Scale bar, 10 m. It is well accepted that apoptotic cells are rapidly cleared by phagocytesin vivowithout intracellular content release (Lauberet al.2004). Although exogenous TNF failed to cause significant cell death in enriched preOL cultures, it became cytotoxic when preOLs were in contact with astrocytes. Collectively, our results demonstrate oligodendroglial TNFR1 in mediating inflammatory destruction of preOLs and suggest a previously unrecognized role for Leukadherin 1 astrocytes in promoting TNF toxicity to preOLs. Keywords:oligodendrocyte precursors, cell death, microglia, white matter injury, inflammation, periventricular leukomalacia == Introduction == Inflammatory responses associated with activation of microglia within the central nervous system (CNS) have been implicated in many pathological conditions including white matter disorder periventricular leukomalacia (PVL). PVL is the major form of cerebral white matter injury and the most important determinant of the neurologic morbidity in premature infants (Volpe 2003). Pathologically, it is characterized by focal necrotic lesions deep in the cerebral white matter that correlates with the development of cerebral palsy, and by diffuse white matter lesions leading to subsequent myelination abnormalities that likely contribute to cognitive and behavioral deficits frequently observed in survivors of prematurity (Volpe 2003). Diffuse PVL lesions are now recognized as the predominant form of brain injury of prematurity with hallmarks of prominently activated astrocytes and microglia and dying premyelinating oligodendrocytes (preOLs) (Hayneset al.2003). Although the role of these reactive astrocytes and microglia remains unclear, they are likely involved in preOL injury by releasing highly reactive oxygen/nitrogen species and proinflammatory cytokines, such as tumor necrosis factor (TNF) (Hayneset al.2005,Back 2006,Rezaie & Dean 2002). In fact, preOLs, the primary OL lineage cells in human cerebral white matter during the peak risk period for PVL (Backet al.2001), are intrinsically highly sensitive to oxidative/nitrative (Backet al.1998,Backet al.2002) and excitotoxic insults (McDonaldet al.1998,Follettet al.2000). Hypoxia/ischemia and maternal/fetal infection are the two primary triggers for PVL (Volpe 2001). Accumulating experimental and clinical evidence suggests a strong link between the gram negative bacterial component lipopolysaccharide (LPS) and PVL. Many animal studies employing systemic, local, or intrauterine LPS administration and hypoxia/ischemia have demonstrated selective injury to developing cerebral white matter, leading to hypomyelination (reviewed by (Hagberget al.2002)). Proinflammatory cytokine TNF is a central player in cerebral ischemia and neuroinflammation, and may contribute to the pathogenesis of PVL. TNF is known to exert most of its biological functions by signaling through cognate receptors TNF receptor 1 (TNFR1, p55) and/or receptor 2 (TNFR2, p75). Increased TNF production and TNFR1/2 expression have been reported for human PVL (Deguchiet al.1996,Kadhimet al.2006). Furthermore, transgenic animal studies have demonstrated that overexpression of TNF in astrocytes results in OL apoptosis and demyelination (Akassoglouet al.1998), implying a pathogenic effect of Leukadherin 1 local TNF signaling in the CNS. Using anin vitromodel for inflammatory injury to preOLs, we demonstrated that LPS induces selective preOL death indirectly by activating microglia (Liet al.2008,Liet al.2005). We further identified that peroxynitrite underlies the potent direct killing capability of activated microglia to preOLs and that astrocytes can shift the killing mechanism to one dependent on TNF signaling (Liet al.2008). However, neither the cellular source for TNF production nor the TNF receptor mediating preOL death was identified. Since primary glial cells all express TNF receptors (Doppet al.1997), they can all engage in TNF signaling. Here, we systematically dissected the cellular source for TNF production, determined Rabbit Polyclonal to OR2T2 the TNF receptor required for LPS toxicity, and further identified an essential role for oligodendroglial TNFR1 in TNF-mediated killing of preOLs in mixed glial cultures. Most importantly, we provided the first evidence demonstrating that astrocytes sensitize preOLs to TNF toxicity in a contact-dependent manner. == Materials and Methods == == Animals and reagents == Wildtype B6.129SF2/J and C57BL/6J mice, transgenic eGFP mice (003291, background C57BL/6J), and TNF (background B6.129SF2/J) and TNFR1 (background C57BL/6J) knockout mice were obtained from the Jackson Leukadherin 1 Laboratory (Bar Harbor, ME). Recombinant TNF was obtained from R&D Systems (Minneapolis, MN). PDGF and basic FGF were from PeproTech (Rocky Hill, NJ). Recombinant GFP adenovirus was from Gene Transfer Vector Core, University of Iowa. Rabbit polyclonal antibodies against Iba1 were purchased from Wako Chemicals (Richmond, VA). Rat antimouse TNF (clone MP6-XT22) was obtained from eBioscience (San Diego, CA). Olig2 antibody was a generous gift from Dr. Richard Lu (University of Texas.