| 1.ObjectiveThe damage of retinal cells is an important factor which drop eyesight,so the research of the regeneration and repairing of retinal cells will provide a basic theory to clinic treatment.A basic definition of "stem cell" is a cell that both self-renews and gives rise tomultiple mature cell types.It contains a symmetric cell division and a asymmetric cell division in which one daughter cell remains multipotent and "stem-like".Neural stem cells(NSCs)are multipotent cells which, by definition,can differentiate into any type of neural cell,including neurons, astrocytes,and oligodendrocytes.Retinal stem cells(RSCs)are multipotent central nervous system(CNS)precursors that give rise to most all retinal cell classes,as well as self-renewing to generate more retinal progenitors during the course of development.RSCs are present in the embryonic eyecup of all vertebrate species and remain active in lower vertebrates throughout life.All vertebrate retinas consist of seven major of cells,including retinal ganglion cells,cones,rods,bipolar cells,amacrine cells,horizontal cells and Muller cells.Various retinal neurons are produced from retinal stem cells.The order of retinal stem cell development is strict and regular.The first cells produced in the developing retina are the retinal ganglion cells followed by cones and amacrine cells.The horizontal cells are born shortly thereafter in some species(chick and mammals)while in others(fish and amphibians) they are born a bit later.Rods appear in the next wave of cell birth,with most bipolar cells and Muller glia being generated during the last phase of retinal neurogenesis. Although this birth sequence occurs in a stereotyped pattern,there is considerable overlap among the different classes of cells types.Retinal neurons differentiate in a central to peripheral sequence,such that cells closest to central retina are the first to be born and differentiate,while those at the periphery generally lag behind.A large body of literature exists in which the processes of neurogenesis and differentiation within the retina have been described in considerable detail.In addition,a wide range of species have been used as model systems for studying retinal and visual system development,each system being of comparative interest because of its own unique attributes.In the post literatures,mammals,however,exhibit little RSC activity in adulthood and thus little capacity for retinal growth or regeneration.Until 2000, Tropepe reported the identification of stem cells in the adult mouse,rat,bovine,and human ciliary marginal zone.Single pigmented ciliary margin cells clonally proliferate in vitro to form sphere colonies of cells that can differentiate into retinal-specific cell types,including rod photoreceptors,bipolar neurons,and Muller glia.Adult retinal stem cells are localized to the pigmented ciliary margin and not to the central and peripheral retinal pigmented epithelium,indicating that these cells may be homologous to those found in the eye germinal zone of other nonmammalian vertebrates.The now putative and classic marker for neural stem cells is the intermediate filament nestin,a component of the cytoskeleton of immature neuroepithelial cells. Researchers find it useful for confirming the presence in vitro of highly plastic neural progenitors.The cellular role of nestin is not well-understood.The Nestin expression was first detected in neuroepithelial stem cells.It is a intermediate filament protein which expressed in neuroepithelial stem cells in the early developing neural system.Now it is used for a special marker to identify neural stem cells.However,many tissues such as developing muscles,new endothelium cells, hepatic astrocytes,pancreatic canula cells express nestin.Now the investigations about retinal stem cells most come from the differentiation,culture and identification of retinal stem cells.But it’s location, number and changes along with the development of species have not been studied sufficiently.This study selects SD(Sprague Dawley)rats as objects,by using these technics like an immunocytochemical method and a flow cytometry,we observed the changes of Nestin expression in the eyes of SD rats during their postnatal various growth period,in order to find out he location and number of retinal stem cells. Along with growth of SD rats after their birth,the relation between Nestin expression and growth of retinal neural cells of SD rat was observed.And not only in retinae of SD rats,but in other tissues of SD rats were observed that Nestin has any expressions and whether its expression changes are consistent with that in retinae.2.Materials and Methods2.1 Expression changes of Nestin in SD rat retinae during SD rats postnatal various growth period.Positive expressions of Nestin in retinae of SD rats were detected using an immunocytochemical method and a flow cytometry.2.1.1 To Select 4 rats for each group which contains male or female SD rats that have lived 1 day,3 days,5 days,1 week,two weeks,three weeks,one month after birth. The animals were decapitated,eyelids and skin of the head were removed completely. An incision was made crossly on cornea and the heads were placed in FAA liquid for 12h.The lens and vitreous body were enucleated and the heads were placed in FAA liquid for another 2-3 days in 4℃icebox.The SD rat heads aged one week longer need decalcium for a week.The head was dissected vertically and redundant tissues were removed.Eight pieces of eye materials with orbit tissues were prepared and placed in 60%,70%,80%alcohol liquid for 30 minutes,90%,100%Ⅰ,100%Ⅱalcohol liquid for 20 minutes for dehydration.Then the pieces were placed in dimethylbenzene for 10 minutes,in terebinth for 24h for transparence,placed in 70℃olefin potⅠ,Ⅱfor 1h,before wraped by olefin and transformed to wax.The sliceds were dyed using a HE and an immunocytochemical method.The Nestin expression was detected by an immunocytochemical method,the degree of stainings,extent of distribution and density of positive cells were observed under light microscope.The positive cells contain granule in cytoplasm which represent brown-yellow color.(-):negative,no expression;(+):feint pigmented, represent feint yellow,distribute narrowly,definite positive under high microscope; (++):moderate pigmented,brown-yellow,definite positive under low microscope; (+++):pigmented abundantly,represent brown deeply;(++++):pigmented intensely, represent deep brown intensely.The slice pictures using an immunocytochemical method were gathered through Nikon E800 microscope(Japan),six high microscope field’s pictures under 40 multiple object lens were selected randomly and analyzed to record all counted Nestin positive cells.2.1.2 To Select 8 rats for each group which contains male or female SD rats that have lived 1 day,3 days,5 days,1 week,two weeks,three weeks,one month after birth. The animals were decapitated,eyelids and skin of the head were removed completely. A 0.1ml collagenaseⅣwas injected into vitreous body along edge of cornea to digest retinae for about 30 minutes.The cornea was scissored crossly,lens and vitreous body were picked off.Retinae were washed repeatedly by collagenaseⅣ liquid from injector,centrifugated at low speed,marked by antibody,tested by flow cytometer.2.2 Expression changes of Nestin in SD rat extraocular muscles in postnatal different growth stages.The materials were picked at different time,an immunocytochemical method and an image analysis method were used as same as first part.The results of data were recorded as form of Mean±Std.deviation((?)±S)and analyzed using SPSS11.0.One-way ANOVA were operated between each groups.If equal variances assumed,the method of a Student-Newman-Keuls(SNK)was used.If equal variances not assumed,a Tamhane’s method was operated.It is considered of significant difference when P＜0.05.3.Result3.1 Nestin expression changes of SD rats retinae in different postnatal period.3.1.1 Immunocytochemical staining showed that positive stainings for Nestin were observed in retinae after SD rats had been born for 1d,3d,5d,7d,2w,3w and 1 month. Nestin was showed extensive positive expression in SD rat entire retina layers at one day and three days group after birth.Eight SD rats of one day group showed Nestin positive at level ++++.Six SD rats of three days group showed Nestin positive at level ++++,the other two at level +++.Five and seven days group showed extensive positive in retinal pigment epithelium layer,ganglion cell layer,nerve fiber layer and inner nuclear layer,weak in outer nuclear layer,rod and cone cell layer.Seven SD rats of five days group showed retina Nestin positive at level +++,one at ++.Six SD rats of seven days group showed retina Nestin positive +++,two at ++.Two weeks group showed moderate expression in nerve fiber layer,few in inner nuclear layer, five SD rats showed retina Nestin positive at ++,three at +.Three weeks group showed a few expression in nerve fiber layer,appeared by chance in inner nuclear layer,six SD rats showed retina positive at +,two at -.A month group showed expression only in nerve fiber layer by chance,no expression at other layers.Four SD rats showed retina positive at +,four showed negative.The control groups of all periods showed retina Nestin negative.3.1.2 The results of the image analysis showed the Nestin positive cells in SD rat retinae in postnatal 1d,3d,5d,7d,2w,3w,1 month are 3197.67±401.16,2649.17±517.84、1513.00±148.38,1510.50±151.91,510.83±132.24,156.33±40.30,21.33±10.78. Between groups,except one day group compared with three days group,five days group compared with seven days group that showed P＞0.05.All other group’s comparison showed P＜0.05 which showed significant difference.3.1.3 A flow cytometryThe numbers of retina cells were counted by a flow cytometer,the percent of Nestin positive cells at 1d,3d,5d,7d,2w,3w,1 month are 11.77±1.75%,9.48±0.96%, 7.83±0.23%,6.75±1.09%,3.48±0.42%,2.43±0.40%,0.85±0.28%.The negative control group was 0.06±0.02%.3.2 Expression changes of Nestin in SD rat’s extraocular muscles in different postnatal period.3.2.1 Immunocytochemical staining showed that extraocular muscles also contain yellow-dyed positive Nestin expression in cytoplasm besides SD rat retinae. But their expressions decreased during the SD rats’development,they begin with ++++ which expressed strongly at one day after birth to the end with - which have no positive expression a month later after birth.One day group contains six SD rats whose extraocular muscles expressed Nestin ++++,two expressed +++;three days group contains four SD rats whose extraocular muscles expressed Nestin ++++,three expressed +++,one expressed ++;five days group contains seven SD rats whose extraocular muscles expressed Nestin +++,one expressed ++;seven days group contains five SD rats whose extraocular muscles expressed Nestin +++,three expressed ++;two weeks group contains three SD rats whose extraocular muscles expressed Nestin ++,five expressed +;three weeks group contains four SD rats whose extraocular muscles expressed +,four expressed -;a month group contains all SD rats whose extraocular muscles expressed Nestin -.3.2.2 The results of the image analysis showed the Nestin positive cells in SD rat extraocular muscles in postnatal 1d,3d,5d,7d,2w,3w and 1month are 905.00±149.98,701.17±124.64,558.67±108.88,453.67±95.51,265.50±110.90, 91.83±22.92,0.All groups had value of P＜0.05 compared between each other that showed significant difference except five days group compared with seven days group that had value of P＞0.05.4.Conclusion4.1 To detect the Nestin expression in SD rats at 1d,3d,5d,7d,2w,3w and 1month after birth,we have finished the experiment showing that Nestin positive expression in SD rats retinae changed obviously with time at 1d,3d,5d,7d,2w,3w,1month after birth.The Nestin positive expression in retinae indicated that retinal stem cell decreased gradually from each layer of retinae which expressed extensively to nerve fiber layer which expressed singularly.The trend was matched by the image analysis of immunocytochemical slices and the result of a flow cytometry.The Nestin positive expression of retinal stem cell decreased when SD rats growed order.4.2 Nestin not only expressed positively in retinae,extraocular muscles also contain positive Nestin sign,and their expression decreased along with growth of SD rats. No positive expression could be detected in SD rat extraocular muscles in postnatal one month.4.3 The Variation of Nestin expression in SD rat retinal neural stem cells in postnatal different growth stages matched well with their changes in SD rat extraocular muscles. |
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