Assessment of endothelium and inflammatory response at the onset of reperfusion injury in hand surgery

Background Activation of the endothelium, complement activation and generation of cytokines are known events during ischemia-reperfusion (I/R) that mediate tissue injury. Our aim was to elucidate their respective participation at the onset of the reperfusion phase. Tourniquet application in hand surgery causes short-term ischemia, followed by reperfusion and was therefore used as the model in this study. Methods Ten patients were included in the study after obtaining informed consent. A tourniquet was placed on the upper arm and inflated to 250 mmHg for 116 ± 16 min, during which the surgery was performed. Venous blood and tissue samples from the surgical area were taken at baseline as well as 0, 2, and 10 min after reperfusion and analyzed for the following parameters: Endothelial integrity and/or activation were analyzed by measuring heparan sulfate and syndecan-1 in serum, and vWF, heparan sulfate proteoglycan as well as CD31on tissue. Complement activation was determined by C3a and C4d levels in plasma, levels of C1-inhibitor in serum, and IgG, IgM, C3b/c, and C4b/c deposition on tissue. Cytokines and growth factors IL-5, IL-6, IL-7, IL-8, IL-10, IL-17, G-CSF, GM-CSF, MCP-1, TNFα, VEGF, and PDGF bb were measured in the serum. Finally, CK-MM levels were determined in plasma as a measure for muscle necrosis. Results Markers for endothelial activation and/or integrity as well as complement activation showed no significant changes until 10 min reperfusion. Among the measured cytokines, IL-6, IL-7, IL-17, TNFα, GM-CSF, VEGF, and PDGF bb were significantly increased at 10 min reperfusion with respect to baseline. CK-MM showed a rise from baseline at the onset of reperfusion (p < 0.001) and dropped again at 2 min (p < 0.01) reperfusion, suggesting ischemic muscle damage. Conclusions In this clinical model of I/R injury no damage to the endothelium, antibody deposition or complement activation were observed during early reperfusion. However, an increase of pro-inflammatory cytokines and growth factors was shown, suggesting a contribution of these molecules in the early stages of I/R injury.

The role of purinergic signaling in the liver and in transplantation: effects of extracellular nucleotides on hepatic graft vascular injury, rejection and metabolism

Extracellular nucleotides (e.g. ATP, UTP, ADP) are released by activated endothelium, leukocytes and platelets within the injured vasculature and bind specific cell-surface type-2 purinergic (P2) receptors. This process drives vascular inflammation and thrombosis within grafted organs. Importantly, there are also vascular ectonucleotidases i.e. ectoenzymes that hydrolyze extracellular nucleotides in the blood to generate nucleosides (viz. adenosine). Endothelial cell NTPDase1/CD39 has been shown to critically modulate levels of circulating nucleotides. This process tends to limit the activation of platelet and leukocyte expressed P2 receptors and also generates adenosine to reverse inflammatory events. This vascular protective CD39 activity is rapidly inhibited by oxidative reactions, such as is observed with liver ischemia reperfusion injury. In this review, we chiefly address the impact of these signaling cascades following liver transplantation. Interestingly, the hepatic vasculature, hepatocytes and all non-parenchymal cell types express several components co-ordinating the purinergic signaling response. With hepatic and vascular dysfunction, we note heightened P2- expression and alterations in ectonucleotidase expression and function that may predispose to progression of disease. In addition to documented impacts upon the vasculature during engraftment, extracellular nucleotides also have direct influences upon liver function and bile flow (both under physiological and pathological states). We have recently shown that alterations in purinergic signaling mediated by altered CD39 expression have major impacts upon hepatic metabolism, repair mechanisms, regeneration and associated immune responses. Future clinical applications in transplantation might involve new therapeutic modalities using soluble recombinant forms of CD39, altering expression of this ectonucleotidase by drugs and/or using small molecules to inhibit deleterious P2-mediated signaling while augmenting beneficial adenosine-mediated effects within the transplanted liver.

Amelioration of lung reperfusion injury by L- and E- selectin blockade

OBJECTIVE: Reperfusion injury is the main reason for early graft failure after lung transplantation. Inhibition of the adherence of polymorphonuclear leukocytes to activated endothelium by blocking L- and E-selectins (antibody EL-246) could potentially inhibit reperfusion injury. METHODS: Reperfusion injury was induced in a left lung autotransplant model in sheep. After hilar stripping the left lung was flushed with Euro-Collins solution and preserved for 2 h in situ at 15 degrees C. After reperfusion right main bronchus and pulmonary artery were occluded leaving the animal dependent on the reperfused lung (control, n = 6). Pulmonary function was assessed by alveolo-arterial oxygen difference (AaDO2) and pulmonary vascular resistance (PVR), the chemiluminescence of isolated neutrophils, as well as the release of beta-N-acetyl-glucosaminidase (beta-NAG) served as indicator of neutrophilic activation. Extravascular lung water was an indicator for pulmonary edema formation. EL-246 group animals (n = 6) were treated additionally with 1 mg/kg BW of EL-246 given prior and during reperfusion. RESULTS: After 3 h of reperfusion five control animals developed alveolar edema compared to one animal in the EL-246 group (P = 0.08). AaDO2 (mm Hg) was significantly higher in the control compared to the EL-246 group (510 +/- 148 vs. 214 +/- 86). PVR (dyn x s x cm(-5)) was significantly increased in the control compared to the EL-246 group (656 +/- 240 vs. 317 +/- 87). Neutrophilic activation was significantly lower in the EL-246 group. Extravascular lung water was significantly lower compared to control (6.88 +/- 1.0 vs. 13.4 +/- 2.8 g/g blood-free lung weight). CONCLUSIONS: Treatment with EL-246 results in improved pulmonary function and less in vivo PMN activation in this experimental model. Further studies are necessary to evaluate the possible role of selectin blockade in amelioration of reperfusion injury in human lung transplantation.

Helium breathing provides modest antiinflammatory, but no endothelial protection against ischemia-reperfusion injury in humans in vivo

BACKGROUND: The noble gas helium is devoid of anesthetic effects, and it elicits cardiac preconditioning. We hypothesized that inhalation of helium provides protection against postocclusive endothelial dysfunction after ischemia-reperfusion of the forearm in humans. METHODS: Eight healthy male subjects were enrolled in this study with a crossover design. Each volunteer was randomly exposed to 15 min of forearm ischemia in the presence or absence of helium inhalation. Helium was inhaled at an end-tidal concentration of 50 vol% from 15 min before ischemia until 5 min after the onset of reperfusion ("helium conditioning"). Hyperemic reaction, a marker of nitric oxide bioavailability and endothelial function, was determined at 15 and 30 min of reperfusion on the forearm using venous occlusion plethysmography. Expression of the proinflammatory markers CD11b, ICAM-1, PSGL-1, and L-selectin (CD62L) on leukocytes and P-selectin (CD62P), PSGL-1, and CD42b on platelets were measured by flow cytometry during reperfusion. RESULTS: Ischemia-reperfusion consistently reduced the postocclusive endothelium-dependent hyperemic reaction at 15 and 30 min of reperfusion. Periischemic inhalation of helium at 50 vol% did not improve postocclusive hyperemic reaction. Helium decreased expression of the proinflammatory marker CD11b and ICAM-1 on leukocytes and attenuated the expression of the procoagulant markers CD42b and PSGL-1 on platelets. CONCLUSIONS: Although inhalation of helium diminished the postischemic inflammatory reaction, our data indicate that human endothelium, which is a component of all vital organs, is not amenable to protection by helium at 50 vol% in vivo. This is in contrast to sevoflurane, which protects human endothelium at low subanesthetic concentrations.

Locally targeted cytoprotection with dextran sulfate attenuates experimental porcine myocardial ischaemia/reperfusion injury

AIMS: Intravascular inflammatory events during ischaemia/reperfusion injury following coronary angioplasty alter and denudate the endothelium of its natural anticoagulant heparan sulfate proteoglycan (HSPG) layer, contributing to myocardial tissue damage. We propose that locally targeted cytoprotection of ischaemic myocardium with the glycosaminoglycan analogue dextran sulfate (DXS, MW 5000) may protect damaged tissue from reperfusion injury by functional restoration of HSPG. METHODS AND RESULTS: In a closed chest porcine model of acute myocardial ischaemia/reperfusion injury (60 min ischaemia, 120 min reperfusion), DXS was administered intracoronarily into the area at risk 5 min prior to reperfusion. Despite similar areas at risk in both groups (39+/-8% and 42+/-9% of left ventricular mass), DXS significantly decreased myocardial infarct size from 61+/-12% of the area at risk for vehicle controls to 39+/-14%. Cardioprotection correlated with reduced cardiac enzyme release creatine kinase (CK-MB, troponin-I). DXS abrogated myocardial complement deposition and substantially decreased vascular expression of pro-coagulant tissue factor in ischaemic myocardium. DXS binding, detected using fluorescein-labelled agent, localized to ischaemically damaged blood vessels/myocardium and correlated with reduced vascular staining of HSPG. CONCLUSION: The significant cardioprotection obtained through targeted cytoprotection of ischaemic tissue prior to reperfusion in this model of acute myocardial infarction suggests a possible role for the local modulation of vascular inflammation by glycosaminoglycan analogues as a novel therapy to reduce reperfusion injury.

Dextran sulfate modulates MAP kinase signaling and reduces endothelial injury in a rat aortic clamping model

OBJECTIVE: Mitogen-activated protein kinases (MAPKs), including JNK, p38, and ERK1/2, noticeably influence ischemia/reperfusion injury (IRI). The complement inhibitor dextran sulfate (DXS) associates with damaged endothelium denudated of its heparan sulfate proteoglycan (HSPG) layer. Other glycosaminoglycan analogs are known to influence MAPK signaling. Hypothetically therefore, targeted intravascular cytoprotection by DXS may function in part through influencing MAPK activation to reduce IRI-induced damage of the vasculature. METHODS: IRI of the infrarenal aorta of male Wistar rats was induced by 90 minutes clamping followed by 120 minutes reperfusion. DXS (5 mg/mL) or physiologic saline (NaCl controls) was infused locally into the ischemic aortic segment immediately prior to reperfusion. Ninety minutes ischemia-only and heparinase infusion (maximal damage) experiments, as well as native rat aorta, served as controls. Aortas were excised following termination of the experiments for further analysis. RESULTS: DXS significantly inhibited IRI-induced JNK and ERK1/2 activation (P = .043; P =.005) without influencing the p38 pathway (P =.110). Reduced aortic injury, with significant inhibition of apoptosis (P = .032 for DXS vs NaCl), correlated with decreased nuclear factor kappaB translocation within the aortic wall. DXS treatment clearly reduced C1q, C4b/c, C3b/c, and C9 complement deposition, whilst preserving endothelial cell integrity and reducing reperfusion-induced HSPG shedding. Protection was associated with binding of fluorescein labeled DXS to ischemically damaged tissue. CONCLUSIONS: Local application of DXS into ischemic vasculature immediately prior to reperfusion reduces complement deposition and preserves endothelial integrity, partially through modulating activation of MAPKs and may offer a new approach to tackle IRI in vascular surgical procedures. CLINICAL RELEVANCE: The purpose of the present study was to determine the role of dextran sulfate (DXS), a glycosaminoglycan analog and complement inhibitor, in modulating intracellular MAPK signaling pathways, reducing complement activation and ultimately attenuating ischemia/reperfusion injury (IRI) in a rat aortic-clamping model, in part a surrogate model to study the microvasculature. The study shows a role for DXS in ameliorating endothelial injury by reducing IRI-mediated damage and intravascular, local inflammation in the affected aortic segment. DXS may be envisaged as an endothelial protectant in vascular injury, such as occurs during vascular surgical procedures.

Deletion of CD39 on natural killer cells attenuates hepatic ischemia/reperfusion injury in mice

Natural killer (NK) cells play crucial roles in innate immunity and express CD39 (Ecto-nucleoside triphosphate diphosphohydrolase 1 [E-NTPD1]), a rate-limiting ectonucleotidase in the phosphohydrolysis of extracellular nucleotides to adenosine. We have studied the effects of CD39 gene deletion on NK cells in dictating outcomes after partial hepatic ischemia/reperfusion injury (IRI). We show in mice that gene deletion of CD39 is associated with marked decreases in phosphohydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate to adenosine monophosphate on NK cells, thereby modulating the type-2 purinergic (P2) receptors demonstrated on these cells. We note that CD39-null mice are protected from acute vascular injury after single-lobe warm IRI, and, relative to control wild-type mice, display significantly less elevation of aminotransferases with less pronounced histopathological changes associated with IRI. Selective adoptive transfers of immune cells into Rag2/common gamma null mice (deficient in T cells, B cells, and NK/NKT cells) suggest that it is CD39 deletion on NK cells that provides end-organ protection, which is comparable to that seen in the absence of interferon gamma. Indeed, NK effector mechanisms such as interferon gamma secretion are inhibited by P2 receptor activation in vitro. Specifically, ATPgammaS (a nonhydrolyzable ATP analog) inhibits secretion of interferon gamma by NK cells in response to interleukin-12 and interleukin-18, providing a mechanistic link between CD39 deletion and altered cytokine secretion. CONCLUSION: We propose that CD39 deficiency and changes in P2 receptor activation abrogate secretion of interferon gamma by NK cells in response to inflammatory mediators, thereby limiting tissue damage mediated by these innate immune cells during IRI.

GnRH analogs do not protect ovaries from chemotherapy-induced ultrastructural injury in Hodgkin's lymphoma patients

To determine the protective effect of gonadotropin-releasing hormone analogs (GnRHa) using several ultrasound and endocrine markers to detect ultrastructural ovarian damage in Hodgkin's lymphoma patients.

Do we really need 24-h observation for patients with minimal brain injury and small intracranial bleeding? The Bernese Trauma Unit Protocol

Traumatic brain injury is one of the most common reasons for admission to hospital emergency departments. However, optimal diagnosis and treatment protocols remain controversial. The aim of this study is to assess whether a specific group of patients can be discharged from the hospital without 24-h neurological observation.