Viera Danielisová

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.

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. Anat Rec, 292:2002–2012, 2009.

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.

Improvement of energy state and basic modifications of neuropathological damage in rabbits as a result of graded postischemic spinal cord reoxygenation

The role of graded postischemic reoxygenation applied at the end of 20 min of spinal cord ischemia was studied with respect to the intraspinal pO2 tension, energy state, and histopathological sequelae. Graded postischemic reoxygenation can induce a positive shift in the intraspinal pO2 tension, but normal postischemic reoxygenation with normotensive pO2 blood tension inevitably causes the postischemic intraspinal pO2 overshoot. Graded postischemic reoxygenation significantly improves the energy state expressed by higher adenosine triphosphate (ATP), phosphocreatine (PCr) and glucose levels. Using the Nauta impregnating degenerating method, clear histopathological differences were found in the L3-S3 segments after 20 min of ischemia. Apparently divergent damage was observed when normal reoxygenation or graded postischemic reoxygenation was used. Diametrically different histopathological outcomes were obtained with normal reoxygenation and graded postischemic reoxygenation 2 and 4 days postoperatively.

Postischemic hypoxia improves metabolic and functional recovery of the spinal cord

We studied the effect of graded postischemic reoxygenation on the tissue concentrations of adenylates, glucose, and lactate in the rabbit lumbar spinal cord after 10, 20, and 30 minutes of ischemia. In comparison with recirculation without manipulated Pao2, a decrease of Pao2 to 40 to 45 mm Hg upon reestablishment of blood circulation after ischemia led to an amelioration of the energy metabolism in the spinal cord tissue as determined by measuring the ATP concentration and energy charge. The protective effect of postischemic hypoxia was also reflected by the improvement of neurologic functions in animals after 10 and 20 minutes of ischemia.

Comparative Effects of theN-Methyl-d-aspartate Antagonist MK-801 and the Calcium Channel Blocker KB-2796 on Neurologic and Metabolic Recovery after Spinal Cord Ischemia

Abstract NMDA receptor antagonists have been demonstrated to be neuroprotective in focal cerebral ischemia and are supposed to prevent neurotoxic intracellular calcium increase. Another mechanism of calcium influx during ischemia involves activation of voltage-activated calcium channels, although the efficacy of calcium channel blockers against ischemia-induced damage varies. The purpose of this study was to determine the contributions of the excitotoxic mechanism and of calcium channel activation to metabolic and functional damage to rabbit spinal cord after ischemia induced by occlusion of the abdominal aorta. All metabolic parameters determined (ATP, energy charge, and lactate) completely recovered at 4 days following 20 min of ischemia when NMDA receptor antagonist MK-801 (1 mg/kg given iv) or calcium channel blocker KB-2796 (50 mg/kg given ip) was administered either prior to or after ischemia. Significant metabolic recovery was also observed after 30 min of ischemia with MK-801 administered before occlusion and KB-2796 given early in recirculation. Similarly, neurologic functions followed by functional performance in the hindlimbs were completely recovered following 20 and 30 min of ischemia and 4 days of recovery. This study demonstrates that although MK-801 or KB-2796 does not prevent paraplegia due to spinal cord ischemia in the rabbit, both drugs can influence the rate of recovery after ischemic injury.

Iron deposition after transient forebrain ischemia in rat brain.

In this study, we investigated the iron deposition in the cerebral cortex, hippocampus CA1 area and corpus striatum pars dorsolateralis in a rat model of cerebral ischemia. Forebrain ischemia was induced by four-vessel occlusion for 20 min. Using iron histochemistry, regional changes were examined from 1 to 8 weeks of postischemic recirculation. Neuronal death was demonstrated in pyramidal cells of the hippocampal CA1 area and in the dorsolateral part of the corpus striatum, which are known as areas most vulnerable to ischemia. Iron deposition in hippocampal CA1 area was coupled to delayed pyramidal cell death. Perls reaction with DAB intensification revealed of the 1 week iron deposits in the CA1 area, which gradually increased and formed clusters by 8 weeks. In the corpus striatum, strong iron staining was observed in injured cellular layer pars dorsolateralis 1 week after recirculation. Granular iron was deposited in the cytoplasm of pyramidal cells in layers III and V of the frontal cortex after 2 weeks of recirculation. In contrast to the hippocampus and striatum, the cerebral cortex did not develop severe neuronal cell death and atrophy immediately after the ischemic insult, which suggest that the neuronal cell death in the cerebral cortex occurs extremely late.

Neuroprotection and antioxidants.

Ischemia as a serious neurodegenerative disorder causes together with reperfusion injury many changes in nervous tissue. Most of the neuronal damage is caused by complex of biochemical reactions and substantial processes, such as protein agregation, reactions of free radicals, insufficient blood supply, glutamate excitotoxicity, and oxidative stress. The result of these processes can be apoptotic or necrotic cell death and it can lead to an irreversible damage. Therefore, neuroprotection and prevention of the neurodegeneration are highly important topics to study. There are several approaches to prevent the ischemic damage. Use of many modern therapeutical methods and the incorporation of several substances into the diet of patients is possible to stimulate the endogenous protective mechanisms and improve the life quality.

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.