Modifications of gene expression detected in peripheral blood after brain ischemia treated with remote postconditioning.

BACKGROUND: A stroke is an acute damage to a certain area of a nerve tissue of the brain. In developed countries, it ranks second among the most often causes of death and is also the leading cause of disability. Recent findings emphasize the significant neuroprotective effect of conditioning on the course and rate of recovery after ischemic attack; however the molecular mechanism of ischemic tolerance induced by conditioning is still not completely explored. METHODS AND RESULTS: The purpose of this study is an identification of changes in gene expression induced by stimulation of reaction cascades after activation of the neuroprotective mechanism using an experimental rat model of global ischemia. The induction of neuroprotective cascades was stimulated by the application of early and delayed form of remote ischemic postconditioning. The quantitative qRT-PCR method was used to assess the rate of change in ADM, BDNF, CDKN1A, CREB, GADD45G, IL6, nNOS, and TM4SF1 gene expression levels 72 h after ischemic attack. The detected results confirm the neuroprotective effect of both forms of postconditioning. Participation of neuroprotection-related gene expression changes was observed once as an early one (CREB, GADD45G), once as a delayed one (ADM, IL6), or both (BDNF, CDKN1A, nNOS, TM4SF1) postconditioning forms, depending on the particular gene. CONCLUSIONS: Our results characterize impact of ischemic tolerance on the molecular level. We predict ischemic tolerance to be consisted of complex combination of early and delayed remote postconditioning.

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.

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.

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.

Delayed bradykinin postconditioning modulates intrinsic neuroprotective enzyme expression in the rat CA1 region after cerebral ischemia: a proteomic study.

Pyramidal cells in the CA1 brain region exhibit an ischemic tolerance after delayed postconditioning; therefore, this approach seems to be a promising neuroprotective procedure in cerebral postischemic injury improvement. However, little is known about the effect of postconditioning on protein expression patterns in the brain, especially in the affected hippocampal neurons after global cerebral ischemia. This study is focused on the examination of the ischemia-vulnerable CA1 neuronal layer and on the acquisition of protection from delayed neuronal death after ischemia. Ischemic-reperfusion injury was induced in Wistar rats and bradykinin was applied 2 days after the ischemic insult in an attempt to overcome delayed cell death. Analysis of complex peptide CA1 samples was performed by automated two dimensional liquid chromatography (2D-LC) fractionation coupled to tandem matrix assisted laser desorption/ionization time-of-flight (MALDI TOF/TOF) mass spectrometry instrumentation. We devoted our attention to differences in protein expression mapping in ischemic injured CA1 neurons in comparison with equally affected neurons, but with bradykinin application. Proteomic analysis identified several proteins occurring only after postconditioning and control, which could have a potentially neuroprotective influence on ischemic injured neurons. Among them, the prominent position occupies a regulator of glutamate level aspartate transaminase AATC, a scavenger of glutamate in brain neuroprotection after ischemia-reperfusion. We identified this enzyme in controls and after postconditioning, but AATC presence was not detected in the ischemic injured CA1 region. This finding was confirmed by two-dimensional differential electrophoresis followed by MALDI-TOF/TOF MS identification. Results suggest that bradykinin as delayed postconditioning may be associated with modulation of protein expression after ischemic injury and thus this procedure can be involved in neuroprotective metabolic pathways.

Postconditioning Effectively Prevents Trimethyltin Induced Neuronal Damage in the Rat Brain.

Trimethyltin (TMT) is a toxic substance formerly used as a catalyst in the production of organic substances, as well as in industry and agriculture. TMT poisoning has caused death or severe injury in many dozens of people. The toxicity of TMT is mediated by dose dependent selective damage to the limbic system in humans and other animals, specifically the degeneration of CA1 neurons in the hippocampus. The typical symptoms include memory loss and decreased learning ability. Using knowledge gained in previous studies of global ischaemia, we used delayed postconditioning after TMT intoxication (8 mg/kg i.p.), consisting of applying a stressor (BR, bradykinin 150 µg/kg i.p.) 24 or 48 hours after the injection of TMT. We found that BR had preventive effects on neurodegenerative changes as well as learning and memory deficits induced by TMT intoxication.

Clinical usage of ischemic tolerance-where are its limits?

Ischemic tolerance is a robust internal defense mechanism of all living organisms. The effectiveness of this mechanism has been repeatedly demonstrated in experiments, but a comprehensive review of the clinical applicability of this phenomenon in practice has not yet been published. The results in clinical practice sound ambiguous and unconvincing in comparison with the results of experimental studies. Also, in many localities, the effect of ischemic tolerance was not clinically proven. For the reasons mentioned, the authors analyze the possible causes of the mentioned discrepancies and provide a comprehensive insight into the possible relevant clinical use of this phenomenon in practice for different groups of patients.

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.

Quantitative analysis of selected genetic markers of induced brain stroke ischemic tolerance detected in human blood.

A brain stroke is a serious disease and the second leading cause of death in the European Union. Carotid stenosis accounts for 15% of all ischemic cerebral strokes. However, there is currently no effective screening for carotid disease. Analysis of the DNA from peripheral blood is increasingly being used for several disease diagnoses. The potentially beneficial therapeutic method of inducing tissue tolerance to ischemia has so far been studied mainly in animal models. The aim of this study is to investigate changes in the gene expression of selected markers of brain ischemia during carotid endarterectomy, considered in this study as an activator of ischemic tolerance. During the carotid endarterectomy, there is a short-term occlusion of the internal carotid artery. Using the RT-qPCR method, we detected changes in the early identified gene markers of brain ischemia (ADM, CDKN1A, GADD45G, IL6, TM4SF1) in peripheral blood during sub lethal cerebral ischemia caused by carotid endarterectomy. Patients underwenting surgical procedure were divided into three groups: asymptomatic, symptomatic, and those who underwent carotid endarterectomy after an acute stroke. The results were compared to a negative/control group. Carotid endarterectomy had an impact on the expression of all monitored biomarkers. We observed statistically significant changes (p value 0.05-0.001) when comparing the groups among themselves, as well as the presence of ischemic tolerance of brain tissue to ischemic attacks. In conclusion, ADM, GADD45G, and TM4SF1 were affected in symptomatic patients, GADD45G and IL6 in acute patients, and CDKN1A and ADM in asymptomatic group after application of carotid endarterectomy.

An effective combination of two different methods of postconditioning.

Ischemic tolerance based on the synthesis of protective proteins acquires its full strength by repeated exposure to stress, and "the end effector of tolerance" may paradoxically be activated by the second or lethal stress, particularly in the case of preconditioning. That happens when an additional nonspecific stressor is applied either before (preconditioning) or after (postconditioning) the period of lethal ischemia. A combination of antioxidants with pre or postconditioning prevents the acquisition of tolerance, and in the case of more severe attacks repeated stress can lead to accumulation of damage. Our attempt to weaken ischemic injury to hippocampal CA1 with antioxidants applied after lethal stress, i.e. before delayed postconditioning, was ineffective. We then tried using rapid postconditioning consisting of 30-s reperfusion alternating with 15-s ischemia repeated three times and applied immediately at the end of lethal ischemia as a tool decreasing post-ischemic production of reactive oxygen species, and combining that with delayed postconditioning consisting of an i.p. injection of Bradykinin 2 days after lethal ischemia. This approach once more confirmed the efficacy of both rapid as well as delayed postconditioning but, more importantly, it demonstrated the possibility of effectively combining these two procedures. Our findings further confirm that in cases of delayed neuronal death, which is practically pathologically-induced apoptosis, there exists a 2-day-wide therapeutic window that can be effectively exploited.

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.

Aminoguanidine administration ameliorates hippocampal damage after middle cerebral artery occlusion in rat.

The effects of a selective inducible nitric oxide synthase inhibitor aminoguanidine (AG) on neuronal cells survival in hippocampal CA1 region after middle cerebral artery occlusion (MCAO) were examined. Transient focal cerebral ischemia was induced in rats by 60 or 90 min of MCAO, followed by 7 days of reperfusion. AG treatment (150 mg/kg i.p.) significantly reduced total infarct volumes: by 70% after 90 min MCAO and by 95% after 60 min MCAO, compared with saline-treated ischemic group. The number of degenerating neurons in hippocampal CA1 region was also markedly lower in aminoguanidine-treated ischemic groups compared to ischemic groups without AG-treatment. The number of iNOS-positive cells significantly increased in the hippocampal CA1 region of ischemic animals, whereas it was reduced in AG-treated rats. Our findings demonstrate that aminoguanidine decreases ischemic brain damage and improves neurological recovery after transient focal ischemia induced by MCAO.

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.

Effects of one-day reperfusion after transient forebrain ischemia on circulatory system in the rat.

Although ischemia/reperfusion injury remains incompletely understood, it appears that reactive oxygen species produced mainly during postischemic recirculation play a critical role. The present study examined the impact of forebrain ischemia and subsequent one-day reperfusion on several blood parameters. We determined glutamate concentration in whole blood, measured Cu/Zn- and Mn-SOD (superoxide dismutase) activity in blood cells as well as plasma, and investigated the prevalence of single and double strand breaks of lymphocyte DNA. The results of our experiment showed that the concentration of glutamic acid in whole blood was increased by about 25%. Antioxidant activity of total SOD and Cu/Zn-SOD was reduced in blood cells and plasma. Mn-SOD activity in blood cells was not affected by ischemic insult and one-day reperfusion, but we detected its significantly lower activity in samples of plasma. We observed a weakly reduced level of double and a significantly elevated level of single strand breaks of lymphocyte DNA. In conclusion, one day of recovery after the ischemic attack failed to return peripheral circulatory system to physiological conditions. Reduced antioxidant capacity in the blood and an elevated level of excitotoxic amino acid glutamate may cause lymphocyte DNA damage, and probably contribute to insufficient postischemic recovery of brain tissue.

Remote ischemic postconditioning as well as blood plasma from double-conditioned donor ameliorate reperfusion syndrome in skeletal muscle.

The aim of this study was to verify the possibility of preparation and effectiveness of the use of blood plasma containing an effector of ischemic tolerance activated by applying two sublethal stresses to a donor. As sublethal stresses, two periods of 20-minute hindlimb ischemia were used with a two-day interval between them. Active plasma was isolated six hours after the second hindlimb ischemia. The effectiveness of active plasma as well as remote postconditioning was tested after three hours of tourniquet-induced ischemia on the gastrocnemius muscle. The wet/dry ratio of gastrocnemius muscle (degree of tissue oedema), nitroblue tetrazolium reduction (tissue necrosis), and CatWalk test (hind limb functionality) were evaluated 24 h after the end of ischemia. Three hours of ischemia increased muscle oedema and necrosis in comparison to control by 26.72% (p < 0.001) and 41.58% (p < 0.001) respectively. Remote ischemic postconditioning as well as injection of conditioned blood plasma significantly prevented these changes, even when they were applied one or three hours after the end of ischemia. Equally effective double-conditioned plasma appears to have better prospects in life-threatening situations such as stroke and myocardial infarction.

Transient forebrain ischemia impact on lymphocyte DNA damage, glutamic acid level, and SOD activity in blood.

AIMS: Brain ischemia-reperfusion injury remains incompletely understood but appears to involve a complex series of interrelated biochemical pathways caused mainly by a burst of reactive oxygen species (ROS). In the present work we studied the impact of postischemic condition in the early phase of reperfusion on plasma and blood cells. METHODS: Transient forebrain ischemia was induced in Wistar rats by four-vessel occlusion model. Blood samples collected during postischemic reperfusion 20, 40, 60, 90, and 120 min after ischemia were used for assessing breaks of lymphocyte DNA, fluorimetric measurement of whole blood glutamate concentration, and spectrophotometrical determination of SOD activity in plasma and blood cells. RESULTS: Our results showed the most interesting changes of all observed parameters mainly at 40 and 120 min of reperfusion, when we observed peak DNA damage of lymphocytes and highest glutamate level and total and Cu/Zn SOD activity. At those time points, Mn SOD activity was low in plasma, as well as in blood cells. On the contrary, at 60 and 90 min, all studied parameters were approximately at the level of control. CONCLUSION: Ischemia/reperfusion injury has influence on blood cells and has at least two waves of impact on DNA damage of peripheral lymphocytes, affects activity of major antioxidant enzymes SODs, as well as blood glutamic acid level. Elevation of Mn SOD activity probably plays an important role in the processes of elimination of postischemic damage in blood cells.

Bradykinin postconditioning protects pyramidal CA1 neurons against delayed neuronal death in rat hippocampus.

AIMS: The present study was undertaken to evaluate possible neuroprotective effect of bradykinin against delayed neuronal death in hippocampal CA1 neurons if applied two days after transient forebrain ischemia in the rat. METHODS: Transient forebrain ischemia was induced in male Wistar rats by four-vessel occlusion for 8 min. To assess efficacy of bradykinin as a new stressor for delayed postconditioning we used two experimental groups of animals: ischemia 8 min and 3 days of survival, and ischemia 8 min and 3 days of survival with i.p. injection of bradykinin (150 microg/kg) applied 48 h after ischemia. RESULTS: We found extensive neuronal degeneration in the CA1 region at day 3 after ischemia/reperfusion. The postischemic neurodegeneration was preceded by increased activity of mitochondrial enzyme MnSOD in cytoplasm, indicating release of MnSOD from mitochondria in the process of delayed neuronal death. Increased cytosolic cytochrome c and subsequently caspase-3 activation are additional signs of neuronal death via the mitochondrial pathway. Bradykinin administration significantly attenuated ischemia-induced neuronal death, and also suppressed the release of MnSOD, and cytochrome c, and prevented caspase-3 activation. CONCLUSIONS: Bradykinin can be used as an effective stressor able to prevent mitochondrial failure leading to apoptosis-like delayed neuronal death in postischemic rat hippocampus.

Postconditioning and anticonditioning: possibilities to interfere to evoked apoptosis.

The aim of this study was to validate the ability of postconditioning, used 2 days after kainate intoxication, to protect selectively vulnerable hippocampal CA1 neurons against delayed neuronal death. Kainic acid (8 mg/kg, i.p.) was used to induce neurodegeneration of pyramidal CA1 neurons in rat hippocampus. Fluoro Jade B, the specific marker of neurodegeneration, and NeuN, a specific neuronal marker were used for visualization of changes 7 days after intoxication without and with delayed postconditioning (norepinephrine, 3.1 mumol/kg i.p., 2 days after kainate administration) and anticonditioning (Extract of Ginkgo biloba, 40 mg/kg p.o used simultaneously with kainate). Morris water maze was used on 6th and 7th day after kainate to test learning and memory capabilities of animals. Our results confirm that postconditioning if used at right time and with optimal intensity is able to prevent delayed neuronal death initiated not only by ischemia but kainate intoxication, too. The protective effect of repeated stress-postconditioning was suppressed if extract of Ginkgo biloba (EGb 761, 40 mg/kg p.o.) has been administered together with kainic acid. It seems that combination of lethal stress and antioxidant treatment blocks the activation of endogenous protecting mechanism known as ischemic tolerance, aggravates neurodegeneration and, after repeated stress is able to cause cumulative damage. This observation could be very valuable in situation when the aim of treatment is elimination of unwanted cell population from the organism.

Effect of L-carnitine on postischemic inhibition of protein synthesis in the rat brain.

The purpose of this study was to investigate effects of carnitine administration on protein synthesis recovery after transient cerebral ischemia. Rats received L-carnitine in two doses of 16 mmol/kg i.p. 15 min before ischemia and just on the onset of reperfusion. Transient forebrain ischemia was induced by 4-vessel occlusion for 15 min, followed by 30 min or 7 days of reperfusion. Protein synthesis rate, reinitiation ability and neurodegeneration in the frontal cortex and hippocampus were measured by the incorporation of radioactively labelled leucine into polypeptide chains in postmitochondrial supernatants and by Fluoro-Jade B staining. A protective effect was observed, on protein synthesis as well as the number of surviving neurons, in the L-carnitine-treated groups. Our results indicate that L-carnitine can exert a protective effect in the development of reperfusion-induced injury. L-carnitine significantly reduced the ischemia/reperfusion-induced inhibition of translation and neurodegeneration in the neocortex as well as in the highly sensitive hippocampus and dorsolateral striatum. We expect that the ability of L-carnitine to keep translational machinery on facilitates efficacy of postischemic remodulation of gene expression.

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.