Biomedical subjects

Jozef Burda

Publications and source records attributed to Jozef Burda.

15 recordsLinked to original sources

PubMed2007

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.

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PubMed2006

Fluoro-Jade B staining following zymosan microinjection into the spinal cord white matter.

1. The fluorescein derivate Fluoro-Jade B (FJB), which primarily labels dead or dying neurons, was used to study the acute focal inflammation in the spinal cord white matter. Inflammation was induced by microinjection of the yeast particulate zymosan to evaluate the biological effects of intraspinal macrophages activation without the confounding effects of physical trauma. 2. A single bolus of zymosan (Sigma, 75 nL) was stereotaxically injected at the thoracic level into the lateral white matter of rat spinal cord. A standard Fluoro-Jade B staining protocol was applied to spinal cord sections at 6, 12, 24 h and 2, 4 days postinjection. Neutral Red, NADPH-diaphorase, Iba1-IR, and DAPI staining protocols accomplished examination of the cells participating in the acute inflammatory response. 3. Zymosan caused formation of clearly delineated inflammation lesions localized in the lateral white matter of the spinal cord. Fluoro-Jade B stained cells in the area of inflammation were not observed at 12 h postinjection while mild FJB staining appeared at 24 h and intense staining was observed at 2 and 4 days postinjection. 4. This study shows that the acute response to zymosan-induced inflammation in the rat spinal cord white matter causes a gradual appearance of phagocytic microglia/macrophages and delayed FJB staining of the inflammatory cells. 5. FJB, a reliable marker of dying neurons, is a more universal agent than formerly believed. One possible explanation for the gradual appearance of FJB-stained cells in the area of inflammation is that specific time is required for sufficient levels of proteins and/or myelin debris of axonal origin to appear in the cytoplasm of phagocytic microglia/macrophages.

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PubMed2006

Mapping of rat hippocampal neurons with NeuN after ischemia/reperfusion and Ginkgo biloba extract (EGb 761) pretreatment.

1. The neuroprotective effect of Ginkgo biloba extract (EGb 761) against transient forebrain ischemia following 7 days of reperfusion was studied in male Wistar rats after four-vessel occlusion for 20 min. 2. NeuN, a neuronal specific nuclear protein was used for immunohistochemical detection of surviving pyramidal neurons in the hippocampus, as well as counterstaining with hematoxylin in the same sections for detection of neurons that underwent delayed neuronal death and for glial nuclei staining. GFAP immunohistochemistry was used for detection of astrocytes in the studied area of CA1 region. 3. In the group of rats pretreated 7 days with Ginkgo biloba extract (EGb 761), following 20 min of ischemia and 7 days of reperfusion without EGb 761, increased number of NeuN immunoreactive cells were counted in the most vulnerable CA1 pyramidal layer of hippocampus. On the other hand, the group of rats with 7 days of EGb 761 pretreatment following 20 min of ischemia and 7 days of reperfusion with EGb 761 showed decreased number of surviving NeuN immunoreactive CA1 pyramidal cells in comparison with the first above-mentioned experimental group. 4. Increased number of reactive astrocytes immunolabeled for GFAP (Glial fibrilary acidic protein) was observed in both experimental groups in the stratum oriens and stratum lacunosum and moleculare. 5. Twenty minutes of ischemia is lethal for most population of CA1 pyramidal cell layer. Our results showed that prophylactic oral administration of Ginkgo biloba extract (EGb 761) in the dose 40 mg/kg/day during the 7 days protects the most vulnerable CA1 pyramidal cells against 20 min of ischemia.

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PubMed2006

The changes in endogenous antioxidant enzyme activity after postconditioning.

1. The aim of this work was to study potential mechanisms participating in postischemic protection of selectively vulnerable CA1 neurons in the hippocampus. Experiments were focused on measuring changes in endogenous antioxidant enzyme activity. 2. Forebrain cerebral ischemia was induced in a rat by four-vessel occlusion. Ten minutes of ischemia induces so-called delayed neuronal death in selectively vulnerable CA1 region 3 days later. After 7 days of reperfusion, 71.6% of neurons succumb to neurodegeneration. When 5 min of ischemia was used as postconditioning, 2 days after 10 min of cerebral ischemia, delayed neuronal death in CA1 was almost completely (89.9%) prevented. 3. Searching for mechanisms of protection, we measured the activity of endogenous antioxidant enzymes. Activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were measured in the hippocampus, striatum and cortex by spectrophotometric methods after 10 min of ischemia used as the preconditioning. Two days after the preconditioning or the sham operation, second ischemia was induced for 5 min. We observed significant increase of total SOD activity in all studied regions of the brain 5 h after postconditioning (5 min of ischemia). SOD activity decreased to control values after 24 h. 4. In some experiments, we used intraperitoneal injections of norepinephrine (3.1 microM/kg) or 3-nitropropionic acid (20 mg/kg) as postconditioning, instead of ischemia. All three treatments resulted in significant increase of SOD activity, but norepinephrine was the most effective. The same effect as was seen for total SOD activity could be observed for CuZn-SOD as well as Mn-SOD activity. Similarly, considerable increase in the activity of catalase was detected 5 h after postconditioning (5 min of ischemia). It is interesting that the greatest changes were established in selectively vulnerable hippocampus and striatum. As in the case of SOD, the highest levels of CAT activity were induced by norepinephrine, while lower but significant increase in CAT activity was induced by 3-nitropropionic acid.5. Our results suggest that endogenous antioxidants SOD and CAT could play considerable neuroprotective role after postconditioning.

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PubMed2006

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.

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PubMed2005

Changes of endogenous antioxidant enzymes during ischemic tolerance acquisition.

The purpose of this study was to investigate the role of superoxide dismutase (SOD) and catalase (CAT) in brain ischemic tolerance induced by ischemic preconditioning. Forebrain cerebral ischemia was induced in rat by four vessel occlusion. The activities of the antioxidant enzymes CuZn-SOD, Mn-SOD and CAT were measured in the hippocampus, striatum and cortex after 5 min of ischemia used as a preconditioning and subsequent reperfusion, by spectrophotometric methods. In all ischemia-reperfusion groups (5 h, 1 and 2 days of reperfusion), CuZn-SOD activities were found to be increased if compared to the sham operated controls. The increase was significant (P < 0.05) in all reperfusion groups, particularly after 5 h of reperfusion (3 times) in all studied brain regions; the largest increase was detected in the more vulnerable hippocampus and striatum. Very similar changes were found in Mn-SOD activity. The activity of CAT was increased too, but reached the peak of postischemic activity 24 h after ischemia. Our attempt to understand the mechanisms of increased SOD and CAT activities by application of protein synthesis inhibitor cycloheximide showed that this increase was caused by de novo synthesis of enzymes during first hours after ischemia. Our findings indicate that both major endogenous antioxidant enzymes SOD and CAT are synthesized as soon as 5 h after ischemia. In spite of significant upregulation of these enzymes a large number of neurons in selectively vulnerable CA1 region of hippocampus undergoes to neurodegeneration within 7 days after ischemia.

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PubMed2005

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.

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PubMed2005

The effect of normovolemic hemodilution on c-Fos protein immunoreactivity in the postischemic rat brain cortex.

The hemodilution effect was evaluated in the parietal, temporal, and basal regions of postischemic rat brain neocortex by the immunohistochemical detection of the c-Fos protein. Rats were subjected to the four-vessel-occlusion and the hemodilution was used in one group of animals. Coronal sections of rat brains were used for immunohistochemical processing of c-Fos protein after different postischemic reperfusion intervals. The number of c-Fos positive neuronal nuclei was significantly decreased in the hemodilution group after 4 h of reperfusion in the parietal and basal neocortex compared to the reperfusion without hemodilution.

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PubMed2004

Ischaemic preconditioning in the rat brain: effect on the activity of several initiation factors, Akt and extracellular signal-regulated protein kinase phosphorylation, and GRP78 and GADD34 expression.

Translational repression induced during reperfusion of the ischaemic brain is significantly attenuated by ischaemic preconditioning. The present work was undertaken to identify the components of the translational machinery involved and to determine whether translational attenuation selectively modifies protein expression patterns during reperfusion. Wistar rats were preconditioned by 5-min sublethal ischaemia and 2 days later, 30-min lethal ischaemia was induced. Several parameters were studied after lethal ischaemia and reperfusion in rats with and without acquired ischaemic tolerance (IT). The phosphorylation pattern of the alpha subunit of eukaryotic initiation factor 2 (eIF2) in rats with IT was exactly the same as in rats without IT, reaching a peak after 30 min reperfusion and returning to control values within 4 h in both the cortex and hippocampus. The levels of phosphorylated eIF4E-binding protein after lethal ischaemia and eIF4E at 30 min reperfusion were higher in rats with IT, notably in the hippocampus. eIF4G levels diminished slightly after ischaemia and reperfusion, paralleling calpain-mediated alpha-spectrin proteolysis in rats with and without IT, but they did not show any further decrease after 30 min reperfusion in rats with IT. The phosphorylated levels of eIF4G, phosphatidylinositol 3-kinase-protein B (Akt) and extracellular signal-regulated kinases (ERKs) were very low after lethal ischaemia and increased following reperfusion. Ischaemic preconditioning did not modify the observed changes in eIF4G phosphorylation. All these results support that translation attenuation may occur through multiple targets. The levels of the glucose-regulated protein (78 kDa) remained unchanged in rats with and without IT. Conversely, our data establish a novel finding that ischaemia induces strong translation of growth arrest and DNA damage protein 34 (GADD34) after 4 h of reperfusion. GADD34 protein was slightly up-regulated after preconditioning, besides, as in rats without IT, GADD34 levels underwent a further clear-cut increase during reperfusion, this time as earlier as 30 min and coincident with translation attenuation.

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PubMed2004

Iron deposition in the brain following the ischemia in a rat model of ischemic tolerance.

Preconditioning of the brain by short-term ischemia increases brain tolerance to the subsequent severer ischemia. In this study, we investigated iron deposition in the cerebral cortex and the ischemic tolerance in a rat model of cerebral ischemia. Forebrain ischemia was induced by four-vessel occlusion for 5 min as ischemic preconditioning. Two days after preconditioning or after the sham-operation, the second ischemia was induced for 20 min. Changes in the cerebral cortex were examined after 1 to 8 weeks of recirculation following 20 min ischemia with or without preconditioning using the iron histochemistry. Granular deposits of the iron were found in the cytoplasm of the pyramidal cells in the layers III and V of the frontal cortex after 1 week of recirculation. When the rats were exposed to 5 min ischemia 2 days before 20 min lasting ischemia, the deposition of iron in the cytoplasm of the pyramidal cells in layers III and V of the frontal cortex was significantly lower during all periods of reperfusion. Preconditioning 5 min ischemia followed by 2 days of reperfusion before 20 min ischemia also prevented degeneration of the pyramidal neurons in layers III and V of the frontal cortex.

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PubMed2003

Peripheral nerve injury influences the disinhibition induced by focal ischaemia in the rat motor cortex.

Photothrombotic lesions were produced in the rat primary motor cortex, and the brain excitability was assessed in a paired-pulse stimulation protocol by transcranial recording, in parallel at 16 points of the frontal cortex, including the insulted and the surrounding areas. The cortical lesion reduced the inhibition in the extended frontal cortex, with a delay of a few minutes. Unilateral facial nerve transection, however, accelerated the widespread disinhibition. Although the mechanism is not clear in detail, both peripheral and central injury-induced disinhibition may have a significant impact on the recovery of the function.

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PubMed2003

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.

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PubMed2002

Cerebral postischemic reperfusion-induced demethylation of the protein phosphatase 2A catalytic subunit.

Brain reperfusion after a period of global ischemia induces changes in the phosphorylation state of a great number of proteins. Neuronal responses to ischemia and reperfusion are quite different depending on the brain region, and phosphorylation changes may be implicated in this tissue-specific response. For this reason, we have used both biochemical and immunohistochemical methods to investigate the potential role of PP2A, the most abundant Ser/Thr phosphatase in the brain, in ischemic injury. PP2A activity as measured with phosphorylase a as substrate was slightly inhibited after 30 min ischemia followed by 30 min reperfusion, and this inhibition correlated with an increased S6K1 and ERK1/2 phosphorylation. Using a monoclonal antibody unable to recognize the methylated form of PP2Ac, we demonstrated that the catalytic subunit of PP2A (PP2Ac) was highly methylated in the brain. In addition, the postischemic reperfusion-induced changes in PP2Ac methylation were studied in sections from cerebral cortex, hippocampus and striatum. Regional differences in PP2Ac methylation were observed within control brains, and the postischemic reperfusion caused a generalized demethylation of PP2Ac. Those regions in the control brains containing highest levels of methylated PP2Ac were the most intensively demethylated after reperfusion and corresponded to the regions most vulnerable to ischemic damage.

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PubMed2002

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. Perl's 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.

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PubMed2002

Prevention of in vivo excitotoxicity by a family of trialkylglycines, a novel class of neuroprotectants.

Excitotoxicity has been implicated in the etiology of ischemic stroke and chronic neurodegenerative disorders. Hence, the development of novel neuroprotectant molecules that ameliorate excitotoxic brain damage is vigorously pursued. We used a neuroprotection-based cellular assay to screen a synthetic combinatorial library of N-alkylglycine trimers. Two compounds (6-1-2 and 6-1-10) that efficiently prevented excitotoxic neurodegeneration in vitro and in vivo were identified. Both molecules protected primary cultures of cerebellar neurons against glutamate-induced neuronal death with an efficiency equivalent to N-methyl-D-aspartate (NMDA) receptor antagonists. These trialkylglycines did not block appreciably the NMDA receptor channel, or attenuated glutamate-induced increase of Ca(2+), or affect the glutamate-nitric oxide-cGMP pathway. Intraperitoneal injection of both peptoids in mice attenuated > or = 80% ammonia-induced, NMDA receptor-mediated animal death. Furthermore, these two molecules reduced by > or = 50% the neurodegeneration in striatum in a rat model of cerebral ischemia. Neuroprotection against ischemia was associated with decreased activation of caspase-3, reflecting prevention of apoptotic neuronal death. Collectively, the results reported indicate that these trialkylglycines are new neuroprotectant leads with important in vivo activity against excitotoxicity, and that they act on a novel, yet-unrecognized cellular target. These lead compounds may become tolerated drugs for the treatment of acute and chronic neurodegenerative diseases with fewer side effects than NMDA receptor antagonists.

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