Background 23G pars plana vitrectomy has been widely applied to treat diabetic retinopathy (OR).Researching the influence of 23G pars plana vitrectomy on corneal endothelium cell has a great clinical significance.Objective To observe the influence of 23G pars plana vitrectomy on corneal endothelial cells in phakic eyes of diabetes and non-diabetes patients.Methods A retrospective study was designed.One hundred and twenty-four eyes of 124 patients with vitreoretinopathy were included in Affiliated Hospital of Qingdao University from August 2012 to June 2013.The patients were assigned to DR group (52 eyes) and non-DR group (72 eyes).23G pars plana vitrectomy was performed on all the patients under their informed consent.Endothelial cell density,corneal thickness(CT),coefficient of variation (CV) of cellular area,standard deviation (SD) of average cellular area and percentage of hexagonal endothelial cells were measured before and 1 day,3 days,1 week,2 week,1 month and 3 months after surgery with Topcon SP-3000P corneal specular microscope.Results No significant differences were found in the central corneal endothelial cell density between the DR group and non-DR group at various time points (Fgroup =2.148,P=0.150;Ftime =0.900,P=0.504).The CV of endothelial cells,SD of endothelial cellular area and CT in the first day after surgery were higher than preoperation (P =0.000,0.011,0.033),while the percentage of hexagonal endothelial cells was declined (P =0.001).The CV of endothelial cells and the percentage of hexagonal endothelial cells recovered in postoperative 1 month in the DR group.In the non-DR group,the CV of endothelial cells and CT elevated in postoperative 1 day in comparison with preoperation (P =0.002,0.003),and the percentage of hexagonal endothelial cells reduced (P =0.000).These abnormalities returned to a preoperative level in a week after surgery.Conclusions 23G pars plana vitrectomy results in a reversible morphology damage of corneal endothelial cells.These damage may be more severe with a longer duration in DR patients compared with non-DR patients.

Objective To observe the changes of retinal morphology and function of macular-off rhegmatogenous retinal detachment (RRD) after scleral bulking.Methods In this prospective study,42 eyes of 41 patients who underwent scleral bulking were enrolled.There were 26 males (27 eyes) and 15 females (15 eyes),with an average age of (33.78± 11.21) years.Best corrected visual acuity (BCVA),intraocular pressure,indirect ophthalmoscope,visual fields,optical coherence tomography (OCT) and B scan of ocular ultrasound were measured for all patients.The average BCVA was 0.29±0.18.The retinal detachment time was (21.12±3.71) days.The mean visual field defect (MD) was (13.54±6.44) dB.The mean loss variance (LV) was (8.43±2.11) dB.All the patients were performed cryotherapy and sub-choroidal fluid drain out.The mean follow up was 12.4 months (from 6 to 24 months).At two weeks,1,3,6,12 months after surgery,the changes of BCVA,visual fields,retinal morphology and subretinal fluid were observed.Results Indirect ophthalmoscope combined with B scan showed the time of retinal reattachment was (7.32±2.53) days.Subretinal fluid was found completely absorbed by OCT with a mean of (7.82±3.52) months.At 12 months after surgery,subretinal fluid was completely absorbed in 37 eyes (88.10％).In these 37 eyes,15 eyes had normal retinal microstructure,5 eyes had neuroepithelial cystoid edema; 12 eyes had disrupted inner segment/outer segment (IS/OS) junction,and 5 eyes had disrupted IS/ OS and external limiting membrane (ELM).BCVA at 6 months after surgery was no significant difference with that at 12 months after surgery (t=-0.636,P=0.529).At 12 months after surgery,there were4 retinal patterns on OCT examination,including normal retinal microstructure,neuroepithelial cystoid edema,IS/OS line disruption,and IS/OS and ELM disruption.The BCVA difference among these 4 groups was significant (F =52.42,P ＜ 0.05).The BCVA difference between eyes with or without residual subretinal fluid was significant (t=-5.747,P=0.000).At 1,2 weeks and 1,3,6,12 months after surgery,the MDwere (11.38±2.53),(10.14±2.19),(9.17±2.13),(6.63±1.70),(5.71±1.89),(5.14± 1.69) dB respectively,with a significant difference between these time-points (F=63.528,P =0.00).However,the MD at 6 months after surgery was no significant difference with that at 12 months after surgery (t=1.442,P=0.157).At 12 months after surgery,there were 12 eyes with normal MD,30eyes with higher MD.There was no significant difference between surgery eyes with higher MD and fellow eyes in MD (t =-1.936,P =0.06).The MD value was positively correlated to the time of retinal detachment in patients with normal retinal microstructure (r=0.84,P =0.00).There were differences in LV during different periods after surgery (F=57.25,P =0.00).Conclusions The retinal microstructure,visual acuity,visual fields were gradually improved after scleral bulking.The patients had better vision with normal retinal microstructure.The time of retinal detachment positively correlated with visual fields damage.

ObjectiveTo investigate the expression of hypoxia inducible factor 1 (HIF-1α) in rats' retinae during the embryonic and earlier postnatal period.MethodsThe retinal expression patterns of HIF-1α protein and mRNA of embryonic day 12 (E12), E16, E20, and postnatal day 1(P1) and P5 rats were determined by immunohistochemical staining and reverse transcription polymerase chain reaction (RT-PCR).ResultsHIF-1α protein was detected in the neural epithelial layer and the pigment epithelial layer at all those 5 timepoints, with higher expression in the ganglion cell layer and the inner plexiform layer, and seems limited to the ganglion cell layer when retina became more mature. Embryonic rat retina had higher expression of HIF-1α protein and mRNA than postnatal retina, the difference was significant (P<0. 01). ConclusionThe expression of HIF-1α in rats' retinae differs from embryonic to earlier postnatal stages.

BACKGROUND: Hypoxia inducible factor-1 α(HIF-1 α) is not only related to physiological reaction of hypoxia,but also takes part in normal embryonic development.OBJECTIVE: To study the alteration of HIF- 1 α in retina during rat development.DESIGN,TIME AND SETTING: Randomized contrast animal study,which was performed in the Shandong Provincial Molecular Virus Key Laboratory,Medical College of Qingdao University between January and September 2007.MATERIALS: Adult Wistar rats,nulliparity,clean grade,and weighing 200 250 g were used in this study.METHODS: Male and female rats were caged as the ratio of 1 : 1.Embryos were obtained at 12-day,16-day,and 20-day pregnancy.Eyeballs were obtained from newborn rats by anesthesia at 1-day,5-day,10-day,and 12-month birth.Retina was separated and made into paraffin section.MAIN OUTCOME MEASURES: Expressions of HIF 1 α protein and HIF-1 α mRNA in retina were measured by immunohistochemistry and semiquantitative reverse transcription-polymerase chain reaction at various dine points of embryonic development.RESULTS: HIF-1 α positively expressed at stratum neuroepitheliale retinae and purpurogenous membrane in the embryonic phase.Additionally,HIF-1 α still positively expressed at stratum neuroepitheliale retinae and purpurogenous membrane,especially in ganglionic cells and inner plexiform layer,in early development.With the gradual development,the positive expression was mainly located at stratum ganglionare retinae.HIF-1 α protein and mRNA expressions were the highest in the embryonic phase,lower in the development,and the lowest in the adult period.There were significantly differences among these three phases (P ＜ 0.01).CONCLUSION: HIF-1 α decreases gradually in retina and its expression is mainly located at stratum ganglionare retinae.

Objective o observe the expression of Notch1 and Delta-like ligand 4 (Dll4) on the fibrovascular membranes in proliferative diabetic retinopathy (PDR),and investigate its relationship with vascular endothelial growth factor receptor 2 (VEGFR2).Methods Fifty-seven PDR patients (60 eyes) who underwent vitrectomy were enrolled in this study.The PDR patients were divided into non-injection group (30 patients,32 eyes) and injection group (27 patients,28 eyes).The eyes in injection group received intravitreal injection with ranibizumab at 2 to 7 days before surgery.The preretinal fibrovascular membranes were obtained from the PDR patients during vitrectomy.Eighteen epiretinal membranes were obtained from the non-diabetic patients was served as controls.The real-time polymerase chain reaction (RT-PCR) and immunohistochemical methods were used to detecting the expression ofNotch1,Dll4 and VEGFR2.In the meantime,the numbers of the nucleus of vascular endothelial cells in the membranes stained with hematoxylin were counted.Results The immunohistochemical staining revealed that there were positive expression ofNotch1,Dll4 and VEGFR2 in all PDR membranes,regardless of the injection of the ranibizumab.The levels ofNotch1,Dll4 and VEGFR2 protein in non-injection group were higher than those of injection group (t=3.45,6.01,4.08;P=0.030,0.008,0.023).In injection group,the number of endothelial cells in the membranes reduced (17.17 ± 2.48) compared with that of the non-injection group (41.50± 5.57).There was significant difference in the number of endothelial cells in the membranes between the two groups (t=9.58,P＜0.05).RT-PCR showed that the differences of the mRNA expression ofNotch1,Dll4 and VEGFR2 were all statistically significant among the PDR group and control group (H=12.50,12.50,12.02;P＜0.05).The expression ofNotch1,Dll4 and VEGFR2 in the PDR membranes was higher than that of epiretinal membranes from non-diabetic patients.In the PDR group,the expression of Notch1,Dll4 and VEGFR2 of non-injection group was higher than that of injection group.Spearman correlation analysis showed that the expression of mRNA between VEGFR2 and Dll4 (r=0.83),VEGFR2 and Notch1 (r=0.81),Notch1 and Dll4 (r=0.87) were all significantly correlated (P＜0.05).Conclusions The expression of Notch1 and Dll4 in the PDR membranes are higher than that of the control group,and it is positively correlated with the expression of the VEGFR2.Notchl and Dll4 play a regulatory rule in the neovascularization in PDR,the acting way may be correlated with VEGFR2.

Objective To observe the efficacy of intravitreal injection of ranibizumab (IVR) for different patterns of optical coherence tomography (OCT) of diabetic macular edema and the relationship between integrity of ellipsoidal zone and visual acuity outcomes.Methods Eighty-five IVR treated eyes were enrolled.The examination of BCVA was according to Early Treatment Diabetic Retinopathy Study,and the results were recorded as logarithm of the minimum angle of resolution (logMAR).Frequency-domain OCT was used to measure the central foveal thickness (CFT) and the integrity of ellipsoidal zone.All eyes were classified as diffuse macular edema (DRT group,31 eyes),cystoid macular edema (CME group,29 eyes),and serous retinal detachment (SRD group,25 eyes).All the patients were treated with intravitreal injection of 0.05 ml (0.5 mg) ranibizumab.The mean follow-up time was (9.21+3.56) months after IVR treatment.The changes of BCVA and CFT in 3 groups were compared and analyzed after 3,6 and 12 months.According to visual acuity at different ranges,the relationship between integrity of ellipsoidal zone and BCVA was analyzed.Results Compared with the average logMAR BCVA before treatment,except for 12 months after treatment in group SRD (t=2.104,P=0.053),the average logMAR BCVA after IVR at 3 months,6 months and 12 months improved in DRT group (t=7.847,6.771,6.426;P=0.000,0.000,0.000),CME group (t=8.560,6.680,5.082;P=0.000,0.000,0.000) and SRD group (t=5.161,3.968,2.104;P=0.000,0.001,0.053).The average logMAR BCVA of the DRT group was lesser than that in CME and SRD group after 12 months treatment (t=-2.043,-3.434;P=0.030,0.001).The average CFT after IVR at 3 months,6 months and 12 months reduced significantly in DRT group (t=12.746,10.687,9.425;P=0.000,0.000,0.000),CME group (t=13.400,11.460,10.169;P=0.000,0.000,0.000),and SRD group (t=1 1.755,10.100,9.173;P=0.000,0.000,0.000).After 12 months of treatment,the average CFT of the SRD group was thicker than that in DRT group and CME group (t=-3.251,-1.227;P=0.003,0.025);there was significant difference in the integrity of ellipsoidal zone among 3 groups (x2=1.267,P=0.531).The results showed that there were significant differences in the integrity of ellipsoidal zone with different ranges of BCVA before and after 12 months treatment (x2=20.145,41.035;P=0.000,0.000).Conclusions IVR could significantly improve the visual acuity of different patterns of DME,reduced the CFT,and had the best efficacy in the DRT group.There was relationship between the integrity of ellipsoidal zone and the visual acuity outcomes.

Objective To observe the effect of different concentration netrin-1 on retinal vascular permeability in diabetes mellitus (DM) rats.Methods Eighty adult Sprague-Dawley rats were randomly divided into 8 groups,10 rats in each group,including normal control group (group A),normal+balanced salt solution (BSS) group (group B),normal+netrin-1 (500 μg/ml) group (group C) and DM group (50 rats in 5 subgroups).DM rats were induced by intraperitoneal injection of streptozocin.Three months after intraperitoneal injection,10 DM rats in the control group were injected with BSS (group D).Forty DM rats were injected with 5 μl of different concentrate netrin-1,and were divided into DM+netrin-1 10 μg/ml group (group E),DM+netrin-1 50 μg/ml group (group F),DM+netrin-1 100 μg/ml group (group G),DM+netrin-1 500 μg/ml group (group H)according to the different concentration.Non-DM rats in group C were injected with netrin-1 500 μg/ml.The expression of occludin was determined by immunohistochemistry for protein,and by real-time fluorescence quantitative reverse transcription polymerase chain reaction for mRNA level.Retinal vascular permeability was measured by Evans blue infusion.Results The expression of occludin protein and mRNA in group D were less than group A (t=27.71,8.59;P=0.00,0.00).However,the retinal vascular permeability increased in group D (t=-42.72,P=0.00).The expression of occluding protein,occludin mRNA and retinal vascular permeability showed significant differences between group D,E,F,G and H (F=146.31,16.54,67.77;P=0.00,0.00,0.00).Compared the group B with group C,there was no significant differences between the expression of occludin protein,occludin mRNA and the retinal vascular permeability (t=-1.13,0.93,1.04;P=0.27,0.36,0.31).The concentrate of netrin-1 showed a significant positive correlation to the expression level of occludin and occludin mRNA (r=0.73,0.81;P=0.00,0.00),but negative correlation to the vascular permeability (r=-0.61,P=0.00).Conclusion Netrin-1 can reduce the DM rats' retinal vascular permeability,which depended on the concentration of netrin-1.

BACKGROUND:Studies have found that erythropoietin has a protective effect on embryonic development of the retina, but there are rare studies concerning the embryonic development of the lens. OBJECTIVE: To observe the difference in erythropoietin expression in the lens from Wistar rats at different embryonic periods and to investigate the effect and significance of erythropoietin in the embryonic development of the lens. METHODS:Clean Wistar rats with pregnancy for 10 days (n=5), 12 days (n=5), 14 days (n=5), 16 days (n=5), 18 days (n=5), 20 days (n=5) were randomly colected and divided into six groups. Every two embryonic ratsof the different 30 pregnant rats were obtained randomly by the caesarean operation under ketamine-induced anesthesia. The eye tissues of al the embryonic rats were isolated and cut into sections. The expression of erythropoietin protein and mRNA in rat lens was detected by immunohistochemistry and reverse t ranscription-PCR, respectively. RESULTS AND CONCLUSION:Erythropoietin was distinctly expressed at the six different embryo stages, and the expression of erythropoietin protein and mRNA gradualy increased from embryonic day 10 to embryonic day 16, and decreased from embryonic day 10 to embryonic day 20. There were significant differences between the six groups. These findings indicate that the expression of embryonic appears in a low to high to low fashion during the embryonic development of Wistar rats, which may be closely associated with the developing procedure of lens.

Diabetic retinopathy is a serious complication of diabetes and is the leading cause of blindness in people with diabetes.At present,there are many views on the pathogenesis of diabetic retinopathy,including the changes of retinal microenvironment caused by high glucose,the formation of advanced glycation end products,oxidative stress injury,inflammatory reaction and angiogenesis factor.These mechanisms produce a common pathway that leads to retinal degeneration and microvascular injury in the retina.In recent years,cell regeneration therapy plays an increasingly important role in the process of repairing diseases.Different types of stem cells have neurological and vascular protection for the retina,but the focus of the target is different.It has been reported that stem cells can regulate the retinal microenvironment and protect the retinal nerve cells by paracrine production,and can also reduce immune damage through potential immunoregulation,and can also differentiate into damaged cells by regenerative function.Combined with the above characteristics,stem cells show the potential for the repair of diabetic retinopathy,this stem cell-based regenerative therapy for clinical application provides a pre-based evident.However,in the process of stem cell transplantation,homogeneity of stem cells,cell delivery,effective homing and transplantation to damaged tissue is still a problem of cell therapy.

Objective To observe the effect of netrin-1 on retinal Müller cells in diabetes mellitus (DM) rats. Methods Fifty Sprague-Dawley rats were randomly divided into the normal control group (group A), normal + balanced salt solution (BSS) group (group B), normal+netrin-1 group (group C), DM+BSS group (group D) and DM+netrin-1 group (group E), with 10 rats in each group. DM rats were induced by intraperitoneal injection of Streptozotocin (60 mg/kg). The expression level of glial fibrillary acidic protein (GFAP) on retinal Müller cells was determined by immunohistochemistry, the level of GFAP mRNA was analyzed by real-time fluorescence quantitative reverse transcription polymerase chain reaction. Results Immunohistochemistry showed that GFAP was distributed in retinal ganglion cells and retinal nerve fiber layer in group A, B and C. Compared to group B, GFAP staining was brighter in the group D. There were significant differences in the expression of GFAP protein and mRNA among groups A-E (F=203.43, 72.91; P=0.00, 0.00), they were higher in group D than group A (t=-26.01, 22.26; P=0.00, 0.00), and group E (t=-10.78, 3.93; P=0.00, 0.00). They were higher in group E than group A (t=7.00, -9.82; P=0.00,0.00). There were no significant differences in between group A and group C (t=-0.29, 0.50; P=0.77, 0.62). Conclusion The expression of GFAP in Müller cells of DM rats could be decreased by injecting netrin-1 into vitreous.