reduction of the life-and-death signal [11–16]. Since this temporal pattern resembles that exhibited during the progression towards brain death by patients died of organophosphate poisoning [6], they are designated respectively the pro-life and pro-death phase in this model of brain death [10]. We further demonstrated that nitric oxide (NO) generated by NO synthase I (NOS I) in RVLM, followed by activation of the soluble guanylyl cyclase/cGMP/ protein kinase G (PKG) cascade, is responsible for the pro-life phase; peroxynitrite formed by a reaction between NOS II-derived NO and superoxide anion underlies the pro- death phase [13,14]. That Mev elicits hypoxia in RVLM [15] and that both NOS I [17,18] and NOS II [19,20] are hypoxia responsive gene products offer an important clue in our continual search for the cellular and molecular mechanisms that underlie brain death. Because of the reduction in the amount of oxygen that reaches brain tissues, hypoxia in brain tissue may trigger a series of adaptive responses in neurons. In this regard, the transcription factor hypoxia-inducible factor 1 (HIF-1) operates as a master regulator of cellular responses to hypoxia via activation of a multitude of oxygen-sensitive gene products [21–24]. We demonstrated recently [25] that one of the hypoxia responsive gene products upregulated transcriptionally by HIF-1a [26] in RVLM is heme oxygenase-1, which plays a pro-life role in experimental brain death by sustaining central cardiovascular regulatory function via activation of heat shock protein 70 [27]. A new system of post-translational protein modification that is also activated by transient cerebral ischemia [28,29] or hypoxia [30], in addition to phosphorylation and ubiquitin conjugation, is massive protein conjugation by the small ubiquitin-like modifier (SUMO). Of note is that HIF-1a is among the many transcription factors that are sumoylated [31]. Acute hypoxia induces expression of SUMO-1 [32], although the effects of sumoylation of HIF-1a on its stability and transcriptional activity are controversial [33–36]. It follows that sumoylation of HIF-1a in RVLM in response to hypoxia may play a modulatory role on brain stem cardiovascular regulation during experimental brain death. The present study assessed the hypothesis that sumoylation of HIF-1a in RVLM participates in brain stem cardiovascular regulation during brain death by interacting with NOS I/PKG or NOS II/peroxynitrite signaling pathway. We demonstrated that sumoylation of HIF-1a in RVLM plays a preferential pro-life role by ameliorating cardiovascular regulatory failure during brain death via selective upregulation of NOS I/PKG signaling cascade. Results Biphasic cardiovascular responses in experimental brain death Figure 1 shows that microinjection bilaterally of Mev (10 nmol) into RVLM in animals pretreated with vehicle elicited a progressive hypotension that became significant 100 min after application, accompanied by indiscernible alterations in heart rate (HR). Concurrent changes in power density of the LF component of SAP signals revealed two distinct phases of Mev-induced cardiovascular responses. The pro-life Phase I entailed a significantly augmented LF power that endured 80–100 min. The pro-death Phase II, which lasted the remainder of our 180- min observation period, exhibited further and significant reduction in the power density of this spectral component to below baseline to reflect failure of brain stem cardiovascular regulatory functions that precedes brain death [10]. Sumoylation of HIF-1a in Experimental Brain Death Sumoylation of proteins in RVLM ameliorates failure of brain stem cardiovascular regulation associated with experimental brain death The most fundamental premise for the present study is that sumoylation of proteins in RVLM is causally involved in brain stem cardiovascular regulation during brain death. Our first series of experiments employed loss-of-function by immunoneutraliza- tion to establish this premise. Figure 1 shows that compared to normal rabbit or mouse serum, pretreatment with microinjection bilaterally into RVLM of an antiserum (1:20) against SUMO-1 or Ubc9, the only known conjugating enzyme for the sumoylation pathway [37,38] 30 min before local application of Mev (10 nmol), significantly and selectively enhanced the progressive hypotension and reduced the increase in power density of LF component of SAP signals during Phase I Mev intoxication, without affecting HR. In addition, the hypotension and reduced LF power already exhibited during Phase II were significantly potentiated. Preferential augmentation of protein sumoylation in RVLM during the pro-life phase We next evaluated the logical possibility that the augmentation of sumoylated proteins in RVLM underpins its ameliorating effect on Mev-elicited central cardiovascular regulatory dysfunction, noting that acute hypoxia induces expression of SUMO-1 [32]. In this particular set of experiments, Mev was given intravenously because the dimension of this neural substrate does not allow for simultaneous placement of the glass micropipette used for microinjection and the probe used for sensing tissue oxygen and blood flow. Our laboratory demonstrated previously [16] that systemic administration of Mev induces cardiovascular responses by acting on RVLM. Given at an i.v. dose of 640 mg kg21, the elicited changes in SAP, HR and LF power were compatible both in magnitude and time-course with those induced by microinjec- tion of Mev (10 nmol) directly to RVLM. Figure 2A shows that the tissue oxygen tension in RVLM underwent, after a short latency, a drastic drop by 1265 mmHg (n = 7) that lasted 8–10 min after i.v. administration of Mev. This was followed by a sustained hypoxia at 462 mmHg (n = 7) below baseline over the remainder of our 180-min observation period. Intriguingly, the local blood flow responded with an increase that mirrored the abrupt hypoxia, followed by a sustained reduction of approximately 550660 blood perfusion units (BPU) (n = 7). Western blot analysis (Figure 2B) revealed a significant augmentation of proteins that were conjugated to SUMO-1 in both cytosolic and nuclear fractions of extracts from ventrolateral medulla during early Phase I when the drastic drop in tissue oxygen level in RVLM took place. This increase sustained throughout Phase I, and returned to baseline during Phase II Mev intoxication. Ubc9 exhibited similar temporal expression pattern (Figure 2B). On the other hand, the level of proteins conjugated by SUMO-1 or expression of Ubc9 in ventrolateral medulla of animals that received artificial cerebrospinal fluid (aCSF) was comparable to sham controls (Figure 2B and 2C). Double immunofluorescence staining coupled with laser scanning confocal microscopy revealed that the results on SUMO-1 demonstrated in our biochemical analyses on protein extracts from ventrolateral medulla indeed took place at the neuronal level. Against a clearly defined nucleus and nucleolus in cells stained positively with the neuronal marker, neuron-specific nuclear protein (NeuN), there was an increased presence of SUMO-1- immunoreactivity in the cytoplasm and nucleus of RVLM neurons during early and late Phase I when compared with sham control or PLoS ONE | www.plosone.org 2 March 2011 | Volume 6 | Issue 3 | e17375