Cardiovascular Collapse/Methamphetamine Fatality Figure 3. Presence of methamphetamine in RVLM neurons after intravenous administration. (A–C). Changes in concentration of METH in serum (A), ventrolateral medulla (B) or extracellular fluid collected from RVLM (C) of rats that died of intravenous administration of METH (at arrow). Values are mean 6 SEM, n = 4–9 animals per experimental group. *P,0.05 versus saline group at corresponding time-points in the post hoc Scheff e´ multiple-range test. ND, not detected. (D–E). Representative laser scanning confocal microscopic images showing cells in RVLM that were immunoreactive to METH (METH-IR; green fluorescence) and additionally stained positively for a neuronal marker, neuron-specific nuclear protein (NeuN; red fluorescence) in rats that received saline (D) or died of METH (48 mg/kg, i.v.; E) group. These results are typical of 4 animals from each experimental group. Scale bar, 20 mm. White arrow, METH-IR cells in RVLM; white arrowhead, RVLM neurons which were double stained with METH-IR and NeuN. doi:10.1371/journal.pone.0030589.g003 biogenesis and enhanced oxidative stress also play a crucial role in METH-induced neurotoxic effects in SH-SY5Y cell line [24]. Depressive effects of METH on the activity of mitochondrial respiratory chain complexes have also been reported [25]. In spontaneously hypertensive rats, the increased superoxide anions in RVLM can also take origin from NADPH oxidase [26]. Whether this source of ROS is involved in METH-induced oxidative stress in RVLM requires further elucidation. Postmortem studies showed that cerebral edema [27] or brain stem hematoma [15] is a common clinical phenotype in patients died of METH intoxication. The present study demonstrated that damage to RVLM in the form of necrotic cell death is a contributing factor to METH-induced mortality. A critical determinant of the eventual cell death fate resides in intracellular ATP concentration. Whereas ATP is required for the development of apoptosis, ATP depletion is associated with necrosis [28]. Thus, the significant depletion of intracellular ATP content induced by lethal doses of METH, together with the characteristic morpho- logical features under H&E stain and the uptake of PI, are indicative of damage of cell membrane integrity, leading to necrotic cell death in RVLM. The absence of activated caspase-3 and histone-associated DNA fragments, two hallmark experimen- tal indices for apoptosis, in RVLM of rats that died of METH further supports the validity of this notion. We previously found that lethal dose of organophosphate pesticide mevinphos induced anoxia-induced bioenergetics failure and subsequent necrotic cell death in RVLM, resulting in cardiovascular collapse prior to cessation of life. On the other hand, sub-lethal dose of mevinphos elicited hypoxia-dependent and mitochondria-dependent apoptot- ic cell death in RVLM that result in dysfunction of brain stem cardiovascular regulation and hypotension [29–32]. It follows that, subject to confirmation, sub-lethal doses of METH may only induce hypoxia in RVLM, leading to apoptotic cell death that is accompanied by brain stem cardiovascular dysfunction without imminent death. In addition to mitochondria, the endoplasmic reticulum is reported recently to be another target of ROS [33]. Whereas the contribution of endoplasmic reticulum to METH-induced necrotic cell death in RVLM cannot be ruled out, we noted that ROS- mediated endoplasmic reticulum stress is mainly responsible for apoptosis induced by proapoptotic inducer in cancer cells [33,34]. Because of the similarities in cellular programming for apoptosis and autophagy [35,36], the lack of participation of apoptotic cell death in RVLM during METH-induced cardiovascular collapse observed in the present study deemed the involvement of autophagy in this process minimal. The effectiveness of CoQ10, Mito-TEMPO or IM-54 to sustain survival, antagonize arterial pressure collapse, asystole and disappearance of power density of the LF spectral component, and blunt mitochondrial dysfunction, bioenergetics failure, oxidative stress or necrotic cell death in RVLM induced by lethal doses of METH provided the most crucial documentation that these events are causally related. CoQ10 is an essential component of the mitochondrial electron transport chain [37], and the concentration of CoQ10 in the mitochondrial membrane is related to the rate of electron transfer and respiratory function [38]. By enhancing quantitatively the availability of freely mobile electron carriers and qualitatively the efficacy of electron transfer across the mitochondrial respiratory chain [39], CoQ10 may also avail more oxygen to the neurons for oxidative phosphorylation. Mito- TEMPO is a recently developed mitochondria-targeted antioxi- dant and superoxide dismutase mimetic [40] that prevents loss of mitochondrial membrane potential or increase in mitochondrial permeability transition pore opening and necrosis in epithelial cells that are subject to ATP depletion [41]. Mito-TEMPO also decreases mitochondrial superoxide level, prevents mitochondrial oxidative damage or mitochondrial dysfunction and attenuates hypertension elicited in mice by angiotensin II or DOCA salt [40]. IM-54 is a recently developed tool for investigating the molecular mechanism of oxidative stress-induced necrosis. It inhibits necrotic cell death in hydrogen peroxide-treated HL60 cells or in anticancer agent chrysophanol-treated J5 human liver cancer cells [42], but not in etoposide-induced apoptotic cell death [43]. We are aware that heart failure is another common phenomenon observed in patients died of METH, and METH may act directly on the heart on intravenous administration [6,8,9]. As such, the possibility exists for depression of cardiac functions, rather than failure of brain stem cardiovascular regulation, to be primarily responsible for our observed METH-induced cardiovascular collapse. However, our results deemed this possibility minimal. Pretreatments with CoQ10, Mito-TEMPO or IM-54, at a dose that antagonized mitochondrial dysfunction (Figure 7), bioenergetic failure (Figure 6), oxidative stress (Figure 8) or necrotic cell death (Figure 6) in RVLM, significantly antagonized the cessation in arterial pressure or heart rate and disappearance of the LF spectral component (Figure 9) induced by intravenous administration of a lethal dose of METH, along with sustained survival. Subcutaneous administration of METH (9 mg/kg) has been reported to elicit hyperthermia and structural or functional abnormalities in rat nucleus accumbens [44], cortex, hippocam- pus, thalamus and hypothalamus [45]. In particular, acute brain edema induced by the inflow of simple ions promoted by a breakdown of blood-brain barrier presents serious consequences [46]. Since animals that died of METH did not manifest significant changes of tissue temperature in RVLM, we reason that the neurotoxicity we observed in this neural substrate may not be the result of local hyperthermia. On the other hand, it is plausible that the cellular consequences induced by METH in the forebrain structures may also take place in RVLM. Further experiments are required to address this issue. In conclusion, the present study revealed that lethal doses of METH elicit cardiovascular collapse by inducing an acute loss of functionality in RVLM via necrotic cell death, which leads to the cessation of brain stem cardiovascular regulation that resembles that in brain death. We further showed that the underlying cellular events include mitochondrial dysfunction that leads to depletion of ATP production (bioenergetic failure) and upregulation of mitochondrial superoxide anion (oxidative stress) because of sustained anoxia and zero tissue perfusion in RVLM. This information should offer novel leads for devising clinical PLoS ONE | www.plosone.org 6 January 2012 | Volume 7 | Issue 1 | e30589