Idiopathic macular hole (IMH) refers to full thickness defects of retina in macular area with no clear reasons.The management of IMH includes vitrectomy combined with internal limiting membrane (ILM) peeling and pharmacological vitreolysis.But ILM peeling may damage the inner retina;novel techniques,such as inverted ILM flap technique and foveola non-peeling ILM surgery,autologous ILM transplantation had made the method of ILM peeling more diversified with less damage.Pharmacological vitreolysis targeting fibronectin and laminin is considered to work in a two-step mechanism,involving both vitreoretinal separation and vitreous liquefaction.Furthermore,IMH judgment and prognosis indicators like ellipsoid zone,macular hole index,hole formation factor,diameter hole index and tractional hole index based on spectral domain optical coherence tomography enriched the assessment of macular hole diameter,depth and shape.How to make full use of new interventions to reduce the incidence of macular hole and obtain a better visual acuity with closed holes is an important direction for future research.

Objective:To study the therapeutic effect and mechanism of autologous platelet-rich plasma (PRP) on rabbit traumatic optic neuropathies (TON) and retina.Methods:Forty adult New Zealand white rabbits were selected to establish the optic nerve clamp injury model in their right eyes.According to the random number table method, 36 New Zealand white rabbits with effective model were randomly divided into model control group, normal saline control group and PRP group, 12 for each group.Another 12 healthy rabbits served as the normal control group.Rabbit autologous blood was collected to prepare PRP.The retrobulbar 20 μl PRP/20 μl saline solution injection was administered every two days near the injury after modeling according to grouping.The injection was carried out for 10 times.There was no other interference administrated to the model control group except the normal anti-infective treatment.No interference was given to the normal control group.At 30 and 60 days after modeling, the eyeballs and optic nerves of right eyes were harvested through sacrificing the animals by anesthetic overdose, three eyes for each time.Histopathological assessments were performed to observe the morphological changes of retina and optic nerve, and to evaluate the changes of retinal ganglion cells (RGCs) density and retinal nerve fiber layer (RNFL) thickness.Immunohistochemistry was used to assess the expressions of apoptosis factors caspase-3 and B cell lymphoma-2(Bcl-2). Quantitative real-time PCR and Western blot were used to detect the mRNA and protein expressions of brain-derived neurotrophic factor (BDNF) and growth associated protein-43 (Gap-43). This study protocol was approved by the Experimental Animal Ethics Committee of Wuhan University (No.E2019072805). The use and care of animals complied with ARVO statement.Results:The thickness of RNFL and number of RGCs at 30 days and 60 days after modeling were (6.60±1.16) μm, (6.89±1.21) μm, (13.00±1.00)/field of vision, (20.00±2.65)/field of vision in the PRP group, respectively, and were (4.80±0.43)μm, (2.18±0.23)μm, (6.33±0.58)/field of vision, (10.33±1.53)/field of vision in the model control group, respectively.The number of RGCs in the PRP group at 60 days was higher than that at 30 days after modeling, the number of RGCs in the PRP group was higher than that in the model control group, the thickness of RNFL in the PRP group was higher than that in the model control group; and the differences were statistically significant (all at P<0.05). At 30 and 60 days after modeling, the positive expression A value of caspase-3 protein in the normal saline group and model control group were higher than those in the normal control group and PRP group, while the positive expression A value of Bcl-2 protein in the PRP group was higher than those in the model control group and normal saline group, and the differences were statistically significant (all at P<0.05). The mRNA level and protein content of BDNF and Gap-43 in the retina and optic nerves at 30 days and 60 days after modeling in the PRP group were higher than those in the model control group, and the differences were statistically significant (all at P<0.05), but the mRNA and protein expression levels of BDNF and Gap-43 in different tissues in the PRP group at 60 days after modeling were lower than those at 30 days after modeling ( P<0.05). Conclusions:PRP can effectively inhibit the apoptosis of RGCs and the secondary injury of the retina after optic nerve injury, promote cell anti-apoptosis effect of RGCs, thereby retard the damage of the retina and optic nerve after TON, and also promote the repair of optic nerve and retina through upregulating the expression of nerve growth factors.

Objective To evaluate the correlation of oxygen saturation of retinal vessels and diabetic retinopathy (DR) stages or HbA1c level in patients with DR. Methods Cross sectional study. A total of 102 patients (102 eyes) with DR and 20 age-matched healthy controls (20 eyes) (normal control group) were enrolled in this study. DR patients were divided into mild and moderate non-proliferative DR (NPDR) group (55 patients), severe NPDR group (26 patients) and proliferative DR (PDR) group (21 patients). DR patients were also divided into 3 groups according to the HbA1c level including HbA1c>9%(8 patients), HbA1c 7%–9%(33 patients) and HbA1c<7% group (61 patients). The oxygen saturation of retinal vessel was measured by spectrophotometric oximetry unit in the retinal vessels with a diameter greater than 60μm in the area around the optic disc. Results The retinal artery oxygen saturation of patients in severe NPDR group was significantly higher than that in mild to moderate NPDR group and normal control group (F=13.670, P<0.05). The retinal vein oxygen saturation of patients in PDR group was significantly higher than that in mild to moderate NPDR group and normal control group (F=6.379, P<0.05). The difference between retinal artery and vein oxygen saturation of patients in severe NPDR group was significantly bigger than that in mild to moderate NPDR group and PDR group (F=5.536, P<0.05). The retinal artery and vein oxygen saturation in patients of HbA1c>9%group were significantly higher than that in HbA1c 7%–9%group and HbA1c<7%group (F=9.989, 10.208;P<0.05). The differences between retinal artery and vein oxygen saturation were same between patients in HbA1c>9%, HbA1c 7%<9%and HbA1c<7%group (F=1.836, P>0.05). Conclusions The retinal artery and vein oxygen saturation in DR patients are related to the DR stages. Severe NPDR patients show the highest retinal artery oxygen saturation as well as biggest difference between retinal artery and vein oxygen saturation. There is also a trend that retinal vein oxygen saturation increases with higher DR stages. In addition, there is a positive correlation between the levels of HbA1c and retinal vessel oxygen saturation.

Optogenetics is a novel technique which combines optics with genetics.Using genetic means,a selected opsin protein is ectopically expressed in target neurons,which are then stimulated by light to moderate the neuronal circuit,as a consequence to regulate the animal's behaviors.Retinal degeneration like retinitis pigmentosa and aged macular degeneration causes visual impairment and eventual blindness.Optogenetics techniques have opened the door to creating artificial photoreceptors in the remaining retinal circuits of retinal degeneration retinas via gene therapy.However,there are still limitations in optogenetics technique,for example,potential risk in virus infection,the choice of target cells and the low visual resolution of the experiment animal.It has been reported that vision was successfully restored to a certain extent in animal model using optogenetics technique.With higher photosensitivity of opsin protein,longer activation kinetics and higher transfection efficiency of virus vector,optogenetics techniques' application in ophthalmology will be improved.

Objective To observe the influence of down-regulation of HtrA1 expression by small interfering RNA on light-injured human retinal pigment epithelium (RPE) cells.Methods Cultured human RPE cells(8th-12th generations)were exposed to the blue light at the intensity of (2000 ± 500) Lux for 6 hours to establish the light injured model.Light injured cells were divided into HtrA1 siRNA group,negative control group and blank control group.HtrA1 siRNA group and negative control group were transfected with HtrA1 siRNA and control siRNA respectively.The proliferation of cells was assayed by CCK-8 method.Transwell test was used to detect the invasion ability of these three groups.Flow cytometry was used to detect the cell cycle and apoptosis.The expression of HtrA1 and vascular endothelial growth factor (VEGF)-A was detected by real time-polymerase chain reaction and Western blot respectively.Results The mRNA and protein level of HtrA1 in the light injured cells increased significantly compared to that in normal RPE cells (t=17.62,15.09;P＜0.05).Compared with negative control group and blank control group,the knockdown of HtrA1 in HtrA1 siRNA group was associated with reduced cellular proliferation (t=6.37,4.52),migration (t =9.56,12.13),apoptosis (t =23.37,29.08) and decreased mRNA (t=17.36,11.32,7.29,4.05) and protein levels (t=12.02,15.28,4.98,6.24) of HtrA1 and VEGF-A.Cells of HtrA1 siRNA group mainly remained in G0/G1 phase,the difference was statistically significant (t=6.24,4.93;P ＜0.05).Conclusion Knockdown of HtrA1 gene may reduce the proliferation,migration capability and apoptosis of light-injured RPE cells,and decrease the expression of VEGF-A.

Background Human retinal pigment epithelial(RPE) cells play a critical role in the pathogenesis of proliferative vitreoretinopathy(PVR) and other related ocular diseases.Research demonstrated that epidermal growth factor(EGF) stimulates activation of RPE cells and therefore causes PVR,and integrin α_5 mediates the adhesion of cells and EGF.Objective This study aims to investigate the role of mitogen-activated protein kinase(MAPK) in regulation of EGF on integrin α_5 expression in human RPE cells.MethodsHuman RPE cells strain(CRL2302) was cultured in DMEM/F12 with 10% calf serum and passaged in 0.25% trypsin.Cultured cells were divided into DMEM/F12control group,20μmol/L PD98059 +10 ng/mL EGF＋DMEM/F12(PD98059) group and 10 ng/mL EGF＋DMEM/F12(EGF) group.The expression of integrin α_5 protein in CRL2302 cells was detected by RT-PCR and calculated as α_5 mRNA/β-actin mRNA,and the expression of integrin α_5 mRNA in CRL2302 cells was detected evaluated by flow cytometry.The MAPK phosphorylation level in each group of human RPE cells was tested by Western blot.ResultsThe expression value of integrin α_5 mRNA was 0.93±0.06 in the control group,1.06±0.07 in PD98059 group and 1.97±0.09 in EGF group,showing a significant difference among the three groups(F=458.896,P<0.01).The fluorescence intensity of integrin α5 protein in CRL2302 cells after 24 hours was 1.94±0.22,4.56±0.25,2.39± 0.14 in three groups respectively with a significant difference(F=21.05,P<0.05).After 30 minutes of culture,Western blot result showed that the strongest phosphorylation levels of ERK1/2 activation in EGF group and the weakest phosphorylation levels of ERK1/2 activation in the control group;While that in PD98059 group was significantly stronger than control group and weaker than EGF group(F=143.14,P<0.01).ConclusionEGF stimulates activation of ERK1/2 pathway in human RPE cells and the expressions of integrin α_5 mRNA and protein in human RPE cells in vitro.

Objective To investigate the inhibitory effects and related mechanism of S100A4 gene silencing on oxygen-induced retinal neovascularization.Methods 7-day-old C57BL/6J mice were randomly divided into 5 groups including normal group,normal-S100A4 group,oxygen induced retinopathy (OIR) group,OIR-S100A4 group,OIR-green fluorescent protein (GFP) group.To establish the OIR model,mice from all groups except normal one were exposed to (75±2) ％ oxygen for 5 days and then to room air.In the OIR-S100A4 group and OIR-GFP group,the OIR mice were given an intravitreal injection of 1 μl of 1.0 × 109 PFU/ml adenovirus of Ad-S100A4-RNAi or Ad-GFP at P12,and then returned to normoxia for the next 5 days.In the OIR group,OIR was induced in C57bl/6J mice from P7 to P17.In the normal-S100A4 group,the normal P12 mice were give an intravitreal injection of 1 μl of Ad-S100A4-RNAi adenovirus,and maintained in room air from P12 to P17.In normal group,newborn mouse litters were maintained in room air from P0 to P17 without any treatment.Mice in all five groups were euthanized at P17,and retinas were collected for biochemical assays and morphological study.Retinal neovascularization (RNV) was evaluated by counting the number of pre-retinal neovascular cells and the whole mount immunofluorescent staining of the mouse retina.Protein and mRNA expression levels of S100A4,cAMP responsive element binding protein (CREB),B cell lymphoma-2 (bcl-2),Caspase-3 were determined with western blot and real-time PCR.Results The number of pre-retinal neovascular cell nuclei in retinas from OIR-S100A4 group were obviously lower than those in the retinas from OIR group and OIR-GFP group (t=13.61,14.64;P＜0.05).In OIR-S100A4 group,the retinal neovascular tufts area and the vaso-oblitertion area were both significantly smaller than those in OIR group and OIR-GFP group (P＜0.05).Protein level of CREB and bcl-2 were significantly down-regulated in OIR-S100A4 group than those in OIR and OIR-GFP group (P＜0.05).On the contrary,protein levels of Caspase-3 were up-regulated in OIR-S100A4 group than those in OIR and OIR-GFP group (P＜0.05).Conclusion Ad-S100A4-RNAi transfer ameliorates RNV in mouse model of OIR maybe through down-regulating the expression of bcl-2 and CREB,and up-regulating the Caspase-3.

Objective To observe the effect of epidermal growth factor (EGF) on integrin α5 expression and its influence on human retinal pigment epithelium (RPE) cells. Methods Human RPE cells were treated in vitro with 0.1, 1.0, 10.0, 20.0 and 100.0 ng/ml of EGF, the mRNA and protein of integrin α5 was measured by reverse transcription polymerase chain reaction(RT-PCR)and flow cytometry. Human RPE cells were cultured under 4 conditions including DMEM/F12, DMEM/F12 + 10 ng/ml EGF, DMEM/F12 + 10 ng/ml EGF+ rabbit anti-human integrin α5 antibody (1: 100), DMEM/F12 + 10 ng/ml EGF+ rabbit anti-human vimentin antibody (1: 100), and their proliferation and migration were measured by methyl-thiazole tetrazolium(MTT)and Boyden chamber. Results The integrin α5 mRNA level of human RPE cells was not changed after 12 hours of EGF stimulation (F=0.618, P = 0. 687), however it was induced in a dose-dependent manner after 24 and 48 hours of EGF stimulation (F=465. 303, 212. 340; P= 0. 000, 0. 000). The protein level of integrinα5 was higher in 10 ng/ml EGF stimulation compared with the control group and 0. 1 ng/ml group (P<0. 01). MTT and Boyden chamber showed that the integrin α5 expression increased the proliferation and migration of human RPE cells. Conclusion EGF can induce integrin α5 expression, thus increase the proliferation and migration of human RPE cells.

Objective To observe the changes of visual acuity?multifocal electroretinogram (mfERG) and optical coherence tomogram(OCT) before and after successful idiopathic macular hole (IMH) surgery, and evaluate the efficiency of the IMH surgery. Methods A total of 28 eyes of 28 patients with IMH who underwent vitrectomy during February 2001 and May 2002 in our hospital were collected. visual acuity, mfERG, and OCT were examined preoperatively and 1, 3, 6, and 12 months postoperatively, respectively. The results were analyzed statistically compared with 33 eyes in control group. Results (1) OCT showed that 27 eyes (96.43%) had anatomic closure of the macular hole 1 month after the surgery without recurrence in 12 months. Just 1 eye (3.57%) failed in the closure within 1 year. (2) The visual acuity was much higher in the eyes 1 year after surgery, especially within 6 months postoperatively, than that before the vitrectomy(P

Objective To observe the manifestation of fundus angiographs of polypoidal choroidal vasculopathy (PCV). Methods Twelve PCV patients involved 7 males and 5 females aging from 40 to 69 year old (average 56.4). Fundus examination, fundus fluorescein angiography (FFA), and indocyanine green angiography (ICGA) were performed on 12 patients (12 eyes) with PCV, out of whom 5 underwent optical coherenece tomography (OCT). Results In 12 eyes, deep and (or) superficial hemorrhage and yellow hard exudations were found, including orange-red lesions in 6 and pre-retinal hemorrhage in 2. The results of FFA discovered orange-red spotty fluorescence in 6 eyes and choroidal vascular network in 4 eyes. At the late phase, leakage of polypoidal hyperfluorescence spot in all of the eyes except 2 without leakage were found. The images of ICGA showed typical dotted or clustered polypoidal hyperfluorescence in 12 eyes at the late phase. OCT disclosed protrusion of the retinal pigment epitelium (RPE) with a bumpy surface at polypoidal structure in 4 eyes and no change in 1 eye. Conclusions PCV mainly affects the elderly persons and mostly on unilateral eyes. Macular hemorrhage, serous RPED, and (or) neuroepithelial detachment with yellow hard exudations are the main manifestations. Branching choroidal vascular net with ployplike terminal anourysmal dilations can be discovered in FFA and ICGA.