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Activated protein C improves pial microcirculation in experimental endotoxemia in rats
Microvascular Research 83 (2012) 276–280
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Activated protein C improves pial microcirculation in experimental endotoxemia in rats Juan Zhou a, b, 1, Dragan Pavlovic d, 1, Julia Willecke d, Claudius Friedel e, Sara Whynot a, Orlando Hung a, c, Vladimir Cerny a, f, Henry Schroeder e, Michael Wendt d, Romesh Shukla a, Christian Lehmann a, b, c, d,⁎ a
Department of Anesthesia, Dalhousie University, Halifax, NS, Canada Department of Microbiology and Immunology, Dalhousie University, Halifax, NS, Canada Department of Pharmacology, Dalhousie University, Halifax, NS, Canada d Department of Anesthesiology and Intensive Care Medicine, Ernst Moritz Arndt University Greifswald, Germany e Department of Neurosurgery, Ernst Moritz Arndt University Greifswald, Germany f Department of Anesthesiology and Intensive Care Medicine, Charles University Prague, Faculty of Medicine Hradec Kralove, Hradec Kralove, Czech Republic b c
a r t i c l e
i n f o
Article history: Accepted 1 March 2012 Available online 9 March 2012
a b s t r a c t Introduction: The brain is one of the first organs affected clinically in sepsis. Microcirculatory alterations are suggested to be a critical component in the pathophysiology of sepsis. The aim of this study was to investigate the effects of recombinant human activated protein C (rhAPC) on the pial microcirculation in experimental endotoxemia using intravital microscopy. Our hypothesis is rhAPC protects pial microcirculation in endotoxemia. Methods: Endotoxemia was generated in Lewis rats with intravenous injection of lipopolysaccharide (LPS, 5 mg/kg i.v.). Dura mater was removed through a cranial window to expose pial vessels on the brain surface. The microcirculation, including leukocyte–endothelial interaction, functional capillary density (FCD) and plasma extravasation of pial vessels was examined by fluorescent intravital microscopy (IVM) 2 h after administration of LPS, LPS and rhAPC or equivalent amount of saline (used as Control group). Plasma cytokine levels of interleukin 1 alpha (IL1-α), tumor necrosis factor-α (TNF-α), interferon γ (IFN-γ), Monocyte chemotactic protein-1 (MCP-1) and Granulocyte-macrophage colony-stimulating factor (GM-CSF) were evaluated after IVM. Results: LPS challenge significantly increased leukocyte adhesion (773 ± 190 vs. 592 ± 152 n/mm2 Control), decreased FCD (218 ± 54 vs. 418 ± 74 cm/cm 2 Control) and increased proinflammatory cytokine levels (IL1α: 5032 ± 1502 vs. 8 ± 21 pg/ml; TNF-α: 1823 ± 1007 vs. 168 ± 228 pg/ml; IFN-γ: 785 ± 434 vs. 0 pg/ml; GM-CSF: 54 ± 52 vs. 1 ± 3 pg/ml) compared to control animals. rhAPC treatment significantly reduced leukocyte adhesion (599 ± 111 n/mm2), increased FCD (516 ± 118 cm/cm2) and reduced IL-1α levels (2134 ± 937 pg/ml) in the endotoxemic rats. Conclusion: APC treatment significantly improves pial microcirculation by reducing leukocyte adhesion and increasing FCD. © 2012 Elsevier Inc. All rights reserved.
Introduction Sepsis is the leading cause of death in surgical intensive care units. In response to systemic inflammation, large amounts of proinflammatory cytokines are released into the circulation, which cause multiple organ failure (Streck et al., 2008; Wang and Ma, 2008). The brain is one of the initial organs affected in sepsis (septic encephalopathy). Abbreviations: rhAPC, Recombinant human activated protein C; FCD, Functional capillary density; DCD, Dysfunctional capillary density; NCD, Nonfunctional capillary density; IVM, Fluorescent intravital microscopy. ⁎ Corresponding author at: Dalhousie University, 5850 College Street, 6H Tupper Building, Halifax, NS, B3H 1X5, Canada. E-mail address: [email protected] (C. Lehmann). 1 First and second author equally contributed to the manuscript. 0026-2862/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2012.03.003
Although the mechanisms are not completely elucidated, disruption of the blood brain barrier and disturbed microcirculation have been proposed as major factors for the pathogenesis of septic encephalopathy (Pytel and Alexander, 2009). The brain has an immunologic advantage due to its anatomical separation from the immune system by the blood brain barrier (BBB), a lack of lymphatic drainage system, and low expression of histocompatibility complex antigens. The BBB plays an important role in controlling entry of inflammatory cells and macromolecules into the brain. Entry is achieved via selective permeability; a feature mediated by tight junctions of microvascular endothelial cells and associated astrocytes (Pytel and Alexander, 2009). Conflicting evidence exists showing both increased permeability of the BBB in septic encephalopathy (Mayhan, 1998; Siami et al., 2008) and unchanged BBB
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permeability with intact tight junctions of endothelial cell in endotoxemic animals (Bickel et al., 1998; Rosengarten et al., 2008). Activated protein C (APC) is an endogenous protein converted from its inactive precursor, protein C by a thrombin–thrombomodulin complex on the endothelial surface (Esmon, 2000). It promotes fibrinolysis, inhibits thrombosis and also exhibits anti-inflammatory properties. Several clinical studies in pediatric and adult patients with sepsis found decreased plasma protein C levels in comparison to controls (Boldt et al., 2000; Donati et al., 2009; Venkataseshan et al., 2007). Early data in a subset of sepsis patients suggested a survival benefit by APC substitution (Bernard et al., 2001). However, this could not be confirmed in recent studies (Angus, 2012). The effect of rhAPC on brain microcirculation has not been clearly demonstrated. In the present study we examined the effects of rhAPC on brain microcirculation in an in vivo endotoxemia rat model by observing leukocyte and vascular endothelia interactions, capillary perfusion and leakage of pial vessels — a microvasculature accessible for fluorescent intravital microscopy (IVM) through a cranial window. Material and methods Animals Thirty male Lewis rats (weight 250–300 g) were purchased from Charles River Laboratories (Sulzfeld, Germany) and maintained in the University Hospital Greifswald animal care facility. Animals were provided with water and rodent chow ad libitum under standard 12-h light/dark rhythmic conditions. All animal experimental procedures were performed in accordance with the guidelines of German animal safety legislations and approved by the Institutional Review Board of Animal Care. Anesthesia and preparation Anesthesia was induced with intraperitoneal (i.p.) injection of sodium pentobarbital (65 mg/kg, Synopharm, Barsbuttel, Germany) and maintained by repeated intravenous (i.v.) injections of pentobarbital (15 mg/kg). To maintain stable body temperature, rats were placed in the dorsal position on a heating pad. Core temperature was monitored by a rectal probe. Animals received a tracheotomy to permit airway access; however, spontaneous ventilation using room air was used during the experiments. The femoral artery was cannulated with a polyethylene catheter (PE 50, internal diameter 0.58 mm, external diameter 0.96 mm, Portex, Hythe, Kent, UK) for continuous measurement of arterial blood pressure (MAP) and heart rate (HR) using a Hewlett Packard monitor (Model M1092A, Saronno, Italy). The femoral vein was also cannulated with a polyethylene catheter (internal diameter 0.28 mm, external diameter 0.96 mm, Portex, Hythe, Kent, UK) for fluid maintenance (normal saline: 0.9% Sodium Chloride at a continuous rate of 2 ml/h), bolus administration of lipopolysaccharide (LPS, Escherichia coli, serotype O26:B6, Sigma-Aldrich, Steinheim, Germany) and/or rhAPC. Animals were randomly assigned to one of the three groups (n = 10 per group): 1) LPS group: received 5 mg/kg LPS; 2) LPS + APC group: received 5 mg/kg LPS followed by 2 mg/kg rhAPC immediately (Xigris®; Lilly Deutschland GmbH, Bad Homburg, Germany); 3) Control group: received normal saline at an equal volume of LPS and APC. LPS or normal saline was administrated at time 0 and rhAPC was given at time 10 min right after LPS administration. Cranial window surgery After stabilization in a stereotactic frame, a midline incision of the scalp was made. The underlying soft tissue was removed and bleeding stopped. Using a low speed drill, a cranial window (approximately 3 × 3 mm) was made right of the middle sagittal line between the
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bregma and lambdoid sutures. Constant irrigation with an artificial cerebrospinal fluid (as described in (Sarker et al., 2000)) was used during drilling to avoid brain overheating. Then, dura mater was removed using micro-iris scissors (Codman & Shurtleff Inc., Raynham, MA, USA) and the cerebral surface was superfused with a continuous flow of artificial cerebrospinal fluid at a rate of 0.5 ml/h. Intravital microscopy (IVM) Two hours after injection of LPS or normal saline, rats were injected with 0.05% Rhodamine 6 G (Sigma, Deisenhofen, Germany) solution (0.75 mg/kg i.v.) for leukocyte staining and 5% fluorescein isothiocyanate (FITC)-BSA (Sigma) solution (50 mg/kg i.v.) for visualizing plasma extravasation. Intravital microscopy through the cranial window was performed to observe leukocyte–endothelium interaction, capillary perfusion and plasma extravasation. The fluorescence microscope (Carl Zeiss, Jena, Germany) consisted of an epifluorescence Axiotech Vario; a light source HBO 50; oculars 10× and objective lens 20 ×/0.5; a filter type #10 and #20. Five video sequences (30 s each) of randomly chosen pial vessels were captured by a black and white-CCD video camera (BC-12, AVT-Horn Aalen, Germany) and recorded by a S-VHS video tape recorder (Panasonic NVSV120EG-S, Matsushita Audio Video, Germany) connected to a black and white monitor (14-inch PM-159, Ikegami Electronics, Germany). Leukocytes interacting with endothelial cells in pial blood vessels (diameter b 100 μm) were classified as either adherent or rolling leukocytes. Leukocytes adherent to the endothelia for 30 s were defined as adherent leukocytes. Leukocyte adhesion is expressed as the number of adherent leukocytes per mm 2 of endothelial surface, calculated from diameter and length of the vessel segment studied, assuming cylindrical geometry. Leukocytes which interacted with but did not adhere to the endothelia were defined as rolling leukocytes. Roller flow is defined as the number of nonadherent, rolling leukocytes passing through the observed vessel segment within a 30-s window. To evaluate vascular perfusion, functional capillary density (FCD: continuous flow) was analyzed. FCD is the length of capillaries with observable erythrocyte perfusion in relation to a predetermined rectangular field (unit: cm/cm 2) as defined by Schmid-Schoenbein et al. (1977). In addition, dysfunctional capillary density (DCD: intermittent perfusion) and nonfunctional capillary density (NCD: no flow) were also analyzed. To measure plasma extravasation across pial vessels, the intensity of FITC fluorescence both inside and outside of the venous vessels was quantified. This ratio is defined as plasma extravasation. All evaluations, including leukocyte adhesion, roller flow, capillary density and plasma extravasation, were performed in a blinded fashion. Cytokine analysis Blood samples were taken at the end of the experiments (at time 135 min after LPS or normal saline administration). Plasma was separated and stored at −80 °C. Cytokine levels were measured using a rat flowcytomix multiplex (Bender Medsystems, Vienna, Austria) for interleukin 1α (IL-1α), tumor necrosis factor-α (TNF-α), interferon γ (IFN-γ), Monocyte chemotactic protein-1 (MCP-1) and Granulocytemacrophage colony-stimulating factor (GM-CSF) according to the manufacturer's instructions. Statistical analysis All the data were analyzed with statistical software, Prism (Graphpad Inc., La Jolla, CA, USA), by which normal distribution of data was tested using the Kolmogorov–Smirnov test. Mean arterial pressure, heart rate and core temperature were analyzed by a two-way analysis of variance (repeated measures in the factor of time), followed by the Bonferroni test. Other data were analyzed by a one-way analysis of
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variance, followed by the Newman–Keuls multiple comparison test. Data are presented as mean ± standard deviation. Probability (p) values less than 0.05 were considered significant.
Results Vital parameters Mean arterial pressure (MAP) and heart rate (HR) remained consistent in control animals (Figs. 1a,b). LPS administration reduced MAP and elevated HR significantly (p b 0.05). rhAPC treatment right after LPS challenge did not change MAP and HR significantly in comparison to endotoxemic animals (Figs. 1a,b). Core body temperature of animals remained within normal limits (37.2 ± 0.8 °C) in all groups of animals throughout the experiment without significant differences between groups (Fig. 1c).
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Leukocyte–endothelium interaction The number of leukocytes firmly adherent to the endothelia is shown in Fig. 2a. Challenge with LPS for 2 h increased (773 ± 190 vs. 592 ± 152 n/mm 2 Control, p b 0.05) leukocytes adherent to the endothelia. Treatment with rhAPC right after LPS administration significantly reduced (599 ± 111 vs. 773 ± 190 n/mm 2 LPS, p b 0.05) leukocyte adhesion compared to that in LPS challenged animals. Fig. 2b shows the number of rolling leukocytes in a 30-s observed period in control, LPS and rhAPC treated animals. LPS challenge decreased (p b 0.05) the number of rolling leukocytes, whereas rhAPC treatment did not change the roller flow in the endotoxemic rats. Functional, dysfunctional and nonfunctional capillary density To investigate capillary flow within the microcirculation, functional, dysfunctional and nonfunctional capillary density was studied. rhAPC treatment improved the FCD, and reduced dysfunctional capillary density (DCD) and nonfunctional capillary density (NCD) in endotoxemic animals significantly (p b 0.05, Fig. 3). Plasma extravasation
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LPS challenge significantly increased (p b 0.05) the levels of inflammatory cytokines such as IL-1α, TNF-α, IFN-γ and GM-CSF compared to the control groups (Table 1). However, rhAPC treatment decreased IL-1α significantly in the endotoxemic rats (p b 0.05). The
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There was a trend of increased plasma extravasation in LPS challenged rats compared to control animals whereas rhAPC treatment reduced plasma extravasation compared to LPS challenged rats (Fig. 4). However, the changes of extravasation between these groups did not reach statistical significance (p > 0.05).
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Fig. 1. Vital parameters. a) Mean arterial pressure (MAP, mm Hg), b) heart rate (HR, beats/min) and c) rectal temperature (Temperature, °C) in control (Control), endotoxemic (LPS) and rhAPC treated endotoxemic (LPS + APC) animals (n = 10 per group, mean ± standard deviation). * significant differences: Control vs. LPS and Control vs. LPS + APC groups (p b 0.05). LPS or saline was administered at time 0 and rhAPC was administered at time 10 min after LPS injection.
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Fig. 2. a) Leukocyte adhesion (n/mm2) and b) roller flow (n/min) in the pial microvasculature of control (Control), endotoxemic (LPS) and rhAPC treated endotoxemic (LPS+ APC) rats (n = 10 per group, mean ± standard deviation). * significant difference: Control vs. LPS.# significant difference: LPS vs. LPS + APC (p b 0.05). LPS or saline was administered at time 0 and rhAPC was administered at time 10 min after LPS injection.
J. Zhou et al. / Microvascular Research 83 (2012) 276–280
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Fig. 3. a) Functional capillary density (FCD, cm/cm2), b) dysfunctional capillary density (DCD, cm/cm2) and c) nonfunctional capillary density (NCD, cm/cm2) in the pial vessels of control (Control), endotoxemic (LPS) and rhAPC treated endotoxemic (LPS + APC) rats (n= 10 per group, mean± standard deviation). * significant difference: Control vs. LPS.# significant difference: LPS vs. LPS + APC (pb 0.05). LPS or saline was administered at time 0 and rhAPC was administered at time 10 min after LPS injection.
levels of MCP-1 did not show significant differences between LPS, APC and control groups (Table 1).
Discussion We demonstrated, in this study, that rhAPC treatment significantly attenuates deterioration of pial microcirculation during sepsis by reducing leukocyte adhesion to endothelia and improving functional capillary density of pial vessels. In addition, rhAPC treatment significantly decreased systemic IL-1α release. Proposed mechanisms relevant to sepsis 1.00 0.95
PE %
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8.58 ± 21.12 168.2 ± 228.0 0 0 796.1 ± 378.6 1.0 ± 2.89
5032 ± 1502* 1823 ± 1007* 785.1 ± 433.6* 7.0 ± 14.06 445.1 ± 503.1 54.00 ± 51.72*
2134 ± 937.1*# 1678 ± 1053* 838.1 ± 504.2* 7.5 ± 15.61 454.6 ± 533.2 80.50 ± 73.64*
Plasma cytokine concentration (pg/ml) in control (Control), endotoxemic (LPS) and rhAPC treated endotoxemic (LPS + APC) rats (n = 10 per group, mean ± standard deviation). * indicates p b 0.05 compared to control. # indicates p b 0.05 compared to LPS. IL-1α: interleukin 1α, TNF-α: tumor necrosis factor-α, IFN-γ: interferon γ, MCP1: Monocyte chemotactic protein-1 and GM-CSF: Granulocyte-macrophage colonystimulating factor.
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Fig. 4. Plasma exvasation (PE, %) in the pial vessels of control (Control), endotoxemic (LPS) and rhAPC treated endotoxemic (LPS + APC) rats (n = 10 per group, mean ± standard deviation).
may include anticoagulation, fibrinolysis, anti-inflammation and preservation of endothelial permeability barrier function (Macias et al., 2005). In vitro experiments showed that rhAPC inhibits leukocyte response to chemotactic signals (Sturn et al., 2003) and suppresses E-selectin mediated cell adhesion to the vascular endothelia (Iba et al., 2005). In addition, rhAPC suppresses cytokine-induced upregulation of adhesion molecules in cultured endothelial cells and diminishes the expression of inflammatory cytokines and chemokines (Galley et al., 2008; Hoffmann et al., 2004). Furthermore, rhAPC directly binds to β integrins and inhibits human neutrophil adhesion to matrix proteins therefore preventing neutrophil migration (Elphick et al., 2009). Such in vitro evidence supports our in vivo observation that rhAPC treatment in endotoxemic rats reduces leukocyte adhesion to endothelia in pial vessels and this reduction may be due to the reduced expression of adhesion molecules on leukocytes and endothelial cells. Similar results were also reported in several other endotoxemia experimental models (Hoffmann et al., 2004; Iba et al., 2005; Lehmann et al., 2006, 2008a). We have previously demonstrated that rhAPC treatment in healthy non-endotoxemic rats does not show any effect on vital parameters, and on leukocyte adhesion, vessel perfusion and cytokine secretion in intestinal (Lehmann et al., 2008a) and mesenteric microcirculation (Lehmann et al., 2008b), suggesting that rhAPC alone do not have toxic effect. Functional capillary density (FCD) is another indicator used to evaluate the function of microcirculation in sepsis. Microcirculation abnormality in sepsis includes decreases in FCD and increases in the proportion of nonperfused or intermittently perfused capillaries. APC treatment in sepsis improved microcirculation alterations in septic patients (De Backer et al., 2006). In our study we demonstrated that rhAPC treatment significantly reversed the impairment of capillary density by increasing FCD and decreasing NCD and DCD. Although APC is an anti-coagulator and plays a role in preventing clot formation in the microcirculation, it is believed that both antiinflammatory and anti-coagulatory are responsible for its effect on capillary perfusion. Plasma extravasation represents a clinically relevant characteristic of microcirculation deterioration due to tissue edema and decreased perfusion pressures caused by relative hypovolemia (Lehmann et al., 2004). In our experiments, plasma extravasation tended to increase in endotoxemia rats 2 h after LPS administration and decreased after rhAPC treatment. However, none of the changes reached statistical significance. Since we have previously demonstrated that LPS increased plasma extravasation significantly at 4 h after LPS administration (Zhou et al., 2011), the results in the present study suggest that 2-h endotoxemia is not severe enough to generate significant plasma extravasation in pial vessels. Systemic inflammatory cytokine levels have been shown to be elevated in sepsis patients, including IL-1, IL-6, IFN-γ and TNF-α (Kawasaki et al., 1999; Wang and Ma, 2008) and may be reduced by APC treatment due to its anti-inflammatory effect. In animal models, APC significantly inhibited ischemia/reperfusion-induced increase of
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TNF-α and IL-8 (Mizutani et al., 2000) and prevented pulmonary vascular injury by inhibiting cytokine production (Murakami et al., 1997). APC treatment also reduced LPS induced increase of TNF-α and IL-6 production (Iba et al., 2005). However, APC did not reduce the cytokine production of IL-1 and IL-8 by human neutrophils stimulated by LPS in vitro (Galley et al., 2008) and APC only reduced IL-1 but not TNF-α production in endotoxemic rats (Lehmann et al., 2006, 2008a, 2008b). These findings suggest that the effect of APC on inflammatory cytokine production is unclear and observed differences could be related to the experimental model used. In summary we have demonstrated, using intravital microscopy, that administration of endotoxin LPS significantly deteriorates brain microcirculation. Recombinant human APC treatment significantly attenuated the deterioration of brain microcirculation induced by endotoxin and reduces IL-1α production. The beneficial effects of rhAPC may play an important role in the management of patients with sepsis due to the fundamental role of the microcirculation in the pathogenesis of sepsis and multiple organ dysfunction. Acknowledgments We wish to express our gratitude to Sabine Will and Annette Wegner for their excellent contributions in the preparation of the experiments and Roswitha Dressler and Raila Busch for the cytokine measurements. References Angus, D.C., 2012. Drotrecogin alfa (activated) … a sad final fizzle to a roller-coaster party. Crit. Care 16, 107. Bernard, G.R., Vincent, J.L., Laterre, P.F., LaRosa, S.P., Dhainaut, J.F., Lopez-Rodriguez, A., et al., 2001. Efficacy and safety of recombinant human activated protein C for severe sepsis. N. Engl. J. Med. 344, 699–709. Bickel, U., Grave, B., Kang, Y.S., del Rey, A., Voigt, K., 1998. No increase in blood–brain barrier permeability after intraperitoneal injection of endotoxin in the rat. J. Neuroimmunol. 85, 131–136. Boldt, J., Papsdorf, M., Rothe, A., Kumle, B., Piper, S., 2000. Changes of the hemostatic network in critically ill patients — is there a difference between sepsis, trauma, and neurosurgery patients? Crit. Care Med. 28, 445–450. De Backer, D., Verdant, C., Chierego, M., Koch, M., Gullo, A., Vincent, J.L., 2006. Effects of drotrecogin alfa activated on microcirculatory alterations in patients with severe sepsis. Crit. Care Med. 34, 1918–1924. Donati, A., Romanelli, M., Botticelli, L., Valentini, A., Gabbanelli, V., Nataloni, S., et al., 2009. Recombinant activated protein C treatment improves tissue perfusion and oxygenation in septic patients measured by near-infrared spectroscopy. Crit. Care 13 (Suppl. 5), S12. Elphick, G.F., Sarangi, P.P., Hyun, Y.M., Hollenbaugh, J.A., Ayala, A., Biffl, W.L., et al., 2009. Recombinant human activated protein C inhibits integrin-mediated neutrophil migration. Blood 113, 4078–4085. Esmon, C.T., 2000. Regulation of blood coagulation. Biochim. Biophys. Acta 1477, 349–360. Galley, H.F., El Sakka, N.E., Webster, N.R., Lowes, D.A., Cuthbertson, B.H., 2008. Activated protein C inhibits chemotaxis and interleukin-6 release by human neutrophils without affecting other neutrophil functions. Br. J. Anaesth. 100, 815–819.
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Microvascular Research journal homepage: www.elsevier.com/locate/ymvre
Activated protein C improves pial microcirculation in experimental endotoxemia in rats Juan Zhou a, b, 1, Dragan Pavlovic d, 1, Julia Willecke d, Claudius Friedel e, Sara Whynot a, Orlando Hung a, c, Vladimir Cerny a, f, Henry Schroeder e, Michael Wendt d, Romesh Shukla a, Christian Lehmann a, b, c, d,⁎ a
Department of Anesthesia, Dalhousie University, Halifax, NS, Canada Department of Microbiology and Immunology, Dalhousie University, Halifax, NS, Canada Department of Pharmacology, Dalhousie University, Halifax, NS, Canada d Department of Anesthesiology and Intensive Care Medicine, Ernst Moritz Arndt University Greifswald, Germany e Department of Neurosurgery, Ernst Moritz Arndt University Greifswald, Germany f Department of Anesthesiology and Intensive Care Medicine, Charles University Prague, Faculty of Medicine Hradec Kralove, Hradec Kralove, Czech Republic b c
a r t i c l e
i n f o
Article history: Accepted 1 March 2012 Available online 9 March 2012
a b s t r a c t Introduction: The brain is one of the first organs affected clinically in sepsis. Microcirculatory alterations are suggested to be a critical component in the pathophysiology of sepsis. The aim of this study was to investigate the effects of recombinant human activated protein C (rhAPC) on the pial microcirculation in experimental endotoxemia using intravital microscopy. Our hypothesis is rhAPC protects pial microcirculation in endotoxemia. Methods: Endotoxemia was generated in Lewis rats with intravenous injection of lipopolysaccharide (LPS, 5 mg/kg i.v.). Dura mater was removed through a cranial window to expose pial vessels on the brain surface. The microcirculation, including leukocyte–endothelial interaction, functional capillary density (FCD) and plasma extravasation of pial vessels was examined by fluorescent intravital microscopy (IVM) 2 h after administration of LPS, LPS and rhAPC or equivalent amount of saline (used as Control group). Plasma cytokine levels of interleukin 1 alpha (IL1-α), tumor necrosis factor-α (TNF-α), interferon γ (IFN-γ), Monocyte chemotactic protein-1 (MCP-1) and Granulocyte-macrophage colony-stimulating factor (GM-CSF) were evaluated after IVM. Results: LPS challenge significantly increased leukocyte adhesion (773 ± 190 vs. 592 ± 152 n/mm2 Control), decreased FCD (218 ± 54 vs. 418 ± 74 cm/cm 2 Control) and increased proinflammatory cytokine levels (IL1α: 5032 ± 1502 vs. 8 ± 21 pg/ml; TNF-α: 1823 ± 1007 vs. 168 ± 228 pg/ml; IFN-γ: 785 ± 434 vs. 0 pg/ml; GM-CSF: 54 ± 52 vs. 1 ± 3 pg/ml) compared to control animals. rhAPC treatment significantly reduced leukocyte adhesion (599 ± 111 n/mm2), increased FCD (516 ± 118 cm/cm2) and reduced IL-1α levels (2134 ± 937 pg/ml) in the endotoxemic rats. Conclusion: APC treatment significantly improves pial microcirculation by reducing leukocyte adhesion and increasing FCD. © 2012 Elsevier Inc. All rights reserved.
Introduction Sepsis is the leading cause of death in surgical intensive care units. In response to systemic inflammation, large amounts of proinflammatory cytokines are released into the circulation, which cause multiple organ failure (Streck et al., 2008; Wang and Ma, 2008). The brain is one of the initial organs affected in sepsis (septic encephalopathy). Abbreviations: rhAPC, Recombinant human activated protein C; FCD, Functional capillary density; DCD, Dysfunctional capillary density; NCD, Nonfunctional capillary density; IVM, Fluorescent intravital microscopy. ⁎ Corresponding author at: Dalhousie University, 5850 College Street, 6H Tupper Building, Halifax, NS, B3H 1X5, Canada. E-mail address: [email protected] (C. Lehmann). 1 First and second author equally contributed to the manuscript. 0026-2862/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2012.03.003
Although the mechanisms are not completely elucidated, disruption of the blood brain barrier and disturbed microcirculation have been proposed as major factors for the pathogenesis of septic encephalopathy (Pytel and Alexander, 2009). The brain has an immunologic advantage due to its anatomical separation from the immune system by the blood brain barrier (BBB), a lack of lymphatic drainage system, and low expression of histocompatibility complex antigens. The BBB plays an important role in controlling entry of inflammatory cells and macromolecules into the brain. Entry is achieved via selective permeability; a feature mediated by tight junctions of microvascular endothelial cells and associated astrocytes (Pytel and Alexander, 2009). Conflicting evidence exists showing both increased permeability of the BBB in septic encephalopathy (Mayhan, 1998; Siami et al., 2008) and unchanged BBB
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permeability with intact tight junctions of endothelial cell in endotoxemic animals (Bickel et al., 1998; Rosengarten et al., 2008). Activated protein C (APC) is an endogenous protein converted from its inactive precursor, protein C by a thrombin–thrombomodulin complex on the endothelial surface (Esmon, 2000). It promotes fibrinolysis, inhibits thrombosis and also exhibits anti-inflammatory properties. Several clinical studies in pediatric and adult patients with sepsis found decreased plasma protein C levels in comparison to controls (Boldt et al., 2000; Donati et al., 2009; Venkataseshan et al., 2007). Early data in a subset of sepsis patients suggested a survival benefit by APC substitution (Bernard et al., 2001). However, this could not be confirmed in recent studies (Angus, 2012). The effect of rhAPC on brain microcirculation has not been clearly demonstrated. In the present study we examined the effects of rhAPC on brain microcirculation in an in vivo endotoxemia rat model by observing leukocyte and vascular endothelia interactions, capillary perfusion and leakage of pial vessels — a microvasculature accessible for fluorescent intravital microscopy (IVM) through a cranial window. Material and methods Animals Thirty male Lewis rats (weight 250–300 g) were purchased from Charles River Laboratories (Sulzfeld, Germany) and maintained in the University Hospital Greifswald animal care facility. Animals were provided with water and rodent chow ad libitum under standard 12-h light/dark rhythmic conditions. All animal experimental procedures were performed in accordance with the guidelines of German animal safety legislations and approved by the Institutional Review Board of Animal Care. Anesthesia and preparation Anesthesia was induced with intraperitoneal (i.p.) injection of sodium pentobarbital (65 mg/kg, Synopharm, Barsbuttel, Germany) and maintained by repeated intravenous (i.v.) injections of pentobarbital (15 mg/kg). To maintain stable body temperature, rats were placed in the dorsal position on a heating pad. Core temperature was monitored by a rectal probe. Animals received a tracheotomy to permit airway access; however, spontaneous ventilation using room air was used during the experiments. The femoral artery was cannulated with a polyethylene catheter (PE 50, internal diameter 0.58 mm, external diameter 0.96 mm, Portex, Hythe, Kent, UK) for continuous measurement of arterial blood pressure (MAP) and heart rate (HR) using a Hewlett Packard monitor (Model M1092A, Saronno, Italy). The femoral vein was also cannulated with a polyethylene catheter (internal diameter 0.28 mm, external diameter 0.96 mm, Portex, Hythe, Kent, UK) for fluid maintenance (normal saline: 0.9% Sodium Chloride at a continuous rate of 2 ml/h), bolus administration of lipopolysaccharide (LPS, Escherichia coli, serotype O26:B6, Sigma-Aldrich, Steinheim, Germany) and/or rhAPC. Animals were randomly assigned to one of the three groups (n = 10 per group): 1) LPS group: received 5 mg/kg LPS; 2) LPS + APC group: received 5 mg/kg LPS followed by 2 mg/kg rhAPC immediately (Xigris®; Lilly Deutschland GmbH, Bad Homburg, Germany); 3) Control group: received normal saline at an equal volume of LPS and APC. LPS or normal saline was administrated at time 0 and rhAPC was given at time 10 min right after LPS administration. Cranial window surgery After stabilization in a stereotactic frame, a midline incision of the scalp was made. The underlying soft tissue was removed and bleeding stopped. Using a low speed drill, a cranial window (approximately 3 × 3 mm) was made right of the middle sagittal line between the
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bregma and lambdoid sutures. Constant irrigation with an artificial cerebrospinal fluid (as described in (Sarker et al., 2000)) was used during drilling to avoid brain overheating. Then, dura mater was removed using micro-iris scissors (Codman & Shurtleff Inc., Raynham, MA, USA) and the cerebral surface was superfused with a continuous flow of artificial cerebrospinal fluid at a rate of 0.5 ml/h. Intravital microscopy (IVM) Two hours after injection of LPS or normal saline, rats were injected with 0.05% Rhodamine 6 G (Sigma, Deisenhofen, Germany) solution (0.75 mg/kg i.v.) for leukocyte staining and 5% fluorescein isothiocyanate (FITC)-BSA (Sigma) solution (50 mg/kg i.v.) for visualizing plasma extravasation. Intravital microscopy through the cranial window was performed to observe leukocyte–endothelium interaction, capillary perfusion and plasma extravasation. The fluorescence microscope (Carl Zeiss, Jena, Germany) consisted of an epifluorescence Axiotech Vario; a light source HBO 50; oculars 10× and objective lens 20 ×/0.5; a filter type #10 and #20. Five video sequences (30 s each) of randomly chosen pial vessels were captured by a black and white-CCD video camera (BC-12, AVT-Horn Aalen, Germany) and recorded by a S-VHS video tape recorder (Panasonic NVSV120EG-S, Matsushita Audio Video, Germany) connected to a black and white monitor (14-inch PM-159, Ikegami Electronics, Germany). Leukocytes interacting with endothelial cells in pial blood vessels (diameter b 100 μm) were classified as either adherent or rolling leukocytes. Leukocytes adherent to the endothelia for 30 s were defined as adherent leukocytes. Leukocyte adhesion is expressed as the number of adherent leukocytes per mm 2 of endothelial surface, calculated from diameter and length of the vessel segment studied, assuming cylindrical geometry. Leukocytes which interacted with but did not adhere to the endothelia were defined as rolling leukocytes. Roller flow is defined as the number of nonadherent, rolling leukocytes passing through the observed vessel segment within a 30-s window. To evaluate vascular perfusion, functional capillary density (FCD: continuous flow) was analyzed. FCD is the length of capillaries with observable erythrocyte perfusion in relation to a predetermined rectangular field (unit: cm/cm 2) as defined by Schmid-Schoenbein et al. (1977). In addition, dysfunctional capillary density (DCD: intermittent perfusion) and nonfunctional capillary density (NCD: no flow) were also analyzed. To measure plasma extravasation across pial vessels, the intensity of FITC fluorescence both inside and outside of the venous vessels was quantified. This ratio is defined as plasma extravasation. All evaluations, including leukocyte adhesion, roller flow, capillary density and plasma extravasation, were performed in a blinded fashion. Cytokine analysis Blood samples were taken at the end of the experiments (at time 135 min after LPS or normal saline administration). Plasma was separated and stored at −80 °C. Cytokine levels were measured using a rat flowcytomix multiplex (Bender Medsystems, Vienna, Austria) for interleukin 1α (IL-1α), tumor necrosis factor-α (TNF-α), interferon γ (IFN-γ), Monocyte chemotactic protein-1 (MCP-1) and Granulocytemacrophage colony-stimulating factor (GM-CSF) according to the manufacturer's instructions. Statistical analysis All the data were analyzed with statistical software, Prism (Graphpad Inc., La Jolla, CA, USA), by which normal distribution of data was tested using the Kolmogorov–Smirnov test. Mean arterial pressure, heart rate and core temperature were analyzed by a two-way analysis of variance (repeated measures in the factor of time), followed by the Bonferroni test. Other data were analyzed by a one-way analysis of
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variance, followed by the Newman–Keuls multiple comparison test. Data are presented as mean ± standard deviation. Probability (p) values less than 0.05 were considered significant.
Results Vital parameters Mean arterial pressure (MAP) and heart rate (HR) remained consistent in control animals (Figs. 1a,b). LPS administration reduced MAP and elevated HR significantly (p b 0.05). rhAPC treatment right after LPS challenge did not change MAP and HR significantly in comparison to endotoxemic animals (Figs. 1a,b). Core body temperature of animals remained within normal limits (37.2 ± 0.8 °C) in all groups of animals throughout the experiment without significant differences between groups (Fig. 1c).
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Leukocyte–endothelium interaction The number of leukocytes firmly adherent to the endothelia is shown in Fig. 2a. Challenge with LPS for 2 h increased (773 ± 190 vs. 592 ± 152 n/mm 2 Control, p b 0.05) leukocytes adherent to the endothelia. Treatment with rhAPC right after LPS administration significantly reduced (599 ± 111 vs. 773 ± 190 n/mm 2 LPS, p b 0.05) leukocyte adhesion compared to that in LPS challenged animals. Fig. 2b shows the number of rolling leukocytes in a 30-s observed period in control, LPS and rhAPC treated animals. LPS challenge decreased (p b 0.05) the number of rolling leukocytes, whereas rhAPC treatment did not change the roller flow in the endotoxemic rats. Functional, dysfunctional and nonfunctional capillary density To investigate capillary flow within the microcirculation, functional, dysfunctional and nonfunctional capillary density was studied. rhAPC treatment improved the FCD, and reduced dysfunctional capillary density (DCD) and nonfunctional capillary density (NCD) in endotoxemic animals significantly (p b 0.05, Fig. 3). Plasma extravasation
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LPS challenge significantly increased (p b 0.05) the levels of inflammatory cytokines such as IL-1α, TNF-α, IFN-γ and GM-CSF compared to the control groups (Table 1). However, rhAPC treatment decreased IL-1α significantly in the endotoxemic rats (p b 0.05). The
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There was a trend of increased plasma extravasation in LPS challenged rats compared to control animals whereas rhAPC treatment reduced plasma extravasation compared to LPS challenged rats (Fig. 4). However, the changes of extravasation between these groups did not reach statistical significance (p > 0.05).
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Fig. 1. Vital parameters. a) Mean arterial pressure (MAP, mm Hg), b) heart rate (HR, beats/min) and c) rectal temperature (Temperature, °C) in control (Control), endotoxemic (LPS) and rhAPC treated endotoxemic (LPS + APC) animals (n = 10 per group, mean ± standard deviation). * significant differences: Control vs. LPS and Control vs. LPS + APC groups (p b 0.05). LPS or saline was administered at time 0 and rhAPC was administered at time 10 min after LPS injection.
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Fig. 2. a) Leukocyte adhesion (n/mm2) and b) roller flow (n/min) in the pial microvasculature of control (Control), endotoxemic (LPS) and rhAPC treated endotoxemic (LPS+ APC) rats (n = 10 per group, mean ± standard deviation). * significant difference: Control vs. LPS.# significant difference: LPS vs. LPS + APC (p b 0.05). LPS or saline was administered at time 0 and rhAPC was administered at time 10 min after LPS injection.
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Fig. 3. a) Functional capillary density (FCD, cm/cm2), b) dysfunctional capillary density (DCD, cm/cm2) and c) nonfunctional capillary density (NCD, cm/cm2) in the pial vessels of control (Control), endotoxemic (LPS) and rhAPC treated endotoxemic (LPS + APC) rats (n= 10 per group, mean± standard deviation). * significant difference: Control vs. LPS.# significant difference: LPS vs. LPS + APC (pb 0.05). LPS or saline was administered at time 0 and rhAPC was administered at time 10 min after LPS injection.
levels of MCP-1 did not show significant differences between LPS, APC and control groups (Table 1).
Discussion We demonstrated, in this study, that rhAPC treatment significantly attenuates deterioration of pial microcirculation during sepsis by reducing leukocyte adhesion to endothelia and improving functional capillary density of pial vessels. In addition, rhAPC treatment significantly decreased systemic IL-1α release. Proposed mechanisms relevant to sepsis 1.00 0.95
PE %
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LPS
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8.58 ± 21.12 168.2 ± 228.0 0 0 796.1 ± 378.6 1.0 ± 2.89
5032 ± 1502* 1823 ± 1007* 785.1 ± 433.6* 7.0 ± 14.06 445.1 ± 503.1 54.00 ± 51.72*
2134 ± 937.1*# 1678 ± 1053* 838.1 ± 504.2* 7.5 ± 15.61 454.6 ± 533.2 80.50 ± 73.64*
Plasma cytokine concentration (pg/ml) in control (Control), endotoxemic (LPS) and rhAPC treated endotoxemic (LPS + APC) rats (n = 10 per group, mean ± standard deviation). * indicates p b 0.05 compared to control. # indicates p b 0.05 compared to LPS. IL-1α: interleukin 1α, TNF-α: tumor necrosis factor-α, IFN-γ: interferon γ, MCP1: Monocyte chemotactic protein-1 and GM-CSF: Granulocyte-macrophage colonystimulating factor.
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Fig. 4. Plasma exvasation (PE, %) in the pial vessels of control (Control), endotoxemic (LPS) and rhAPC treated endotoxemic (LPS + APC) rats (n = 10 per group, mean ± standard deviation).
may include anticoagulation, fibrinolysis, anti-inflammation and preservation of endothelial permeability barrier function (Macias et al., 2005). In vitro experiments showed that rhAPC inhibits leukocyte response to chemotactic signals (Sturn et al., 2003) and suppresses E-selectin mediated cell adhesion to the vascular endothelia (Iba et al., 2005). In addition, rhAPC suppresses cytokine-induced upregulation of adhesion molecules in cultured endothelial cells and diminishes the expression of inflammatory cytokines and chemokines (Galley et al., 2008; Hoffmann et al., 2004). Furthermore, rhAPC directly binds to β integrins and inhibits human neutrophil adhesion to matrix proteins therefore preventing neutrophil migration (Elphick et al., 2009). Such in vitro evidence supports our in vivo observation that rhAPC treatment in endotoxemic rats reduces leukocyte adhesion to endothelia in pial vessels and this reduction may be due to the reduced expression of adhesion molecules on leukocytes and endothelial cells. Similar results were also reported in several other endotoxemia experimental models (Hoffmann et al., 2004; Iba et al., 2005; Lehmann et al., 2006, 2008a). We have previously demonstrated that rhAPC treatment in healthy non-endotoxemic rats does not show any effect on vital parameters, and on leukocyte adhesion, vessel perfusion and cytokine secretion in intestinal (Lehmann et al., 2008a) and mesenteric microcirculation (Lehmann et al., 2008b), suggesting that rhAPC alone do not have toxic effect. Functional capillary density (FCD) is another indicator used to evaluate the function of microcirculation in sepsis. Microcirculation abnormality in sepsis includes decreases in FCD and increases in the proportion of nonperfused or intermittently perfused capillaries. APC treatment in sepsis improved microcirculation alterations in septic patients (De Backer et al., 2006). In our study we demonstrated that rhAPC treatment significantly reversed the impairment of capillary density by increasing FCD and decreasing NCD and DCD. Although APC is an anti-coagulator and plays a role in preventing clot formation in the microcirculation, it is believed that both antiinflammatory and anti-coagulatory are responsible for its effect on capillary perfusion. Plasma extravasation represents a clinically relevant characteristic of microcirculation deterioration due to tissue edema and decreased perfusion pressures caused by relative hypovolemia (Lehmann et al., 2004). In our experiments, plasma extravasation tended to increase in endotoxemia rats 2 h after LPS administration and decreased after rhAPC treatment. However, none of the changes reached statistical significance. Since we have previously demonstrated that LPS increased plasma extravasation significantly at 4 h after LPS administration (Zhou et al., 2011), the results in the present study suggest that 2-h endotoxemia is not severe enough to generate significant plasma extravasation in pial vessels. Systemic inflammatory cytokine levels have been shown to be elevated in sepsis patients, including IL-1, IL-6, IFN-γ and TNF-α (Kawasaki et al., 1999; Wang and Ma, 2008) and may be reduced by APC treatment due to its anti-inflammatory effect. In animal models, APC significantly inhibited ischemia/reperfusion-induced increase of
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TNF-α and IL-8 (Mizutani et al., 2000) and prevented pulmonary vascular injury by inhibiting cytokine production (Murakami et al., 1997). APC treatment also reduced LPS induced increase of TNF-α and IL-6 production (Iba et al., 2005). However, APC did not reduce the cytokine production of IL-1 and IL-8 by human neutrophils stimulated by LPS in vitro (Galley et al., 2008) and APC only reduced IL-1 but not TNF-α production in endotoxemic rats (Lehmann et al., 2006, 2008a, 2008b). These findings suggest that the effect of APC on inflammatory cytokine production is unclear and observed differences could be related to the experimental model used. In summary we have demonstrated, using intravital microscopy, that administration of endotoxin LPS significantly deteriorates brain microcirculation. Recombinant human APC treatment significantly attenuated the deterioration of brain microcirculation induced by endotoxin and reduces IL-1α production. The beneficial effects of rhAPC may play an important role in the management of patients with sepsis due to the fundamental role of the microcirculation in the pathogenesis of sepsis and multiple organ dysfunction. Acknowledgments We wish to express our gratitude to Sabine Will and Annette Wegner for their excellent contributions in the preparation of the experiments and Roswitha Dressler and Raila Busch for the cytokine measurements. References Angus, D.C., 2012. Drotrecogin alfa (activated) … a sad final fizzle to a roller-coaster party. Crit. Care 16, 107. Bernard, G.R., Vincent, J.L., Laterre, P.F., LaRosa, S.P., Dhainaut, J.F., Lopez-Rodriguez, A., et al., 2001. Efficacy and safety of recombinant human activated protein C for severe sepsis. N. Engl. J. Med. 344, 699–709. Bickel, U., Grave, B., Kang, Y.S., del Rey, A., Voigt, K., 1998. No increase in blood–brain barrier permeability after intraperitoneal injection of endotoxin in the rat. J. Neuroimmunol. 85, 131–136. Boldt, J., Papsdorf, M., Rothe, A., Kumle, B., Piper, S., 2000. Changes of the hemostatic network in critically ill patients — is there a difference between sepsis, trauma, and neurosurgery patients? Crit. Care Med. 28, 445–450. De Backer, D., Verdant, C., Chierego, M., Koch, M., Gullo, A., Vincent, J.L., 2006. Effects of drotrecogin alfa activated on microcirculatory alterations in patients with severe sepsis. Crit. Care Med. 34, 1918–1924. Donati, A., Romanelli, M., Botticelli, L., Valentini, A., Gabbanelli, V., Nataloni, S., et al., 2009. Recombinant activated protein C treatment improves tissue perfusion and oxygenation in septic patients measured by near-infrared spectroscopy. Crit. Care 13 (Suppl. 5), S12. Elphick, G.F., Sarangi, P.P., Hyun, Y.M., Hollenbaugh, J.A., Ayala, A., Biffl, W.L., et al., 2009. Recombinant human activated protein C inhibits integrin-mediated neutrophil migration. Blood 113, 4078–4085. Esmon, C.T., 2000. Regulation of blood coagulation. Biochim. Biophys. Acta 1477, 349–360. Galley, H.F., El Sakka, N.E., Webster, N.R., Lowes, D.A., Cuthbertson, B.H., 2008. Activated protein C inhibits chemotaxis and interleukin-6 release by human neutrophils without affecting other neutrophil functions. Br. J. Anaesth. 100, 815–819.
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