Hemin uptake and release by neurons and glia.

Hemin accumulates in intracerebral hematomas and may contribute to cell injury in adjacent tissue. Despite its relevance to hemorrhagic CNS insults, very little is known about hemin trafficking by neural cells. In the present study, hemin uptake and release were quantified in primary murine cortical cultures, and the effect of the hemin-binding compound deferoxamine (DFO) was assessed. Net uptake of (55)Fe-hemin was similar in mixed neuron-glia, neuron, and glia cultures, but was 2.6-3.6-fold greater in microglia cultures. After washout, 40-60% of the isotope signal was released by mixed neuron-glia cultures into albumin-containing medium within 24 h. Inhibiting hemin breakdown with tin protoporphyrin IX (SnPPIX) had minimal effect, while release of the fluorescent hemin analog zinc mesoporphyrin was quantitatively similar to that of (55)Fe-hemin. Isotope was released most rapidly by neurons (52.2 ± 7.2% at 2 h), compared with glia (15.6 ± 1.3%) and microglia (17.6 ± 0.54%). DFO did not alter (55)Fe-hemin uptake, but significantly increased its release. Mixed cultures treated with 10 µM hemin for 24 h sustained widespread neuronal loss that was attenuated by DFO. Concomitant treatment with SnPPIX had no effect on either enhancement of isotope release by DFO or neuroprotection. These results suggest that in the presence of a physiologic albumin concentration, hemin uptake by neural cells is followed by considerable extracellular release. Enhancement of this release by DFO may contribute to its protective effect against hemin toxicity.

Interactions between NO, CO and an endothelium-derived hyperpolarizing factor (EDHF) in maintaining patency of the ductus arteriosus in the mouse.

BACKGROUND AND PURPOSE: Prenatal patency of ductus arteriosus is maintained by prostaglandin (PG) E(2), possibly along with nitric oxide (NO) and carbon monoxide (CO), and cyclooxygenase (COX) deletion upregulates NO. Here, we have examined enzyme source and action of NO for ductus patency and whether NO and CO are upregulated by deletion of, respectively, heme oxygenase 2 (HO-2) and COX1 or COX2. EXPERIMENTAL APPROACH: Experiments were performed in vitro and in vivo with wild-type and gene-deleted, near-term mouse fetuses. KEY RESULTS: N(G)-nitro-L-arginine methyl ester (L-NAME) contracted the isolated ductus and its effect was reduced by eNOS, but not iNOS, deletion. L-NAME contraction was not modified by HO-2 deletion. Zinc protoporphyrin (ZnPP) also contracted the ductus, an action unaffected by deletion of either COX isoform. Bradykinin (BK) relaxed indomethacin-contracted ductus similarly in wild-type and eNOS-/- or iNOS-/-. BK relaxation was suppressed by either L-NAME or ZnPP. However, it reappeared with combined L-NAME and ZnPP to subside again with K(+) increase or K(+) channel inhibition. In vivo, the ductus was patent in wild-type and NOS-deleted fetuses. Likewise, no genotype-related difference was noted in postnatal closure. CONCLUSIONS AND IMPLICATIONS: NO, formed mainly via eNOS, regulates ductal tone. NO and CO cooperatively mediate BK-induced relaxation in the absence of PGE(2). However, in the absence of PGE(2), NO and CO, BK induces a relaxant substance behaving as an endothelium-derived hyperpolarizing factor. Ductus patency is, therefore, sustained by a cohort of agents with PGE(2) and NO being preferentially coupled for reciprocal compensation.

Ferritin induction protects cortical astrocytes from heme-mediated oxidative injury.

Hemin is released from hemoglobin after CNS hemorrhage and may contribute to its cytotoxic effect. In a prior study, we demonstrated that heme oxygenase-1 induction protected murine cortical astrocytes from hemoglobin toxicity. Since heme metabolism releases iron, this observation suggested that these cells are able to effectively sequester and detoxify free iron. In this study, we tested the hypotheses that astrocytes increased ferritin synthesis after exposure to heme-bound iron, and that this induction protected cells from subsequent exposure to toxic concentrations of hemin. Incubation with low micromolar concentrations of hemin, hemoglobin, or ferrous sulfate increased ferritin expression, as detected on immunoblots stained with a polyclonal antibody that was raised against horse spleen ferritin. Time course studies demonstrated an increase in ferritin levels within 2 h. Weak and scattered cellular staining was detected by immunohistochemistry in control, untreated cultures, while diffuse immunoreactivity was observed in cultures exposed to heme-bound iron. An enhanced ferritin band was detected on immunoblots from cultures that were treated with purified apoferritin, consistent with astrocytic ferritin uptake. Immunoreactivity after apoferritin treatment was not altered by concomitant treatment with cycloheximide. Pretreatment with apoferritin protected astrocytes from hemin toxicity in a concentration-dependent fashion between 1 and 4 mg/ml. At the highest concentration, cell death due to a 6-h exposure to 30 microM hemin was decreased by about 85%. A protective effect was also produced by induction of endogenous ferritin with nontoxic concentrations of ferrous sulfate, hemoglobin, or hemin. These results suggest that cortical astrocytes respond to exogenous heme-bound or free iron by rapidly increasing ferritin synthesis. The combined action of heme oxygenase-1 and ferritin may be a primary astrocytic defense against heme-mediated injury.

Activation of extracellular signal-regulated kinases potentiates hemin toxicity in astrocyte cultures.

Hemin is present in intracranial hematomas in high micromolar concentrations and is a potent, lipophilic oxidant. Growing evidence suggests that heme-mediated injury may contribute to the pathogenesis of CNS hemorrhage. Extracellular signal-regulated kinases (ERKs) are activated by oxidants in some cell types, and may alter cellular vulnerability to oxidative stress. In this study, the effect of hemin on ERK activation was investigated in cultured murine cortical astrocytes, and the consequence of this activation on cell viability was quantified. Hemin was rapidly taken up by astrocytes, and generated reactive oxygen species (ROS) within 30 min. Increased immunoreactivity of dually phosphorylated ERK1/2 was observed in hemin-treated cultures at 30-120 min, without change in total ERK. Surprisingly, ERK activation was not attenuated by concomitant treatment with antioxidants (U74500A or 1,10-phenanthroline) at concentrations that blocked ROS generation. Cell death commenced after 2 h of hemin exposure and was reduced by antioxidants and by the caspase inhibitor Z-VAD-FMK. Cytotoxicity was also attenuated by MEK inhibition with PD98059 or U0126 at concentrations that were sufficient to prevent ERK activation. Whereas the effect of Z-VAD-FMK on cell survival was transient, the effect of MEK inhibitors was long-lasting. MEK inhibitors had no effect on cellular hemin uptake or subsequent ROS generation. The present results suggest that hemin activates ERK in astrocytes via a mechanism that is independent of ROS generation. This activation sensitizes astrocytes to hemin-mediated oxidative injury.

Magnesium deprivation decreases cellular reduced glutathione and causes oxidative neuronal death in murine cortical cultures.

The vulnerability of cultured cortical neurons to oxidative injury is an inverse function of the extracellular Mg2+ concentration. In order to test the hypothesis that depolarization-enhanced release of reduced glutathione (GSH) contributes to this phenomenon, we assessed the effect of Mg2+ deprivation on cellular and medium glutathione levels. Incubation of mixed neuronal and glial cultures in Mg2+-free medium resulted in a decline in cellular total glutathione (GSx) within 8 h, without change in oxidized glutathione (GSSG); no effect was seen in pure glial cultures. This decrease in cellular GSx was associated with a progressive increase in GSx but not GSSG in the culture medium. Cellular GSH loss was not attenuated by concomitant treatment with antioxidants (ascorbate, Trolox, or deferoxamine), but was prevented by the NMDA receptor antagonist MK-801. Mg2+ deprivation for over 24 h produced neuronal but not glial death, with release of about 40% of neuronal lactate dehydrogenase by 48-60 h. Most of this cytotoxicity was prevented by treatment with either antioxidants or MK-801. These results suggest that Mg2+ deprivation causes release of neuronal reduced glutathione via a mechanism involving excessive NMDA receptor activation. If prolonged, cellular GSH depletion ensues, leading to oxidative neuronal death.

Heme oxygenase-1 induction protects murine cortical astrocytes from hemoglobin toxicity.

Exposure to micromolar concentrations of hemoglobin (Hb) results in the oxidative death of cultured cortical neurons, but glia are resistant. The role of heme oxygenase-1 (HO-1) induction on this glial resistance was investigated. Within two hours of exposure to 5 microM Hb, immunoblotting demonstrated an increase in HO-1 in confluent glial cultures. Consistent with prior observations, 23-30 h Hb exposure had little or no effect on glial viability, as assessed by lactate dehydrogenase release. Concomitant treatment with the HO inhibitors tin protoporphyrin IX or the D-amino acid peptide rvnlrialry resulted in release of 40-71% of glial lactate dehydrogenase; protein synthesis inhibition with cycloheximide produced a similar effect. These results are consistent with the hypothesis that HO-1 induction protects cortical astrocytes from Hb toxicity.

Potentiation of excitotoxic injury by high concentrations of extracellular reduced glutathione.

Glutathione is present in the central nervous system in millimolar concentrations, and is a predominant intracellular antioxidant and detoxicant. In addition, glutathione is released into the extracellular space via a depolarization-enhanced process. Although the role of extracellular glutathione has not been precisely defined, a growing body of experimental evidence suggests that it has multifaceted electrophysiological effects. At low micromolar concentrations, glutathione depolarizes neurons by binding to its own receptors and modulates glutamatergic excitatory neurotransmission by displacing glutamate from its ionotropic receptors. At higher concentrations, reduced glutathione may increase N-methyl-D-aspartate receptor responses by interacting with its redox sites. In this study, the effect of extracellular glutathione on excitotoxic neuronal injury was quantitatively assessed in murine cortical cell cultures. Neuronal death due to 20-25 h exposure to 6-9 microM N-methyl-D-aspartate was not altered by 10-100 microM reduced glutathione but was markedly enhanced by 300-1000 microM reduced glutathione; kainate neurotoxicity was unaffected. Two related compounds that lack a sulfhydryl group, oxidized glutathione and S-hexylglutathione, had no significant effect on N-methyl-D-aspartate neurotoxicity alone but completely blocked the effect of reduced glutathione. Mercaptoethanol, a sulfhydryl reducing agent that increases N-methyl-D-aspartate receptor responses by interacting with redox sites, increased N-methyl-D-aspartate neurotoxicity to a degree comparable to that of reduced glutathione; this effect was also blocked by equimolar S-hexylglutathione or oxidized glutathione. Addition of reduced glutathione to mercaptoethanol did not further increase N- methyl-D-aspartate-induced neuronal death. These results suggest that release of reduced glutathione from central nervous system cells that are subjected to traumatic or ischemic insults may enhance excitotoxic neuronal loss. Although multiple mechanisms may account for this phenomenon, the high concentrations required suggest that it is at least partly mediated by reduction of N-methyl-D-aspartate receptor redox sites.

Extracellular reduced glutathione increases neuronal vulnerability to combined chemical hypoxia and glucose deprivation.

In addition to its intracellular antioxidant role, reduced glutathione (GSH) is released by CNS cells and may mediate or modulate excitatory neurotransmission. Although extracellular GSH levels rise in the ischemic cortex, its effect on the viability of energy-compromised neurons has not been defined. In this study, we tested the hypothesis that exogenous GSH would increase the vulnerability of cultured cortical neurons to azide-induced chemical hypoxia combined with glucose deprivation. Thirty minutes azide exposure in a glucose-free buffer was tolerated by most neurons, with release of less than 10% of neuronal LDH over the subsequent 21-25 h. Concomitant treatment with 10-100 microM GSH increased cell death in a concentration-dependent fashion, to 71.6+/-5.1% of neurons at 100 microM; GSH alone was nontoxic. Injury was blocked by the selective N-methyl-d-aspartate (NMDA) antagonist MK-801 but not by the AMPA/kainate antagonist NBQX. The sulfhydryl reducing agent mercaptoethanol (10-100 microM) mimicked the action of GSH; however, the zinc chelator ethylenediaminetetraacetic acid (EDTA) was ineffective. Two GSH analogues that lack a sulfhydryl group, S-hexylglutathione (SHG) and oxidized glutathione (GSSG), were inactive per se but attenuated the effect of both GSH and mercaptoethanol. These results suggest that micromolar concentrations of GSH enhance neuronal loss due to energy depletion by altering the extracellular redox state, resulting in increased NMDA receptor activation.

Toxic effect of hemoglobin on spinal cord neurons in culture.

The vulnerability of spinal cord neurons to hemoglobin was quantitatively assessed in primary cultures derived from fetal mice. Exposure to hemoglobin for 28 h in a serum-free medium resulted in concentration-dependent neuronal death, with an EC50 of 0.9 microM; glia were not injured. Neuronal death was decreased by the ferric iron chelator deferoxamine, the alpha-tocopherol analogue Trolox C, ascorbate, and exogenous catalase, but was potentiated by superoxide dismutase. Neuronal death was also increased by depletion of cellular glutathione with the gamma-glutamylcysteine synthetase inhibitor buthionine sulfoxamine; inhibition of endogenous catalase with 3-amino-1,2,4-triazole had no significant effect. These results suggest that hemoglobin is toxic to spinal neurons via an iron-dependent, oxidative mechanism involving a hydrogen peroxide intermediate, and support the hypothesis that hemoglobin release may contribute to neuronal loss after spinal cord trauma.

The effect of magnesium on oxidative neuronal injury in vitro.

The effect of magnesium on the oxidative neuronal injury induced by hemoglobin was assessed in murine cortical cell cultures. Exposure to 5 microM hemoglobin in physiologic (1 mM) magnesium for 26 h resulted in the death of about one-half the neurons and a sixfold increase in malondialdehyde production; glia were not injured. Increasing medium magnesium to 3 mM reduced neuronal death by about one-half and malondialdehyde production by about two-thirds; neuronal death and lipid peroxidation were approximately doubled in 0.3 mM magnesium. Comparable results were observed in spinal cord cultures. The NMDA antagonist MK-801 weakly attenuated hemoglobin neurotoxicity in low-magnesium medium, but tended to potentiate injury in physiologic magnesium. Incubation in low-magnesium medium alone for 24 h reduced cellular glutathione by approximately 50% in mixed neuronal and glial cultures but by only 10% in pure glial cultures. The iron-dependent oxidation of phosphatidylethanolamine liposomes was attenuated in a concentration-dependent fashion by 2.5-10 mM magnesium; a similar effect was provided by 0.01-0.1 mM cobalt. However, oxidation was weakly enhanced by 0.5-1 mM magnesium. These results suggest that the vulnerability of neurons to iron-dependent oxidative injury is an inverse function of the extracellular magnesium concentration. At high concentrations, magnesium inhibits lipid peroxidation directly, perhaps by competing with iron for phospholipid binding sites. At low concentrations, enhancement of cell death may be due to the combined effect of increased NMDA receptor activity, glutathione depletion, and direct potentiation of lipid peroxidation.

Estrogens attenuate neuronal injury due to hemoglobin, chemical hypoxia, and excitatory amino acids in murine cortical cultures.

A growing body of evidence supports the hypothesis that estrogens may be beneficial in Alzheimer's disease and other neurodegenerative processes. Less is known of their therapeutic potential in acute CNS insults. In this study, we assessed the effect of estrogens in three injury paradigms that may be relevant to CNS hemorrhage, trauma, and ischemia. Supraphysiologic concentrations of 17beta-estradiol, estrone, or equilin attenuated neuronal loss due to prolonged exposure to the pro-oxidant hemoglobin, with complete protection at 10 microM. Most of this effect persisted despite concomitant treatment with the antiestrogen ICI 182,780 or the protein synthesis inhibitor cycloheximide. In contrast, the non-estrogenic steroid methylprednisolone, which is currently in clinical use in spinal cord injury, reduced neuronal loss by only about 30%. High concentrations of equilin or estrone also attenuated the submaximal neuronal injury induced by 3.5-4.5 h exposure to the cytochrome oxidase inhibitor sodium azide, with near complete protection at 30 microM. Estrogens had a weaker and somewhat variable effect on pure excitotoxic injury, reducing neuronal loss due to 24 h kainate exposure by about half, and due to 24 h NMDA exposure by 15-65%; similar neuroprotection was provided by the antioxidant 21-aminosteroid U74500A. These results suggest that estrogens may be beneficial in acute CNS injuries associated with oxidative and excitotoxic stress. Investigation of high dose estrogen therapy in in vivo models of CNS hemorrhage, trauma, and ischemia is warranted.

The vulnerability of spinal cord neurons to excitotoxic injury: comparison with cortical neurons.

The neurotoxicity of the glutamate receptor agonists N-methyl-D-aspartate (NMDA), (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and kainate was quantitatively assessed in murine spinal cord and cortical cultures prepared under identical conditions. Compared with cortical neurons, spinal neurons were less vulnerable to NMDA (EC50 for 24 h exposure about 30 microM versus 10 microM in cortical cultures) and more vulnerable to AMPA (EC50 5 microM versus 12 microM) and kainate (EC50 20 microM versus 50 microM). Neurons subject to kainate-activated cobalt uptake, a marker of calcium-permeable AMPA/kainate channels, were resistant to NMDA in both systems; these cells were significantly more prevalent in spinal cord cultures. Both the AMPA/kainate antagonist GYKI-52466 and the NMDA antagonist MK-801 attenuated spinal cord neuronal loss due to glucose deprivation; however, GYKI-52466 was more effective. These results support the hypothesis that AMPA/kainate receptor activation may play a significant role in excitotoxic injury to spinal cord neurons.

Hemoglobin potentiates excitotoxic injury in cortical cell culture.

Excessive activation of glutamate receptors may contribute to neuronal loss after a traumatic or ischemic central nervous system insult. Such injuries are often associated with hemorrhage and extravasation of hemoglobin, a prooxidant and putative neurotoxin. In this study, we investigated the effect of nontoxic concentrations of hemoglobin on the neurotoxicity of the synthetic glutamate receptor agonists NMDA, AMPA, and kainate in primary murine cortical cultures. Continuous exposure to each excitotoxin alone for 24-28 h produced concentration-dependent neuronal death (EC(50) about 12 mu M for AMP(+)A, 50 mu M for kainate, and 12 mu M for NMDA). Hemoglobin 0.25-1.0 mu M consistently potentiated the neurotoxicity of low concentrations of AMPA and kainate, increasing neuronal loss by about 150% at 6 mu M AMPA and by about 90% at 30 mu M kainate. This effect was attenuated by the iron chelator deferoxamine and the alpha-tocopherol analogue trolox. Hemoglobin coexposure had less impact on slowly triggered NMDA neurotoxicity, significantly increasing neuronal death only at agonist concentrations that alone produced little or no injury. Hemoglobin pretreatment had no effect on the rapidly triggered excitotoxicity induced by brief exposure to high concentrations of NMDA. These results suggest that hemoglobin may contribute to neuronal loss after CNS hemorrhage by exacerbating excitotoxicity. At moderate levels of agonist exposure, this effect may be somewhat selective for the AMPA/kainate component of injury.

Ultrastructure of excitotoxic neuronal death in murine cortical culture.

Ischemic and traumatic brain injury are likely to involve neuronal injury triggered by glutamate receptor overactivation. Although excitotoxic neuronal injury has been widely studied in the setting of primary culture, the extent to which these in vitro injury paradigms resemble in vivo ischemic injury morphologically has not previously been well studied. We studied glutamate receptor mediated neuronal death by transmission electron microscopy and light microscopy. Morphologic characteristics of neurons injured by 10 min exposure to 500 microM glutamate include rapid swelling of mitochondria and endoplasmic reticulum, and cytoplasmic and nuclear lucency. Both alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and kainic acid caused vacuolation, dilatation of the endoplasmic reticulum, cytoplasmic condensation and random condensation of chromatin with preserved mitochondria. None of these injuries was ameliorated by cycloheximide or actinomycin D; all were significantly lessened by aurintricarboxylic acid. Gel electrophoresis showed no increase in DNA fragmentation over control. The morphologic changes seen with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and kainate are distinct from the changes induced by glutamate. Excitotoxic injury in this system due to high concentrations of glutamate resembles necrosis while the other agonists produce a different form of cell death which is neither necrosis nor apoptosis.

Traumatic neuronal injury in cortical cell culture is attenuated by 21-aminosteroids.

The effect of the 21-aminosteroids U74500A and U74389F, alone and in combination with the NMDA receptor antagonist MK-801, on traumatic neuronal injury was quantitatively assessed in murine neocortical cell cultures. Consistent with prior observations, a mechanical insult to the culture monolayer resulted in widespread neuronal death over the following 24 h. Treatment with either U74500A or U74389F provided moderate protection, reducing neuronal death as measured by lactate dehydrogenase release by 25-50%. This effect was most consistent when these agents were preincubated for 2 h prior to injury. Combined treatment with a 21-aminosteroid plus the NMDA receptor antagonist MK-801 reduced injury more than either drug alone. Approximately 40% of the neuronal death occurring in the presence of MK-801 was blocked by concomitant treatment with 10 microM U74500A or U74389F. These results suggest that free radicals may contribute to cell death in this in vitro model of traumatic neuronal injury.

Assessment of hemoglobin-dependent neurotoxicity: alpha-alpha crosslinked hemoglobin.

Adult human hemoglobin A0 (HbA0) has been shown to be neurotoxic, and we wish to report on similar studies conducted using a modified hemoglobin, which has been crosslinked between the alpha subunits (alpha-alpha Hb). Cortical cell cultures were prepared from fetal Swiss-Webster mice at 15-16 days gestation. Mature cultures (days in vitro, 12-16) were exposed to alpha-alpha Hb in a defined medium for 24-48 hours at 37 degrees C. Low micromolar amounts of alpha-alpha Hb were neurotoxic in a concentration-dependent fashion. This toxicity was attenuated by the antioxidants Trolox and U-74500A and by the iron chelator deferoxamine. The hemoglobin-binding protein, haptoglobin, also completely blocked alpha-alpha Hb-dependent neurotoxicity. The latter result was unexpected because complex formation between alpha-alpha Hb and haptoglobin was not detected using assays of haptoglobin fluorescence and hemoglobin peroxidase activity.

The effect of NMDA, AMPA/kainate, and calcium channel antagonists on traumatic cortical neuronal injury in culture.

A traumatic insult was delivered to murine cortical neuronal and glial cell cultures by tearing the cell layer with a stylet in a grid pattern. Consistent with prior observations, neurons adjacent to a tear developed immediate swelling, and then went on to degenerate over the next several hours. Delivery of multiple tears produced enough cell death that measurable levels of lactate dehydrogenase accumulated in the bathing medium 24 h later, correlating well with the extent of cell death as assessed by Trypan blue exclusion and cell counts. 50-75% of this trauma-induced cell death was blocked by the NMDA receptor antagonist MK-801. 10-100 microM CNQX also attenuated neuronal degeneration, but this neuroprotective effect was likely due to attenuation of NMDA receptor-mediated toxicity, since the more specific AMPA/kainate antagonist NBQX was ineffective. CNQX also did not augment the protective effect of MK-801. High concentrations of nimodipine or nifedipine produced modest neuroprotective effects; either dihydropyridine when combined with MK-801 reduced injury more than MK-801 alone. These results suggest that traumatic neuronal death in this in vitro model is mediated in part by excessive activation of NMDA receptors, and in part by mechanisms sensitive to high concentrations of dihydropyridines, but not by AMPA/kainate receptors.

NMDA neurotoxicity in murine cortical cell cultures is not attenuated by hemoglobin or inhibition of nitric oxide synthesis.

The role of nitric oxide in N-methyl-D-aspartate (NMDA) neurotoxicity was investigated in murine cortical cell cultures. Exposure of cultures to 300 microM NMDA for 5 min resulted in death of 50-80% of neurons over the subsequent 24 h. This injury was not attenuated by hemoglobin, the nitric oxide synthase (NOS) inhibitors NG monomethyl-L-arginine (MMA) or N omega-nitro-L-arginine (NA), or L-arginine depletion. Hemoglobin and NOS inhibitors consistently prevented the increase in cyclic guanosine monophosphate (cGMP) seen after NMDA exposure. These results suggest that NMDA neurotoxicity in this cell culture system is mediated, at least in part, by mechanisms other than NOS activation.

Neurotoxicity of hemoglobin in cortical cell culture.

Hemoglobin (Hb) has been demonstrated to be neurotoxic when injected into the cerebral cortex in vivo. However, associated systemic factors such as ischemia and epileptogenesis have limited investigations of Hb toxicity in the intact central nervous system (CNS). In this study, the neurotoxicity of human Hb was assessed in mixed neuronal and glial neocortical cell cultures derived from fetal mice. Exposure of cultures to Hb for 24-28 h produced widespread and concentration-dependent neuronal death (EC50 1-2.5 microM), without injuring glia. Brief exposures (1-2 h) were not toxic. Neuronal death was completely blocked by the 21-aminosteroid U74500A, the antioxidant Trolox, and the ferric iron chelator deferoxamine. The results of these experiments suggest that, in this system, Hb is a potent neurotoxin, and that Hb neurotoxicity may contribute to secondary injury processes after trauma and intracranial hemorrhage.

Glutamate neurotoxicity in spinal cord cell culture.

The neurotoxicity of glutamate was investigated quantitatively in mixed neuronal and glial spinal cord cell cultures from fetal mice at 12-13 days of gestation. Five-minute exposure to 10-1000 microM glutamate produced widespread acute neuronal swelling, followed by neuronal degeneration over the next 24 h (EC50 for death about 100-200 microM); glia were not injured. Glutamate was neurotoxic in cultures as young as four days in vitro, although greater death was produced in older cultures. By 14-20 days in vitro, 80-90% of the neuronal population was destroyed by a 5-min exposure to 500 microM glutamate. Acute neuronal swelling following glutamate exposure was prevented by replacement of extracellular sodium with equimolar choline, with minimal reduction in late cell death. Removal of extracellular calcium enhanced acute neuronal swelling but attenuated late neuronal death. Both acute neuronal swelling and late degeneration were effectively blocked by the noncompetitive N-methyl-D-aspartate receptor antagonist dextrorphan and by the novel competitive antagonist CGP 37849. Ten micromolar 7-chlorokynurenate also inhibited glutamate neurotoxicity; protection was reversed by the addition of 1 mM glycine to the bathing medium. These observations suggest that glutamate is a potent and rapidly acting neurotoxin on cultured spinal cord neurons, and support involvement of excitotoxicity in acute spinal cord injury. Similar to telencephalic neurons, spinal neurons exposed briefly to glutamate degenerate in a manner dependent on extracellular Ca2+ and the activation of N-methyl-D-aspartate receptors.