Visualization Of Morphological Dynamics Of Lecs And Fibers Within The Whole Lens By Phase Contrast Microscopy

PrefaceLens is a transparent organ constituted of two kind of cells:lens epithelial cells and lens fiber cells. The unique morphology and regular arrangement of these cells has an important role in the maintenance of lens transparency. Cataract formation is due to the morphology changes of these cells, which increases light scattering and absorption and causes the lens transparency decreased. The studies of lens cell morphology has an important significance to understand the physiology and pathology of the lens.Traditional research methods of morphological study of the lens are to observe the lens tissue sections under light microscope and electron microscope. However, chemical and physical factors may cause morphological damage of the lens cells and these methods can not obtain continuous observation of the same sample. The cultured cells once dissociated from their physiological location, lose their intrinsic interactions with the environment. Once dissociated from their physiological location, the cultured cells lose their initial cell shape and become unsuitable for the morphological study Due to the lack of appropriate means of observation, there is no study of the characteristics of morphological dynamics of LECs and fibers within the whole lens.In this study, by adjusting the condenser annulus of the inverted phase contrast microscope, we are able to observe the cellar dynamics within the whole lens. We present the images of mitosis and apoptosis of the LECs and the dynamics of posterior fiber cells after injury, providing morphological information of lens cell physiology and pathology.Part I Characteristics of morphological dynamics of LECs within the whole lens under a phase contrast microscopePurpose To observe the characteristics of mitosis and apotosis of LECs within the whole lens under a phase contrast microscope.Methods Female Wistar(200-250g) rat lenses were kept in organ culture. The culture dish was put under the inverted phase contrast microscope(Leica DM13000B), adjusting the condenser annulus to gain the most clear observation. Cultured lenses were divided into three groups:In group I(IGF-I), the rat lenses was incubated in IGF-I (20ng/ml) for15hours to to induce cell mitosis. In group II(UVA), the lens front surface was irradiated for10min by UVA to induce cell apoptosis. In group III(Control group), the lenses were cultured for7days without any treatment.Results Using the condenser annulus PH0with the high power objective lens(20×,40×) could obtain the best observation of epithelial cells. Small, rounded humps (arrow) were present all over the anterior surface and the normal epithelial cells were not distinguishable under the microscope. The control group did not show any obvious morphological changes. Numerous mitotic figures were present all over the anterior surface after15hours’ incubation with20ng/ml IGF-I. The number, location, and stages of the dividing cells were readily observed. The nuclei of the apoptotic cells moved aside, and round caves were left in the original sites. Fluorescent staining showed that all of the LECs in the radiation area were dead.Conclusions LECs within the whole lens showed unique morphology characteristics in the process of proliferation and apoptosis under a phase contrast Microscope.Part Ⅱ Characteristics of morphological dynamics of damaged fiber cells within the whole lens under a phase contrast microscopePurpose To observe the characteristics of damaged fiber cells within the whole lens under a phase contrast microscope.Methods Female Wistar(200-250g) rat lenses were kept in organ culture. Cultured lenses were divided into four groups:In group Ⅰ(UVA), the lens posterior surface was irradiated for10min to induce fiber damage. In group Ⅱ(H2O2), the rat lenses was incubated in H2O2(100μM) for48hours. In group Ⅲ(Galactose), the rat lenses was incubated in galactose (150μM) for72hours. In group Ⅳ(Controlled group), the lenses were cultured for7days without any treatment. The cellular morphological dynamics within the whole lens were monitored by inverted phase contrast microscopy.Results The control group did not show any obvious morphological changes after7days’ culture, the shape and arrangement of fiber cells were clearly revealed. The fiber cells show two kinds of morphological changes after UVA radiation. The fibers on the radiation area aggregated into globules.However, on the adjacent area the extracellular space between fibers became enlarged, the fibers themselves did not break down. After48hours incubated in H2O2(100μM), the equator area appeared circular damage. After72hour s incubated in galactose (150μM), the surface fibers showed radiation-like damage.Conclusions Damaged fiber cells within the whole lens showed unique morphology characteristics under a phase contrast microscope.