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While stroke research represents the primary interface between circulation and brain research, the hemostasis system also carries a pivotal role in the mechanism of vascular brain injury. The complex interrelated events triggered by the energy crisis have a specific spatial and temporal pattern arching from the initial damage to the final events of brain repair. The complexity of the pathophysiology make it difficult to model this disease, therefore it is challenging to find appropriate therapeutic targets. The ever-persistent antagonism between the positive results of drug candidates in the experimental stroke models and the failures of the clinical trials prompts changes in the research strategy, especially in the field of potential neuroprotective therapies. System biology approach could initiate new directions in the future for both preclinical and clinical research. Incentive methods aimed at anti-apoptosis mechanisms and the augmentation of post-ischemic brain repair could benefit the facts, that these processes can be targeted much longer following the cell-necrosis in the hyper-acute phase. Sequential monitoring of candidate genes and proteins responsible for stroke progression and post-stroke repair seems to be useful both in therapeutic target-identification, and in clinical testing. Understanding the mechanism behind the effect of selegiline and other drugs capable of activating the anti-apoptotic gene expression could help to find new approaches to enhance the regenerative potential in the remodeling of neuronal and microvascular networks. [ABSTRACT FROM AUTHOR]

The release of [3H]dopamine ([3H]DA) and [3H]noradrenaline ([3H]NA) in acutely perfused rat striatal and cortical slice preparations was measured at 37 °C and 17 °C under ischemic conditions. The ischemia was simulated by the removal of oxygen and glucose from the Krebs solution. At 37 °C, resting release rates in response to ischemia were increased; in contrast, at 17 °C, resting release rates were significantly reduced, or resting release was completely prevented. The removal of extracellular Ca2+ further increased the release rates of [3H]DA and [3H]NA induced by ischemic conditions. This finding indicated that the Na+/Ca2+ exchanger (NCX), working in reverse in the absence of extracellular Ca2+, fails to trigger the influx of Ca2+ in exchange for Na+ and fails to counteract ischemia by further increasing the intracellular Na+ concentration ([Na+]i). KB-R7943, an inhibitor of NCX, significantly reduced the cytoplasmic resting release rate of catecholamines under ischemic conditions and under conditions where Ca2+ was removed. Hypothermia inhibited the excessive release of [3H]DA in response to ischemia, even in the absence of Ca2+. These findings further indicate that the NCX plays an important role in maintaining a high [Na+]i, a condition that may lead to the reversal of monoamine transporter functions; this effect consequently leads to the excessive cytoplasmic tonic release of monoamines and the reversal of the NCX. Using HPLC combined with scintillation spectrometry, hypothermia, which enhances the stimulation-evoked release of DA, was found to inhibit the efflux of toxic DA metabolites, such as 3,4-dihydroxyphenylacetaldehyde (DOPAL). In slices prepared from human cortical brain tissue removed during elective neurosurgery, the uptake and release values for [3H]NA did not differ from those measured at 37 °C in slices that were previously maintained under hypoxic conditions at 8 °C for 20 h. This result indicates that hypothermia preserves the functions of the transport and release mechanisms, even under hypoxic conditions. Oxidative stress (H2O2), a mediator of ischemic brain injury enhanced the striatal resting release of [3H]DA and its toxic metabolites (DOPAL, quinone). The study supports our earlier findings that during ischemia transmitters are released from the cytoplasm. In addition, the major findings of this study that hypothermia of brain slice preparations prevents the extracellular calcium concentration ([Ca2+]o)-independent non-vesicular transmitter release induced by ischemic insults, inhibiting Na+/Cl−-dependent membrane transport of monoamines and their toxic metabolites into the extracellular space, where they can exert toxic effects. [ABSTRACT FROM AUTHOR]

Purpose: Endothelial cells have an essential role in the maintenance of cardiac function. Cardiac endothelial cells in response of different cardiovascular risk factors become activated and this activation is often followed by endothelial dysfunction. The change of endothelial phenotype may influence interactions with surrounding cardiac fibroblasts, myocytes, pericytes.The aim of our work is to produce in vitro culture system with high cell purity to investigate endothelial cells in different heart diseases. Poor quality of these tissue samples indicates the optimalization of isolation and culture conditions for both cardiac microvascular and endocardial endothelial cells (CMVE and EE cells).Methods: Human heart samples were obtained from valve surgery or explanted hearts during transplantation. CMVE cells were isolated from left ventricle with collagenase and trypsin digestion. EE cells were digested with collagenase from mitral valve. After separation the pellets were seeded to culture dishes and grown in a medium containing endothelial cell growth supplement. These basic methods would be changed as follows. The time of enzymatic digestion was changed. The collected cell suspensions were preincubated to allow fibroblasts to attach to the bottom of culture dish. The decanting medium was centrifuged repeatedly and collected cells were cultured in specific endothelial medium completed with sera from different origins. CMVE cells were treated with puromycin to eliminate pericytes. Cells were seeded on different artificial extracellular surface components (collagen, laminin, gelatin, fibronectin). The CMVE and EE cells were characterized by uptake of acetylated LDL and immunostaining of vonWillebrand factor, VE-cadherin and CD31. Fibroblasts were identified by vimentin staining.Results: Primary cells isolated with basic methods exhibited different cell morfologies, they were multilayered and only the 4% and 10% of the cells were endothelial cells, CMVE and EE respectively, separated by flourescent-activated cell sorting (FACS). The attachment of fibroblasts and the puromycin treatment were effective in the primary cell cultures. Numerous cell colonies were observed within 2-3 days on gelatin coated surface and confluence was achieved within 10-14 days.Conclusion: With use of these culture conditions the endothelial cell rate is increased in primery cultures and further separation by FACS or immunomagnetic methods will be more effective. These optimalized cell culture systems provide possibility to investigate endothelial functions in different heart diseases. [ABSTRACT FROM PUBLISHER]