Abstract Recent studies demonstrated that LPS treated Sprague-Dawley rats induced a reduction (Phase I), followed by an augmentation (Phase II) and a secondary decrease (Phase III) in the power density of vasomotor components (0-0.8 Hz) in systemic arterial pressure signals. The molecular mechanisms underlie the progression toward death in the brain stem is unclear. In order to find out the differentially expressed genes between LPS-treated RVLM and saline-treated RVLM, we used suppression subtractive hybridization,a method commonly used to search differentially expressed genes, and subtractive cDNA library construction. At present, we have found some differentially expressed genes and these genes are up-regulation expression. These genes may be involved in the progression toward death in the rat brain stem.

Recent studies have discovered that LPS-treated Sprague-Dawley rats induced a reduction (phase I), followed by an augmentation (phase II), and decrease again (phase III) in the power density of the vasomotor component (0-0.8 Hz) in systemic arterial pressure (SAP). It was reported that the vasomotor components were related to the brain stem, even closely related to the rostral ventrolateral medulla (RVLM). But the molecular mechanism involved in the death progression of rat brain stem is mostly unknown. We used suppression subtractive hybridization (SSH) and library construction to find differentially expressed genes between phase I and phase II of LPS-treated RVLM. At present, we have found some genes that are differentially expressed between phase I and phase II of LPS-treated RVLM. Some genes are up-regulation expression and others are down-regulation expression. Thus, these genes may be involved in the molecular mechanism of the death progression in the rat brain stem.

The rostral ventrolateral medulla (RVLM) is the origin of a ¡§life-and-death¡¨ signal identifies from systemic arterial blood pressure spectrum that reflects failure of central cardiovascular regulation during brain stem death. It is also a target site where endogenous angiotensin II acts on angiotensin II type 1 receptors (AT1R) to increase blood pressure (BP); or on type 2 receptors (AT2R) to inhibit baroreceptor reflex (BRR) response. This study investigated the roles of AT1R and AT2R and their signaling pathways in RVLM for ¡§life-and-death¡¨ signal response during experimental brain stem death, using organophosphate mevinphos (Mev) as the experimental insult. In Sprague-Dawley rats, Mev (640 £gg/kg, i.v.) elicited an increase (pro-life phase) followed by a decrease (pro-death phase). Real-time PCR analysis revealed that whereas AT1R level underwent a 10% increase at pro-life phase, AT2R exhibited a significance increase of up to 40% at pro-death phase. Western blot analysis revealed that whereas AT1R level underwent a 20% increase at pro-life phase, AT2R exhibited a significant increase of up to 50% at pro-death phase. Pretreatment with microinjection of an AT1R antagonist losartan (2 nmol) into RVLM elicited abrupt death because of drastic hypotension through inhibiting NADPH oxidase and its downstream superoxide anion. Pretreatment with NADPH oxidase inhibitor DPI (1.5 nmol) inhibited NADPH oxidase avtiviting and superoxide anion production and decreased ¡§life-and-death¡¨ signal at pro-life phase; using superoxide anion inhibitor tempol (5 nmol) potentiated blood pressure and ¡§life-and-death¡¨ signal at pro-death phase. However, pretreatment with an AT2R antagonist PD123319 (2 nmol) potentiated the ¡§life-and-death¡¨ signal and antagonized hypotension during pro-death phase through inhibiting protein phosphotase 2A (PP2A) then activating extracellular signal-regulated kinase 1/2 (ERK1/2). Similar to AT2R antagonist PD123319, pretreatment with PP2A inhibitor okadaic acid (0.5 fmol) inhibit PP2A, leading to activation of ERK1/2, potentiate ¡§life-and-death¡¨ signal and antagonized hypotension during pro-death phase. These results suggest that AT1R in RVLM plays a ¡§pro-life¡¨ role through NADPH oxidase/superoxide anion during experimental brain stem death by maintaining BP and ¡§life-and-death¡¨ signal; AT2R plays a ¡§pro-death¡¨ role through PP2A/ERK1/2 by inhibiting BP and ¡§life-and-death¡¨ signal, and superoxide may also plays a ¡§pro-life and pro-death¡¨ role at pro-death phase.

Brain stem cardiovascular regulatory dysfunction during brain stem death is underpinned by an upregulation of nitric oxide synthase II (NOS II) in rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from blood pressure of comatose patients that disappears before brain stem death ensues. At the same time, the ubiquitin-proteasome system (UPS) is involved in the synthesis and degradation of NOS II. We assessed the hypothesis that the UPS participates in brain stem cardiovascular regulation during brain stem death by engaging in both synthesis and degradation of NOS II in RVLM. In a clinically relevant experimental model of brain stem death using Sprague-Dawley rats, pretreatment by microinjection into the bilateral RVLM of proteasome inhibitors (lactacystin or proteasome inhibitor II) antagonized the hypotension and reduction in the life-and-death signal elicited by intravenous administration of Escherichia coli lipopolysaccharide (LPS). On the other hand, pretreatment with an inhibitor of ubiquitin-recycling or UCH-L1 potentiated the elicited hypotension and blunted the prevalence of the life-and-death signal. Real-time polymerase chain reaction, Western blot, electrophoresis mobility shift assay, chromatin immunoprecipitation and co-immunoprecipitation experiments further showed that the proteasome inhibitors antagonized the augmented nuclear presence of NF-£eB or binding between NF-£eB and nos II promoter and blunted the reduced cytosolic presence of phosphorylated I£eB. The already impeded NOS II protein expression by proteasome inhibitor II was further reduced after gene-knockdown of NF-£eB in RVLM. In animals pretreated with UCH-L1 inhibitor and died before significant increase in nos II mRNA occurred, NOS II protein expression in RVLM was considerably elevated. We conclude that UPS participates in the defunct and maintained brain stem cardiovascular regulation during experimental brain stem death by engaging in both synthesis and degradation of NOS II at RVLM. Our results provide information on new therapeutic initiatives against this fatal eventuality.