W. Y. Lee, C. J. Park, T. J. Shin, K. W. Yum, T. G. Yoon, K. S. Seo, H

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Only tetracaine and not other local anaesthetics induce apoptosis in rat cortical astrocytes W.Y. Lee, C.J. Park, T.J. Shin, K.W. Yum, T.G. Yoon, K.S. Seo, H.J. Kim British Journal of Anaesthesia Volume 103, Issue 5, Pages 719-725 (November 2009) DOI: 10.1093/bja/aep237 Copyright © 2009 British Journal of Anaesthesia Terms and Conditions

Fig 1 Comparison of cell death induced by lidocaine, ropivacaine, bupivacaine, levobupivacaine, and tetracaine treatment for 18 h in primary cultured astrocytes. Cell death was assessed by LDH assay. Bupivacaine and tetracaine caused significant cell death at a concentration of 1 mM compared with the control. In addition, tetracaine also enhanced significant cell death even at a concentration of 300 µM compared with the control. The diagrams show the results of five independent experiments. The top of each box represents the median. Error bar indicates inter-quartile ranges of the distribution. *P<0.01 is statistically different from the control. British Journal of Anaesthesia 2009 103, 719-725DOI: (10.1093/bja/aep237) Copyright © 2009 British Journal of Anaesthesia Terms and Conditions

Fig 2 Cytotoxicity of tetracaine with an LDH assay in primary cultured astrocytes. Dose-responsiveness was observed during the 18 h tetracaine treatment (a). The time course of cell death was obtained after treatment with tetracaine 400 µM (b). The LD50 of tetracaine on primary astrocytes was calculated as ∼325 µM (c). The diagrams show the results of five independent experiments. Median and inter-quartile ranges are shown. British Journal of Anaesthesia 2009 103, 719-725DOI: (10.1093/bja/aep237) Copyright © 2009 British Journal of Anaesthesia Terms and Conditions

Fig 3 Morphological changes induced by tetracaine treatment in primary cultured astrocytes. Untreated and healthy astrocytes (a) were polygonal in shape. However, astroglial cell death occurred at 6 h (b) and 9 h after exposure to tetracaine 400 µM (c). After 18 h, they appeared to transform to a shrunken and elongated cell shape and lose their processes. Also many of the primary cultured astrocytes had detached from the bottom of the culture plate (d). Arrows indicate tetracaine-induced condensed and segmented nuclei. Scale bar indicates 100 µm. British Journal of Anaesthesia 2009 103, 719-725DOI: (10.1093/bja/aep237) Copyright © 2009 British Journal of Anaesthesia Terms and Conditions

Fig 4 Tetracaine induced apoptosis in primary cultured astrocytes. Hoechst 33258 and PI nuclear staining showed significant nuclear alterations in primary cultured astrocytes after tetracaine 400 µM treatment. Untreated control cultures (a), and cells exposed to tetracaine 400 µM for 1 h (b), 3 h (c), and 6 h (d) are shown. Arrows indicate tetracaine-induced condensed, coalesced, and segmented nuclei with bright red fluorescence. Scale bar indicates 30 µm. British Journal of Anaesthesia 2009 103, 719-725DOI: (10.1093/bja/aep237) Copyright © 2009 British Journal of Anaesthesia Terms and Conditions

Fig 5 Procaspase-3 and PARP cleavage in tetracaine-induced apoptosis of primary cultured astrocytes. The degradation of PARP was evident at 2 h after exposure to tetracaine 400 µM. Activation of caspase-3 was seen 3 h after incubation with tetracaine 400 µM. Representative results from three experiments are shown. Data are expressed as median and ranges. British Journal of Anaesthesia 2009 103, 719-725DOI: (10.1093/bja/aep237) Copyright © 2009 British Journal of Anaesthesia Terms and Conditions

Fig 6 ROS production and cell death in primary cultured cells exposed to tetracaine astrocyte apoptosis. The level of intracellular ROS, abruptly increased 10 min after tetracaine 400 µM treatment, and peaked in primary cultured astrocytes at 30 min (a). Cell death, induced by tetracaine 400 µM for 18 h, was inhibited by the co-treatment with NAC or trolox (b). Data from five independent experiments. Median and inter-quartile range are shown. *P<0.05 is statistically different from the control. British Journal of Anaesthesia 2009 103, 719-725DOI: (10.1093/bja/aep237) Copyright © 2009 British Journal of Anaesthesia Terms and Conditions