Abstract
Abstract: :
Purpose: To determine the effects of leukemia inhibitory factor (LIF), a known neuronal survival factor and inhibitor of stem cell differentiation, on retinal vascular development and on endothelial cell proliferation in vitro. Methods: Sprague Dawley rat pups were injected intraperitoneally (IP) with 0.1 ml of LIF (1 µg/ml) or PBS at postnatal age day 3 (P3). At P6, pups were euthanized, and dissected retinal wholemounts were either stained with lectin to visualize endothelial cells, or with activated caspase–3 and propidium iodide (PI) to stain apoptotic and nucleated cells respectively. Lectin stained wholemounts were analyzed to assess avascular and vascular areas using ImageTool. Images of the inner nuclear layer (INL) were captured using confocal laser microscopy in the caspase/PI stained retinas and counts of caspase positive cells (apoptotic) or PI stained condensed nuclei were made. Cultured human retinal microvascular endothelial cell (hREC) were plated at 18x10E3. At d5, hREC were changed to serum free medium and exposed to LIF (100 or 200 ng/ml) on d5, and cell counts made at d7. Results: At P6, LIF injected animals had a significantly larger peripheral retinal avascular area compared to PBS injected pups (2.4 vs 1.7 mm2 respectively, p=0.022 T–test, n=13). The LIF injected pups had significantly more apoptotic cells (23.2 vs. 4.7; p<0.001 T–test) and PI stained condensed nuclei (28.2 vs. 20.5; p=0.05, T–test) in the INL compared to PBS injected. LIF had no effect on hREC proliferation in vitro compared to control. Conclusions: LIF resulted in reduced retinal vascularization and increased cell apoptosis in the INL in rat pups injected IP at P3. It did not affect hREC proliferation in vitro at the doses tested.
Keywords: apoptosis/cell death • retinal development • cytokines/chemokines