Proliferating Changes Of Retinal Stem Cells In The Proliferating Marginal Region Of RCS Rat Retina Regulated By Shh/ptc Signaling

Retinitis pigmentosa ( RP )1, 2 results in the apoptosis of photoreceptor and retinal pigment epithelial ( RPE ) dysfunction which led to loss of vision. RP is one of the most common hereditary diseases caused visual loss and blindness. Etiopathogenisis of the RP have not been clarified yet. Once the RP developed, it is hard to repair the loss of photoceptor, and there is currently no effective measures to cure RP. Hence, stem cell transplantation has been focused extensively and is considered one of the promising treatment measures in clinic field3-6.Proliferating cells, that fulfill the minimum criteria of being multipotent and possessing the ability to self-renew, can be defined as stem cells7-9. A large number of different sources of the RSCs have been reported 10-12. The retinal margin ( or ciliary margin zone, CMZ ) and pars plana of the ciliary body are considered to be proliferating marginal region of the retinas, and the putative area containing stem cells for a lifetime13 . It is reported, in low vertebrate, this area is a potential source of renewable cells from which stem cells could proliferate and differentiate into new neurons to replace those injured following retinal damage11, 14-17. But there is hardly any report about proliferating marginal regions of mammal, especially the proliferation changes of RSCs during development of RP. In recent study, we observed the cell proliferation in the proliferating marginal region of RCS rat retina and its mechanisms.We aimed to clarify the role of RSCs in pathologic process of RP.It is revealed that Sonic Hedgehog-patched signaling ( Shh/ptc signaling ) 18, one of the signalings that regulated cell proliferation during the development of the eye prenatal and worked as a key regulator of neurogenesis19-23, was also involved in cell proliferation of RSCs during postnatal stage. Besides, evidences24, 25 gathered from in vivo and in vitro researches show expression of Ptc1, Smo and Gli1, which are the key factors of Shh/ptc signaling, at stem cell in retinal margin and pars plana of the ciliary body, it indicates that Shh/ptc signaling is involved in cell proliferation and differentiation of RSCs. However, during development of RP, the relationship of Shh/ptc signaling expression and the activity of RSCs has not been clarified yet. The ralatinship is important to understand the pathologic mechanism of RP and RSCs regeneration, and also provides possible strategy for RSCs transplantation and treatment of retinal degenerative diseases.On this basis, we initially investigated the thickness changes of retinal outer nuclear layer ( ONL ), and examined the proliferation of stem cells within the retinal proliferating marginal region, during developing Royal College of Surgeons ( RCS ) rats, an animal model for retinal degeneration, using ohistochemistry on frozen sections; the mRNA of Shh, Ptch1, Smo and Gli1 in Shh/ptc signaling were detected by fluorescent quantitation PCR in the same region; analyzed the relationship between expression of Shh/ptc signaling and proliferation of RSCs, and deduced whether Shh/ptc signaling played a significant role in RP progression and RSCs proliferation. Our study included 2 part:Part 1: The proliferating changes of RSCs at proliferating marginal region in RCS rats retinas with agingAs retinal degenerating of RCS rats, .the ONL thickness declined gradually, with disorder of cell arrangement and apoptosis of photoreceptors, almost disappeared at PN ( postnatal ) 90d and lost the photoreceptive function. Meanwhile, during the disease development, the number of RSCs and proliferating cells were significantly higher in RCS rats at PN 30d and reached the peak at PN 60d, then at PN 90d declined to level of that in PN 15d group. There were more positve cells at PN 30d and PN 60d RCS rats than that in Long-Evan's rats age-matched ( P<0.01 ), in which positve cells could hardly be detected and decline gradually with aging.Part 2: The expression changes of Shh/ptc signaling at proliferating marginal regions in RCS rats and the relationship with RSCs proliferation.Results shows mRNA expression of Shh, Ptch1, Smo and Gli1 are almost up-regulated in PN 60d RCS rats, compared with PN 15d, 30d, 90d RCS rats.Regarding to age-matched Long-Evan's rats, mRNA expression of Shh, Ptch1, Smo and Gli1 were, more or less, up-regulated in PN 60d RCS rats ( Gli1, P<0.05; Shh, Smo, P<0.01 ). Interestingly, the changes of up-regulation and down-regulation of Shh/ptc signaling are extremely coincident with tendence of RSCs proliferating changes during the development of RP.Conclusion:1. As retinal degenerating of RCS rats, the ONL thickness declined gradually. RCS rat was thought to be a stable model.2. RSCs could be activated to proliferate at proliferating marginal region during a short time as retinas of RCS rats degenerating significantly at later stage ( PN 60d ).3. Shh/ptc signaling was up-regulated at proliferating marginal region during a short time while retinal degeneration of RCS rats at later stage ( PN 60d ).4. Shh/ptc signaling might play an important role in RSCs proliferation in proliferating marginal region at later stage ( PN 60d ) of RCS rats stimulated by RP.