Human neural progenitor cells promote photoreceptor survival in retinal explants

Different types of progenitor and stem cells have been shown to provide neuroprotection in animal models of photoreceptor degeneration. The present study was conducted to investigate whether human neural progenitor cells (HNPCs) have neuroprotective properties on retinal explants models with calpain- and caspase-3-dependent photoreceptor cell death. In the first experiments, HNPCs in a feeder layer were co-cultured for 6 days either with postnatal rd1 mouse or normal rat retinas. Retinal histological sections were used to determine outer nuclear layer (ONL) thickness, and to detect the number of photoreceptors with labeling for calpain activity, cleaved caspase-3 and TUNEL. The ONL thickness of co-cultured rat and rd1 retinas was found to be almost 10% and 40% thicker, respectively, compared to controls. Cell counts of calpain activity, cleaved caspase-3 and TUNEL labeled photoreceptors in both models revealed a 30-50% decrease when co-cultured with HNPCs. The results represent significant increases of photoreceptor survival in the co-cultured retinas. In the second experiments, for an identification of putative survival factors, or a combination of them, a growth factor profile was performed on conditioned medium. The relative levels of various growth factors were analyzed by densitometric measurements of growth factor array membranes. Following growth factors were identified as most potential survival factors; granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GMCSF), insulin-like growth factor II (IGF-II), neurotrophic factor 3 (NT-3), placental growth factor (PIGF), transforming growth factors (TGF-β1 and TGF-β2) and vascular endothelial growth factor (VEGF-D). HNPCs protect both against calpain- and caspase-3-dependent photoreceptor cell death in the rd1 mouse and against caspase-3-dependent photoreceptor cell death in normal rat retinas in vitro. The protective effect is possibly achieved by a variety of growth factors secreted from the HNPCs.     

α2-肾上腺素受体激动剂右旋美托嘧啶对缺血性脑血管病的保护作用

缺血性脑血管病是常见病、多发病,其致残率和死亡率均高,严重影响着人类的身体健康和生活质量。当脑缺血缺氧时,神经元会出现损伤和死亡。现有大量研究证明α2-肾上腺素受体激动剂能保护大脑神经元免受缺血／再灌注的损伤,但其发挥神经保护作用的确切机制尚不完全清楚,可能与α2-肾上腺素受体的激活有关。右旋美托嘧啶（dexmedetomidine）是一种高选择性的α2-肾上腺素受体激动剂,具有一定的神经保护作用。本文就缺血性脑血管病病理生理演变过程、右旋美托嘧啶的分子药理学及其发挥神经保护作用的机制作一综述。     

Erythropoietin protects neurons against chemical hypoxia and cerebral ischemic injury by up-regulating Bcl-xL expression

Erythropoietin (EPO) promotes neuronal survival after cerebral ischemia in vivo and after hypoxia in vitro. However, the mechanisms underlying the protective effects of EPO on ischemic/hypoxic neurons are not fully understood. The present in vitro experiments showed that EPO attenuated neuronal damage caused by chemical hypoxia at lower extracellular concentrations (10(- 4)-10(-2) U/ml) than were previously considered. Moreover, EPO at a concentration of 10(-3) U/ml up-regulated Bcl-xL mRNA and protein expressions in cultured neurons. Subsequent in vivo study focused on whether EPO rescued hippocampal CA1 neurons from lethal ischemic damage and up-regulated the expressions of Bcl-xL mRNA and protein in the hippocampal CA1 field of ischemic gerbils. EPO was infused into the cerebroventricles of gerbils immediately after 3 min of ischemia for 28 days. Infusion of EPO at a dose of 5 U/day prevented the occurrence of ischemia-induced learning disability. Subsequent light microscopic examinations showed that pyramidal neurons in the hippocampal CA1 field were significantly more numerous in ischemic gerbils infused with EPO (5 U/day) than in those receiving vehicle infusion. The same dose of EPO infusion caused significantly more intense expressions of Bcl-xL mRNA and protein in the hippocampal CA1 field of ischemic gerbils than did vehicle infusion. These findings suggest that EPO prevents delayed neuronal death in the hippocampal CA1 field, possibly through up-regulation of Bcl-xL, which is known to facilitate neuron survival.     

Activated microglial cells migrate towards sites of excitotoxic neuronal injury inside organotypic hippocampal slice cultures

The aim of this study was to analyse microglial reactions to excitotoxic N ‐methyl‐ d ‐aspartic acid (NMDA)‐induced degeneration of rat dentate and hippocampal neurons in vitro . We used a migration model combining the techniques of microglial single cell culture and organotypic hippocampal slice culture (OHSC). Site‐specific oxidative damage in OHSCs was induced by pretreatment with 50 μ m NMDA. Neuronal injury determined by propidium iodide (PI) uptake included the hippocampal cell layers of the dentate gyrus (DG) and the cornu ammonis (CA). Fluorescence‐prelabelled microglial cells with ameboid morphology were transferred onto the OHSC and migrated predominantly to the prelesioned cell layers of DG and CA when compared with unlesioned areas of the OHSC. In NMDA pretreated slices, microglial cells clustered around degenerating granule cells in the DG and pyramidal cells in the CA. This effect was significantly inhibited in unlesioned slice cultures and in NMDA‐exposed cultures that were pretreated with the NMDA‐antagonist MK‐801. Our observations suggest that microglia – attracted by the presence of stimuli provided by NMDA‐induced neuronal death – migrate specifically towards these lesioned neurons.     

NMDA and Kainate-evoked Release of Nitric Oxide and Classical Transmitters in the Rat Striatum: In Vivo Evidence that Nitric Oxide May Play a Neuroprotective Role

The effects of N-methy-d -aspartate (NMDA), kainate, S-α-amino-3-hydroxyd-5-methyl-4-isoxazole propionate (AMPA) and KCI on striatal nitric oxide (NO), acetylcholine (ACh), dopamine (DA), serotonin (5-HT), aspartate (ASP), glutamate (GLU) and γ-aminobutyric acid (GABA) release were measured in anaesthetized rats in vivo by microdialysis and in vitro in organotypic slice cultures. Local NMDA (1–100 μM) infusion by retrodialysis dose-dependently increased levels of classical transmitters, NO₂-, NO₃-, ctrulline and arginine at similar thresholds (10 γM) Similar patterns of NMDA-evoked (50 μM) release were seen in striatal cultures. NMDA-evoked changes were all calcium-dependent and blocked by NMDA (APV or MK-801) but not AMPN/kainate (DNQX) receptor antagonists, excepting DA which could be prevented by both. In vivo, kainate increased NO₂^-, NO₃^-, CIT and ARG levels at 50 and 100 μM but was less potent than NMDA. Kainate also evoked significant Ach₁ DA and GLU release dose-dependently starting at 1–10 μM whereas 5-HT, ASP and GABA required 50 or 100 μM doses. Kainate effects were inhibited by DNQX, but not by APV, and were calcium-dependent. AMPA failed to alter NO₂^-, NO₃^-, CIT or ARG levels at 50 or 100 μM doses but dose-dependently increased ACh and DA. Similar results were seen with kainate (50 μM) and AMPA (50 μM) in vitro KCI evoked NO₂^-, NO₃^-, CIT and ARG release as well as that of the classical transmitters in vivo and in vitro. In vivo administration of the NO synthase inhibitor L-nitroarginine (L-NARG; 100 μM) significantly reduced NO₂^-, NO₃^- and CIT levels and prevented NMDA, kainate or KCI-evoked increases. It also potentiated ACh, ASP, GLU and GABA release and reduced that of DA in response to 50 μM NMDA whereas treatment with an NO-donor (SNAP; 10 μM) significantly reduced evoked ACh, ASP and GLU release. The NO synthase inhibitor L-NARG potentiated kainate-evoked ACh release and reduced that of DA, although less potently than NMDA, but it had no effect on KCI-evoked transmitter release. Overall, these results show that both NMDA and kainate increase striatal NO release at similar dose-thresholds as for classical transmitter release suggesting that NO is dynamically released under physiological and not just pathological conditions. Reduction of striatal NO levels also potentiates calcium-dependent transmitter release in response to NMDA and, to a lesser extent, kainate, whereas increasing them reduces it. This is consistent with a role for NO as a neuroprotective agent in this region acting to desensitize NMDA receptors.     

Actions of the sodium channel inhibitor 202W92 on rat midbrain dopaminergic neurons

Excessive glutamatergic activity is implicated in Parkinson's disease (PD) and sodium channel blockade, resulting in inhibition of glutamate release, is a potential therapeutic approach to PD therapy. Beneficial effects of riluzole and lamotrigine have been reported in animal models of PD, but these compounds have relatively low potency as sodium channel inhibitors and also inhibit N and P/Q-type calcium channels. 202W92, a structural analog of lamotrigine, is a potent sodium channel inhibitor, with no effect on N, P/Q-type channels. Here we present the effects of 202W92 on single patch-clamped dopaminergic neurons. 202W92 (> or =10 microM) inhibited spontaneous action potential firing and reduced amplitude and frequency of evoked action potentials. It also inhibited the frequency of 4-aminopyridine (4-AP)- and electrically evoked excitatory postsynaptic currents (EPSCs) and GABAergic inhibitory postsynaptic currents (IPSCs), with >80% inhibition at 10 microM (IC(50) 1.5 microM). EPSC and IPSC amplitudes were partially inhibited. 202W92 did not affect postsynaptic responses to locally applied glutamate and GABA, nor spontaneously occurring mini-IPSCs. These actions of 202W92 are compatible with sodium channel inhibition and depression of transmitter release.     

Mechanism of neuroprotection by donepezil pretreatment in rat cortical neurons chronically treated with donepezil

Previously, we showed that in rat cortical neurons, chronic donepezil treatment (10 microM, 4 days) up-regulates nicotinic receptors (nAChR) and makes neurons more sensitive to the neuroprotective effect of donepezil. Here we examined the mechanism of donepezil-induced neuroprotection in neurons chronically treated with donepezil. The mechanism of neuroprotection was examined under different conditions of exposure to glutamate, acute and moderate, that induce cell death associated with necrotic and apoptotic cell death, respectively. Concomitant treatment with antagonists of nAChRs but not muscarinic receptors inhibited donepezil pretreatment-induced neuroprotection against acute glutamate treatment-induced death. Donepezil pretreatment prevented acute glutamate- and ionomycin-induced neurotoxicity, but not S-nitrosocysteine-induced neurotoxicity, suggesting that donepezil protects neurons via nAChR at levels before nitric oxide synthase activation against acute glutamate neurotoxicity. Concomitant treatment with antagonists of nAChR or phosphatidylinositol 3-kinase (PI3K) signaling inhibitors significantly inhibited neuroprotection against moderate glutamate neurotoxicity and decreased the phosphorylation level of Akt. Neuroprotection was also inhibited by treatment with inhibitor of mitogen-activated protein kinase (MAPK) kinase. These results suggest that donepezil protects neurons against moderate glutamate neurotoxicity via nAChR-PI3K-Akt and MAPK signaling pathways. This study provides novel insight into the mechanism of donepezil-induced neuroprotection that involves nAChR up-regulation.