Simultaneous Measurement of Cytosolic and Mitochondrial Ca2+ During Ischemia in Mice Whole-Brain Slice Preparation and I
We developed a conventional imaging method to measure Ca2+ 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
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Abstract We developed a conventional imaging method to measure Ca2+ 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 T. Nishiyama (*) Department of Anesthesiology, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku, Tokyo 160-0023, Japan Department of Anesthesiology, Hachioji Medical Center, Tokyo Medical University, 1163 Tate-machi Hachioji City, Shinjuku, Tokyo 193-0998, Japan Department of Anesthesiology, Kimura Hospital, Tokyo, Japan e-mail: [email protected] E. Hasegawa Laboratory of Biochemistry, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi Hachioji, Tokyo 192-0392, Japan S. Yanagi, R. Hamada, N. Matsumura, M. Tomino, and Y. Muromachi Department of Anesthesiology, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku, Tokyo 160-0023, Japan Y. Kudo Department of Anesthesiology, Tokyo Medical University, Tokyo, Japan K. Hatakeyama Department of Anesthesiology, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku, Tokyo 160-0023, Japan Department of Anesthesiology, Hachioji Medical Center, Tokyo Medical University, 1163 Tate-machi Hachioji City, Shinjuku, Tokyo, 193-0998, Japan Department of Anesthesiology, Toda Central General Hospital, Toda, Japan Department of Anesthesiology, Toda Chuo General Hospital, Toda, Japan H. Uchino, MD, PhD Department of Anesthesiology, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku, Tokyo 160-0023, Japan Department of Anesthesiology, Hachioji Medical Center, Tokyo Medical University, 1163 Tate-machi Hachioji City, Shinjuku, Tokyo 193-0998, Japan
Hela cells, we applied it to mouse whole-brain slice preparation, which was exposed to oxygen- and glucosedeprived 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 Ca2+concentration), while the fluorescence due to Rhod-2 (excited by 560 ± 10 nm) increased (indicating the increase in mitochondrial Ca2+ 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+/Ca2+ exchanger, on the abnormal Ca2+ increase in cytosol. We confirmed that the present method will be useful for future pathological and pharmacological studies on ischemia-induced brain damage. Keywords Ischemia • Cytosol • Mitochondria • Fluorescence Ca2+ indicator • Rhod-2 • FuraRed • Cyclosporine A SEA0400 • Na+/Ca2+exchanger • Mitochondrial permeability transition
Introduction Brain tissue has been known to require a very large amount of energy. Embolism or infarction of cerebral blood vessels and also accidental hypoxia and hypoglycemia cause encephalopathies, which are sometimes fatal or have severe sequelae depending upon the s
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