In vivo Activation of Cell Proliferation in Postnatal Mammalian Retina and Ciliary Epithelium

Abstract

Abstract: : Purpose: Neural stem cells/progenitors isolated from embryonic retina and adult ciliary epithelium proliferate in vitro in the presence of EGF and/or FGF2. We are studying the ability of exogenous growth factors to promote cell proliferation in vivo in order to determine whether or not regeneration is a possibility in postnatal mammalian retina. Methods: Postnatal day 7 (PN7) rats were anesthetized and growth factors (insulin 1µg/µl, FGF2 20ng/µl) and BrdU (1µg/µl) were delivered into right eye by intraocular injection using a glass micropipette attached to a 10µl Hamilton syringe. The left eye was used as a control and injected with PBS and BrdU. Animals were sacrificed at different time points (2^nd, 7^th and 14^th day of injection) and eyes were enucleated for fixation using 4% paraformaldehyde. Immunohistochemical analysis was carried out on cryostat eye sections. Results: BrdU positive cells were readily detected in the periphery of the retina and in pigmented ciliary epithelium on 2^nd day of intraocular injection. However, the proportion of proliferating cells was higher in growth factor treated eyes than in the controls. BrdU⁺ cells expressed retinal progenitor markers Chx10 and Pax6. BrdU⁺ cells were detected in the periphery of retina on 7^th day and 14^th day of injection and their proportion remained higher in growth factor treated eyes than in the controls. However, the proportion of BrdU⁺ cells co-expressing proliferation marker Ki-67 decreased significantly on 7^th day suggesting that the majority of BrdU positive cells were likely to be postmitotic and did not represent proliferating cells at that stage. On 14^th day of injection only 1 or 2 BrdU⁺ and Ki-67⁺ cells could be detected within the retinal periphery/section. Conclusions: Our results suggest that exogenous growth factors promote cell proliferation in postnatal retina and ciliary epithelium.

Keywords: retinal development • growth factors/growth factor receptors • cell-cell communication