Neocortical Cell Cultures Research Articles

NMDA induces NO release from primary cell cultures of human fetal cerebral cortex

We and others have previously reported that N-methyl- d-aspartate (NMDA) induces nitric oxide (NO) release from the rat cerebral cortex in vivo. It is crucial to determine if this phenomenon also exists in human brain tissue. In this study, we investigated the interactions of NMDA and NO in human primary neocortical cell cultures obtained from elective abortions. Extracellular NO concentration was monitored through Nafion- and porphyrine-coated carbon fiber electrodes, which have previously been demonstrated sensitive and selective responses to NO. We found that local application of NMDA induced NO release from neocortical neuron-enriched cultures but not from glial cultures. Pretreatment with N G-nitro- l-arginine methyl ester hydrochloride ( l-NAME), a nitric oxide synthase inhibitor, or MK801 significantly attenuated NMDA-induced NO overflow. This is the first time, to our knowledge, that extracellular NO concentration evoked by exogenous NMDA has been directly measured from the fetal human cortical neurons.

Potentiation of oxygen-glucose deprivation-induced neuronal death after induction of iNOS.

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. Astrocytic iNOS was induced by exposure of murine cortical cultures to interferon gamma in combination with either interleukin-1 beta 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. 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-1 beta, 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. 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.

Correlation of CGS 19755 neuroprotection against in vitro excitotoxicity and focal cerebral ischemia.

The in vivo neuroprotective effect and brain levels of cis-4-phosphonomethyl-2-piperidine carboxylic acid (CGS 19755), a competitive N-methyl-D-aspartate (NMDA) antagonist, were compared with its in vitro neuroprotective effects. The dose-response for in vitro neuroprotection against both NMDA toxicity and combined oxygen-glucose deprivation (OGD) was determined in murine neocortical cultures. Primary cultures of neocortical cells from feta mice were injured by exposure to 500 microM NMDA for 10 min or to OGD for 45 min. The effect of CGS 19755 in both injury paradigms was assessed morphologically and quantitated by determination of lactate dehydrogenase release. Near complete neuroprotection was found at high doses of CGS 19755. The ED50 for protection against NMDA toxicity was 25.4 micro M, and against OGD the ED50 was 15.2 microM. For the in vivo paradigm rabbits underwent 2 h of left internal carotid, anterior cerebral, and middle cerebral artery occlusion followed by 4 h reperfusion; ischemic injury was assessed by magnetic resonance imaging and histopathology. The rabbits were treated with 40 mg/kg i.v. CGS 19755 or saline 10 min after arterial occlusion. CSF and brain levels of CGS 19755 were 12 microM and 5 microM, respectively, at 1 h, 6 microM and 5 microM at 2 h, and 13 microM and 7 microM at 4 h. These levels were neuroprotective in this model, reducing cortical ischemic edema by 48% and ischemic neuronal damage by 76%. These results suggest that a single i.v. dose penetrates the blood-brain barrier, attaining sustained neuroprotective levels that are in the range for in vitro neuroprotection.

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 μM U74500A or U74389F. These results suggest that free radicals may contribute to cell death in this in vitro model of traumatic neuronal injury.

Combined oxygen and glucose deprivation in cortical cell culture: calcium-dependent and calcium-independent mechanisms of neuronal injury

Murine neocortical cell cultures were transiently deprived of both oxygen and glucose, producing widespread neuronal swelling in less than 60 min, followed by neuronal degeneration over the ensuing several hours, despite return to normal medium. Cultured glia (> 95% astrocytes) were irreversibly injured only by oxygen-glucose deprivation exposures exceeding 4-6 hr. Replacing either Na+ or Cl- with impermeant ions blocked acute neuronal swelling but did not prevent delayed neuronal degeneration. While neuronal swelling and death were increased by removing Ca2+ from the exposure medium, combined removal of extracellular Ca2+ together with Na+ or Cl- substitution blocked both acute and delayed injury. If acute swelling was limited by a hyperosmolar medium, then neuronal loss depended on extracellular [Ca2+]. Oxygen-glucose deprivation was associated with a large increase in extracellular glutamate concentration. Both early swelling and later neuronal degeneration were blocked by addition of NMDA receptor antagonists to the exposure medium but not by the AMPA/kainate receptor antagonist 6-cyano-7-dinitroquinoxaline-2,3-dione (CNQX), dihydropyridines nifedipine or nimodipine, or TTX. Oxygen-glucose deprivation induced substantial neuronal uptake of tracer 45Ca2+ from the exposure medium that was reduced by NMDA receptor antagonists and closely paralleled the degree of subsequent neuronal loss. These observations suggest the presence of two distinct components of hypoxic injury, each involving NMDA receptor activation and each capable of leading to neuronal death. Acute swelling is mediated by influx of Na+, Cl-, and water, and is enhanced by removal of extracellular Ca2+. Delayed neuronal degeneration depends on the presence of extracellular Ca2+ and correlates closely with cellular uptake of 45Ca2+.

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 (EC 50 1–2.5 μM), 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.

Adrenocorticotropin interferes with transforming growth factor-beta-induced growth inhibition of neocortical cells from the human fetal adrenal gland.

The effects of transforming growth factor-beta (TGF-beta) and ACTH on growth, as indicated by [3H]thymidine incorporation into DNA, of primary cultures of neocortical cells from the human fetal adrenal gland were studied. TGF-beta inhibited, in a dose- and time-dependent manner, the growth of fetal neocortical cells, and ACTH significantly blunted the inhibitory effects of TGF-beta on growth of these cells. ACTH did not block the inhibitory effects of TGF-beta on growth of fetal adrenal fibroblasts or liver cells; neither ACTH nor TGF-beta had any effect on growth of fetal kidney cells. Thus, it appears that growth regulation of the neocortex may differ strikingly from that of the fetal zone of the human fetal adrenal, in which ACTH and TGF-beta have been reported recently to have additive inhibitory effects on cell proliferation.

7-Chlorokynurenate Blocks NMDA Receptor-Mediated Neurotoxicity in Murine Cortical Culture.

We examined the neuroprotective actions of the glycine site N-methyl-D-aspartate (NMDA) antagonist, 7-chlorokynurenate, in murine neocortical cell cultures. Cultures exposed for 5 min to 100 - 500 microM NMDA in the absence of added glycine developed substantial neuronal degeneration over the next 24 h. The addition of 10 microM glycine did not increase submaximal NMDA-induced neuronal injury, suggesting that endogenous glycine levels were sufficient to saturate its receptor sites on NMDA receptor complexes. Addition of 3 - 300 microM 7-chlorokynurenate produced concentration-dependent reduction in this neuronal damage with an IC50 of approximately 30 microM. Some injury reduction was seen even if the drug was added after completion of the NMDA exposure. The protective effect of 100 microM 7-chlorokynurenate could be overcome by adding 10 - 1000 microM glycine (glycine median effective concentration (EC50) approximately 100 microM) or 1 mM D-serine. As predicted by its ability to block NMDA receptor-mediated injury, 10 - 300 microM 7-chlorokynurenate also produced concentration-dependent reduction in the neuronal loss induced by 50 - 60 min exposure to combined glucose and oxygen deprivation. These data support the suggestion that pharmacologic interference with the binding of glycine to the NMDA receptor complex represents a potentially effective approach to blocking NMDA receptor-induced neurotoxicity in ischemia.

Traumatic neuronal injury in vitro is attenuated by NMDA antagonists

Pure traumatic neuronal injury was modeled in dispersed neocortical cell cultures derived from fetal mice. A plastic stylet was used to tear the neuronal and glial cell layer; medium oxygen content, pH, and glucose remained unchanged. Adjacent to this local disruption, many neurons developed acute swelling and went on to degenerate over the next day, but glia were relatively spared. If the same mechanical insult was delivered in the presence of the N-methyl- d-aspartate (NMDA) antagonists dextrorphan or d-2-amino-5-phosphonoval-erate, resultant neuronal degeneration was markedly reduced. The protective effect of these NMDA antagonists was concentration-dependent between 1 and 100 μM, with EC 50 near 10 μM for both compounds. Present findings suggest that endogenous excitatory amino acids may participate significantly in the propagation of central neuronal cell loss in response to a purely mechanical insult.

Dextrorphan and dextromethorphan attenuate glutamate neurotoxicity

The dextrorotatory morphinan opioid, dextrorphan, which has recently been reported to block the excitation of cortical neurons by N-methyl- d-aspartate, was found at 10-100 μM concentrations to attenuate both morphological and chemical evidence of glutamate neurotoxicity in murine neocortical cell cultures; a similar effect was found with its methyl ester derivative, dextromethorphan. Given other data suggesting that glutamate neurotoxicity may participate in the pathogenesis of the central neuronal loss associated with certain human neurological diseases, the present observations raise the possibility that these clinically tested opioids, or related compounds, may eventually prove to have some clinical therapeutic utility.

Ketamine protects cultured neocortical neurons from hypoxic injury

The general anesthetic ketamine, which has recently been reported to block the excitation of cortical neurons by N-methyl- d-aspartate (NMDA), was found to markedly reduce neuronal loss in murine neocortical cell cultures exposed to a hypoxic atmosphere or to cyanide. These observations may be relevant to attempts to find pharmacological means of minimizing hypoxic brain damage in the clinical setting.