Tatiana Ravingerova

Oxytocin exerts protective effects on in vitro myocardial injury induced by ischemia and reperfusion.

Among the cardiovascular pathologies, ischemic heart disease is a serious medical problem that can result in cardiac injury and (or) heart failure. The aim of the present study was to test the hypothesis that neuropeptide oxytocin induces cardioprotective effects on ischemia-reperfusion-induced myocardial damage. The functional parameters of isolated Langendorff-perfused rat hearts were recorded before and after global 25 min ischemia and subsequent reperfusion. The infarct size was determined by a computerized planimetric method. The results showed that oxytocin produced negative chronotropic effect even at low concentrations (90-125 nmol/L). Perfusion with oxytocin before ischemia resulted in significant reduction of the infarct size (p<0.01), which was about 66% smaller than that in the control group. To evaluate the functional mechanisms involved, further experiments were performed under conditions of constant heart rate. The lower dose of oxytocin (90 nmol/L), which was ineffective in spontaneously beating hearts, induced a significant decrease of contractility. Elimination of the negative chronotropic effect of oxytocin prevented its cardioprotective action. In conclusion, our results demonstrated an attenuation of the infarct size in oxytocin-treated hearts, indicating a cardioprotective effect of oxytocin. The data suggest that the negative chronotropic action of oxytocin participates in its protective effects on ischemia-reperfusion-induced myocardial injury.

Effect of streptozotocin-induced diabetes on daily expression of per2 and dbp in the heart and liver and melatonin rhythm in the pineal gland of Wistar rat.

The circadian system is a flexible framework allowing a proper adjustment of physiological functions to the regularly changing environment. Pathways that are used to synchronize components of the circadian system have been shown to be susceptible to pathophysiological conditions. In our study, we investigated effects of streptozotocin (STZ)-induced diabetes mellitus on function of the circadian system at the level of melatonin synthesis and expression of per2 and dbp in the heart and liver in 8-week-old Wistar rats. Rhythmic pattern of clock gene per2 and transcription factor dbp in controls and STZ-treated animals was determined. Streptozotocin administration had a more substantial effect on per2 expression in the liver than in the heart. Pronounced phase advance in the rhythm of dbp expression in both the liver and the heart was observed. The melatonin rhythm reflecting the phase of the master clock was not affected by STZ application. Changes in per2 and dbp expression in the heart and liver imply alterations in input pathway or peripheral oscillators with possible consequences on function of analysed organs. (Mol Cell Biochem 270: 223–229, 2005)

Reperfusion-induced arrhythmias in isolated rat heart: an index of cellular damage or viability of cardiomyocytes?

Susceptibility of rat myocardium to reperfusion-induced arrhythmias after sustained (4 h) and brief (10 min) durations of regional ischaemia in relation to structurally-related impairment of heart function was studied in isolated Langendorff-perfused rat hearts. Reperfusion arrhythmias after 4 h ischaemia were characterized by low severity accompanied by only partial restoration of coronary flow upon reperfusion (no-reflow). Electron microscopy examination of the ischaemic hearts revealed severely ultrastructure of cardiac myocytes and capillary endothelium. These deteriorations were not reversed upon reperfusion. On the contrary, brief ischaemia resulted in high susceptibility of the heart to severe arrhythmias upon reperfusion with persisting hyperaemia. Ischaemia-induced minor changes in myocardial ultrastructure were reversed upon reperfusion. In conclusion, sustained ischaemia in the rat heart with absent collaterals renders myocardial tissue non-viable with consequent impaired recovery of heart function and loss of excitability of the myocardium upon reperfusion. Accordingly, high susceptibility to arrhythmias may indicate the preservation of viable myocardial cells.

Prevention by 7-oxo-prostacyclin of the calcium paradox in rat heart: Role of the sarcolemmal (Na,K)-ATPase

It is demonstrated a fast and significant depression in the sarcolemmal (Na,K)-ATPase activity that occurs as early as 25 sec after the onset of Ca2+ depletion, and participates in the development of Ca2+-paradox in the rat heart. Pretreatment of the animals with 7-oxo-prostacyclin (PG12) 24–48 h prior to the experiment prevented fairly the Ca2+-depletion-induced depression in (Na,K)ATPase activity and the accompanying structural and functional damage to the heart and sarcolemma during Ca2+-depletion as well as the development of Ca2+-paradox during the subsequent Ca2+-repletion. Pretreatment with PGI, was chosen intentionally because previous experiments revealed, that in its late effect the drug is acting via stabilizing the membranes due induction of high activity of (Na,K)-ATPase that has increased affinity to ATP. From results obtained the following may be concluded: If during the phase of Ca2+-deprivation, the capability of heart sarcolemma to maintain sodium extrusion remains preserved, the expected aggravation of Ca2+-overload injury to Ca2+-paradox that would develop during Ca2+-repletion, may be definitely prevented. Sufficiently preserved (Na,K)-ATPase activity, hand in hand with stabilized sarcolemmal structure, may prevent an accumulation of sodium beneath the sarcolemma and consequently also an overexcessive entry of Ca2+ into the myocytes.

The late effect of 7-oxo prostacyclin protects the rat heart against calcium paradox

Protective effect of a stable derivative of prostacyclin (7-oxo PGI2) was studied on the model of calcium overload (Ca paradox) 48 h after i.m. administration of the drug in a dosage of 50 micrograms/kg. In isolated rat heart perfused at 37 degrees C and a constant perfusion pressure of 65 mm Hg (Langendorff preparation) Ca paradox was induced by a 3 min perfusion with a calcium-free Krebs-Henseleit (KH) solution and followed by a 10 min perfusion with KH containing normal concentration of calcium. Late protective effect of 7-oxo PGI2 was manifested by: i. improved recovery of heart function (developed pressure and coronary flow) after Ca paradox, ii. better preservation of macroergic phosphates content, iii. better preserved cardiac ultrastructure (sarcolemma) already during Ca depletion phase. One of the proposed mechanisms of the protection afforded by the pretreatment with 7-oxo PGI2 may be that the cell membrane and possibly the intercalated discs become less affected by calcium depletion, resulting in less contracture-mediated membrane damage upon calcium repletion.

Lidocaine does not prevent the calcium paradox in rat hearts: A laser microprobe mass analysis (LAMMA) study

The calcium paradox stands for the cell damage that occurs when isolated hearts are perfused with a Ca(2+)-free solution followed by perfusion with a Ca(2+)-containing solution. Although it is generally accepted that a massive Ca2+ influx during the Ca(2+)-repletion phase is responsible for the cell damage, there is no consensus about what makes the heart susceptible to the calcium paradox during the Ca(2+)-depletion phase. It has been suggested that the extent of the calcium paradox is primarily determined by accumulation of Na+ during Ca2+ depletion and a subsequent accumulation of Ca2+ via reverse Na(+)-Ca2+ exchange during Ca2+ repletion. According to another theory, weakening of intercalated disc junctions during Ca2+ depletion and contracture-mediated disruption of the cell membrane during Ca2+ repletion are responsible for the calcium paradox. In the present study we further investigated the possible role of Na+ in the development of the calcium paradox. During Ca2+ depletion, lidocaine was used to inhibit Na+ entry through the Na+ channels. Isolated rat hearts were perfused with Krebs Henseleit buffer (KH) containing 1.4 mM Ca2+ for 15 min, followed by 10 min of Ca(2+)-free perfusion and 10 min of reperfusion with Ca2+. In the treated group 0.1 mM lidocaine was present throughout the experiment. At the end of each experiment, Ca2+ cytochemistry was performed and the intracellular Ca2+ content was analyzed by laser microprobe mass analysis (LAMMA). The results show that during Ca2+ depletion, the intracellular Ca2+ content did not change significantly. Ca2+ repletion, however, gave rise to a full calcium paradox irrespective of the presence of lidocaine: massive cell damage and Ca2+ accumulation in the mitochondria. The results provide further evidence that intracellular Na+ accumulation during Ca2+ depletion is not involved in the occurrence of the calcium paradox.

Cardioprotection of ischaemic preconditioning is associated with inhibition of translocation of MLKL within the plasma membrane

Necroptosis, a form of cell loss involving the RIP1‐RIP3‐MLKL axis, has been identified in cardiac pathologies while its inhibition is cardioprotective. We investigated whether the improvement of heart function because of ischaemic preconditioning is associated with mitigation of necroptotic signaling, and these effects were compared with a pharmacological antinecroptotic approach targeting RIP1. Langendorff‐perfused rat hearts were subjected to ischaemic preconditioning with or without a RIP1 inhibitor (Nec‐1s). Necroptotic signaling and the assessment of oxidative damage and a putative involvement of CaMKII in this process were analysed in whole tissue and subcellular fractions. Ischaemic preconditioning, Nec‐1s and their combination improved postischaemic heart function recovery and reduced infarct size to a similar degree what was in line with the prevention of MLKL oligomerization and translocation to the membrane. On the other hand, membrane peroxidation and apoptosis were unchanged by either approach. Ischaemic preconditioning failed to ameliorate ischaemia–reperfusion‐induced increase in RIP1 and RIP3 while pSer229‐RIP3 levels were reduced only by Nec‐1s. In spite of the additive phosphorylation of CaMKII and PLN because of ditherapy, the postischaemic contractile force and relaxation was comparably improved in all the intervention groups while antiarrhythmic effects were observed in the ischaemic preconditioning group only. Necroptosis inhibition seems to be involved in cardioprotection of ischaemic preconditioning and is comparable but not intensified by an anti‐RIP1 agent. Changes in oxidative stress nor CaMKII signaling are unlikely to explain the beneficial effects.

Molecular hydrogen potentiates beneficial anti-infarct effect of hypoxic postconditioning in isolated rat hearts: a novel cardioprotective intervention1

Generation of free radicals through incomplete reduction of oxygen during ischemia-reperfusion (I/R) is well described. On the other hand, molecular hydrogen (H2) reduces oxidative stress due to its ability to react with strong oxidants and easily penetrate cells by diffusion, without disturbing metabolic redox reactions. This study was designed to explore cardioprotective potential of hypoxic postconditioning (HpostC) against I/R (30 min global I - 120 min R) in isolated rat hearts using oxygen-free Krebs-Henseleit buffer (KHB). Furthermore, the possibility to potentiate the effect of HpostC by H2 using oxygen-free KHB saturated with H2 (H2 + HpostC) was tested. HPostC was induced by 4 cycles of 1-minute perfusion with oxygen-free KHB intercepted by 1-minute perfusion with normal KHB, at the onset of reperfusion. H2 + HPostC was applied in a similar manner using H2-enriched oxygen-free KHB. Cardioprotective effects were evaluated on the basis of infarct size (IS, in % of area at risk, AR) reduction, post-I/R recovery of heart function, and occurrence of reperfusion arrhythmias. HPostC significantly reduced IS/AR compared with non-conditioned controls. H2 present in KHB during HPostC further decreased IS/AR compared with the effect of HPostC, attenuated severe arrhythmias, and significantly restored heart function (vs. controls). Cardioprotection by HpostC can be augmented by molecular hydrogen infusion.

High arrythmogenesis during early reperfusion of ischaemic myocardium: participation of oxygen free radicals.

The early period of reperfusion of ischaemic myocardium leads to a high incidence of severe tachyarrhythmias including ventricular fibrillation (VF), accompanied by a sudden transitional dysfunction. Oxygen free radicals (OFR) have been identified as one of the principal factors responsible for reperfusion-induced events. However, direct evidence for participation of OFR in the arrhythmogenic mechanisms upon reperfusion is still lacking. In the present study, in isolated Langendorff-perfused rat hearts subjected to 30 min global ischaemia, the onset of reperfusion induced 100% incidence of both ventricular tachycardia (VT) and VF with their gradual cessation during 5 min of reperfusion. Generation of H2O2 in the myocardium in the first minutes of reperfusion was demonstrated by means of cerium cytochemistry. There was an increased density of cerium perhydroxide precipitate distributed throughout the myocytes and endothelial cells, confirmed by X-ray microanalysis. The mechanism of the arrhythmogenic effect of OFR may involve the inhibition of the sarcolemmal Na+/K+ ATPase activity, as was revealed by subjecting the isolated sarcolemmal fraction of rat heart to the action of an oxy-radical generating system (H2O2 + FeSO4).

Comparison of different osmotic therapies in a mouse model of traumatic brain injury

BACKGROUND Inflammation in the affected region, increased intracranial pressure, consequent oedema and congestion contribute to the negative outcome of traumatic brain injury. Osmotic therapies are recommended for improvement in cognitive and motor functions. Aim of the present study was to evaluate the effect of osmotic therapies in a mice model of traumatic brain injury. METHODS Experimental closed head injury was performed in adult Swiss albino mice by the weight-drop method. Different group of animals were treated with normal saline (G1), mannitol (G2), mannitol+glycerin (G3) and Neurotol (G4). Neurological Severity Score (NSS) was recorded at different time-points upto a period of six days. Effect of treatments on cerebral oedema, learning and memory function, motor function and co-ordination were evaluated by gravimetry, Morris water maze and beam walk test respectively. Histopathology was performed to evaluate the treatment effects on microscopic complications arising from primary closed head injury (CHI). RESULTS All the treatments showed a marked improvement in the evaluated parameters as compared with the vehicle control group. It was evident that G3 and G4 had a distinct advantage over mannitol therapy. Based on the NSS score, Neurotol proved to be comparatively safe and more efficacious than either mannitol or a combination of mannitol+glycerol. The effect of Neurotol could have been enhanced by the presence of VRP011 (a Mg+2 salt). CONCLUSIONS Neurotol is safe and exhibits better efficacy as compared with other treatments for the management of traumatic brain injury.