Possible role of increased brain methylation in methionine sulfoximine epileptogenesis: effects of administration of adenosine and homocysteine thiolactone

An intraventricular pulse of [14COOH]L-methionine to mice pretreated with the convulsant L-methionine-dl-sulfoximine (MSO) resulted in significantly higher than control specific radioactivity values of cerebral [14COOH]L-methionine (Met), [14COOH]S-adenosyl-L-methionine (AdoMet) and [14COOH]S-adenosyl-L-homocysteine (AdoHcy). MSO administration (3 hr) also decreased brain steady-state levels of Met, AdoMet, and AdoHcy. Following an intraventricular pulse of [3H-methyl]L-methionine, the levels of [3H-methyl]phosphatidylmonomethylethanolamine and of membrane associated and soluble [3H-methyl]carboxylmethylated proteins were increased over corresponding saline-treated controls. The activity of cerebral histamine N-methyltransferase was also increased after MSO treatment. The administration of a combination of adenosine and homocysteine thiolactone to MSO-pretreated animals counteracted the MSO-induced decreases in brain Met, AdoMet, and AdoHcy as well as the increase in histamine N-methyltransferase activity. In addition, administration of adenosine together with homocysteine thiolactone decreased the incidence of, and increased the latency to MSO seizures, with the most effective anticonvulsant action occurring when cerebral AdoHcy levels were at their highest.     

The use of different experimental models of hyperhomocysteinemia in neurochemical studies

We reviewed experimental models in which an increase in the content of homocysteine in rat blood was achieved by chronic (1 month) consumption of methionine with drinking water (1 g/kg weight) or forced oral injection of methionine solution (0.1–0.15 g in a small volume of water). The effectiveness of the model of hyperhomocysteinemia was evaluated using the homocysteine content and intensity of lipid peroxidation in the blood serum. It was found that strictly dosed consumption of methionine during its forced oral administration was not accompanied by side effects (dehydration or pediculosis) and also results in less substantial, as compared to consumption with drinking water, deviations in the blood content of homocysteine. One of the advantages of this experimental model is the clear changes in the content of catecholamines in the rat hypothalamus.     

Effects of glutamate and sulphur containing amino acids on ammonia toxicity and methionine sulphoximine convulsions in mice.

Arginine and large doses of glutamate (greater than 500 mg/kg) were found to reduce ammonia toxicity transiently in mice. Smaller doses of glutamate (greater than or equal to 200 mg/kg) were effective when administered with glucose. Cysteic acid, homocysteine and methionine, but not taurine reduced ammonia toxicity. All 4 amino acids reduced the number of convulsions induced by methionine sulphoximine. It is proposed that taurine has a general anticonvulsant action and that cysteic acid, homocysteine and methionine may have a specific effect on the action of ammonia on the central nervous system.     

48-hour sleep-wake cycles in manic-depressive illness: naturalistic observations and sleep deprivation experiments

Wrist motor activity and sleep were monitored longitudinally in 15 rapidly cycling and 52 nonrapidly cycling manic-depressive patients. The majority of patients experienced one or more consecutive 48-hour sleep-wake cycles (alternate nights with no sleep) when they switched out of depression into mania of hypomania. During a depressive phase, nine rapidly cycling patients were asked to simulate a 48-hour sleep-wake cycle by remaining awake for 40 hours (one night's total sleep deprivation). Eight switched out of depression, and seven were rated as manic or hypomanic; indicating that sleep loss (such as occurs with spontaneous 48-hour sleep-wake cycles) may help to trigger switches from depression to mania. The 48-hour sleep-wake cycles in patients may depend on a mechanism that is normally present in all humans, since normal persons also spontaneously experience near-48 hour sleep-wake cycles in certain experimental conditions.     

Amino acid uptake in vivo by the mouse brain and by various regions of the rabbit brain after drug-induced convulsions.

The levels of the free amino acids of the mouse brain were studied after convulsions induced by Metrazol; a decrease of level of taurine and a significant increase of level of alanine, phenylalanine and isoleucine were found. The net uptake of L-histidine by the mouse brain in vivo was similar under normal conditions and after Metrazol-induced generalized convulsions; that of L-methionine was much higher after convulsions and there was no uptake of L-aspartic acid, either under physiological conditions or after convulsions. The net uptake of L-histidine by various regions of the rabbit brain in vivo after convulsions was significantly higher than control values in the cortical areas, while that of L-methionine was significantly higher in the subcortical ones; there was no uptake of L-aspartic acid by the rabbit brain in normal condition, whereas after convulsions a small entry seemed to occur in the subcortical areas. These results indicate that cerebral permeability processes of amino acids are somewhat altered during convulsive phenomena, as already described elsewhere for ions andproteins.     

Sleep disturbance induced by substance P in mice

Substance P (SP) and neurokinins have been implicated in modulating pain and mood but little is known about their effect on sleep-wake behavior. The purpose of the present study was to examine the possible involvement of SP in sleep-wake mechanisms without activation of painful responses. Electrophysiological recordings of the sleep-wake cycle were conducted in C57BL/6J male mice that had intracerebral ventricular cannula inserted for drug administration. Initially, in order to determine the highest dose of SP that would not induce nociceptive response, 10 animals per group received administration of either SP doses or artificial cerebrospinal fluid (CSF-sham group) through the cannula and were assessed by the hot plate test. The sleep-wake cycle of two other groups of mice was recorded for 24 h before (baseline) and after receiving CSF (n=10) or SP-1 mM (n=11), dose that had been determined in the previous hot plate test. SP interfered with sleep, when compared to baseline and to sham group, by reducing sleep efficiency, increasing latency of sleep and the number of awakening bouts. To examine the reversal of SP effects, eight mice were administered with an NK1 receptor antagonist before SP administration. Prior administration of the NK1 antagonist prevented the disturbances in sleep. CONCLUSIONS: The results suggest that SP produces disturbances in sleep, likely mediated by the NK1 receptor.     

Restoring the Molecular Clockwork within the Suprachiasmatic Hypothalamus of an Otherwise Clockless Mouse Enables Circadian Phasing and Stabilization of Sleep-Wake Cycles and Reverses Memory Deficits

The timing and quality of sleep-wake cycles are regulated by interacting circadian and homeostatic mechanisms. Although the suprachiasmatic nucleus (SCN) is the principal clock, circadian clocks are active across the brain and the respective sleep-regulatory roles of SCN and local clocks are unclear. To determine the specific contribution(s) of the SCN, we used virally mediated genetic complementation, expressing Cryptochrome1 (Cry1) to establish circadian molecular competence in the suprachiasmatic hypothalamus of globally clockless, arrhythmic male Cry1/Cry2-null mice. Under free-running conditions, the rest/activity behavior of Cry1/Cry2-null controls expressing EGFP (SCN^Con) was arrhythmic, whereas Cry1-complemented mice (SCN^Cry1) had coherent circadian behavior, comparable to that of Cry1,2-competent wild types (WTs). In SCN^Con mice, sleep-wakefulness, assessed by electroencephalography (EEG)/electromyography (EMG), lacked circadian organization. In SCN^Cry1 mice, however, it matched WTs, with consolidated vigilance states [wake, rapid eye movement sleep (REMS) and non-REMS (NREMS)] and rhythms in NREMS δ power and expression of REMS within total sleep (TS). Wakefulness in SCN^Con mice was more fragmented than in WTs, with more wake-NREMS-wake transitions. This disruption was reversed in SCN^Cry1 mice. Following sleep deprivation (SD), all mice showed a homeostatic increase in NREMS δ power, although the SCN^Con mice had reduced NREMS during the inactive (light) phase of recovery. In contrast, the dynamics of homeostatic responses in the SCN^Cry1 mice were comparable to WTs. Finally, SCN^Con mice exhibited poor sleep-dependent memory but this was corrected in SCN^Cry1mice. In clockless mice, circadian molecular competence focused solely on the SCN rescued the architecture and consolidation of sleep-wake and sleep-dependent memory, highlighting its dominant role in timing sleep.SIGNIFICANCE STATEMENT The circadian timing system regulates sleep-wake cycles. The hypothalamic suprachiasmatic nucleus (SCN) is the principal circadian clock, but the presence of multiple local brain and peripheral clocks mean the respective roles of SCN and other clocks in regulating sleep are unclear. We therefore used virally mediated genetic complementation to restore molecular circadian functions in the suprachiasmatic hypothalamus, focusing on the SCN, in otherwise genetically clockless, arrhythmic mice. This initiated circadian activity-rest cycles, and circadian sleep-wake cycles, circadian patterning to the intensity of non-rapid eye movement sleep (NREMS) and circadian control of REMS as a proportion of total sleep (TS). Consolidation of sleep-wake established normal dynamics of sleep homeostasis and enhanced sleep-dependent memory. Thus, the suprachiasmatic hypothalamus, alone, can direct circadian regulation of sleep-wake.     

同型半胱氨酸与糖尿病周围神经病变的关系

目的：了解血浆同型半胱氨酸(Hcy)与2型糖尿病周围神经病变间的关系及叶酸对Hcy水平的影响和对糖尿病周围神经病变的防治作用。方法：24只糖尿病KKAy小鼠分为3组：糖尿病诱发饮食组(KA组)；高蛋氨酸饮食组(KB组)；喂养高蛋氨酸饮食的基础上加用叶酸组(Kc组)。16只C57小鼠为正常对照组，分为2组：糖尿病诱发饮食组(CA组)；高蛋氨酸饮食组(CB组)。测定血糖、体重、血浆HCY、叶酸浓度，并观察光镜下及电镜下坐骨神经结构变化。结果：糖尿病小鼠喂饲蛋氨酸后出现高Hcy血症，对照组无此改变。糖尿病小鼠的坐骨神经出现不同程度的缺血性改变，且与Hcy水平相关。叶酸可减轻坐骨神经损害的程度。结论：高同型半胱氨酸为糖尿病微血管病变的危险因素，叶酸可起到干预作用。     

No added value of the methionine loading task in assessment for venous thrombosis and cardiovascular disease risk

Homocysteine is a risk factor for cardiovascular disease and venous thrombosis. Clinical guidelines differ in their recommendation whether or not to measure homocysteine after methionine loading. In this study, we investigated the added value of the methionine loading test next to fasting homocysteine levels for identifying subjects at risk for venous thrombosis or cardiovascular disease, using Receiver Operating Characteristic (ROC) curves.The analysis was performed in 185 patients with recurrent venous thrombosis, 130 patients with cardiovascular disease and 601 controls.The discriminatory power of the fasting homocysteine measurement alone for identifying subjects at risk of venous thrombosis expressed as the area under the ROC curve (AUC) was 0.61 (95%CI 0.56-0.66). Using both a fasting homocysteine measurement and a methionine loading test together yielded a similar AUC of 0.65 (95%CI 0.60-0.69), indicating no added value of methionine loading next to fasting homocysteine measurement in identifying subjects at risk for thrombosis. Similar results where found for cardiovascular disease,with an AUC of 0.62 (95%CI 0.57-0.67) for the fasting homocysteine measurement alone and an AUC of 0.62 (95% CI0.57-0.67) for the combination of both the fasting and the post-load homocysteine measurement. The methionine loading test has no added value next to measuring fasting homocysteine levels for identifying subjects at risk for venous thrombosis or cardiovascular disease and for that reason should not be used in clinical practice.     

Low fasting methionine concentration as a novel risk factor for recurrent venous thrombosis

Hyperhomocysteinemia is a risk factor for venous thrombosis, but the underlying mechanism is unclear. If the thiol-group of homocysteine interferes with components of the clotting system, we expect that high cysteine will be also a risk factor for venous thrombosis. If high homocysteine reflects a disturbed methyl-group donation by S-adenosylmethionine, we expect that low methionine will be a risk factor for thrombosis. We performed a case-control study in 185 patients with recurrent venous thrombosis and in 500 control subjects. We determined methionine, homocysteine, cysteine and assessed the associated thrombotic risk. Low fasting methionine was associated with an increased risk on recurrent venous thrombosis [OR(bottom vs. top quartile) = 3.3 (95%CI 1.9-5.7)]. Low methionine remained a risk factor [OR(bottom vs. top quartile) = 3.5 (95%CI 2.0-6.0)] after adjusting for homocysteine and cysteine, whereas the thrombotic risk for homocysteine was lost [OR = 1.0 (95%CI 0.6-1.9)] after adjustment. Cysteine yielded a highest odds ratio of 2.1(top vs. bottom quartile) (95%CI 1.0-4.0) after adjustment. In conclusion, we found that low fasting methionine is a risk factor for recurrent venous thrombosis. This risk association was stronger for methionine than for homocysteine or cysteine. This supports the hypothesis that impaired methylation may be involved in the pathogenesis of venous thrombosis.     

The effect of methylphenidate on the sleep-wake cycle of brain-injured patients undergoing rehabilitation

BACKGROUND AND PURPOSE: A number of neuro-stimulants are routinely used as part of post-acute care of hospitalized brain-injured patients. To our knowledge, the effect of these stimulants on the sleep-wake cycles of brain-injured patients undergoing rehabilitation has not been addressed. We examined the effect of one of the most commonly used neuro-stimulants, methylphenidate, on the sleep-wake behavior of brain-injured patients undergoing rehabilitation at a dedicated brain injury clinic. PATIENTS AND METHOD: For this study, records of patients admitted between January and December 1999 were scrutinized retrospectively for the data on observationally defined sleep-wake distribution. A total of 30 patients diagnosed with traumatic brain injury were identified as having been observed for a full 24h a day for at least 10 days. Some of these patients (n=17) were administered methylphenidate on clinical grounds. They served as the experimental group, while the unmedicated patients (n=13) served as controls. For the present analysis, the sleep-wake cycles were arbitrarily designated as nighttime and daytime, respectively. A cumulative sleep-wake quantity in a 24-h period was also observed. RESULT: The average number of hours of sleep during a 24-h period was not significantly different for the two cohorts. Similar trends emerged for the nighttime and daytime observations. On the whole, methylphenidate appears not to have unfavorable effects on sleep-wake cycles, presently defined as nighttime, daytime and 24-h, in the traumatic brain injury population. CONCLUSION: This study sought to gain better understanding of the effect of methylphenidate on daytime sleepiness and nighttime sleep, and the data suggest that administration of methylphenidate does not appear to have an adverse effect on sleep-wake quantity.     

Effect of the intracerebroventricular and systemic administration of L-serine on the concentrations of D- and L-serine in several brain areas and periphery of rat

To gain further insight into the metabolic mechanism of endogenous D-serine, the effect of the intracerebroventricular and intraperitoneal administration of L-serine on the concentrations of D- and L-serine in several brain areas and periphery was investigated. The intracerebroventricular injection of L-serine caused a rapid and marked increase in the L-serine levels in almost all brain regions of adult rats. This administration also produced a gradual increase in the D-serine levels in the forebrain, whereas a slight but significant elevation of D-serine was found in the cerebellum and pons-medulla. The intraperitoneal administration of L-serine caused a marked increase in the L-serine levels in all brain regions of both infant and adult rats. The treatment induced a significant augmentation of the D-serine levels in all brain regions of infant rats with higher concentrations in the cerebellum and cortex, whereas no significant change was observed in the cerebellum and pons-medulla of adult rats. These in vivo observation, together with the fact that immunohistochemical studies have indicated that both D-serine and serine racemase are highly concentrated in Bergmann glia of developing cerebellum, suggest that D-serine can be synthesized not only in the forebrain but also in the hindbrain by serine racemase. Furthermore, because the drastic decline in the cerebellar D-serine level coincides well with a dramatic increase in the cerebellar D-amino acid oxidase during early postnatal development, synthesized D-serine may be metabolized by D-amino acid oxidase in the hindbrain of adult rats.     

Homocysteine and thrombosis: from basic science to clinical evidence

Homocysteine is a sulfhydryl-containing amino acid formed during the metabolism of methionine. Rapidly accumulating evidence links elevated homocysteine levels to thrombosis via several mechanisms such as increased tissue factor expression, attenuated anticoagulant processes, enhanced platelet reactivity, increased thrombin generation, augmented factor V activity, impaired fibrinolytic potential, and vascular injury, including endothelial dysfunction. Molecular mechanisms underlying prothrombotic actions of homocysteine are incompletely understood and involve oxidative stress, DNA hypomethylation, and proinflammatory effects. Current evidence from retrospective and prospective studies supports the concept that higher total plasma homocysteine concentration is associated with increased risk of coronary artery disease, stroke, and venous thromboembolism. Hyperhomocysteinemia is currently considered a relatively weak prothrombotic factor. It is still unclear whether administration of vitamins, that reduce homocysteine levels acting as cofactors of the enzymes involved in the methionine metabolism, may decrease the risk of arterial and/or venous thromboembolic events. Ongoing clinical trials might help clarify this issue.     

Methylation status in females with rett syndrome

The authors studied methionine and creatine metabolism in females with Rett syndrome. Plasma metabolites (including methionine, homocysteine, guanidinoacetate) and urine creatine/creatinine ratios in 29 females with Rett syndrome were within the age-appropriate range. Although the authors have not been able to identify any abnormalities, it can be speculated that patients with Rett syndrome may benefit from dietary intervention to increase the supply of labile methyl groups to affected tissues.     

The novel brain neuropeptide, orexin-A, modulates the sleep-wake cycle of rats

Orexin-A is a novel neuropeptide initially isolated from hypothalamic extracts but now known to be present in fibres distributed throughout the rat CNS including many regions associated with sleep-wake regulation. The recognition of a particularly dense innervation of orexinergic nerves in the locus coeruleus, together with the observed increase in firing rate of locus coeruleus neurons following application of orexin-A in vitro, further highlighted a potential involvement of the peptide in modulating the arousal state. The present study was undertaken to determine the effects of intracerebroventricularly (ICV) administered orexin-A on the sleep-wake cycle of conscious rats using electroencephalographic and electromyographic recordings. When administered at the onset of the normal sleep period, orexin-A (1, 10 or 30 microg/rat ICV) produced a dose-dependent increase in the time rats spent awake during the second and third hours after dosing. The enhancement of arousal was accompanied by a marked reduction in paradoxical sleep and deep slow wave sleep at the highest dose. The latency to the first occurrence of paradoxical sleep was also prolonged. This overall profile of increased arousal and decreased paradoxical sleep is consistent with a high rate of firing of locus coeruleus neurons as would be expected to occur following ICV administration of orexin-A. It is concluded that orexin-A may play an important physiological role in regulating the sleep-wake state, a hypothesis considerably strengthened by the recently reported narcoleptic phenotype of prepro-orexin (the precursor for orexin-A) knockout mice.     

Cocaine exposure during adolescence affects anxiety in adult mice

Psychostimulant drugs such as cocaine have profound and long-lasting neurobiological effects, which may affect anxiety or social behaviors. These actions could be greater when cocaine is administered during a developmental period such as adolescence. The present work attempts to further clarify the long-lasting effects of cocaine administration on mice, examining three major variables: age; pattern of drug administration; and housing conditions. Adolescent (postnatal day 26) or early adult mice (postnatal day 46) were exposed to a daily or binge cocaine administration and 15 days later their behavior was evaluated, the mice being housed either in isolation or in groups during this stage. After a period free of drug, the behaviors evaluated were: spontaneous and cocaine-induced motor activity; anxiety, using the elevated plus maze; the social profile, assessed in a social interaction test. Daily cocaine administration increased avoidance and flee in isolated adolescent-treated mice and decreased social contacts in those which were grouped. On the other hand, the binge pattern modified the anxiety of the grouped adolescent-treated mice evidenced by the increase in time spent on the open arms of the plus maze. An increase in spontaneous and cocaine-induced motor activity was shown in animals after a daily pre-treatment. The results are discussed in terms of presenting cocaine-induced behavioral changes within a specific temporal window and depending on the three variables studied.     

Sex-specific hippocampal metabolic signatures at the onset of systemic inflammation with lipopolysaccharide in the APPswe/PS1dE9 mouse model of Alzheimer’s disease

Systemic inflammation enhances the risk and progression of Alzheimer’s disease (AD). Lipopolysaccharide (LPS), a potent pro-inflammatory endotoxin produced by the gut, is found in excess levels in AD where it associates with neurological hallmarks of pathology. Sex differences in susceptibility to inflammation and AD progression have been reported, but how this impacts on LPS responses remains under investigated. We previously reported in an APP/PS1 model of AD that systemic LPS administration rapidly altered hippocampal metabolism in males. Here, we used untargeted metabolomics to comprehensively identify hippocampal metabolic processes occurring at onset of systemic inflammation with LPS (100 µg/kg, i.v.) in APP/PS1 mice, at an early pathological stage, and investigated the sexual dimorphism in this response. Four hours after LPS administration, pathways regulating energy metabolism, immune and oxidative stress responses were simultaneously recruited in the hippocampi of 4.5-month-old mice with a more protective response in females despite their pro-inflammatory and pro-oxidant metabolic signature in the absence of immune stimulation. LPS induced comparable behavioural sickness responses in male and female wild-type and APP/PS1 mice and comparable activation of both the serotonin and nicotinamide pathways of tryptophan metabolism in their hippocampi. Elevations in N-methyl-2-pyridone-5-carboxamide, a major toxic metabolite of nicotinamide, correlated with behavioural sickness regardless of sex, as well as with the LPS-induced hypothermia seen in males. Males also exhibited a pro-inflammatory-like downregulation of pyruvate metabolism, exacerbated in APP/PS1 males, and methionine metabolism whereas females showed a greater cytokine response and anti-inflammatory-like downregulation of hippocampal methylglyoxal and methionine metabolism. Metabolic changes were not associated with morphological markers of immune cell activation suggesting that they constitute an early event in the development of LPS-induced neuroinflammation and AD exacerbation. These data suggest that the female hippocampus is more tolerant to acute systemic inflammation.     

The S-adenosyl homocysteine hydrolase inhibitor 3-deaza-adenosine prevents oxidative damage and cognitive impairment following folate and vitamin E deprivation in a murine model of age-related, oxidative stress-induced neurodegeneration

Deficiencies in folate promote neurodegeneration and potentiate the influence of other risk factors for neurodegeneration. This is accomplished at least in part by increasing levels of the neurotoxin homocysteine (HC). The S-adenosyl homocysteine (SAH) hydrolase inhibitor 3-deaza-adenosine (DZA) prevents HC accumulation following folate deprivation. We tested the ability of dietary supplementation with DZA to counteract the deleterious influence of folate deprivation. Folate deficiency has previously been shown to potentiate the impact of apolipoprotein E (ApoE); ApoE-/- mice deprived of folate demonstrated increased oxidative damage in brain tissue and impaired cognitive performance as compared to normal mice or to ApoE-/- mice receiving folate. Herein, we demonstrate that dietary supplementation with DZA prevented both the increase in oxidative damage and impaired cognition characteristic of ApoE-/- mice following folate deprivation. These findings suggest that manipulation of the methionine cycle by DZA can counteract folate deficiency. Because folate deprivation, increased HC, and apolipoprotein E deficiency are all risk factors for Alzheimer’s disease, these findings also underscore that DZA might be useful in a therapeutic approach to delay neurodegeneration in Alzheimer’s disease.     

Effects of homocysteine on the dopaminergic system and behavior in rodents

Long-term treatment of levodopa for Parkinson's disease (PD) patients is known to elevate homocysteine level in their plasma. The present study was designed to examine the possible neurotoxic effects of the increased homocysteine level on the dopaminergic system. Homocysteine was administered into Sprague-Dawley male rats intracerebroventricularly or C57BL/6 mice intraperitoneally. Following homocysteine injection the locomotor activities, the levels of dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and immunohistochemistry of dopaminergic neurons were examined. The results obtained indicate that homocysteine administration (1 or 2 micromol, i.c.v.) into the rat brains for 5 days significantly decreased the locomotor activities and dopamine as well as its metabolites, DOPAC and HVA, in the rat striatal regions. Two different doses of homocysteine (50 and 100mg/100g, i.p. daily) were administered into mice for 36 days to evaluate the effect of systemic treatment of homocysteine on the dopaminergic neurons of the brain. The intraperitoneal injections of two doses of homocysteine significantly increased homocysteine levels in the striatal regions of mouse brains by 21.5 and 39.2%, while reducing dopamine turnover rates in the striatal regions by decreasing (DOPAC+HVA)/DA, 23.7 and 51.6%, respectively. Accordingly, homocysteine decreased locomotor activities significantly by decreasing movement time by 29 and 38%, total distance by 32 and 42%, and numbers of movement by 28 and 41%, respectively. Moreover, homocysteine decreased tyrosine hydroxylase immunoreactivity in substantia nigra of mouse brain. The data obtained indicate that the potential of homocysteine to be toxic to the dopaminergic system. Consequently, long-term levodopa therapy for PD may accelerate the progression of PD, at least in part by elevated homocysteine.     

Hyperhomocysteinemia increases damage on brain slices exposed to in vitro model of oxygen and glucose deprivation: prevention by folic acid

In the present study we evaluate the effects of homocysteine on cellular damage using hippocampal slices from Wistar rats exposed to oxygen and glucose deprivation (OGD, followed by reoxygenation), an in vitro model of hypoxic-ischemic events. For chronic treatment, we induced elevated levels of homocysteine in blood (500 microM), comparable to those of human homocystinuria, and in brain (60 nmol/g wet tissue) of young rats by subcutaneous injections of homocysteine (0.3-0.6 micromol/g of body weight), twice a day with 8 h intervals, from the 6 th to the 28 th postpartum day and controls received saline. Rats were sacrificed 1, 3 or 12 h after the last injection. For acute treatment, 29-day-old rats received one single injection of homocysteine (0.6 micromol homocysteine/g body weight) or saline and were sacrificed 1h later. In another set of experiments rats were pretreated with Vitamins E (40 mg/kg) and C (100 mg/kg) or folic acid (5 mg/kg) during 1 week; 12 h after the last administration they received a single injection of homocysteine or saline and were sacrificed 1 h later. Results showed that both chronic (1 h after homocysteine administration) and acute hyperhomocysteinemia increased the cellular damage measured by LDH released to de incubation medium, suggesting an increase of tissue damage caused by OGD. Pretreatment with folic acid completely prevented the damage caused by acute hyperhomocysteinemia, whereas Vitamin E just partially prevented such effect. These findings may be relevant to explain, at least in part, the higher susceptibility of hyperhomocysteinemic patients to be susceptible to ischemic events and point to a possible preventive treatment.