Figure 2. Methamphetamine dose-dependently and time- dependently elicited anoxia and cessation of microperfusion in rostral ventrolateral medulla. Temporal changes in tissue partial pressure of oxygen and blood flow detected in rostral ventrolateral medulla (RVLM) of animals that died of intravenous administration of METH (at arrow). Values are mean 6 SEM, n = 5–9 animals per experimental group. *P,0.05 versus saline group at corresponding time-points in the post hoc Scheffe´ multiple-range test. B, baseline. doi:10.1371/journal.pone.0030589.g002 regulation that resembles that in brain death because of necrotic cell death in RVLM underpins METH-induced cardiovascular collapse. We further demonstrated that the repertoire of interposing cellular events triggered by METH in RVLM includes sustained anoxia and zero tissue perfusion that leads to bioenergetics failure and oxidative stress because of mitochondrial dysfunction (Figure 10). The experimental design of the present study addresses specifically the mechanisms of METH-induced cardiovascular collapse rather than to model consequences of human METH Cardiovascular Collapse/Methamphetamine Fatality abuse. As such, we chose intravenous administration of METH in our study because this is a common route [18] that is employed by abusers to rapidly raise the blood concentration of METH to euphoric levels. At the same time, we chose to employ high doses of METH that would result in cardiovascular collapse and fatality to mimic observations from clinical conditions [19,20]. Compared to its primary metabolite amphetamine, METH is more lipid- soluble, with enhanced transport across the blood-brain barrier and stability against enzymatic degradation by monoamine oxidase [21]. Indeed, our pharmacokinetic data indicated that 10 min after intravenous administration, the concentration of METH in the extracellular microenvironment of RVLM was already 10 times higher than that in serum, approaching a plateau within 20 min. With the high tissue concentration detected in ventrolateral medulla and the demonstrated presence of METH in RVLM neurons, the present study established RVLM as a relatively unknown though vital brain targets for this psychostim- ulant. The present results were highly reminiscent of those in a recent study from our laboratory [14] that showed that a crucial determinant for successful revival of an arrested heart is that spontaneous circulation must resume before brain death com- mences, and that maintained functional integrity of RVLM holds the key to this vital time-window. Our results showed that a time- window represented experimentally by the power density of the LF component of the SBP spectrum also exists between the administration of METH and death, and was inversely related to the doses used. We further demonstrated that sustained anoxia, which took place as early as 2 min after intravenous administra- tion of METH at 48 mg/kg, followed 4–6 min later by cessation of tissue perfusion, accounts for the loss of functionality of RVLM. Of note is that this time-window parallels the time taken for extracellular concentration of METH in RVLM to reach plateau. Given that RVLM is long known to be responsible for the maintenance of sympathetic vasomotor tone and stable blood pressure [13], and is the origin of a life-and-death signal [12] that disappears in patients confirmed of brain death [11], the selective vulnerability of this neural substrate to METH-induced toxicity is to be anticipated. Neuronal functions are critically dependent on a continuous supply of oxygen because their primary source of metabolic energy is oxidative phosphorylation in the mitochondrion. The mito- chondrial respiratory chain located in the inner membrane of mitochondria comprises of Complexes I, II, III, IV and V (ATP synthase). Orderly transfer of electrons through this chain produces energy in the form of ATP. A disruption of electron transfer not only decreases ATP production but also leaks electrons from the inner membrane to the matrix that results in the formation of reactive oxygen species (ROS) primarily in the form of superoxide anions. The mitochondrion is also the primary site of oxygen consumption in the cell, and as such is highly susceptibility to anoxia. The drastic reduction in ATP production and increase in ADP level or ADP/ATP ratio, alongside significant depression of the activity of most of the mitochondrial respiratory chain Complexes, reduction of the capacity of electron transport between Complexes I to III (NCCR) and II to III (SCCR), and augmentation of mitochondria-derived superoxide anion in RVLM of rats that died of METH strongly support the notion that bioenergetic failure and oxidative stress triggered by anoxia and cessation of tissue perfusion precipitate the eventual cessation of neuronal functions in RVLM. We noted that METH induces mitochondrial damage via production of ROS in rat hippocampal neural progenitor cells [22] and human dopaminer- gic neuroblastoma SH-SY5Y cells [23]. Aberrant mitochondrial PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e30589