Leon Zagrean

Oxidative damage following cerebral ischemia depends on reperfusion ‐ a biochemical study in rat

The extent of brain injury during reperfusion appears to depend on the experimental pattern of ischemia/reperfusion. The goals of this study were: first, to identify the rate of free radicals generation and the antioxidant activity during ischemia and reperfusion by means of biochemical measurement of lipid peroxidation (LPO) and both enzymatic (superoxid dismutase ‐ SOD, catalase ‐ CAT, glutathion peroxidase ‐ GPx) and non‐enzymatic antioxidants activity (glutathione ‐ GSH); and second, to try to find out how the pattern of reperfusion may influence the balance between free radical production and clearance. Wistar male rats were subject of four‐vessel occlusion model (Pulsinelly & Brierley) cerebral blood flow being controlled by means of two atraumatic arterial microclamps placed on carotid arteries. The level of free radicals and the antioxidant activity were measured in ischemic rat brain tissue homogenate using spectrophotometrical techniques. All groups subjected to ischemia shown an increase of LPO and a reduction of the activity of enzymatic antioxidative systems (CAT, GPx, SOD) and non‐enzymatic systems (GSH). For both groups subjected to ischemia and reperfusion, results shown an important increase of LPO but less significant than the levels found in the group with ischemia only. Statistically relevant differences (p<0.01) between continuous reperfusion and fragmented reperfusion were observed concerning the LPO, CAT, SOD and GSH levels, oxidative aggresion during fragmented reperfusion being more important.

High-throughput analysis of gangliosides in defined regions of fetal brain by fully automated chip-based nanoelectrospray ionization multi-stage mass spectrometry

Gangliosides (GGs), a large group of sialylated glycosphingolipids, are considered biomarkers of human brain development, aging and certain diseases. Determination of individual GG components in complex mixtures extracted from a human brain represents a fundamental prerequisite for correlating their specificity with the specialized function of each brain area. In the context of modern glycomics, detailed investigation of GG expression and structure in human brain requires a continuous development and application of innovative methods able to improve the quality of data and speed of analysis. In this work, for the first time, a high-throughput mapping and sequencing of gangliosides in human fetal brain was performed by a novel mass spectrometry (MS)-based approach developed recently in our laboratory. Three GG mixtures extracted and purified from different regions of the same fetal brain in the 36th gestational week: frontal neocortex (NEO36), white matter of the frontal lobe (FL36) and white matter of the occipital lobe (OL36) were subjected to comparative high-throughput screening and multi-stage fragmentation by fully automated chip-based nanoelectrospray ionization (nanoESI) high capacity ion trap (HCT) MS. Using this method, in only a few minutes of signal acquisitions, over 100 GG and asialo-GG species were detected and identified in the three mixtures. Obtained data revealed for the first time that differences in GG expression in human fetal brain are dependent on phylogenetic development rather than topographic factors. While a significant variation of GG distribution in NEO36 vs FL36 was observed, no significant differences in GG expression in white matter of frontal vs occipital lobe were detected. Additionally, the largest number of species was identified in NEO36, which correlates with the functional complexity of neocortex as the newest brain region. In the last stage of analysis, using MS2–MS3 molecular ion fragmentation at variable amplitudes, a NEO36-associated GD1b isomer could clearly be discriminated. Present results indicate that the combination of fully automated chipESI with HCT MS n is able to provide ultra-fast, sensitive and reliable analyses of complex lipid-linked carbohydrates from which the pattern of their expression and structure in a certain type of bio-matrix can be determined.

Coffee drinking enhances the analgesic effect of cigarette smoking.

Nicotine (from cigarette smoke) and caffeine (from coffee) have analgesic effects in humans and experimental animals. We investigated the combined effects of coffee drinking and cigarette smoking on pain experience in a group of moderate nicotine-dependent, coffee drinking, young smokers. Pain threshold and pain tolerance were measured during cold pressor test following the habitual nocturnal deprivation of smoking and coffee drinking. Smoking increased pain threshold and pain tolerance in both men and women. Coffee drinking, at a dose that had no independent effect, doubled the increase in pain threshold induced by smoking. The effect could not be explained by a cumulative raise in blood pressure. Our data suggest that caffeine enhances the analgesic effect of nicotine.

Brain Chondroitin/Dermatan Sulfate, from Cerebral Tissue to Fine Structure: Extraction, Preparation, and Fully Automated Chip-Electrospray Mass Spectrometric Analysis

Chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans (GAGs) are covalently linked to proteins, building up a wide range of proteoglycans, with a prevalent expression in the extracellular matrix (ECM). In mammalian tissues, these GAG species are often found as hybrid CS/DS chains. Their structural diversity during chain elongation is produced by variability of sulfation in the repeating disaccharide units. In central nervous system, a large proportion of the ECM is composed of proteoglycans; therefore, CS/DS play a significant role in the functional diversity of neurons, brain development, and some brain diseases. A requirement for collecting consistent data on brain proteoglycan glycosylation is the development of adequate protocols for CS/DS extraction and detailed compositional and structure analysis. This chapter will present a strategy, which combines biochemical tools for brain CS/DS extraction, purification, and fractionation, with a modern analytical platform based on chip-nanoelectrospray multistage mass spectrometry (MS) able to provide information on the essential structural elements such as epimerization, chain length, sulfate content, and sulfation sites.

Endogenous adenosine A1 receptor activation underlies the transient post-ischemic rhythmic delta EEG activity

OBJECTIVE Emergence of slow EEG rhythms within the delta frequency band following an ischemic insult of the brain has long been considered a marker of irreversible anatomical damage. Here we investigated whether ischemic adenosine release and subsequent functional inhibition via the adenosine A(1) receptor (A(1)R) contributes to post-ischemic delta activity. METHODS Rats were subjected to episodes of non-injuring transient global cerebral ischemia (GCI) under chloral hydrate anesthesia. RESULTS We found that a GCI lasting only 10s was enough to induce a brief discharge of rhythmic delta activity (RDA) with a peak frequency just below 1 Hz quantified as an increase by twofold of the 0.5-1.5 Hz spectral power. This post-ischemic RDA did not occur following administration of the A(1)R antagonist 8-cyclopentyl-1,3-dipropylxanthine. Nevertheless, a similar RDA could be induced in rats not subjected to GCI, by systemic administration of the A(1)R agonist N(6)-cyclopentyladenosine. CONCLUSIONS Our data suggest that A(1)R activation at levels that occur following cerebral ischemia underlies the transient post-ischemic RDA. SIGNIFICANCE It is likely that the functional, thus potentially reversible, synaptic disconnection by A(1)R activation promotes slow oscillations in the cortical networks. This should be accounted for in the interpretation of early post-ischemic EEG delta activity.

The neuroprotective effect of intranasally applied leptin against hypoxic neuronal injury

Hypoxia may result from hypoperfusion, as seen in the cardio-respiratory arrest. Subsequent to the acute neuronal damage, the delayed neuronal death ensues, and further neurons die within hours or days thereafter. An effective neuroprotective therapeutic agent should counteract one or, ideally, all well-established neuronal death pathways, i.e., excitotoxicity, oxidative stress and apoptosis. All these three mechanisms propagate through distinctive and mutual exclusive signal transduction pathway and contribute to the neuronal loss following the initial hypoxic-ischemic brain injury. Thus, the ideal therapeutic intervention against the hypoxic-ischemic neuronal injury should aim to prevent all three mechanisms of the neuronal death in a concerted effort. Recent studies demonstrated that intranasally administered leptin results in supra-physiological leptin levels at various regions of the brain (including hippocampus) within 30min of administration. We consider leptin to be an ideal neuroprotective agent, having targeted excitotoxicity (directly, by inhibiting AMDA and NMDA) oxidative stress (indirectly, by HIF1 mediation) and apoptosis (directly, by activating ERK 1/2 pathway) and hypothesize that intranasally administered leptin has neuroprotective effect against the neuronal hypoxic injury. If our hypothesis is confirmed, leptin administered before and/or soon after hypoxic injury, may be effective in minimizing the devastating sequelae of such event.

Electro‐cortical signs of early neuronal damage following transient global cerebral ischemia in rat

During recovery after a transient global cerebral ischemia (TGCI), rat electrocorticogram (ECoG) shows epochs of synchronized activity (SA) alternating with epochs of low amplitude background activity (BA). The aim of this study was to compare the changes in these electrical activities during a 30‐min recovery period that followed either a noninjuring (3 minutes, N=10) or an injuring (10 minutes, N=10) TGCI. During TGCI there was a 3 fold reduction in amplitudes of both SA and BA but no changes in frequency. During reperfusion following a 3 minutes TGCI, the amplitudes of both SA and BA recovered to about 70%. During the reperfusion that followed a 10 minutes TGCI, BA showed no recovery, whereas SA recovered to about 40%. During the 30 min reperfusion, there was a timedependent decrease in the frequency of SA, but independent on the duration of TGCI. In contrast, the frequency of the BA did not change during reperfusion. Our data indicate that following cerebral ischemia the recovery of SA can take place independently of BA. The lack of recovery in BA may indicate early subcortical neuronal damage.

Trans-resveratrol enriched maternal diet protects the immature hippocampus from perinatal asphyxia in rats

Trans-resveratrol (tRESV), a polyphenol with antioxidant properties, is common in many food sources, hence easily accessible for study as a maternal dietary supplement in perinatal asphyxia (PA). Hypoxic-ischemic encephalopathy secondary to PA affects especially vulnerable brain areas such as hippocampus and is a leading cause of neonatal morbidity. The purpose of this study is to identify new epigenetic mechanisms of brain inflammation and injury related to PA and to explore the benefit of tRESV enriched maternal diet. The hippocampal interleukin 1 beta (IL-1b), tumour necrosis factor alpha (TNFα) and S-100B protein, at 24-48h after 90min of asphyxia were assessed in postnatal day 6 rats whose mothers received either standard or tRESV enriched diet. The expression of non-coding microRNAs miR124, miR132, miR134, miR146 and miR15a as epigenetic markers of hippocampus response to PA was determined 24h post-asphyxia. Our results indicate that neural response to PA could be epigenetically controlled and that tRESV reduces asphyxia-related neuroinflammation and neural injury. Moreover, tRESV could increase, through epigenetic mechanisms, the tolerance to asphyxia, with possible impact on the neuronal maturation. Our data support the neuroprotective quality of tRESV when used as a supplement in the maternal diet on the offsprings outcome in PA.

Maternal High-Fat Diet Modifies the Immature Hippocampus Vulnerability to Perinatal Asphyxia in Rats

Background: High-fat diet (HFD) is a detrimental habit with harmful systemic consequences, including low-grade, long-lasting inflammation. During pregnancy, HFD can induce developmental changes. Moreover, HFD-related maternal obesity might enhance the risk of peripartum complications including hypoxic-ischemic encephalopathy secondary to perinatal asphyxia (PA). Objectives: Following our previous results showing that PA increases neuroinflammation and neuronal injury in the immature hippocampus and modifies hippocampal epigenetic programming, we further aimed to establish the impact of maternal HFD on offspring hippocampus response to PA. Methods: We assessed hippocampal tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1b) and S-100B protein (S-100B), 24–48 h after PA exposure in postnatal day 6 Wistar rats, whose mothers received either the standard diet or HFD. The expression of small non-coding microRNA species miR124, miR132, miR134, miR146, and miR15a, as epigenetic markers for the maternal dietary influence on immature hippocampus response after PA, was determined 24 h after asphyxia exposure. Metabolic activity was measured using resazurin test in hippocampal cell suspension obtained 24 h after PA. Results: Our results indicate that maternal HFD additionally increases hippocampal TNFα, IL-1b, and S-100B after PA. Also, PA associated with maternal HFD induces miR124 upregulation and miR132 downregulation relative to PA only. Metabolic activity was increased in hippocampal cells from pups whose mothers received HFD. Conclusion: HFD increases the PA-induced neuroinflammation and neuronal injury, and epigenetically influences homeostatic synaptic plasticity and neuronal tolerance to asphyxia, processes associated with a higher hippocampal cellular metabolism.

Neurobiology of Postischemic Recuperation in the Aged Mammalian Brain

Old age is associated with an enhanced susceptibility to stroke and poor recovery from brain injury, but the cellular processes underlying these phenomena are not well understood. Potential mechanism underlying functional recovery after brain ischemia in aged subjects include neuroinflammation, changes in brain plasticity-promoting factors, unregulated expression of neurotoxic factors, or differences in the generation of scar tissue that impedes the formation of new axons and blood vessels in the infarcted region. Studies suggest that behaviorally, aged rats were more severely impaired by ischemia than were young rats and showed diminished functional recovery. Both in old and young rats, the early intense proliferative activity following stroke leads to a precipitous formation of growthinhibiting scar tissue, a phenomenon amplified by the persistent expression of neurotoxic factors. Recent evidence shows that the human brain can respond to stroke with increased progenitor proliferation in aged patients, opening the possibilities of utilizing this intrinsic attempt for neuroregeneration of the human brain as a potential therapy for ischemic stroke.