Sumoylation of HIF-1a in Experimental Brain Death Figure 5. Activation of HIF-1a in RVLM ameliorates failure of central cardiovascular regulation associated with experimental brain death. Temporal changes in MSAP, HR or power density of LF component of SAP signals in rats that received pretreatment by microinjection bilaterally into RVLM of NRS (1:20), anti-HIF-1a, HIF-1b or HIF-2a antiserum (1:20), 30 min before local application (at arrow) of aCSF or Mev (10 nmol) to the bilateral RVLM. Values are mean 6 SEM, n = 5–7 animals per experimental group. *P,0.05 versus NRS+aCSF group, and +P,0.05 versus NRS+Mev group at corresponding time-points in the post hoc Scheff e´ multiple-range test. B, baseline. doi:10.1371/journal.pone.0017375.g005 Animals Adult male Sprague-Dawley rats (264 to 345 g, n = 285) purchased from the Experimental Animal Center of the National Science Council, Taiwan were used. Rats received preparatory surgery under an induction dose of pentobarbital sodium (50 mg kg21, i.p.). During the experiment, animals received continuous intravenous infusion of propofol (20–25 mg kg21 h21; Zeneca, Macclesfield, UK), which provided satisfactory anesthetic maintenance while preserving the capacity of central cardiovascular regulation [53]. They were allowed to breathe spontaneously with room air, and body temperature was maintained at 37uC by a heating pad. Mev intoxication model of brain death SAP signals recorded from the femoral artery were subject to simultaneous on-line and real-time power spectral analysis [11– 14,25,27]. We were particularly interested in the LF (0.25–0.8 Hz) component of the SAP spectrum because it takes origin from RVLM [8] and the waxing and waning of the LF power reflects the prevalence of the pro-life and pro-death phases during the progression towards brain death in patients who succumbed to organophosphate poisoning [10]. HR was derived instantaneously from SAP signals. Since Mev induces comparable cardiovascular responses when given systemically or directly to RVLM [16], we routinely microinjected Mev bilaterally into RVLM to elicit site- specific effects [11–14,25,27]. The only exception was when Mev was given intravenously in conjunction with evaluation of tissue hypoxia in RVLM. Temporal changes in pulsatile SAP, mean SAP (MSAP), HR and power density of the LF component were routinely followed for 180 min after the administration of Mev, 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 [11–14,25,27] via a glass micropipette connected to a 0.5-ml Hamilton microsyringe (Reno, NV). 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. Test PLoS ONE | www.plosone.org 9 March 2011 | Volume 6 | Issue 3 | e17375