title: Midazolam activates the intrinsic pathway of apoptosis independent of benzodiazepine and death receptor signaling.
abstract: OBJECTIVE
  Midazolam has neurotoxic properties when administered neuraxially in vivo. Furthermore, midazolam induces neurodegeneration in neonatal animal models in combination with other general anesthetics. Therefore, this study focuses on the mechanism of neurotoxicity by midazolam in neuronal and nonneuronal cells. The study aims to evaluate the apoptotic pathway and to investigate the protective effects of the benzodiazepine antagonist flumazenil and the caspase inhibitor N-(2-quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)-methylketone.
  
  
  METHODS
  The apoptosis-inducing effect of preservative-free midazolam on human lymphoma and neuroblastoma cell lines was evaluated using flow cytometric analysis of early apoptotic stages (annexin V/7AAD) and caspase 3 activation. B-cell lymphoma (Bcl2) protein overexpressing and caspase 9-deficient lymphoma cells were used to determine the role of the mitochondrial (intrinsic) pathway. Caspase 8-deficient and Fas-associated protein with death domain (FADD)-deficient cells were used to evaluate the death receptor (extrinsic) pathway. The protective effects of flumazenil and the caspase inhibitor N-(2-quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)-methylketone were investigated in neuroblastoma cells and primary rat neurons using metabolic activity assays (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) and immunofluorescence microscopy.
  
  
  RESULTS
  Midazolam induced apoptosis in all investigated cell types in a concentration-dependent manner, indicated by flow cytometry. Bcl2-overexpression and caspase 9 deficiency protected against toxicity, whereas caspase 8 or FADD deficiency had no effect. Pancaspase inhibition had a strong protective effect, whereas flumazenil did not inhibit midazolam-induced apoptosis.
  
  
  CONCLUSIONS
  Midazolam induces apoptosis via activation of the mitochondrial pathway in a concentration-dependent manner. The mechanism of midazolam toxicity switches from caspase-dependent apoptosis to necrosis with increasing concentrations. The induction of apoptosis and necrosis by midazolam is presumably unrelated to GABAA receptor pathway signaling.
authors.affiliation: Department of Anesthesiology, Academic Medical Center, University of Amsterdam, The Netherlands.
authors.forename: Markus F
authors.initials: MF
authors.lastname: Stevens
authors.forename: Robert
authors.initials: R
authors.lastname: Werdehausen
authors.forename: Nina
authors.initials: N
authors.lastname: Gaza
authors.forename: Henning
authors.initials: H
authors.lastname: Hermanns
authors.forename: David
authors.initials: D
authors.lastname: Kremer
authors.forename: Inge
authors.initials: I
authors.lastname: Bauer
authors.forename: Patrick
authors.initials: P
authors.lastname: Küry
authors.forename: Markus W
authors.initials: MW
authors.lastname: Hollmann
authors.forename: Sebastian
authors.initials: S
authors.lastname: Braun
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001569:Benzodiazepines / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D019169:Jurkat Cells; D008874:Midazolam / Q000494:pharmacology*; D011247:Pregnancy; D051381:Rats; D017208:Rats, Wistar; D053218:Receptors, Death Domain / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology
chemical_list: D053218:Receptors, Death Domain; D001569:Benzodiazepines; D008874:Midazolam