Cultured neurons for testing antihypoxic drug effects.

Cultured neurons of chick embryo cerebral hemispheres were used as an in vitro system for investigating the influence of several drugs on neuronal cell viability and metabolic activity under hypoxic conditions. Hypoxia was induced by addition of sodium cyanide to the nutrient medium, which led to a rapid depletion of energy stores. The ATP level of the cells and the protein content of the cultures were used to characterize the degree of neuronal damage after cytotoxic hypoxia and recovery, respectively, recovery lasting 15 min or 3 days. Various calcium antagonists, NMDA-antagonists, central depressants, central stimulants, nootropics, and miscellaneous drugs were tested. NMDA-antagonists and central depressants consistently protected the neurons against alterations caused by hypoxia. However, only one (flunarizine) out of five calcium antagonists, two (naftidrofuryl, pyritinol) out of 13 nootropics, the kappa-agonist ketazocine, and the ATPase inhibitor ouabaine exerted neuroprotection. The in vitro model seems to be suitable for testing neuroprotective drug effects and to be a valuable supplement for in vivo experiments, especially when the cellular mechanism of drug action has to be clarified.

Effects of delayed administration of methohexital and ketamine on posthypoxic cell damage of primary neuronal cultures.

Primary neuronal cultures from chick embryo cerebral hemispheres received NaCN (cytotoxic hypoxia) for 120 min and were then allowed to recover. Methohexital (300 mumol/l) or ketamine (30 mumol/l) given either before or during the hypoxic period elevated adenosine triphosphate (ATP) content of the cultures 15 min after hypoxia. Prehypoxic administration of ketamine also preserved the structural integrity and ATP content of neurons 20 h later, while methohexital did not. Ketamine elevated ATP content as measured 20 h after hypoxia even when administered 15 min after beginning of recovery. Pharmacokinetic reasons for contradictory effects of ketamine on neuronal cell loss in vivo ischemia studies and our in vitro experiments are discussed.

Cultured neurons for testing cerebroprotective drug effects in vitro.

An attempt was made to establish hypoxic cultured neurons from chick embryo hemispheres as an in vitro model for testing cerebroprotective drug effects. Hypoxia was induced by adding 1 mmol/l KCN to the incubation medium of the cells. After various time periods (15-120 min), the cyanide-containing medium was replaced by fresh medium so that the cells could recover from hypoxia. High-energy phosphate levels of the cells and the protein content of the cultures were determined as indicators of the cell viability and response. The cells showed a rapid and pronounced decrease in the levels of high-energy phosphates during cytotoxic hypoxia, and after removal of cyanide the energy state was restored again within few minutes. The protein content of the cultures remained unchanged. Pentobarbital inhibited the breakdown of the high-energy phosphates during hypoxia and accelerated their restitution during the recovery period. The results suggest that hypoxic cultured neurons could be a useful model for testing cerebroprotective drug effects.