Influence of high dose tumescent local anaesthesia with prilocaine on systemic interleukin (IL)-6, IL-8 and tumour necrosis factor-α.

BACKGROUND AND OBJECTIVE: Tumescent local anaesthesia (TLA) with high prilocaine doses leads to formation of methemoglobin (MHb) which is known to be a potent activator of pro-inflammatory endothelial cell response in vitro. As TLA is widely used for large dermatological resections, the aim of this study was to investigate the effects of high prilocaine doses on the systemic inflammatory response in vivo and its clinical relevance. METHODS: This prospective study examines the influence of MHb on serum interleukin (IL)-6, IL-8 and tumour necrosis tumour necrosis (TNF)-α levels up to 72 h after application of TLA with prilocaine in doses higher than 600 mg. RESULTS: A total of 30 patients received prilocaine in a median dose of 1500 mg (range: 880-4160 mg) for large resections. Peak prilocaine serum concentration was reached 4 h (0.72 ± 0.07 µg/mL), the maximum concentration of MHb (7.43 ± 0.87%) and IL-6 (28.4 ± 4.1 U/L) 12 h after TLA application. TNF-α and IL-8 release were not found significantly increased. Three patients developed MHb concentrations >15%. CONCLUSIONS: This clinical study shows for the first time that a high prilocaine serum concentration leads in vivo to elevated systemic levels of IL-6 but not of IL-8 and TNF-α because of initial high MHb levels. Because of possible and unpredictable high MHb concentrations, TLA should only be performed with prilocaine in doses of 2.5 mg/kg. In general, new solutions of TLA are necessary to achieve adequate anaesthesia for large dermatological resections to decrease the risk of methemoglobinaemia.

Anaesthesia of small rodents during magnetic resonance imaging.

The use of experimental animals for magnetic resonance studies requires anaesthesia to provide immobility and acquire signals with minimal stress and maximal reproducibility. However, the conduct of anaesthesia within a magnetic resonance imaging (MRI) suite implicates many problems, because most of the anaesthetic and monitoring equipment contains ferromagnetic substances. To decrease disturbances during anaesthesia and make data interpretation more accurate, it is mandatory that investigators become familiar with methods and physiologic effects of anaesthesia under these special conditions. This article is intended to give an overview of anaesthetic medication, administration routes and practical instructions for anaesthesia in small rodents during MRI.

[The role of endothelial progenitor cells in sepsis]. / Die Rolle endothelialer Vorläuferzellen in der Sepsis.

In sepsis and septic shock a series of immunological events are initiated that alter endothelial function in the macrocirculation and microcirculation. Endothelial swelling, deformation and apoptosis with detachment from the vasculature occur and endothelial cells (EC) appear in the circulation. Simultaneous to these pathological processes, reconstitution of the endothelial layer is initiated which can occur via migration and proliferation of surrounding mature ECs. However, terminally differentiated ECs have a low proliferative potential, hence their capacity to substitute damaged endothelium is limited. Therefore, adequate vascular repair requires additional support. Many studies have now convincingly demonstrated that vascular maintenance, repair, angiogenesis and neovascularization are partly mediated by recruitment of endothelial progenitor cells (EPCs) from the basal membrane. However, it seems that EPCs play a pivotal role not only in re-endothelialization after vascular damage, but also after severe inflammation. Recently, evidence was found that EPCs are increasingly mobilized during sepsis and that this mobilization is associated with clinical outcome. In septic patients the number of EPCs was significantly higher than in controls and was correlated with survival and the concentration of cytokines. In summary EPCs may exert an important function as an endogenous repair mechanism to maintain the integrity of the endothelial layer by replacing denuded parts of the microcirculation or by stimulation of EC proliferation. Therefore, EPC enumeration seems to be a valuable prognostic and diagnostic marker for the outcome in these patients and the induction of enhanced EPC mobilization a therapeutic option.

Hypothermic preservation of lung allograft inhibits cytokine-induced chemoattractant-1, endothelial leucocyte adhesion molecule, vascular cell adhesion molecule-1 and intracellular adhesion molecule-1 expression.

Organ dysfunction is a major clinical problem after lung transplantation. Prolonged cold ischaemia and reperfusion injury are believed to play a central role in this complication. The influence of cold preservation on subsequent warm reperfusion was studied in an isolated, ventilated and perfused rat lung. Rat lungs were flushed with cold Perfadex-solution and stored at 4 degrees C for different time periods. Thereafter lungs were perfused and ventilated for up to 3 h. Physiological parameters, production of inflammatory mediators and leucocyte infiltration were measured before and after perfusion. Lungs subjected to a cold ischaemia time of up to 6 h showed stable physiological conditions when perfused for 3 h. However, cold-ischaemia time beyond 6 h resulted in profound tissue oedema, thereby impairing ventilation and perfusion. Warm reperfusion and ventilation per se induced a strong inflammatory response, as demonstrated by a significant up-regulation of chemokines and adhesion molecules (cytokine-induced chemoattractant-1, intracellular adhesion molecule and endothelial leucocyte adhesion molecule), accompanied by enhanced leucocyte infiltration. Although the up-regulation of inflammatory mediators was blunted in lungs that were subjected to cold ischaemia, this did not influence leucocyte infiltration. In fact, cold ischaemia time correlated with leucocyte sequestration. Although cold preservation inhibits the expression of inflammatory mediators it does not affect leucocyte sequestration during warm reperfusion. Cold preservation might cause impairment of the endothelial barrier function, as evidenced by tissue oedema and profound leucocyte infiltration.

Heterogeneity in lipopolysaccharide responsiveness of endothelial cells identified by gene expression profiling: role of transcription factors.

Interindividual differences of endothelial cells in response to endotoxins might contribute to the diversity in clinical outcome among septic patients. The present study was conducted to test the hypothesis that endothelial cells (EC) with high and low proinflammatory potential exist and to dissect the molecular basis underlying this phenomenon. Thirty human umbilical vein endothelial cell (HUVEC) lines were stimulated for 24 h with lipopolysaccharide (LPS) and screened for interleukin (IL)-8 production. Based on IL-8 production five low and five high producers, tentatively called types I and II responders, respectively, were selected for genome-wide gene expression profiling. From the 74 genes that were modulated by LPS in all type II responders, 33 genes were not influenced in type I responders. Among the 41 genes that were increased in both responders, 17 were expressed significantly stronger in type II responders. Apart from IL-8, significant differences in the expression of proinflammatory related genes between types I and II responders were found for adhesion molecules [intercellular adhesion molecule (ICAM-1), E-selectin)], chemokines [monocyte chemoattractant protein (MCP-1), granulocyte chemotactic protein (GCP-2)], cytokines (IL-6) and the transcription factor CCAAT/enhancer binding protein-delta (C/EBP-delta). Type I responders also displayed a low response towards tumour necrosis factor (TNF)-alpha. In general, maximal activation of nuclear factor (NF)-kappaB was achieved in type I responders at higher concentrations of LPS compared to type II responders. In the present study we demonstrate that LPS-mediated gene expression differs quantitatively and qualitatively in types I and II responders. Our results suggest a pivotal role for common transcription factors as a low inflammatory response was also observed after TNF-alpha stimulation. Further studies are required to elucidate the relevance of these findings in terms of clinical outcome in septic patients.

Release of CXC-chemokines by human lung microvascular endothelial cells (LMVEC) compared with macrovascular umbilical vein endothelial cells.

In the present study, the sensitivity of LMVEC and human umbilical vein endothelial cells (HUVEC) to lipopolysaccharide (LPS) and the proinflammatory cytokines IL-1, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) was compared. To this end, the production of the CC- (MCP-1), CXC- (IL-8, ENA-78, Groalpha, NAP-2, GCP-2) and CX3C (fractalkine) chemokines was studied. A low basal production of these chemokines was observed in both cell types. TNF-alpha, IL-1 and LPS up-regulated all chemokines tested. IFN-gamma however was only able to up-regulate MCP-1 production. LMVEC were more sensitive to IL-1 and LPS compared with HUVEC, since LMVEC produced significantly more MCP-1, ENA-78 and Groalpha (P < 0. 01) under these conditions. Maximal production of MCP-1 in LMVEC was achieved with TNF-alpha (28.4 ng/ml, P < 0.01), whereas IL-1 was the most potent stimulator of ENA-78 (2.78 ng/ml, P < 0.001) and Groalpha (29.2 ng/ml, P < 0.001). IL-8 production in LMVEC cells was maximal after LPS stimulation (28.4 ng/ml), but lower than on HUVEC (P < 0.01). LPS, TNF-alpha and IL-1 stimulation strongly up-regulated all chemokine mRNA. No quantitative differences in mRNA expression between LMVEC and HUVEC were detected for MCP-1 and Groalpha after LPS stimulation. mRNA expression of ENA-78, GCP-2, CX3C and NAP-2 was however higher in LMVEC under LPS stimulation. In contrast, IL-8 mRNA was slightly more expressed in HUVEC under these conditions. These results further support the hypothesis that the microvascular lung endothelium plays an active role in the induction and perpetuation of acute lung injury.

Modulation of chemokine production in lung microvascular endothelial cells by dopamine is mediated via an oxidative mechanism.

Serum concentrations of catecholamines are high in patients with sepsis or acute respiratory distress syndrome (ARDS). Because chemokines mediate the recruitment of neutrophils into inflammatory sites, we addressed the question of whether dopamine (DA) is able to influence chemokine production in endothelial cells under basal and proinflammatory conditions. To this end, lung microvascular endothelial cells (LMVEC) were stimulated or not for 24 h with the bacterial toxins lipopolysaccharide (LPS) (1 microg/ml) or lipoteichonic acid (LTA) (10 microg/ml) in the presence or absence of various concentrations of DA (1-100 microg/ml). Whereas under basal and stimulatory conditions, the addition of DA to endothelial cells dose-dependently increased IL-8 production, the production of ENA-78 and Gro-alpha was significantly inhibited (P < 0.01). This effect could still be demonstrated when the cells were stimulated for up to 3 h with LPS before DA administration. Similar findings were detected for the mRNA expression of these chemokines. The influence of DA on chemokine production was not receptor mediated and could be prevented by antioxidants or radical scavengers. Moreover, addition of H(2)O(2) to endothelial cells gave results similar to those observed with DA stimulation, suggesting a pivotal role for reactive oxygen species in DA-mediated modulation of chemokine production in endothelial cells. Our data thus demonstrate that DA administration results in the induction of oxidative stress, with profound effects on endothelial chemokine production.