Chronic intrathecal infusion of mibefradil, ethosuximide and nickel attenuates nerve ligation-induced pain in rats Y.L. Chen, M.L. Tsaur, S.W. Wang,

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Chronic intrathecal infusion of mibefradil, ethosuximide and nickel attenuates nerve ligation-induced pain in rats Y.L. Chen, M.L. Tsaur, S.W. Wang, T.Y. Wang, Y.C. Hung, C.S. Lin, Y.F. Chang, Y.C. Wang, S.J. Shiue, J.K. Cheng British Journal of Anaesthesia Volume 115, Issue 1, Pages 105-111 (July 2015) DOI: 10.1093/bja/aev198 Copyright © 2015 The Author(s) Terms and Conditions

Fig 1 Effects of T-type Ca2+ channel blockers on the development of nerve ligation-induced mechanical allodynia. Intrathecal infusion of mibefradil, ethosuximide or NiCl2 attenuated the development of L5/6 spinal nerve ligation-induced mechanical allodynia (a–c), without affecting mechanical sensitivity in naive rats. Bar above the X-axis represents intrathecal infusion with saline or tested agents. Data are presented as median and IQR. *P<0.05 vs Sham/Saline group; +P<0.05 vs Ligation/Saline group (Mann–Whitney U-test, n=6–8 per group). British Journal of Anaesthesia 2015 115, 105-111DOI: (10.1093/bja/aev198) Copyright © 2015 The Author(s) Terms and Conditions

Fig 2 Effects of T-type Ca2+ channel blockers on the development of nerve ligation-induced thermal hyperalgesia. Intrathecal infusion of mibefradil, ethosuximide or NiCl2 attenuated the development of L5/6 spinal nerve ligation-induced thermal hyperalgesia (a–c), without affecting the thermal sensitivity of naive rats. Bar above the X-axis represents intrathecal infusion with saline or tested agents. Data are presented as median and IQR. *P<0.05 vs Sham/Saline group; +P<0.05 vs Ligation/Saline group (Mann–Whitney U-test, n=6–8 per group). British Journal of Anaesthesia 2015 115, 105-111DOI: (10.1093/bja/aev198) Copyright © 2015 The Author(s) Terms and Conditions

Fig 3 Representative expression patterns of CaV3.2 in a single rat L5 dorsal root ganglion using double immunofluorescence. a–d: Co-localization of CaV3.2 with parvalbumin (a), IB4 (b), CGRP (c) and VR1 (d) in the cell bodies of dorsal root ganglion neurones. The arrowhead indicates the co-localization of CaV3.2 and marker and the arrow indicates a cell expressing CaV3.2 but not marker. Scale bar: 75 µM. British Journal of Anaesthesia 2015 115, 105-111DOI: (10.1093/bja/aev198) Copyright © 2015 The Author(s) Terms and Conditions

Fig 4 Representative expression of CaV3.2 in a single rat L5 spinal dorsal horn using double immunofluorescent staining. The arrowhead indicates the co-localization of CaV3.2 and marker. CaV3.2 is expressed in neurones (a), but not microglia (b) or astrocytes (c). Scale bar: 32 µm (A), 50 µm (b and c). British Journal of Anaesthesia 2015 115, 105-111DOI: (10.1093/bja/aev198) Copyright © 2015 The Author(s) Terms and Conditions

Fig 5 Representative western blots and summarized bar graphs depicting the expression level of CaV3.2 protein in right L5/6 DRG (a) and spinal cord (b) sampled from sham (Sham) and nerve-ligated (Ligation) rats seven days after surgery. Top blots are CaV3.2 protein; lower bottom blots are control protein actin. The ordinate of the graph is the expression level of the CaV3.2 protein, taking the sham group as 100%. *P<0.05 compared with the sham group (Student’s t-test, n=4 per group). British Journal of Anaesthesia 2015 115, 105-111DOI: (10.1093/bja/aev198) Copyright © 2015 The Author(s) Terms and Conditions