(Stroke. 1996;27:1586-1591.)
© 1996 American Heart Association, Inc.
Articles |
Potentiation of Oxygen-Glucose Deprivation–Induced Neuronal Death After Induction of iNOS
the Department of Neurology and Center for the Study of Nervous System Injury, Washington University School of Medicine, St Louis, Mo.
Correspondence to Dennis W. Choi, MD, PhD, Department of Neurology and Center for the Study of Nervous System Injury, Washington University School of Medicine, 660 S Euclid Ave, St Louis, MO 63110. E-mail choid@neuro.wustl.edu.
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Abstract |
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Background and Purpose Previous studies have shown that brain ischemia and other insults can induce a marked increase in inducible nitric oxide synthase (iNOS) expression in astrocytes and some immune cells, but the biological significance of this phenomenon has not been elucidated. The purpose of the present study was to determine whether this induction of astrocyte iNOS alters neuronal vulnerability to severe hypoxic insults.
Methods Astrocytic iNOS was induced by exposure of murine cortical cultures to interferon gamma in combination with either interleukin-1ß or lipopolysaccharide. Cultures were exposed to combined oxygen-glucose deprivation. The extracellular concentration of glutamate was measured by high-performance liquid chromatography. N-Methyl-D-aspartate (NMDA) receptor activity was assessed by measurement of 45Ca2+ influx; neuronal death was assessed by morphological examination and quantitated by measurement of lactate dehydrogenase efflux to the bathing medium.
Results In murine neocortical cell cultures containing neurons and astrocytes, neuronal injury induced by combined oxygen-glucose deprivation was not reduced by the addition of the nitric oxide synthase inhibitors NG-nitro-L-arginine or LG-nitro-arginine methyl ester. However, after induction of astrocyte iNOS activity with interferon gamma plus lipopolysaccharide or interleukin-1ß, oxygen-glucose deprivation–induced neuronal injury was markedly enhanced and nitric oxide synthase inhibitors became protective. This iNOS-mediated potentiation was associated with a large increase in both extracellular glutamate accumulation and 45Ca2+ influx into neurons. The potentiation could be blocked by MK-801 but not CNQX, suggesting critical involvement of NMDA receptor activation.
Conclusions These results support the idea that nitric oxide production mediated by induced astrocytic iNOS can potentiate NMDA receptor–mediated neuronal death consequent to hypoxic-ischemic insults.
Key Words: anoxia • astrocytes • hypoxia • neuronal death • nitric oxide
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Nitric oxide is synthesized from L-arginine by NOS, an enzyme with constitutive and inducible isoforms (for review, see References 1 and 2). In brain, two distinct constitutive, Ca2+-dependent isoforms are expressed, one in distinct neurons3 (nNOS; type I) and another in endothelial cells4 (eNOS; type III). Normally, the constitutive forms of NOS produce NO transiently in response to agonist stimulation.2 5 Expression of the Ca2+-independent, inducible isoform6 (iNOS; type II) can be induced in some brain cells after cytokine stimulation in vitro7 8 9 10 or inflammation in vivo.11 12 13 14 15 Once induced, iNOS is continuously active, producing large quantities of NO.16
Convincing evidence has implicated NO derived from nNOS in the pathogenesis of NMDA receptor–mediated excitotoxicity in vitro17 and focal hypoxic-ischemia brain damage in vivo.18 In addition, some19 20 but not all21 22 studies have shown protective effects of nonselective NOS inhibitors in animal models of brain hypoxia-ischemia, although effects on blood flow were not excluded.
We previously reported that NO derived from astrocyte iNOS, induced by exposure to IFN-
plus IL-1ß, can also contribute to NMDA receptor–mediated excitotoxicity in vitro.23 This contribution may be relevant to brain injury in vivo. NADPH-diaphorase staining, a histochemical marker for NOS,24 is induced in murine astrocytes after cerebral stab wounds,25 transient focal cerebral ischemia,12 and excitatory amino acid injection.12 Immunohistochemical studies have confirmed specifically that the NADPH-diaphorase activity in astrocytes represented iNOS12 13 ; increased iNOS expression was also found in some macrophages/microglia.12
Implication of injury-induced iNOS expression in the pathogenesis of ischemic brain injury was recently provided by a study showing that the iNOS selective inhibitor, aminoguanidine, attenuated cerebral damage even when administered 24 hours after a permanent focal cerebral ischemic insult in rats.26 In that model, iNOS expression was detected in neutrophils but not astrocytes.15 However, as summarized above, astrocytic iNOS expression has been detected in other brain injury models. Thus, we set out to determine whether induction specifically of astrocyte iNOS could alter neuronal vulnerability to anoxic insults. This material has been published in abstract form.27
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Materials and Methods |
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Cell Culture
Primary cortical cell cultures containing both neuronal and glial elements were prepared from fetal mice at 15 to 16 days of gestation in accordance with institutional guidelines as set forth by the National Institutes of Health, as described previously.23 28 Dissociated cortical cells were plated at approximately three hemispheres per 24-multiwell (15-mm) vessels (Primaria; Falcon) on a previously established bed of glia (see below) in modified Eagle's MEM (Earle's salts, supplied bicarbonate- and glutamine-free) supplemented with horse serum (5%, vol/vol), fetal calf serum (5%, vol/vol), glutamine (2 mmol/L), bicarbonate (26.2 mmol/L), and glucose (15 mmol/L; total=20 mmol/L). Cultures were maintained at 37°C in a humidified, 5% CO2-containing incubator. Nonneuronal cell division was halted after 5 to 7 days in vitro by 1 to 3 days of exposure to 10 µmol/L cytosine arabinosine. Cultures were subsequently fed twice weekly with a medium identical to the plating medium except lacking fetal calf serum, and they were used for experimentation between 13 and 16 days in vitro.
Astrocyte cultures were prepared similarly from 1- to 3-day-old mice neocortices. Cortices were plated at 0.5 to 1 hemisphere per 24-multiwell vessel in a medium similar to that described above but containing 10% (vol/vol) horse serum, 10% (vol/vol) fetal calf serum, and 10 ng/mL epidermal growth factor. Purity of cultures (>97%) was ascertained morphologically and immunocytochemically as described.9
Induction of iNOS
Astrocytic iNOS was induced as described previously.9 23 Briefly, 24 to 30 hours before oxygen-glucose deprivation, cells were exposed to IFN- (200 to 400 U/mL) in combination with IL-1ß (250 pg/mL) or LPS (5 µg/mL) in an incubation medium of MEM containing 10 µmol/L glycine and 1% (vol/vol) serum (0.5% [vol/vol] fetal calf serum plus 0.5% [vol/vol] horse serum) at 37°C in an incubator containing 5% CO2.
Combined Oxygen-Glucose Deprivation
Cultures were placed in an anaerobic chamber (Forma Scientific) that contained a gas mixture of 5% CO2, 10% H2, and 85% N2 (<0.2% O2).29 Culture medium was replaced by thorough exchange with deoxygenated, glucose-free balanced salt solution containing the following (mmol/L): 116 NaCl, 5.4 KCl, 0.8 MgSO4, 1 NaH2PO4, 26.2 NaHCO3, 1.8 CaCl2, 0.01 glycine, and 0.6 L-arginine; plates were then placed in a 37°C humidified incubator within the chamber for 40 to 50 minutes. Cultures were subsequently removed from the incubator, the exposure medium was exchanged with oxygenated MEM, and cultures were returned to a 37°C 5% CO2–containing normoxic (21% O2) incubator. The medium was supplemented with L-arginine to prevent this amino acid substrate of NOS from being limiting.
Assessment of Neuronal Injury
Neuronal cell death was estimated by examination of cultures under phase-contrast microscopy and quantitated by measurement of LDH released by damaged or destroyed cells in the bathing media 20 to 24 hours after experimentation.30 The LDH signal corresponding to near-complete neuronal death without glial degeneration (total LDH) was measured in sister cultures exposed to 500 µmol/L NMDA for 24 hours. Basal LDH levels determined in sister cultures subjected to sham wash (generally <15% of total LDH) were subtracted from values obtained in experimental conditions to yield the signal specific to experimental injury.
Measurement of NO
NO production was determined by measurement of nitrite,31 a stable oxidative breakdown product of NO, immediately before oxygen-glucose deprivation. Nitrite levels were assayed by mixing 100-µL portions of culture medium with 100 µL of Greiss reagent (1:1; 1% [wt/vol] sulfanilamide in 60% [vol/vol] acetic acid plus 0.1% [wt/vol] napthylenediamine dihydrochloride in distilled water), and absorbance at 540 nm was determined on a microtiter plate reader (UVmax; Molecular Devices).
HPLC Quantitation of Extracellular Glutamate
Samples of the bathing medium collected before the termination of oxygen-glucose deprivation were assayed for glutamate by means of phenylisothiocyanate derivatization, HPLC reverse-phase separation, and ultraviolet detection at a wavelength of 254 nm.32 Then 200 µL of buffer was derivatized with 100 µL of phenylisothiocyanate, methanol, and triethylamine (1:4:2) and dried under vacuum. Before the HPLC run, the samples were reconstituted in solvent consisting of 0.14 mol/L sodium acetate, 0.05% (vol/vol) triethylamine, and 6% (vol/vol) acetonitrile and brought to pH 6.4 with glacial acetic acid. The column was washed with 60% (vol/vol) acetonitrile/40% (vol/vol) water between samples.
Measurement of Cellular 45Ca2+ Accumulation
Cultures were deprived of oxygen and glucose as described above, with the inclusion of specified drugs and 45Ca2+ (New England Nuclear; final activity, 2 µCi/mL) in the exposure medium.29 Immediately after the deprivation period, the exposure solution was washed out with four rinses of balanced salt solution to remove any residual extracellular 45Ca2+, and the cells were lysed by addition of warm 0.2% (wt/vol) sodium dodecyl sulfate. An aliquot of cell lysate was added to scintillation fluid for ß-counting. We have previously demonstrated that the cellular accumulation of 45Ca2+ in this model is mostly neuronal.33
Reagents
Culture medium was purchased from GIBCO as a 10X concentrated stock lacking bicarbonate and glutamine; serum was obtained from Hyclone Laboratories Inc. NMDA, phenylisothiocyanate, and amino acid standards were obtained from Sigma Chemical Co. MK-801 and N-Arg were purchased from Research Biochemicals Inc. NAME was obtained from Biomol, and CNQX was purchased from Tocris Neuramin. Recombinant mouse IL-1ß and IFN- were obtained from Genzyme, and LPS was obtained from Difco Co.
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Mixed neuronal and astrocyte cortical cell cultures exposed to LPS (5 µg/mL) plus IFN-
(400 U/mL) for 24 to 30 hours responded with an increase in NO production, reflected as accumulated nitrite in the bathing medium (Fig 1
). This increased production of NO did not itself cause any neuronal damage, as assessed by phase-contrast microscopy and quantitated by the release of LDH into the bathing medium (Fig 1). We showed previously that this enhanced NO production reflected the new expression of the inducible isoform of NOS (iNOS) in astrocytes but not neurons23 ; exposure to IL-1ß (250 pg/mL) plus IFN- (200 U/mL) was also effective in inducing astrocytic iNOS expression.9 23 No iNOS expression was observed in unstimulated control cultures,23 as further indicated here by lack of NO production (Fig 1).
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Mixed cultures exposed for 40 to 50 minutes to combined oxygen-glucose deprivation developed intermediate levels of neuronal death without astrocyte death by the next day.29 This death was not significantly reduced by addition of the NOS inhibitors NAME (5 mmol/L) or N-Arg (1 mmol/L) (Fig 2). However, after induction of astrocyte iNOS by LPS/IFN- or IL-1ß/IFN-, a treatment that is not itself toxic, the same exposure to oxygen-glucose deprivation caused markedly greater neuronal death, and this potentiated death was now sensitive to NOS inhibitors (Fig 2).
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Consistent with previous data,29 neuronal loss induced by combined oxygen-glucose deprivation in control cultures was attenuated by addition of the NMDA antagonist MK-801 (10 µmol/L), but not the AMPA/kainate receptor antagonist CNQX (30 µmol/L) (Fig 3), to the exposure medium. The potentiated oxygen-glucose deprivation–induced neuronal injury seen after iNOS induction was also attenuated by MK-801 but not CNQX (Fig 3). A similar pharmacological profile was observed after induction of astrocytic iNOS with IFN-/IL-1ß (data not shown).
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In addition to increasing oxygen-glucose deprivation–induced neuronal death, astrocytic iNOS induction also increased the associated buildup of glutamate in the bathing medium and the neuronal accumulation of 45Ca2+33 measured at the end of the deprivation period (Figs 3 and 4
). This increase in glutamate buildup was not due to early neuronal cell death/lysis. Although neurons were swollen, they were intact under phase-contrast microscopy, and little LDH release occurred by that time point (data not shown). Consistent with its neuroprotective action, MK-801 but not CNQX attenuated both the buildup of extracellular glutamate (Fig 3) and the increase in neuronal 45Ca2+ accumulation (Fig 4).
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A good correlation between NO formation, extracellular glutamate accumulation, and neuronal death persisted when sufficient N-Arg (1.5 mmol/L) was added to reduce NO formation by approximately half (Table).
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Discussion |
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Excess efflux of glutamate into the extracellular space with resultant excitotoxic neuronal injury has been proposed to contribute importantly to the pathogenesis of hypoxic-ischemic neuronal damage.34 35 In addition, elevated concentrations of cytokines have been found in brain after both cerebral ischemia and trauma.36 37 38 39 The possibility that these cytokines may participate in subsequent neurodegeneration has been raised, perhaps in part through the induction of NOS (iNOS), leading to increased NO.12 13 14 15
The central finding of the present in vitro study is that induction of astrocytic iNOS, achieved by exposure to the proinflammatory mediator IFN- in combination with either IL-1ß or LPS, enhances cortical neuronal vulnerability to death after combined oxygen-glucose deprivation. This enhanced death was associated with increases in NO formation, extracellular glutamate buildup, and neuronal 45Ca2+ accumulation.
Consistent with the study of Demerle-Pallardy and colleagues40 in whole brain cultures, NOS inhibitors did not affect the neuronal loss induced by combined oxygen-glucose deprivation in control cultures, presumably indicating lack of participation of nNOS. In contrast, Cazevielle and colleagues41 did see a protective effect of an NOS inhibitor on neuronal injury induced by anoxia in near-pure cortical neuronal cultures. Similar variability has been reported with regard to the effect of NOS inhibitors against excitotoxic injury in cultures, with the positive findings of Dawson and coworkers17 confirmed in some42 43 but not all in vitro systems,40 44 the latter including our system.28
In our view, the most likely explanations for the variability of nNOS contribution to oxygen-glucose deprivation–induced or exogenous excitotoxin–induced neuronal death seen in different culture preparations are a greater presence of astrocytes or a smaller number of NOS-containing neurons in cultures in which NOS inhibitors are not neuroprotective. Astrocytes may be a target for neuronally produced NO,45 and this may limit its availability to participate in neurotoxic reactions. As we have previously noted, at least our culture system has fewer nNOS-containing (NADPH-diaphorase–positive) neurons than that of other systems.17 28 The ability of astrocytic iNOS induction to bring out an NOS inhibitor–sensitive component of excitotoxic23 or oxygen-glucose deprivation–induced (present results) neuronal death supports the idea that a threshold level of NO production is required to contribute to this death. In addition, present results go beyond simple augmentation of culture NO levels to support the specific hypothesis that cytokine-induction of astrocytic iNOS, as occurs in brain after acute insults, can markedly potentiate neuronal vulnerability to oxygen-glucose deprivation–induced death.
While NO has been reported to be toxic to cells through several different mechanisms,46 47 48 49 we demonstrate here that NO triggered by iNOS induction may contribute to an enhancement of oxygen-glucose deprivation–induced neuronal death through the potentiation of NMDA receptor– but not AMPA/kainate receptor–mediated excitotoxicity. This is consistent with our previous finding that direct NMDA but not kainate neurotoxicity was potentiated by prior induction of astrocytic iNOS.23
Further studies will be needed to determine the exact mechanisms by which enhanced astrocytic NO production can potentiate the NMDA receptor–mediated excitotoxicity induced by oxygen-glucose deprivation. The possibility that this potentiation occurs strictly downstream from NMDA receptor activation is unlikely given that extracellular glutamate (measured early, before neuronal cell lysis) is also increased. While bath glutamate concentrations never exceeded the low micromolar range, higher concentrations may have accumulated local to synaptic regions. Furthermore, the glutamate concentrations needed to kill energy-depleted neurons are far less than those required to kill healthy neurons.50 Thus, we propose that iNOS-derived NO may contribute to oxygen-glucose deprivation–induced excitotoxicity in part by reducing the cellular reuptake of glutamate (perhaps by oxidative stress51 ) or by enhancing excitatory amino acid release.52 53
Interestingly, MK-801 attenuated the buildup of extracellular glutamate induced by oxygen-glucose deprivation in both control (Reference 54 and this study) and in iNOS-induced cultures. This observation suggests the existence of a positive feedback loop, whereby NMDA receptor activation increases the buildup of extracellular glutamate, which further increases NMDA receptor activation. One possible mechanism might involve presynaptic NMDA receptors, acting to enhance glutamate release55 56 with further amplification by NO.52 57 58 Alternatively, such a loop could be provided through postsynaptic NMDA receptors, acting to injure or Na+/Ca2+-load neuronal cells sufficiently to impair glutamate uptake and enhance glutamate efflux.
Regardless of the exact mechanisms linking astrocytic iNOS activity, NMDA receptor activation, and neuronal death, the present study supports the intriguing possibility that astrocytic iNOS induction after an initial brain insult—including ischemia—could have the dangerous consequence of enhancing excitotoxic brain injury after subsequent ischemic insults.
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Selected Abbreviations and Acronyms |
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-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
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Acknowledgments |
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This study was supported by National Institutes of Health research grant NS-32636 (Dr Choi). We thank Aghdas Jafari for technical support.
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Footnotes |
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From the Department of Neurology and Center for the Study of Nervous System Injury, Washington University School of Medicine, St Louis, Mo.
Received February 13, 1996; revision received April 23, 1996; accepted May 22, 1996.
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Editorial Comment
Stroke and Neurovascular RegulationMassachusetts General HospitalHarvard Medical SchoolBoston, Mass
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Events occurring over hours to days after hypoxic-ischemic injury are poorly understood and importantly relate to programmed cell death,1R inflammation,2R tissue reorganization, neuronal plasticity, and recovery of function.3R Not surprisingly, glial cells, the most abundant cell type in brain, have been implicated in several of these processes. The accompanying article by Hewett and colleagues describes a potentially important mechanism of cell killing that requires the expression of an enzyme, iNOS, that is both transcriptionally and translationally dependent.4R In the presence of an elevated basal NO synthesis by iNOS, injury after oxygen-glucose deprivation is worsened. Their findings complement existing reports emphasizing the importance of nNOS5R to cytotoxicity during the earliest stages of ischemia, plus the importance of eNOS to sustain vascular hemodynamics in ischemic tissue.6R 7R Together they suggest the potential therapeutic merits of applying selective inhibitors of either nNOS (type I) or iNOS (type II) proteins at distinct times during the evolution of hypoxic-ischemic injury.
Hewett and colleagues also propose a novel mechanism of NO toxicity based on the ability of this gaseous modulator to enhance glutamate release or block its uptake. The concept is important and warrants testing in in vivo models of brain injury, despite the fact that NMDA receptor blockers reportedly exhibit a relatively short (
6 hours) therapeutic window in focal cerebral ischemia.
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Cultures were treated as described in Fig 1 except that the NOS inhibitor was N-Arg (1.5 mmol/L). Data are mean±SEM of 4 to 16 cultures per condition, pooled from four separate experiments. Nitrite levels were determined 24 to 30 hours after preexposure to the indicated agents, then oxygen-glucose deprivation (OGD) was performed. Glutamate accumulation was measured at the end of OGD; neuronal injury was assessed 20 to 24 hours thereafter. Percent inhibition is the reduction of LPS/IFN-–stimulated values blocked by N-Arg.
*Values greater than oxygen-glucose deprivation alone; 
N-Arg–induced diminution of the cytokine-induced potentiation, as determined by one-way ANOVA followed by the Student-Newman-Keuls multiple comparisons test (P<.05).
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1R. Chopp M, Chan PH, Hsu CY, Cheung ME, Jacobs TP. DNA damage and repair in central nervous system injury: National Institute of Neurological Disorders and Stroke workshop summary. Stroke.. 1996;27:363-369.
2R. Feuerstein GZ, Wang X, Yue TL, Barone FC. Inflammatory cytokines and stroke: emerging new strategies for stroke therapeutics. In: Moskowitz MA, Caplan LR, eds. Cerebrovascular Diseases: Nineteenth Princeton Stroke Conference. Boston, Mass: Butterworth-Heinemann; 1995:75-92.
3R. Johansson BB, Grabowski M. Functional recovery after brain infarction: plasticity and neural transplantation. Brain Pathol.. 1994;4:85-95.[Medline] [Order article via Infotrieve]
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Nathan C, Xie Q. Regulation of biosynthesis of nitric oxide. J Biol Chem.. 1994;269:13725-13728.
5R. Huang Z, Huang PL, Panahian N, Dalkara T, Fishman MC, Moskowitz MA. Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science.. 1994;265:1883-1885.
6R. Huang Z, Huang PL, Ma J, Meng W, Ayata C, Fishman MC, Moskowitz MA. Enlarged infarcts in endothelial nitric oxide synthase knockout mice are attenuated by nitro-L-arginine. J Cereb Blood Flow Metab. In press.
7R. Lo EH, Hara H, Rogowska J, Trocha M, Pierce AR, Huang PL, Fishman MC, Wolf GL, Moskowitz MA. Temporal correlation mapping analysis of the hemodynamic penumbra in mutant mice deficient in endothelial nitric oxide synthase gene expression. Stroke.. 1996;27:1382-1386.
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