Ketamine induces apoptosis via the mitochondrial pathway in human lymphocytes and neuronal cells  S Braun, N Gaza, R Werdehausen, H Hermanns, I Bauer, M.E. Durieux, M.W. Hollmann, M.F. Stevens  British Journal of Anaesthesia  Volume 105, Issue 3, Pages 347-354 (September 2010) DOI: 10.1093/bja/aeq169 Copyright © 2010 The Author(s) Terms and Conditions

Fig 1 The two major pathways of apoptosis. The intrinsic or mitochondrial pathway of apoptosis (left side) involves mitochondrial dysfunction, release of cytochrome c (cyt c) and the subsequent activation of caspase-9 (casp-9) at the apoptosome. The antiapoptotic protein Bcl-2 inhibits the release of cytochrome c from the mitochondrion. The extrinsic or death receptor pathway (right side) is initiated by binding of death ligands to the death receptor and subsequent recruitment of the adapter protein FADD and caspase-8 (casp-8) into the death-inducing signalling complex (DISC). Both apoptosis pathways converge at the activation of effector caspase-3 (casp-3), which cleaves several cellular proteins, finally leading to the typical alterations of apoptosis such as DNA fragmentation in the nucleus. British Journal of Anaesthesia 2010 105, 347-354DOI: (10.1093/bja/aeq169) Copyright © 2010 The Author(s) Terms and Conditions

Fig 2 Cell survival of Jurkat T-lymphoma cells after 24 h exposure was measured by flow cytometry revealing the percentages of overall cell death and early apoptotic cells (Annexin V/7-AAD +/−) and late apoptotic or necrotic cells (Annexin V/7-AAD +/+). (a) Concentration-dependent toxicity and apoptosis induction by ketamine in Jurkat T-lymphoma cells. STS was used as a positive control. (b) Wilde-type (wt), Bcl-2 overexpressing (Bcl2+), and caspase-9-deficient (cas9−) cells were exposed to control medium (left) or 2 mM ketamine (right). (c) Wilde-type (wt), caspase-8-deficient (cas8−), and FADD-deficient (FADD−) cells were exposed to the control medium (left) or 2 mM ketamine (right). Data are presented as mean (sd). *P<0.05 compared with the negative control; n.s., not significant (n=3). British Journal of Anaesthesia 2010 105, 347-354DOI: (10.1093/bja/aeq169) Copyright © 2010 The Author(s) Terms and Conditions

Fig 3 Apoptosis induction in neuroblastoma cells (SHEP) after 24 h exposure to negative control, STS as a positive control and increasing concentrations of ketamine. In (a), apoptosis induction was measured by flow cytometry revealing the percentages of overall cell death and early apoptotic cells (Annexin V/7-AAD +/−) and late apoptotic or necrotic cells (Annexin V/7-AAD +/+). In (b), activation of caspase-3 was measured as a marker for apoptosis by flow cytometry. Data are presented as mean (sd). *P<0.05 compared with the negative control (n=3). British Journal of Anaesthesia 2010 105, 347-354DOI: (10.1093/bja/aeq169) Copyright © 2010 The Author(s) Terms and Conditions

Fig 4 Apoptosis induction in neuroblastoma cells (SHEP) after 24 h exposure to negative control, STS as a positive control and increasing concentrations of ketamine with or without the pancaspase inhibitor Q-VD (10 µM). Flow cytometry revealing the percentages of overall cell death and early apoptotic cells (Annexin V/7-AAD +/−) and late apoptotic or necrotic cells (Annexin V/7-AAD +/+). Data are presented as mean (sd). *P<0.05. British Journal of Anaesthesia 2010 105, 347-354DOI: (10.1093/bja/aeq169) Copyright © 2010 The Author(s) Terms and Conditions

Fig 5 Comparison of the neurotoxicity of S(+)-ketamine and its racemate in equimolar concentrations after 24 h exposure in Jurkat T-lymphoma cells (a) and SHEP neuroblastoma cells (b) measured by flow cytometry revealing the percentages of overall cell death and early apoptotic cells (Annexin V/7-AAD +/−) and late apoptotic or necrotic cells (Annexin V/7-AAD +/+). Data are presented as mean (sd). *P<0.05 (n=3). British Journal of Anaesthesia 2010 105, 347-354DOI: (10.1093/bja/aeq169) Copyright © 2010 The Author(s) Terms and Conditions