West Nile virus (WNV) is a neurotropic flavivirus that causes

Apoptosis is an important mechanism of West Nile virus (WNV) pathogenesis within the central nervous system (CNS).N euronal apoptosis is an important mechanism of virus-induced pathogenesis within the central nervous system (CNS) (1). During West Nile virus (WNV) encephalitis, the proapoptotic executioner caspase, caspase 3, is activated, and mice lacking caspase 3 have reduced neuronal death and tissue injury following West Nile virus infection (2). Despite the importance of apoptosis in WNV pathogenesis, the exact pathways involved in triggering apoptotic cell death in the CNS have not yet been defined.Activation of initiator caspases 8 (3, 4) and 9 (3) occurs in cultured neuronal cells infected with WNV, and inhibition of these initiator caspases leads to reduced cleavage of the caspase 3 substrate poly(ADP-ribose) polymerase (PARP) (3, 4). These studies indicate that both extrinsic and intrinsic apoptotic signaling pathways are activated in vitro following WNV infection and are consistent with in vitro studies demonstrating that mitochondrial apoptotic signaling protein Bax is upregulated in neuronal cells following WNV infection (5) and that cytochrome c is released from the mitochondria (3). However, it remains to be seen whether the same apoptotic pathways are also activated in the intact brain during WNV encephalitis.Innate and adaptive immune responses also influence WNV pathogenesis within the CNS. Toll-like receptors 3 and 7 (TLR3 and TLR7) and the cytoplasmic proteins encoded by retinoic acidinducible gene I (RIG-I) and melanoma-differentiation-associated gene 5 (MDA5) are important for detection of WNV within the CNS, and defects in these, or additional, components of the interferon (IFN) response show enhanced viral burden and increased lethality (6). Although IFN restricts infection, pathogenic WNV strains attenuate IFN function at several steps of the induction and signaling cascade, allowing the virus to establish infection (6). Studies performed in mice (7-11) and humans (12-14) have also highlighted the role of CD8 ϩ T cells and the associated importance of pathways involving Fas ligand (10) and tumor necrosis factor (TNF)-related apoptosis-inducing factor ligand (TRAIL) (15) effector mechanisms in containing WNV CNS infection.In this report, we demonstrate that genes involved in death receptor (DR) apoptotic signaling are upregulated in the mouse brain following WNV infection. We also show for the first time that the activity of the DR-associated initiator caspase, caspase 8, is increased in the brain following WNV infection. WNV-induced activation of caspase 8 in the CNS is associated with cleavage of the proapoptotic Bcl-2 family protein Bid and with activation of caspase 9, suggesting that the caspase 8-dependent cleavage of Bid promotes intrinsic apoptotic signaling within the brains of infected animals. Utilization of WNV-infected ex vivo brain slice cultures (BSC), a novel model of WNV encephalitis, revealed that inhibition of caspase 8 decreased virus-induced activation of caspase 3 an...

West Nile virus (WNV) is a neurotropic flavivirus that can cause significant neurological disease. Mouse models of WNV infection demonstrate that a proinflammatory environment is induced within the central nervous system (CNS) after WNV infection, leading to entry of activated peripheral immune cells. We utilized spinal cord slice cultures (SCSC) to demonstrate that anti-inflammatory mechanisms may also play a role in WNV-induced pathology and/or recovery. Microglia are a type of macrophage that function as resident CNS immune cells. Similar to mouse models, infection of SCSC with WNV induces the upregulation of proinflammatory genes and proteins that are associated with microglial activation, including the microglial activation marker Iba1 and CC motif chemokines CCL2, CCL3, and CCL5. This suggests that microglia assume a proinflammatory phenotype in response to WNV infection similar to the proinflammatory (M1) activation that can be displayed by other macrophages. We now show that the WNV-induced expression of these and other proinflammatory genes was significantly decreased in the presence of minocycline, which has antineuroinflammatory properties, including the ability to inhibit proinflammatory microglial responses. Minocycline also caused a significant increase in the expression of anti-inflammatory genes associated with alternative anti-inflammatory (M2) macrophage activation, including interleukin 4 (IL-4), IL-13, and FIZZ1. Minocycline-dependent alterations to M1/M2 gene expression were associated with a significant increase in survival of neurons, microglia, and astrocytes in WNV-infected slices and markedly decreased levels of inducible nitric oxide synthase (iNOS). These results demonstrate that an anti-inflammatory environment induced by minocycline reduces viral cytotoxicity during WNV infection in CNS tissue. West Nile virus (WNV) causes substantial morbidity and mortality, with no specific therapeutic treatments available. Antiviral inflammatory responses are a crucial component of WNV pathology, and understanding how they are regulated is important for tailoring effective treatments. Proinflammatory responses during WNV infection have been extensively studied, but anti-inflammatory responses (and their potential protective and reparative capabilities) following WNV infection have not been investigated. Minocycline induced the expression of genes associated with the anti-inflammatory (M2) activation of CNS macrophages (microglia) in WNV-infected SCSC while inhibiting the expression of genes associated with proinflammatory (M1) macrophage activation and was protective for multiple CNS cell types, indicating its potential use as a therapeutic reagent. This culture system can uniquely address the ability of CNS parenchymal cells (neurons, astrocytes, and microglia) to respond to minocycline and to modulate the inflammatory environment and cytotoxicity in response to WNV infection without peripheral immune cell involvement.

Objective: Polyethylene glycol (PEG), a hydrophilic polymer, can immediately repair neuronal membranes and inhibit free radical production following trauma. The aim of this study is to examine whether PEG can directly repair mitochondria in the event of trauma. Method: Purified brain mitochondria from guinea pigs were used. Mitochondrial function was assessed by biochemical methods and structural changes were observed by both fluorescence light microscopy and coherent anti-Stokes Raman scattering microscopy. Results: We present evidence suggesting that PEG is capable of directly reducing injury to mitochondria independent of plasma membrane repair. Specifically, the suppression of oxygen consumption rate of purified mitochondria due to H₂O₂ and/or calcium can be significantly reversed by 12.5 mM PEG. PEG also significantly reduced mitochondrial swelling due to similar injury. Furthermore, we have shown that such PEG-mediated mitochondrial protection is dependent on the molecular weight of PEG, suggesting a direct physical blockade of mitochondrial permeability transitional pore by PEG. Conclusion: These findings, coupled with previous evidence that PEG enters the cytosol following mechanical trauma, strongly indicate that there are at least 2 avenues of PEG-mediated cytoprotection in mechanically injured spinal cords: repair of plasma membrane and protection of mitochondria.