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Background Increase in ophthalmic optical medical instruments and microsurgical applications leads to retinal photochemical damage and other problems delivery of a variety of devices,so the in-depth study and understanding of its pathogenesis after retina light damage can provide a reference for the clinical treatment of related diseases.Objective This study was to investigate the therapeutic effect and relative mechanism of erythropoietin (EPO) on mouse retina photic injury by studying the expression of matrix metalloproteinases-2 (MMP-2)and MMP-9.Methods Fifty-two SPF BALB/c mice were randomized into normal control group,simple light-induced group and EPO pretreatment group by balloting method.The mice of simple light-induced group and EPO pretreatment group were continuously irradiated with 6000 lx diffuse light for 4 hours in a home-made box to establish the models of light-induced damage;while recombinant human EPO (rhEPO)of 5000 U/kg was intraperitoneally injected prior to the light exposure in the EPO pretreatment group.The expressions of MMP-2 and MMP-9 were examined at 6,12,36,72,96 hours and 7 days following light-exposure by immunohistochemistry.Results Edema and structural disorder of RPE cells appeared inthe simple light-induced group after light-exposure and aggravated with lapse of light-exposure time,but no similar change was seen until 7 days in the EPO pretreatment group.The immunohistochemistry findings showed that the expression of MMP-2(A value)in RPE cells was less in the normal mice.However,a large quantity of positive cells appeared in RPE layer 36 hours after light-exposure.Compared with the simple light-induced group,the positive expression of MMP-2 protein in EPO pretreatment group was significantly decreased,showing statistically significant differences among these three groups and different time points (Fgroup =3.68,P =0 04; Ftime =9.13,P=0.00).There was hardly any MMP-9 expression in the retina of the normal mice.In simple light-induced group,a few of positive cells appeared in RPE layer 6 hours after light-exposure and reached its peak 12 hours following light-exposure.The gradually down-regulation of MMP-9 expression happened 96 hours later following light-irradiation.The expression tendency of MMP-9 in EPO pretreatment group was similar to the simple light-induced group.Significant differences in expressions of MMP-9 were found among different groups and time points (Fgroup=3.61,P =0.04;Ftime =16.91,P=0.00).Conclusions MMP-2 and MMP-9 may be involved in the mechanism of retina photic injury by down-regulating the expression of MMP-2 and MMP-9 in RPE cells.
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BackgroundRetinal pigment epithelial (RPE)cell senescence damage the metabolism of photoreceptor,leading to retinal dysfunction and loss of vision.To understand RPE cell senescence mechanism will contribute to the study of age-related macular degeneration ( AMD).ObjectiveThe present study was to prepare the ageing RPE cell model with passage and explore its potential mechanisms.MethodsThis study was approved by the Ethic Committee of Qingdao University Medical College,and the informed consent was obtained from each gravida.Six human eyeballs were obtained from artificial labor fetusl with the gestational age 16-28 weeks.RPE cells were isolated,cultured and passaged in vitro to establish the cell replicative aging model.The third to twelfth cells were collected to be used to this experiment.Human keratin was used to identify the cells by immunochemistry,and MTT method was utilized to assess the proliferation and viability of different generations of cells as the A490 value.The cellular cycles and transmembrane potential (△ψm)of mitochondrion (△ψm) with passage were detected and compared using Flow Cytometry. Results Cultured and passaged cells showed the hexagon in shape with the melanin in 1-2 generations of cells and presented with the brown staining in cytoplasm for human keratin.The melanin was absent in the third generation cells.Vibrant growth statues were seen from the 3-6 generations cells and thereafter the proliferation ability reduced.The cells of G0/G1 phase were gradually elevated with the passage from 3 - 12 generations with a percentage of 68.40％ in the third generation of cells to 87.33％ in the twelfth generation of cells,showing a significant difference among various generations ( F =180.43,P =0.00),and that of the sixth,ninth and twelfth generation of cells was significant higher than the third,sixth and ninth generation respectively (t =4.002,P＜0.05 ; t=12.885,P＜0.01 ;t=16.387,P＜0.01 ).MTT assay showed that of RPE cells were significantly declined with the passage ( F =38.77,P =0.00),and the A490 value of the ninth,twelfth generations of cells was considerably lower in comparison with sixth and ninth generation respectively ( t =5.991,11.983,P＜0.01 ).From 3 through 12 generations of cells,the staining intensity of rhodamine 123 was gradually decreased ( F =121.68,P =0.00 ),and the staining intensity in the sixth,ninth and twelfth generation of cells was significant lower than that the third,sixth and ninth generation respectively(t=6.918,7.620,11.207,P＜0.01 ).Conclusions A replicative aging model can be successfully created by the passage in vitro using human fetal RPE cells.The reduce of transmembrane potential and damage of mitochondria might be one of mechanisms of senescence of RPE cells.
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Background Erythropoietin (EPO) was proved to be express in hematopoietic tissue and nervous system and play the effects of stimulating blood cell production and protecting nervous tissue.Researches showed that EPO is expressed in the embryon brain of animal.However,whether EPO exist in nervous-derived retina and its action on retina with the development is concerned. Objective This research was to investigate the expression of EPO during the embryonic development period of rat retina and explore the role of EPO in retina development process.Methods Clean Wistar rats with pregnancy for 12 days,16 days and 20 days were collected,and the embryonic 12-day rats (E12 d,5 rats),embryonic 16-day rats (E16 d,5 rats) and embryonic 20-day rats ( E20 d,5 rats) were obtained by caesarean operation,and 5 12-month W istar rats were used as controls.The rats were sacrificed by cervical dislocation and the retinal sections were prepared in the different-embryo-phase (12 d,16 d,20d) and growth phase.The expression of EPO protein and mRNA in rat retina was detected by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR),respectively.The feed and use of the animals followed the Regulation for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results EPO was positively expressed in the cytoplasm and nuclei in the neuroepithelial layer and pigment epithelium of every-embryo-phase rats but only in retinal ganglion cell layer in 12-month-old rats.The gray scale values of EPO expression in retina were 105.55±10.35,99.35± 8.71,83.27± 7.84and 30.30± 3.80 in E12 d rats,E16 d rats,E20 d rats and 12-month-old rats respectively with a statistically significant difference (F=76.13,P＜0.01 ).RT-PCR revealed that the relative values of EPO mRNA expression in retina were 0.876±0.10,0.861 ±0.09 and 0.256±0.03 in E16 d rats,E20 d rats and 12-month-old rats respectively,presenting a elevated value in embryonic rats compared with adult rats ( P =0.00).Gel imaging deletion showed that the A value of EPO amplification products was highest in E16 d rats and lowest in adult rats.Conclusions The expression of EPO appears a high to low fashion during the embryonic development of Wistar rats,which is closely associated with the developing procedure of retina.
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AIM: To explore the relationship between the expression of caspase-2 and caspase-3 and the apoptosis in retinal ischemia/reperfusion (I/R) injury of rats, as well as the therapeutic effects of brain derived neurotrophic factor (BDNF)on the ischemic and reperfused retina.METHODS: This experiment was conducted at the laboratory of Affiliated Hospital of Qingdao University Medical College from February 2007 to July 2007. The models of retinal ischemia/reperfusion injury were made by transiently elevating intraocular pressure. A total of 28 rats were divided into Normal and Operative Groups. Operative group was divided into six subgroups. In each subgroup there were four rats. The left eyes of rats were used for I/R and the right eyes were used for intravitreal injection of brain-derived neurotrophic factor (BDNF) as treatment group. After reperfusion we divided our subgroups according to the reperfusion time as 1, 6, 12, 24, 48, 72 hours. The retinal ganglion cell number was counted by using optic microscope(BX-51,Olympus). Apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL) method, and the expression of caspase-2,caspase-3 was studied by enzyme linked immunosorbent assay (ELISA) and strept avidin-biotin complex (SABC)immunohistochemistry.RESULTS: No positive apoptotic cells were observed in the normal rats' retinae, but there were a significant number of positive apoptosis cells in 6-24 hours after transient ischemia followed by a decrease at 48 hours. The number of apoptotic cells reached a maximum at 24 hours after ischemia .The expression of caspase-2 gradually increased as early as at 6 hours, reached a peak at 24 hours, then decreased between 48 and 72 hours. Similarly, caspase-3 has the same rule with caspsae-2 in the time courses of expression in retinal tissues.BDNF administered before reperfusion inhibited the expression of apoptosis and ameliorated the retinal tissue damage. It also decreased caspase-2 and caspase-3 expression in ischemic/reperfused retina.CONCLUSION: Retinal ischemia-reperfusion can induce apoptosis of cells in the retina. BDNF rescues retinal ganglion cells (RGCs) from retinal ischemia/reperfusion injury through down-regulation of cell apoptosis and caspase-2 and caspase-3 expression. BDNF have a neuroprotective effect on retina.
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· AIM: To explore the relationship between the expression of Fas/FasL and the apoptosis in retinal ischemia/reperfusion injury of rats, as well as the therapeutic effects of basic fibroblast growth factor (bFGF)on the ischemic retina.injury were made by transiently elevating introcular pressure. A total of 28 rats were divided into Normal Group and Operative Group. The latter were subdivided into 1, 6, 12, 24, 48 and 72h after reperfusion, in which the left eyes of the rats were in the ischemia/reperfusion groups and the right ones were in the treatment groups (bFGF intracameral injection). Apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL) method, and theexpression of Fas/FasL ligand was studied by strept avidin-biotin complex (SABC) immunohistochemistry.mai rats' retinae, but there were a significant number of TUNEL positive cells in 6-24h after transient ischemia followed by a decrease at 48h. The number of TUNEL positive cells reached a maximum at 24h after ischemia.The expression of Fas gradually increased as early as at 6h, reached a peak at 24h, then decreased at 48h. Similarly, the expression of Fas ligand was at peak in 24-48h in GCL and INL of retina. bFGF ministered before reperfusion inhibited apoptotsis and ameliorated the tissue damage. It also diminished Fas and FasL expression in ischemic/reperfused retina.siently elevated IOP induced apoptosis of cells in the retina. Fas/FasL may have an important role in the early events of the apoptotic pathways. bFGF can rescue RGCs from retinal ischemia/reperfusion injury through down-regulation of Fas and Fas ligand expression and may represent an important mechanism for therapeutic neuroprotection.
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<p><b>BACKGROUND</b>Basic fibroblast growth factor (bFGF) plays important roles in retina degeneration, light injury, mechanical injury, especially in retina ischemia-reperfusion injury (RIRI). This study was to investigate the therapeutical effect of bFGF on RIRI and its mechanisms.</p><p><b>METHODS</b>Experimental RIRI was induced by increasing intraocular pressure (IOP) in the eyes of 48 rats. These rats were divided into normal control, ischemia-reperfusion and bFGF-treated groups. Histological and ultrastructural changes of in the retina of different groups were observed, and the number of retinal ganglion cells (RGCs) was quantitatively analyzed under microscopy. Apoptotic cells were detected using the TdT-dUTP terminal nick-end labeling (TUNEL) method. The expression of caspase-3 was determined by streptavidin peroxidase (SP) immunohistochemistry. Atomic absorption spectrum method was used to evaluate the intracellular calcium changes.</p><p><b>RESULTS</b>At the early stage of retinal ischemia-reperfusion injury, retina edema in the treated group was significantly eliminated compared with the untreated ischemic animals. RGCs in the bFGF-treated group was more than those in the untreated ischemic group during the post-reperfusion stages. In ischemic group, apoptotic cells could be found at 6th hour after reperfusion and reached the peak at 24 hours. At 72nd hour no apoptotic cells could be found.The changes in caspase-3 expression had a similar manner. The intracellular calcium of rat retina began to increase at 1st hour, reached the peak at 24 hours, and began to decrease at 72 hours. The change of the three markers in the treatment group showed a similar pattern, but they were all relatively less obvious.</p><p><b>CONCLUSION</b>Apoptosis may play a vital role in RIRI. bFGF may has therapeutical effects on RIRI by inhibiting the increase of intracellular calcium and caspase-3 expression.</p>