ABSTRACT
ObJeetive To report a simple and efficient method to label two different types of retinal ganglion cells (RGCs) in mouse retina.Methods Eyeballs were harvested from normal adult C57 BL/6 J mouse,the retinas were isolated,four radial cuts were done,the retinas were pasted on the nitrocellulose membrane with the ganglion cell layer upturned.The immunofluorescence double staining and laser confocal nmicroscope was used to reveal conventional retinal ganglion cells and intrinsically photosensitive retinal ganglion cells (ipRGCs) using Brn3a and Melanopsin.Results The double staining results of whole mount retina showed that conventional RGCs and melanopsin immunopositive ipRGCs had a complementary distribution in mouse retina,these two subtypes of RGCs were predominantly present in the ganglion cell layer.The numbers of ipRGCs was just about 1％-2％ of conventional RGCs,and the axons of ipRGCs toward the direction of the optic disc,several dendrites toward the inner plaximem layer.Conclusion The immunofluorescence double staining of whole mount retina is a simple,stable and efficient method to label two different types of mouse RGCs.
ABSTRACT
Objective To study the estradiolNGF regulatory cascade in retinal endothelial cells. Methods Retinachoroids vascular endothelial cell line RF/6A from rhesus was cultured in M199 medium contain 20% FBS.mRNA of ER,NGF and its receptors,TrkA and P75 NTR were detected with RTPCR.The cells were incubated with NGF,VEGF,antibodies against NGF or VEGF,and K252a(100nmol/L),the specific inhibitor of TrkA,separately or in different combination.MTT based cell counting assay was used to study the viability of the cells.The apoptosis was evaluated by FACS,mass migration by wound healing assay,and tubogenesis by AngioMatrix assay. Results We amplified the specific fragments of cDNA of ER,NGF and NGF receptor TrkA using RTPCR.10?nmol/L1??mol/L estradiol augments the proliferation and increases the viability of RF/6A in a dosage dependent manner.In the wound healing based migration assay,we found the similar alteration.This effects of estradiol was partially blocked by NGF neutralized antibody and K252a.Apoptosis rates were at the similar level among the groups.For the tubogenesis of RF/6A,we found no augmentation by NGF,and no blocked augmentation by estradiol.Conclusion NGF,first identified as the survival factor of nerve system,now also seemed to be an activator of retinal endothelial cell,is under the regulation of estrogen.The proliferation and migration,but not the tubogenesis of retinal endothelial cells are regulated by this regulatory cascade.
ABSTRACT
Objective The purpose of this experiment is to study the in vivo differentiation and myelin formation of rat striatal neural precursor cells after transplantation into homogeneous retina,observe the order of myelination and its influence on the structure of retina,establish an animal model of CNS myelin formation in vivo. Methods Passage cultured striatal neural precursor cells from embryonic Sprague-Dawley rats were transplanted into the vitreous cavity of neonatal rats.In different stages after transplantion,myelin formation in retina was observed under light and electron microscope and analysed with different stained methods.Results Bundles of myelin appeared in parts of retina 4 weeks later.The distribution and morphology of myelined area expanded with prolonged survival time after cell transplantation.Oligodendrocyte wrapped the naked axons and formed normal myelin limited in the nerve fiber layer.Myelination influenced the distribution of local retinal ganglion cells.Conclusion Striatal neural precursor cells could differentiate into oligodendrocytes and formed myelin after transplanted into retina and the naked axons in retina promoted the myelin formation.This model provides a new method to study the myelin formation and myelin-axon interaction in vivo.