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 β-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.

Cardiac arrest is one of the leading causes of death among adults in older age. Understanding mechanisms how organism responds to ischemia at global level is essential for the prevention and ischemic patient's treatment. In this study, we used a global cerebral ischemia induced by four-vessel occlusion as an established animal model for ischemic stroke to investigate metabolic changes after 24 h reperfusion, when transitions occur due to the onset of delayed neuronal death. We also focused on the endogenous phenomenon known as ischemic tolerance by the pre-ischemic treatment. The experiments were carried out on blood plasma samples as easily available and metabolically reflecting the overall changes in injured organism. Our results imply that disturbed glycolysis pathway, as a consequence of ischemic injury, leads to the increased level of ketone bodies (acetone, acetoacetate and β-hydroxybutyrate) along with increased utilization of triacylglycerols in plasma of ischemic and ischemically preconditioned rats. Complementary to, a decreased level of glycolytic intermediates (lactate, pyruvate, acetate) with increased level of glucose was found in ischemic and preconditioned animals. The protective effect of ischemic preconditioning on metabolome recovery was demonstrated by significantly increased level of creatine compared to ischemic, non-preconditioned rats. We also document that acetoacetate, pyruvate, lactate, and leucine have the best discriminatory power between ischemic and control plasma. Conclusively, our results provide evidence that NMR spectra analysis can identify specific group of metabolites present in plasma with the capability for discrimination between individual groups of animals. In addition, an excellent feasibility for the statistical discrimination among ischemic, preconditioned, and control rats can be applied regardless of native or deproteinated plasma and also regardless of noesy or cpmg NMR acquisition.

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.

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.

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.