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Desmopressin improves intestinal functional capillary density and decreases leukocyte activation in experimental endotoxemia
YMVRE-03364; No. of pages: 7; 4C: Microvascular Research xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Microvascular Research journal homepage: www.elsevier.com/locate/ymvre
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K Wafa a, C Lehmann a,b,c,d,⁎, L Wagner d, I Drzymulski d, A Wegner d, D Pavlovic e 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 Pathophysiology, American School of Medicine, European University, Belgrade, Serbia b c
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Background: Blood flow to the intestine is decreased in sepsis in favor of vital organs resulting in ischemic damage of the gut mucosa. Once the mucosa is damaged, increased translocation of intestinal bacteria to the systemic circulation may occur. This in turn aggravates the inflammatory response contributing to the development of multi-organ failure. Desmopressin is a synthetic analog of vasopressin, an anti-diuretic hormone which has been shown to induce vasodilation and is thought to be implicated in immunomodulation. In this study, we investigate the effects of desmopressin on the intestinal microcirculation during sepsis in an experimental endotoxemia model in rats using intravital microscopy. In addition, we investigate the effects of desmopressin on systemic inflammation. Methods: Forty Lewis rats were subdivided into four groups, where rats received intravenous saline (control), desmopressin (1 μg/kg/ml), lipopolysaccharide (5 mg/kg) or lipopolysaccharide followed by desmopressin. Inflammatory response was assessed by quantifying the number of temporary and firmly adherent leukocytes in submucosal venules. Capillary perfusion was determined by assessing the number of functional, nonfunctional and dysfunctional capillaries in the intestinal wall layers (muscularis longitudinalis, muscularis circularis and mucosa). Additionally, inflammatory cytokine levels were determined by multiplex assays. Results: The number of firmly adhering leukocytes in V1 venules of rats receiving lipopolysaccharide and treated with desmopressin was significantly reduced compared to lipopolysaccharide only group (LPS: 259 ± 25.7 vs. LPS + DDAVP: 203 ± 17.2; n/mm2; p b 0.05). Additionally, desmopressin treatment improved impaired intestinal microcirculation by improving functional capillary density following lipopolysaccharide administration in all examined layers of the intestinal wall. We also observed a significant decrease in TNF-α levels in rats which received desmopressin in endotoxemia compared to untreated rats (LPS: 383 ± 64.2; LPS + DDAVP: 261.3 ± 22; pg/ml; p b 0.05). Conclusion: Desmopressin administration improved intestinal capillary perfusion and reduced inflammatory response in rat endotoxemia. © 2013 Published by Elsevier Inc.
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Desmopressin improves intestinal functional capillary density and decreases leukocyte activation in experimental endotoxemia
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Introduction
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Sepsis affects all aspects of the microcirculation including blood, endothelial, and smooth muscle cells (Ince, 2005). Microcirculatory dysfunction in sepsis has serious clinical implications. If left untreated it may result in multiorgan dysfunction (Deitch, 1992). Specifically during sepsis, blood flow to the intestine is decreased in favor of vital organs. The blood flow prioritization results in ischemic damage of the epithelial mucosa layer which serves as a barrier between the circulation and
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⁎ Corresponding author at: Department of Anesthesia, Pain Management and Perioperative Medicine, QE II Health Sciences Centre, 10 West Victoria, 1276 South Park St., Halifax, NS B3H 2Y9, Canada. Fax: +1 902 423 9454. E-mail address: [email protected] (C. Lehmann).
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the bacterial flora allowing the translocation of bacteria from the gut to the systemic circulation. This phenomenon is known as the “second hit”, eventually causing multi-system organ failure. Improvement of the intestinal microcirculation would increase oxygen delivery and decrease risk of mortality due to the “second hit” phenomenon (Deitch, 1992). Desmopressin (DDAVP, 1-deamino-8-D-arginine vasopressin) is a synthetic analog of the nonapeptide antidiuretic pituitary hormone, vasopressin (Favory et al., 2009; Trigg et al., 2012). While vasopressin mediates its effects mainly via the vasopressin 1, 2 and 3 receptors (V1R, V2R, V3R), desmopressin only acts on the V2R (Birnbaumer, 2000). The V2R is mainly expressed in the kidney collecting duct and in endothelial cells. Activation of the V2R receptor by DDAVP in endothelial cells causes the release of von Willebrand factor (VWF) and
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Please cite this article as: Wafa, K., et al., Desmopressin improves intestinal functional capillary density and decreases leukocyte activation in experimental endotoxemia, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.09.001
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Animals
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Forty male Lewis rats (body weight 280 ± 70 g; Charles River Laboratories, Sulzfeld, Germany) were housed in chip-bedded cages and kept on a 12 h light/dark cycle with ad lib food (Rat chow, Altromin, Lage, Germany) and water. Housing conditions were maintained at 22 °C with a 55–60% humidity environment. All animal procedures were performed according to the guidelines set by the German animal safety legislation and were approved by the institutional Animal Care Committee. Upon completion of experiments, rats were sacrificed by receiving an overdose of pentobarbital by intravenous administration and bilateral pneumothorax.
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Anesthesia was induced by intraperitoneal administration of pentobarbital (60 mg/kg, Pentobarbital Natrium, Fargon, Barsbüttel, Germany). Anesthesia was maintained by intravenous pentobarbital injections (5 mg/kg) when required. The animals were positioned in a supine position and the neck was shaved and disinfected. To secure the airways, rats received a tracheostomy and were allowed to breathe room air. Polyethylene catheters (vein: inner diameter 0.28 mm, outer diameter 0.61 mm; artery: inner diameter 0.58 mm, outer diameter 0.96 mm; Smiths Medical, Kent, UK) were inserted in the left external jugular vein and common carotid artery for the administration of fluids, endotoxin and fluorescent dyes. The rats were placed on a hot plate (LHG hotplate HAS 01/AL, Harry Gestigkeit GmbH, Düsseldorf, Germany) to maintain a body temperature of 37 ± 0.5 °C and the temperature was monitored by using a rectal thermometer. Finally, the abdominal area was shaved and disinfected and a median laparotomy was performed from the xiphoid process to the symphysis 30 min prior to intravital microscopy.
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Animals were randomly assigned to one of four groups (n = 10 per group): control (saline), DDAVP (desmopressin, 1 μg/kg/ml intravenously, [deamino-Cys1, D-Arg8]-Vasopressinacetat; Minirin®, Ferring
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Intravital microscopy
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Intravital microscopy was performed using an epifluorescent microscope (Axiotech Vario, Carl Zeiss, Jena, Germany) with a light source (HBO 50, Carl Zeiss, Jena, Germany), ocular (10×) and lens (20×/0.5 Achroplan, Carl Zeiss, Jena, Germany). The microscope contained both type #20 and #10 filters (Carl Zeiss, Jena, Germany) for the visualization of rhodamine 6G and fluorescein isothiocyanate albumin, respectively. These fluorescent dyes were used to visualize leukocytes and vessels, 2 h after starting experiments, on a segment of the terminal ileum 1 cm proximal from the ileocecal valve. To facilitate the microscopic evaluation of approximately 1 cm2 of the intestinal surface a 76 × 26 mm transparent glass cover slip (Menzel, Braunschweig, Germany) was used. To avoid dehydration due to exposure to ambient air, areas of the intestine not being used were covered with gauze and continuously superfused with 37 °C isotonic saline. In addition, to avoid any mechanical damage to areas of the gut which were not in direct contact with the cover slip the “hanging drop” technique (Pavlovic et al., 2006) was used and the tissue was warmed to 37 °C. To minimize phototoxic effects due to light exposure, repeated observation by intravital fluorescence microscopy on the same area of the intestine was avoided. Video images were captured by a black-and-white CCD video camera (BC-12, AVT-Horn, Aalen, Germany) connected to a black-and-white monitor (PM-159, Ikegami Electronics, Neuss, Germany) and recorded by a Panasonic NV-SV120EG-S recorder (Matsushita Audio Video, Tokyo, Japan). Videos were reviewed at a later date for data analysis. Fifteen minutes before intravital fluorescence microscopy was initiated rats received 200 μl of a 0.05% rhodamine 6G solution (Sigma) to visualize all leukocyte subpopulations and 200 μl of a 5% fluorescein isothiocyanate albumin dissolved in saline (sigma) to distinguish blood cells from plasma. The animal was then placed on a specialized heated microscope stage and the microscope was focused on the submucosa of the prepared intestinal section. Fields containing non-branching collecting venules of at least 300 μm in length and postcapillary venules were recorded for 30 s per field. Additionally, video recordings 30 s long were completed of random fields of the capillaries within each of the longitudinal and circular muscle layers. To capture video recordings of the mucosa a section of the intestinal lumen approximately 2 cm in length (antimesenteric) was opened using a microcautery knife. The intestine was then flushed with warm isotonic saline (37 °C), placed on a supporting device and six videos 30 s in length of randomly chosen mucosa sections were recorded. To ensure that cauterization did not influence the results video capture of the mucosa was only conducted on mucosa directly bordering the mesentery. All video analysis was blinded and took place offline on a video monitor. Analyzed parameters included the following: number of adherent leukocytes (number of leukocytes during an observation period which remained firmly attached to the intestinal endothelial for at least 30 s), number of rolling leukocytes (the number of leukocytes which during an observation period of 30 s pass in a rolling motion through) and functional capillary density. Functional capillary density is a measure of the microcirculatory perfusion of the tissue. Functional capillary density determination was completed as described previously (Al-Banna et al., 2013). In brief, three layers of the intestinal wall were examined; muscularis longitudinalis, muscularis circularis and the mucosa. Capillaries were defined to be either functional, dysfunctional
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Arzneimittel GmbH, Kiel, Germany), LPS (lipopolysaccharide, 5 mg/kg intravenously from Escherichia coli, serotype O26:B6; Sigma, Steinheim, Germany) and LPS + DDAVP (animals treated with DDAVP 1 μg/kg/ml intravenously 15 min after LPS administration). Intravenous administration of compounds (saline, DDAVP, LPS or LPS + DDAVP) was initiated 15 min after the insertion of the vein and artery catheters to allow the animals to recover. The laparotomy was performed 90 min post compound administration and was followed by intravital fluorescence microscopy procedure.
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tissue-type plasminogen activator (tPA) (Jochberger et al., 2009; Landry and Oliver, 2001). In addition, DDAVP increases plasma levels of coagulation factor VIII and increases the adhesion of platelets to vascular walls (Franchini, 2007). DDAVP is known to have strong vasodilator effects thought to be attributed to the cAMP-dependent activation of endothelial NO synthase (eNOS) (Kaufmann et al., 2003). cAMP signaling has also been associated with immune suppression specifically attenuating LPS and TNF-α mediated immune response implicating DDAVP and other cAMP activating compounds in attenuating the inflammatory response (Chassin et al., 2007). In this study, we investigate the effects of DDAVP on the intestinal microcirculation in experimental endotoxemia in rats. Due to the important role, which the intestinal microcirculation plays in sepsis pathophysiology, it would be clinically advantageous to improve the microcirculation of the gut in patients with sepsis. Given that DDAVP is a known antidiuretic substance with vasodilator, anti-inflammatory and hemostatic effects, we believe that the administration of this compound in sepsis may improve intestinal microcirculation and improve survival (Birnbaumer, 2000). To our knowledge, this is the first study to determine the effects of DDAVP on the intestinal microcirculation in a model of sepsis. Here, we study the effects of DDAVP on leukocyte recruitment and functional capillary density in the intestinal microvasculature by intravital microscopy. In addition, we investigate the levels of inflammatory cytokines in response to DDAVP administration in experimental endotoxemia in rats.
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Please cite this article as: Wafa, K., et al., Desmopressin improves intestinal functional capillary density and decreases leukocyte activation in experimental endotoxemia, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.09.001
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The numbers of firmly adherent and rolling leukocytes were determined in collecting (V1) and post-capillary (V3) venules of the intestinal submucosa.
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For macrocirculation, blood pressure and heart rate were continuously monitored using the arterial catheter (Hewlett Packard, Saronno, Italy).
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To quantify plasma levels of select cytokines and chemoattractants, arterial blood samples (1 ml) were collected from animals in heparinized tubes prior to IVM and after IVM completion (180 min). Upon
Unless otherwise stated, all data comparisons were completed using an unpaired two-tailed t-test, or a one-way analysis of variance (ANOVA). Significance obtained by ANOVA was further subjected to a Newman–Keuls test for post-hoc analysis. Data is normally distributed, expressed as mean ± SEM and was considered statistically significant when the difference in mean values between groups had a P value less than 0.05.
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In control animals MAP and HR were maintained at a constant level ranging from 110 to 130 mmHg and 420 to 445 bpm for MAP and HR respectively (Fig. 1). MAP and HR values for rats receiving DDAVP were comparable to those of control rats. Rats receiving LPS or LPS
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collection, blood was immediately centrifuged at 5000 rpm for 10 min at 4 °C and plasma was collected and stored at −80 °C for later processing. To determine the levels of Interleukin-1 α (IL-1α), Monocyte chemoattractant protein-1 (MCP-1), Tumor necrosis factor α (TNFα), Interferon γ (INFγ), Granulocyte macrophage–colony stimulating factor (GM–CSF) and Interleukin-4 (IL-4) plasma samples were analyzed using a Procarta Multiplex Cytokine Assay kit (Affymetrix, Fremont, CA) according to the manufacturer's instructions (Luminex, Bio-Rad, Mississauga, ON).
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or non-functional. Functional capillaries are capillaries with red blood cells moving continuously through the microvessels (detection facilitated by FITC-dextran-labeled plasma) over an observation period of 30 s. During the observation time, red blood cell flow has to be more prominent than the intervals of the pure plasma flow. Dysfunctional capillaries are capillaries where at least one erythrocyte crosses the capillary length observed. The intervals of plasma flow are longer than those with erythrocyte perfusion. Non-functional capillaries are capillaries where erythrocytes oscillate in direction without passing the observed capillary length.
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Fig. 1. Mean arterial pressure (MAP, A) and heart rate (HR, B) of rats receiving saline (control), LPS, DDAVP and LPS + DDAVP. Values are expressed as mean ± SEM. One way ANOVA, Newman–Keuls test for post-hoc analysis, *p b 0.05 compared to control and DDAVP groups, and data was collected from 10 rats per group (N = 10).
Please cite this article as: Wafa, K., et al., Desmopressin improves intestinal functional capillary density and decreases leukocyte activation in experimental endotoxemia, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.09.001
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Figs. 2 and 3 summarizes the effects of the administration of DDAVP, LPS and LPS plus DDAVP on the number of temporary adherent leukocytes and firmly adherent leukocytes in V1 and V3 venules of the intestinal submucosa, compared to controls, 2 h post injections. DDAVP administration did not significantly alter the number of temporary or firmly adherent leukocytes in both the V1 and V3 venules compared to controls. LPS challenge significantly decreased the number of temporary adherent leukocyte levels and increased the numbers of firmly adherent leukocytes in both the V1 and V3 venules. The same was
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and DDAVP demonstrated similar MAP and HR values at baseline. Upon injection of LPS or LPS plus DDAVP MAP dropped to ~70–80 mmHg at the 15 min mark but steadily recovered to values comparable to those in the control and DDAVP groups by 2 h post injection. While the LPS and LPS plus DDAVP MAP values remained significantly different from control and DDAVP groups, they recovered and remained within acceptable physiological levels. Heart rate levels for the LPS and LPS plus DDAVP groups increased significantly compared to control and DDAVP groups 60 min post injections and remained elevated for the recorded duration (3 h).
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Fig. 2. Number of temporary adherent (rolling) leukocytes in V1 (A) and V3 (B) venules of rats receiving saline (control), LPS, DDAVP and LPS + DDAVP. Values are expressed as mean ± SEM. One way ANOVA, Newman–Keuls test for post-hoc analysis, *p b 0.05 compared to control and DDAVP groups, data was collected from 10 rats per group (N = 10).
Fig. 3. Number of adhering leukocytes in V1 (A) and V3 (B) venules of rats receiving saline (control), LPS, DDAVP and LPS + DDAVP. Values are expressed as mean ± SEM. One way ANOVA, Newman–Keuls test for post-hoc analysis, *p b 0.05 compared to control and DDAVP groups, #p b 0.05 compared to LPS, and data was collected from 10 rats per group (N = 10).
observed in V1 and V3 venules of rats with LPS plus DDAVP. In LPS animals treated with DDAVP we observed a significant decrease in firmly adherent leukocytes compared to untreated LPS animals in the V1 venules.
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Functional, dysfunctional and non-functional capillary density
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In the control group there was a high number of functional capillaries in all three layers (muscularis longitudinalis, 100 cm/cm2; muscularis circularis, 63 cm/cm2; mucosa, 295 cm/cm2) and a low number of dysfunctional (muscularis longitudinalis, 5 cm/cm2; muscularis circularis, 20 cm/cm2; mucosa, 95 cm/cm2) and non-functional capillaries (muscularis longitudinalis, 2 cm/cm2; muscularis circularis, 18 cm/cm2; mucosa, 30 cm/cm2). DDAVP animals had similar values as those observed in control rats (Fig. 4). LPS administration decreased capillary perfusion in the small intestine in all three layers significantly by decreasing functional capillary density (muscularis longitudinalis, 38 cm/cm2; muscularis circularis, 20 cm/cm2; mucosa, 70 cm/cm2) and increasing dysfunctional (muscularis longitudinalis, 40 cm/cm2; muscularis circularis, 38 cm/cm2; mucosa, 150 cm/cm2) and nonfunctional capillary density (muscularis longitudinalis, 45 cm/cm2; muscularis circularis, 70 cm/cm2; mucosa, 200 cm/cm2). Treatment of endotoxemic animals with DDAVP restored capillary perfusion in most of the intestinal layers examined. DDAVP restored functional capillary levels in both the muscularis longitudinalis and muscularis circularis layers of the small intestine and close to control levels in the mucosa. While treatment with DDAVP did not effectively reduce the levels of dysfunctional capillaries in the examined layers (except for the muscularis longitudinalis) of the small intestine it significantly
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Please cite this article as: Wafa, K., et al., Desmopressin improves intestinal functional capillary density and decreases leukocyte activation in experimental endotoxemia, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.09.001
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Fig. 4. Number of functional (A, D, G), dysfunctional (B, E, H) and non-functional capillaries (C, F, I) in the muscularis longitudinalis (A–C), muscularis circularis (D–F) and mucosa layers (G–I) of the small intestine. Values are expressed as mean ± SEM. One way ANOVA, Newman–Keuls test for post-hoc analysis, *p b 0.05 compared to control, #p b 0.05 compared to LPS, and data was collected from 10 rats per group (N = 10).
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Analysis of selected inflammatory cytokines (IL-1α, TNFα, INFγ, GM–CSF and IL-4) from plasma collected at the 3 h mark revealed significant elevated levels in response to LPS when compared to the control group (Fig. 5). Levels of the chemoattractant MCP-1 were decreased. Administration of DDAVP alone yielded cytokine and chemoattractant molecule levels similar to control. From the cytokines investigated, treatment of LPS animals with DDAVP modulated TNF-α levels decreasing them compared to untreated LPS animals.
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In this study, we demonstrate the ability of DDAVP to protect the intestinal microcirculation in experimental endotoxemia by improving functional capillary density. Accordingly, DDAVP was observed to
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reduced the levels of non-functional capillaries in the three examined layers. It was most effective in the muscularis longitudinalis where non-functional capillaries were reduced to control levels.
decrease the number of dysfunctional and non-functional capillaries in the intestine. Intestinal microcirculation has been demonstrated to play an important role in the pathogenesis of sepsis and septic shock (Deitch, 1990a, 1990b, 1992; Legrand et al., 2010; Lundy and Trzeciak, 2009). To our knowledge, this is the first study to experimentally demonstrate the effects of DDAVP on intestinal capillary perfusion. Several studies investigated the role of DDAVP on liver, kidney and pancreatic microcirculation. There are some discrepancies in the literature with regards to the effects of DDAVP on capillary perfusion. Banafsche et al. reported that DDAVP has a negative impact on hepatic microcirculation (Banafsche et al., 2002). In a prospective randomized trial investigating the effects of DDAVP on kidney graft function it was determined that primary graft failure from donors who had been treated with DDAVP was significantly higher compared to control (Hirschl et al., 1996). However, in a retrospective trial there was no significant change found in renal function in donors treated with DDAVP (Guesde et al., 1998). Alternatively, in a retrospective trial by Decraemer et al. which studied the effects of DDAVP and vasopressin on graft function after pancreas transplantation concluded that both drugs had no effect on graft function.
Please cite this article as: Wafa, K., et al., Desmopressin improves intestinal functional capillary density and decreases leukocyte activation in experimental endotoxemia, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.09.001
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Fig. 5. Select inflammatory mediators levels in rats receiving saline (control), LPS, DDAVP and LPS + DDAVP. Interleukin-1α (IL-1α), A; Monocyte chemoattractant protein-1 (MCP-1), B; Tumor necrosis factor α (TNFα), C; Interferon-γ (INFγ), D; Granulocyte macrophage–colony stimulating factor (GM–CSF), E; Interleukin-4 (IL-4), F. Values are expressed as mean ± SEM. One way ANOVA, Newman–Keuls test for post-hoc analysis, *p b 0.05 compared to control, #p b 0.05 compared to LPS, and data was collected from 10 rats per group (N = 10).
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Focus of our study was the impact of DDAVP on intestinal capillary perfusion and leukocyte adhesion. Given that DDAVP is known to exert a dose dependent vasodilation (Kaufmann et al., 2003) we adjusted the DDAVP dose in pilot experiments so that there would be no effect on macrocirculation potentially affecting the microcirculation. Therefore, in our study we did not observe an effect of DDAVP on mean arterial pressure and heart rate. This is not the first study to test a single dose of DDAVP (Rybaltowski et al., 2011). With our experimental model and
the half-life of DDAVP being approximately 3 h (Agerso et al., 2004) we were able to see the effects of DDAVP administration on intestinal microcirculation by 105 min after drug administration. The study of Banafsche et al. showed an increase in the number of temporary and firmly adherent leukocytes in response to DDAVP administration (Banafsche et al., 2002). The authors attributed this observation to an increased expression of adhesion molecules (P-selectin, ICAM-1, VCAM-1) as well as the possible release of vWF.
Please cite this article as: Wafa, K., et al., Desmopressin improves intestinal functional capillary density and decreases leukocyte activation in experimental endotoxemia, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.09.001
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In conclusion, this study provided evidence that DDAVP improves intestinal microcirculation in experimental endotoxemia. We demonstrate that at low doses DDAVP exhibits anti-inflammatory properties without affecting the macrocirculation. Further experiments are required to elucidate the exact mechanism by which DDAVP attenuates the inflammatory response.
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In our experiments while DDAVP did not significantly increase the number of temporary adherent leukocytes, we observed a trend to higher values compared to controls. This result is in agreement with Banafsche et al. and other studies which demonstrate that DDAVP increases the number of temporary adherent leukocytes (Kanwar et al., 1995). DDAVP is also known to induce erythrocyte adherence to microvasculature (Tsai et al., 1990). However, DDAVP was not associated with blockage of the microcirculation despite reporting increased adherence of red blood cells to the microvasculature. We observed the same effect whereby DDAVP administration did not impact the intestinal microcirculation despite the observed trend in the increase in intestinal temporary adherent leukocytes numbers. Additionally, MAP values for rats receiving DDAVP were comparable to those of control rats. While Banafsche et al. used the same concentrations of DDAVP and the same method of administration in their single dose data they administered the DDAVP only 15 min prior to IVM, whereas we administered the DDAVP 90 min prior to IVM. This could indicate that the increase in temporary leukocyte adherence is a transient phenomenon. It is also possible that rat strain differences could be responsible for the discrepancy between these two studies. Interestingly, DDAVP significantly decreased leukocyte adhesion in V1 venules during endotoxemia. We believe this phenomenon could be associated with the observed improvement of the intestinal capillary perfusion. Given the larger size of V1 venules compared to V3 venules, there would be an increased shear force due to blood flow in V1 venules compared to V3 venules. By improving blood flow leukocytes are less capable of adhering to the endothelium. Attenuation of NF-κB signaling as well as upregulation of cAMP levels could also be responsible for decreased leukocyte adhesion. In human epithelial A549 cells an increase in cAMP has been demonstrated to inhibit the TNF induced vascular cell adhesion molecule 1 release (Chassin et al., 2007; Lee et al., 2004). Additionally, Boyd et al. have demonstrated that stimulation of the V2R by vasopressin in the lung reduced pulmonary inflammation as measured by IL-6 levels (Boyd et al., 2008). This phenomenon was attributed to the attenuation of NF-κB signaling by V2R stimulation by vasopressin. Sepsis is the systemic inflammatory response to an infection (Liu and Malik, 2006). In agreement with the literature, our endotoxemia model demonstrates an enhanced inflammatory response by the release of TNF-α, IL-1α, IL-4, INFγ, and GM–CSF. Interestingly, in our model treatment of endotoxemia by DDAVP resulted in a significant decrease of TNF-α plasma levels suggesting an anti-inflammatory role. NF-κB signaling has been established to mediate the transcriptional activation of proinflammatory cytokines, chemokines and adhesion molecules (Baeuerle and Baichwal, 1997; Pahl, 1999). Some of these molecules include TNF, IL-1, IL-2, IL-3, IL-5, IL-6, IL-12, and IL-18. We believe the attenuation of NF-κB signaling by DDAVP might explain the significant decrease in TNF-α levels obtained with DDAVP treatment of LPS induced endotoxemia. Additionally, activation of cAMP signaling has been associated with immune suppression specifically attenuating LPS and TNF-α mediated immune response (Chassin et al., 2007). However, further studies are required to elucidate the exact mechanism responsible for DDAVP related anti-inflammatory effects.
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Please cite this article as: Wafa, K., et al., Desmopressin improves intestinal functional capillary density and decreases leukocyte activation in experimental endotoxemia, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.09.001
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Contents lists available at ScienceDirect
Microvascular Research journal homepage: www.elsevier.com/locate/ymvre
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K Wafa a, C Lehmann a,b,c,d,⁎, L Wagner d, I Drzymulski d, A Wegner d, D Pavlovic e 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 Pathophysiology, American School of Medicine, European University, Belgrade, Serbia b c
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Background: Blood flow to the intestine is decreased in sepsis in favor of vital organs resulting in ischemic damage of the gut mucosa. Once the mucosa is damaged, increased translocation of intestinal bacteria to the systemic circulation may occur. This in turn aggravates the inflammatory response contributing to the development of multi-organ failure. Desmopressin is a synthetic analog of vasopressin, an anti-diuretic hormone which has been shown to induce vasodilation and is thought to be implicated in immunomodulation. In this study, we investigate the effects of desmopressin on the intestinal microcirculation during sepsis in an experimental endotoxemia model in rats using intravital microscopy. In addition, we investigate the effects of desmopressin on systemic inflammation. Methods: Forty Lewis rats were subdivided into four groups, where rats received intravenous saline (control), desmopressin (1 μg/kg/ml), lipopolysaccharide (5 mg/kg) or lipopolysaccharide followed by desmopressin. Inflammatory response was assessed by quantifying the number of temporary and firmly adherent leukocytes in submucosal venules. Capillary perfusion was determined by assessing the number of functional, nonfunctional and dysfunctional capillaries in the intestinal wall layers (muscularis longitudinalis, muscularis circularis and mucosa). Additionally, inflammatory cytokine levels were determined by multiplex assays. Results: The number of firmly adhering leukocytes in V1 venules of rats receiving lipopolysaccharide and treated with desmopressin was significantly reduced compared to lipopolysaccharide only group (LPS: 259 ± 25.7 vs. LPS + DDAVP: 203 ± 17.2; n/mm2; p b 0.05). Additionally, desmopressin treatment improved impaired intestinal microcirculation by improving functional capillary density following lipopolysaccharide administration in all examined layers of the intestinal wall. We also observed a significant decrease in TNF-α levels in rats which received desmopressin in endotoxemia compared to untreated rats (LPS: 383 ± 64.2; LPS + DDAVP: 261.3 ± 22; pg/ml; p b 0.05). Conclusion: Desmopressin administration improved intestinal capillary perfusion and reduced inflammatory response in rat endotoxemia. © 2013 Published by Elsevier Inc.
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Desmopressin improves intestinal functional capillary density and decreases leukocyte activation in experimental endotoxemia
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Introduction
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Sepsis affects all aspects of the microcirculation including blood, endothelial, and smooth muscle cells (Ince, 2005). Microcirculatory dysfunction in sepsis has serious clinical implications. If left untreated it may result in multiorgan dysfunction (Deitch, 1992). Specifically during sepsis, blood flow to the intestine is decreased in favor of vital organs. The blood flow prioritization results in ischemic damage of the epithelial mucosa layer which serves as a barrier between the circulation and
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⁎ Corresponding author at: Department of Anesthesia, Pain Management and Perioperative Medicine, QE II Health Sciences Centre, 10 West Victoria, 1276 South Park St., Halifax, NS B3H 2Y9, Canada. Fax: +1 902 423 9454. E-mail address: [email protected] (C. Lehmann).
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the bacterial flora allowing the translocation of bacteria from the gut to the systemic circulation. This phenomenon is known as the “second hit”, eventually causing multi-system organ failure. Improvement of the intestinal microcirculation would increase oxygen delivery and decrease risk of mortality due to the “second hit” phenomenon (Deitch, 1992). Desmopressin (DDAVP, 1-deamino-8-D-arginine vasopressin) is a synthetic analog of the nonapeptide antidiuretic pituitary hormone, vasopressin (Favory et al., 2009; Trigg et al., 2012). While vasopressin mediates its effects mainly via the vasopressin 1, 2 and 3 receptors (V1R, V2R, V3R), desmopressin only acts on the V2R (Birnbaumer, 2000). The V2R is mainly expressed in the kidney collecting duct and in endothelial cells. Activation of the V2R receptor by DDAVP in endothelial cells causes the release of von Willebrand factor (VWF) and
0026-2862/$ – see front matter © 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.mvr.2013.09.001
Please cite this article as: Wafa, K., et al., Desmopressin improves intestinal functional capillary density and decreases leukocyte activation in experimental endotoxemia, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.09.001
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Animals
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Forty male Lewis rats (body weight 280 ± 70 g; Charles River Laboratories, Sulzfeld, Germany) were housed in chip-bedded cages and kept on a 12 h light/dark cycle with ad lib food (Rat chow, Altromin, Lage, Germany) and water. Housing conditions were maintained at 22 °C with a 55–60% humidity environment. All animal procedures were performed according to the guidelines set by the German animal safety legislation and were approved by the institutional Animal Care Committee. Upon completion of experiments, rats were sacrificed by receiving an overdose of pentobarbital by intravenous administration and bilateral pneumothorax.
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Anesthesia was induced by intraperitoneal administration of pentobarbital (60 mg/kg, Pentobarbital Natrium, Fargon, Barsbüttel, Germany). Anesthesia was maintained by intravenous pentobarbital injections (5 mg/kg) when required. The animals were positioned in a supine position and the neck was shaved and disinfected. To secure the airways, rats received a tracheostomy and were allowed to breathe room air. Polyethylene catheters (vein: inner diameter 0.28 mm, outer diameter 0.61 mm; artery: inner diameter 0.58 mm, outer diameter 0.96 mm; Smiths Medical, Kent, UK) were inserted in the left external jugular vein and common carotid artery for the administration of fluids, endotoxin and fluorescent dyes. The rats were placed on a hot plate (LHG hotplate HAS 01/AL, Harry Gestigkeit GmbH, Düsseldorf, Germany) to maintain a body temperature of 37 ± 0.5 °C and the temperature was monitored by using a rectal thermometer. Finally, the abdominal area was shaved and disinfected and a median laparotomy was performed from the xiphoid process to the symphysis 30 min prior to intravital microscopy.
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Animals were randomly assigned to one of four groups (n = 10 per group): control (saline), DDAVP (desmopressin, 1 μg/kg/ml intravenously, [deamino-Cys1, D-Arg8]-Vasopressinacetat; Minirin®, Ferring
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Intravital microscopy was performed using an epifluorescent microscope (Axiotech Vario, Carl Zeiss, Jena, Germany) with a light source (HBO 50, Carl Zeiss, Jena, Germany), ocular (10×) and lens (20×/0.5 Achroplan, Carl Zeiss, Jena, Germany). The microscope contained both type #20 and #10 filters (Carl Zeiss, Jena, Germany) for the visualization of rhodamine 6G and fluorescein isothiocyanate albumin, respectively. These fluorescent dyes were used to visualize leukocytes and vessels, 2 h after starting experiments, on a segment of the terminal ileum 1 cm proximal from the ileocecal valve. To facilitate the microscopic evaluation of approximately 1 cm2 of the intestinal surface a 76 × 26 mm transparent glass cover slip (Menzel, Braunschweig, Germany) was used. To avoid dehydration due to exposure to ambient air, areas of the intestine not being used were covered with gauze and continuously superfused with 37 °C isotonic saline. In addition, to avoid any mechanical damage to areas of the gut which were not in direct contact with the cover slip the “hanging drop” technique (Pavlovic et al., 2006) was used and the tissue was warmed to 37 °C. To minimize phototoxic effects due to light exposure, repeated observation by intravital fluorescence microscopy on the same area of the intestine was avoided. Video images were captured by a black-and-white CCD video camera (BC-12, AVT-Horn, Aalen, Germany) connected to a black-and-white monitor (PM-159, Ikegami Electronics, Neuss, Germany) and recorded by a Panasonic NV-SV120EG-S recorder (Matsushita Audio Video, Tokyo, Japan). Videos were reviewed at a later date for data analysis. Fifteen minutes before intravital fluorescence microscopy was initiated rats received 200 μl of a 0.05% rhodamine 6G solution (Sigma) to visualize all leukocyte subpopulations and 200 μl of a 5% fluorescein isothiocyanate albumin dissolved in saline (sigma) to distinguish blood cells from plasma. The animal was then placed on a specialized heated microscope stage and the microscope was focused on the submucosa of the prepared intestinal section. Fields containing non-branching collecting venules of at least 300 μm in length and postcapillary venules were recorded for 30 s per field. Additionally, video recordings 30 s long were completed of random fields of the capillaries within each of the longitudinal and circular muscle layers. To capture video recordings of the mucosa a section of the intestinal lumen approximately 2 cm in length (antimesenteric) was opened using a microcautery knife. The intestine was then flushed with warm isotonic saline (37 °C), placed on a supporting device and six videos 30 s in length of randomly chosen mucosa sections were recorded. To ensure that cauterization did not influence the results video capture of the mucosa was only conducted on mucosa directly bordering the mesentery. All video analysis was blinded and took place offline on a video monitor. Analyzed parameters included the following: number of adherent leukocytes (number of leukocytes during an observation period which remained firmly attached to the intestinal endothelial for at least 30 s), number of rolling leukocytes (the number of leukocytes which during an observation period of 30 s pass in a rolling motion through) and functional capillary density. Functional capillary density is a measure of the microcirculatory perfusion of the tissue. Functional capillary density determination was completed as described previously (Al-Banna et al., 2013). In brief, three layers of the intestinal wall were examined; muscularis longitudinalis, muscularis circularis and the mucosa. Capillaries were defined to be either functional, dysfunctional
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Arzneimittel GmbH, Kiel, Germany), LPS (lipopolysaccharide, 5 mg/kg intravenously from Escherichia coli, serotype O26:B6; Sigma, Steinheim, Germany) and LPS + DDAVP (animals treated with DDAVP 1 μg/kg/ml intravenously 15 min after LPS administration). Intravenous administration of compounds (saline, DDAVP, LPS or LPS + DDAVP) was initiated 15 min after the insertion of the vein and artery catheters to allow the animals to recover. The laparotomy was performed 90 min post compound administration and was followed by intravital fluorescence microscopy procedure.
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tissue-type plasminogen activator (tPA) (Jochberger et al., 2009; Landry and Oliver, 2001). In addition, DDAVP increases plasma levels of coagulation factor VIII and increases the adhesion of platelets to vascular walls (Franchini, 2007). DDAVP is known to have strong vasodilator effects thought to be attributed to the cAMP-dependent activation of endothelial NO synthase (eNOS) (Kaufmann et al., 2003). cAMP signaling has also been associated with immune suppression specifically attenuating LPS and TNF-α mediated immune response implicating DDAVP and other cAMP activating compounds in attenuating the inflammatory response (Chassin et al., 2007). In this study, we investigate the effects of DDAVP on the intestinal microcirculation in experimental endotoxemia in rats. Due to the important role, which the intestinal microcirculation plays in sepsis pathophysiology, it would be clinically advantageous to improve the microcirculation of the gut in patients with sepsis. Given that DDAVP is a known antidiuretic substance with vasodilator, anti-inflammatory and hemostatic effects, we believe that the administration of this compound in sepsis may improve intestinal microcirculation and improve survival (Birnbaumer, 2000). To our knowledge, this is the first study to determine the effects of DDAVP on the intestinal microcirculation in a model of sepsis. Here, we study the effects of DDAVP on leukocyte recruitment and functional capillary density in the intestinal microvasculature by intravital microscopy. In addition, we investigate the levels of inflammatory cytokines in response to DDAVP administration in experimental endotoxemia in rats.
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The numbers of firmly adherent and rolling leukocytes were determined in collecting (V1) and post-capillary (V3) venules of the intestinal submucosa.
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For macrocirculation, blood pressure and heart rate were continuously monitored using the arterial catheter (Hewlett Packard, Saronno, Italy).
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To quantify plasma levels of select cytokines and chemoattractants, arterial blood samples (1 ml) were collected from animals in heparinized tubes prior to IVM and after IVM completion (180 min). Upon
Unless otherwise stated, all data comparisons were completed using an unpaired two-tailed t-test, or a one-way analysis of variance (ANOVA). Significance obtained by ANOVA was further subjected to a Newman–Keuls test for post-hoc analysis. Data is normally distributed, expressed as mean ± SEM and was considered statistically significant when the difference in mean values between groups had a P value less than 0.05.
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In control animals MAP and HR were maintained at a constant level ranging from 110 to 130 mmHg and 420 to 445 bpm for MAP and HR respectively (Fig. 1). MAP and HR values for rats receiving DDAVP were comparable to those of control rats. Rats receiving LPS or LPS
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collection, blood was immediately centrifuged at 5000 rpm for 10 min at 4 °C and plasma was collected and stored at −80 °C for later processing. To determine the levels of Interleukin-1 α (IL-1α), Monocyte chemoattractant protein-1 (MCP-1), Tumor necrosis factor α (TNFα), Interferon γ (INFγ), Granulocyte macrophage–colony stimulating factor (GM–CSF) and Interleukin-4 (IL-4) plasma samples were analyzed using a Procarta Multiplex Cytokine Assay kit (Affymetrix, Fremont, CA) according to the manufacturer's instructions (Luminex, Bio-Rad, Mississauga, ON).
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or non-functional. Functional capillaries are capillaries with red blood cells moving continuously through the microvessels (detection facilitated by FITC-dextran-labeled plasma) over an observation period of 30 s. During the observation time, red blood cell flow has to be more prominent than the intervals of the pure plasma flow. Dysfunctional capillaries are capillaries where at least one erythrocyte crosses the capillary length observed. The intervals of plasma flow are longer than those with erythrocyte perfusion. Non-functional capillaries are capillaries where erythrocytes oscillate in direction without passing the observed capillary length.
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Fig. 1. Mean arterial pressure (MAP, A) and heart rate (HR, B) of rats receiving saline (control), LPS, DDAVP and LPS + DDAVP. Values are expressed as mean ± SEM. One way ANOVA, Newman–Keuls test for post-hoc analysis, *p b 0.05 compared to control and DDAVP groups, and data was collected from 10 rats per group (N = 10).
Please cite this article as: Wafa, K., et al., Desmopressin improves intestinal functional capillary density and decreases leukocyte activation in experimental endotoxemia, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.09.001
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Figs. 2 and 3 summarizes the effects of the administration of DDAVP, LPS and LPS plus DDAVP on the number of temporary adherent leukocytes and firmly adherent leukocytes in V1 and V3 venules of the intestinal submucosa, compared to controls, 2 h post injections. DDAVP administration did not significantly alter the number of temporary or firmly adherent leukocytes in both the V1 and V3 venules compared to controls. LPS challenge significantly decreased the number of temporary adherent leukocyte levels and increased the numbers of firmly adherent leukocytes in both the V1 and V3 venules. The same was
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and DDAVP demonstrated similar MAP and HR values at baseline. Upon injection of LPS or LPS plus DDAVP MAP dropped to ~70–80 mmHg at the 15 min mark but steadily recovered to values comparable to those in the control and DDAVP groups by 2 h post injection. While the LPS and LPS plus DDAVP MAP values remained significantly different from control and DDAVP groups, they recovered and remained within acceptable physiological levels. Heart rate levels for the LPS and LPS plus DDAVP groups increased significantly compared to control and DDAVP groups 60 min post injections and remained elevated for the recorded duration (3 h).
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Fig. 2. Number of temporary adherent (rolling) leukocytes in V1 (A) and V3 (B) venules of rats receiving saline (control), LPS, DDAVP and LPS + DDAVP. Values are expressed as mean ± SEM. One way ANOVA, Newman–Keuls test for post-hoc analysis, *p b 0.05 compared to control and DDAVP groups, data was collected from 10 rats per group (N = 10).
Fig. 3. Number of adhering leukocytes in V1 (A) and V3 (B) venules of rats receiving saline (control), LPS, DDAVP and LPS + DDAVP. Values are expressed as mean ± SEM. One way ANOVA, Newman–Keuls test for post-hoc analysis, *p b 0.05 compared to control and DDAVP groups, #p b 0.05 compared to LPS, and data was collected from 10 rats per group (N = 10).
observed in V1 and V3 venules of rats with LPS plus DDAVP. In LPS animals treated with DDAVP we observed a significant decrease in firmly adherent leukocytes compared to untreated LPS animals in the V1 venules.
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In the control group there was a high number of functional capillaries in all three layers (muscularis longitudinalis, 100 cm/cm2; muscularis circularis, 63 cm/cm2; mucosa, 295 cm/cm2) and a low number of dysfunctional (muscularis longitudinalis, 5 cm/cm2; muscularis circularis, 20 cm/cm2; mucosa, 95 cm/cm2) and non-functional capillaries (muscularis longitudinalis, 2 cm/cm2; muscularis circularis, 18 cm/cm2; mucosa, 30 cm/cm2). DDAVP animals had similar values as those observed in control rats (Fig. 4). LPS administration decreased capillary perfusion in the small intestine in all three layers significantly by decreasing functional capillary density (muscularis longitudinalis, 38 cm/cm2; muscularis circularis, 20 cm/cm2; mucosa, 70 cm/cm2) and increasing dysfunctional (muscularis longitudinalis, 40 cm/cm2; muscularis circularis, 38 cm/cm2; mucosa, 150 cm/cm2) and nonfunctional capillary density (muscularis longitudinalis, 45 cm/cm2; muscularis circularis, 70 cm/cm2; mucosa, 200 cm/cm2). Treatment of endotoxemic animals with DDAVP restored capillary perfusion in most of the intestinal layers examined. DDAVP restored functional capillary levels in both the muscularis longitudinalis and muscularis circularis layers of the small intestine and close to control levels in the mucosa. While treatment with DDAVP did not effectively reduce the levels of dysfunctional capillaries in the examined layers (except for the muscularis longitudinalis) of the small intestine it significantly
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Fig. 4. Number of functional (A, D, G), dysfunctional (B, E, H) and non-functional capillaries (C, F, I) in the muscularis longitudinalis (A–C), muscularis circularis (D–F) and mucosa layers (G–I) of the small intestine. Values are expressed as mean ± SEM. One way ANOVA, Newman–Keuls test for post-hoc analysis, *p b 0.05 compared to control, #p b 0.05 compared to LPS, and data was collected from 10 rats per group (N = 10).
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Analysis of selected inflammatory cytokines (IL-1α, TNFα, INFγ, GM–CSF and IL-4) from plasma collected at the 3 h mark revealed significant elevated levels in response to LPS when compared to the control group (Fig. 5). Levels of the chemoattractant MCP-1 were decreased. Administration of DDAVP alone yielded cytokine and chemoattractant molecule levels similar to control. From the cytokines investigated, treatment of LPS animals with DDAVP modulated TNF-α levels decreasing them compared to untreated LPS animals.
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In this study, we demonstrate the ability of DDAVP to protect the intestinal microcirculation in experimental endotoxemia by improving functional capillary density. Accordingly, DDAVP was observed to
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reduced the levels of non-functional capillaries in the three examined layers. It was most effective in the muscularis longitudinalis where non-functional capillaries were reduced to control levels.
decrease the number of dysfunctional and non-functional capillaries in the intestine. Intestinal microcirculation has been demonstrated to play an important role in the pathogenesis of sepsis and septic shock (Deitch, 1990a, 1990b, 1992; Legrand et al., 2010; Lundy and Trzeciak, 2009). To our knowledge, this is the first study to experimentally demonstrate the effects of DDAVP on intestinal capillary perfusion. Several studies investigated the role of DDAVP on liver, kidney and pancreatic microcirculation. There are some discrepancies in the literature with regards to the effects of DDAVP on capillary perfusion. Banafsche et al. reported that DDAVP has a negative impact on hepatic microcirculation (Banafsche et al., 2002). In a prospective randomized trial investigating the effects of DDAVP on kidney graft function it was determined that primary graft failure from donors who had been treated with DDAVP was significantly higher compared to control (Hirschl et al., 1996). However, in a retrospective trial there was no significant change found in renal function in donors treated with DDAVP (Guesde et al., 1998). Alternatively, in a retrospective trial by Decraemer et al. which studied the effects of DDAVP and vasopressin on graft function after pancreas transplantation concluded that both drugs had no effect on graft function.
Please cite this article as: Wafa, K., et al., Desmopressin improves intestinal functional capillary density and decreases leukocyte activation in experimental endotoxemia, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.09.001
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Fig. 5. Select inflammatory mediators levels in rats receiving saline (control), LPS, DDAVP and LPS + DDAVP. Interleukin-1α (IL-1α), A; Monocyte chemoattractant protein-1 (MCP-1), B; Tumor necrosis factor α (TNFα), C; Interferon-γ (INFγ), D; Granulocyte macrophage–colony stimulating factor (GM–CSF), E; Interleukin-4 (IL-4), F. Values are expressed as mean ± SEM. One way ANOVA, Newman–Keuls test for post-hoc analysis, *p b 0.05 compared to control, #p b 0.05 compared to LPS, and data was collected from 10 rats per group (N = 10).
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Focus of our study was the impact of DDAVP on intestinal capillary perfusion and leukocyte adhesion. Given that DDAVP is known to exert a dose dependent vasodilation (Kaufmann et al., 2003) we adjusted the DDAVP dose in pilot experiments so that there would be no effect on macrocirculation potentially affecting the microcirculation. Therefore, in our study we did not observe an effect of DDAVP on mean arterial pressure and heart rate. This is not the first study to test a single dose of DDAVP (Rybaltowski et al., 2011). With our experimental model and
the half-life of DDAVP being approximately 3 h (Agerso et al., 2004) we were able to see the effects of DDAVP administration on intestinal microcirculation by 105 min after drug administration. The study of Banafsche et al. showed an increase in the number of temporary and firmly adherent leukocytes in response to DDAVP administration (Banafsche et al., 2002). The authors attributed this observation to an increased expression of adhesion molecules (P-selectin, ICAM-1, VCAM-1) as well as the possible release of vWF.
Please cite this article as: Wafa, K., et al., Desmopressin improves intestinal functional capillary density and decreases leukocyte activation in experimental endotoxemia, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.09.001
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Agerso, H., et al., 2004. Pharmacokinetics and renal excretion of desmopressin after intravenous administration to healthy subjects and renally impaired patients. Br. J. Clin. Pharmacol. 58, 352–358. Al-Banna, N.A., et al., 2013. Acute administration of antibiotics modulates intestinal capillary perfusion and leukocyte adherence during experimental sepsis. Int. J. Antimicrob. Agents 41, 536–543. Baeuerle, P.A., Baichwal, V.R., 1997. NF-kappa B as a frequent target for immunosuppressive and anti-inflammatory molecules. Adv. Immunol. 65, 111–137. Banafsche, R., et al., 2002. Desmopressin impairs hepatic microcirculation: impact on liver graft quality. Transplant. Proc. 34, 2310–2311. Birnbaumer, M., 2000. Vasopressin receptors. Trends Endocrinol. Metab. 11, 406–410. Boyd, J.H., et al., 2008. Vasopressin decreases sepsis-induced pulmonary inflammation through the V2R. Resuscitation 79, 325–331. Chassin, C., et al., 2007. Hormonal control of the renal immune response and antibacterial host defense by arginine vasopressin. J. Exp. Med. 204, 2837–2852. Deitch, E.A., 1990a. Bacterial translocation of the gut flora. J. Trauma 30, S184–S189. Deitch, E.A., 1990b. The role of intestinal barrier failure and bacterial translocation in the development of systemic infection and multiple organ failure. Arch. Surg. 125, 403–404. Deitch, E.A., 1992. Multiple organ failure. Pathophysiology and potential future therapy. Ann. Surg. 216, 117–134. Favory, R., et al., 2009. Investigational vasopressin receptor modulators in the pipeline. Expert Opin. Investig. Drugs. 18, 1119–1131. Franchini, M., 2007. The use of desmopressin as a hemostatic agent: a concise review. Am. J. Hematol. 82, 731–735. Guesde, R., et al., 1998. Administration of desmopressin in brain-dead donors and renal function in kidney recipients. Lancet 352, 1178–1181. Hirschl, M.M., et al., 1996. Effect of desmopressin substitution during organ procurement on early renal allograft function. Nephrol. Dial. Transplant. 11, 173–176. Ince, C., 2005. The microcirculation is the motor of sepsis. Crit. Care 9 (Suppl. 4), S13–S19. Jochberger, S., et al., 2009. The vasopressin and copeptin response to infection, severe sepsis, and septic shock. Crit. Care Med. 37, 476–482. Kanwar, S., et al., 1995. Desmopressin induces endothelial P-selectin expression and leukocyte rolling in postcapillary venules. Blood 86, 2760–2766. Kaufmann, J.E., et al., 2003. Desmopressin (DDAVP) induces NO production in human endothelial cells via V2 receptor- and cAMP-mediated signaling. J. Thromb. Haemost. 1, 821–828. Landry, D.W., Oliver, J.A., 2001. The pathogenesis of vasodilatory shock. N. Engl. J. Med. 345, 588–595. Lee, J.H., et al., 2004. Intercellular adhesion molecule-1 mediates cellular cross-talk between parenchymal and immune cells after lipopolysaccharide neutralization. J. Immunol. 172, 608–616. Legrand, M., et al., 2010. The response of the host microcirculation to bacterial sepsis: does the pathogen matter? J. Mol. Med. 88, 127–133. Liu, S.F., Malik, A.B., 2006. NF-kappa B activation as a pathological mechanism of septic shock and inflammation. Am. J. Physiol. Lung Cell. Mol. Physiol. 290, L622–L645. Lundy, D.J., Trzeciak, S., 2009. Microcirculatory dysfunction in sepsis. Crit. Care Clin. 25, 721–731 (viii). Pahl, H.L., 1999. Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene 18, 6853–6866. Pavlovic, D., et al., 2006. Thermostatic tissue platform for intravital microscopy: “the hanging drop” model. J. Microsc. 224, 203–210. Rybaltowski, M., et al., 2011. In vivo imaging analysis of the interaction between unusually large von Willebrand factor multimers and platelets on the surface of vascular wall. Pflugers Arch. 461, 623–633. Trigg, D.E., et al., 2012. A systematic review: the use of desmopressin for treatment and prophylaxis of bleeding disorders in pregnancy. Haemophilia 18, 25–33. Tsai, H.M., et al., 1990. Desmopressin induces adhesion of normal human erythrocytes to the endothelial surface of a perfused microvascular preparation. Blood 75, 261–265.
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In conclusion, this study provided evidence that DDAVP improves intestinal microcirculation in experimental endotoxemia. We demonstrate that at low doses DDAVP exhibits anti-inflammatory properties without affecting the macrocirculation. Further experiments are required to elucidate the exact mechanism by which DDAVP attenuates the inflammatory response.
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In our experiments while DDAVP did not significantly increase the number of temporary adherent leukocytes, we observed a trend to higher values compared to controls. This result is in agreement with Banafsche et al. and other studies which demonstrate that DDAVP increases the number of temporary adherent leukocytes (Kanwar et al., 1995). DDAVP is also known to induce erythrocyte adherence to microvasculature (Tsai et al., 1990). However, DDAVP was not associated with blockage of the microcirculation despite reporting increased adherence of red blood cells to the microvasculature. We observed the same effect whereby DDAVP administration did not impact the intestinal microcirculation despite the observed trend in the increase in intestinal temporary adherent leukocytes numbers. Additionally, MAP values for rats receiving DDAVP were comparable to those of control rats. While Banafsche et al. used the same concentrations of DDAVP and the same method of administration in their single dose data they administered the DDAVP only 15 min prior to IVM, whereas we administered the DDAVP 90 min prior to IVM. This could indicate that the increase in temporary leukocyte adherence is a transient phenomenon. It is also possible that rat strain differences could be responsible for the discrepancy between these two studies. Interestingly, DDAVP significantly decreased leukocyte adhesion in V1 venules during endotoxemia. We believe this phenomenon could be associated with the observed improvement of the intestinal capillary perfusion. Given the larger size of V1 venules compared to V3 venules, there would be an increased shear force due to blood flow in V1 venules compared to V3 venules. By improving blood flow leukocytes are less capable of adhering to the endothelium. Attenuation of NF-κB signaling as well as upregulation of cAMP levels could also be responsible for decreased leukocyte adhesion. In human epithelial A549 cells an increase in cAMP has been demonstrated to inhibit the TNF induced vascular cell adhesion molecule 1 release (Chassin et al., 2007; Lee et al., 2004). Additionally, Boyd et al. have demonstrated that stimulation of the V2R by vasopressin in the lung reduced pulmonary inflammation as measured by IL-6 levels (Boyd et al., 2008). This phenomenon was attributed to the attenuation of NF-κB signaling by V2R stimulation by vasopressin. Sepsis is the systemic inflammatory response to an infection (Liu and Malik, 2006). In agreement with the literature, our endotoxemia model demonstrates an enhanced inflammatory response by the release of TNF-α, IL-1α, IL-4, INFγ, and GM–CSF. Interestingly, in our model treatment of endotoxemia by DDAVP resulted in a significant decrease of TNF-α plasma levels suggesting an anti-inflammatory role. NF-κB signaling has been established to mediate the transcriptional activation of proinflammatory cytokines, chemokines and adhesion molecules (Baeuerle and Baichwal, 1997; Pahl, 1999). Some of these molecules include TNF, IL-1, IL-2, IL-3, IL-5, IL-6, IL-12, and IL-18. We believe the attenuation of NF-κB signaling by DDAVP might explain the significant decrease in TNF-α levels obtained with DDAVP treatment of LPS induced endotoxemia. Additionally, activation of cAMP signaling has been associated with immune suppression specifically attenuating LPS and TNF-α mediated immune response (Chassin et al., 2007). However, further studies are required to elucidate the exact mechanism responsible for DDAVP related anti-inflammatory effects.
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