Petra Hartmann

Anti-inflammatory effects of phosphatidylcholine in neutrophil leukocyte-dependent acute arthritis in rats

We investigated the effects of exogenous phosphatidylcholine (PC) and non-steroidal diclofenac supplementation on polymorphonuclear cell influx in carrageenan-induced arthritis in rats. The microcirculatory consequences were evaluated by a novel method developed for direct intravital microscopic observation of the synovial membrane. Arthritis was induced by injection of a mixture of 2% lambda-carrageenan and 4% kaolin into the knee joints and the animals were treated orally with PC (150 mg/kg twice daily), sodium diclofenac (0.5mg/kg twice daily) or saline vehicle. Intravital videomicroscopy was used to investigate the leukocyte-endothelial interactions directly in the synovial membrane at 6h after the challenge. The inflammation-induced thermal and mechanical secondary hyperalgesic reactions were assessed at 24h, and the knee volume changes at 48h after the insult. The development of arthritis was accompanied by a significant increase in the number of adherent leukocytes in the synovial postcapillary venules, but this increase was reduced significantly (by approximately 40%) by PC, and slightly (by 22%) by diclofenac treatment. The perivascular infiltration of the neutrophil leukocytes and the intercellular adhesion molecule-1 (ICAM-1) expressions were reduced only by PC treatment. The significant decrease (45%) in the thermal nociceptive latency, the 3-fold increase in the mechanical touch sensitivity and the knee cross-sectional area (which was increased by 35% by the arthritis induction) were significantly ameliorated by both treatments. The present study demonstrated the anti-inflammatory effects of PC in experimental arthritis. The therapeutic potential may be linked to the reduction of neutrophil leukocyte-mediated microcirculatory inflammatory reactions.

Protective effects of a phosphatidylcholine-enriched diet in lipopolysaccharide-induced experimental neuroinflammation in the rat.

Our goal was to characterize the neuroprotective properties of orally administered phosphatidylcholine (PC) in a rodent model of systemic inflammation. Sprague-Dawley rats were killed at 3 h, 1 day, 3 days, or 7 days after i.p. administration of lipopolysaccharide (LPS) to determine the plasma levels of tumor necrosis factor &agr; (TNF-&agr;) and interleukin 6 cytokines. The control group and one group of LPS-treated animals were nourished with standard laboratory chow, whereas another LPS-treated group received a special diet enriched with 1% PC for 5 days before the administration of LPS and thereafter during the 7-day observation period. Immunohistochemistry was performed to visualize the bromodeoxyuridine and doublecortin-positive neuroprogenitor cells and Iba1-positive microglia in the hippocampus, whereas the degree of mucosal damage was evaluated on ileal and colon biopsy samples after hematoxylin-eosin staining. The activities of proinflammatory myeloperoxidase and xanthine-oxidoreductase and the tissue nitrite/nitrate (NOx) level were additionally determined, and the cognitive functions were monitored via Morris water maze testing. The inflammatory challenge transiently increased the hippocampal NOx level and led to microglia accumulation and decreased neurogenesis. The intestinal damage, mucosal myeloperoxidase, xanthine-oxidoreductase, and NOx changes were less pronounced, and long-lasting behavioral alterations were not observed. Phosphatidylcholine pretreatment reduced the plasma TNF-&agr; and hippocampal NOx changes and prevented the decreased neurogenesis. These data demonstrated the relative susceptibility of the brain to the consequences of transient peripheral inflammatory stimuli. Phosphatidylcholine supplementation did not reduce the overall extent of peripheral inflammatory activation, but efficiently counteracted the disturbed hippocampal neurogenesis by lowering circulating TNF-&agr; concentrations.

Inhaled Methane Limits the Mitochondrial Electron Transport Chain Dysfunction during Experimental Liver Ischemia-Reperfusion Injury

Background Methanogenesis can indicate the fermentation activity of the gastrointestinal anaerobic flora. Methane also has a demonstrated anti-inflammatory potential. We hypothesized that enriched methane inhalation can influence the respiratory activity of the liver mitochondria after an ischemia-reperfusion (IR) challenge. Methods The activity of oxidative phosphorylation system complexes was determined after in vitro methane treatment of intact liver mitochondria. Anesthetized Sprague-Dawley rats subjected to standardized 60-min warm hepatic ischemia inhaled normoxic air (n = 6) or normoxic air containing 2.2% methane, from 50 min of ischemia and throughout the 60-min reperfusion period (n = 6). Measurement data were compared with those on sham-operated animals (n = 6 each). Liver biopsy samples were subjected to high-resolution respirometry; whole-blood superoxide and hydrogen peroxide production was measured; hepatocyte apoptosis was detected with TUNEL staining and in vivo fluorescence laser scanning microscopy. Results Significantly decreased complex II-linked basal respiration was found in the normoxic IR group at 55 min of ischemia and a lower respiratory capacity (~60%) and after 5 min of reperfusion. Methane inhalation preserved the maximal respiratory capacity at 55 min of ischemia and significantly improved the basal respiration during the first 30 min of reperfusion. The IR-induced cytochrome c activity, reactive oxygen species (ROS) production and hepatocyte apoptosis were also significantly reduced. Conclusions The normoxic IR injury was accompanied by significant functional damage of the inner mitochondrial membrane, increased cytochrome c activity, enhanced ROS production and apoptosis. An elevated methane intake confers significant protection against mitochondrial dysfunction and reduces the oxidative damage of the hepatocytes.

L-alpha-glycerylphosphorylcholine reduces the microcirculatory dysfunction and nicotinamide adenine dinucleotide phosphate-oxidase type 4 induction after partial hepatic ischemia in rats

BACKGROUNDnWe set out to investigate the microcirculatory consequences of hepatic ischemia-reperfusion (IR) injury and the effects of L-alpha-glycerylphosphorylcholine (GPC), a deacylated phospholipid derivative, on postischemic hepatocellular damage, with special emphasis on the expression of nicotinamide adenine dinucleotide phosphate oxidase type 4 (NOX4), which is predominantly expressed in hepatic microvessels.nnnMATERIALS AND METHODSnAnesthetized male Sprague-Dawley rats were subjected to 60-min ischemia of the left liver lobes and 180-min reperfusion, with or without GPC treatment (50 mg/kg intravenously 5 min before reperfusion, n = 6 each). A third group (n = 6) served as saline-treated control. Noninvasive online examination of the hepatic microcirculation was performed hourly by means of modified spectrometry. Plasma tumor necrosis factor (TNF-α), high-mobility group box 1 protein (HMGB1), plasma aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase levels, tissue xanthine oxidoreductase (XOR) and myeloperoxidase (MPO) activities, and expressions of NOX2 and NOX4 proteins were determined.nnnRESULTSnLiver IR resulted in significant increases in NOX2 and NOX4 expressions and XOR and MPO activities, and approximately 2-fold increases in the levels of the inflammatory cytokines TNF-α and HMGB1. The microvascular blood flow and tissue oxygen saturation decreased by ∼20% from control values. GPC administration ameliorated the postischemic microcirculatory deterioration and reduced the liver necroenzyme levels significantly; the NOX4 expression, MPO activity, and HMGB1 level were also decreased, whereas the NOX2 expression, TNF-α level, and XOR activity were not influenced by GPC pretreatment.nnnCONCLUSIONSnNOX4 activation is a decisive component in the IR-induced microcirculatory dysfunction. Exogenous GPC ameliorates the inflammatory activation, and preserves the postischemic microvascular perfusion and liver functions, these effects being associated with a reduced hepatic expression of NOX4.

A novel murine model for the in vivo study of transdermal drug penetration

Enhancement of the transdermal penetration of different active agents is an important research goal. Our aim was to establish a novel in vivo experimental model which provides a possibility for exact measurement of the quantity of penetrated drug. The experiments were performed on SKH-1 hairless mice. A skin fold in the dorsal region was fixed with two fenestrated titanium plates. A circular wound was made on one side of the skin fold. A metal cylinder with phosphate buffer was fixed into the window of the titanium plate. The concentration of penetrated drug was measured in the buffer. The skin fold was morphologically intact and had a healthy microcirculation. The drug appeared in the acceptor buffer after 30u2009min, and its concentration exhibited a continuous increase. The presence of ibuprofen was also detected in the plasma. In conclusion, this model allows an exact in vivo study of drug penetration and absorption.

Anti-inflammatory effects of limb ischaemic preconditioning are mediated by sensory nerve activation in rats

We have shown that ischaemic preconditioning ameliorates both the local periosteal and the systemic leukocyte activation evoked by limb ischaemia–reperfusion. We hypothesized that the activation of chemosensitive afferent nerves by transient ischaemia contributes to the protective mechanisms of ischaemic preconditioning via a calcitonin gene-related peptide (CGRP)-dependent mechanism. In Sprague–Dawley rats, 60-min complete limb ischaemia was followed by 180xa0min of reperfusion. In further experiments, the CGRP analogue hCGRP (0.3xa0μgxa0kg−1) or ischaemic preconditioning (2u2009×u200910-min ischaemia/10-min reperfusion) was applied prior to the ischaemia–reperfusion insult. Ischaemic preconditioning was performed in three subgroups in which animals received the CGRP receptor antagonist CGRP8–37 (30xa0μgxa0kg−1xa0h−1), the chemosensitive afferent nerve inactivator resiniferatoxin (3u2009×u200915xa0μgxa0kg−1, sc), or vehicle. The effects of CGRP8-37 and resiniferatoxin on ischaemia–reperfusion without ischaemic preconditioning were also evaluated. In the tibial periosteum of rats, intravital fluorescence microscopy and immunohistochemistry revealed significant attenuations of ischaemia-reperfusion-induced post-ischaemic leukocyte–endothelial interactions (rolling and adherence in the postcapillary venules) and tissue intracellular adhesion molecule expression following ischaemic preconditioning or hCGRP administration. Administration of CGRP8-37 or pretreatment of animals with resiniferatoxin reversed the anti-inflammatory effects of limb ischaemic preconditioning, but failed to affect the microcirculatory consequences of ischaemia–reperfusion without ischaemic preconditioning. The results suggest that activation of the chemo- (capsaicin-) sensitive afferent nerves is involved in the mechanisms of microcirculatory anti-inflammatory protection provided by limb ischaemic preconditioning. Controlled activation of chemosensitive C-fibres or the CGRP receptors by the induction of ischaemic preconditioning or other means may furnish therapeutic benefit by ameliorating the periosteal microcirculatory consequences of tourniquet ischaemia.

Laser-Doppler microvascular measurements in the peri-implant areas of different osseointegrated bone conductor implant systems

The objective of this study was to evaluate the impact of hydroxyapatite coating of newly designed osseointegrated fixtures’ abutments on the postoperative complication rates. The integrity of peri-implant microcirculation was used as a marker to compare tissue viability after different surgical techniques. Laser-Doppler Flowmetry (LDF) measures alone, and coupled with heat provocation tests were applied to test the different microcircular patterns. Measures for 17 consecutively implanted patients (8 women, 9 men, ages ranged from 18 to 77xa0years) were recruited; seven with soft tissue reduction (STR); and 10 with soft tissue preservation (STP).Thirteen non-operated retro-auricular areas were examined as naive controls. In isotherm conditions the baseline blood flow remained stable in all groups. The naive control patients demonstrated significant changes of blood flux in the intact skin. The non-implanted yet previously operated contralateral sides of the patients demonstrated marginally lower (pxa0=xa00.09) blood flux index. The STR sides however, showed significantly lower (average 217xa0%) provoked blood flux compared to controls (pxa0<xa00.001). At the STP sides a maladaptation could be observed (average 316xa0%) compared to the contralateral sides (pxa0=xa00.53). STP sides demonstrated a significantly better blood flow improvement compared to the STR sides (pxa0=xa00.02). These results suggest a favorable postoperative condition of vascular microcirculation after STP, than after STR surgery. The possibly faster wound healing and lower potential complication rate may widen the inclusion criteria and maybe beneficial for the patient compliance with a better quality-of-life.

Remote ischemic preconditioning differentially affects NADPH oxidase isoforms during hepatic ischemia–reperfusion

AIMSnWe investigated the function of major superoxide-generating enzymes after remote ischemic preconditioning (IPC) and hepatic ischemia-reperfusion (IR), with the specific aim of assessing the importance of the most relevant NADPH oxidase (NOX) isoforms, NOX2 and NOX4, in the mechanism of action.nnnMAIN METHODSn60-min partial liver ischemia was induced in Sprague-Dawley rats in the presence or absence of remote IPC (2 × 10-min limb IR), and hepatic microcirculatory variables were determined through intravital video microscopy and lightguide spectrophotometry during reperfusion. Inflammatory enzyme activities (myeloperoxidase (MPO) and xanthine oxidoreductase (XOR)), cytokine production (TNF-α and HMGB-1), liver necroenzyme levels (AST, ALT and LDH) and NOX2 and NOX4 protein expression changes (Western blot analysis) were assayed biochemically.nnnKEY FINDINGSnIn this setting, remote IPC significantly decreased the IR-induced hepatic NOX2 expression, but the NOX4 expression remained unchanged. The remote IPC provided significant, but incomplete protection against the leukocyte-endothelial cell interactions and flow deterioration. Hepatocellular damage (AST, ALT and LDH release), cytokine levels, and XOR and MPO activities also diminished.nnnSIGNIFICANCEnRemote IPC limited the IR-induced microcirculatory dysfunction, but the protective effect did not affect all NOX homologs (at least not NOX4). The residual damage and inflammatory activation could well be linked to the unchanging NOX4 activity.

Periosteal microcirculatory action of chronic estrogen supplementation in osteoporotic rats challenged with tourniquet ischemia.

AIMSnTransient ischemia of osteoporotic bones during elective orthopedic surgery or fracture repair carries risks for serious complications, and estrogen loss or replacement has a potential to influence ischemia-reperfusion-induced inflammatory activation. To clarify this, we investigated the periosteal inflammatory changes in a clinically relevant time frame in ovariectomized rats, an experimental model of postmenopausal bone loss. Furthermore, the effects of chronic estrogen supplementation on the postischemic local and systemic inflammatory reactions were assessed.nnnMAIN METHODSnBilateral ovariectomy or sham operation was performed in 3-month-old female Sprague-Dawley rats. Five months later, estrogen replacement therapy with 17β-estradiol (20 μg(-1) kg(-1) day(-1)) or vehicle treatment was initiated. The microcirculatory inflammatory consequences of 60-min total hindlimb ischemia followed by 180-min reperfusion were examined 11 months after ovariectomy and were compared with those in 3-month-old animals.nnnKEY FINDINGSnThe osteoporosis that developed 5 months after ovariectomy was significantly ameliorated by estrogen replacement therapy. Both in ovariectomized and in non-ovariectomized animals, ischemia-reperfusion elevated the neutrophil adherence ~3-fold in the postcapillary venules of the periosteum (intravital microscopy), with an ~50-60% increase in intravascular neutrophil activation (CD11b; FACS analysis), an enhanced TNF-α release (ELISA) and periosteal expression of ICAM-1 (the endothelial ligand of CD11b; immunohistochemistry). Exogenous 17β-estradiol considerably reduced TNF-α release and the number of neutrophil-endothelial interactions in the periosteum, without affecting the CD11b and ICAM-1 expression changes.nnnSIGNIFICANCEnOsteoporosis itself does not increase the magnitude of the limb ischemia-reperfusion-associated periosteal inflammatory reaction. Chronic estrogen supplementation, however, reverses osteoporosis and significantly ameliorates the microcirculatory consequences of transient ischemia.

Excessive alcohol consumption induces methane production in humans and rats

Various studies have established the possibility of non-bacterial methane (CH4) generation in oxido-reductive stress conditions in plants and animals. Increased ethanol input is leading to oxido-reductive imbalance in eukaryotes, thus our aim was to provide evidence for the possibility of ethanol-induced methanogenesis in non-CH4 producer humans, and to corroborate the in vivo relevance of this pathway in rodents. Healthy volunteers consumed 1.15u2009g/kg/day alcohol for 4 days and the amount of exhaled CH4 was recorded by high sensitivity photoacoustic spectroscopy. Additionally, Sprague-Dawley rats were allocated into control, 1.15u2009g/kg/day and 2.7u2009g/kg/day ethanol-consuming groups to detect the whole-body CH4 emissions and mitochondrial functions in liver and hippocampus samples with high-resolution respirometry. Mitochondria-targeted L-alpha-glycerylphosphorylcholine (GPC) can increase tolerance to liver injury, thus the effects of GPC supplementations were tested in further ethanol-fed groups. Alcohol consumption was accompanied by significant CH4 emissions in both human and rat series of experiments. 2.7u2009g/kg/day ethanol feeding reduced the oxidative phosphorylation capacity of rat liver mitochondria, while GPC significantly decreased the alcohol-induced CH4 formation and hepatic mitochondrial dysfunction as well. These data demonstrate a potential for ethanol to influence human methanogenesis, and suggest a biomarker role for exhaled CH4 in association with mitochondrial dysfunction.