Mark P. Goldberg

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.

Dextromethorphan reduces neocortical ischemic neuronal damage in vivo

The dextrorotatory morphinan dextromethorphan (DM), a clinically tested antagonist of the N-methyl-D-aspartate (NMDA) receptor-channel complex, was tested in an in vivo model of acute transient focal cerebral ischemia. Rabbits were randomly assigned to pretreatment with a 20 mg/kg i.v. bolus followed by 10 mg/kg/h of 0.4% DM in normal saline (NS), or with an equivalent volume of NS alone. They then underwent 1 h occlusion of the left internal carotid artery an anterior cerebral artery followed by 4 h of reperfusion. DM-treated animals showed a significant decrease in the percentage of severe neocortical ischemic neuronal damage (10.5%), as compared to NS-treated animals (49.6%).

Adenosine reduces cortical neuronal injury induced by oxygen or glucose deprivation in vitro

The endogenous neuromodulatory purine, adenosine, substantially attenuated neuronal degeneration when added to dissociated cortical cell cultures acutely deprived of either oxygen or glucose. The protective effect of adenosine, was concentration-dependent between 30 and 1000 microM (EC50 about 100 microM), and could be mimicked by the stable adenosine analogue N6-cyclohexyladenosine (10 microM). Unlike postsynaptic glutamate receptor antagonists, which also block these forms of neuronal injury, adenosine did not alter the neurotoxicity of exogenously applied glutamate.

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-phosphonovalerate, resultant neuronal degeneration was markedly reduced. The protective effect of these NMDA antagonists was concentration-dependent between 1 and 100 microM, with EC50 near 10 microM 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.

Antagonism of non-NMDA receptors augments the neuroprotective effect of NMDA receptor blockade in cortical cultures subjected to prolonged deprivation of oxygen and glucose.

A 30-60 min period of oxygen and glucose deprivation induced widespread degeneration of cultured murine neocortical neurons. Neuronal degeneration could be blocked by adding the selective NMDA antagonist MK-801 to the bathing medium; however, if the deprivation period was prolonged to 90-105 min, the neuroprotective effect of MK-801 was overcome. The non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) at 1-100 microM concentrations also failed to protect neurons against this prolonged insult, but the combination of CNQX with either MK-801 or D-APV produced marked neuroprotection. This synergistic action of CNQX was not due to enhanced blockade of NMDA receptors, as it was not mimicked by combining MK-801 with D-APV or 7-chlorokynurenate. These observations support the idea that combined NMDA and non-NMDA receptor blockade may have value in ameliorating the neuronal loss associated with prolonged ischemic insults in vivo.

Glucose deprivation neuronal injury in cortical culture

Murine cortical cell cultures deprived of glucose for 6-8 h developed extensive neuronal degeneration, apparent both morphologically and by efflux of lactate dehydrogenase to the bathing medium. This neuronal damage could be substantially reduced by addition of D-2-amino-5-phosphonovalerate (D-APV), in a concentration-dependent (IC50 about 2 microM) and stereospecific (D-APV more potent than L-APV) fashion. A similar neuron-protective effect could also be obtained with several other NMDA antagonists, 2-amino-7-phosphonoheptanoate, phencyclidine, MK-801, ketamine, and (+)-SKF 10,047, as well as with the broad spectrum glutamine antagonist kynurenate. In contrast, little protection could be obtained with gamma-D-glutamylaminomethyl sulfonate and L-glutamate diethyl ester, compounds which have been reported to act primarily at non-NMDA receptors. These observations support the hypothesis that glucose deprivation-induced cortical neuronal injury is largely mediated by NMDA receptors, and suggest that cell culture methodology can be useful in the quantitative characterization of that injury.

Oxygen or Glucose Deprivation-Induced Neuronal Injury in Cortical Cell Cultures Is Reduced by Tetanus Toxin

We examined glutamate-mediated neurotoxicity in cortical cell cultures pretreated with 1-5 micrograms/ml tetanus toxin to attenuate the Ca(2+)-dependent release of neurotransmitters. Efficacy of the tetanus toxin pretreatment was suggested by blockade of electrical burst activity induced by Mg2+ removal and by reduction of glutamate efflux induced by high K+. Tetanus toxin reduced neuronal injury produced by brief exposure to elevated extracellular K+ or to glutamate, situations in which release of endogenous excitatory neurotransmitter is likely to play a role. Furthermore, although glutamate efflux evoked by anoxic conditions may occur largely via Ca(2+)-independent transport, tetanus toxin attenuated both glutamate efflux and neuronal injury following combined oxygen and glucose deprivation. With prolonged exposure periods, the neuroprotective efficacy of tetanus toxin was comparable to that of NMDA receptor antagonists. Presynaptic inhibition of Ca(2+)-dependent glutamate release may be a valuable approach to attenuating hypoxic-ischemic brain injury.

Dextrorphan and dextromethorphan attenuate hypoxic injury in neuronal culture

The dextrorotatory opioid derivatives, dextrorphan and dextromethorphan, can attenuate hypoxic injury in cortical cell cultures. This effect is concentration-dependent in the micromolar range, and not strongly stereospecific, as it can also be demonstrated with the levorotatory enantiomer of dextrorphan, levorphanol. The possibility that these clinically available compounds may have therapeutic utility in hypoxic or ischemic encephalopathy warrants further investigation.

Hypoxic neuronal injury in vitro depends on extracellular glutamine

Hypoxic neuronal injury (HNI) in cortical cell cultures was enhanced in a concentration-dependent fashion by the presence of 500 microM to 2 mM (EC50 about 500 microM) glutamine in the medium, concentrations approximating those normally present in cerebrospinal fluid (CSF). Regardless of the glutamine concentration, glutamate receptor antagonists 2-amino-5-phosphonovalerate or dextrorphan could substantially reduce HNI. Thus, the availability of extracellular glutamine could be a determinant of hypoxic neuronal injury in vivo, most likely reflecting its importance in the synthesis of the neurotransmitter excitotoxins glutamate and aspartate.

Acute brain injury, NMDA receptors, and hydrogen ions: Observations in cortical cell cultures

Excess stimulation of NMDA receptors by endogenous glutamate likely contributes to the neuronal cell loss associated with several types of acute brain injury in vivo (Meldrum, 1985; Rothman and Olney, 1987, Choi, 1988), including ischemia (Simon et al., 1984), hypoglycemia (Wieloch, 1985), epilepsy (Labuyere et al., 1986) and trauma (Faden and Simon, 1988). Among the experiments supporting this statement are those studying the controlled delivery of insults to dispersed neuronal and glial cells in primary culture. Demonstration that a given pharmacological manipulation is neuroprotective in such cultures establishes that a beneficial effect can be produced directly on brain parenchyma, without involvement of systemic metabolism or alterations in blood flow. While organizational features of the intact nervous system are not expressed in cell culture, many intrinsic aspects of neuronal and glial cell behavior do appear to be qualitatively preserved. In particular, basic mechanisms relevant to glutamate transmission and glutamate neurotoxicity are present in cultured brain cells.