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.

Ischemic Tolerance-A Way to Reduce the Extent of Ischemia-Reperfusion Damage.

Individual tissues have significantly different resistance to ischemia-reperfusion damage. There is still no adequate treatment for the consequences of ischemia-reperfusion damage. By utilizing ischemic tolerance, it is possible to achieve a significant reduction in the extent of the cell damage due to ischemia-reperfusion injury. Since ischemia-reperfusion damage usually occurs unexpectedly, the use of preconditioning is extremely limited. In contrast, postconditioning has wider possibilities for use in practice. In both cases, the activation of ischemic tolerance can also be achieved by the application of sublethal stress on a remote organ. Despite very encouraging and successful results in animal experiments, the clinical results have been disappointing so far. To avoid the factors that prevent the activation of ischemic tolerance, the solution has been to use blood plasma containing tolerance effectors. This plasma is taken from healthy donors in which, after exposure to two sublethal stresses within 48 h, effectors of ischemic tolerance occur in the plasma. Application of this activated plasma to recipient animals after the end of lethal ischemia prevents cell death and significantly reduces the consequences of ischemia-reperfusion damage. Until there is a clear chemical identification of the end products of ischemic tolerance, the simplest way of enhancing ischemic tolerance will be the preparation of activated plasma from young healthy donors with the possibility of its immediate use in recipients during the initial treatment.

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.

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.

Effect of Bradykinin Postconditioning on Ischemic and Toxic Brain Damage.

Brain damage caused by ischemia or toxic agents leads in selectively vulnerable regions to apoptosis-like delayed neuronal death and can result in irreversible damage. Selectively vulnerable neurons of the CA1 area of hippocampus are particularly sensitive to ischemic damage. We investigated the effects of bradykinin (BR) postconditioning on cerebral ischemic and toxic injury. Transient forebrain ischemia was induced by four-vessel occlusion for 10 min and toxic injury was induced by trimethyltin (TMT, 8 µg/kg i.p.). BR as a postconditioner at a dose of 150 µg/kg was applied intraperitoneally 48 h after ischemia or TMT intoxication. Experimental animals were divided into groups according to the length of survival (short-3 and 7 days, and long-28 days survival) and according to the applied ischemic or toxic injury. Glutamate concentration was lowered in both CA1 and dentate gyrus areas of hippocampus after the application of BR postconditioning in both ischemic and toxic brain damage. The number of degenerated neurons in the hippocampal CA1 region was significantly lower in BR-treated ischemic and toxic groups compared to vehicle group. The behavioral test used in our experiments confirms also the memory improvement in conditioned animals. The rats' ability to form spatial maps and learn was preserved, which is visible from our Barnes maze results. By using the methods of delayed postconditioning is possible to stimulate the endogenous protective mechanisms of the organism and induce the neuroprotective effect. In this study we demonstrated that BR postconditioning, if applied before the onset of irreversible neurodegenerative changes, induced neuroprotection against ischemic or toxic injury.

Ionizing radiation as preconditioning against transient cerebral ischemia in rats.

Induction of ischemic tolerance (IT), the ability of an organism to survive an otherwise lethal ischemia, is the most effective known approach to preventing postischemic damage. IT can be induced by exposing animals to a broad range of stimuli. In this study we tried to induce IT of brain neurons using ionizing radiation (IR). A preconditioning (pre-C) dose of 10, 20, 30 or 50 Gy of gamma rays was used 2 days before an 8 min ischemia in adult male rats. Ischemia alone caused the degeneration of almost one half of neurons in CA1 region of hippocampus. However, a significant decrease of the number of degenerating neurons was observed after higher doses of radiation (30 and 50 Gy). Moreover, ischemia significantly impaired the spatial memory of rats as tested in Morris's water maze. In rats with a 50 Gy pre-C dose, the latency times were reduced to values close to the control level. Our study is the first to reveal that IR applied in sufficient doses can induce IT and thus allow pyramidal CA1 neurons to survive ischemia. In addition, we show that the beneficial effect of IR pre-C is proportional to the radiation dose.

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.

Bradykinin preconditioning affects the number of degenerated neurons and the level of antioxidant enzymes in spinal cord ischemia in rabbits.

Bradykinin preconditioning has been used for acquisition of tolerance after spinal cord ischemia. Rabbits were preconditioned intraperitoneally with bradykinin 48 h prior to 20 min of abdominal aorta ligation followed by 24 and 48 h of reperfusion. The activities of SOD and catalase were measured and Fluoro Jade B (FJB)-positive degenerated neurons were evaluated. The outcomes of Tarlov scoring system used to assess neurological functions showed significant improvement in bradykinin groups compared to the ischemic group. The number of FJB-positive degenerated neurons was decreased in ventral horns of both bradykinin groups. Significantly decreased activities of total SOD and mitochondrial Mn-SOD were also detected in both bradykinin groups versus ischemic group while CuZn-SOD and catalase activities were significantly decreased only in the bradykinin group after 24h of reperfusion versus ischemic group. These findings suggest that one of the possibilities of the neuroprotective effect of delayed bradykinin preconditioning against spinal cord ischemic injury could be realized by mitochondrial protection and decreased synthesis of Mn-SOD as well as by promotion of neuronal survival.

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.

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.

Development of a pattern in biochemical parameters in the core and penumbra during infarct evolution after transient MCAO in rats.

The period from stroke initiation to the cessation of penumbra damage spread represents a therapeutic window when expansion can be alleviated. In the present work, we studied some biochemical parameters helpful for the estimation of infarct progression and thus for the application of interventions. We designed four groups: the control group and three groups of animals after middle cerebral artery occlusion with reperfusion periods of 2h, 1day or 3days. In the ischaemic core and penumbra, fluorimetric and spectrophotometric methods for investigating total MnSOD and MAO-A/B activity as well as level of the glutamate were used. Protein synthesis was assessed by in vitro measurements of (14)C-leucine incorporation. Noticeable differences between core and penumbra biochemical parameters were shown. In the core, protein synthesis was transiently inhibited two hours and three days after ischaemia (36%). Glutamate and total SOD activity peaked on the first day, but on the third day after MCAO, rapidly decreased by about 44% and 33.6%, respectively. In the penumbra, ischaemia led to higher protein synthesis (78%), elevations in glutamate and rapid activation of MnSOD (by about 884%) one day after insult. On the third day, protein synthesis and MnSOD were still significantly elevated (36% and 388%, respectively), while glutamate levels returned to baseline. In addition, the impact of ischaemia on MAO-A/B activity in the penumbra was confirmed. In conclusion, biochemical parameter screening could be helpful to assess cell damage progress and the possibility of rescue. These regions reflect different biochemical patterns that seem to be clearly established on the first day after transient MCAO. Moreover, the first day of post-ischaemic reperfusion in the present model of stroke seems to be the breakpoint, i.e. the time at which expanding cell death from the infarct core to the penumbra can be at least partially eliminated.

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.

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.

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.

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.

Ischemic tolerance: the mechanisms of neuroprotective strategy.

The phenomenon of ischemic tolerance perfectly describes this quote "What does not kill you makes you stronger." Ischemic pre- or postconditioning is actually the strongest known procedure to prevent or reverse neurodegeneration. It works specifically in sensitive vulnerable neuronal populations, which are represented by pyramidal neurons in the hippocampal CA1 region. However, tolerance is effective in other brain cell populations as well. Although, its nomenclature is "ischemic" tolerance, the tolerant phenotype can also be induced by other stimuli that lead to delayed neuronal death (intoxication). Moreover, the recent data have proven that this phenomenon is not limited to application of sublethal stimuli before the lethal stress but reversed arrangement of events, sublethal stress after lethal insult, is rather equally effective. A very important term is called "cross conditioning." Cross conditioning is the capability of one stressor to induce tolerance against another. So, since pre- or post-conditioners can be used plenty of harmful stimuli, hypo- or hyperthermia and some physiological compounds, such as norepinephrine, bradykinin. Delayed neuronal death is the slow development of postischemic neurodegeneration. This allows an opportunity for a great therapeutic window of 2-3 days to reverse the cellular death process. Moreover, it seems that the mechanisms of ischemic tolerance-delayed postconditioning could be used not only after ischemia but also in some other processes leading to apoptosis.

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.