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Wadgaonkar, Rajendra
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Fall 2011
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2011-07-21
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Doctoral Dissertation
Adobe PDF, 3.97 MB
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Sepsis is the number one killer in hospital critical care units, and the 10th leading cause of death in the United States. Despite such staggering statistics, the pathogenesis of the central nervous system (CNS) dysfunction during sepsis is poorly understood. CNS dysfunction usually results in neuroinflammation and encephalopathy. During sepsis blocking of ceramide (Cer), tumor necrosis factor (TNFα), platelet-activating factor (PAF) and prostaglandins have not proven to be effective in clinical trials. This suggests that several parallel and interacting molecular mechanisms are involved. Regulation of sphingolipid metabolism is a likely mechanism in maintaining the cellular processes, such as cell differentiation, proliferation, migration, cell survival and cell death, in the CNS. Sphingosine kinase (SPHK) is the enzyme in the sphingolipid pathway that converts sphingosine (Sph) into sphingosine-1-phosphate (S-1-P). At least two sphingosine kinase isoforms have been cloned and characterized - sphingosine kinase 1 (SPHK1) and sphingosine kinase 2 (SPHK2). We hypothesized that SPHK1 induction and/or SPHK2 inhibition may represent novel treatment targets for sepsis and septic encephalopathy (SE). Bacterial endotoxin - lipopolysaccharide (LPS) was used to establish a septic brain injury model in vivo and study neuroinflammatory parameters. Neuroinflammation was induced by intracerebral injection of LPS 1mg/kg into the lateral ventricles of wild type (WT) and SphK1-/- mice; control animals received sterile saline. Neuroinflammatory parameters were assayed such as white matter rarefactions, development of cytotoxic edema, astrogliosis, oligodendrocytes precursors loss, and cytokines levels. The data showed significant upregulation of the neuroinflammatory response in SphK1-/- mice. We tested intraperitoneal injection of LPS at two doses 5mg/kg and 10 mg/kg. Heart rates were measured, white blood cells were counted, antigenic markers of microglial and astrocytic activation and oligodendrocytes progenitors were assayed by immunohistochemistry and Western Blot. Levels of TNFα and IL-6 were assayed by reverse transcriptase PCR (rt-PCR) and level of pro-apoptotic protein - caspase-8 was assayed by Western Blot. These data showed that LPS at a dose of 10mg/kg is more sufficient to induce severe septic brain injury. In addition, SphK1-/- mice had persistent upregulation of neuroinflammatory parameters in the absence of exogenous induction of neuroinflammation. These data proved that SPHK1-/- mice are more susceptible to LPS induced brain injury and revealed a protective role of SphK1 in the regulation of neuroinflammation. These data also strongly suggest that SphK1 regulates basal levels of neuroinflammatory proteins as well.
Citation
Grin'kina, Natalia (2011). Sphingosine kinase 1 in septic brain injury. [Doctoral dissertation, SUNY Downstate Health Sciences University]. SUNY Open Access Repository. https://soar.suny.edu/handle/20.500.12648/15908
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Doctoral Dissertation