Microscopic Image Analysis Of The K-NPPase Enzyme Hitochemistry On Cellular Membrane Of Rabbit's IPE And RPE

PurposeIris pigment epithelium cells have the same embryonic origin and similar cell morphology as RPE cells, and easy to harvest, so it is expectable to substitute RPE cells for autologous transplantation to treat age-related macular degeneration, retinitis pigmentosa, and macular dystrophy etc. We observed the distribution of K-NPPase stained grains on the cellular membrane of rabbit's IPE and RPE by electronic image analysis. We can deduce the distribution of Na+,K+-ATPase by analysis of the distance of K-NPPase stained grains in order to provid a theoretics for IPE cells instead of RPE cells to transplantation.Material and methods.Animals:Choosed healthy 6 pigment and 6 non-pigment rabbits(provided by animal ministry of china medical university), their weight from 2.5Kg-3.0Kg,average 0.5 years old. Regardless male and formale. Both eyes of each rabbit were enucleated immediately , andfixed by injecting 4 % paraformaldyhyde-0.5 % glutaraldehyde containing 6% sucrose into the eyeballs and immersed them in room temperature, 45 minutes.The eyeballs were opened and made into 2×4mm sections ,and incubated with magnesium of p-nitrobenzphosphate , 37℃, 45 minutes.The eyeballs ' insections were rinsed with 0.1-mol/L cacodylata buffer twice ,10 minutes. Postfixed with 1%OsO4 at room tempreture in 0.1-mol/L cacodylata buffer for 45 mintutes, after dehydration in a grade series of alcohol and acetones, and then embedded them in Epon 812 one night. Semithin sections were made and observed by light microscope to find the requisite parts. Ultrathin sections were stained with uranyl acetate and observed under transmission electron microscope.Analysis procedure: Adopted Metamorph/DP10/BX51 system analysis each transmission electron microscope photographs' scale , choosed IPE cellular membrane of microvilli and RPE cells' summit where adjoint photoreceptor outer segments and was reported the density of grains is most. Drawing distance of two neighbouring points stochastic. Mean and standard deviation (x眘) were calculated. Comparision between the IPE groups and the RPE groups, the pigment groups and the non-pigment groups ,were performed using t test.ResultsBoth IPE cellular membrane of microvilli and RPE cells' summit where adjoint photoreceptor outer segments can be observed with K-NPPase grains. In the comparison between RPE and IPE ,there were no significantly difference between them, 0.5>P>0.2.ConclusionThere are K-NPPase enzyme grains aggradation on both IPE and RPE cellular membrane, there are no significant difference between the two groups of average datum. That is to say, there are enough distribution of Na+,K+-ATPase on both IPE and RPE cellular membrane. And autologous IPE might be perfect as a substitute for RPE cells after transplantation into the subretinal space of patients, and exert material transportation function.In the comparison between pigment groups and non-pigment groups, Both IPE and RPE cellular membrane have aggradated by K-NPPase enzyme grains.