C Lehmann

Anti-inflammatory effects of cannabinoid CB2 receptor activation in endotoxin-induced uveitis

Cannabinoid CB2 receptors mediate immunomodulation. Here, we investigated the effects of CB2 receptor ligands on leukocyte‐endothelial adhesion and inflammatory mediator release in experimental endotoxin‐induced uveitis (EIU).

Clinical evaluation of the intestinal microcirculation using sidestream dark field imaging - Recommendations of a round table meeting

INTRODUCTION In clinical setting, Sidestream Dark Field (SDF) imaging has provided unprecedented insights into the gut microcirculation mainly by studying the intestinal mucosa of patients with ileostomies. Visualizing microvascular structure and function of ileal mucosa at the bedside brings unique opportunity for clinical research, particularly in critically ill patients. Several papers that were focused on intestinal microcirculation, used different methods of assessment because an accepted scoring systems does not exist so far and it is no surprise that it is rather difficult to compare the results from these studies. The present paper presents recommendations concerning specific aspects of image acquisition and proposes some parameters for the description of the intestinal microcirculation in human studies, as suggested by the participants of a round table meeting. METHODS The round table meeting participants reviewed all relevant literature, discussed various aspects of image acquisition by SDF technology in patients with ileostomy and parameters for the description of intestinal mucosa microcirculation. Selected key conditions for high quality and reproducible image recordings were identified. To evaluate quality of intestinal microcirculation, selected parameters and scoring system were suggested and described. RESULTS For image acquisition in ileostomies, five key points were proposed: optimal timing, optimal SDF device probe positioning, optimal stabilization, optimal number and length of acquired video recordings, and optimal avoidance of pressure artefacts. With regard to image analysis, simplified set of quantitative and qualitative parameters for the description of the intestinal mucosa microcirculation for the clinical studies has been proposed: vessels per villus, microvascular flow index, proportion of perfused villi, and borders of villi. The proposed parameters can be included in a semi-quantitative scoring system; however, this scoring system needs further validation. This simplified analysis does not require sophisticated software and can be performed manually on the video screen. CONCLUSION We propose a simple methodology for image acquisition and suggest specific microvascular parameters to analyze SDF imaging studies of the intestinal mucosa microcirculation in patients with ileostomy. Proposed scoring system needs to be validated in further clinical studies.

Cannabinoid 2 receptor activation reduces leukocyte adhesion and improves capillary perfusion in the iridial microvasculature during systemic inflammation

BACKGROUND Leukocyte adhesion to the endothelium and decreased microvascular blood flow causing microcirculatory dysfunction are hallmarks of systemic inflammation. We studied the impact of cannabinoid receptor activation on the iridial microcirculation, which is accessible non-invasively in vivo, in systemic inflammation induced by endotoxin challenge. METHODS 40 Lewis rats were used in the experiments. Endotoxemia was induced by 2 mg/kg i.v. lipopolysaccharide (LPS). Cannabinoid receptors (CBRs) were stimulated by i.v. administration of WIN 55212-2 (WIN; 1 mg/kg). CB1R antagonist (AM281; 2.5 mg/kg i.v.) or CB2R antagonist (AM630; 2.5 mg/kg i.v.) treatment prior to WIN was applied to identify the anti-inflammatory effects underlying each CBR subtype. Leukocyte-endothelial interactions were examined in rat iridial microvas culature by intravital microscopy at baseline and 1 and 2 h post-LPS. Additionally, systemic (mean arterial pressure, heart rate) and local (laser Doppler flow) hemodynamic variables were measured prior to and during cannabinoid treatments. RESULTS Endotoxemia resulted in severe inflammation as shown by significantly increased numbers of adherent leukocytes at 1 and 2 h observation time post-LPS challenge and decreased microcirculatory blood flow at 2 h within the iridial microcirculation. WIN treatment significantly reduced leukocyte adhesion in iridial microvessels with a diameter greater and less than 25 μm during endotoxemia (p <  0.05). Pre-treatment of animals by CB1R antagonist, AM281, did not affect WIN effects on LPS-induced leukocyte adhesion. When pre-treated with the CB2R antagonist, AM630, a reversal of the WIN-induced reduction in leukocyte adhesion was noticed in vessels with a diameter of less than 25 μm (p <  0.05). Cannabinoid treatment significantly increased the local iridial microcirculatory blood flow 2 hours after systemic LPS administration (p <  0.05). CONCLUSIONS Systemic administration of the CBR agonist, WIN, decreased leukocyte-adhesion and improved iridial microvascular blood flow. This effect is most likely mediated by CB2R activation. Our findings indicate that the iris microvasculature can serve as a model to study the microcirculation during systemic inflammation and help to identify potential therapies to treat microcirculatory dysfunction in diseases such as sepsis.

Leukocyte-endothelial interactions within the ocular microcirculation in inflammation and infection.

Leukocyte-endothelial interactions within the microvasculature represent a hallmark of inflammation regardless of whether the inflammation results from non-infectious or infectious triggers. In this review, we highlight features of leukocyte recruitment in ocular disease and postulate mechanisms by which the infiltrating cells may lead to the progression of the ocular inflammatory response, including cytokine and chemokine production, T cell or non-T cell responses. Additionally, ex-vivo and in vivo methods used to study the general features of the immune response are discussed, with a specific focus on intravital imaging, which allows real-time non-invasive examination of leukocyte-endothelial interactions in the ocular microvasculature. At the present time there are still significant gaps in our understanding of the process of leukocyte recruitment in vivo in different microvascular beds. Further studies using non-invasive imaging approaches, such as intravital microscopy, provide an opportunity to study dynamic tissue-specific leukocyte-endothelial interactions in vivo and identify novel targets for early intervention in the inflammatory process. This knowledge is essential to the rational use of therapeutics to resolve inflammation in ocular disease.

Effects of N-acetylcysteine and tirilazad mesylate on intestinal functional capillary density, leukocyte adherence, mesenteric plasma extravasation and cytokine levels in experimental endotoxemia in rats.

INTRODUCTION The studys objective was to determine the effects of the administration of N-acetylcysteine (NAC) and of tirilazad mesylate (TM) on intestinal functional capillary density, mesenteric plasma extravasation, leukocyte adherence and on cytokine release during experimental endotoxemia in rats. METHODS In a prospective, randomized, controlled animal study, 80 male Wistar rats were examined in 2 test series. Both series were divided into 4 groups. Group 1 served as control group (CON group). Group 2 (LPS group), group 3 (NAC group) and group 4 (TM group) received endotoxin infusions (10 mg/kg over 2 h). In NAC group 150 mg/kg body weight NAC was administered after the first 30 minutes of endotoxemia intravenously. In TM group, 10 mg/kg body weight TM was administered after the first 30 minutes of endotoxemia intravenously. Animals of the series 1 underwent studies of leukocyte adherence on submucosal venular endothelium of the small bowel wall and intestinal functional capillary density (FCD) in the intestinal mucosa and the circular as well as the longitudinal muscle layer by intravital fluorescence microscopy (IVM). Plasma levels of interleukin 1beta (IL-1beta), interferone gamma (IFN-gamma) and soluble intercellular adhesion molecule1 (s-ICAM 1) as well as white blood cell count (WBC) were estimated. In the animals of the series 2 mesenteric plasma extravasation was determined by IVM and plasma levels of tumor necrosis factor alpha (TNF-alpha), IL-4, IL-6, IL-10 and malondialdehyde (MDA) were estimated. RESULTS After LPS administration, FCD in the villi intestinales was unchanged and in the longitudinal muscularis layer it was increased. There was no effect of NAC or TM administration on FCD.Although the plasma extravasation was not significantly influenced by LPS administration, TM administration resulted in a lower plasma extravasation in the TM group compared to the other groups. After endotoxin challenge, the firmly adherence of leukocytes to vascular endothelium as a parameter of leukocyte activation in endotoxemia was increased but NAC or TM administration had no influence on leukocyte adherence. The plasma levels of IL-1beta, IL-6, IL-10, TNF-alpha, IFN-gamma and sICAM-1 were increased in the endotoxemic groups (LPS group, NAC group and TM group) and the WBC was decreased compared to controls. IL-4 levels were unchanged during observation period. Plasma MDA levels were not influenced by LPS administration compared to controls. The administration of NAC resulted in lower sICAM-1 and MDA levels compared to the LPS group. The IL-1beta, IL-6, IL-10, TNF-alpha and IFN-gamma plasma levels were not influenced by NAC or TM administration. CONCLUSIONS In this posttreatment sepsis model in rats, NAC administration resulted in lower sICAM-1 and MDA levels compared to the LPS treated animals. TM administration reduced the plasma extravasation in this model.

Impact of antibiotics on the microcirculation in local and systemic inflammation

The main function of antibiotics is related to their capacity to eliminate a microorganism. In addition to the antimicrobial function of antibiotics, they are known to have anti-inflammatory and vasomodulatory effects on the microcirculation. The ability of non-antimicrobial derivatives of antibiotics to control inflammation illustrates the distinct anti-microbial and anti-inflammatory roles of antibiotics. In this review, we discuss the impact of antibiotics on leukocyte recruitment and the state of the microcirculation. Literature reporting the effect of antibiotics in non-infectious inflammatory conditions is reviewed as well as the studies demonstrating the anti-inflammatory effects of antibiotics in animal models of infection. In addition, the effect of the antibiotics on the immune system is summarized in this review, in order to postulate some mechanisms of action for the proand anti-inflammatory contribution of antibiotics. Literature reported the effect of antibiotics on the production of cytokines, chemotaxis and recruitment of leukocytes, production of reactive oxygen species, process of phagocytosis and autophagy, and apoptosis of leukocytes. Yet, all antibiotics may not necessarily exert an anti-inflammatory effect on the microcirculation. Thus, we suggest a model for spectrum of anti-inflammatory and vasomodulatory effects of antibiotics in the microcirculation of animals in local and systemic inflammation. Although the literature suggests the ability of antibiotics to modulate leukocyte recruitment and microperfusion, the process and the mechanism of action are not fully characterized. Studying this process will expand the knowledge base that is required for the selection of antibiotic treatment based on its anti-inflammatory functions, which might be particularly important for critically ill patients.

Desmopressin improves intestinal functional capillary density and decreases leukocyte activation in experimental endotoxemia

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 (1μg/kg/ml), lipopolysaccharide (5mg/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, non-functional 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/mm(2); p<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<0.05). CONCLUSION Desmopressin administration improved intestinal capillary perfusion and reduced inflammatory response in rat endotoxemia.

Short time impact of different hydroxyethyl starch solutions on the mesenteric microcirculation in experimental sepsis in rats

BACKGROUND Fluid resuscitation plays a crucial role in the therapy of severe sepsis and septic shock. The use of colloids in sepsis is controversial at present. The aim of our study was to evaluate the effects of second and third generation colloids on the mesenteric microcirculation in early experimental sepsis. METHODS Male Lewis rats (n=64) were used. Animals underwent sham surgery or colon ascendens stent insertion for sepsis induction by peritonitis. Sixteen hours after the surgery animals were randomly assigned to receive one of the following fluid regimens intravenously: 16ml/kg Ringers lactate, 64ml/kg Ringers lactate, 16ml/kg 130/0.4 hydroxyethyl starch, and 16ml/kg 200/0.5 hydroxyethyl starch. Intravital microscopy of the mesenteric microcirculation (plasma extravasation; leukocyte-endothelial interactions) and arterial blood gas analysis were performed before and after fluid resuscitation. RESULTS In animals with experimental sepsis plasma extravasation was significantly increased compared to control animals (p<0.05). There were no significant differences in plasma extravasation between septic animals receiving crystalloids and or colloid. Furthermore, the type of administered fluid did not influence the number of adhering leucocytes during the observation period. CONCLUSION The short time impact of different hydroxyethyl starch solutions on the microcirculation of the mesentery is not different from crystalloids in colon ascendens stent peritonitis-induced experimental sepsis in rats.

Metabolomic analysis as biomarker to study steroid hormone administration in sepsis

Sepsis is a life-threatening disease requiring rapid diagnosis and treatment. Steroid hormones (e.g., estradiol, dehydroepiandosterone) have been suggested to reduce the hyper-inflammatory response of the immune system and to improve outcome in sepsis. We hypothesize that the impact of steroid hormones on the metabolic profile (metabolomic fingerprint) can be used to study and guide steroid hormone administration in sepsis. Potential biomarker candidates are sphingomyelines and phosphatidylcholines.

Impact of Combined C1 Esterase Inhibitor/Coagulation Factor XIII or N-Acetylcysteine/Tirilazad Mesylate Administration on Leucocyte Adherence and Cytokine Release in Experimental Endotoxaemia

We determined the effects of combinations of C1 esterase inhibitor (C1-INH) with factor XIII and of N-acetylcysteine (NAC) with tirilazad mesylate (TM) during lipopolysaccharide (LPS)-induced endotoxaemia in rats. Forty Wistar rats were divided into four groups: the control (CON) group received no LPS; the LPS, C1-INH + factor XIII and NAC + TM groups received endotoxin infusions (5 mg/kg per h). After 30 min of endotoxaemia, 100 U/kg C1-INH + 50 U/kg factor XIII was administered to the C1-INH + factor XIII group, and 150 mg/kg NAC + 10 mg/kg TM was administered in the NAC + TM group. Administration of C1-INH + factor XIII and NAC + TM both resulted in reduced leucocyte adherence and reduced levels of interleukin-1β (IL-1β). The LPS-induced increase in IL-6 levels was amplified by both drug combinations. There was no significant effect on mesenteric plasma extravasation. In conclusion, the administration of C1-INH + factor XIII and NAC + TM reduced endothelial leucocyte adherence and IL-1β plasma levels, but increased IL-6 levels.