Dénes Garab

Methane biogenesis during sodium azide-induced chemical hypoxia in rats.

Previous studies demonstrated methane generation in aerobic cells. Our aims were to investigate the methanogenic features of sodium azide (NaN(3))-induced chemical hypoxia in the whole animal and to study the effects of l-α-glycerylphosphorylcholine (GPC) on endogenous methane production and inflammatory events as indicators of a NaN(3)-elicited mitochondrial dysfunction. Group 1 of Sprague-Dawley rats served as the sham-operated control; in group 2, the animals were treated with NaN(3) (14 mg·kg(-1)·day(-1) sc) for 8 days. In group 3, the chronic NaN(3) administration was supplemented with daily oral GPC treatment. Group 4 served as an oral antibiotic-treated control (rifaximin, 10 mg·kg(-1)·day(-1)) targeting the intestinal bacterial flora, while group 5 received this antibiotic in parallel with NaN(3) treatment. The whole body methane production of the rats was measured by means of a newly developed method based on photoacoustic spectroscopy, the microcirculation of the liver was observed by intravital videomicroscopy, and structural changes were assessed via in vivo fluorescent confocal laser-scanning microscopy. NaN(3) administration induced a significant inflammatory reaction and methane generation independently of the methanogenic flora. After 8 days, the hepatic microcirculation was disturbed and the ATP content was decreased, without major structural damage. Methane generation, the hepatic microcirculatory changes, and the increased tissue myeloperoxidase and xanthine oxidoreductase activities were reduced by GPC treatment. In conclusion, the results suggest that methane production in mammals is connected with hypoxic events associated with a mitochondrial dysfunction. GPC is protective against the inflammatory consequences of a hypoxic reaction that might involve cellular or mitochondrial methane generation.

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

BACKGROUND We 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. MATERIALS AND METHODS Anesthetized 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. RESULTS Liver 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. CONCLUSIONS NOX4 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.

Peripheral inflammatory activation after hippocampus irradiation in the rat

Abstract Purpose: To detect the possible biochemical signs of inflammatory activation in the peripheral circulation in a rodent model of hippocampus irradiation, and to examine the effects of L-alpha-glycerylphosphorylcholine (GPC) in this experimental protocol. Materials and methods: Anesthetized Sprague-Dawley rats were subjected to 40 Gy cobalt irradiation of both hemispheres of the hippocampus, with or without GPC treatment (50 mg/kg intravenously (i.v.), 5 min before the irradiation, n = 6, each). A third group (n = 6) served as saline-treated control. Blood samples were obtained 3 h after the end of irradiation in order to examine the changes in plasma histamine, tumor necrosis factor-alpha (TNF-α), interleukin 1-beta, interleukin 6 (IL-6) and interleukin 10 (IL-10); liver tissue samples were taken to determine adenosine triphosphate (ATP) concentrations. Results: The hepatic ATP levels were significantly declined, while plasma concentrations of circulating TNF-α, IL-6, IL-10 and histamine were significantly increased after hippocampus irradiation. GPC treatment significantly reduced the irradiation-induced release of cytokines and histamine, and the liver ATP level was maintained at the control value. Conclusions: Targeted brain irradiation produced measurable pro- and anti-inflammatory cytokine changes in the systemic circulation. GPC supplementation provides significant protection against irradiation-induced peripheral pro-inflammatory activation and ATP depletion.

Increase in Alzheimer's related markers preceeds memory disturbances: Studies in vasopressin-deficient Brattleboro rat

Alzheimers disease (AD) is the most common form of dementia in the elderly. For more effective therapy early diagnostic markers could be beneficial. Therefore we compared one year old rats with adults and examined if changes in possible brain markers of AD preceeded memory decline. We also tested if vasopressin-deficient animals were useful model of AD as vasopressin has well known positive effect on memory and AD patient has decreased vasopressin production. We compared adult (3 month) and old (12 month), normal and vasopressin-deficient Brattleboro rats. To receive a comprehensive picture about their memory we examined their social discrimination, object discrimination and conditioned learning abilities (shuttle box). Amyloid precursor protein (APP), mitogen-activated protein kinase 1 (MAPK1), β-actin and tryptophan 2,3-dioxygenase 2 (TDO2) mRNA levels was measured by quantitative PCR. There was no difference between the memory of adult and aged groups. The vasopressin-deficient rats at both ages showed a weaker performance in the course of social and object discrimination tests and a higher escape failure during the shuttle box experiment. The brain marker mRNAs of the elder animals were higher than the levels of the adults, but the absence of vasopressin had no influence on them. Thus, the one year old rats showed elevated levels of AD-related markers, but memory deficits were observable only in vasopressin deficient animals. Vasopressin does not seem to have pathogenic role in AD. Changes in the studied markers might predict later symptoms, although further studies are required for confirmation.

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

AIMS We 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. MAIN METHODS 60-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. KEY FINDINGS In 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. SIGNIFICANCE Remote 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.

AIMS Transient 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. MAIN METHODS Bilateral 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. KEY FINDINGS The 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. SIGNIFICANCE Osteoporosis 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.

Limb ischemia-reperfusion differentially affects the periosteal and synovial microcirculation.

BACKGROUND Joints are privileged compartments that enjoy increased protection against the inflammatory reactions affecting the extremities. We hypothesized that the functional characteristics of the microvasculature would contribute to the differential defensive potential of the synovial membrane. METHODS We investigated the synovial microcirculatory reactions and compared them with those of the tibial periosteum in response to 60 min of total limb ischemia, followed by 180 min of ischemia-reperfusion (IR) in rats. Carrageenan/kaolin-induced knee monoarthritis, a neutrophil-driven synovial inflammation model, served as the positive control. RESULTS IR brought about a significant reduction in red blood cell velocity in the capillaries and increases in rolling and adherence of the neutrophil leukocytes in the postcapillary venules (intravital microscopy), in adhesion molecule expression (intercellular adhesion molecule-1 immunohistochemistry) and in xanthine oxidoreductase activity in the periosteum. These changes were also pronounced in carrageenan/kaolin-induced monoarthritis but were almost completely absent in the synovium after the IR challenge. Most importantly, even after IR and in carrageenan/kaolin monoarthritis, the synovial microcirculation was characterized by significantly greater red blood cell velocities than that in the periosteum under resting conditions. CONCLUSIONS The ischemic duration, which significantly affected the functional integrity of the periosteal microcirculation, did not bring about a marked deterioration in that of the synovial membrane, suggesting that the synovial microcirculation is less endangered to the consequences of short-term tourniquet exposure than the periosteum. The greater microcirculatory red blood cell velocities and lower IR-induced endothelial expression of intercellular adhesion molecule-1 in the synovial membrane might explain the greater resistance of this compartment to the inflammatory consequences of IR.

A Novel Method for In Vivo Visualization of the Microcirculation of the Mandibular Periosteum in Rats

The periosteum plays an important role in bone physiology, but observation of its microcirculation is greatly limited by methodological constraints at certain anatomical locations. This study was conducted to develop a microsurgical procedure which provides access to the mandibular periosteum in rats.

Capsaicin-induced rapid neutrophil leukocyte activation in the rat urinary bladder microcirculatory bed

This study was initiated to investigate the involvement of neutrophil leukocyte activation in neurogenic inflammation, a process also involved in human urinary pathologies, elicited in the rat urinary bladder by the local administration of capsaicin, the archetypal TRPV1 agonist. The contribution of afferent nerves and sensory neuropeptides to leukocyte activation in the urinary bladder microcirculatory bed was examined.

Estrogen-dependent efficacy of limb ischemic preconditioning in female rats†

Our aim was to examine the effects of ischemic preconditioning (IPC) on the local periosteal and systemic inflammatory consequences of hindlimb ischemia‐reperfusion (IR) in Sprague–Dawley rats with chronic estrogen deficiency (13 weeks after ovariectomy, OVX) in the presence and absence of chronic 17beta‐estradiol supplementation (E2, 20 µg kg−1, 5 days/week for 5 weeks); sham‐operated (non‐OVX) animals served as controls. As assessed by intravital fluorescence microscopy, rolling and the firm adhesion of polymorphonuclear neutrophil leukocytes (PMNs) gave similar results in the Sham + IR and OVX + IR groups in the tibial periosteal microcirculation during the 3‐h reperfusion period after a 60‐min tourniquet ischemia. Postischemic increases in periosteal PMN adhesion and PMN‐derived adhesion molecule CD11b expressions, however, were significantly reduced by IPC (two cycles of 10′/10′) in Sham animals, but not in OVX animals; neither plasma free radical levels (as measured by chemiluminescence), nor TNF‐alpha release was affected by IPC. E2 supplementation in OVX animals restored the IPC‐related microcirculatory integrity and PMN‐derived CD11b levels, and TNF‐alpha and free radical levels were reduced by IPC only with E2. An enhanced estrogen receptor beta expression could also be demonstrated after E2 in the periosteum. Overall, the beneficial periosteal microcirculatory effects of limb IPC are lost in chronic estrogen deficiency, but they can be restored by E2 supplementation. This suggests that the presence of endogenous estrogen is a necessary facilitating factor of the anti‐inflammatory protection provided by limb IPC in females. The IPC‐independent effects of E2 on inflammatory reactions should also be taken into account in this model.