AB025.Machine learning applied to the oxygen induced retinopathy model

Background:The oxygen induced retinopathy rodent model is widely used,notably for the assessment of developmental dystrophies in preclinical studies of vascular retinal diseases.Typically,the quantification of vessel tufts and avascular regions is computed manually from flat mounted retinas imaged using fluorescent probes that highlight the vascular network.However,such manual measurements are time-consuming and hampered by user variability and bias,thus a rapid and objective alternative is required.Methods:We employ a machine learning approach to segment and characterize vascular tufts.The proposed quantitative retinal vascular assessment(QuRVA)technique uses quadratic discrimination analysis and morphological techniques to provide reliable measurements of vascular density and pathological vascular tuft regions,devoid of user intervention within seconds.Our algorithms allow also delineating the whole vasculature network,and identifying and analyzing avascular regions.Results:Our first experiment shows the high degree of error and variability of manual segmentations.In consequence,we developed a set of algorithms to perform this task automatically.We benchmark and validate the results of our analysis pipeline using the consensus of several manually curated segmentations using commonly used computer tools.We describe the method,provide details for reproducing the algorithm,and validate all aspects of the analysis.Conclusions:Manual and semi-automated procedures for tuft detection present strong fluctuations among users,demonstrating the need for fast and unbiased tools in this highly active research field with tremendous implications for basic research and industry.

The protective effect of 17 beta-estradiol on oxygen-induced retinopathy and its relation with the changes of malondialdehyde

Objective： Retinopathy of prematurity is becoming obvious with the improvement of neonatal ambulance. However there is still not a good treatment. The present study is to observe the effect of 17 beta-estradiol （E2） on oxygen-induced retinopathy （OIR）, and explore the relationship between the changes of avascular area and malondialdehyde （MDA） in retina. Methods： Newborn oxygen-exposed mice underwent subcutaneous injections of different dose of E2 （0.1 μg, 1.0 μg, 10.0 μg ）, tamoxifen or phosphate buffered saline （PBS; controls）everyday from post-natal day （p）7 to p17. At p17, retinal flat mounts were scored for the percentage of avascular/total retinal area, and pathological changes during revascularization. The MDA concentration in the retina was determined also. In the most efficacious E2 group （10.0 μg）, 100.0 μg tamoxifen was also administered, and the percentage of capillary-free/total retinal area determined, and the retinal malondialdehyde concentration assayed. Results： The mean percentage of capillary-free area over total retinal area was 0（PBS, in room air）, 34.197±1.301（PBS, in hyperoxia）, 23.685±0.407 （0.1 μg E2）, 14.648±0.355 （1.0 μg E2）, 4.693±0.450 （10.0 μg E2） and 32.240±0.654 （10.0 μg E2 ＋100.0 μg tamoxifen）. The difference was significant （F = 2778.759, P 〈 0.01）, and the difference between any two groups were also significant （all P value were less than 0.01）. The predilection of tufts and clusters during revascularization was mainly aggregated in zones 2 and 3, but the difference of retinal neovascular clusters and tufts in fourth zone among different groups were significant [clusters （F = 44.719, P 〈 0.01） vs tufts （F = 39.997,P 〈 0.01）]. The mean MDA concentration were 0.711 ±0.037（PBS, in room air）, 2.084±0.066 （PBS, in hyperoxia）, 1.829±0.091（0.1 μg E2）, 1.152± 0.067（1.0 μg E2）, 0.796 ±0.027（10.0 μg E2）, 1.988 ± 0.049（10.0μg E2 ＋100.0 μg tamoxifen） （F = 628.103, P 〈 0.01）. The difference between any two groups were also significant （all P value were less than 0.05）. The close relation between the percentage of avascular/total retinal area and MDA concentration was also verified （r = 0.981, P 〈 0.01）. Conclusion： Oxidative stress responses play a pivotal role in OIR, by means of receptor pathway. E2 can alleviate oxidative stress reaction, and thus ameliorate the severity of oxygen induced retinopathy.

Effect of endothelial progenitor cells derived from human umbilical cord blood on oxygen-induced retinopathy in mice by intravitreal transplantation

AIM： To investigate the effect of endothelial progenitor cells（EPCs） labeled by carboxy fluorescein diacetate succinimidyl ester（CFSE） on murine oxygen-induced retinopathy（OIR） by intravitreal transplantation.METHODS： After isolated from human umbilical cord blood mononuclear cells, EPCs were cultivated and then labeled with CFSE in vitro. C57BL/6J mice were placed to75% hyperoxia chamber from P7 to P12 to establish OIR model. At P12, OIR mice were intravitreally injected with1 μL suspension contained 2×105EPCs（EPCs group） or isometric phosphate buffered saline（PBS group）. The contralateral eye of each mice received no injection（OIR group）. Evans blue angiography and frozen section were examined to track the labeled cells in OIR group at P15 and P19. Using retina paraffin sections and adenosinediphos phatase staining at P12 and P19, the effect of EPCs on OIR mice was evaluated quantitatively and qualitatively. RESULTS： The retinas from EPCs group with less non-perfusion area and fewer peripheral tufts wereobserved at P19, comparing with that from PBS or OIR group. The retinopathy in EPCs group receded earlier with less non-ganglion cells and neovascular nuclei,together with relatively regular distribution. The counts of the neovascular nuclei at P19 were reduced by 44% or45%, compared with those of OIR group or PBS group respectively. Three days after EPCs injection, a large number of EPCs appeared in the vitreous cavity and adhered to the retinal surface. While at one week, the cells gathered between the internal plexiform layer and the inner limiting membrane, and some EPCs appeared in retinal vessels.CONCLUSION： EPCs transplantation can participate in the reparative procedure of the neovascularization in OIR.

Inhibition of retinal angiogenesis by gold nanoparticles via inducing autophagy

AIM： To investigate the effect of gold nanoparticles on retinal angiogenesis in vitro and in vivo, and to reveal the possible mechanism.METHODS： Seed growth method was used to synthesize gold nanoparticles（GNPs）. The size, zeta potential, absorption spectrum and morphology of GNPs were identified using Malvern Nano-ZS, multimode reader（Bio Tek synergy2） and transmission electron microscope. Cell viability was analyzed using cell counting kit-8 method and cell growth was assessed with EdU kit. Transwell chamber was used to investigate cell migration. Tube formation method was used to assess the angiogenic property in vitro. Oxygen induced retinopathy（OIR） model was used to investigate the effect of GNPs on retinal angiogenesis. Confocal microscope and Western blot were used to study the possible mechanism of GNPs inhibited angiogenesis.RESULTS： The GNPs synthesized were uniform and well dispersed. GNPs of 10 μg/mL and 20 μg/mL were able to inhibit human umbilical vein endothelial cells proliferation（50% and 72% separately, P〈0.001）, migration（54% and 83% separately, P〈0.001） and tube formation（52% and 90% separately, P〈0.001）. Further data showed that GNPs were able to improve the retinopathy in an OIR model. The possible mechanism might be that GNPs were able to induce autophagy significantly（P〈0.05）.CONCLUSION： The present study suggests that GNPs are able to inhibit retinal neovascularization in vitro and in vivo. GNPs might be a potential nanomedicine for the treatment of retinal angiogenesis.