after a time lag, on diagnosis of brain death [10]. This suggests that permanent impairment of the brain stem cardiovascular regulatory machinery should precede the inevitable death [11]. Another crucial clue arises from the identification by our laboratory of a common prognostic determinant among comatose patients [11] who succumbed to systemic inflammatory response syndrome, severe brain injury or organophosphate poisoning. A dramatic reduction or loss of the power density of the low-frequency (LF) component (0.04–0.15 Hz in human) in the power spectrum of arterial pressure signals consistently takes place before significant hypotension and the eventual asystole occur. We also demonstrated [12] that the origin of this life-and-death signal resides in the rostral ventrolateral medulla (RVLM), which is long known to be responsible for the maintenance of sympathetic vasomotor tone and stable blood pressure [13]. The third clue comes from our demonstration that successful resuscitation of an arrested heart depends on maintained functionality of RVLM [14], suggesting that failure of brain stem cardiovascular regulation, rather than the heart, holds the key to cardiovascular collapse. Cerebrovascular pathology, most commonly hypoxia and hemorrhage, is present in 20% of cases in METH-elicited death [4,15]. Of particular interests is that our laboratory found previously in experimental brain death models that the degree of tissue hypoxia in RVLM is a crucial determinant of the severity of central circulatory regulatory dysfunction [14,16]. An immediate corollary that arises, which forms the hypothesis for the present study, is that cessation of brain stem cardiovascular regulation that resembles that in brain death because of the loss of functionality in RVLM may underlie the cardiovascular collapse elicited by METH. This hypothesis was validated in the present study. We further demonstrated that sustained anoxia and cessation of local blood flow, followed by acute bioenergetic failure because of mitochondrial dysfunction that leads to necrotic cell death in RVLM underlie METH-induced shutdown of central cardiovas- cular regulation that precedes cardiovascular collapse. Results Lethal doses of METH induce cardiovascular collapse Intravenous administration of METH (24, 48 or 60 mg/kg) elicited a dose-dependent decrease in survival rate (Figure 1A). At a dose of 24 mg/kg, METH was effective in reducing the survival rate by 60% within 20 min; increasing the doses to 48 or 60 mg/ kg further exacerbated the mortality rate to 90% or 100% by 20 min. In rats that died, there was a dose-dependent collapse of arterial pressure (Figure 1B) and heart rate (Figure 1C) that paralleled the time-course of mortality, with a progressive reduction in response latency. The LF component of the systolic blood pressure (SBP) spectrum underwent a transient increase that exhibited a dose-dependent reduction in peak power and response latency (Figure 1D), followed by the disappearance of this life-and- death signal that also paralleled temporally with mortality. However, survival rate (Figure 1A) or cardiovascular responses (Figure 1B–D) were not affected by intravenous administration of saline (vehicle control). Anoxia and cessation of tissue perfusion in RVLM Our laboratory demonstrated previously [14] that anoxia and zero tissue perfusion in RVLM precede sustained cessation of LF power and cardiovascular collapse seen during experimental brain death. Figure 2 shows that in rats that succumbed to METH (24 or 48 mg/kg, i.v.), there was also an abrupt anoxia and cessation of local microvascular perfusion in RVLM. Of note was that those events exhibited a latency that was shorter than the cardiovascular Cardiovascular Collapse/Methamphetamine Fatality responses induced by the same doses of METH. On the other hand, administration of saline induced insignificant changes in tissue oxygen level and local blood flow in RVLM. On the other hand, simultaneous measurement of in situ temperature revealed insignificant differences in RVLM tissue temperature between saline-control group (34.560.2uC), rats that survived after METH at 24 mg/kg (34.860.3uC) and rats that died of METH at 24 (33.860.5uC) or 48 mg/kg (33.760.3uC). RVLM as a site of action for intravenously administered METH A crucial prerequisite for RVLM to be a site of action for METH is for the psychostimulant to reach this neural substrate on systemic administration. In addition to a dose-dependent increase of METH in serum (Figure 3A), we detected the same trend in tissue samples (Figure 3B) and extracellular fluid (Figure 3C) collected from RVLM in rats that died of intravenous adminis- tration of METH (24 or 48 mg/kg). Double immunofluorescence staining coupled with laser scanning confocal microscopy further showed that in addition to a clearly defined nucleus and nucleolus in cells stained positively with the neuronal marker, neuron- specific nuclear protein (NeuN) (Figures 3D,E), METH-immuno- reactivity was present in the cytoplasm and nucleus of RVLM neurons (Figure 3E) from animals that died of METH. Necrotic cell death in RVLM Hematoxylin and eosin (H&E) staining revealed that present in RVLM of rats that died of METH (48 mg/kg, i.v.) were necrosis- appearing cells (Figure 4) characterized by shrinkage of cytoplasm, chromatolysis (disruption of Nissl bodies), karyolysis (nuclear fading) and karyorrhexis (nuclear fragmentation). On the other hand, 81.961.1% of cells in RVLM of saline-control rats exhibited clearly hematoxylin-labeled nucleolus, intact nuclear membrane, nucleus and cytoplasm were observed (Figure 4), as opposed to 13.861.9% in the METH (48 mg/kg, i.v.) group. Likewise, the ratio of RVLM cells that manifested chromatolysis (23.761.2% versus 3.760.7%), karyolysis (40.662.5% versus 7.661.1%) and karyorrehexis (21.961.4% versus 6.860.5%) in the METH group was higher than those in the saline-control group. In contrast, activated caspase-3 and histone-associated DNA fragments, two hallmark experimental indices for apoptosis, were absent from RVLM of rats that received saline or died of METH (Figure 5). Bioenergetic failure that leads to necrotic cell death because of mitochondrial dysfunction in RVLM Because ATP is the primary source of metabolic energy in neurons, neurons tend to undergo necrosis in response to anoxic stress [17]. We found that animals died of METH (24 mg/kg, i.v.) exhibited a drastic reduction in ATP production and an increase in ADP level or ADP/ATP ratio in RVLM (Figure 6A), alongside elevated PI uptake indicative of necrotic cell death (Figure 6B). In addition to maintaining survival, all those four responses were reversed by microinjection bilaterally of Mito-TEMPO (500 pmol) or CoQ10 (7 nmol) into RVLM (Figures 6A,B); and IM-54 (3 pmol) blunted the elevated PI level (Figure 6B). We further observed a depression of the activities of Complexes I, II, III and V (Figures 6A–C,E), NADH cytochrome c reductase (NCCR) or succinate cytochrome c reductase (SCCR) (Figures 7F,G), but not Complex IV (Figure 6D), in the mitochondrial respiratory chain in RVLM of rats that succumbed to METH (24 mg/kg, i.v.). Again, those depressive actions were antagonized by CoQ10 (7 nmol) or Mito-TEMPO (500 pmol) (Figures 7A–C,E–G). PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e30589