Abstract
Abstract: :
Purpose: Application of fibroblast growth factor (FGF-2) to the cut axons has been shown to promote the survival of axotomized retinal ganglion cells (RGCs) of the frog, Rana pipiens. Recently the ERK signal transduction cascade has been shown to be activated by FGF-2 treatment of retinal neurons in vitro. The purpose of this research is to test the hypothesis that the in vivo survival effects of FGF-2 also involve the activation of this signaling pathway. Methods: The optic nerve of Rana pipiens was cut between eyeball and chiasm and treated immediately with FGF-2. After 24hrs, 7 days or 15 days the experimental retinas and optic nerves were dissected (n = 3). Frozen sections of retinal tissue were prepared for immunocytochemical staining with polyclonal antibodies against ERK1, ERK2 and phosphorylated ERK1/2. Western blots were performed using protein extractions from each group of dissected retinas. Immunoblotted protein signals (MEK1/2, phosphorylated MEK1/2, ERK1, ERK2 and phosphorylated ERK1/2) were analyzed by densitometry. Each experiment was performed in triplicate. Results: Phosphorylated ERK1/2 immunoreactivity is increased in retinal ganglion cell bodies following axotomy and further increased after FGF-2 treatment. Axotomy transiently upregulates the ERK1/2 activator, MEK1/2, for up to 24 hours after injury, followed by a gradual decrease over 7 days. FGF-2 treatment increases and sustains this activation for up to 15 days after axotomy. Following the activation of MEK1/2, phosphorylated ERK1/2 levels increase 24 hours after axotomy and eventually decrease after 7 days. In contrast, FGF-2 treatment prolongs significantly the increased levels of phosphorylated ERK1/2 for up to 15 days. Treatment with the specific MEK1/2 inhibitor, U0126, in axotomized animals blocks activation of the ERK signal transduction pathway, decreasing phosphorylated ERK1/2 levels to below control values. Conclusions: Axotomy transiently increases the activation of the ERK signal transduction cascade in the retina. Immunostaining suggests that RGCs contain increased amounts of activated ERK1/2. FGF-2 application after axotomy increases and prolongs this activation of the ERK signal transduction cascade in RGCs. These data are consistent with the hypothesis that this pathway is involved in the survival effect of FGF-2 in vivo.
Keywords: ganglion cells • growth factors/growth factor receptors • regeneration