Mechanisms Involved in the Ischemic Tolerance in Brain: Effect of the Homocysteine

Abstract

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.

Introduction

Co-morbidities have long been known to increase risk for myocardial infarction and stroke (Lehotsky et al. 2009a; Kwon et al. 2014). A number of conventional risk factors for ischemic stroke are known, such as earlier occurrence of stroke, prior transient ischemic attack (TIA), arterial disease, atrial fibrillation, unproper diet and/or obesity and physical inactivity (Dirnagl et al. 2009). It has been reported that hyperhomocysteinemia (hHCy) may also be associated with the incidence of ischemic brain stroke, mainly due to pleiotropic activity of homocysteine (Hcy) and acceleration of atherosclerotic changes (Refsum et al. 1998; Petras et al. 2014; Steele et al. 2013; Kwon et al. 2014; Williams et al. 2014). In fact, Hcy suppresses NO production by endothelial cells and platelets, and increases generation of reactive oxygen species (ROS) by the release of arachidonic acid from platelets. It also inhibits glutathione peroxidase, and thus stimulates proliferation of endothelial cells (see Petras et al. 2014, for review). In addition, Hcy has been shown to inhibit methyltransferases, to suppress DNA reparation and to facilitate apoptosis when accumulated inside the cells. Autooxidation of Hcy metabolites results in H₂0₂ accumulation (Boldyrev et al. 2013) and long term incubation of neurons with Hcy metabolites induces necrotic cell death (Zieminska et al. 2003). Consequently, co-morbid Hcy level has been shown to be co-morbidly elevated in neurodegenerative and acute disorders of the CNS, e.g. Alzheimer´s disease or Parkinson’s disease (Dionisio et al. 2010). Thus, incorporation of animal models more consistent with the clinical population afflicted by stroke are urgently needed for proper exploration of the disease ´s etiology. In fact, only limited number of literature data can be found to describe the mutual influence of co-morbid hHCy to ischemic damage on animal models of ischemic stroke.

The brain is highly susceptible to ischemia, and numerous endogenous mechanisms exist to protect neural tissue from its effects. Although these mechanisms are naturally stimulated in response to stroke, they may also be artificially induced by non-damaging techniques to produce a protective state known as ischemic tolerance (Dirnagl et al. 2009). It represents the phenomenon of innate, an evolutionally conserved endogenous neuro-protection/plasticity which can be induced by various paradigmas/stressors in a rapid or delayed fashion against a subsequent injurious/lethal event. Preconditioning is one of the recognized neuroprotective strategy by which ischemic tolerance is induced prior to stroke. It could be potentially used as a preventative measure in a high risk individuals or as a precaution against secondary stroke following medical procedures such as aneurysm repair or cardiac surgery (Dirnagl et al. 2009; Lehotsky et al. 2009a; Thompson et al. 2013). Initial pre-clinical studies of this phenomenon relied primarily on brief periods of ischemia or hypoxia as the ischemic preconditioning (IPC) stimuli, but it was later realized that many other stressors, including pharmacological agents are also effective. Considerably more experimentation is needed to thoroughly validate the efficacy of any already identified preconditioning agent to protect ischemic brain. However, the fact that some of these agents are already clinically used, implies that the growing enthusiasm for translational success in the field of pharmacologic preconditioning may be well justified. In the case of naturally occurring human strokes which cannot be predicted, maneuver of postconditioning may be a therapeutic strategy that could be used afterward to accelerate or enhance protective mechanisms or as a precaution against stroke recurrence (Danielisova et al. 2014). Some evidence suggests, however, that, although preconditioning may be beneficial in the short term, long term structural changes in the brain indicate that tissue damage is merely being postponed (Lehotsky et al. 2009a; Thompson et al. 2013). Clinical studies are needed to test the safety and efficacy of these novel strategies in humans (Dirnagl et al. 2009). Although molecular mechanisms of IPC are not fully understood, it has been shown to influence several pathways in intracellular signaling including receptor activities, MAP, and other kinases and apoptotic mechanisms. Potential mediators of more clinically relevant postconditioning maneuver include inhibition of metalloproteinase 9 expression and subsequent suppression of the extracellular matrix degradation. Interestingly, ischemic tolerance in the brain can also be stimulated remotely, for example, by application of a tourniquet to one of the limbs. Remote preconditioning has even been shown to have beneficial effects in human patients with subarachnoid hemorrhage, but further studies are still needed (Cox-Limpens et al. 2014; Dirnagl et al. 2009; Lehotsky et al. 2009a; Thompson et al. 2013).

Only limited number of experiments can be found in the literature to describe the mutual influence of co-morbid hHCy to ischemic damage on animal models of human stroke. We summarize the recent data, and we present here an overview on the Hcy metabolism and on the genetic and the metabolic causes of hHCy-related neurotoxicity. In this context, the review also documents that combination of experimental hHCy with ischemic insult and/or with the preischemic challenge affects the extent of neuronal degeneration as well as the intracellular signaling involved in the preconditioning phenomenon.

Homocysteine Cellular Toxicity

Hcy is an intermediate sulfhydryl- containing amino acid derived from methionine with recognized toxicity to neuronal and vascular endothelial cells (Fig. 1). It originates from a dietary protein through S-adenosyl methionine conversion (Medina et al. 2001). Association between elevated Hcy and vascular diseases has been recognized in early sixties. Interestingly, patients with severe hHcy exhibit a wide range of clinical manifestations including neurological abnormalities, such as cerebral atrophy, dementia, and seizures (Obeid and Hermann 2006). Numerous epidemiological studies have recognized the association of folate deficiency and hHCy with increased risk of vascular disease and ischemic stroke (Herrmann and Obeid 2011; Petras et al. 2014).

Fig. 1.

Pathways and the mechanisms of Hcy toxicity leading to the stroke. SAM-S-adenosyl methionine, SAH-S-adenosyl Hcy, hHCy hyperhomocysteinemia. Increased dietary intake of methionine and deficiency of vitamine B₆, B₁₂, and folate leads in the predisposed individual to hHCy. Prolonged elevated level of Hcy initiates complex processes which include oxidative stress, protein homocysteinylation, and Ca²⁺ dysregulation. These events in parallel with epigenetic changes can finalize to apoptosis and blood–brain dysregulation manifested as stroke

Hcy is metabolized from the methionine by three independent alternative pathways: remethylation, transmethylation to methionine, or transsulfuration to cysteine. Though mutations or polymorphisms in the key genes of Hcy metabolism pathways have been well elucidated in stroke, emerging evidences suggested that epigenetic mechanisms, such as DNA methylation, chromatin remodeling, RNA editing, noncoding RNAs (ncRNAs), and microRNAs (miRNAs) might equally play an important role in the stroke development (Dirnagl et al. 2009; Kalani et al. 2014). Clinical studies suggest that genetic variations of genes involved in the metabolic pathways, such as methylentetrahydrofolate reductase (MTHFR), cystathionine β-synthase (CBS), DNA methyltransferase (DNMT), and nicotinamide N-methyl-transferase (NNMT) might increase the risk of stroke during hHcy. Nutritional supplements, e.g. folic acid (a co-factor in one-carbon metabolism), regulate the epigenetic alterations and may play an important role in the maintenance of neuronal integrity (Kalani et al. 2013; Obeid and Hermann 2006).

Homocysteine Metabolism

Hcy metabolism (methyl group transfer and remethylation) requires vitamin B₁₂, folic acid for N-5-methyltetrahydrofolate-Hcy methyltransferase. Additionally, the transsulfuration of Hcy depends on the vitamin B₆ (Fig. 2). In the brain, the Hcy metabolism differs from other organs. The transsulfuration pathway is not active and the remethylation pathway using betaine is absent (Smulders et al. 2006; Petras et al. 2014). Thus, the capacity for Hcy metabolism is largely dependent on the supplies of folate and cobalamin. The glial cells possess very low stores of vitamin B₁₂ that are quickly depleted during the negative balance. The toxicity of the Hcy to CNS neurons is widely recognized in affecting both the neuronal survival rate and the ability of neurons to transmit signal and thus to form functional neural networks demonstrating that it has an effect that goes beyond neuronal survival. Increased Hcy levels in humans are associated with several disorders, that affect the CNS, such as epilepsy, stroke, Alzheimer’s disease, dementia, as well as with classical homocystinuria (Seshadri et al. 2002; Kwon et al. 2014; Petras et al. 2014).

Fig. 2.

Overview of the Hcy metabolism and the role of dietary vitamins. Dietary methionine acts as a methyl donor via conversion of S-adenosyl methionine (SAM) to S-adenosyl Hcy (SAH). SAH by release of Adenosine produces Hcy. Methionine is directly converted to Hcy in the presence of methyl tetrahydrofolic acid (methyl THF) and vitamine B₆. Conversion of Hcy to cysteine requires vitamine B₆

Causes of Hyperhomocysteinemia

The reference total plasma Hcy range in humans is 5–10 µmol/l. Several types of hHcy are classified in relation to the total plasma Hcy concentration such as: moderate (for concentrations between 16 and 30 µmol/l), intermediate (for concentrations of 31–100 µmol/l), and severe (for concentrations higher than 100 µmol/l) appeared with homocystinuria (Herrmann and Obeid 2011). hHCy can be the result of perturbed Hcy metabolism in conditions such as dietary deficiencies in folic acid, vitamin B₆, and/or vitamin B₁₂ (Fig. 1). The hHCy can be caused by genetic deficiencies in methionine and Hcy metabolism, most likely by methylenetetrahydrofolate reductase (MTHFR) deficiencies. While Hcy is formed in all tissues, its detoxification occurs only in the liver/kidney through the transsulfuration pathway. So, in other tissues such as the blood vessels and the brain, remethylation is the only alternative available. With significant reduction in MTHFR activity Hcy cannot be remethylated to methionine, hence accumulates within the nervous system. Interestingly, the role of the MTHFR C677T polymorphism as a risk factor for ischemic stroke has been established in different laboratories and also in different genetic cohorts (Kim et al. 2013; Li and Qin 2014).

Neurotoxicity of Homocysteine

The neurotoxicity of Hcy involves several etiopathogenic mechanisms, including Hcy induced glutamate receptor mediated neurotoxicity (da Cunha et al. 2012; Kwon et al. 2014). Notably, glutamatergic excitotoxicity is also associated with the brain damage caused by ischemic insult. Hcy was shown as an inductor of caspase-dependent neuronal apoptosis, by a mechanism involving DNA damage, poly-ADP-ribose polymerase (PARP), and mitochondrial dysfunction by caspase-3 activation. Hcy appears to be also critical in the glial–vascular interface as part of the blood–brain barrier. The importance of astrocytes in the regulation of brain metabolism and in particular in the brain energy metabolism has been documented (Verkhratsky and Toescu 2006). Therefore, increased level of the Hcy leads to an enhanced excitatory glutamatergic neurotransmission in different brain areas, whereby neuronal damage derives from excessive Ca²⁺ influx and reactive oxygen generation.

In fact, dysregulation in redox balance and oxidative stress have been suggested as a primary mechanism responsible for hHcy-related pathogenesis (Herrmann and Obeid 2011; Petras et al. 2014). Several sets of studies showed that dysequilibrium in redox reactions may be a key factor in the development of vascular hypertrophy, thrombosis, and atherosclerosis in hyperhomocysteinemic animals (Dayal et al. 2004; Markovic et al. 2011). ROS are generated during oxidation of the free thiol group of Hcy, when Hcy binds via a disulphide bridge with plasma proteins—mainly albumin—or with other low-molecular plasma thiols, or with a second Hcy molecule. Several mechanisms have been proposed for Hcy induced oxidative stress. They include: (i) inhibition of the activity of cellular antioxidant enzymes, (ii) Hcy autooxidation, (iii) nitric oxide synthase (NOS)-dependent generation of superoxide anion via uncoupling of endothelial NOS (eNOS), (iv) disruption of extracellular superoxide dismutase from endothelial surfaces, and (v) activation of NADPH oxidases. Interestingly, concomitant formation of strong oxidant peroxynitrite leads to tyrosine nitration which causes the alteration in protein function and induces cellular dysfunction (Postea et al. 2006). Autooxidation of Hcy metabolites results in H₂0₂ accumulation, and long term incubation of neurons with Hcy metabolites induces necrotic cell death (Zieminska et al. 2003; Boldyrev et al. 2013).

Hcy is also metabolized to the thioester Hcy-thiolactone in an error-editing reaction in protein biosynthesis when Hcy is erroneously selected in place of methionine by methionyl-tRNA-synthetase. Human and experimental studies have shown that Hcy-thiolactone contributes to Hcy patho-biology, which is caused by protein N-homocysteinylation through the formation of amine bonds with protein lysine residues (Jakubowski 2011) which impairs or alters the structure and function. Interestingly, temporal lobe epilepsy as the most common type of epilepsy in adult is usually associated with a poor response to antiepileptic drugs. Several clinical studies have reported that increased plasma Hcy levels may provoke seizures. In agreement with this finding it was suggested that systemic administration of Hcy at high doses is able to induce convulsions in mice and it can be suggested that similar detrimental effects might occur in patients affected by temporal lobe epilepsy (Baldelli et al. 2010). Hcy-derived chemically reactive metabolites are suggested to play an important role in Hcy–induced seizures.

The series of papers from this and other laboratories (Danielisová et al. 2007; Urban et al. 2009; Lehotsky et al. 2009b; Pavlíková et al. 2009) found that ischemia/reperfusion injury (IRI) in rats initiates time dependent dysregulation of redox balance in cortex and hippocampus. The insult also leads to the differences in gene expression at both the mRNA and protein levels. In addition, studies documented that the redox balance and gene expression is affected by preconditioned preischemic treatment (Stetler et al. 2014). However, the literature data on describing the mutual influence of IRI and hHCy as known risk factor to ischemic damage are very limited (Kwon et al. 2014). Results of Petras et al. (2014) which describe the effect of chronic dietary supplementation of Hcy for 2 weeks (to initiate hHCy) show the induction of significant increase of lipoperoxidative and protein oxidative products in rat hippocampus. Experimental hHCy was induced by subcutaneous administration of Hcy in saline solution (0,45 µmol/g body weight) twice a day at 8 h interval for 14 days (Pavlikova et al. 2011; Petras et al. 2014). It is well know that Hcy crosses the blood/brain barrier and presents a peak in the cerebrum and parietal cortex between 15 and 60 min after subcutaneous injection. Plasma Hcy concentration in rats treated this way achieved levels similar to those found in homocystinuric patients (moderate hHCy). Groups of rats were classified as follows (Pavlikova et al. 2011; Petras et al. 2014): (1) sham-operated control (naive) animals, (2) sham-operated control (preconditioning) animals, (3) the animals that underwent 15 min ischemia (naive), (4) the animals with induced 5 min IPC following 15 min ischemia, (5) sham-operated hyperhomocysteinemic control animals, (6) the hyperhomocysteinemic animals that underwent 15 min ischemia, and (7) the hyperhomocysteinemic animals with induced preconditioning animals following 15 min ischemia. Experiments clearly show that hHCy induces significant increase of lipoperoxidative and protein oxidative products, the results which correlate well with previously published studies (da Cunha et al. 2012). Moreover, as detected by the numbers of Fluro Jade B positive- and TUNEL positive cells (as indicator of neuronal degeneration) (Lehotský et al. 2011, 2014; Kovalska et al. 2012; Pavlikova et al. 2011), the proportion of degenerated cells over intact neurons clearly increases in hippocampus of hyperhomocysteinemic animals and almost competes with the levels reached after ischemic insult in non-treated, naive group. Autooxidation of Hcy metabolites results in H₂0₂ accumulation, and long term incubation of neurons with Hcy metabolites induces necrotic cell death (Zieminska et al. 2003; Boldyrev et al. 2013). Likewise as was shown by Ataie et al. (2013), the intracerebroventricular injection of Hcy induces typical apoptotic appearance in the substantia nigra cells which is followed by the Parkinson’s disease-like behavior in rats. The concomitantly Hcy induced accumulated ROS may form hydroxyl radicals as the most potent, powerful free radical with the ability to remove electrons from other molecules in the most cellular components including lipids, proteins, carbohydrates, and DNA (Kolling et al. 2011). Thus, as seen from previous and also from our results using hyperhomocysteinic model rats (Lehotský et al. 2011, 2014; Murín et al. 2014; Pavlikova et al. 2011; Petras et al. 2014), the elevated level of Hcy manifests significant neurotoxic effect. This effect is probably due to hHCy- induced oxidative imbalance and cellular stress. Interestingly, the plasma Hcy level is significantly associated with alterations of hippocampal volume as was detected in human study on Alzheimer’s patients and patients with mild cognitive impairment. This has a direct adverse effect which is not mediated by cerebral beta amyloid deposition and vascular burden, but likely to oxidative imbalance (Choe et al. 2014; Kwon et al. 2014).

In another study on hyperhomocysteinic model in rats, Pavlikova et al. (2011) have documented remarkable differences in the level of mRNA expression for calcium pump in secretory pathways (SPCA1) which were followed also on protein level. This protein plays a pivotal role in normal neural development and migration (Sepulveda et al. 2008) and SPCA1 deficiency leads to stress of Golgi apparatus presented with membranous structural changes and redox imbalance in neurons. There are no literature data on how the Hcy might affect the expression profile of the Ca²⁺-transport proteins in neuronal cells. In fact, the general mechanism of transcriptional regulation of SPCA1 gene is not yet fully understood. The transcription factors Sp1 and YY1 were shown to be involved in the gene regulation by the cis-enhancing elements in 5′-untranslated regions (Kawada et al. 2005). hHCy often results in intracellular Ca²⁺ mobilization, and endoplasmic reticulum (ER) stress (Kalani et al. 2013; Petras et al. 2014), which often follow with the subsequent development of apoptotic events, endothelial dysfunction and remodeling of the extracellular matrix also in brain parenchyma. Interestingly, Hcy itself by metabolic interfering with the level of S-adenosylmethionine (donor of methyl group) has also been reported to induce modulation of gene expression through alteration of the methylation status (Dionisio et al. 2010).

Notably, another etiopathogenic process, such as protein structure modifications, has been detected which might lead to Hcy induced neurotoxity. Two main types of homocysteinylation have been indicated: S-homocysteinylation and N-homocysteinylation, both of which are considered as post-translational protein modifications. The degree of the protein homocysteinylation increases with increased plasma Hcy level (Kolling et al. 2011), and the conversion of Hcy to Hcy-thiolactone followed by protein N-homocysteinylation largely contributes to manifestations of Hcy toxicity. In fact, homocysteinylation can cause functional protein modification and even enhanced protein degradation leading to cell damage (Jakubowski 2004). In this context, Petras et al. (2014) observed the 57.9 % decrease of Mn²⁺ activated superoxide dismutase (Mn-SOD) activity in cortical mitochondria in the 14 days hHCy model in rats. This catalytic activity is included in the first line of cellular defense against oxidative injury and has been shown suppressed in the hHcy group (9.34 ± 1.901 U/mg proteins) compared to the control group (22.186 ± 4.017 U/mg proteins). On the other hand, using Western blot and immunohistochemical analysis, authors determined the 13.6 % increase in the SOD protein level in hHcy group compared to the controls group. These results might be explained by an increased post-translational modifications of Mn-SOD due to higher level of HCy, which likely by the enzyme homocysteinylation and thiolation might contribute to its inactivation. In the same study (Petras et al. 2014), authors detected a 12.46 % increase of catalase (CAT) activity in the hHcy group (180.068 ± 3.57 nmol/min/mg proteins) compared to the control group (157.62 ± 1.14 nmol/min/mg proteins) likely as a response to the increased level of ROS. As was shown recently by Li et al. (2014), the imbalance among antioxidant enzymes caused by hHcy might alter ROS elimination, and thus lead to the increasing amount of free radicals and likely to Hcy inducible endoplasmic reticular stress which can finalize to the functional consequences in rat hippocampus.

Effect of hHCy on Ischemic/Reperfusion Injury and Ischemic Tolerance Induced by IPC

Apart from the clearly recognized clinical effect of co-morbidity to the stroke incidence and severity, the literature data which deals with the experimental approach to study mutual influence of co-morbid hHCy to ischemic damage on animal models of human stroke is very sparse (Sato et al. 1998; Thompson et al. 2013; Stetler et al. 2014). Ischemic insult/reperfusion insult is generally linked with the degeneration of majority (more than 64 %) of hippocampal neurons (Dirnagl et al. 2009; Kovalska et al. 2012). As has been shown in experiments (Pavlikova et al. 2011; Lehotský et al. 2011; Kovalska et al. 2012), which combine 14 days hHCy with 15 min forebrain ischemia/reperfusion, the hippocampal area manifested morphologically changed neurons and disturbances of glial cells. However, this maneuver promoted the percentage of morphologically intact and probably non-degenerated cells to larger extent in comparison to the naive ischemic/reperfusion insult. Interestingly, Sato et al. (1998) documented a dose dependent protection of hippocampal CA1 neurons by S-adenosyl-l-methionine (SAMe) after transient forebrain ischemia in rats. The effect was suppressed by simultaneous administration of S-adenosyl-l-Hcy, a potent inhibitor in transmethylation. Authors suggested that it is necessary for the prevention of delayed neuronal death to enhance cerebral SAMe level and to activate transmethylation using SAMe as a methyl donor in postischemic brain.

The preischemic maneuver is known to rescue the majority of hippocampal neurons to the extent of more than 75 % of all neurons (Lehotsky et al. 2009a; Kovalska et al. 2012). If we combine 14 days hHCy with preconditioning (Pavlikova et al. 2011; Lehotský et al. 2011, 2014, Kovalska et al. 2012; Murín et al. 2014; Petras et al. 2014), this maneuver leads to the larger suppression of cell degeneration to the extent not exceeding 5 % of the total number of neurons. It apparently seems that at least in this hyperhomocysteinic model in rats, the protective effect of IPC stimulus is corroborated by till now non-understood mechanism. Interestingly, a novel epigenetic mechanism has been described which regulates gene expression and also encompasses gene environment interactions and functional networks. The emerging evidences show that several epigenetic mechanisms are also involved in stroke pathogenesis and tolerance ethiology. One of the critical enzyme regulating DNA methylation is DNA-N-methyl transferase and high level of Hcy leads to increase in the level of S-adenosyl methionine. Consequent higher activity of this enzyme might cause hypermethylation of the genes and silencing of functional genes (Kalani et al. 2013).

As shown earlier, the preischemic challenge affords not only preservation of majority of hippocampal neurons but it also activates recovery of the rate of SPCA1 mRNA and protein expression in rats (Pavlíková et al. 2009; Stetler et al. 2014). Interestingly, hyperhomocysteinemic animals manifested suppression of the SPCA mRNA expression and this suppression was de-repressed by the IPC maneuver by 2.5 times. In fact, the literature data on how Hcy might affect the expression profile of the Ca²⁺-transport proteins in neuronal cells are very sparse. One can only speculate whether this process is somehow associated with the disturbed Ca²⁺ homeostasis which is initiated and follows ischemic insult (Dirnagl et al. 2009; Kalani et al. 2013). The mechanism of transcriptional regulation of Ca²⁺ transporting genes in neuronal cells is not yet fully understood. However, it should be noted in this context, that the one-carbon unit metabolism pathway which is involved in the regulation of Hcy is also part of the process which methylates functional proteins, histones, RNA, and DNA. To this end, demethylation of S-adenosyl methionine which gives rise to S-adenosyl-Hcy, is the sole source of de novo methyl groups for the cells. Dysregulation of this step might have a broad implication on many cellular processes including modulation of functional gene expression as well as the epigenetic regulation (Dionisio et al. 2010; Kalani et al. 2013, 2014). Another pathophysiologicaly relevant aspect which arises from the effect of hHCy is that human body evolves the ability to eliminate one of the metabolites of Hcy, Hcy-thiolactone. A high density lipoprotein (HDL)—associated enzyme, Hcy—thiolactonase/paraoxonase 1 (PON1) is able to hydrolyze this toxic metabolite (Hcy-thiolactone) in human serum (Domagala et al. 2006). It has been suggested that PON1 protects mice against Hcy-thiolactone neurotoxicity by hydrolyzing it also in the brain (Borowczyk et al. 2012).

Taken together, documented responses of neuronal cells to preischemic challenge in 14 days hyperhomocysteinemic model in rats (Fig. 3) might suggest for the correlation of several ethiological factors such as antioxidant defense (Borowczyk et al. 2012), as well as the mechanisms of Ca²⁺ transport and epigenetic mechanisms, such as DNA methylation and chromatin remodeling in the phenomenon of ischemic damage and ischemic tolerance (Dirnagl et al. 2009; Lehotsky et al. 2009a; Kalani et al. 2013; Thompson et al. 2013; Stetler et al. 2014).

Fig. 3.

Mechanisms leading to the Hcy neurotoxicity and the protection induced by IPC in hyperhomocysteinemic conditions. (+): increased number of cells (activity), (−): decreased number of cells (activity). Hcy induced neurotoxicity includes dysregulation in redox balance, lipo- and protein oxidation and Ca²⁺ pump dysfunction which is detected in sensitive cells by increased Fluoro-Jade B staining and TUNEL analysis. IPC suppresses oxidative dysregulation which leads to lower Fluoro-Jade B and TUNEL positivities in sensitive cells

Conclusion and Challenges

Elevated level of Hcy is now recognized as a risk factor of human ischemic stroke. Plasma hHCy leads to an increase in cerebrovascular permeability and causes thiolation and homocysteinylation to proteins and enzymes also in the brain parenchyma. As a consequence, these post-translational modifications affect the function and activity of enzymes involved in the free radical protection, such as SOD, catalase or glutathione peroxidase. Hcy metabolism leads also to the redox imbalance and to increased oxidative stress. Altogether, this finalizes to the elevated neuronal lipoperoxidation and cellular protein oxidation, all the products clearly recognized in the process of brain damage. This paper also highlights the neurotoxic effect of 14 days experimental hHCy and the protective effect of preischemic challenge in rats. This maneuver itself and also combined with hHCy can affect the extent of neuronal degeneration as well as the intracellular signaling and epigenetic regulation. It is our hope 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.