PURPOSE:To examine the role of apoptosis in photoreceptor cells degeneration process in Pathologic myopia. METHODS: Nine human eyes with pathologic myopia were studied by histopathologic and TDT-mediated biotin-dUTP nick-end labelling (TUNEL) techniques. RESULT:The characteristic DNA fragmentation of apoptosis was observed in scattered photoreceptor cells in 4 of 9 eyes. CONCLUSION:The results suggested that apoptosis is one of the pathways of photoreceptor cells death in retinal degeneration of pathologic myopia. （Chin J Ocul Fundus Dis,1996,12: 144-146 ）
Objective To investigate the correlation of ascorbic acid distribution and retinal susceptibility to iron toxicity of the retina.Methods Autoclaved iron particles of 5 mg and 15 mg were implanted into the vitreous cavities of 32 Spragu-Dawley (SD) rats and 9 rabbits, respectively. The retinal sections of rats and rabbits were examined after hemotoxylin-eosin (HE) staining. Apoptos is of rabbits′retinal neurons was investigated by TdT-mediated dUTP-biotin nick-end labeling (TUNEL). Chinoy′s method was used to observe the distribution of as corbic acid in the retinae of the 2 kinds of animals.Results In rats, histological and structural densification was observed only in the photoreceptor cells after implantation of the iron particles. In rabbits, however, histological and structural destruction as well as TUNEL-positive nuclei were observed in all neuronal layers of the retina 3 days after the implantation of the iron particles. Silver granules reduced by ascorbic acid from silver nitrate were observed only in the outer nuclear layer in normal rats retinae, while they were observed evenly throu ghout all layers of rabbits′retinae. Conclusions The suscept ibility of retina to iron toxicity is correlated to the distribution of ascorbic acid in retina. (Chin J Ocul Fundus Dis，2003，19：269-332)
Objective To observe the distribution of human photoreceptor cells at the posterior pole, detect the change of density of the cells affected by eccentricity, and analyze the relationship between the density distribution and the visual sensitivity. Methods Twenty human eye cups with the cornea removed were fixed in 4% polyformaldehyde for 1-4 weeks, and the retinal mounts were observed by differential interference contrast microscope to reveal the retinal cellular configuration and density. The inner segments of photoreceptor cells were first observed from the center to the temporal peripheral part of the retinal mounts. Results The highest density of visual cone cells was at the central fovea (134 000-267 000/mm2, mean 198 090/mm2; CV value:18.2%). The density and individual variation decreased rapidly in the peripheral area. The high density area of rod cells was at the 4 mm of the eccentricity, with the highest value of 72 610-182 350/mm2 and with the high density between 3 and 5 mm. Conclusions The inner segment of photoreceptor cells was monolayer, which may tell the cellular absolute value. The high density of retinal cone cells at the central fovea provide the basis of sensitive central visual acuity, which relates to the individual variation and development. The rod cells have the peak density at the eccentricity with 4 mm, and this area has the greatest sensitivity of dim vision.
Objective To investigate the protective effect of recombinant erythropoietin (EPO) on the photoreceptor cells in rat with retinal detachment (RD).Methods One hundred and sixtytwo normal male rats were randomly divided into normal control (NC) group, RD model group, RD+phosphate buffer solution （RD+PBS） group, RD+EPO 100 ng group, RD+EPO 200 ng group and RD+EPO 400 ng group. Three days after RD, activated caspase3 and bclXL were detected by Western blot and/or immunofluorescence, and apoptosis were measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate digoxigenin nick-end labeling（TUNEL）. Fourteen and 28 days and two months after RD, the outer nuclear layer （ONL）thickness was measured by histopathologic method.Results Western bolt indicated that the protein level of activated caspase-3 and bcl-XL between six groups were statistically significant（F=35.96, 30.75；P＜0.01）. The number of TUNEL positive cells and activated caspase-3 positive cells are consistent with each other in different groups. Fourteen days and two months after RD，the differences of ONL thickness between six groups were statistically significant（F=21.52，96.25；P＜0.01）.Conclusion Supplement of EPO after RD can alleviate apoptosis by inhibiting of the caspase-3 activity and increasing the expression of bcl-XL,thus exerts protective effect on photoreceptor cells.
Objective To demonstrate if apoptosis is one of the mechanisms of siderotic retinopathy. Methods Autoclaved iron particles were implanted in the vitreous cavities of 32 eyes of SD rats.Glass chips were implanted in 10 control eyes.The experimental eyes were enucleated at various time intervals from days 1 to 15.Retinal degeneration was examined using the TdT-mediated,dUTP-biotin nickend labeling(TUNEL)method.Electrophoresis on agarose gel was used to detect internucleosomal DNA fragmentation.Results TUNEL-positive nuclei were observed only in the outer nuclear layer beginning on day 2.The nuclei spread throughout the outer nuclear layer by the end of day 3.No TUNEL-positive nuclei were observed in other layers throughout the experimental perios.Analysis of DNA,extracted from the retinas by electrophoresis on agarose gel,revealed a typical ladder pattern of internucleosoma DNA cleavage in the experimental eyes.ConclusionApoptosis of photoreceptors occurs at the early phase of iron-induced retinopathy in the rats.
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 observe the frequency domain optical coherence tomography (SD-OCT) features of Henle fiber layer (HFL) of health adults in china by changing the angle of the measurement beam. Methods Twenty-four subjects (28 eyes) who showed no abnormalities on routine eye examination were included in the study, including 15 males (16 eyes) and 9 females (12 eyes) with an average age of (35.51±3.54) years old, and mean refraction power of (-0.89±1.15) D. All subjects underwent corrected visual acuity, intraocular pressure, slit lamp microscope, direct ophthalmoscope, visual field and SD-OCT examination. The macular area was scanned by Zeiss Cirrus SD-OCT (5 HD line) single line scan mode. Based on the entry position of the SD-OCT beam through the pupil, the subjects were divided into 3 groups, including group A (center of the pupil), group B (near the temporal edge of the pupil) and group C (near the nasal edge of the pupil). The thickness of outer plexiform layer (OPL), HFL, and outer nuclear layer (ONL) were measured at 0.75 mm, 1.50 mm from the fovea. ResultsWhen entry position of the SD-OCT beam was near the temporal edge of the pupil (group B); there were two layer structures with different signal intensities in the weak reflectivity zones in front of the external limiting membrane (ELM). The signal of the inner layer was slightly higher than the outer layer. The OPL thickness at the decreased side (nasal) increased significantly compared with the other side, but the ONL thickness was significantly thinner than other side. When entry position of the SD-OCT beam was near the nasal edge of the pupil (group C), there were also two layer structures with different signal intensities in the weak reflectivity zones in front of the ELM. The signal of the outer layer was slightly higher than the inner layer. The OPL thickness at the decreased side (temporal) increased significantly compared with the other side, but the ONL thickness was significantly thinner than other side. The OPL thickness at the decreased side was significantly different between these 3 groups (P < 0.01). ConclusionsSD-OCT provided the possibility of distinguishing HFL from the actual ONL by changing the angle of the measurement beam. This finding has great clinical significance for related diseases affecting HFL or ONL.
Objective To investigate the feasibility of gene transfection into retinal pigment epithelial (RPE) cells and photoreceptors (PRs) in vivo electroporation. Methods A total of 147 Sprague-Dawley (SD) rats were divided into 5, 10, 15, 20, 25, 30 and 35 V group according to different voltage. The right eyes of rats underwent the injection of eukaryotic expressive plasmid of enhanced green fluorescent protein (EGFP) pEGFP-N1 into subretinal space as experimental eyes; the left eyes were injected with TE buffer as control eyes. Each group was divided into RPE and RP subgroups according to different transfection direction. There were same parameters of 99 ms pulse width, 0.5 s pulse interval and 5 consecutive pulses except different voltage in groups. With a negative charge in the electric field was transfected into RPE cell layer, reverse electrode set to be transfected into PR cell layer. Retina mounts were made on seven days after transfection and the fluorescence of EGFP was photographed by fluorescent microscope. The expression of EGFP mRNA and protein were detected by reverse transcription polymerase chain reaction technique (RT-PCR) and Western blot.Results On seven days after transfection, in RPE subgroups, there were no specific fluorescence expressions in RPE cell layer and retina mounts of control eyes, while there were fluorescence expressions in experimental eyes. Western blot showed that the grayscale ratio of EGFP protein and beta;actin protein bands rose with the increased voltage. RT-PCR showed that each group produced positive amplification bands, and the relative ratio of gray level of EGFP mRNA and GADPH mRNA amplified bands gradually increased with the increased voltage.Conclusion Electroporation is an effective method for gene delivery into RPE cells in vivo.
Objective To observe the effects of local macular foveal photoreceptor defects on visual acuity.Methods Thirty-one patients (31 eyes) with photoreceptor defect in macular fovea (case group) diagnosed by spectral domain optical coherence tomography (SD-OCT) and 30 patients (30 eyes) age- and diopter- matched normal subjects (control group) were enrolled in this study. There were 22 eyes with full photoreceptor defects and 9 eyes with outer segment defects in case group. All subjects were examined for best corrected visual acuity (BCVA), slit-lamp microscopy, direct ophthalmoscope and SD-OCT. Independent sample t-test was used to compare central foveal thickness (CFT) between case group and control group. Difference of logMAR BCVA, CFT, maximum width and height of photoreceptor defects, defected area and residual retinal thickness in macular between patients with full photoreceptor defects and outer segment defects were also compared.Results The CFT of case group and control group were (225.32plusmn;19.70),(240.02plusmn;10.70) mu;m, the difference was not statistically significant (t=-1.96, P＞0.05). In full photoreceptor defects and outer segment defects patients, the mean logMAR BCVA were 0.22plusmn;0.31, 0.32plusmn;0.43; the mean CFT were (224.09plusmn;20.57), (228.33plusmn;18.17) mu;m; the maximum width of photoreceptor defects were (131.32plusmn;108.18), (143.22plusmn;66.93) mu;m; the mean defected area were (0.022plusmn;0.054), (0.019plusmn;0.019) mm2; the mean maximum height of photoreceptor defects were (77.41plusmn;6.62), (44.89plusmn;4.26) mu;m; the mean residual retinal thickness were (87.00plusmn;20.31), (128.33plusmn;23.54) mu;m respectively. There was no statistical significance between full photoreceptor defects and outer segment defects patients in the mean logMAR BCVA, CFT, maximum width of photoreceptor defects and defected area (t=-0.76, -0.538, -0.305, 0.166; P＞0.05), but there were significant difference in mean maximum width of photoreceptor defects and residual retinal thickness (t=12.72, -4.91;P＜0.05). Conclusions The local photoreceptor defects in macular fovea can lead to decrease of visual acuity. The wider the photoreceptor defects, the worse the visual acuity.
Objective:To observe the intervention effect of the tetra methylpyraz ine on the rds mice with retinitis pigmentosa. Methods:A total of 84 rds mice were randomly divided into 2 groups, with 42 mice in each group. The mice in the experimental group underwent intraperitoneal cavity injection with hydrochlor i c tetramethylpyrazine (80 mg/kg, twice per day) at the date of birth and till 35 days after birth, whereas the normal saline was injected into the intraperito n eal cavity of rats in the control group. The mice were sacrificed 0, 3, 7, 14, 2 1, 28, 35 days after birth, and the eyeballs were enucleated for the routine pat hologic examination with the light microscope. The apoptosis of photoreceptor ce ll nuclei was detected by terminal deoxynucleotidyl transferasemediated dUTP n i ck endlabeling (TUNEL) technigue and the expression of bcl2 in retina was de tect by immunohistochemistry method. Results:The results of li ght microscopy s howed that the layer number of retinal photoreceptor cell nuclei with tetramethy lpyrazine treatment was increased 14, 21, 28, 35 days after the treatment compar ed with that in the control group（P＜0.01）. The results of electron-micro scope suggested that tetramethylpyrazine might reduce lesions in the photoreceptor cells and the destruction of the disc member, mitochondrion，and outer limiting me mbrane in the photoreceptor outer segment in rds mice. The apoptosis of the phot oreceptor cell nuclei reduced in rds mice 3, 7, 14, 21, 28 and 35 days after the treatment compared with that in the control group （P＜0.01）. The express ion of bcl-2 in the matrix of retinal photoreceptor cell nuclei and its inner and o u ter segments increased significantly in rds mice 3，7, 14, 21, 28 and 35 days af ter the treatment （P＜0.05）. Conclusions:Tetramethylpyra zine might reduce ret inal photoreceptor apoptosis by upregulating the expression of bcl-2 in the m at rix of retinal photoreceptor cell nuclei or its inner and outer segments in rds mice.