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First published online February 4, 2005
Journal of Experimental Biology 208, 681-686 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01436

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Intracellular calcium and survival of tadpole forebrain cells in anoxia

Michael S. Hedrick^1,2,*, Christian S. Fahlman¹ and Philip E. Bickler¹

¹ Department of Anesthesia, University of California, San Francisco, CA 94143-0542, USA
² Department of Biological Sciences, California State University, Hayward, Hayward, CA 94542-3083, USA

^* Author for correspondence at address 2 (e-mail: mhedrick{at}csuhayward.edu)

Accepted 7 December 2004

The frog brain survives hypoxia with a slow loss of energy charge and ion homeostasis. Because hypoxic death in most neurons is associated with increases in intracellular calcium ([Ca²⁺]_i), we examined the relationship between [Ca²⁺]_i and survival of a mixed population of isolated cells from the forebrain of North American bullfrog Rana catesbeiana tadpoles. Forebrain cells from stage V-XV tadpoles were isolated by enzymatic digestion and loaded with one of three different calcium indicators (Fura-2, Fura 2-FF and BTC) to provide estimates of [Ca²⁺]_i accurate at low and high [Ca²⁺]_i. Propidium iodide (PI) fluorescence was used as an indicator of cell viability. Cells were exposed to anoxia (100% N₂) and measurements of [Ca²⁺]_i and cell survival made from 1 h to 18 h. Intracellular [Ca²⁺] increased significantly after 3-6 h anoxia (P<0.05), regardless of the type of Ca²⁺ indicator used; however, there were substantial differences in the measurements of [Ca²⁺]_i with the different indicators, reflecting their varying affinities for Ca²⁺. Resting [Ca²⁺]_i was approximately 50 nmol l^-1 and increased to about 9-30 µmol l^-1 after 4-6 h anoxia. The significant increase in [Ca²⁺]_i during anoxia was not associated with significant increases in cell death, with 85-95% survival over this time period. Cells exposed to anoxia for 18 h, or those made anoxic for 4-6 and reoxygenated for 12 h to 16 h, had survival rates greater than 70%, but survival was significantly less than normoxic controls. These results indicate that large increases in [Ca²⁺]_i are not necessarily associated with hypoxic cell death in vertebrate brain cells.

Key words: cell calcium, amphibian, Rana catesbeiana, neuroprotection, anoxia, Fura 2-FF, BTC, Fura-AM, propidium iodide