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.

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.

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.

Delayed postconditionig initiates additive mechanism necessary for survival of selectively vulnerable neurons after transient ischemia in rat brain.

1. The aim of this study was to validate the role of postconditioning, used 2 days after lethal ischemia, for protection of selectively vulnerable brain neurons against delayed neuronal death. 2. Eight, 10, or 15 min of transient forebrain ischemia in rat (four-vessel occlusion model) was used as initial lethal ischemia. Fluoro Jade B, the marker of neurodegeneration, and NeuN, a specific neuronal marker were used for visualization of changes 7 or 28 days after ischemia without and with delayed postconditioning. 3. Our results confirm that postconditioning if used at right time and with optimal intensity can prevent process of delayed neuronal death. At least three techniques, known as preconditioners, can be used as postconditioning: short ischemia, 3-nitropropionic acid and norepinephrine. A cardinal role for the prevention of death in selectively vulnerable neurons comprises synthesis of proteins during the first 5 h after postconditioning. Ten minutes of ischemia alone is lethal for 70% of pyramidal CA1 neurons in hippocampus. Injection of inhibitor of protein synthesis (Cycloheximide), if administered simultaneously with postconditioning, suppressed beneficial effect of postconditioning and resulted in 50% of CA1 neurons succumbing to neurodegeneration. Although, when Cycloheximide was injected 5 h after postconditioning, this treatment resulted in survival of 90% of CA1 neurons. 4. Though postconditioning significantly protects hippocampal CA1 neurons up to 10 min of ischemia, its efficacy at 15 min ischemia is exhausted. However, protective impact of postconditioning in less-sensitive neuronal populations (cortex and striatum) is very good after such a damaging insult like 15 min ischemia. This statement also means that up to 15 min of ischemia, postconditioning does not induce cumulation of injuries produced by the first and the second stress.

Evidence for a role of second pathophysiological stress in prevention of delayed neuronal death in the hippocampal CA1 region.

In ischemic tolerance experiment, when we applied 5-min ischemia 2 days before 30-min ischemia, we achieved a remarkable (95.8%) survival of CA1 neurons. However, when we applied 5-min ischemia itself, without following lethal ischemia, we found out 45.8% degeneration of neurons in the CA1. This means that salvage of 40% CA1 neurons from postischemic degeneration was initiated by the second pathophysiological stress. These findings encouraged us to hypothesize that the second pathophysiological stress used 48 h after lethal ischemia can be efficient in prevention of delayed neuronal death. Our results demonstrate that whereas 8 min of lethal ischemia destroys 49.9% of CAI neurons, 10 min of ischemia destroys 71.6% of CA1 neurons, three different techniques of the second pathophysiological stress are able to protect against both: CA1 damage as well as spatial learning/memory dysfunction. Bolus of norepinephrine (3.1 micromol/kg i.p.) used two days after 8 min ischemia saved 94.2%, 6 min ischemia applied 2 days after 10 min ischemia rescued 89.9%, and an injection of 3-nitropropionic acid (20 mg/kg i.p.) applied two days after 10 min ischemia protected 77.5% of CA1 neurons. Thus, the second pathophysiological stress, if applied at a suitable time after lethal ischemia, represents a significant therapeutic window to opportunity for salvaging neurons in the hippocampal CA1 region against delayed neuronal death.