Systemic inflammation induces apoptosis with variable vulnerability of different brain regions
Introduction
Sepsis and its complications are the leading causes of mortality accounting for 10–50% of deaths on intensive care units (Tran et al., 1990, Brun-Buisson, 2000). In recent years, the characterization of inflammatory cascades leading to multi-organ failure (MOF) and ultimately death has attracted much interest. Since most studies focussed on peripheral organs such as lung, liver, gut and kidney, the participation of the brain during sepsis seems less clear. Septic encephalopathy (SE) represents a brain dysfunction due to sepsis and systemic inflammatory response syndrome (SIRS). SE has been reported to occur in 8–70% of septic patients, depending on the inclusion criteria employed (Sprung et al., 1990, Young et al., 1990). Additionally, it is the most common encephalopathy in intensive care units (Bleck et al., 1993). Importantly, SE is associated with a significantly higher mortality rate of septic patients (Sprung et al., 1990). Despite being a critical factor for brain function and overall survival, the pathophysiological mechanisms leading to SE remain to be elucidated.
Induction of the inducible isoform of nitric oxide (NO)-producing enzymes (iNOS) is a key feature of sepsis and SIRS. Because iNOS is independent of Ca2+, its activity is sustained and can last for days (Xie and Nathan, 1994), resulting in NO-mediated cell death and tissue damage (Kim et al., 2001). iNOS expression in response to sepsis has been detected in peripheral tissue (Thiemermann, 1997), but also in neuronal and glial cells in different animal models of peripheral inflammation (Jacobs et al., 1997, Satta et al., 1998, Wong et al., 1996). It has been shown that neurons are remarkably sensitive to NO and undergo cell death once exposed to sustained NO generation (Boje and Arora, 1992, Chao et al., 1992, Leist et al., 1997, Heneka et al., 1998). NO generated by cytokine-activated murine microglial cells is highly neurotoxic (Peterson et al., 1994) and activated glia has a remarkable capacity to kill cocultured neurons even when activated glial cells are present at relatively low numbers (Chao et al., 1992, Chao et al., 1996, Hu et al., 1997, Kingham et al., 1999, Bal-Price and Brown, 2000, Bal-Price and Brown, 2001). In addition to these observations, a causal role of iNOS expression for neuronal apoptosis has been demonstrated after microinjection of IFN-gamma/LPS (Matsuoka et al., 1999) or in artificially infected animals (Quan et al., 1999).
Several findings suggest that NO induces apoptosis by regulating pro- and antiapoptotic protein levels. During ischemia, NO is involved in apoptosis of parenchymal brain cells in vivo (Elibol et al., 2001) and in vitro (Tamatani et al., 1998a) by influencing Bcl-2 and Bax protein levels. Exposure of hippocampal neurons to the NO releasing compound sodium nitroprusside (SNP) leads to down-regulation of Bcl-2 and up-regulation of Bax protein levels followed by caspase-3-like cleavage activity (Tamatani et al., 1998b). Inhibition of NO production by N(G)-monomethyl-l-arginine, in astrocytes exposed to hypoxia/reoxygenation, increased neuronal survival and reversed the down-regulation of Bcl-2 and up-regulation of Bax protein levels in cocultured neurons (Tamatani et al., 1998a). A role of NO for neuronal cell death is further supported by Matsuoka et al. (1999), who reported neuronal apoptosis in the rat hippocampus after microinjection of IFN-y/LPS, that was diminished by the NOS inhibitor N(G)-nitro-l-arginine dose-dependently (l-NA).
To investigate how the brain is affected during experimental sepsis, we determined expression patterns of iNOS, micro- and astroglial activation markers, terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL), Bax and Bcl-2-immunoreactivity.
Section snippets
Materials
Phosphate-buffered saline (PBS) was purchased from GIBCO Life Technologies (Karlsruhe, Germany). Bacterial lipopolysaccharide (LPS) (E. coli, 0127:B8) was from Sigma (Deisenhofen, Germany). Acetic acid, formaldehyde, methanol and all other chemicals were of analytical grade and purchased from Merck (Darmstadt, Germany).
Animals and animal procedures
Male Wistar rats (Interfauna, Tuttlingen, Germany) weighing 250–300 g were housed in groups of four under standard conditions at a temperature of 22 °C (±1 °C) and a 12 h light:12 h
Results
Approximately, 40% of rats did not survive the LPS challenge during the observation period. Treatment with NMMA did not change the survival rate.
In the surviving animals intraperitoneal administration of LPS induced a time-dependent increase of glial activation markers and iNOS immunoreactivity in most of the examined brain regions. The increase of iNOS-positive cells was observed in all brain areas, except the cortex (Fig. 1, Fig. 2) and reached the level of statistical significance at 24 h
Discussion
The present study investigates the effects of peripheral inflammation on the brain and hereby reveals that activation of micro- and astroglial cells during experimental sepsis is followed by apoptosis of parenchymal brain cells. This sepsis-induced neuronal cell death is at least in part, caused by the generation of nitric oxide and is accompanied by changes in the Bax and Bcl-2 protein regulation.
Immunoactivation of brain cells in response to experimental sepsis was demonstrated by a
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