None of the immunohistochemical controls exhibited specific immunoreactivity. While most of the immunoreactions were carried out using 7 mm paraffin sections, free-floating vibratome sections (60 m) were used for nestin, and VEGF receptor (flt-1andflk-1) immunostaining. on astrogliosis. By contrast infusion of NA toflt-1significantly decreased astroglial mitogenicity and scar formation and caused some increase in endothelial degeneration. Neutralization of theflt-1receptor function, but notflk-1, caused significant reduction in the astroglial expression of the growth factors, CNTF and FGF by seven days. These data suggest that after CNS injury, endogenous VEGF upregulation (by astrocytes) induces angiogenesis and, by autocrine signaling, increases both astrocyte proliferation and facilitates expression of growth factors. It is likely that VEGF plays an important role in aspects of astroglial scar formation. Keywords:Astrocytes, gliosis, angiogenesis, VEGF, CNTF, bFGF, nestin == Introduction == The CNS responds immediately to trauma by triggering a series of cellular events that are designed to repair the vasculature and wall off the injured area (Fitch and Silver 1997;Fawcett and Asher 1999;McGraw et al. 2001). Revascularization and repair of the blood-brain barrier reestablish metabolic and trophic support to the injured tissue (de Paermentier et al. 1986;Krum and Felbinac Rosenstein 1988;Orita Felbinac et al. 1989). Activated astrocytes form a gliotic scar to provide structural support, and isolate the injury site by reestablishing the glia limitans, thus restricting the migration of inflammatory cells into healthy tissue (Stichel and Mller 1998;Sofroniew 2005). Such reactive gliosis is characterized by astroglial proliferation, hypertrophy, process extension, and increased synthesis of intermediate filaments, including GFAP and vimentin, as well as production of many bioactive molecules (Eddleston and Mucke 1993;Silver and Miller 2004). Astroglial reactivity is stimulated by a number of factors, including thrombin, bFGF, CNTF, TGF, TGF1, TGF2, TNF, and IL-1 (Fawcett 1997;Campbell 2001;Swartz et al. 2001), several of which are microglial-derived cytokines (Liberto et al. 2004), and by trauma-induced release of purines such as ATP (Neary and Kang 2005). One of the cytokines with seminal involvement in the process of brain repair is vascular endothelial growth Felbinac factor (VEGF), which is upregulated during many pathological events in the CNS, including ischemia (Kovacs et al. 1996;Cobbs et al. 1998;Lennmyr et al. 1998;Issa et al. 1999;Lee et al. 1999;Pichiule 1999;Plate et al. 1999;Jin et al. 2000), cold lesions (Nag et al. 1997;Papavassiliou et al. 1997), spinal cord injuries (Bartholdi et al. 1997;Tsao et al. 1999;Vaquero et al. 1999), brain contusion (Skold et al. 2005) and direct wounding (Krum and Rosenstein 1998,1999). VEGF Felbinac is a secreted dimeric protein that is fundamentally important for angiogenesis during development, tissue regeneration/repair and tumor growth throughout the body (Leung et al. 1989;Klagsbrun and DAmore 1996;Thomas 1996;Ferrara et al. 2003). The major isoform present in most mammalian tissues is VEGF165, a 45 kDa heparin binding, secreted glycoprotein (Ferrara et al. 2003). VEGF binds to the tyrosine kinase receptorsflt-1(VEGF-R1) and (predominantly)flk-1(KDR/VEGF-R2) that are expressed by vascular endothelial cells and triggers the mitotic and migratory processes necessary for angiogenesis in the periphery (Shibuya and Claesson-Welsh 2006). VEGF is also a vascular maintenance factor that promotes endothelial cell survival Rabbit Polyclonal to LIMK1 by acting through theflk-1receptor (Yang, W. and de Bono 1997;Darland et al. 2003;Huang et al. 2003). In the intact adult CNS, VEGF protein expression is limited to the choroid plexus, area postrema, and cerebellar granule cells (Monacci et al. 1993), and VEGF receptors are normally expressed at very low levels (Peters et al. 1993;Kremer et al. 1997;Soker et al. 1998). In the injured CNS, VEGF is implicated in post-traumatic angiogenesis, which is dependent upon the upregulation of endothelialflk-1(Krum and Rosenstein 1998;Rosenstein et al. 1998;Issa et al. 1999;Krum and Rosenstein 1999;Plate et al. 1999;Proescholdt et al. 1999;Silverman et al. 1999;Jin et al. 2000;Beck et al. 2002;Harrigan et al. 2002;Krum et al. 2002;Mani et al. 2003;Croll et al. 2004;Skold et al. 2005). Concomitantly, VEGF protein is also strongly upregulated in astroglia and inflammatory cells near the damaged area (Bartholdi et al. 1997;Nag et al. 1997;Papavassiliou et al. 1997;Krum and Rosenstein 1998,1999;Tsao et al. 1999;Vaquero et al. 1999;Salhia et al. 2000;Chodobski et al. 2003;Skold et al. 2005); in ischemia models, neurons have also been reported to express VEGF (Kovacs et al. 1996;Cobbs et.