Objective To investigate the mechanism of the toxic effect of N methyl N-nitrosourea (MNU) on photoreceptor cell apoptosis of rat’s retina. Methods Thirty 50-day-old female Sprague-Dawley ( SD ) rats were intraperitoneally injected with MNU (60 mg/kg) and were put to death by dislocation of cervical vertebra 12, 24, 48, and 72 hours and 7 days after the injection, respectively. The photoreceptor cell apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and transmission electron microscope. The expression of proliferating cell nuclear antigen (PCNA), vimentin and glial fibrillary acidic protein (GFAP) was detected at different time after injection by immunohistochemical methods. Results The apoptotic index of the retina in the posterior pole was (33. 6±2. 3), (46. 5±5. 7), (20. 1±5. 3), (8. 2±3. 6) and (2. 5±1. 3~//oo at the 12th,24th, 48 th, and 72nd hour and on the 7th day, respectively, after injection. Karyopyknosis was found in most photoreeeptor cells 24 hours after injection. The expression of PCNA was found in internal granularlayer and between internal granular layer and choroid 24 hours after injection, reached the peak after 72 hours, and reduced obviously after 7 days. The positive expression of GFAP and vimentin was found in internal and external granular layer 24 hours after injection, reached the peak after 72 hours, and reducedobviously after 7 days.Conclusion MNU may selectively lead the photoreceptor cell apoptosis and proliferation of Mvller cells. (Chin J Ocul Fundus Dis，2004，20：33-36)
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.
Cilia are hair-like protuberance on cells of the human body that play a vital role in organs generation and maintenance. Abnormalities of ciliary structure and function affect almost every system of the body, such as the brain, eyes, liver, kidney, bone, reproductive system and so on. Retinal photoreceptor cells are one of sensory neurons which convert light stimuli into neurological responses. This process, called phototransduction, takes place in the outer segments (OS) of rod and cone photoreceptors. OS are specialized sensory cilia, and disruptions in cilia genes, which are causative in a growing number of non-syndromic retinal dystrophies, such as retinitis pigmentosa, Leber’s congenital amaurosis. These syndromes are genetically heterogeneous, involving mutations in a large number of genes. They show considerable clinical and genetic overlap. At present, there are few researches on retinal ciliopathies and clinical treatment strategy. This review shows a comprehensive overview of ciliary dysfunction and visual development related diseases, which contributes to understand the characteristics of these diseases and take early intervention in clinic.
ObjectiveTo observe the morphological characteristics of photoreceptor necroptosis in experimental retinal detachment, and explore the mechanism. MethodsA total of 60 Sprague-Dawley male rats were included in this study. Retinal detachment were induced in the right eyes with 1% sodium hyaluronate (50 μl) injection (experimental group), while the left eyes received no treatment (control group). At 3 days after modeling, the morphological characteristics of photoreceptor cell were observed by electron microscopy. Cleaved Caspase 8 and phosphorylation receptor-interacting protein 1 (p-RIP1) were measured by Western blot and immunoprecipitation. ResultsAt 3 days after modeling, photoreceptor necroptosis showed the following morphological features: chromatin condensation, severe vacuolation, early loss of plasma membrane integrity, and many autophagosomes. Western blot showed that the protein expression of cleaved Caspase 8 were 0.78±0.03, 0.06±0.01 in experimental group and control group respectively, which was significantly different (F=4 023.21, P < 0.05). Immunoprecipitation showed that the protein expression of p-RIP1 were 0.23±0.03, 0.14±0.02 in experimental group and control group respectively, which was significantly different (F=56.44, P < 0.05). ConclusionsPhotoreceptor necroptosis showed chromatin condensation, severe vacuolation, early loss of plasma membrane integrity, and many autophagosomes. Necroptosis activation was associated with the increase of RIP1 phosphorylation.
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 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.