Opposing effects of thapsigargin on the survival of developing cerebellar granule neurons in culture

Elevated levels of potassium (K⁺) promote maturation and survival of cerebellar granule neurons in culture. When switched from a culture medium containing high K⁺ (25 mM) to one with low K⁺ (5 mM) mature granule neurons undergo death by apoptosis. The mechanism by which high K⁺ promotes neuronal survival and conversely inhibits apoptosis) is unclear. Several pieces of evidence indicate that an increase in intracellular calcium (Ca²⁺) resulting from depolarization mediated-influx of extracellular Ca²⁺ is necessary. We examined the effect of thapsigargin on granule neuron cultures. Thapsigargin is an inhibitor of the endoplasmic reticular Ca²⁺ ATPase causing a depletion of Ca²⁺ from internal stores. this treatment would therefore be expected to raise intracellular cytosolic Ca²⁺ without membrane depolarization. We find that treatment of mature neurons with thapsigargin at doses 5 nM inhibits death resulting from the lowering of extracellular K⁺. The survival effect of thapsigargin was not affected by inhibitors of extracellular Ca²⁺ influx including nifedipine, verapamil, methoxyverapamil, Mg²⁺, and Ni²⁺, nor was it inhibited by the NMDA receptor antagonist, MK801. We have further examined whether thapsigargin could substitute for elevated K⁺ during the maturation of granule cells. Unexpectedly, treatment of younger (immature) neuronal cultures with the same dose of thapsigargin (5 nM) induced cell death. DNA fragmentation analysis suggested that death was due to apoptosis and not toxicity. As observed with the survival effect on mature neurons, the lethal effect of thapsigargin on immature granule cells was not prevented by inhibitors of Ca²⁺ influx.     

Agmatine induces glutamate release and cell death in cultured rat cerebellar granule neurons

We investigated the effect of agmatine on cell viability of rat cerebellar granule neurons in a high-K+ (27.5 mM) medium. Exposure of cultured rat cerebellar granule neurons to agmatine (200-800 microM) resulted in a significant decrease in cell viability. Agmatine-induced neuronal death began to occur 6-12 h after addition, and gradually progressed. The agmatine neurotoxicity was attenuated by N-methyl-D-aspartate (NMDA) receptor antagonists and by enzymatic degradation of L-glutamate with glutamic pyruvic transaminase. Furthermore, a significant increase in extracellular L-glutamate concentration was detected before cell death occurred. In addition, agmatine-induced glutamate release and cell death were both blocked by pretreatment with botulinum toxin C, which is known to specifically inhibit the exocytosis. The agmatine neurotoxicity was not observed when extracellular K+ concentration was lower (10 mM). These results suggest that agmatine induces glutamate release through the exocytosis and thereby causes NMDA receptor-mediated neuronal death in conditions in which extracellular K+ concentrations are elevated.     

Mastoparan-induced apoptosis of cultured cerebellar granule neurons is initiated by calcium release from intracellular stores

We have recently reported that mastoparan, a peptide toxin isolated from wasp venom, induces apoptosis in cultured cerebellar granule neurons that can be blocked by cholera toxin, an activator of G_s. Measurements of intracellular free calcium concentration ([Ca²⁺]_i) reveal that mastoparan induces a dramatic elevation of [Ca²⁺]_i that is frequently followed by enhanced leakage of fura-2 out of the neurons, suggesting that this rise in [Ca²⁺]_i may be due to a more generalized change in membrane permeability. However, the mastoparan-induced initial elevation of [Ca²⁺]_i is maintained in the absence of extracellular Ca²⁺, suggesting that the rise of [Ca²⁺]_i is from intracellular stores. This conclusion is supported by the observation that depletion of [Ca²⁺]_i stores by pretreatment with either caffeine or thapsigargin attenuates both the rise in [Ca²⁺]_i and cell death induced by mastoparan. Phospholipase C (PLC) inhibitors, neomycin and U73122 block mastoparan-induced increases of [Ca²⁺]_i and protect against neuronal death. Pretreatment with cholera toxin, but not pertussis toxin, reduced the mastoparan-induced rise in [Ca²⁺]_i. Taken together, our data suggest that mastoparan initiates cell death in cerebellar granule neurons by inducing Ca²⁺ release from intracellular stores, probably via activation of PLC and IP₃. A secondary or parallel process results in disruption of plasma membrane integrity and may be ultimately responsible for the death of these neurons by mastoparan.     

Depolarization or glutamate receptor activation blocks apoptotic cell death of cultured cerebellar granule neurons

Cerebellar granule neurons can be readily maintained in culture if depolarized with high concentrations of K⁺ or subtoxic concentrations of various excitatory amino acids. We now report that these depolarizing stimuli promote cerebellar granule neuron survival by blocking their programmed death via apoptosis. Cerebellar granule neurons maintained in depolarizing conditions and then changed to non-depolarizing conditions, exhibit the morphological and biochemical features of apoptosis, including cytoplasmic blebbing, condensation and aggregation of nuclear chromatin internucleosomal DNA fragmentation. Inhibitors of RNA or protein synthesis greatly attenuate cell death induced by non-depolarizing culture conditions. In contrast, cerebellar granule neurons, when exposed to fresh serum-containing medium or to high concentrations of glutamate, exhibit a delayed-type of neurotoxicity which is non-apoptotic in nature. Given the actions of excitatory amino acid receptor agonists in preventing apoptosis of cultured cerebellar granule neurons, we hypothesize that the functional innervation of postmigratory granule neurons during cerebellar development may prevent further elimination of these neurons by blocking their programmed death.     

Adenosine and ADP prevent apoptosis in cultured rat cerebellar granule cells

Cerebellar granule cells (CGCs) explanted in vitro undergo death via apoptosis when the concentration of potassium is shifted from 25 mM to 5 mM. We report that adenosine and ADP, which act as neurotransmitters and neuromodulators in the brain, exert in cultured cerebellar granule cells a specific and marked antiapoptotic action with half-maximal effect in the 10–100 μM range. The action of adenosine is partly inhibited by the A1AR antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and is mimicked by the A1AR agonist 2-chloro-N6-cyclopentyladenosine (CCPA), while ADP effect, that is completely blocked by the P2x, P2y receptors noncompetitive antagonist suramine, is restored in the presence of the selective P2x purinoceptors agonist β,γ-methylene- -ATP. These findings demonstrate that adenosine and ADP markedly inhibit the program of cell death in cerebellar granule cells and suggest that such an action is mediated via interaction with, respectively, A1 and P2x receptors.     

Pseudomonas fluorescens lipopolysaccharide inhibits both delayed rectifier and transient A-type K+ channels of cultured rat cerebellar granule neurons

Pseudomonas fluorescens is a Gram-negative bacillus closely related to the pathogen P. aeruginosa known to provoke infectious disorders in the central nervous system (CNS). The endotoxin lipopolysaccharide (LPS) expressed by the bacteria is the first infectious factor that can interact with the plasma membrane of host cells. In the present study, LPS extracted from P. fluorescens MF37 was examined for its actions on delayed rectifier and A-type K(+) channels, two of the main types of voltage-activated K(+) channels involved in the action potential firing. Current recordings were performed in cultured rat cerebellar granule neurons at days 7 or 8, using the whole-cell patch-clamp technique. A 3-h incubation with LPS (200 ng/ml) markedly depressed both the delayed rectifier (I(KV)) and transient A-type (I(A)) K(+) currents evoked by depolarizations above 0 and -40 mV, respectively. The percent decrease of I(KV) and I(A) ( approximately 30%) did not vary with membrane potential, suggesting that inhibition of both types of K(+) channels by LPS was voltage-insensitive. The endotoxin did neither modify the steady-state voltage-dependent activation properties of I(KV) and I(A) nor the steady-state inactivation of I(A). The present results suggest that, by inhibiting I(KV) and I(A), LPS applied extracellulary increases the action potential firing in cerebellar granule neurons. It is concluded that P. fluorescens MF37 may provoke in the CNS disorders associated with sever alterations of membrane ionic channel functions.     

AMPA receptor subunit mRNAs and intracellular [Ca] in cultured mouse and rat cerebellar granule cells

Cultured mouse cerebellar granule cells differ from their rat counterparts in that they survive well when grown in non-depolarising medium (5 mM K⁺). However, when chronically stimulated by added glutamate agonists, including (RS)α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), rat cerebellar granule cells also survive well in non-depolarising medium. We hypothesised that the relatively good survival of mouse cerebellar granule cells in the absence of added glutamate agonists might reflect AMPA receptors resistant to desensitisation. These receptors might be stimulated by endogenous glutamate. We tested this hypothesis by comparing cultured mouse and rat cerebellar granule cells grown in depolarising (25 mM K⁺) and non-depolarising (5 mM K⁺) medium. We studied the AMPA-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), using the fluorescent Ca²⁺ chelator, Fluo-3, and the relative concentrations of mRNAs for the four AMPA receptor subunits, GluR1–4. GluR1–4 mRNAs were measured by restriction enzyme analysis of a PCR product containing cDNA with a composition proportional to the four subunit mRNAs. We found that the [Ca²⁺]_i-response to AMPA receptor activation in cultured cerebellar granule cells is determined mainly by the desensitisation properties of the AMPA receptors rather than by their ion permeability. We also found that mouse cerebellar granule cells express AMPA receptors which are more resistant to desensitisation than the corresponding rat AMPA receptors. Thus, relatively slow AMPA receptor desensitisation kinetics may contribute to the survival of mouse cerebellar granule cells in non-depolarising medium.     

Troglitazone inhibits both post-glutamate neurotoxicity and low-potassium-induced apoptosis in cerebellar granule neurons

Both excitotoxicity and apoptosis contribute to neuronal loss in various neurodegenerative diseases such as Alzheimer's disease as well as stroke, and a drug inhibiting both types of cell death may lead to practical treatment for these diseases. Post-treatment with troglitazone, a potent and specific activator of peroxisome proliferator-activated receptor (PPAR)-gamma attenuated the cell death of cerebellar granule neurons, triggered by glutamate exposure. The inhibitory effect of troglitazone against glutamate excitotoxicity, in vitro, was observed even when added 2.5 h after the end of glutamate exposure, a time when glutamate antagonists are no longer neuroprotective. However, troglitazone did not block the glutamate-induced elevation of calcium influx, suggesting that troglitazone interfered with downstream consequences of excitotoxic glutamate receptor overactivation. In addition, troglitazone also suppressed low-potassium-induced apoptosis in cerebellar granule neurons in a phosphatidylinositol 3-kinase independent manner. In conclusion, although the mechanisms of troglitazone's neuroprotective effects are unknown, the post-treatment-neuroprotective effect and the dual-inhibitory-activity against both excitotoxicity and apoptosis may provide a novel therapy for various neurodegenerative diseases.     

Survival promotion of rat cerebellar granule neurons by co-culture with pontine explant

Cultured cerebellar granule neuron (CGN) of the rat is the most frequently used model system for analysis of activity-dependent neuronal survival. CGNs do not survive longer than 2 weeks in a standard culture medium unless KCl (or other excitants such as glutamate) is added. It is assumed that KCl represents synaptic activity, but no tests have been made on whether the survival of CGNs really depends on the synaptic input. Here we co-cultured CGNs with an explant of the pons including the basilar pontine nucleus (BPN), which is one of the input sources of CGNs in vivo, to confirm if synaptic input is really a determinant for the survival of these cells. In this co-culture system, the viability of CGNs was significantly increased without the addition of KCl. The survival promotion was confined to the population of CGNs having contact with neurites of BPN and was cancelled by an application of tetrodotoxin or antagonists of glutamate receptors, indicating that the survival depended on synaptic activity. Explants of other glutamatergic tissues including the hippocampus failed to promote the survival, although neurites grew out from these explants as vigorously as from the BPN explants. Calcium and FM1-43 imaging examinations revealed that the CGNs had formed functional synapses with the BPN explant but not with the hippocampal explant. These results, confirming the assumption that synaptic activity determines neuronal survival, provide evidence for presynaptic contribution to the survival.     

L-type voltage-gated calcium channels modulate kainic acid neurotoxicity in cerebellar granule cells

This study reports on the regulation of kainate neurotoxicity in cerebellar granule cells by calcium entry through voltage-gated calcium channels and by calcium release from internal cellular stores. Kainate neurotoxicity was prevented by the AMPA selective antagonist LY 303070 (10 microM). Kainate neurotoxicity was potentiated by cadmium, a general voltage-gated calcium channel blocker, and the L-type voltage-gated calcium channel blocker nifedipine. The antagonists of intracellular Ca2+ ([Ca2+]i) release, thapsigargin and ryanodine, were also able to potentiate kainate neurotoxicity. Kainate treatment elevated [Ca2+]i concentration with a rapid initial increase that peaked at 1543 nM and then declined to plateau at approximately 400 nM. Nifedipine lowered the peak response to 764 nM and the plateau response to approximately 90 nM. Thapsigargin also lowered the kainate-induced increase in [Ca2+]i (640 nM peak, 125 nM plateau). The ryanodine receptor agonist caffeine eliminated the kainate-induced increase in [Ca2+]i, and reduced kainate neurotoxicity. Kainate neurotoxicity potentiated by nifedipine was not prevented by RNA or protein synthesis inhibitors, nor by the caspase inhibitors YVAD-CHO and DEVD-CHO. Neither DNA laddering nor the number of apoptotic nuclei were increased following treatment with kainate and nifedipine. Increased nuclear staining with the membrane impermeable dye propidium iodide was observed immediately following kainate treatment, indicating a loss of plasma membrane integrity. Thus, kainate neurotoxicity is prevented by calcium entry through L-type calcium channels.     

Regulation of neurotransmitter enzyme by quisqualate subtype glutamate receptors in cultured cerebellar and hippocampal neurons

The possible involvement of ionotropic and metabotropic quisqualate (QA) receptors in neuronal plasticity was studied in cultured glutamtergic cerebellar or hippocampal cells in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When cerebellar of hippocampal neurons were treated with QA, it elevated the specific activity of glutaminase in a dose-dependent manner. The half-maximal effect was obtained at about 0.1 μM, the maximum increase was at about 1 μM, but levels higher than 10 μM QA produced progressive reduction in glutaminase activity. In contrast, QA had little effects on the activities of lactate dehydrogenase and aspartate aminotransferase and the amount of protein, indicating that the increase in glutaminase was relatively specific. The QA-mediated increase in glutaminase was mimicked by the ionotropic QA receptor agonist -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; EC₅₀, about 0.5 μM), but not by the metabotropic QA receptor agonist trans-(±)-1-aino-cyclopentyl-1,3,dicarboxyalte (t-ACPD; up to 0.5 mM). The specific ionotropic QA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) inhibited QA- and AMPA-mediated increases in glutaminase activity in a dose-dependent manner, whereas other glutamate receptor antagonists, -2-amino-5-phosphonovalerate, γ- -glutamyl aminomethyl sulphonic acid and γ- -glutamyl diethyl ester were ineffective. The elevation of neurotransmitter enzyme was Ca²⁺-dependent. The increase in Ca²⁺ influx essentially through the activation of L-type voltage-operated Ca²⁺ channels, and not the mobilization of internal Ca²⁺ stores, was responsible for these QA receptor-mediated long-term plastic changes in hippocampal and cerebellar neurons.     

Glutamine transport in cerebellar granule cells in culture

In the present study, uptake of glutamine by rat cerebellar granule cells, a predominantly glutamatergic nerve cell population, has been investigated. Glutamine is taken up by granule cells via at least three transport systems, A, ASC and L. The L-type low affinity system (K_m=2.6 mM) is the major transport system in the absence of Na⁺. The systems A and ASC represent the Na⁺-dependent transport routes, both with almost identical high affinity for glutamine (K_m=0.26 mM). Similar transport systems for glutamine are also found in cerebral cortical neurons, a predominantly GABAergic nerve cell population, and cerebral cortical astrocytes. The glutamine transport properties in granule cells, however, show a series of differences from that of cortical neurons and astrocytes: (1) uptake of glutamine by granule cells is primarily mediated by system A (54%), while contributions by system A in cortical neurons and astrocytes are less than 30%; (2) granule cells exhibit strikingly higher transport efficiency for glutamine (V_max/K_m=20 min⁻¹ for system A as compared to the V_max/K_m ratio of 5 min⁻¹ in cortical neurons and astrocytes), and (3) the initial uptake rates and the steady-state accumulation levels of glutamine are two- to threefold higher in granule cells than that of cortical neurons and astrocytes. These results taken together suggest that in accordance with the important need to replenish the neurotransmitter pool of glutamate, glutamatergic neurons exhibit highly efficient transport systems to accumulate glutamine, one of the major precursors of glutamate.     

NMDA receptors contribute to the survival promoting effect of high potassium in cultured cerebellar granule cells

The present report further analyzes the survival promoting effect of high potassium, a condition that mimics neural activity in cultured cerebellar granule cells, an excellent model to study trophic mechanisms induced by depolarization and trophic factors. We found that the survival promoting effect measured at 7 days in vitro (DIV 7) of depolarizing potassium concentrations (25 mM KCl), added at DIV2, is partially prevented by adding at DIV 2 the non-competitive NMDA blocker MK801 (10 microM). The concentration of MK801 used blocks completely the survival promoting effect of a supramaximal effective concentration of NMDA (100 microM). The addition at DIV 2 of anti-brain derived neurotrophic factor (anti-BDNF) antibody, failed to modify the effect of high potassium. The present report provides evidences that in cultured cerebellar granule cells, high potassium-induced survival promoting effect is due in part by the activation of NMDA receptors. The effect does not require the presence of BDNF.     

Glucose starvation stimulates Zn2+ toxicity in cultures of cerebellar granule neurons

Zinc chloride (0.02 mM, 3 h) did not have any influence on the survival of cerebellar granule neurons (CGNs) incubated in balanced salt solution (BSS). However, in the absence of glucose ZnCl₂ caused severe neuronal damage, decreasing cell survival to 12 ± 2%. Either the blockade of ionotropic glutamate NMDA-receptors with MK-801 or APV or supplementation the medium with ruthenium red (mitochondrial Ca²⁺ uniporter blocker) almost entirely protected CGNs from the toxic effect of ZnCl₂ during glucose deprivation (GD). However, NBQX (AMPA/kainate glutamate receptor blocker) did not show protective effect. Measurements of intracellular calcium ions concentration using fluorescent probe (Fluo-4 AM) and zinc ions (FluoZin-3 AM) demonstrated that 1.5 h-exposure to GD induced intensive increase of Fluo-4 fluorescence and small increase of FluoZin-3 fluorescence in neurons. The supplementation of medium with ZnCl₂ caused equal increase of FluoZin-3 fluorescence at both GD and normoglycemia, whereas the potentiation of Fluo-4 fluorescence by zinc was observed only under GD and could be prevented by MK-801. However, neither MK-801 nor NBQX could influence [Zn²⁺]_i increase caused by zinc addition under GD, while ruthenium red did cause significant increase of [Zn²⁺]_i. This data implies that zinc ions during GD induce an additional overload of CGNs with calcium ions that get transported through activated NMDA-channel. Zinc and calcium ions accumulate in mitochondria and amplify individual destructive action on these organelles leading to neuronal death.     

Elevation of intracellular Ca2+ modulates A-currents in rat cerebellar granule neurons

In the brain, the transient-inactivating voltage-gated potassium channel currents (called I(K(A)) or A-currents) are activated at subthreshold membrane potentials to control the excitability of neurons. In the current study, the effect of intracellular calcium on the A-current and the action mechanism of intracellular calcium was investigated by using the whole-cell voltage-clamp technique. Elevation of intracellular calcium by addition of 2 mM CaCl2 in the pipette solution significantly modulated both the peak amplitude and the kinetics of the A-current in rat granule neurons. The peak amplitudes of the A-current were 1,060 +/- 87 pA and 1,972 +/- 16 pA under conditions of no Ca2+ and elevated intracellular Ca2+, respectively. The time to peak, the time course of fast inactivation, and the steady-state inactivation property of the A-current were all significantly altered by elevating the intracellular Ca2+. Replacement of the Ca2+ in the pipette solution with the same concentration of Co2+ did not mimic the effects of intracellular Ca2+ on the A-current amplitude and kinetics. These effects are similar to the behavior of the reconstituted Kv4/KChIP (K(V) channel-interacting proteins) current induced by expression of KChIP and Kv4 together in a cell expression system. Application of 10 microM arachidonic acid, which can bind to the Kv4/KChIP complex, inhibited the A-current and eliminated the effects of intracellular Ca2+ on the A-current, suggesting that KChIP may be involved in the effects of Ca2+ on the A-current. Collectively, our results indicate that elevated intracellular Ca2+ modulates the amplitude, fast activation, and steady-state inactivation characteristics of the A-current in rat cerebellar granule neurons, and this may occur via KChIP.     

Action of thyroxine on the survival and neurite maintenance of cerebellar granule neurons in culture

Developmental hypothyroidism causes severe impairments in the cerebellum. To understand the role of thyroid hormones (THs) in cerebellar development, we examined the effect of three different THs, thyroxine (T4), 3,5,3′‐triidothyronine (T3), and 3,3′,5′‐triiodothyronine (reverse T3; rT3), on the survival and morphology of cerebellar granule neurons (CGNs) in culture and found novel actions specific to T4. Rat CGNs obtained at postnatal day 6 were first cultured for 2 days in serum‐containing medium with 25 mM K⁺ (K25), then switched to serum‐free medium with physiological 5 mM K⁺ (K5) or with K25 and cultured for an additional 2 or 4 days. CGNs underwent apoptosis in K5 but survived in K25. Addition of T4 at concentrations of 100–200 nM but not T3 or rT3 rescued CGNs from cell death in K5 in a dose‐dependent manner. Furthermore, 200 nM T4 was also effective in maintaining the neurites of CGNs in K5. In K5, T4 suppressed tau phosphorylation at two developmentally regulated sites as well as phosphorylation of c‐jun N‐terminal kinase (JNK) necessary for its activation and localization to axons. These results suggest that, during cerebellar development, T4 exerts its activity in cell survival and neurite maintenance in a manner distinct from the other two thyroid hormones through regulating the activity and localization of JNK. © 2014 Wiley Periodicals, Inc.     

高钾可诱导培养的小脑颗粒神经元凋亡

采用四唑盐比色、琼脂糖凝胶电泳、乙二酸荧光素染色和Hoechst332 58染色等方法研究高钾对原代培养的大鼠小脑颗粒神经元的毒性作用及其机制。结果发现 :①高钾诱导神经元死亡呈剂量 ( 50～ 10 0mmol/L)和时间依赖性 ;②神经元死亡呈现明显的凋亡特征 :胞体缩小 ,染色质浓缩 ,DNA“梯形”条带形成和蛋白质合成抑制剂 (cycloheximide ,1.0mg/L)可阻断其毒性等 ;③MK 80 1( 2 μmol/L)、尼莫地平 ( 10 μmol/L)、硫酸镁 ( 2 0mmol/L)可阻断高钾的大部分毒性作用。结果提示 :高钾可能通过刺激内源性谷氨酸释放从而诱导小脑颗粒神经元凋亡     

磷酸化C—JUN不参与谷氨酸诱导的小脑颗粒神经元凋亡

观察磷酸化 C- JUN与谷氨酸诱导的小脑颗粒神经元凋亡的关系。在培养的小脑颗粒神经元建立谷氨酸凋亡模型 ;采用 MTT法分析细胞存活率 ,相差显微镜观察形态学 ,DNA凝胶电泳法分析细胞凋亡和原位细胞荧光免疫组织化学法检测磷酸化 C- JUN。结果显示 ,谷氨酸诱导大鼠小脑颗粒神经元细胞体积缩小 ,突触断裂、消失 ,DNA电泳呈典型的“梯状”条带 ;谷氨酸处理 2 4 h后细胞存活率为 2 8.6%± 5.2 %。神经元在谷氨酸处理 5,30 min及 1 ,2 ,4,8,1 6和 2 4 h后均未检测到有磷酸化 C- JUN阳性细胞 ,与去极化组 ( 2 5mmol/L KCl)相同。而复极化组 ( 5mmol/L KCl)则在 30 min检测到大量的磷酸化 C- JUN阳性细胞 ,4h荧光最强并持续。处理 4h后 ,40 0倍荧光显微镜下 ,复极化组、去极化组和谷氨酸组的磷酸化 C- JUN阳性细胞数分别为 1 2 4± 1 7,8± 3,5± 3。上述结果提示 ,谷氨酸诱导小脑颗粒神经元凋亡 ,磷酸化 C- JUN不参与谷氨酸诱导的大鼠小脑颗粒神经元凋亡。     

Neuroprotective effects of phenyl-t-butyl-nitrone in gerbil global brain ischemia and in cultured rat cerebellar neurons

We examined the ability of phenyl-t-butyl-nitrone (PBN), an electron spin trapper, to attenuate ischemia-induced forebrain edema and hippocampal CA1 neuronal loss in gerbils, and to protect rat cerebellar neurons in primary culture from glutamate-induced toxicity. PBN, given i.p. at 75 or 150 mg/kg 30 min before ischemia (5 min occlusion), increased survival (at 7 days) of CA1 neurons from 60 +/- 14 (vehicle-treated, n = 17) to 95 +/- 15 (P less than 0.05, n = 15) and 145 +/- 3 (P less than 0.01, n = 15), respectively. When gerbils were treated with PBN (50 mg/kg, i.p.) immediately and 6 h after reperfusion, followed by b.i.d. for an additional 2 days, CA1 neurons survival improved from 35 +/- 9 (vehicle, n = 20, 6 min occlusion) to 106 +/- 17 (P less than 0.01, n = 13). In gerbils exposed to a more severe ischemia (10 min), pretreatment with 150 mg/kg PBN increased the survival of CA1 neurons from 6 +/- 6 (vehicle) to 27 +/- 10 (P less than 0.05, n = 11). Pretreatment with PBN, at 150 mg/kg, reduced forebrain edema (following 15 min ischemia) by 24.7% (P less than 0.01, n = 16). PBN at 50 mg/kg, i.p. had no hypothermic effect and at 75 or 150 mg/kg caused a transient hypothermia. The presence of PBN in the brain was confirmed in microdialysis samples and brain tissue extract using HPLC. In vitro, PBN protected rat cerebellar neurons against 100 microM glutamate-induced toxicity with an EC50 value of 2.7 mM. Our results further support the concept that free radicals contribute to brain injury following ischemia and suggest the potential therapeutic application of electron spin trappers in stroke.