Structural plasticity of synapses in hippocampal slices after oxygen-glucose deprivation

We analyzed structural rearrangements of synaptic contacts in the stratum radiatum of the CA1 area of cultured rat hippocampal slices under conditions of the development of potentiation of synaptic transmission induced by short-term (10 min) oxygen-glucose deprivation (OGD). Studies were carried out using electron microscopy and 3D reconstruction of cellular compartments. Within the 1st h after OGD, we observed increases in the volume of pre-synaptic terminals and post-synaptic spines and also in the area of postsynaptic densities (PSDs) in both asymmetric excitatory and symmetric inhibitory synapses, especially in the case were the PSD was perforated. We also observed significant activation of glial cells (increases in their volume and area of contacts of their processes with the components of synapses). Therefore, OGD results in activationassociated structural rearrangements of both excitatory and inhibitory synapses of the hippocampal CA1 area. Such rearrangements are accompanied by a clearly pronounced reaction of the glia, which correlates with an important role of the latter in modulation of the functioning of neurons.     

Mechanism of guanosine-induced neuroprotection in rat hippocampal slices submitted to oxygen-glucose deprivation

Guanine derivates have been implicated in many relevant extracellular roles, such as modulation of glutamate transmission, protecting neurons against excitotoxic damage. Guanine derivatives are spontaneously released to the extracellular space from cultured astrocytes during oxygen-glucose deprivation (OGD) and may act as trophic factors, glutamate receptors blockers or glutamate transport modulators, thus promoting neuroprotection. The aim of this study was to evaluate the mechanisms involved in the neuroprotective role of the nucleoside guanosine in rat hippocampal slices submitted to OGD, identifying a putative extracellular binding site and the intracellular signaling pathways related to guanosine-induced neuroprotection. Cell damage to hippocampal slices submitted to 15 min of OGD followed by 2 h of reperfusion was decreased by the addition of guanosine (100 microM) or guanosine-5'-monophosphate (GMP, 100 microM). The neuroprotective effect of guanosine was not altered by the addition of adenosine receptor antagonists, nucleosides transport inhibitor, glutamate receptor antagonists, glutamate transport inhibitors, and a non-selective Na(+) and Ca(2+) channel blocker. However, in a Ca(2+)-free medium (by adding EGTA), guanosine was ineffective. Nifedipine (a Ca(2+) channel blocker) increased the neuroprotective effect of guanosine and 4-aminopyridine, a K(+) channel blocker, reversed the neuroprotective effect of guanosine. Evaluation of the intracellular signaling pathways associated with guanosine-induced neuroprotection showed the involvement of PKA, PKC, MEK and PI-3 K pathways, but not CaMKII. Therefore, this study shows guanosine is acting via K(+) channels activation, depending on extracellular Ca(2+) levels and via modulation of the PKA, PKC, MEK and/or PI-3 K pathways.     

Iron toxicity in organotypic cultures of hippocampal slices: role of reactive oxygen species

Free iron has been assumed to potentiate oxygen toxicity by generating reactive oxygen species (ROS) via the iron-catalyzed Haber-Weiss reaction, leading to oxidative stress. ROS-mediated iron cytotoxicity may trigger apoptotic cell death. In the present study, we used iron treatment of organotypic cultures of hippocampal slices to study potential mechanisms involved in iron-induced neuronal damage. Exposure of mature hippocampal slices to ferrous sulfate resulted in concentration- and time-dependent cell death. After iron treatment, markers of ROS formation and lipid peroxidation, i.e. intensity of dichlorofluorescein (DCF) fluorescence and levels of thiobarbiturate reactive substances (TBARS), were significantly increased. Levels of cytochrome c were increased while levels of pro-caspase-9 and pro-caspase-3 were decreased in cytosolic fractions of iron-treated hippocampal slice cultures. Treatment of cultured slices with a synthetic catalytic ROS scavenger, EUK-134, provided between 50 and 70% protection against various parameters of cell damage and markers of oxidative stress. In addition, inhibition of caspase-3 activity by Ac-DEVDcho partially protected cells from iron toxicity. The combination of EUK-134 and Ac-DEVDcho resulted in an almost complete blockade of iron-induced damage. These results indicate that iron elicits cellular damage predominantly by oxidative stress, and that ROS-mediated iron toxicity may involve cytochrome c- and caspase-3-dependent apoptotic pathways.     

Lactate and glucose as energy substrates during, and after, oxygen deprivation in rat hippocampal acute and cultured slices

The effects of raised brain lactate levels on neuronal survival following hypoxia or ischemia is still a source of controversy among basic and clinical scientists. We have sought to address this controversy by studying the effects of glucose and lactate on neuronal survival in acute and cultured hippocampal slices. Following a 1-h hypoxic episode, neuronal survival in cultured hippocampal slices was significantly higher if glucose was present in the medium compared with lactate. However, when the energy substrate during the hypoxic period was glucose and then switched to lactate during the normoxic recovery period, the level of cell damage in the CA1 region of organotypic cultures was significantly improved from 64.3 +/- 2.1 to 74.6 +/- 2.1% compared with cultures receiving glucose during and after hypoxia. Extracellular field potentials recorded from the CA1 region of acute slices were abolished during oxygen deprivation for 20 min, but recovered almost fully to baseline levels with either glucose (82.6 +/- 10.0%) or lactate present in the reperfusion medium (108.1 +/- 8.3%). These results suggest that lactate alone cannot support neuronal survival during oxygen deprivation, but a combination of glucose followed by lactate provides for better neuroprotection than either substrate alone.     

Morphological and functional changes in rat hippocampal slice cultures after short-term oxygen-glucose deprivation

To study effects of short-term cerebral ischemia, hippocampal slice cultures were subjected to oxygen and glucose deprivation (OGD) followed by a period of normoxic reoxygenation. Propidium iodide staining, and MTT/formazan-assay were used to evaluate cell viability and metabolic activity. CA1 pyramidal cells were analyzed at the light- and electron microscopic levels. Cell damage was found to be insignificant during the first hour after 10 min OGD but profound following 4 h, showing delayed neuronal cell damage caused by short-term OGD. Our model can be used to characterize the mechanisms of cell damage caused by mild cerebral ischemia. These data might apply to further development of neuroprotective tools for the treatment of brain diseases.     

缺糖缺氧对体外培养海马神经元的影响

目的：观察缺糖缺氧诱导的培养海马神经元损伤。方法：取培养12d的海马神经元,在缺糖缺氧条件下分别培养0．5～4h后取出,换原神经元培养液在常氧条件下继续培养24h。用0．4％台盼蓝染色,检测神经元坏死,并用TUNEL法检测神经元凋亡,计算存活、坏死和凋亡神经元所占百分率。同时用图像分析仪测定存活、坏死和凋亡神经元的胞体面积、周长和等园直径。结果：培养的海马神经元急性缺糖缺氧后0．5～4h,随缺糖缺氧时间的延长,坏死神经元逐渐增多,缺糖缺氧后0．5～2h再恢复糖和氧供应后24h,凋亡神经元明显增多。图像分析的结果表明,坏死神经元的胞体面积、周长和等园直径均明显大于凋亡神经元。结论：缺糖缺氧可引起海马神经元严重损伤,在急性缺糖缺氧后0．5～4h引起的神经元死亡以坏死为多见,但在缺糖缺氧后0．5～2h再恢复糖和氧供应后24,神经元死亡则以凋亡为多见。     

Purine receptors are required for DHA-mediated neuroprotection against oxygen and glucose deprivation in hippocampal slices

Docosahexaenoic acid (DHA) is important for central nervous system function during pathological states such as ischemia. DHA reduces neuronal injury in experimental brain ischemia; however, the underlying mechanisms are not well understood. In the present study, we investigated the effects of DHA on acute hippocampal slices subjected to experimental ischemia by transient oxygen and glucose deprivation (OGD) and re-oxygenation and the possible involvement of purinergic receptors as the mechanism underlying DHA-mediated neuroprotection. We observed that cellular viability reduction induced by experimental ischemia as well as cell damage and thiobarbituric acid reactive substances (TBARS) production induced by glutamate (10 mM) were prevented by hippocampal slices pretreated with DHA (5 μM). However, glutamate uptake reduction induced by OGD and re-oxygenation was not prevented by DHA. The beneficial effect of DHA against cellular viability reduction induced by OGD and re-oxygenation was blocked with PPADS (3 μM), a nonselective P2X_1–5 receptor antagonist as well as with a combination of TNP-APT (100 nM) plus brilliant blue (100 nM), which blocked P2X₁, P2X₃, P2X_2/3, and P2X₇ receptors, respectively. Moreover, adenosine receptors blockade with A₁ receptor antagonist DPCPX (100 nM) or with A_2B receptor antagonist alloxazine (100 nM) inhibited DHA-mediated neuroprotection. The addition of an A_2A receptor antagonist ZM241385 (50 nM), or A₃ receptor antagonist VUF5574 (1 μM) was ineffective. Taken together, our results indicated that neuroprotective actions of DHA may depend on P2X, A₁, and A_2B purinergic receptors activation. Our results reinforce the notion that dietary DHA may act as a local purinergic modulator in order to prevent neurodegenerative diseases.     

大鼠海马神经元体外缺糖缺氧模型的建立

目的：建立体外培养大鼠海马神经元缺糖缺氧模型。方法：取培养12d的海马神经元,在缺糖缺氧条件下分别培养0．5～4h后取出,换原神经元培养液,在常氧下继续培养24h后测定培养液中乳酸脱氢酶活性。测定神经元形态变化,并计算神经元存活百分率。同时用原位末端标记(TUNEL)法检测神经元凋亡。结果：缺糖缺氧后海马神经元胞体逐渐肿胀,培养液中LDH释放量逐渐增多,细胞存活率逐渐减少。恢复糖和氧供应后24h,凋亡神经元百分率明显增多。结论：用改进的无血清、无糖人工脑脊液成功建立了大鼠海马神经元离体缺糖缺氧模型。     

Possible mediation of quinolinic acid-induced hippocampal damage by reactive oxygen species

Several differences exist between quinolinic acid and N-methyl-D-aspartate (NMDA) in the potency and pharmacology of their neurotoxic actions in the brain, suggesting that quinolinic acid may act by mechanisms additional to the activation of NMDA receptors, possibly involving lipid peroxidation. In the present review, studies are considered which have attempted to determine whether free radicals might contribute to the neuronal damage induced by quinolinic acid. Following Injections into the hippocampus of anaesthetised rats, quinolinic acid induced damage is prevented by melatonin, by an action not blocked by the melatonin receptor blocker luzindole. Deprenyl, but not the non-selective monoamine oxidase inhibitor nialamide, also prevent quinolinic acid-induced damage. In vitro, several groups have shown that quinolinic acid can induce lipid peroxidation of brain tissue The results suggest that free radical formation contributes significantly to quinolinic acid-induced damage in vivo.     

BRCA1 down-regulates cellular levels of reactive oxygen species

Previous studies have shown that the breast cancer suppressor BRCA1 stimulates antioxidant gene expression and protects cells against oxidative stress. To further examine this important function, we tested whether BRCA1 could modulate intracellular levels of reactive oxygen species (ROS). Wild-type BRCA1 (but not a cancer-associated mutant) significantly reduced ROS levels, determined by DCF fluorescence assays by flow cytometry and confocal microscopy. The BRCA1 and REF1 pathways for reduction of ROS levels appear to exhibit cross-talk. BRCA1 also reduced the levels of protein nitration and H₂O₂-induced oxidative damage to DNA. Thus, BRCA1 may protect cellular macromolecules by reducing intracellular ROS levels.     

Guanosine controls inflammatory pathways to afford neuroprotection of hippocampal slices under oxygen and glucose deprivation conditions

Guanosine (GUO) is an endogenous modulator of glutamatergic excitotoxicity and has been shown to promote neuroprotection in in vivo and in vitro models of neurotoxicity. This study was designed to understand the neuroprotective mechanism of GUO against oxidative damage promoted by oxygen/glucose deprivation and reoxygenation (OGD). GUO (100 μM) reduced reactive oxygen species production and prevented mitochondrial membrane depolarization induced by OGD. GUO also exhibited anti‐inflammatory actions as inhibition of nuclear factor kappa B activation and reduction of inducible nitric oxide synthase induction induced by OGD. These GUO neuroprotective effects were mediated by adenosine A₁ receptor, phosphatidylinositol‐3 kinase and MAPK/ERK. Furthermore, GUO recovered the impairment of glutamate uptake caused by OGD, an effect that occurred via a Pertussis toxin‐sensitive G‐protein‐coupled signaling, blockade of adenosine A_2A receptors (A_2AR), but not via A₁ receptor. The modulation of glutamate uptake by GUO also involved MAPK/ERK activation. In conclusion, GUO, by modulating adenosine receptor function and activating MAPK/ERK, affords neuroprotection of hippocampal slices subjected to OGD by a mechanism that implicates the following: (i) prevention of mitochondrial membrane depolarization, (ii) reduction of oxidative stress, (iii) regulation of inflammation by inhibition of nuclear factor kappa B and inducible nitric oxide synthase, and (iv) promoting glutamate uptake.     

七氟醚预处理对大鼠海马脑片缺氧无糖损伤的保护作用:线粒体KATP通道的作用

利用离体海马脑片缺氧无糖(oxygen-glucose deprivation,OGD)损伤模型,探讨七氟醚预处理对神经细胞的保护作用及该作用与线粒体内膜ATP敏感钾通道(mitochondrial ATP-sensitive potassium channels,mitoKATPchannels)的关系,随机将脑片用2%、4%、6%七氟醚,以及6%七氟醚复合mitoKATP通道阻滞剂5-羟基奎酸盐(5-hydroxydecanoic acid,5-HD)预处理30 min,观察OGD损伤14 min复氧1 h期间顺向群峰电位(orthodromic population spike,OPS)的变化,并应用透射电镜观察细胞超微结构的改变.结果表明,与单纯OGD组相比,七氟醚预处理可使海马脑片OPS消失时间明显延长(P＜0.01),使OPS明显恢复,其中4%、6%七氟醚组的恢复率均为71.4%(P＜0.05 vs OGD),相应恢复程度为(61.0±42.3)%和(78.7±21.1)%(P＜0.01),而且6%七氟醚的保护作用可被5-HD取消.OGD组的海马CA1区锥体细胞明显水肿,核膜皱缩、破裂,染色质聚集,线粒体肿胀畸形,嵴断裂或消失,而4%和6%七氟醚组仅见海马CA1区锥体细胞轻度水肿,核膜皱缩不明显,染色质均匀,线粒体轻度肿胀.结果提示,七氟醚预处理对大鼠海马脑片OGD损伤有一定的保护作用,且七氟醚对神经细胞的保护作用与激活mitoKATP通道有关.     

Guanosine prevents nitroxidative stress and recovers mitochondrial membrane potential disruption in hippocampal slices subjected to oxygen/glucose deprivation

Guanosine, the endogenous guanine nucleoside, prevents cellular death induced by ischemic events and is a promising neuroprotective agent. During an ischemic event, nitric oxide has been reported to either cause or prevent cell death. Our aim was to evaluate the neuroprotective effects of guanosine against oxidative damage in hippocampal slices subjected to an in vitro ischemia model, the oxygen/glucose deprivation (OGD) protocol. We also assessed the participation of nitric oxide synthase (NOS) enzymes activity on the neuroprotection promoted by guanosine. Here, we showed that guanosine prevented the increase in ROS, nitric oxide, and peroxynitrite production induced by OGD. Moreover, guanosine prevented the loss of mitochondrial membrane potential in hippocampal slices subjected to OGD. Guanosine did not present an antioxidant effect per se. The protective effects of guanosine were mimicked by inhibition of neuronal NOS, but not of inducible NOS. The neuroprotective effect of guanosine may involve activation of cellular mechanisms that prevent the increase in nitric oxide production, possibly via neuronal NOS.     

Imaging reactive oxygen species in arthritis

Reactive oxygen species (ROS) have been shown to play a role in the pathogenesis of arthritides. Luminol was used as the primary reporter of ROS and photons resulting from the chemiluminescence reaction were detected using a super-cooled CCD photon counting system. Luminol was injected intravenously into groups of animals with different models of arthritis. Imaging signal correlated well with the severity of arthritis in focal and pan-arthritis as determined by histological measurement of ROS by formazan. Measurements were highly reproducible, sensitive, and repeatable. In vivo chemiluminescence imaging is expected to become a useful modality to elucidate the role of ROS in the pathogenesis of arthritides and in determining therapeutic efficacy of protective therapies.     

Mild hypothermia protects hippocampal neurons from oxygen-glucose deprivation injury through inhibiting caspase-3 activation

Mild hypothermia (MH) is thought to be one of the most effective therapeutic methods to treat hypoxic-ischemic encephalopathy (HIE) after cardiac arrest (CA). However, its precise mechanisms remain unclear. In this research, hippocampal neurons were cultured and treated with mild hypothermia and Ac-DEVD-CHO after oxygen-glucose deprivation (OGD). The activity of caspase-3 was detected, in order to find the precise concentration of Ac-DEVD-CHO with the same protective role in OGD injury as MH treatment. Western blot and immunofluorescence staining were conducted to analyze the effects of MH and Ac-DEVD-CHO on the expressions of caspase-3, caspase-8, and PARP. The neuronal morphology was observed with an optical microscope. The lactic acid dehydrogenase (LDH) release rate, neuronal viability, and apoptotic rate were also detected. We found that MH (32 °C) and Ac-DEVD-CHO (5.96 μMol/L) had equal effects on blocking the activation of caspase-3 and the OGD-induced cleavage of PARP, but neither had any effect on the activation of caspase-8, which goes on to activate caspase-3 in the apoptotic pathway. Meanwhile, both MH and Ac-DEVD-CHO had similar effects in protecting cell morphology, reducing LDH release, and inhibiting OGD-induced apoptosis in neurons. They also similarly improved neuronal viability after OGD. In conclusion, caspase-3 serves as a key intervention point of the key modulation site or regulatory region in MH treatment that protects neuronal apoptosis against OGD injury. Inhibiting the expression of caspase-3 had a protective effect against OGD injury in MH treatment, and caspase-3 activation could be applied to evaluate the neuroprotective effectiveness of MH on HIE.     

Imaging reactive oxygen species in asthma

Inflammatory processes in asthma are characterized by an infiltration of inflammatory cells including mononuclear phagocytes. It has been observed that mononuclear phagocytes, alveolar macrophages and blood monocytes, release higher quantities of reactive oxygen species in asthmatic patients than in healthy subjects. Chemiluminescence assays were developed to measure the superoxide anion and the other reactive oxygen species. The chemiluminescence response was first analysed with a luminometer, which made it possible to study cells in suspension before and after PMA-stimulation. Secondly a video-imaging camera was used in experiments on adherent cells before and after stimulation with PMA and/or specific stimulus IgE/anti-IgE. Both techniques showed that human alveolar macrophages, blood monocytes, PMN and lymphocytes were spontaneously primed in vivo and were more easily stimulated in asthma. Analysis of adherent cells in vitro may provide give information on the physiological condition of adherent cells in vivo.     

Measuring mitochondrial reactive oxygen species

This article examines recent methods for measuring reactive oxygen species produced in isolated mitochondria and within live cells, with particular emphasis on the detection of hydrogen peroxide. Protocols for reliable measurements of mitochondrial hydrogen peroxide are presented, while the advantages and pitfalls of these and other methods are discussed. New developments in the detection of lipid peroxidation are outlined. Advice is also provided to aid the interpretation of cellular data with respect to the contribution of oxygen radical production by different components of the mitochondrial respiratory chain.