The cellular and molecular basis of brain stem death remains an enigma. As the origin of a "life-and-death" signal that reflects the progression toward brain stem death, the rostral ventrolateral medulla (RVLM) is a suitable neural substrate for mechanistic delineation of this phenomenon. Here, we evaluated the hypothesis that heat shock proteins (HSPs) play a neuroprotective role in the RVLM during brain stem death and delineated the underlying mechanisms, using a clinically relevant animal model that employed the organophosphate pesticide mevinphos (Mev) as the experimental insult. In Sprague-Dawley rats, proteomic, Western blot, and real-time PCR analyses demonstrated that Mev induced de novo synthesis of HSP60 or HSP70 in the RVLM without affecting HSP90 level. Loss-of-function manipulations of HSP60 or HSP70 in the RVLM using antiserum or antisense oligonucleotide potentiated Mev-elicited cardiovascular depression alongside reduced nitric-oxide synthase (NOS) I/protein kinase G signaling, enhanced NOS II/peroxynitrite cascade, intensified nucleosomal DNA fragmentation, elevated cytoplasmic histone-associated DNA fragments or activated caspase-3, and augmented the cytochrome c/caspase-3 cascade of apoptotic signaling in the RVLM. Coimmunoprecipitation experiments further revealed a progressive increase in the complex formed between HSP60 and mitochondrial or cytosolic Bax or mitochondrial Bcl-2 during Mev intoxication, alongside a dissociation of the cytosolic HSP60-Bcl-2 complex. We conclude that HSP60 and HSP70 confer neuroprotection against Mev intoxication by ameliorating cardiovascular depression via an anti-apoptotic action in the RVLM. The possible underlying intracellular processes include enhancing NOS I/protein kinase G signaling and inhibiting the NOS II/peroxynitrite cascade. In addition, HSP60 exerts its effects against apoptosis by blunting Mev-induced activation of the Bax/cytochrome c/caspase-3 cascade.Whereas brain stem death is currently the clinical definition of death in many countries (1, 2), the cellular and molecular underpinnings of this phenomenon of paramount medical importance are wanting. The invariable prognosis, that asystole takes place within hours or days after the diagnosis of brain stem death (3), strongly suggests that permanent impairment of the brain stem cardiovascular regulatory machinery should precede death. It is therefore intriguing that our laboratory demonstrated previously that a common denominator exists among patients who succumbed to systemic inflammatory response syndrome (4), severe brain injury (5), or organophosphate poisoning (6). We found that a dramatic reduction or loss of the power density of the low frequency (LF) 3 component (0.04 -0.15 Hz in human) in the systemic arterial pressure (SAP) spectrum, which reflects failure of brain stem cardiovascular regulatory functions, invariably precedes death. We further established that this "life-and-death" signal takes origin from the rostral ventrolateral medulla (RVLM) (7), the brain stem site responsible ...

The rostral ventrolateral medulla (RVLM) is the origin of a 'life-and-death' signal that reflects central cardiovascular regulatory failure during brain stem death. Using an experimental endotoxaemia model, we evaluated the hypothesis that the 60 kDa heat shock protein 60 (HSP60) reduces cardiovascular fatality during brain stem death via an anti-apoptotic action in the RVLM. In Sprague-Dawley rats maintained under propofol anaesthesia, proteomic or Western blot analysis revealed a progressive augmentation of HSP60 expression in the RVLM after intravenous administration of Escherichia coli lipopolysaccharide (30 mg kg −1 ). Pretreatment with a microinjection of actinomycin D or cycloheximide into bilateral RVLM significantly blunted this HSP60 increase, whereas real-time PCR showed progressive augmentation of hsp60 mRNA. Intriguingly, superimposed on the augmented expression was a progressive decline in mitochondrial, or elevation in cytosolic, HSP60 in ventrolateral medulla. Loss-of-function manipulations in the RVLM using anti-HSP60 antiserum or antisense hsp60 oligonucleotide exacerbated mortality by potentiating the cardiovascular depression during experimental endotoxaemia, alongside intensified nucleosomal DNA fragmentation, elevated cytoplasmic histone-associated DNA fragments or augmented cytochrome c-caspase-3 cascade of apoptotic signalling in the RVLM. Immunoprecipitation coupled with immunoblot analysis further revealed a progressive increase in the complex formed between HSP60 and mitochondrial or cytosolic Bax or mitochondrial Bcl-2 during endotoxaemia, alongside a dissociation of the cytosolic HSP60-Bcl-2 complex. We conclude that HSP60 redistributed from mitochondrion to cytosol in the RVLM confers neuroprotection against fatal cardiovascular depression during endotoxaemia via reduced activation of the cytochrome c-caspase-3 cascade of apoptotic signalling through enhanced interactions with mitochondrial or cytosolic Bax or Bcl-2.

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Our current understanding on brain death remains limited despite its clinical importance. This study evaluated whether the proteome expressed in the rat rostral ventrolateral medulla (RVLM), a neural substrate that our laboratory identified previously to be intimately related to brain death, is uniquely different from other brain areas, using the cerebral cortex, which is defunct under persistent vegetative state for comparison. We found that a group of antioxidant proteins, including members of the peroxiredoxin (Prx) family (Prx-1, Prx-2, Prx-5, Prx-6), thioredoxin and mitochondrial manganese superoxide dismutase, exhibited significantly higher protein and mRNA expression levels in RVLM when compared to cerebral cortex. Tissue oxygen, ATP contents and ATP synthase subunits α and β in RVLM were also significantly elevated. On the other hand, protein and mRNA levels of members of the ubiquitin-proteasome system, including proteasome subunit α type-1, ubiquitin, uniquitin-conjugating enzyme E2 N, ubiquitin carboxyl-terminal hydrolase isozyme L1 and L3, were comparable in both brain regions. We conclude that a significantly elevated level of antioxidant proteins and mRNA in RVLM is consistent with the exhibition of higher tissue oxygen tension and metabolic energy production in this neural substrate, which together constitute a safeguard mechanism against brain death.