custom designed computer software, we analyzed fibrin microstructure for changes in structural features including fiber density, diameter, branch point density, crossing fibers and void area. We report for the first time that GSNO dose-dependently decreased fibrin density until complete network inhibition was achieved. At low dose GSNO, fiber diameter increased 25%, maintaining clot void volume at approximately 70%. However, at high dose GSNO, abnormal irregularly shaped fibrin clusters with high fluorescence intensity cores were detected and clot void volume increased dramatically. Notwithstanding fibrin clusters, the clot remained stable, as fiber branching was insensitive to GSNO and there was no evidence of fiber motion within the network. Moreover, at the highest GSNO dose tested, we observed for the first time, that GSNO induced formation of fibrin agglomerates. CONCLUSIONS/SIGNIFICANCE: Taken together, low dose GSNO modulated fibrin microstructure generating coarse fibrin networks with thicker fibers; however, higher doses of GSNO induced abnormal fibrin structures and fibrin agglomerates. Since GSNO maintained clot void volume, while altering fiber diameter it suggests that GSNO may modulate the remodeling or inhibition of fibrin networks over an optimal concentration range.

Interactions of multiple gas-transducing systems: hallmarks and uncertainties of CO, NO, and H2S gas biology.

The diverse physiological actions of the "biologic gases," O2, CO, NO, and H2S, have attracted much interest. Initially viewed as toxic substances, CO, NO, and H2S play important roles as signaling molecules. The multiplicity of gas actions and gas targets and the difficulty in measuring local gas concentrations obscures detailed mechanisms whereby gases exert their actions, and many questions remain unanswered. It is now readily apparent, however, that heme-based proteins play central roles in gas-generation/reception mechanisms and provide a point where multiple gases can interact. In this review, we consider a number of key issues related to "gas biology," including the effective tissue concentrations of these gases and the importance and significance of the physical proximity of gas-producing and gas-receptor/sensors. We also take an integrated approach to the interaction of gases by considering the physiological significance of CO, NO, and H2S on mitochondrial cytochrome c oxidase, a key target and central mediator of mitochondrial respiration. Additionally, we consider the effects of biologic gases on mitochondrial biogenesis and "suspended animation." By evaluating gas-mediated control functions from both in vitro and in vivo perspectives, we hope to elaborate on the complex multiple interactions of O2, NO, CO, and H2S.

Fibrinogen decreases cardiomyocyte contractility through an ICAM-1-dependent mechanism.

INTRODUCTION: Cardiomyocytes exposed to inflammatory processes express intracellular adhesion molecule-1 (ICAM-1). We investigated whether fibrinogen and fibrinogen degradation products, including D-dimer, could alter cardiomyocyte contractile function through interaction with ICAM-1 found on inflamed cardiomyocytes. METHODS: In vivo, rats were injected with endotoxin to model systemic inflammation, whereas isolated rat cardiomyocytes were treated with tumor necrosis factor-alpha to model the inflammatory environment seen following exposure to bacterial products such as lipopolysaccharide. RESULTS: In vivo, endotoxin administration profoundly decreased cardiac contractile function associated with a large increase in intracardiac ICAM-1 and perivascular fibrinogen. Confocal microscopy with double-staining of isolated rat cardiomyocytes demonstrated colocalization of ICAM-1 and fibrinogen. This interaction was disrupted through pre-treatment of the cells with an ICAM-1-blocking antibody. Functionally, isolated rat cardiomyocyte preparations exhibited decreased fractional shortening when incubated with fibrinogen, and through the use of synthetic peptides, we determined that residues 117-133 of the fibrinogen gamma chain are responsible for this interaction with ICAM-1. Despite having crosslinked gamma chains, D-dimer retained the ability to decrease cardiomyocyte contractility. CONCLUSION: Site 117-133 of the fibrinogen gamma chain is able to depress cardiomyocyte contractility through binding ICAM-1.

Inhibiting nitric oxide overproduction during hypotensive sepsis increases local oxygen consumption in rat skeletal muscle.

OBJECTIVE: Although nitric oxide (NO) is a known regulator of cardiovascular function, the effect of NO overproduction during sepsis on capillary oxygen transport and local tissue oxygen consumption is not well understood. The objectives of this study were to determine whether sepsis-induced NO overproduction increased capillary stopped-flow and modulated tissue oxygen consumption in skeletal muscle. DESIGN: Prospective, controlled laboratory study. SETTING: Animal laboratory in a university-affiliated research institute. SUBJECTS: Male Sprague-Dawley rats, 165-180 g body weight. INTERVENTIONS: Rats were made septic by cecal ligation and perforation (CLP) and were then ventilated and volume resuscitated (saline). The hind limb extensor digitorum longus (EDL) skeletal muscle was blunt dissected for in vivo microvascular imaging. The inducible NO synthase (iNOS) inhibitor L-N6-(1-iminoethyl)lysine dihydrochloride (L-NIL) was infused (3 mg/kg body weight per hour) starting 1 hr post-CLP to maintain arterial blood and EDL tissue NO(x)(-) (NO2(-) + NO3(-)) at baseline. MEASUREMENTS AND MAIN RESULTS: Red blood cell hemodynamics, hemoglobin oxygen saturation, capillary geometry, and functional capillary density information were used to calculate capillary oxygen flux (the rate of oxygen diffusion from capillary to tissue) and indices of local oxygen delivery and tissue oxygen consumption. Over the first 5 hrs of septic injury, mean arterial pressure decreased while capillary stopped-flow and capillary oxygen flux both increased (p < .05). Inhibiting iNOS/NO overproduction partially restored mean arterial pressure and increased arterial pH. Within the microcirculation, inhibiting NO increased capillary red cell velocity and increased local tissue oxygen consumption (p < .05). Inhibiting NO failed, however, to prevent capillary stopped-flow. CONCLUSIONS: During the onset of sepsis, concurrent with the onset of microvascular dysfunction, there is an iNOS/NO-mediated reduction in local skeletal muscle tissue oxygen consumption.

Albumin resuscitation improves ventricular contractility and myocardial tissue oxygenation in rat endotoxemia.

OBJECTIVE: Fluid resuscitation to improve delivery of oxygen to vital organs is a principal clinical intervention for septic patients. We previously reported that albumin resuscitation in rat endotoxemia improved contractility in isolated cardiomyocytes, but whether this effect occurs in vivo is unknown. We hypothesized that albumin resuscitation would improve decreased ventricular contractility and myocardial tissue oxygenation in vivo. DESIGN: Randomized, controlled, prospective animal study. SETTING: University animal laboratory. SUBJECTS: Male Sprague-Dawley rats (250-350 g). INTERVENTIONS: Rats were randomized into three groups: control with no lipopolysaccharide (n = 8), lipopolysaccharide (10 mg/kg) without albumin resuscitation (n = 8), and lipopolysaccharide with albumin resuscitation (n = 6). Five hours after lipopolysaccharide injection, animals were resuscitated with 10 mL/kg 5% rat albumin in 0.9% saline. Six hours after 10 mL/kg lipopolysaccharide, a pressure-volume conductance catheter (MIKRO-Tip 2.0-Fr, Millar Instruments, Houston, TX) was inserted into the left ventricle to quantify maximum elastance as an index of contractility. Myocardial tissue Po2 was measured using a fiberoptic oxygen probe. MEASUREMENTS AND MAIN RESULTS: Maximum elastance decreased after lipopolysaccharide relative to control (47%, from 5.9 +/- 0.8 to 3.1 +/- 0.4 mm Hg/microL, p < .05). Albumin resuscitation prevented the lipopolysaccharide-induced decrease in maximum elastance (7.0 +/- 1.2 mm Hg/microL, p < .05 vs. lipopolysaccharide). Myocardial tissue Po2 was reduced in endotoxemia compared with control (53%, from 10.1 +/- 0.9 to 4.7 +/- 0.6 mm Hg, p < .05), and albumin resuscitation improved the lipopolysaccharide-induced tissue hypoxia toward the control value (9.0 +/- 1.4 mm Hg, p < .05). CONCLUSIONS: Albumin resuscitation improved decreased ventricular contractility and myocardial oxygenation in endotoxemic rats. This result suggests that albumin resuscitation may improve ventricular dysfunction by improving myocardial hypoxia.

Myocardial hypoxia-inducible HIF-1alpha, VEGF, and GLUT1 gene expression is associated with microvascular and ICAM-1 heterogeneity during endotoxemia.

The systemic inflammatory response to infection is the leading cause of mortality in North American intensive-care units. Although much is known about inflammatory mediators, the relationships between microregional inflammation, microvascular heterogeneity, hypoxia, hypoxia-inducible gene expression, and myocardial dysfunction are unknown. Male Sprague-Dawley rats were injected intraperitoneally with LPS to test the hypothesis that sepsis-induced local inflammation and increased microvascular heterogeneity are spatially and temporally associated with hypoxia, hypoxia-inducible gene expression, and decreased left-ventricular contractility. Using a combination of three-dimensional microvascular imaging, tissue Po(2), and pressure-volume conductance measurements, we found that 5 h after LPS, minimum oxygen-diffusion distances increased (P < 0.05), whereas tissue oxygenation and contractility both decreased (P < 0.05) in the left ventricle. Real-time RT-PCR analysis revealed that the hypoxia-inducible genes hypoxia-inducible factor (HIF)-1alpha, VEGF, and glucose transporter (GLUT) 1 were all upregulated (P < 0.05) in the left ventricle. Tissue regions expressing ICAM-1, obtained by using laser-capture microdissection, had increased HIF-1alpha and GLUT1 (P < 0.05) gene expression. VEGF gene expression was more diffuse. In LPS rats, GLUT1 gene expression correlated (P < 0.05) with left-ventricular contractility. In 5-h hypoxic cardiomyocytes, we found strong transient HIF-1alpha, weak VEGF, and greater prolonged GLUT1 gene expression. By comparison, the HIF-1alpha-GLUT1 gene-induction pattern was reversed in the left ventricle of LPS rats. Together, these results show that LPS induces hypoxia in the left ventricle associated with increased microvascular heterogeneity and decreased contractility. HIF-1alpha and GLUT1 gene induction are related to a heterogeneous ICAM-1 expression and may be cardioprotective during the onset of septic injury.

Endotoxemia increases the clearance of mPEGylated 5000-MW quantum dots as revealed by multiphoton microvascular imaging.

Imaging the microcirculation is becoming increasingly important in assessing life-threatening disease states. To address this issue in a highly light absorbing and light scattering tissue, we use laser scanning multiphoton microscopy and fluorescent 655-nm 5000-MW methoxy-PEGylated quantum dots to image the functional microcirculation deep in mouse hind limb skeletal muscle. Using this approach, we are able to minimize in vivo background tissue autofluorescence and visualize complete 3-D microvascular units, including feeding arterioles, capillary networks, and collecting venules to depths of 150 to 200 microm. In CD1 mice treated with lipopolysaccharide to model an endotoxemic response to bacterial infection, we find that these quantum dots accumulate at microvascular bifurcations and extravasate from the microcirculation in addition to accumulating in organs (liver, spleen, lung, and kidney). The quantum dots are cleared from the circulation with a first-order elimination rate constant seven times greater than under normal conditions, 1.6+/-0.06 compared to 0.23+/-0.05 h(-1), P<0.05, thereby reducing the imaging time window. In vitro experiments using TNFalpha treated isolated leukocytes suggest that circulating monocytes (phagocytes) increased their nonspecific uptake of quantum dots when activated. In combination with multiphoton microscopy, quantum dots provide excellent in vivo imaging contrast of deep microvascular structures.

Toll-like receptor stimulation in cardiomyoctes decreases contractility and initiates an NF-kappaB dependent inflammatory response.

OBJECTIVE: The transmembrane receptor family of Toll-like receptors (TLRs) may play a role in initiating early inflammatory and functional responses to danger signals arising from ischemia-reperfusion and inflammatory stimuli. We determined whether Toll-like receptors are expressed in cardiac tissue and whether stimulation with cognate ligands would result in a pro-inflammatory response and decreased cardiomyocyte contractility. METHODS AND RESULTS: We observed mRNA expression of TLR2, TLR3, TLR4, TLR5, TLR7 and TLR9 in both whole heart tissue and a murine cardiomyocyte cell line (HL-1). Ligand activation of TLR2, TLR4 and TLR5, but not TLR3, TLR7 or TLR9, resulted in cardiomyocyte expression of the inflammatory cytokine IL-6, the chemokines KC and MIP-2, and the cell surface adhesion molecule ICAM-1. Activation of these Toll-like receptors was associated with decreased cardiomyocyte contractility. Using transfection of a nuclear factor kappa B (NF-kappaB)-Luciferase reporter plasmid, we found significantly increased NF-kappaB transcriptional activity in response to TLR2, TLR4 and TLR5 activation in cardiomyocytes. Further, a chemical inhibitor of NF-kappaB, pyrrolidine dithiocarbamate (PDTC), as well as transfection using a dominant negative form of IKKbeta, resulted in profound reduction of the TLR-initiated pro-inflammatory response. CONCLUSIONS: Cardiomyocytes express most known Toll-like receptors. Of these, TLR2, TLR4 and TLR5 signal via NF-kappaB, resulting in decreased contractility and a concerted inflammatory response.

Effect of decreased O2 supply on skeletal muscle oxygenation and O2 consumption during sepsis: role of heterogeneous capillary spacing and blood flow.

One of the main aspects of the initial phase of the septic inflammatory response to a bacterial infection is abnormal microvascular perfusion, including decreased functional capillary density (FCD) and increased blood flow heterogeneity. On the other hand, one of the most important phenomena observed in the later stages of sepsis is an increased dependence of tissue O(2) utilization on the convective O(2) supply. This "pathological supply dependency" is associated with organ failure and poor clinical outcomes. Here, a detailed theoretical model of capillary-to-tissue O(2) transport during sepsis is used to examine the origins of abnormal supply dependency. With use of three-dimensional arrays of capillaries with heterogeneous spacing and blood flow, steady-state O(2) transport is simulated numerically during reductions in the O(2) supply. Increased supply dependency is shown to occur in sepsis for hypoxic (decreased hemoglobin O(2) saturation) and stagnant (decreased blood flow) hypoxia. For stagnant hypoxia, a reduction in FCD with decreasing blood flow is necessary to obtain the observed increase in supply dependency. Our results imply that supply dependency observed under normal conditions does not have its origin at the level of individual capillaries. In sepsis, however, diffusion limitation and shunting of O(2) by individual capillaries occur to a degree that is dependent on the heterogeneity of septic injury and the arrangement of capillary networks. Thus heterogeneous stoppage of individual capillaries is a likely factor in pathological supply dependency.

Microvascular resuscitation as a therapeutic goal in severe sepsis.

Sepsis causes microvascular dysfunction. Increased heterogeneity of capillary blood flow results in local tissue hypoxia, which can cause local tissue inflammation, impaired oxygen extraction, and, ultimately, organ dysfunction. Microvascular dysfunction is clinically relevant because it is a marker for mortality: it improves rapidly in survivors of sepsis but fails to improve in nonsurvivors. This, along with the fact that resuscitation of mean arterial pressure and cardiac output alone fails to improve microvascular function, means that microvascular resuscitation is therefore a therapeutic goal. In animal studies of sepsis, volume resuscitation improves microvascular permeability and tissue oxygenation, and leads to improved organ function, including a reduction in myocardial dysfunction. Microvascular resuscitation strategies include hemodynamic resuscitation using the linked combination of volume resuscitation, judicious vasopressor use, and inotropes and vasodilators. Alternative vasoactive agents, such as vasopressin, may improve microcirculatory function to a greater degree than conventional vasopressors. Successful modulation of inflammation has a positive impact on endothelial function. Finally, targeted treatment of the endothelium, using activated protein C, also improves microvascular function and ultimately increases survival. Thus, attention must be paid to the microcirculation in patients with sepsis, and therapeutic strategies should be employed to resuscitate the microcirculation in order to avoid organ dysfunction and to reduce mortality.

Effect of sepsis on skeletal muscle oxygen consumption and tissue oxygenation: interpreting capillary oxygen transport data using a mathematical model.

Inherent in the inflammatory response to sepsis is abnormal microvascular perfusion. Maldistribution of capillary red blood cell (RBC) flow in rat skeletal muscle has been characterized by increased 1) stopped-flow capillaries, 2) capillary oxygen extraction, and 3) ratio of fast-flow to normal-flow capillaries. On the basis of experimental data for functional capillary density (FCD), RBC velocity, and hemoglobin O2 saturation during sepsis, a mathematical model was used to calculate tissue O2 consumption (Vo2), tissue Po2 (Pt) profiles, and O2 delivery by fast-flow capillaries, which could not be measured experimentally. The model describes coupled capillary and tissue O2 transport using realistic blood and tissue biophysics and three-dimensional arrays of heterogeneously spaced capillaries and was solved numerically using a previously validated scheme. While total blood flow was maintained, capillary flow distribution was varied from 60/30/10% (normal/fast/stopped) in control to 33/33/33% (normal/fast/stopped) in average sepsis (AS) and 25/25/50% (normal/fast/stopped) in extreme sepsis (ES). Simulations found approximately two- and fourfold increases in tissue Vo2 in AS and ES, respectively. Average (minimum) Pt decreased from 43 (40) mmHg in control to 34 (27) and 26 (15) mmHg in AS and ES, respectively, and clustering fast-flow capillaries (increased flow heterogeneity) reduced minimum Pt to 14.5 mmHg. Thus, although fast capillaries prevented tissue dysoxia, they did not prevent increased hypoxia as the degree of microvascular injury increased. The model predicts that decreased FCD, increased fast flow, and increased Vo2 in sepsis expose skeletal muscle to significant regions of hypoxia, which could affect local cellular and organ function.

Oxidative stress and antioxidant therapy: their impact in diabetes-associated erectile dysfunction.

Oxidative stress is believed to affect the development of diabetic-associated vasculopathy, endothelial dysfunction, and neuropathy within erectile tissue. Our hypothesis is that, given adequate concentrations of the oxygen free radical scavenger vitamin E, enhanced levels of circulating nitric oxide (NO) should improve erectile function with the potential for a synergistic effect with a phosphodiesterase type 5 (PDE5) inhibitor. Twenty adult male Sprague-Dawley streptozotocin-induced (60 mg/kg intraperitoneally) diabetic rats were placed in 4 therapeutic groups (n = 5 per group) as follows: 1) peanut oil only (diabetic control), 2) 20 IU of vitamin E per day, 3) 5 mg/kg of sildenafil per day, and 4) vitamin E plus sildenafil using oral gavage for 3 weeks. In addition, 5 age-matched rats served as normal nondiabetic controls (normal). Erectile function was assessed by measuring the rise in intracavernous pressure (ICP) following cavernous nerve electrostimulation. Penile tissue was evaluated for neuronal NO synthase (nNOS), smooth muscle alpha-actin, nitrotyrosine, and endothelial cell integrity. Urine nitrite and nitrate (NOx) concentration was quantified, and electrolytes were tested by a serum biochemistry panel. A significant decrease in ICP was recorded in the diabetic animals, with improvement measured in the animals receiving PDE5 inhibitors either with or without vitamin E; the controls had a pressure of 54.8 +/- 5.3 cm H2O, the vitamin E group had a pressure of 73.5 +/- 6.6 cm H2O, the sildenafil group had a pressure of 78.4 +/- 10.77 cm H2O, and the vitamin E plus sildenafil group had a pressure of 87.9 +/- 5.5 cm H2O (P <.05), compared with the normal cohorts at 103.0 +/- 4.8 cm H2O. Histoexaminations showed improved nNOS, endothelial cell, and smooth muscle cell staining in the vitamin E plus sildenafil group compared to the control animals. Urine NOx increased significantly in all the diabetic groups but was blunted in the vitamin E and vitamin E plus sildenafil groups. A significant increase in positive staining for nitrotyrosine was observed in the vitamin E plus sildenafil group. Vitamin E enhanced the therapeutic effect of the PDE5 inhibitor in this study, supporting the potential use of oxygen free radical scavengers in salvaging erectile function in diabetic patients.

Effect of nitric oxide on capillary hemodynamics and cell injury in the pancreas during Pseudomonas pneumonia-induced sepsis.

Sepsis-induced nitric oxide (NO) overproduction has been implicated in a redistribution of flow from the pancreas making it vulnerable to ischemic injury in septic shock. To test this hypothesis in a remote injury model of normotensive sepsis, we induced Pseudomonas pneumonia in the rat and used intravital video microscopy (IVVM) of the pancreas to measure functional capillary density, capillary hemodynamics [red blood cell (RBC) velocity, lineal density, and supply rate], and lethal cellular damage (propidium iodine staining) at 6 and 24 h after the induction of pneumonia. With pneumonia, plasma nitrite/nitrate [NO2(-)/NO3(-)(NOx(-))] levels were doubled by 21 h (P < 0.05). To assess the effect of NO overproduction on microvascular perfusion, N6-(1-iminoethyl)-L-lysine (L-NIL) was administered to maintain NOx(-) levels at baseline. Pneumonia did cause a decrease in RBC velocity of 23% by 6 h, but by 24 h RBC velocity and supply rate had increased relative to sham by 22 and 38%, respectively (P < 0.05). L-NIL treatment demonstrated that this increase was due to NO overproduction. With pneumonia, there was no change in functional capillary density and only modest increases in cellular damage. We conclude that, in this normotensive pneumonia model of sepsis, NO overproduction was protective of microvascular perfusion in the pancreas.

Bench-to-bedside review: microvascular dysfunction in sepsis--hemodynamics, oxygen transport, and nitric oxide.

The microcirculation is a complex and integrated system that supplies and distributes oxygen throughout the tissues. The red blood cell (RBC) facilitates convective oxygen transport via co-operative binding with hemoglobin. In the microcirculation oxygen diffuses from the RBC into neighboring tissues, where it is consumed by mitochondria. Evidence suggests that the RBC acts as deliverer of oxygen and 'sensor' of local oxygen gradients. Within vascular beds RBCs are distributed actively by arteriolar tone and passively by rheologic factors, including vessel geometry and RBC deformability. Microvascular oxygen transport is determined by microvascular geometry, hemodynamics, and RBC hemoglobin oxygen saturation. Sepsis causes abnormal microvascular oxygen transport as significant numbers of capillaries stop flowing and the microcirculation fails to compensate for decreased functional capillary density. The resulting maldistribution of RBC flow results in a mismatch of oxygen delivery with oxygen demand that affects both critical oxygen delivery and oxygen extraction ratio. Nitric oxide (NO) maintains microvascular homeostasis by regulating arteriolar tone, RBC deformability, leukocyte and platelet adhesion to endothelial cells, and blood volume. NO also regulates mitochondrial respiration. During sepsis, NO over-production mediates systemic hypotension and microvascular reactivity, and is seemingly protective of microvascular blood flow.

Effect of a maldistribution of microvascular blood flow on capillary O(2) extraction in sepsis.

Inherent in the remote organ injury caused by sepsis is a profound maldistribution of microvascular blood flow. Using a 24-h rat cecal ligation and perforation model of sepsis, we studied O(2) transport in individual capillaries of the extensor digitorum longus (EDL) skeletal muscle. We hypothesized that erythrocyte O(2) saturation (SO(2)) levels within normally flowing capillaries would provide evidence of either a mitochondrial failure (increased SO(2)) or an O(2) transport derangement (decreased SO(2)). Using a spectrophotometric functional imaging system, we found that sepsis caused 1) an increase in stopped flow capillaries (from 10 to 38%, P < 0.05), 2) an increase in the proportion of fast-flow to normal-flow capillaries (P < 0.05), and 3) a decrease in capillary venular-end SO(2) levels from 58.4 +/- 20.0 to 38.5 +/- 21.2%, whereas capillary arteriolar-end SO(2) levels remained unchanged compared with the sham group. Capillary O(2) extraction increased threefold (P < 0.05) and was directly related to the degree of stopped flow in the EDL. Thus impaired O(2) transport in early stage sepsis is likely the result of a microcirculatory dysfunction.