Aging Effect On Total Number Of Retinal Neurons And Thickness Of The Whole Retina And Its Sublayers: A Stereological Study

Objectives To investigate that aging effected on retinal morphosis by usingstereological procedures that yield unbiased estimates to quantify the densities, numbersof retinal neurons in six young adult and six old Long-Evans rats, to determine whetherthere are aging-related losses of neurons in the three cellular layers of retina.Methods①Six young (3 months old) and six elderly (2 years old) Long-Evans ratswere used in this study, including 3 male and 3 femal 3-months old rats weighing(278.3±45.79g)and 3 male and 3 female 24-months old rats weighing (731.7±47.92g).②Theright eyeball was dissected from each rat and prepared as a set of serial sagittal sectionsand stained with HE staining. Using the random systematic sampling system, thesections and fields were sampled and examined under a microscope.③To observeretinal morphosis underneath the 100×objective using micro-imaging systerm of Nikon80i.④To estimate the total number of retinal neurons using the stereological methods,including retinal cells in GC, IN and ON layers. To survey diameters of the retinal nervecells nucleus in its sublayers and the thickness of the retina and its sublayers.⑤Thethickness of the retina and its sublayers were difference between the direct and correctformula measurenment methods in using a system random sampling method in thespercial coordinate system using for the retinal modeling.Results①The boundary of retinal sublayers are distinguished by HE staining under(20×magnification) a light microscope between young and elderly rats. The thickness of elderly retina is more significantly decreased than young's. Several cell types reside inthe retinal ganglion cell layer, inner nuclear layer, and outer nuclear layer of the youngand elderly rats, which were observed under the 100×objective. Ganglion cells are thelargest cells in the layer and tend to have an irregular outline, including clear nucleole.There are also a few numbers of shuttling glial cells with trachychromatic nucleus.Amacrine cells tend to be slightly darker than the average ganglion cells, having a moreregular outline, and are smaller on average than ganglion cells. However it is hard todistinguish amacrine cells from the smaller ganglion cells. Compare the three types ofbipolar cells, amacrine cells and horizontal cells with cone and rob cells in outer nuclearlayer are indistinguishable in inner nuclear layer, but there all are a clear nucleus.②Respectively the densities of retinal cells in GC, IN and ON layers were (0.13±0.043)×10~6/mm~3, (0.95±0.307)×10~6/mm~3 and (2.26±0.711)×10~6/mm~3 in young rats, and thesame as those densities were (0.17±0.061)×10~6/mm~3, (1.03±0.188)×10~6/mm~3 and(2.67±0.546)×10~6/mm~3 in elderly rats in a system random sampling method in (77±16)dissectors in young rats, (110±23) dissectors in elderly rats. The total number of retinalsorts of types cells were estimated by the unbias stereological study in young andelderly rats.Respectively the total number of retinal cells in GC, IN and ON layers were(212230.26±28623.350), (1553239.38±228025.736) and (3945630.75±992978.079)inyoung rats, and the same as those cells were (227416.28±48160.453),(1555029.08±412093.320) and (4150317.53±1071902.820)in elderly rats. Averagenecleus size of retinal cells in GC, IN and ON layers per retina is (7.31±1.273)μm,(5.76±1.117)μm and (3.11±0.432)μm in young rats, and (7.03±1.242)μm,(5.56±1.220)μm and (3.18±0.442)μm, in elderly rats. However the densities, totalnumber, necleus size of retinal nerve cells are not significantly decreased in itssublayers in elderly, comparing with young rats, but the result is not statisticallysignificant (P＞0.05). Based on the total number of the cells in the GC, IN and ONlayers that the ratio of the ganglion cells to INL cells to ONL cells in both young and elderly retinae was roughly about 1:7:17.③Compared to that of the young rats, thethickness of the whole retina and most of the sublayers were significantly decreased.Such decrease was proportional so that the proportion of each sublayer to the wholethickness of the retina remained unchanged. That is to the thickness of inner nuclearlayer reduced clearly 42.4%～44.8% and inner plexifrom layer reduced nearly 44.4～46.2% during aging process during normal aging process in large samplings.④Between the direct and correct formula measurement methods in using a systemrandom sampling method in the spercial coordinate system using for the retinalmodeling: Compared to that of the direct measurement, the thickness of the whole retinaand most of the sublayers were significantly changed in the young and elderly rats.There were statistical significance (P＜0.01) between two methods. There weresignificant correlations in the thickness of the whole retina and its most of sublayersbetween using two methods (r=0.956～0.999, P＜0.01).Conclusions①The total number of retinal cells, the thickness of the whole retina andits sunlayers are better to be estimated with stereology study first time.Admittedly themethod of stereology is precise and feasible in our investigation.②The total number,densities and nucleolus diameters several retinal cell types which reside in the retinalganglion cell layer, inner nuclear layer, and outer nuclear layer, were not had anysignificant influence during normal aging process, the thickness of the whole retina andits sunlayers of the young and elderly rats.③This study confirmed that aging has asignificant effect on the thickness of the rat retina. It is better to be affirmed that asystemic random sampling of method can reflex the true process. A system randomsampling method (9～10 disectors) is more practical worth by virtrue of decreasingsampling.④This study confirmed that there are feasibility and repeatability with theanimal empirical studies using the correct fomula of measurement.