Figure 8. Methamphetamine increased production of mito- chondrial superoxide anions in RVLM. (A). Fold changes relative to sham-control group in mitochondrial superoxide anion in sample collected from RVLM of rats died of METH (24 mg/kg, i.v.) or survived with pretreatment by microinjection of Mito-TEMPO (500 pmol) or coenzyme Q10 (CoQ10; 7 nmol) into RVLM prior to METH administra- tion. Values are mean 6 SEM, n = 3–6 animals per experimental group. *P,0.05 versus Vehicle+Saline group, and #P,0.05 versus Vehicle+- METH group in the post hoc Scheff e´ multiple-range test. (B). Representative laser scanning confocal microscopic images superim- posed on phase contrast images showing neurons in RVLM that were immunoreactive to MitoSOX in rats that received saline (B1), died of METH (24 mg/kg, i.v.; B2), or survived with pretreatment by microin- jection of Mito-TEMPO (500 pmol; B3) or CoQ10 (7 nmol; B4) into bilateral RVLM prior to METH administration. These results are typical of 4 animals from each experimental group. Scale bar, 100 mm. Pseudocolor scale bar, white color represented highest and blue color represented lightest fluorescence intensity. NA, nucleus ambiguus. doi:10.1371/journal.pone.0030589.g008 Cardiovascular Collapse/Methamphetamine Fatality Determination of METH in serum, ventrolateral medulla or extracellular fluid from RVLM The level of METH in serum, ventrolateral medulla or perfusate collected from RVLM was determined by a commercial METH indirect ELISA kit (Calbiotech, Spring Valley, CA, USA), in conjunction with a spectrophotometer (Multiskan Spectrum, Thermo Fisher Scientific Waltham, MA, USA) [55,56]. Blood (100 ml) drawn from the femoral vein was centrifuged to obtain serum samples, which were frozen at 280uC until analysis. The perfusate from the extracellular space of RVLM was continuously collected at 10-min interval via a microdialysis probe (CMA/Microdialysis, Stockholm, Sweden) stereotaxically positioned into RVLM and perfused with artificial cerebrospinal fluid (aCSF) at a rate of 1 ml/min. Immunofluorescence staining and confocal microscopy We employed double immunofluorescence staining coupled with laser scanning confocal microscopy [14,57,58] to detect localization of METH in RVLM neurons using a rabbit polyclonal antiserum against METH (Abcam, Cambridge, MA, USA), and a mouse monoclonal antiserum against a specific neuron marker, NeuN (Millipore, Billerica, MA, USA). The secondary antisera (Molecular Probes, Eugene, OR, USA) used included a goat anti-rabbit IgG conjugated with Alexa Fluor 488 for METH, and a goat anti-mouse IgG conjugated with Alexa Fluor 568 for NeuN. Tissues similarly processed but omitting primary antiserum against METH served as our negative controls. Immunoreactivity was viewed under a Fluorview FV10i laser scanning confocal microscope (Olympus, Tokyo, Japan). H&E staining Brain stem fixed in 10% buffered formalin for 48 h were embedded in paraffin wax and processed for H&E staining [59,60]. Four mm-thick tissue sections were dewaxed in xylene, hydrated in decreasing percentages of alcohol and stained with hematoxylin (Merck Millipore, Darmstadt, Germany). This was followed by dehydration in increasing percentages of alcohol until 70%, staining with 0.25% eosin (Merck Millipore) in alcohol, differentiation in 90% alcohol, clearing in xylene and finally coverslipped in mounting medium (Merck Millipore). The stained sections were observed and photographed under an optical microscope (Olympus). Propidium iodide (PI) staining Uptake of PI by RVLM neurons as a marker for necrotic cell death [61,62] was determined by the exhibition of red fluorescence under laser scanning confocal microscopy (Olympus). PI (5 pmol; Sigma-Aldrich, Saint Louis, MO, USA) was microinjected bilaterally into RVLM 30 min before METH or vehicle administration. Experimental indices for apoptosis To quantify apoptosis, a cell death ELISA kit (Roche Applied Science, Indianapolis, IN, USA) that detects apoptotic but not necrotic cell death was used to measure the level of histone-associated DNA fragments in the cytoplasm [57], in conjunction with an ELISA microtiter plate reader (Anthros Labtec, Salzburg, Austria). To quantify activated caspase-3, a caspase-3 fluorescence assay kit (Cayman, Ann Arbor, MI, USA) coupled with a spectrofluorometer (Fluoroskan Ascent FL, Thermo Fisher Scientific Inc.) was used. Microinjection of test agents to RVLM Microinjection bilaterally of test agents into RVLM, each at a volume of 50 nl, was carried out stereotaxically and sequentially PLoS ONE | www.plosone.org 11 January 2012 | Volume 7 | Issue 1 | e30589