Dynamics in Redox-Active Molecules Following Ischemic Preconditioning in the Brain.

It is well known that the brain is quite vulnerable to oxidative stress, initiating neuronal loss after ischemia-reperfusion (IR) injury. A potent protective mechanism is ischemic preconditioning (IPC), where proteins are among the primary targets. This study explores redox-active proteins' role in preserving energy supply. Adult rats were divided into the control, IR, and IPC groups. Protein profiling was conducted to identify modified proteins and then verified through activity assays, immunoblot, and immunohistochemical analyses. IPC protected cortex mitochondria, as evidenced by a 2.26-fold increase in superoxide dismutase (SOD) activity. Additionally, stable core subunits of respiratory chain complexes ensured sufficient energy production, supported by a 16.6% increase in ATP synthase activity. In hippocampal cells, IPC led to the downregulation of energy-related dehydrogenases, while a significantly higher level of peroxiredoxin 6 (PRX6) was observed. Notably, IPC significantly enhanced glutathione reductase activity to provide sufficient glutathione to maintain PRX6 function. Astrocytes may mobilize PRX6 to protect neurons during initial ischemic events, by decreased PRX6 positivity in astrocytes, accompanied by an increase in neurons following both IR injury and IPC. Maintained redox signaling via astrocyte-neuron communication triggers IPC's protective state. The partnership among PRX6, SOD, and glutathione reductase appears essential in safeguarding and stabilizing the hippocampus.

Blood and Brain Metabolites after Cerebral Ischemia.

The study of an organism's response to cerebral ischemia at different levels is essential to understanding the mechanism of the injury and protection. A great interest is devoted to finding the links between quantitative metabolic changes and post-ischemic damage. This work aims to summarize the outcomes of the most studied metabolites in brain tissue-lactate, glutamine, GABA (4-aminobutyric acid), glutamate, and NAA (N-acetyl aspartate)-regarding their biological function in physiological conditions and their role after cerebral ischemia/reperfusion. We focused on ischemic damage and post-ischemic recovery in both experimental-including our results-as well as clinical studies. We discuss the role of blood glucose in view of the diverse impact of hyperglycemia, whether experimentally induced, caused by insulin resistance, or developed as a stress response to the cerebral ischemic event. Additionally, based on our and other studies, we analyze and critically discuss post-ischemic alterations in energy metabolites and the elevation of blood ketone bodies observed in the studies on rodents. To complete the schema, we discuss alterations in blood plasma circulating amino acids after cerebral ischemia. So far, no fundamental brain or blood metabolite(s) has been recognized as a relevant biological marker with the feasibility to determine the post-ischemic outcome or extent of ischemic damage. However, studies from our group on rats subjected to protective ischemic preconditioning showed that these animals did not develop post-ischemic hyperglycemia and manifested a decreased metabolic infringement and faster metabolomic recovery. The metabolomic approach is an additional tool for understanding damaging and/or restorative processes within the affected brain region reflected in the blood to uncover the response of the whole organism via interorgan metabolic communications to the stressful cerebral ischemic challenge.

Alzheimer's Disease-like Pathological Features in the Dorsal Hippocampus of Wild-Type Rats Subjected to Methionine-Diet-Evoked Mild Hyperhomocysteinaemia.

Multifactorial interactions, including nutritional state, likely participate in neurodegeneration's pathogenesis and evolution. Dysregulation in methionine (Met) metabolism could lead to the development of hyperhomocysteinaemia (hHcy), playing an important role in neuronal dysfunction, which could potentially lead to the development of Alzheimer's disease (AD)-like pathological features. This study combines proton magnetic resonance spectroscopy (1H MRS) with immunohistochemical analysis to examine changes in the metabolic ratio and histomorphological alterations in the dorsal rat hippocampus (dentate gyrus-DG) subjected to a high Met diet. Male Wistar rats (420-480 g) underwent hHcy evoked by a Met-enriched diet (2 g/kg of weight/day) lasting four weeks. Changes in the metabolic ratio profile and significant histomorphological alterations have been found in the DG of hHcy rats. We have detected increased morphologically changed neurons and glial cells with increased neurogenic markers and apolipoprotein E positivity parallel with a diminished immunosignal for the N-Methyl-D-Aspartate receptor 1 in hHcy animals. A Met diet induced hHcy, likely via direct Hcy neurotoxicity, an interference with one carbon unit metabolism, and/or epigenetic regulation. These conditions lead to the progression of neurodegeneration and the promotion of AD-like pathological features in the less vulnerable hippocampal DG, which presents a plausible therapeutic target.

Involvement of Proteasomal and Endoplasmic Reticulum Stress in Neurodegeneration After Global Brain Ischemia.

A brief period of transient global brain ischemia leads to selective ischemic neurodegeneration associated with death of hippocampal CA1 pyramidal neurons days after reperfusion. The mechanism of such selective and delayed neurodegeneration is still uncertain. Our work aimed to study the involvement of proteasomal and endoplasmic reticulum (ER) stress in ischemic neurodegeneration. We have performed laser scanning confocal microscopy analysis of brain slices from control and experimental animals that underwent global brain ischemia for 15 min and varying times of reperfusion. We have focused on ubiquitin, PUMA, a proapoptotic protein of the Bcl-2 family overexpressed in response to both proteasomal and ER stress, and p53, which controls expression of PUMA. We have also examined the expression of HRD1, an E3 ubiquitin ligase that was shown to be overexpressed after ER stress. We have also examined potential crosstalk between proteasomal and ER stress using cellular models of both proteasomal and ER stress. We demonstrate that global brain ischemia is associated with an appearance of distinct immunoreactivity of ubiquitin, PUMA and p53 in pyramidal neurons of the CA1 layer of the hippocampus 72 h after ischemic insults. Such changes correlate with a delay and selectivity of ischemic neurodegeneration. Immunoreactivity of HRD1 observed in all investigated regions of rat brain was transiently absent in both CA1 and CA3 pyramidal neurones 24 h after ischemia in the hippocampus, which does not correlate with a delay and selectivity of ischemic neurodegeneration. We do not document significant crosstalk between proteasomal and ER stress. Our results favour dysfunction of the ubiquitin proteasome system and consequent p53-induced expression of PUMA as the main mechanisms responsible for selective and delayed degeneration of pyramidal neurons of the hippocampal CA1 layer in response to global brain ischemia.

Hippocampal metabolic recovery as a manifestation of the protective effect of ischemic preconditioning in rats.

The ever-present risk of brain ischemic events in humans and its full prevention make the detailed studies of an organism's response to ischemia at different levels essential to understanding the mechanism of the injury as well as protection. We used the four-vessel occlusion as an animal model of forebrain ischemia to investigate its impact on the metabolic alterations in both the hippocampus and the blood plasma to see changes on the systemic level. By inducing sublethal ischemic stimuli, we focused on the endogenous phenomena known as ischemic tolerance. NMR spectroscopy was used to analyze relative metabolite levels in tissue extracts from rats' hippocampus and blood plasma in three various ischemic/reperfusion times: 3 h, 24 h, and 72 h. Hippocampal tissues were characterized by postischemically decreased glutamate and GABA (4-aminobutyrate) tissue content balanced with increased glutamine level, with most pronounced changes at 3 h reperfusion time. Glutamate (as well as glutamine) levels recovered towards the control levels on the third day, as if the glutamate re-synthesis would be firstly preferred before GABA. These results are indicating the higher feasibility of re-establishing of glutamatergic transmission three days after an ischemic event, in contrast to GABA-ergic. Tissue levels of N-acetylaspartate (NAA), as well as choline, were decreased without the tendency to recover three days after the ischemic event. Metabolomic analysis of blood plasma revealed that ischemically preconditioned rats, contrary to the non-preconditioned animals, did not show hyperglycemic conditions. Ischemically induced semi-ketotic state, manifested in increased plasma ketone bodies 3-hydroxybutyrate and acetoacetate, seems to be programmed to support the brain tissue revitalization after the ischemic event. These and other metabolites changes found in blood plasma as well as in the hippocampus were observed to a lower extent or recovered faster in preconditioned animals. Some metabolomic changes in hippocampal tissue extract were so strong that even single metabolites were able to differentiate between ischemic, ischemically preconditioned, and control brain tissues.

Metabolic Changes Induced by Cerebral Ischemia, the Effect of Ischemic Preconditioning, and Hyperhomocysteinemia.

1H Nuclear Magnetic Resonance (NMR) metabolomics is one of the fundamental tools in the fast-developing metabolomics field. It identifies and quantifies the most abundant metabolites, alterations of which can describe energy metabolism, activated immune response, protein synthesis and catabolism, neurotransmission, and many other factors. This paper summarizes our results of the 1H NMR metabolomics approach to characterize the distribution of relevant metabolites and their alterations induced by cerebral ischemic injury or its combination with hyperhomocysteinemia in the affected tissue and blood plasma in rodents. A decrease in the neurotransmitter pool in the brain tissue likely follows the disordered feasibility of post-ischemic neurotransmission. This decline is balanced by the increased tissue glutamine level with the detected impact on neuronal health. The ischemic injury was also manifested in the metabolomic alterations in blood plasma with the decreased levels of glycolytic intermediates, as well as a post-ischemically induced ketosis-like state with increased plasma ketone bodies. As the 3-hydroxybutyrate can act as a likely neuroprotectant, its post-ischemic increase can suggest its supporting role in balancing ischemic metabolic dysregulation. Furthermore, the 1H NMR approach revealed post-ischemically increased 3-hydroxybutyrate in the remote organs, such as the liver and heart, as well as decreased myocardial glutamate. Ischemic preconditioning, as a proposed protective strategy, was manifested in a lower extent of metabolomic changes and/or their faster recovery in a longitudinal study. The paper also summarizes the pre- and post-ischemic metabolomic changes in the rat hyperhomocysteinemic models. Animals are challenged with hyperglycemia and ketosis-like state. A decrease in several amino acids in plasma follows the onset and progression of hippocampal neuropathology when combined with ischemic injury. The 1H NMR metabolomics approach also offers a high potential for metabolites in discriminatory analysis in the search for potential biomarkers of ischemic injury. Based on our results and the literature data, this paper presents valuable findings applicable in clinical studies and suggests the precaution of a high protein diet, especially foods which are high in Met content and low in B vitamins, in the possible risk of human cerebrovascular neuropathology.

Methionine Diet Evoked Hyperhomocysteinemia Causes Hippocampal Alterations, Metabolomics Plasma Changes and Behavioral Pattern in Wild Type Rats.

L-methionine, an essential amino acid, plays a critical role in cell physiology. High intake and/or dysregulation in methionine (Met) metabolism results in accumulation of its intermediate(s) or breakdown products in plasma, including homocysteine (Hcy). High level of Hcy in plasma, hyperhomocysteinemia (hHcy), is considered to be an independent risk factor for cerebrovascular diseases, stroke and dementias. To evoke a mild hHcy in adult male Wistar rats we used an enriched Met diet at a dose of 2 g/kg of animal weight/day in duration of 4 weeks. The study contributes to the exploration of the impact of Met enriched diet inducing mild hHcy on nervous tissue by detecting the histo-morphological, metabolomic and behavioural alterations. We found an altered plasma metabolomic profile, modified spatial and learning memory acquisition as well as remarkable histo-morphological changes such as a decrease in neurons' vitality, alterations in the morphology of neurons in the selective vulnerable hippocampal CA 1 area of animals treated with Met enriched diet. Results of these approaches suggest that the mild hHcy alters plasma metabolome and behavioural and histo-morphological patterns in rats, likely due to the potential Met induced changes in "methylation index" of hippocampal brain area, which eventually aggravates the noxious effect of high methionine intake.

Effect of Methionine Diet on Time-Related Metabolic and Histopathological Changes of Rat Hippocampus in the Model of Global Brain Ischemia.

Hyperhomocysteinemia (hHcy) represents a strong risk factor for atherosclerosis-associated diseases, like stroke, dementia or Alzheimer's disease. A methionine (Met)-rich diet leads to an elevated level of homocysteine in plasma and might cause pathological alterations across the brain. The hippocampus is being constantly studied for its selective vulnerability linked with neurodegeneration. This study explores metabolic and histo-morphological changes in the rat hippocampus after global ischemia in the hHcy conditions using a combination of proton magnetic resonance spectroscopy and magnetic resonance-volumetry as well as immunohistochemical analysis. After 4 weeks of a Met-enriched diet at a dose of 2 g/kg of animal weight/day, adult male Wistar rats underwent 4-vessel occlusion lasting for 15 min, followed by a reperfusion period varying from 3 to 7 days. Histo-morphological analyses showed that the subsequent ischemia-reperfusion insult (IRI) aggravates the extent of the sole hHcy-induced degeneration of the hippocampal neurons. Decreased volume in the grey matter, extensive changes in the metabolic ratio, deeper alterations in the number and morphology of neurons, astrocytes and their processes were demonstrated in the hippocampus 7 days post-ischemia in the hHcy animals. Our results suggest that the combination of the two risk factors (hHcy and IRI) endorses and exacerbates the rat hippocampal neurodegenerative processes.

Effect of Methionine Diet on Metabolic and Histopathological Changes of Rat Hippocampus.

Hyperhomocysteinemia (hHcy) is regarded as an independent and strong risk factor for cerebrovascular diseases, stroke, and dementias. The hippocampus has a crucial role in spatial navigation and memory processes and is being constantly studied for neurodegenerative disorders. We used a moderate methionine (Met) diet at a dose of 2 g/kg of animal weight/day in duration of four weeks to induce mild hHcy in adult male Wistar rats. A novel approach has been used to explore the hippocampal metabolic changes using proton magnetic resonance spectroscopy (1H MRS), involving a 7T MR scanner in combination with histochemical and immunofluorescence analysis. We found alterations in the metabolic profile, as well as remarkable histo-morphological changes such as an increase of hippocampal volume, alterations in number and morphology of astrocytes, neurons, and their processes in the selective vulnerable brain area of animals treated with a Met-enriched diet. Results of both methodologies suggest that the mild hHcy induced by Met-enriched diet alters volume, histo-morphological pattern, and metabolic profile of hippocampal brain area, which might eventually endorse the neurodegenerative processes.

Tyrosine nitration of mitochondrial proteins during myocardial ischemia and reperfusion.

Myocardial ischemia reperfusion is associated with mitochondrial dysfunction and increased formation of reactive oxygen/nitrogen species. The main purpose of this study was to assess the role of tyrosine nitration of mitochondrial proteins in postischemic contractile dysfunction known as myocardial stunning. Isolated Langendorff-perfused rat hearts were subjected to 20-min global ischemia followed by 30-min reperfusion. The reperfused hearts showed marked decline in left ventricular developed pressure, maximal rate of contraction (+dP/dt), and maximal rate of relaxation (-dP/dt). Immunofluorescence and ELISA assays demonstrated enhanced protein tyrosine nitration in reperfused hearts. Using two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrometry, eight mitochondrial proteins were identified to be nitrated after ischemia reperfusion. These proteins are crucial in mitochondrial electron transport, fatty acid oxidation, tricarboxylic acid cycle, ATP synthesis, and control of high-energy phosphates. The proteome data also indicated reduced abundance in several of nitrated proteins. The results suggest that these changes may contribute to inhibition of aconitase activity but are unlikely to affect electron transport chain activity. Whether tyrosine nitration of mitochondrial proteins can be considered the contributing factor of postischemic contractile dysfunction remains to be explored.

Global brain ischemia in rats is associated with mitochondrial release and downregulation of Mfn2 in the cerebral cortex, but not the hippocampus.

Mitochondria are crucial for neuronal cell survival and death through their functions in ATP production and the intrinsic pathway of apoptosis. Mitochondrial dysfunction is considered to play a central role in several serious human diseases, including neurodegenerative diseases, such as Parkinson's and Alzheimer's disease and ischemic neurodegeneration. The aim of the present study was to investigate the impact of transient global brain ischemia on the expression of selected proteins involved in mitochondrial dynamics and mitochondria­associated membranes. The main foci of interest were the proteins mitofusin 2 (Mfn2), dynamin­related protein 1 (DRP1), voltage­dependent anion­selective channel 1 (VDAC1) and glucose­regulated protein 75 (GRP75). Western blot analysis of total cell extracts and mitochondria isolated from either the cerebral cortex or hippocampus of experimental animals was performed. In addition, Mfn2 was localized intracellularly by laser scanning confocal microscopy. It was demonstrated that 15­min ischemia, or 15­min ischemia followed by 1, 3, 24 or 72 h of reperfusion, was associated with a marked decrease of the Mfn2 protein in mitochondria isolated from the cerebral cortex, but not in hippocampal mitochondria. Moreover, a translocation of the Mfn2 protein to the cytoplasm was documented immediately after global brain ischemia in the neurons of the cerebral cortex by laser scanning confocal microscopy. Mfn2 translocation was followed by decreased expression of Mfn2 during reperfusion. Markedly elevated levels of the VDAC1 protein were also documented in total cell extracts isolated from the hippocampus of rats after 15 min of global brain ischemia followed by 3 h of reperfusion, and from the cerebral cortex of rats after 15 min of global brain ischemia followed by 72 h of reperfusion. The mitochondrial Mfn2 release observed during the early stages of reperfusion may thus represent an important mechanism of mitochondrial dysfunction associated with neuronal dysfunction or death induced by global brain ischemia.

Histone Hyperacetylation as a Response to Global Brain Ischemia Associated with Hyperhomocysteinemia in Rats.

Epigenetic regulations play an important role in both normal and pathological conditions of an organism, and are influenced by various exogenous and endogenous factors. Hyperhomocysteinemia (hHcy), as a risk factor for several pathological conditions affecting the central nervous system, is supposed to alter the epigenetic signature of the given tissue, which therefore worsens the subsequent damage. To investigate the effect of hHcy in combination with ischemia-reperfusion injury (IRI) and histone acetylation, we used the hHcy animal model of global forebrain ischemia in rats. Cresyl violet staining showed massive neural disintegration in the M1 (primary motor cortex) region as well as in the CA1 (cornu ammonis 1) area of the hippocampus induced by IRI. Neural loss was significantly higher in the group with induced hHcy. Moreover, immunohistochemistry and Western blot analysis of the brain cortex showed prominent changes in the acetylation of histones H3 and H4, at lysine 9 and 12, respectively, as a result of IRI and induced hHcy. It seems that the differences in histone acetylation patterns in the cortical region have a preferred role in pathological processes induced by IRI associated with hHcy and could be considered in therapeutic strategies.

Association of Induced Hyperhomocysteinemia with Alzheimer's Disease-Like Neurodegeneration in Rat Cortical Neurons After Global Ischemia-Reperfusion Injury.

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder that results in massive hippocampal and neocortical neuronal loss leading to dementia and eventual death. The exact cause of Alzheimer's disease is not fully explored, although a number of risk factors have been recognized, including high plasma concentration of homocysteine (Hcy). Hyperhomocysteinemia (hHcy) is considered a strong, independent risk factor for stroke and dementia. However, the molecular background underlying these mechanisms linked with hHcy and ischemic stroke is not fully understood. Paper describes rat model of global forebrain ischemia combined with the experimentally induced hHcy. Global ischemia-reperfusion injury (IRI) was developed by 4-vessels occlusion lasting for 15 min followed by reperfusion period of 72 h. hHcy was induced by subcutaneous injection of 0.45 µmol/g of Hcy in duration of 14 days. The results showed remarkable neural cell death induced by hHcy in the brain cortex and neurodegeneration is further aggravated by global IRI. We demonstrated degeneration of cortical neurons, alterations in number and morphology of tissue astrocytes and dysregulation of oxidative balance with increased membrane protein oxidation. Complementary to, an immunohistochemical analysis of tau protein and ß-amyloid peptide showed that combination of hHcy with the IRI might lead to the progression of AD-like pathological features. Conclusively, these findings suggest that combination of risk factor hHcy with IRI aggravates neurodegeneration processes and leads to development of AD-like pathology in cerebral cortex.

Effect of Hyperhomocysteinemia on Redox Balance and Redox Defence Enzymes in Ischemia-Reperfusion Injury and/or After Ischemic Preconditioning in Rats.

Increased level of homocysteine (hHcy) in plasma is an accompanying phenomenon of many diseases, including a brain stroke. This study determines whether hyperhomocysteinemia (which is a risk factor of brain ischemia) itself or in combination with ischemic preconditioning affects the ischemia-induced neurodegenerative changes, generation of reactive oxygen species (ROS), lipoperoxidation, protein oxidation, and activity of antioxidant enzymes in the rat brain cortex. The hHcy was induced by subcutaneous administration of homocysteine (0.45 µmol/g body weight) twice a day in 8 h intervals for 14 days. Rats were preconditioned by 5 min ischemia. Two days later, 15 min of global forebrain ischemia was induced by four vessel's occlusion. The study demonstrates that in the cerebral cortex, hHcy alone induces progressive neuronal cell death and morphological changes. Neuronal damage was associated with the pro-oxidative effect of hHcy, which leads to increased ROS formation, peroxidation of lipids and oxidative alterations of cortical proteins. Ischemic reperfusion injury activates degeneration processes and de-regulates redox balance which is aggravated under hHcy conditions and leads to the augmented lipoperoxidation and protein oxidation. If combined with hHcy, ischemic preconditioning could preserve the neuronal tissue from lethal ischemic effect and initiates suppression of lipoperoxidation, protein oxidation, and alterations of redox enzymes with the most significant effect observed after prolonged reperfusion. Increased prevalence of hyperhomocysteinemia in the Western population and crucial role of elevated Hcy level in the pathogenesis of neuronal disorders makes this amino acid as an interesting target for future research. Understanding the multiple etiological mechanisms and recognition of the co-morbid risk factors that lead to the ischemic/reperfusion injury and ischemic tolerance is therefore important for developing therapeutic strategies in human brain stroke associated with the elevated level of Hcy.

Role of Homocysteine in the Ischemic Stroke and Development of Ischemic Tolerance.

Homocysteine (Hcy) is a toxic, sulfur-containing intermediate of methionine metabolism. Hyperhomocysteinemia (hHcy), as a consequence of impaired Hcy metabolism or defects in crucial co-factors that participate in its recycling, is assumed as an independent human stroke risk factor. Neural cells are sensitive to prolonged hHcy treatment, because Hcy cannot be metabolized either by the transsulfuration pathway or by the folate/vitamin B12 independent remethylation pathway. Its detrimental effect after ischemia-induced damage includes accumulation of reactive oxygen species (ROS) and posttranslational modifications of proteins via homocysteinylation and thiolation. Ischemic preconditioning (IPC) is an adaptive response of the CNS to sub-lethal ischemia, which elevates tissues tolerance to subsequent ischemia. The main focus of this review is on the recent data on homocysteine metabolism and mechanisms of its neurotoxicity. In this context, the review documents an increased oxidative stress and functional modification of enzymes involved in redox balance in experimentally induced hyperhomocysteinemia. It also gives an interpretation whether hyperhomocysteinemia alone or in combination with IPC affects the ischemia-induced neurodegenerative changes as well as intracellular signaling. Studies document that hHcy alone significantly increased Fluoro-Jade C- and TUNEL-positive cell neurodegeneration in the rat hippocampus as well as in the cortex. IPC, even if combined with hHcy, could still preserve the neuronal tissue from the lethal ischemic effects. This review also describes the changes in the mitogen-activated protein kinase (MAPK) protein pathways following ischemic injury and IPC. These studies provide evidence for the interplay and tight integration between ERK and p38 MAPK signaling mechanisms in response to the hHcy and also in association of hHcy with ischemia/IPC challenge in the rat brain. Further investigations of the protective factors leading to ischemic tolerance and recognition of the co-morbid risk factors would result in development of new avenues for exploration of novel therapeutics against ischemia and stroke.

The Effect of Aging on Mitochondrial Complex I and the Extent of Oxidative Stress in the Rat Brain Cortex.

One of the characteristic features of the aging is dysfunction of mitochondria. Its role in the regulation of metabolism and apoptosis suggests a possible link between these cellular processes. This study investigates the relationship of respiratory complex I with aging-related oxidative stress in the cerebral mitochondria. Deterioration of complex I seen in senescent (26-months old) mitochondria was accompanied by decline in total thiol group content, increase of HNE and HNE-protein adducts as well as decreased content of complex I subunits, GRIM-19 and NDUFV2. On the other hand, decline of complex I might be related with the mitochondrial apoptosis through increased Bax/Bcl-2 cascade in 15-month old animal brains. Higher amount of Bcl-2, Bcl-xL with the lower content of GRIM-19 could maintain to some extent elevated oxidative stress in mitochondria as seen in the senescent group. In the cortical M1 region increased presence of TUNEL+ cells and more than 20-times higher density of Fluoro-Jade C+ cells in 26-months old was observed, suggesting significant neurodegenerative effect of aging in the neuronal cells. Our study supports a scenario in which the age-related decline of complex I might sensitize neurons to the action of death agonists, such as Bax through lipid and protein oxidative stimuli in mitochondria. Although aging is associated with oxidative stress, these changes did not increase progressively with age, as similar extent of lesions was observed in oxidative stress markers of the both aged groups.

Mechanisms involved in the ischemic tolerance in brain: effect of the homocysteine.

Hyperhomocysteinemia (hHCy) is recognized as a co-morbid risk factor of human stroke. It also aggravates the ischemia-induced injury by increased production of reactive oxygen species, and by the homocysteinylation and thiolation of functional proteins. Ischemic preconditioning represents adaptation of the CNS to sub-lethal ischemia, resulting in increased brain tolerance to subsequent ischemia. We present here an overview of recent data on the homocysteine (Hcy) metabolism and on the genetic and metabolic causes of hHCy-related neuropathologies in humans. In this context, the review documents for an increased oxidative stress and for the functional modifications of enzymes involved in the redox balance in experimentally induced hHCy. Hcy metabolism leads also to the redox imbalance and increased oxidative stress resulting in elevated lipoperoxidation and protein oxidation, the products known to be included in the neuronal degeneration. Additionally, we examine the effect of the experimental hHCy in combination with ischemic insult, and/or with the preischemic challenge on the extent of neuronal degeneration as well as the intracellular signaling and the regulation of DNA methylation. The review also highlights that identification of the effects of co-morbid factors in the mechanisms of ischemic tolerance mechanisms would lead to improved therapeutics, especially the brain tissue.

Carbonic anhydrase IX: a promising diagnostic and prognostic biomarker in breast carcinoma.

We examined the expression of carbonic anhydrase IX (CA IX) by immunohistochemical staining using monoclonal antibody M75 (Institute of Virology, Slovak Academy of Sciences, Bratislava) in a group of 38 fibroadenomas and 55 carcinomas of the breast. In each case, the intensity of staining, percentage of labeled cells and subcellular localization of CA IX were assessed. CA IX was detected in 11/38 fibroadenomas (28.9%). Weak cytoplasmic positivity was dominant in these positive cases. Immunohistochemical analysis of 55 carcinomas showed CA IX expression in 34 cases (61.8%). Membrane staining alone was observed in 27/55 carcinomas (49.1%), while cytoplasmic positivity was found in 4/55 cases (7.3%). Combined membrane and cytoplasmic immunostaining of CA IX was detected in 3/55 carcinomas (5.4%). The intensity of immunoreactivity varied from weak to strong. Under 50% of reactive cells were found in 9/38 fibroadenomas (23.6%) and in 29/55 carcinomas (52.7%). More than 50% of reactive cells were found in 2/38 fibroadenomas (5.3%) and in 5/55 carcinomas (9.1%). Statistical analysis confirmed significant differences in the subcellular localization, intensity of immunoreactivity and percentage of labeled cells in fibroadenomas and carcinomas (p<0.05). Our results confirmed the hypothesis that expression of CA IX may represent a valuable tumor biomarker as well as a promising diagnostic and prognostic parameter in breast cancer.

Survivin expression in breast lobular carcinoma: correlations with normal breast tissue and clinicomorphological parameters.

The antiapoptotic protein survivin is rarely expressed in normal adult differentiated tissues, but it is often detected in their malignant counterparts. Immunohistochemically, we evaluated survivin expression in 19 cases of normal breast tissue and 64 cases of lobular breast carcinoma. The intensity of staining, percentage of labeled cells and subcellular location of survivin were assessed. We analyzed the quantitative differences of survivin expression between normal breast tissue and carcinomas. We also correlated survivin expression pattern in carcinomas with clinicomorphological parameters such as age of patients, grade, stage and size of primary tumor, lymph node metastasis, vascular invasion as well as estrogen and progesterone status. Survivin was detected in 10/19 cases of normal breast tissue (52.6%) and in 55/64 cases of lobular breast carcinoma (86%). The statistical analysis confirmed significant correlations between the assessed parameters in normal breast and lobular carcinoma. Furthermore, the expression of estrogen correlated significantly with the subcellular localization and intensity of survivin in carcinoma. However, no significant correlation was shown with regard to other clinicomorphological parameters. Our results suggest that survivin may be a valuable diagnostic marker, as well as a new independent prognostic parameter, in lobular breast carcinoma. Finally, our data support the hypothesis that lobular and ductal breast carcinomas seem to be different clinicomorphological entities.

Intracellular signaling MAPK pathway after cerebral ischemia-reperfusion injury.

The MAPK/ERK/p38 are signal transduction pathways that couple intracellular responses to the external stimuli. Contrary to ERK protein which is part of the survival route, presence of p38 could have an impact on cell injury. Tolerance induced by ischemic preconditioning (IPC) is a phenomenon of tissue adaptation, which results in increased tolerance to lethal ischemia-reperfusion injury (IRI). Paper describes changes in MAPK protein pathways after brain IPC. Ischemia was induced by 4-vessels occlusion and rats were preconditioned by sub-lethal ischemia. Western blot and immunohistochemistry identified ERK/p38 proteins in injured areas. The highest level of the pERK was detected at 24 h in IPC groups. A contrary pattern of MAPK/p38 activation was observed in this group, where the lowest level of p38 was displayed at 24 h after ischemia. This suggests that the MAPK signal transduction might have a potential role in tissues response subjected to IRI and in the phenomenon of tolerance.