ABSTRACT
Background The incidence of retinal vascular diseases increase annually,such as diabetic retinopathy,retinopathy of prematurity and age-related macular degeneration.The key of treatment for these diseases is how to evaluate retinal vascular change effectively and objectively.Retro-orbital injection of fluorescein isothiocyanatedextran (FITC-dextran) is a simple and effective method for observing C57BL/6J mouse retinal vessels.But,whether it is suitable for other mice and rats is seldom reported.Objective This experiment was to assess the feasibility of the observation of retinal vessels by retro-orbital injection of FITC-dextran in different genus of mouse and offer the reference for relevant study.Methods Twelve animals of C57BL/6J mice,Kunming mice,SD rats and Wistar rats were selected,respectively and divided into the experimental group and control group at average.The right eyes of the animals of the experimental group received the retro-orbital injection of 9 ml/kg FITC-dextran,and the right eyes of animals of the control group received PBS solution at the same volume and way.All the animals were sacrificed 10 seconds after injection and both eyes of each animal were obtained for retinal stretched preparation.The retrobulbar tissue and whole-mount retina were viewed under a fluorescence microscope.The use of the animals complied with Regulation for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results Retinal blood vessels labeled by FITC-dextran could be observed in both eyes of C57BL/6J mice and Kunming mice to present with a green fluorescence in experimental group under a fluorescence microscope,but no any fluorescence-labeled retinal blood vessel was exhibited in the control mice.The retinal blood vessel could not be observed in all eyes of SD rats and Wistar rats after the injection of FITC-dextran both in the experimental group and the control group under a fluorescence microscope.The surrounding tissues of the right eyes of mice and rats dyed with green fluorescence of FITC-dextran in the experimental group,however,green fluorescence could not be seen in the surrounding tissues of the left eyes of mice and rats.Conclusions Retro-orbital injection of FITC-dextran is a suitable method of observing the retinal vessels of mouse but not rat.
ABSTRACT
Background To optimize the culture method of human retinal microvascular endothelial cells is very important for the study of retinal angiogenesis disease.Human retinal microvascular endothelial cells have been successfully cultured in previous studies,but further improvement of the culture method to harvest higher yields and purity cells is still needed.Objective This study was to design a modified method to isolate and purify human retinal microvascular endothelial cells much easily and quickly,and to compare the expression of specific markers of vascular endothelial cells,factor Ⅷ and CD31/CD34 in the cells.Methods The use of human donor eyeballs was approved by the Ethic Commission of Zhongshan Ophthalmic Center of Sun Yat-sen University.The retina tissue from healthy donor was isolated and digested by the two-step digestion method with 2％ trypsin and 0.133％ collagenase Ⅳ.Human retinal microvascular endothelial cells were collected and plated in 60 mm dishes coated by 0.1％ fibronectin and cultured in endothelial cell-specialized medium supplemented with 10％ fetal bovine serum,0.3 mg/L β-endothelial cell growth factor (ECGF) and 100 ng/L sodium heparin.During the culturing,the growth situation of the cells was monitored by morphological observation,and immunohistochemical staining was performed to probe vascular endothelial cell-specific membrane protein CD31,CD34 and factor Ⅷ for identification of the cell purity.Results Human retinal microvascular endothelial cells were isolated successfully from the retina by the twostep digestion method.The primary cultured cells adhered to well 72 hours later and achieved confluence with the typical cobblestone appearance 9 to 10 days after cultured.The cells exhibited the blue nuclei and reddish cytoplasm by regular haematoxylin and eosin stain and showed a strong positive response for CD31,CD34 and factor Ⅷ by immunohistochemistry.The positive dye of CD31 and CD34 was lower than Ⅷ factor in both endothelial cells.Conclusions Modified culture method of human retinal microvascular endothelial cells can improve cell culture result and purify target cells.
ABSTRACT
Background Retinopathy of prematurity (ROP) threats the visual function of prematurity patients.The screen of ROP is very important for the early prevention and treatment.Some relevant works have performed in several region and cities in China.But the survey data is still lack in Shandong province.Objective This work was to survey and analyze the screening result of ROP in Jinan city and assess the risk factors for ROP.Methods A cross-sectional study was designed.Two hundreds and eighty-eight eyes of 144 cases with low birth weight or prematurity were clinically examined in Shandong Eye Hospital from June,2008 to June,2010.The screening criteria was established based on the ROP screening guidelines and the outcome implemented by Ministry of Public Health with minor modification,including newborn infants with birth weight of 2500 grams or less and gestational age of 34 weeks or less.The history and systemic status were recorded,and the screening results were compared with those obtained from the national screening criteria.Gestation age and birth weight between the infants with and without ROP were compared using student t-test.The ratios of persistent oxygen inhalation were tested by Fisher' s exact test.Results In all examined infants,30 eyes of 15 cases (10.42％) developed ROP,including 12 eyes of 6 cases suffering from stage Ⅰ,8 eyes of 4 cases from stage Ⅱ,8 eyes of 4 cases from stage Ⅲ with plus disease and 2 eyes of 1 case from stage V.In ROP patients,gestation age ranged from 27 weeks to 33 weeks with the average weeks of 30.85±1.79,and the birth weight ranged from 910 grams to 1980 grams with the average weight (1408.89±259.93)grams.Statistical analysis showed that the gestation age and birth weight in prematurity infants with normal fundus group were (32.38±1.48) weeks ranged from 28 weeks to 35 weeks and (1763.19±338.62) grams ranged from 1310 grams to 2450 grams respectively,showing a shorter gestation age (t=3.71,P＜0.01 ) and a lower birth weight ( t =2.88,P＜0.01 ) in ROP infants.The ratios of persistent oxygen inhalation in normal fundus group and ROP group were 58.91％ and 60.00％,respectively with the insignificant difference between them (P =1.000).Conclusions The incidence of ROP is 10.42％ in the present study,which is consistent with the result from other region in China,suggesting that the national criteria is practical for the ROP screening in Jinan region.Prematurity and low birth weight are identified as the high risk factors of ROP.