Figure 5. Methamphetamine did not induce apoptotic cell death in RVLM. Changes of activated caspase-3 or histone-associated DNA fragments in fold relative to sham-control group in samples collected from RVLM of rats that received saline or died of intravenous administration of METH. Values are mean 6 SEM, n = 5–7 animals per experimental group. P.0.05 among all groups in one-way ANOVA. doi:10.1371/journal.pone.0030589.g005 Materials and Methods Ethics statement All experimental procedures carried out in this study were approved by the Institutional Animal Care and Use Committee of the Kaohsiung Chang Gung Memorial Hospital (CGMH891071), and were in compliance with the guidelines for animal care and use set forth by that committee. All efforts were made to reduce the number of animals used and to minimize animal suffering during the experiment. Animals Specific pathogen-free adult, male, Sprague-Dawley rats (260 to 325 g, n = 200) purchased from the Experimental Animal Center of the National Science Council and BioLASCO, Taiwan, Republic of China, were used. They were housed in an Association for Assessment and Accreditation of Laboratory Animal Care (AAA- LAC) International-accredited animal facility under temperature control (24–25uC) and 12-h light-dark cycle. Standard laboratory rat chow and tap water were available ad libitum. General preparation Under an induction dose of pentobarbital sodium (50 mg/kg, i.p.), rats received preparatory surgery that included tracheal intubation and cannulation of the femoral artery and vein. Cardiovascular Collapse/Methamphetamine Fatality Animals received thereafter an intravenous infusion of propofol (20–25 mg/kg/h; Zeneca, Macclesfield, England), which provided satisfactory maintenance of anesthesia while preserving the capacity of central cardiovascular regulation [47]. During the recording sessions, animals were allowed to breathe spontaneously with room air, and body temperature was maintained at 37uC by a heating pad. Recording of cardiovascular parameters Arterial pressure (AP) signals recorded from the femoral artery were processed by an arterial blood pressure analyzer (APR31a; Notocord, Croissy-Sur-Seine, France) and the SBP signals were subject simultaneously to online and real-time spectral analysis (SPA10a; Notocord). We were particularly interested in the LF (0.25–0.8 Hz) component because it takes origin from RVLM [12], and its power density mirrors the prevalence of baroreflex- mediated sympathetic neurogenic vasomotor discharges that emanate from RVLM [48] and is therefore a reasonable index for brain stem cardiovascular regulation. With particular reference to the present study, the increase or decrease in LF power was also used to reflect lower or higher probability of manifesting brain death [11,49–51]. Heart rate (HR) was derived instantaneously from AP signals. Intravenous administration of METH METH was purchased from the Food and Drug Administra- tion, Department of Health, Executive Yuan, Taipei, Taiwan. It was administered intravenously, with saline serving as the vehicle control. Temporal changes in pulsatile AP, mean AP (MAP), HR and power density of the LF component were routinely followed for 20 min in an on-line and real-time manner [14,16,52], or until the animal succumbed to METH. The survival rate within 240 min was also recorded. Measurement of tissue oxygen tension, microvascular perfusion and temperature in rostral ventrolateral medulla Simultaneous and continuous measurement of tissue oxygen tension, blood flow and temperature (Oxford Optronix, Oxford, England) was carried out by a combined oxygen/temperature/ blood flow probe stereotaxically positioned into the RVLM [14,52]. The coordinates used were: 4.5 to 5 mm posterior to lambda, 1.8 to 2.1 mm lateral to midline and 8.1 to 8.4 mm below dorsal surface of cerebellum. Instantaneous changes in local oxygen tension, compensated for fluctuations in tissue tempera- ture, were processed by an OxyLite monitor (Oxford Optronix). Real-time microvascular red blood cell perfusion in tissue was processed by an OxyFlo monitor (Oxford Optronix). Laser Doppler signals from the tissue were recorded in blood perfusion units (BPU), which is a relative unit defined against a controlled motility standard. Collection of tissues from RVLM We routinely collected tissue samples [52–54] immediately after rats died of METH or 20 min after intravenous administration of saline in rats killed with an overdose of pentobarbital sodium. Tissue samples from both sides of the ventrolateral medulla, at the level of RVLM (0.5–1.5 mm rostral to the obex), were collected by micropunches made with a 1-mm (i.d.) stainless-steel bore to cover the anatomical boundaries of RVLM, with a diameter of 625– 650 mm [52]. In some experiments, medullary tissues collected from anesthetized animals but without receiving experimental manipulations served as the sham controls. PLoS ONE | www.plosone.org 8 January 2012 | Volume 7 | Issue 1 | e30589