CHARACTERIZATION OF A CHEMICAL ANOXIA MODEL IN CEREBELLAR GRANULE NEURONS USING SODIUM-AZIDE - PROTECTION BY NIFEDIPINE AND MK-801

Induction of chemical anoxia, using sodium azide in cerebellar granule cells maintained in primary culture, was evaluated as an in vitro ass ay for screening of potential neuroprotective compounds. The purpose o f this study was to evaluate sodium azide as an alternative to cyanide salts, compounds which, despite their unfavorable characteristics, ar e often used in assays for chemical anoxia. The viability of neuronal cultures after treatment with azide, with or without preincubation wit h calcium channel blockers, tetrodotoxin (TTX), or glutamate receptor antagonists, was monitored by subsequent incubation with the tetrazoli um dye MTT (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) , followed by isopropanol extraction and spectrophotometric quantifica tion of cellularly reduced MTT. The azide-induced degeneration of neur ons was shown to be dependent on the concentration as well as on the d uration of incubation with submaximal concentrations of azide. Incubat ion of the neurons with nifedipine, a blocker of L-type voltage-sensit ive calcium channels (L-VSCC), or with the noncompetitive N-methyl-D-a spartate (NMDA) subtype glutamate receptor antagonist MK-801, prior to addition of submaximal concentrations of azide, significantly attenua ted azide-induced neuronal death. Blockers of N-type and Q-type VSCC ( omega-conotoxin MVIIA and MVIIC, respectively) and the P-type VSCC blo cker omega-agatoxin IVA had no effect in this assay. The sodium channe l blocker TTX was without effect when added to neurons under depolariz ing conditions, but potently and effectively protected cells when expe riments were performed in a nondepolarizing buffer. The results show t hat chemical anoxia induced by incubation of cultured neurons with azi de leads to detrimental effects, which may be quantitatively monitored by the capability of the cells to reduce MTT. This procedure is a sui table method for screening of compounds for possible protective effect s against neuronal death induced by energy depletion. In addition, the results suggest involvement of L-type VSCC as well as of glutamate re ceptors in the pathways leading to neuronal degradation induced by ene rgy depletion in cerebellar granule neurons. This would further suppor t the notion that these pathways might be important in neurodegenerati on induced by cerebral ischemia or anoxia. (C) 1996 Wiley-Liss, Inc.