title: Simultaneous measurement of cytosolic and mitochondrial Ca(2+) during ischemia in mice whole-brain slice preparation and its application to drug evaluation.
abstract: We developed a conventional imaging method to measure Ca(2+) concentration in cytosol (using FuraRed as an indicator) and mitochondria (using Rhod-2 as an indicator), simultaneously, by alternative excitation with specific wave length. After confirming the availability of the method in Hela cells, we applied it to mouse whole-brain slice -preparation, which was exposed to oxygen- and glucose-deprived artificial cerebrospinal fluid (ischemic ACSF) for 12 min. The fluorescence (>570 nm) at the cerebral cortex and hippocampus due to FuraRed (excited by 480 ± 10 nm) decreased (indicating the increase in cytosolic Ca(2+)-concentration), while the fluorescence due to Rhod-2 (excited by 560 ± 10 nm) increased (indicating the increase in mitochondrial Ca(2+) concentration) during exposure to ischemic conditions. We found the characteristic protective effects of cyclosporine A (10(-6) M), a known blocker for mitochondrial permeability transition, and SEA0400 (10(-6) M), a blocker for Na(+)/Ca(2+) exchanger, on the abnormal Ca(2+) increase in cytosol. We confirmed that the present method will be useful for future pathological and pharmacological studies on ischemia-induced brain damage.
authors.affiliation: Department of Anesthesiology, Tokyo Medical University, Tokyo, Japan. nishiyat@tokyo-med.ac.jp
authors.forename: Takahisa
authors.initials: T
authors.lastname: Nishiyama
authors.forename: Erika
authors.initials: E
authors.lastname: Hasegawa
authors.forename: Shigeru
authors.initials: S
authors.lastname: Yanagi
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Kudo
authors.forename: Ryuta
authors.initials: R
authors.lastname: Hamada
authors.forename: Noriko
authors.initials: N
authors.lastname: Matsumura
authors.forename: Mikiko
authors.initials: M
authors.lastname: Tomino
authors.forename: Yukio
authors.initials: Y
authors.lastname: Muromachi
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Hatakeyama
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Uchino
mesh_terms: D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D004195:Disease Models, Animal; D004353:Drug Evaluation, Preclinical; D006367:HeLa Cells; D006575:Heterocyclic Compounds, 3-Ring; D006801:Humans; D007093:Imidazoles; D066298:In Vitro Techniques; D007511:Ischemia / Q000473:pathology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009474:Neurons / Q000648:ultrastructure*; D010647:Phenyl Ethers / Q000494:pharmacology; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors; D013997:Time Factors
chemical_list: D000814:Aniline Compounds; D001572:Benzofurans; D006575:Heterocyclic Compounds, 3-Ring; D007093:Imidazoles; D010647:Phenyl Ethers; C430918:SEA 0400; D019831:Sodium-Calcium Exchanger; C068483:rhod-2; C082877:fura red; D002118:Calcium