Objective To establish a method for primary culture of iris pigment epithelial cells(IPE). MethodsEnzyme-Assisted microdissection was used to isolate and cultivate the IPE cells.An identification was made with microscopic and immunohistochemical observations.Results IPE were successfully sultured and showed on differences with RPE in primary culture and subculture.ConclusionEnzyme-Assisted microdissection is a reliable and quick method for the isolation of IPE.
Objective To study on the ultrastructural characteristic of segments of photoreceptors from neonatal retinas for supporting donor retina choice of retinal transplantation． Methods Photoreceptors from neonatal calf and adult calf were analysed by scanning electron microscopy and transmission electron microscopy． Results Segments of photoreceptors from neonatal calf appeared the mushroom pattern, in which, distal end of outer segment which was ball-shaped formed the head with mushrooms appearance, and the inner segments along with some of outer segments formed the body with mushrooms appearance． Within the outer segment, plasma membranes of adjacent evaginations form a disk subsequently． The a rray of most disks were vertical to the entire length of segments, but some were parallel and slope to．Owing to the incomplete formation, some rim of disk near distal end of outer segment revealed step-shaped appearance． The distal end of outer segment displays some processes consisted of membranous discs, much vesicular material and mitochondria, much rough endoplasmic reticulum (RER) and numerous polyso mes．Segments of photoreceptor connected with outer nuclear layer via the external limiting membrane． Conclusion The typical morphol ogical structures of outer segments suggest the immature and b gowth ability of photoreceptors of the retina of neonatal calf, and therefore the competence for donor material of retinal transplantation． (Chin J Ocul Fundus Dis, 2001,227-229)
ObjectiveTo investigate relationship between ultrastructural changes and expression of basic fibroblast growth factor of diabetic retinopathy in rats.MethodsDiabetes was induced in rats with a single injection of streptozotocin (STZ) and divided into normal control group and 1- , 3- and 5- month diabetes group. The paraffin slide was observed by in-situ hybridization and immunohistochemistry, and retinal ultrastructure was examined by transmission electron microscopy.ResultsNo change of retinal ultrastructure was found in the control group. Different degrees of ultrastructure lesion were found in 1-month diabetic rats with fragmental increase of thickness of basement membrane, swelling of endothelial cells and obvions fingerlike processes in the capillary cavity, disconcentration of heterochromatin both in endothelium and pericyte, and swelling and degeneration of mitochondrion. The edema of endothelial cells of 3-month diabetic rats was more serious than that of 1month ones, and the capillary cavity was nearly occluded. In 5-month diabetic rats, the basement membrane was unevenly thickened, or obviously split. The positive rate of in-situ hybridization in 3-month diabetic rats was 77.8% while the positive rate of immunohistochemical stain was 55.6%, which increased to 88.9% in 5-month diabetic rats.ConclusionsThe occurrence of the ultrastructural changes in STZ rats with diabetic retinopathy is earlier than that of the expression of bFGF.(Chin J Ocul Fundus Dis, 2003,19:348-351)
Objective To observe the enzymic histochemical and ultrastructral changes of cryopreserved human retina. Methods To compare the activity of lactate dehydrogenase (LDH)， succinate dehydrogenase (SDH) and ATPase in cryopreserved retina with those in fresh retina and to observe the histological and ultrastructural changes of cryopreserved retina. Results There was no statistical difference between the activity of LDH，SDH and ATPase in fresh and in cryopreserved retina. Histologically, in the cryopreserved retina, fluid in neural fiber and outer plexiform layers, as well as in cone and rod layer, was sligthly more than normal. The ultrastructure is normal except that the mitochondria was swollen in different degree. Conclusion Cryopreservation may be an effective method for keeping the retinal cells alive for a long period and might free the transplantation from dependance on aviability of fresh dornor tissue. (Chin J Ocul Fundus Dis，2000，16：139-212)
Objective：To observe the histochemical changes of retinal photochemical damage in rats. Methods：The changes of retinal ultrastructure were observed.The concentration of malondaldehyde(MDA) was tested and the activity the histochemical change of cytochrome oxidase (CCO) and (Mg ++ -ATPasw) were evaluated on the retnal photochemical damage in SD rats. Results：At the 6th hour after light exposure,the swelling appwared at the nuclei of photoreceptor,the mitochondria of inner segment.The apical microvilli of RPE disappeared and lysosomes increased in RPE.On the 6th day after light exposure,the changes became more obvious.While on the 14th day after light expose the nuclei of photoreceptors and the inner segments renewed but the arrangement of the disk was lose;and the microvilli appeared of the disk was lose;and the microvilli appeared at the tip of RPE.The Activity of CCO and Mg ++ -ATPase decreased and MDA increased in retina at the 6th hour and on the 6th day and they recovered on the 14th day after light exposure. Conclusion：Lipd peroxidation that broke the cell membrane system of photoreceptor which induced changes of the cell ultrastru cture abd the activity of enzyme might relate to pathogenesis in retinal photochemical damage. (Chin J Ocul Fundus Dis,1998,14:38-40)
Objective To observe the difference between blood brain barrier and blood optic nerve barrier. Methods Twenty normal male SD rat sprime; optic nerve including prelaminar region, lamina cribrosa, retro-laminar region, intraorbital portion, intracanalicular portion, and intracranial portion respectively,and cerebral cortex were removed separately. Ultrastructure of endothelial cells was observed by electron microscopy. Immunohistochemical staining was used to detect the expression of transferrin receptor (OX-26) and metalloproteinase inducer (OX-47) and extravasation of fibrinogen around microvessels. Results The results of electron microscopy showed that endothelial cells of microvessels in each portion of optic nerves and cerebral cortex did share the same tight junctions. However, the number of plasmalemmal vesicles in prelaminar region was significantly more than that in cerebral cortex(P＜0.05);there was no significant difference between other parts of optic nerves (lamina cribrosa, retro- laminar region, intraorbital portion, intracanalicular portion, and intracranial portion)and cerebral cortex in the number of the plasmalemmal vesicles(Pgt;0.05). By immunohistochemical staining，the endothelial cells of microvessels in the prelaminar region showed no expression of the OX-26 and OX-47,but extravasation of fibrinogen around microvessels was found; b positive expression of OX-26 and OX-47 was observed in the endothelial cells of the microvessels in other parts of optic nerves (lamina cribrosa, retro-laminar region, intraorbital portion, intracanalicular portion, and intracranial portion) andcerebral cortex, and no fibrinogen was seen aro und the microvessels. Conclusions There is a significant difference between the endothelial cells of the microvessels in prelaminar region and cerebral cortex in the ultrastructure, markers expression, and permeability, so the microvessel s in prelaminar region lacks the typical blood brain barrier characteristics.The microvessels in other parts of optic nerves (lamina cribrosa, retro-laminar region, intraorbital portion, intracanalicular portion, and intracranial portion) have blood brain barrier properties due to its similar specialties as which in cerebral cortex. (Chin J Ocul Fundus Dis, 2006, 22: 390-393)
Objective To observe the ultrastructural characteristics of human retinal progenitor cells cultured in vitro. Methods Six 5-month-old human fetuses（12 eyes）without eye diseases were selected. Retinal progenitor cells from the retina of one eye of each fetus were cultured in vitro，and observed by transmission electronic microscopy（TEM）; while those from the other eye were directly observed by TEM. Results Abundant heterochromatin were found in the karyon of 5-month embryonic retinal neuroepithelial cells，and the figure of the karyons was irregular.A few scattered initial cells were seen in retinal neuroepithelial layer with large karyon，smooth surface，abundant euchromatin，and distinct nucleolus.The human retinal progenitor cells cultured in vitro had the same ultrastructural characteristics as the initial cells:with huge karyon which almost occupied the whole cell，little cytoplasm，distint nucleolus，abundant euchromatin，and little heterochromatin.The cells clung to each other in the neural globoid cell mass.The size of the outer cells was large，and karyokinesis could be found. Conclusion The cultured human retinal progenitor cells are provided with the same ultrastructure characteristics as the initial cells. (Chin J Ocul Fundus Dis, 2006, 22: 185-187)
Objective To study the global and histological changes of myopia and explore its pathogenic mechanism. Methods Chicks were reared with monocular suture of eyelid. When myopia had been confirmed by optometry, eyeballs were removed and subjected subsequently to measurement and light and electron microscopies. Results Three dimensions in the eyeballs of suture group were all enlarged markedly and the mean diopter was -15.00D. Under the light microscope, rod outer segment elongated and connected With PREC in suture group. With micrometer measure, cartilaginous sclera thickened and retina became thinner. Under electron microscope, rod outer segment elongated and membrane disc was intact. In the cytoplasm of RPEC, the phagosomes containing fractions of the membrane disc of outer segment were remarkably decreased. Conclusion Early form deprivation may affect the drop of membrane disc and cause eyeball enlargement; thus, myopia forms. (Chin J Ocul Fundus Dis,1999,15:20-23)
Objective To observe the morphological changes of dendrite and soma in retinal ganglion cells (RGCs) which subsisted in early diabetic rats. Methods The RGCs of 3-months-course diabetic rats and coeval normal rats were marked by gene gun techniques. To collect RGCs photographs by Leica microscope with Z axis and CCD camera；to observe the changes of diameter, variance of structural features in dendritic field and somata after classification which according to the size and morphology. Thy-1 antibody marks on the retinal RGCs, taking a photograph under fluorescent microscope, counting the changes of retinal RGCs density in early diabetic rat. Results In three-month diabetic rats,the density of retinal RGCs was decreased obviously. Morphological changes of RGCs in the dendritic fields were observed with gene gun technique. There was no severe variation in all kinds of the bole of cell dendrite, in which some only showed crispation partially and sparseness also twisting in the dendritic ramus. The mean diameter of dendritic field and soma in class A of diabetic rats was (401plusmn;86) mu;m, the mean diameter of dendritic field in control group was (315plusmn;72) mu;m,compared with each other, there is statistically significant differences (t=21.249，Plt;0.001); the mean diameter of soma in class A of diabetic rats was (24plusmn;6) mu;m, the mean diameter of soma in control group was (22plusmn;5) mu;m, compared with each other, there is no statistically significant differences (t=0.927，Pgt;0.05); the mean diameter of dendritic field and soma in class B of diabetic rats were (170plusmn;36)、(14plusmn;2) mu;m respectively, in control group were (165plusmn;36)、(16plusmn;2) mu;m, the mean diameter of dendritic field and soma in class C of diabetic group were(265plusmn;78)、(17plusmn;5) mu;m respectively, in control group were (251plusmn;57)、(17plusmn;4) mu;m , compared with each other, there are on statistically significant differences(t=1.357，0.798，0.835，1.104，Pgt;0.05). Conclusions In short-term diabetes, the survived RGCs show good plasticity in adult diabetic rats, especially in class A. The changes of dendrites were more sensitive than the soma, which could be the leading index of the morphologic changes of RGCs in the early stage. The good plasticity showed by the RGCs and the time window from changing in dendrite to cell death provide us many evidences not only for the research but also for the nerve protection in clinic. (Chin J Ocul Fundus Dis,2008,24:249-254)
Objective To investigate the effect of polyethylene glycolbovine hemoglobin (PEG-bHb), which was used as an oxygen carrier in cardioplegic solution, on the protection of isolated rat hearts. Methods The hearts of 32 male SD rats were harvested and transferred to Langendorff circuit. They were divided into 4 groups according to cardiocplegia: St.Thomas group (group A), 1∶2 PEG-bHb group (group B), 1∶4 PEG-bHb group (group C) and 1∶8 PEG-bHb group (group D). After 20min balance period, hearts were perfused with cold (4℃) cardioplegic solutions, and preserved at 30℃ for 60min, then reperfused. Levels of cardiac troponin I (cTn I) and adenosine triphosphate(ATP) contant in coronary effuent were detected, and ultrastructures of myocardium were observed. Results After reperfusion, cTn I contant of group A were higher (F=52.955,Plt;0.05) and ATP contant were lower (F=68.757,Plt;0.05) than those in group B, group C and group D. Myocardial water contant were lower in group B and group C(F=3.048,Plt;0.05). Conclusion PEG-bHb in cardioplegic solutions can provide better myocardial protection during ischemia.