Obesity as a Limiting Factor for Remote Ischemic Postconditioning-Mediated Neuroprotection after Stroke.

Background: Remote ischemic postconditioning (RIPostC) may protect the brain from ischemia/reperfusion (I/R) injury. The association between RIPostC and obesity has not yet been extensively studied. Methods: Twelve-week-old male Zucker diabetic fatty (ZDF; n=68) and Zucker diabetic lean (ZDL; n=51) rats were subjected to focal cerebral ischemia for 90 minutes, followed by 24 hours of reperfusion. RIPostC was performed with 5-minute I/R cycles using a tourniquet on the right hind limb. Results: The results showed a negative association between obesity and neurological impairment in ischemic animals. We observed a 70% greater infarct size in ZDF rats compared with their lean counterparts, as evaluated by 2,3,5-triphenyltetrazolium chloride staining. To measure the total fragmented DNA in peripheral lymphocytes, comet assay was performed. Obese rats exhibited higher levels of DNA damage (by approximately 135%) in peripheral blood lymphocytes even before the induction of stroke. RIPostC did not attenuate oxidative stress in the blood in obese rats subjected to ischemia. Focal cerebral ischemia increased core and penumbra tissue glutamate release in the brain and decreased it in the blood of ischemic ZDL rats, and these changes improved following RIPostC treatment. However, changes in blood and tissue glutamate content were not detected in ischemic ZDF rats or after RIPostC intervention. Conclusion: Our findings suggest that obese animals respond more severely to ischemia-reperfusion brain injury. However, obese animals did not achieve neuroprotective benefits of RIPostC treatment.

Changes in excitatory amino acid transporters in response to remote ischaemic preconditioning and glutamate excitotoxicity.

The successful implementation of remote ischaemic conditioning as a clinical neuroprotective strategy requires a thorough understanding of its basic principles, which can be modified for each patient. The mechanisms of glutamate homeostasis appear to be a key component. In the current study, we focused on the brain-to-blood glutamate shift mediated by glutamate transporters (excitatory amino acid transports [EAATs]) and the effect of remote ischaemic preconditioning (RIPC) as a mediator of ischaemic tolerance. We used model mimicking ischaemia-mediated excitotoxicity (intracerebroventricular administration of glutamate) to avoid the indirect effect of ischaemia-triggered mechanisms. We found quantitative changes in EAAT2 and EAAT3 and altered membrane trafficking of EAAT1 on the cells of the choroid plexus. These changes could underlie the beneficial effects of ischaemic tolerance. There was reduced oxidative stress and increased glutathione level after RIPC treatment. Moreover, we determined the stimulus-specific response on EAATs. While glutamate overdose stimulated EAAT2 and EAAT3 overexpression, RIPC induced membrane trafficking of EAAT1 and EAAT2 rather than a change in their expression. Taken together, mechanisms related to glutamate homeostasis, especially EAAT-mediated transport, represents a powerful tool of ischaemic tolerance and allow a certain amount of flexibility based on the stimulus used.

Remote Ischaemic Preconditioning Accelerates Brain to Blood Glutamate Efflux via EAATs-mediated Transport.

Remote ischaemic conditioning (RIC) becomes an attractive strategy for the endogenous stimulation of mechanisms protecting neurons against ischaemia. Although the processes underlying the RIC are not clearly understood, the homeostasis of glutamate seems to play an important role. The present study is focused on the investigation of the brain to blood efflux of glutamate in a condition mimicking ischaemia-mediated excitotoxicity and remote ischaemic preconditioning (RIPC). The animals were pre-treated with a hind-limb tourniquet one hour before the intraventricular administration of glutamate and its release was monitored as the concentration of glutamate/glutathione in blood and liquor for up to 1 h. The transport mediated by excitatory amino acid transporters (EAATs) was verified by their inhibition with Evans Blue intraventricular co-administration. RIPC mediated the efflux of glutamate exceeding from CSF to blood in the very early stage of intoxication. As a consequence, the blood level of glutamate rose in a moment. EAATs inhibition confirmed the active role of glutamate transporters in this process. In the blood, elevated levels of glutamate served as a relevant source of antioxidant glutathione for circulating cells in RIPC-treated individuals. All of those RIPC-mediated recoveries in processes of glutamate homeostasis reflect the improvement of oxidative stress, suggesting glutamate-accelerated detoxication to be one of the key mechanisms of RIPC-mediated neuroprotection.

Identification of Proteins Responsible for the Neuroprotective Effect of the Secretome Derived from Blood Cells of Remote Ischaemic Conditioned Rats.

We have recently shown that the blood cell-derived secretome of remote ischaemic (RIC)-conditioned individuals provides an external source of neuroprotection. In this study, we identified the bioactive compounds from the total proteins released by those cells. Our main strategy was to separate protein-protein complexes while maintaining their native structure and testing their bioactive properties. Subsequently, we identified up- and downregulated bioactive proteins. We uncovered two bioactive fractions composed of 18 proteins. Most of the protein peaks were unchanged; however, RIC mediated a decrease in two peaks (comprising seven proteins) and an increase in one peak (identified as haptoglobin). When focussing on the biological activity of these proteins, we found positive impacts on the regulation of cellular metabolic processes and an increase in biological processes related to the acute phase response and inflammation in the RIC-treated samples. Although we have identified the 18 proteins that exert the greatest cytoprotection, additional studies are needed to elucidate their particular function and detailed mechanisms of action.

Accelerated capacity of glutamate uptake via blood elements as a possible tool of rapid remote conditioning mediated tissue protection.

Recently, the function of blood cells in remote ischemic conditioning (RIC) mediated neuroprotection was undoubtedly confirmed. In the present paper, we have focused on the role of blood elements in glutamate homeostasis. The blood of remote conditioned (tolerant) animals was incubated ex vivo with 100 µM glutamate, and the quantitative and qualitative changes of excitatory amino acid transporters (EAAT 1, 2, and 3) were determined. We confirmed RIC mediated accelerated sequestration of extracellular glutamate via EAATs and altered distribution of that amino acid between plasma and cell elements compared to non-tolerant counterparts. The activity of EAATs was elevated in erythrocytes and monocytes, while the density of transporters was not affected. Quantitative changes of EAAT1 density were detected solely in platelets where the forced scavenging was independent of EAATs inhibition. Surprisingly, the trafficking of immunovisualised EAAT2 and 3 raised at tolerant erythrocytes and monocytes. We have found that protein synthesis underlined this process. On the other hand, depletion of protein synthesis did not significantly affect the scavenging capacity of those cell populations. Our work has demonstrated that the elevated blood scavenging of glutamate overdose could be one of the potential mechanisms underlying RIC mediated tissue protection.

Brain to blood efflux as a mechanism underlying the neuroprotection mediated by rapid remote preconditioning in brain ischemia.

Glutamate represents the main excitatory neurotransmitter in the mammalian brain; however, its excessive elevation in the extracellular space is cytotoxic and can result in neuronal death. The ischemia initiated brain damage reflects changes in glutamate concentration in peripheral blood. This paper investigated the role of the brain in blood efflux of the glutamate in an improved tolerance of the brain tissue to ischemic conditions. In the rat model of focal brain ischemia, the neuroprotection was initiated by rapid remote ischemic preconditioning (rRIPC). Our results confirmed a strong neuroprotective effect of rRIPC. We observed reduced infarction by about 78% related to improved neuronal survival by about 70% in the ischemic core. The level of tissue glutamate in core and penumbra dropped significantly and decreased to control value also in the core region of the contralateral hemisphere. Despite significant improvement of blood-brain barrier integrity (by about 76%), the additional gain of glutamate content in the peripheral blood was caused by rRIPC. Based on our results, we can assume that neuroprotection mediated by rapid remote ischemic preconditioning could lie in the regulated, whole-brain release of glutamate from nerve tissue to the blood, which preserves neurons from the exposure to glutamate toxicity and results in reduced infarction.

P2X7 Receptors as a Therapeutic Target in Cerebrovascular Diseases.

Shortage of oxygen and nutrients in the brain induces the release of glutamate and ATP that can cause excitotoxicity and contribute to neuronal and glial damage. Our understanding of the mechanisms of ATP release and toxicity in cerebrovascular diseases is incomplete. This review aims at summarizing current knowledge about the participation of key elements in the ATP-mediated deleterious effects in these pathologies. This includes pannexin-1 hemichannels, calcium homeostasis modulator-1 (CALHM1), purinergic P2X7 receptors, and other intermediaries of CNS injury downstream of ATP release. Available data together with recent pharmacological developments in purinergic signaling may constitute a new opportunity to translate preclinical findings into more effective therapies in cerebrovascular diseases.

Rapid remote conditioning mediates modulation of blood cell paracrine activity and leads to the production of a secretome with neuroprotective features.

The indirect use of the protective potential of stem cells in the form of cell secretomes has become an attractive strategy in regenerative medicine. In the present work, we studied the paracrine activity of blood cells that could be modulated towards a neuroprotective nature using in vivo remote conditioning (i.e. tolerant blood cells). The increased neuronal survival mediated by the tolerant secretome was clearly confirmed in vitro in a model of glutamate toxicity in a primary culture of rat cortical neurons and in vivo in a pre- and post-treatment of rats that were subjected to transient occlusion of the middle cerebral artery. Bioinformatic-based analysis of the protein profile revealed higher amounts of proteins released by the tolerant blood cells; 29 proteins were recognised as secreted. More than half of these secreted proteins were involved in the biological processes of the response to the stimulus (GO:0050896) and the response to chemicals (GO:0042221). The protective phenotype was most likely mediated by the synergistic effect of multiple identified proteins, including unique to the tolerant secretome (ceruloplasmin, D-3-phosphoglycerate dehydrogenase) and was promoted by the co-participation of several reaction pathways. The most probably of these pathways were post-translation protein modification, MAP2K and MAPK activation and platelet activation. Taken together, our results demonstrate that properly stimulated blood cells could serve as a source for cell-free-based therapies of regenerative medicine.

Neuroprotection mediated by remote preconditioning is associated with a decrease in systemic oxidative stress and changes in brain and blood glutamate concentration.

It has been shown that ischemia of remote organs can generate resistance to ischemic conditions within sensitive brain tissues. However, only limited information about its mechanism is available. In the present paper, we used hind-limb ischemia by tourniquet to generate early remote ischemic tolerance in rats. The main objective was to investigate the role of glutamate in the process of neuroprotection and discover parameters that are affected in the blood of ischemia-affected animals. Our results showed that pretreatment with a hind-limb tourniquet caused a decrease in neurodegeneration by about 30%. However, we did not observe neurological deficit recovery. When compared to ischemia, glutamate concentration decreased in all observed brain regions (cortex, CA1 and dentate gyrus of hippocampus), regardless of their sensitivity to blood restrictions. In contrast to this, the blood levels raised significantly from 26% to 29% during the first four days of postischemic reperfusion. Pretreatment of animals reduced systemic oxidative stress-as represented by lymphocytic DNA damage-by about 80%, while changes in blood antioxidant enzymes (catalase, superoxide dismutase) were not detected. With these data we can further hypothesize that hind-limb-tourniquet preconditioning could accelerate brain-to-blood efflux of glutamate which could positively impact neuronal survival of ischemia-affected brain regions. Moreover, remote preconditioning improved systemic oxidative stress and did not seem to be affected by enzymatic antioxidant defenses in the blood.

Blockade and knock-out of CALHM1 channels attenuate ischemic brain damage.

Overactivation of purinergic receptors during cerebral ischemia results in a massive release of neurotransmitters, including adenosine triphosphate (ATP), to the extracellular space which leads to cell death. Some hypothetical pathways of ATP release are large ion channels, such as calcium homeostasis modulator 1 (CALHM1), a membrane ion channel that can permeate ATP. Since this transmitter contributes to postischemic brain damage, we hypothesized that CALHM1 activation may be a relevant target to attenuate stroke injury. Here, we analyzed the contribution of CALHM1 to postanoxic depolarization after ischemia in cultured neurons and in cortical slices. We observed that the onset of postanoxic currents in neurons in those preparations was delayed after its blockade with ruthenium red or silencing of Calhm1 gene by short hairpin RNA, as well as in slices from CALHM1 knockout mice. Subsequently, we used transient middle cerebral artery occlusion and found that ruthenium red, a blocker of CALHM1, or the lack of CALHM1, substantially attenuated the motor symptoms and reduced significantly the infarct volume. These results show that CALHM1 channels mediate postanoxic depolarization in neurons and brain damage after ischemia. Therefore, targeting CALHM1 may have a high therapeutic potential for treating brain damage after ischemia.

Blood cells serve as a source of factor-inducing rapid ischemic tolerance in brain.

Ischemic tolerance (IT) has gained attention as an attractive strategy for improving stroke outcome. Recently, it was shown that signal responsible for rapid IT induction (tolerance induction factor - TIF) is transmitted via circulating blood. In this study, we have hypothesized about the role of the blood cell compartment in TIF production. We used hind-limb ischemia to generate TIF as a rapid preconditioning against transient middle cerebral artery occlusion (MCAO). The essential properties of protein synthesis inhibitors actinomycin D and cycloheximide were utilized to obtain the following results: (i) TIF is proteinaceous. Hind-limb ischemia mediates gene expression followed by translation, resulting in the production of TIF. Blocking of each of these two steps in protein synthesis resulted in rapid infarct evolution (281.5 ± 23.37 and 330.4 ± 71.8 mm3 , respectively). (ii) Tourniquet-treated muscle is not a source of TIF. Actinomicine D injected into rat prior to tolerance induction significantly suppressed RNA synthesis in blood cells and muscle tissue. Cross-circulation of those rats (donors) with control animals (recipients) did not mediate significant infarct reduction (272.9 ± 12.45 mm3 ), even when hind-limb ischemia was performed before MCAO in the recipient (223.2 ± 37.51 mm3 ). (iii) Blood cells serve as a source of TIF. Preischemic transfusion of plasma-free, protein-synthesis-inactive blood cells, which were obtained from tolerant animals did not reduce infarct volume in recipients (131 ± 16.1 mm3 ) in a range comparable with their protein-synthesis-active counterparts (17.2 ± 12 mm3 ). We can conclude that blood cells are associated with the induction of rapid IT via production of a bioactive proteinaceous substance.

Scheme of Ischaemia-triggered Agents during Brain Infarct Evolution in a Rat Model of Permanent Focal Ischaemia.

The impact of therapeutic intervention in stroke depends on its appropriate timing during infarct evolution. We have studied markers of brain tissue damage initiated by permanent occlusion of the middle cerebral artery (MCAO) at three time points during which the infarct spread (1, 3 and 6 h). Based on Evans Blue extravasation and immunohistochemical detection of neurons, we confirmed continuous disruption of blood-brain barrier and loss of neurons in the ischaemic hemisphere that peaked at the sixth hour, especially in the core. Glutamate content started to rise dramatically in the entire hemisphere during the first 3 h; the highest level was determined in the core 6 h after MCAO (141 % increase). Moreover, the enzyme antioxidant defence grew by about 42 % since the first hour in the ipsilateral penumbra. Enzymes of the apoptotic pathway as well as mitochondrial enzyme release were detected since the third hour of MCAO in the ischaemic hemisphere; all achieved their maxima in the penumbra during both time periods (except cytochrome C). In conclusion, the preserved integrity of mitochondrial membrane and incompletely developed process of apoptosis may contribute to the better therapeutic outcome after ischaemic attack; however, a whole brain response should not be omitted.

Blood as the carrier of ischemic tolerance in rat brain.

This study provides clear evidence that the factor inducing tolerance to ischemia is transmitted via the circulating blood. By using the remote ischemia and the cross-circulation model, the tolerance to ischemia was transmitted from donor to recipient. For this study, the following experimental groups were designed: I, sham control group; II, group of tolerant hindlimb tourniquet-treated rats; III, positive control group; IV, control for cross-circulation influence; preconditioned animals: V, tolerant animals subjected to middle cerebral artery occlusion (MCAO); VI, tolerant animals cross-circulated with SHC, followed by MCAO; VII, SHC animals cross-circulated with tolerant animals and subsequently subjected to MCAO; VIII, tolerant animals cross-circulated with ischemic rats, followed by MCAO; IX, SHC animals cross-circulated with ischemic animals and subjected to MCAO; postconditioned animals: X, ischemic animals treated with a remote limb tourniquet; XI, ischemic animals cross-circulated with SHC control rats; and XII, ischemic animals cross-circulated with tolerant rats. Results confirmed that remote ischemia induced reduction of infarct volume in the preconditioned (V, 60%) as well as in the postconditioned group (X, 52%). Significant diminution was also observed in group XII (56.6%). In the preconditioned group, decreased infarct volume was detected in groups VI and VII (about 65%) and in group IX (about 50%). The greatest infarct reduction (84%) was induced by the presence of ischemic blood in a tolerant rat before ischemia induction. In summary, the factor inducing tolerance to ischemia is generated by remote ischemia and by ischemia itself; from the site of origin to the rest of the body, it is transported by the systemic blood circulation and can be transferred from animal to animal. The effect of conditioning with two different ischemic events (brain and hindlimb ischemia) led to a cumulative, stronger tolerance response.

Blockade of P2X7 receptors or pannexin-1 channels similarly attenuates postischemic damage.

The role of P2X7 receptors and pannexin-1 channels in ischemic damage remains controversial. Here, we analyzed their contribution to postanoxic depolarization after ischemia in cultured neurons and in brain slices. We observed that pharmacological blockade of P2X7 receptors or pannexin-1 channels delayed the onset of postanoxic currents and reduced their slope, and that simultaneous inhibition did not further enhance the effects of blocking either one. These results were confirmed in acute cortical slices from P2X7 and pannexin-1 knockout mice. Oxygen-glucose deprivation in cortical organotypic cultures caused neuronal death that was reduced with P2X7 and pannexin-1 blockers as well as in organotypic cultures derived from mice lacking P2X7 and pannexin 1. Subsequently, we used transient middle cerebral artery occlusion to monitor the neuroprotective effect of those drugs in vivo. We found that P2X7 and pannexin-1 antagonists, and their ablation in knockout mice, substantially attenuated the motor symptoms and reduced the infarct volume to ~50% of that in vehicle-treated or wild-type animals. These results show that P2X7 receptors and pannexin-1 channels are major mediators of postanoxic depolarization in neurons and of brain damage after ischemia, and that they operate in the same deleterious signaling cascade leading to neuronal and tissue demise.

ATP signaling in brain: release, excitotoxicity and potential therapeutic targets.

Adenosine 5'-triphosphate (ATP) is released as a genuine co-transmitter, or as a principal purinergic neurotransmitter, in an exocytotic and non-exocytotic manner. It activates ionotropic (P2X) and metabotropic (P2Y) receptors which mediate a plethora of functions in the brain. In particular, P2X7 receptor (P2X7R) are expressed in all brain cells and its activation can form a large pore allowing the passage of organic cations, the leakage of metabolites of up to 900 Da and the release of ATP itself. In turn, pannexins (Panx) are a family of proteins forming hemichannels that can release ATP. In this review, we summarize the progress in the understanding of the mechanisms of ATP release both in physiological and pathophysiological stages. We also provide data suggesting that P2X7R and pannexin 1 (Panx1) may form a large pore in cortical neurons as assessed by electrophysiology. Finally, the participation of calcium homeostasis modulator 1 is also suggested, another non-selective ion channel that can release ATP, and that could play a role in ischemic events, together with P2X7 and Panx1 during excitotoxicity by ATP.

Delayed remote ischemic postconditioning protects against transient cerebral ischemia/reperfusion as well as kainate-induced injury in rats.

To test the appropriateness of using delayed remote ischemic postconditioning against damage caused to the hippocampus by ischemia or apoptosis inducing intoxication, we chose 10-min normothermic ischemia induced by four-vessel occlusion or kainate injection (8 mg/kg i.p.) in rats. Ischemia alone caused the number of degenerated CA1 neurons after 7 days lasting reperfusion to be significantly (p<0.001) increased by 72.77%. Delayed remote ischemic postconditioning lasting 20 min was able to prevent massive increase in the neurodegeneration. The group with 10 min of ischemia and postconditioning after 2 days of reperfusion had only 15.87% increase in the number of apoptotic neurons. Seven days after kainic acid injection the number of surviving neurons was 42.8% (p<0.001), but the portion of surviving pyramidal cells in the postconditioning group is more than 98%. Our data show that remote postconditioning, performed with 20 min of tourniquet ischemia applied to the hind limb, is a simple method able to effectively stop the onset of neurodegeneration and prevent occurrence of massive muscle cell necrosis, even when used 2 days after the end of the adverse event. Surviving neurons retained a substantial part of their learning and memory ability.

Bradykinin postconditioning ameliorates focal cerebral ischemia in the rat.

The goal of this study is to investigate the effects of bradykinin (BR) postconditioning on cerebral ischemic injury. Transient focal cerebral ischemia was induced in rats by 60min of middle cerebral artery occlusion (MCAO), followed by 3days of reperfusion. BR as a postconditioner at a dose of 150µg/kg was applied intraperitoneally 3, 6, 24 and 48h after MCAO. BR postconditioning significantly reduced total infarct volumes if applied 3h after MCAO by 95%, 6h after MCAO by 80% and 24h after MCAO by 70% in versus vehicle group. Neurological functions were amarked improvement in the BR groups compared to the ischemia group. The number of degenerated neurons in the hippocampal CA1 region was also significantly lower in BR-treated ischemic groups compared to vehicle group. BR postconditioning prevented the release of MnSOD from the mitochondria and reduced the activity of the total SOD and CAT if it is administrated short time after stroke. Our data proves the ischemic tolerance in the brain induced by BR postconditioning resulted as effective agent against as strong an attack as 60min MCAO even when used many hours after ischemia.

Differential neuroprotective effects of 5'-deoxy-5'-methylthioadenosine.

BACKGROUND: 5'-deoxy-5'-methylthioadenosine (MTA) is an endogenous compound produced through the metabolism of polyamines. The therapeutic potential of MTA has been assayed mainly in liver diseases and, more recently, in animal models of multiple sclerosis. The aim of this study was to determine the neuroprotective effect of this molecule in vitro and to assess whether MTA can cross the blood brain barrier (BBB) in order to also analyze its potential neuroprotective efficacy in vivo. METHODS: Neuroprotection was assessed in vitro using models of excitotoxicity in primary neurons, mixed astrocyte-neuron and primary oligodendrocyte cultures. The capacity of MTA to cross the BBB was measured in an artificial membrane assay and using an in vitro cell model. Finally, in vivo tests were performed in models of hypoxic brain damage, Parkinson's disease and epilepsy. RESULTS: MTA displays a wide array of neuroprotective activities against different insults in vitro. While the data from the two complementary approaches adopted indicate that MTA is likely to cross the BBB, the in vivo data showed that MTA may provide therapeutic benefits in specific circumstances. Whereas MTA reduced the neuronal cell death in pilocarpine-induced status epilepticus and the size of the lesion in global but not focal ischemic brain damage, it was ineffective in preserving dopaminergic neurons of the substantia nigra in the 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP)-mice model. However, in this model of Parkinson's disease the combined administration of MTA and an A2A adenosine receptor antagonist did produce significant neuroprotection in this brain region. CONCLUSION: MTA may potentially offer therapeutic neuroprotection.

Brain-derived neurotrophic factor blood levels in two models of transient brain ischemia in rats.

We monitored possible influence of transient focal and global brain ischemia on BDNF blood level. In both models noticeable fluctuation of BDNF concentration mainly in reperfusion was observed. During the first 90 min, BDNF in total blood and in blood cells continuously decreased in both models but plasma BDNF raised at 40 min and peaked at 90 min of reperfusion. Our data confirm the impact of transient brain ischemia on BDNF levels in the circulatory system, suggest blood cells as a possible source of BDNF and demonstrate the interdependence of blood compartments and physiological state of an affected organism.

Delayed post-conditioning reduces post-ischemic glutamate level and improves protein synthesis in brain.

In the clinic delayed post-conditioning would represent an attractive strategy for the survival of vulnerable neurons after an ischemic event. In this paper we studied the impact of ischemia and delayed post-conditioning on blood and brain tissue concentrations of glutamate and protein synthesis. We designed two groups of animals for analysis of brain tissues and blood after global ischemia and post-conditioning, and one for analysis of blood glutamate after transient focal ischemia. Our results showed elevated blood glutamate in two models of transient brain ischemia and decreases in blood glutamate to control in the first 20min of post-conditioning recirculation followed by a consecutive drop of about 20.5% on the first day. Similarly, we recorded reduced protein synthesis in hippocampus and cortex 2 and 3days after ischemia. However, increased glutamate was registered only in the hippocampus. Post-conditioning improves protein synthesis in CA1 and dentate gyrus and, surprisingly, leads to 50% reduction in glutamate in whole hippocampus and cortex. In conclusion, ischemia leads to meaningful elevation of blood and tissue glutamate. Post-conditioning activates mechanisms resulting in rapid elimination of glutamate from brain tissue and/or in the circulatory system that could otherwise impede brain-to-blood glutamate efflux mechanisms. Moreover, post-conditioning induces protein synthesis renewing in ischemia affected tissues that could also contribute to elimination of excitotoxicity. In addition, the potential of glutamate for monitoring the progress of ischemia and efficacy of therapy was shown.