Cardiovascular Collapse/Methamphetamine Fatality Figure 10. Proposed mechanisms in RVLM that underlies METH-induced cardiovascular collapse. Intravenous administration of METH rapidly reaches RVLM to induce anoxia and cessation of tissue perfusion, followed by bioenergetics failure and oxidative stress because of mitochondrial dysfunction that lead to necrotic cell death. The loss of functionality in RVLM results in cessation of central cardiovascular regulation and the eventual cardiovascular collapse. doi:10.1371/journal.pone.0030589.g010 MitoSOX red mitochondrial superoxide indicator (MitoSOX; Molecular probes) or PI (Sigma-Aldrich), with distilled water (solvent for CoQ10 and Mito-TEMPO) or 0.2% DMSO (solvent for IM-54 and MitoSOX) serving as the vehicle control. To avoid the confounding effects of drug interactions, each animal received only one freshly prepared pharmacological pretreatment. Measurement of ATP or ADP concentration An ApoSENSOR ADP/ATP ratio assay (BioVision, Mountain View, CA, USA) was used to determine ATP or ADP concentration and ADP/ATP ratio [65]. Light emitted from a luciferase mediated reaction and measured by a luminometer (Berthold Detection Systems GmbH, Pforzheim, Germany) was used to calculate the measured values. Assay for mitochondrial electron transport chain complex enzyme activity and electron coupling capacity The activity of individual mitochondrial electron transport chain Complexes was determined immediately after isolation of mitochondrial fraction [16,66] from RVLM. Enzyme activity assay kits (MitoSciences, Eugene, OR, USA) for Complex I, II or IV, and ATP synthase enzyme activity microplate assay kit (MitoSciences) for Complex V were used in conjunction with a spectrophotometer (Thermo Fisher Scientific Inc.); the activity of Complex III was determined as described by Nadanaciva [67]. The activities of NCCR (marker enzyme for electron transport capacity between Complexes I and III) or SCCR (marker enzyme for electron transport capacity between Complexes II and III) were analyzed as reported previously [14]. Total protein in the PLoS ONE | www.plosone.org 13 January 2012 | Volume 7 | Issue 1 | e30589