Adult male Sprague–Dawley rats (275–350 g, n = 129) purchased from the Experimental Animal Center of the National Science Council, Taiwan, Republic of China were used. They were housed in our Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-International accredited Center for Laboratory Animals. All animal care and experimental procedures carried out in this study have been approved by the Institutional Animal Care and Use Committee of the Kaohsiung Chang Gung Memorial Hospital, and were in compliance with the guidelines of this Committee. Animals were housed in groups of two to three in individually ventilated cages, in a temperature-controlled room (22 ± 2°C) with 12 h light/12 h dark cycles (lights on at 07:00 h), with free access to rat chow and water. All efforts were made to minimize animal suffering and to reduce the number of animal used.
After application of an induction dose of pentobarbital sodium (50 mg/kg, i.p.), preparatory surgery, including cannulation of a femoral artery and a femoral vein, together with tracheal intubation, was carried out. During the recording session, which routinely commenced 60 min after the administration of pentobarbital sodium, anesthesia was maintained by intravenous infusion of propofol (Zeneca, Macclesfield, UK) at 20–25 mg/kg/h. We have demonstrated previously  that this scheme provided satisfactory anesthetic maintenance while preserving the capacity of central cardiovascular regulation. Rats were allowed to breathe spontaneously with room air and body temperature of rats was maintained at 37°C with a heating pad.
Animal model of brain stem death
The Mev intoxication model of brain stem death  that we established previously was used. Since Mev induces comparable cardiovascular responses on given systemically or directly to RVLM , we routinely microinjected Mev bilaterally into RVLM to elicit site-specific effects [9, 14–17]. SAP signals recorded from the femoral artery were simultaneously subject to on-line power spectral analysis (SPA10a; Notocord, Croissy-Sur-Seine, France) [9, 14–17, 33]. We were particularly interested in the LF component (0.25–0.8 Hz) in the SAP spectrum because its power density mirrors the prevalence of baroreflex-mediated sympathetic neurogenic vasomotor discharges that emanate from this brain stem site . More importantly, our laboratory demonstrated previously [14–17] that the power density of this spectral signal exhibits biphasic changes that reflect the pro-life and pro-death phases seen during the progression towards brain stem death in patients who succumbed to organophosphate poisoning . Heart rate (HR) was derived instantaneously from SAP signals. Temporal changes in the power density of the LF component, pulsatile SAP, mean SAP (MSAP) and HR were routinely followed for 180 min after Mev administration in an on-line and real-time manner.
Microinjection of test agents
Microinjection bilaterally of test agents into RVLM, each at a volume of 50 nl, was carried out stereotaxically and sequentially [9, 14–17]via a glass micropipette connected to a 0.5-μl Hamilton (Reno, NV, USA) microsyringe. The coordinates used were: 4.5–5 mm posterior to lambda, 1.8–2.1 mm lateral to midline, and 8.1–8.4 mm below the dorsal surface of cerebellum. These coordinates were selected to cover the ventrolateral medulla at which functionally identified sympathetic premotor neurons reside . Test agents used included Mev (kindly provided by Huikwang Corporation, Tainan, Taiwan), two specific JNK inhibitors, JNK inhibitor I (Calbiochem, San Diego, CA, USA)  and JNK inhibitor II (SP600125, Calbiochem) ; two specific p38MAPK inhibitors, p38 MAPK inhibitor III (Calbiochem)  and SB203580 (Calbiochem) ; and negative controls, JNK inhibitor I negative control (Calbiochem)  or SB202474 (Calbiochem) . All test agents used for pretreatment were given 30 min before the administration of Mev. The doses were adopted from previous reports [35–38] that used those test agents for the same purpose as in this study. Application of the same amount of artificial cerebrospinal fluid (aCSF) controlled for possible volume or solvent effect. The composition of aCSF was (mmol/L): NaCl 117, NaHCO3 25, glucose 11, KCl 4.7, CaCl2 2.5, MgCl2 1.2 and NaH2PO4 1.2. To avoid the confounding effects of drug interactions, each animal was subject routinely to only one pharmacological treatment scheme.
Collection of tissue samples from ventrolateral medulla
As in previous studies [14–17], we routinely collected tissue samples for subsequent biochemical evaluations during the peak of the pro-life phase and pro-death phase (Mev group), or 30 or 180 min after microinjection of aCSF into RVLM (vehicle control group). Animals were killed with an overdose of pentobarbital sodium and tissues 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. Medullary tissues collected from anesthetized animals without any treatment served as the sham-controls. The concentration of total proteins extracted from tissue samples was determined by the BCA protein assay (Pierce, Rockford, IL, USA).
ELISA for protein level of JNK, p38MAPK, MAP2K4, MAP2K6 or their phosphorylated forms
Cell lysate from ventrolateral medulla was subject to a commercial kit for enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol to detect the levels of JNK1/2/3 (eBioscience, San Diego, CA, USA), phosphorylated JNK1/2/3 at Thr183/Tyr185 (eBioscience), p38MAPK (eBioscience), phosphorylated p38MAPK at Thr180/Tyr182 (eBioscience), MAP2K4 (Antibodies-online, Atlanta, GA, USA), phosphorylated MAP2K4 at Ser257/Thr261 (TGR BioSciences, Thebarton, Australia), MAP2K6 (MyBioSource, San Diego, CA, USA) or phosphorylated MAP2K6 at Ser207/Thr211 (R and D Systems, Minneapolis, MN, USA). The final absorbance of reaction solution at 450 nm was determined by spectrophotometry using an ELISA microtiter plate reader (Anthros Labtec, Salzburg, Austria), and was expressed as fold changes against baseline-controls.
Nuclear extract from ventrolateral medulla
In some experiments, proteins from the nuclear fraction of the medullary samples were extracted using a commercial kit (Active Motif, Carlsbad, CA, USA). The concentration of protein in the nuclear extracts was again estimated by the BCA Protein Assay (Pierce).
ELISA for activity of transcription factors ATF-2, c-Jun or Elk-1
Nuclear extract from ventrolateral medulla was subject to a sensitive and specific commercial kit (Active Motif) for ELISA according to the manufacturer’s protocol to detect the levels of phosphorylated c-Jun at Ser73, phosphorylated E twenty-six-like transcription factor 1 (Elk-1) at Ser383 or phosphorylated ATF-2 at Thr71. The final absorbance of the reaction solution at 450 nm was determined by spectrophotometry using an ELISA microtiter plate reader (Anthros Labtec), and expressed as fold changes against baseline-controls.
In some animals that were not used for biochemical analysis, the brain stem was removed at the end of the physiological experiment and fixed in 30% sucrose in 10% formaldehyde-saline solution for at least 72 h. Frozen 25-μm sections of the medulla oblongata stained with neural red were used for histological verification of the microinjection sites.
All values are expressed as mean ± SEM. The averaged value of MSAP or HR calculated every 20 min after the administration of test agents or aCSF, the sum total of power density for the LF component in the SAP spectrum over 20 min, or the level or activity of protein or transcriptional factor in RVLM during each phase of experimental brain stem death, were used for statistical analysis. One-way or two-way ANOVA with repeated measures was used, as appropriate, to assess group means. This was followed by the Scheffé multiple-range test for post hoc assessment of individual means. P < 0.05 was considered to be statistically significant.