Miroslava Nemethova

Possible mechanisms involved in the down-regulation of translation during transient global ischaemia in the rat brain.

The striking correlation between neuronal vulnerability and down-regulation of translation suggests that this cellular process plays a critical part in the cascade of pathogenetic events leading to ischaemic cell death. There is compelling evidence supporting the idea that inhibition of translation is exerted at the polypeptide chain initiation step, and the present study explores the possible mechanism/s implicated. Incomplete forebrain ischaemia (30 min) was induced in rats by using the four-vessel occlusion model. Eukaryotic initiation factor (eIF)2, eIF4E and eIF4E-binding protein (4E-BP1) phosphorylation levels, eIF4F complex formation, as well as eIF2B and ribosomal protein S6 kinase (p70(S6K)) activities, were determined in different subcellular fractions from the cortex and the hippocampus [the CA1-subfield and the remaining hippocampus (RH)], at several post-ischaemic times. Increased phosphorylation of the alpha subunit of eIF2 (eIF2 alpha) and eIF2B inhibition paralleled the inhibition of translation in the hippocampus, but they normalized to control values, including the CA1-subfield, after 4--6 h of reperfusion. eIF4E and 4E-BP1 were significantly dephosphorylated during ischaemia and total eIF4E levels decreased during reperfusion both in the cortex and hippocampus, with values normalizing after 4 h of reperfusion only in the cortex. Conversely, p70(S6K) activity, which was inhibited in both regions during ischaemia, recovered to control values earlier in the hippocampus than in the cortex. eIF4F complex formation diminished both in the cortex and the hippocampus during ischaemia and reperfusion, and it was lower in the CA1-subfield than in the RH, roughly paralleling the observed decrease in eIF4E and eIF4G levels. Our findings are consistent with a potential role for eIF4E, 4E-BP1 and eIF4G in the down-regulation of translation during ischaemia. eIF2 alpha, eIF2B, eIF4G and p70(S6K) are positively implicated in the translational inhibition induced at early reperfusion, whereas eIF4F complex formation is likely to contribute to the persistent inhibition of translation observed at longer reperfusion times.

Role of protein synthesis in the ischemic tolerance acquisition induced by transient forebrain ischemia in the rat.

Although ischemic preconditioning of the heart and brain is a well-documented neuroprotective phenomenon, the mechanism underlying the increased resistance to severe ischemia induced by a preceding mild ischemic exposure remains unclear. In this study we have determined the effect of ischemic preconditioning on ischemia/reperfusion-associated translation inhibition in the neocortex and hippocampus of the rat. We studied the effect of the duration on the sublethal ischemic episode (3, 4, 5 or 8 min), as well as the amount of time elapsed between sublethal and lethal ischemia on the cell death 7 days after the last ischemic episode. In addition, the rate of protein synthesis in vitro and expression of the 72-kD heat shock protein (hsp) were determined under the different experimental conditions. Our results suggest that two different mechanisms are essential for the acquisition of ischemic tolerance, at least in the CA1 sector of hippocampus. The first mechanism implies a highly significant reduction in translation inhibition after lethal ischemia, especially at an early time of reperfusion, in both vulnerable and nonvulnerable neurons. For the acquisition of full tolerance, a second mechanism, highly dependent on the time interval between preconditioning (sublethal ischemia) and lethal ischemia, is absolutely necessary; this second mechanism involves synthesis of protective proteins, which prevent the delayed death of vulnerable neurons.

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 μmol/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.

Post-conditioning exacerbates the MnSOD immune-reactivity after experimental cerebral global ischemia and reperfusion in the rat brain hippocampus.

This study monitored the effects of sub‐lethal ischemia (post‐conditioning) applied after a previous ischemic attack by way of the MnSOD immune‐reactivity examined in CA1 and dentate gyrus of the rat hippocampus. The experimental 10 min transient cerebral ischemia was followed by 2 days of reperfusion, the rats then underwent a second ischemia (4 or 6 min post‐conditioning). MnSOD immune‐reactivity was evaluated after 5 h, 1 and 2 days. Results obtained by computer microdensitometric image analysis indicated that 4 min of ischemic post‐conditioning caused higher MnSOD immune‐reactivity than 6 min. However, higher viability of CA1 neurons after stronger (6 min) post‐conditioning when production of MnSOD is lower, as well as differences between MnSOD in CA1 and dentate gyrus indicates another mechanism switching pro‐apoptotic destination of CA1 neurons to anti‐apoptotic.

Activities of endogenous antioxidant enzymes in the cerebrospinal fluid and the hippocampus after transient forebrain ischemia in rat

The activity of SOD and CAT was measured in controls and 5 h after 5, 10 and 15 min of ischemia, as well as 1 or 2 days after 10 min of ischemia in the hippocampus and in the CSF. A significant increase in total SOD activity 5 h after ischemia was caused mainly by increased CuZn-SOD activity. The highest values were measured 5 h after 5 min ischemia (by 160%) and smallest if 15 min (by 40%) of ischemia was used. In comparison to the hippocampus, the activity of SOD in CSF increased equally after all intervals of ischemia. Activities of total SOD and CuZn-SOD after 10 min of ischemia in the hippocampus were significantly increased only after 5 and 24 h of reperfusion but in CSF they were increased after all examined intervals of reperfusion. The activity of CAT was significantly increased in the hippocampus after 5 (by 260%), 10 and 15 min (by 100%) of ischemia. CAT activity in CSF was increased equally after all intervals of ischemia (by 200%). Ischemic attack causes a rapid response in hippocampal tissue as well as in the CSF, represented by an increase in the activity of endogenous antioxidant enzymes SOD and CAT.

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.

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.

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.

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.

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.