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Objective:To investigate the molecular expression and pathological features of endothelial cell (EC) in a murine model of choroidal neovascularization (CNV) based on single-cell RNA sequencing (scRNA-seq).Methods:Six C57BL/6 mice aged 6-8 weeks were randomly divided into two groups, with 3 mice in each group.Bilateral eyeballs were enucleated.The choroidal tissues from the two groups were isolated by shearing the complex and scraping the choroid, respectively.Single-cell suspension was prepared by continuous digestion with trypsin/type Ⅰ collagenase at 37 ℃, and the cell viability and EC ratio were detected by flow cytometry to determine the preparation method of single-cell suspension.Another 6 mice were randomly assigned into the control group and the CNV group, with 3 mice in each group.The CNV model was induced by laser photocoagulation and single-cell suspensions were prepared 7 days after modeling.Gene expression library construction was performed using the Chromi-um (10x Genomics) instrument.High throughput sequencing was performed using the Illumina Novaseq6000 to obtain the expression matrix.The EC subpopulations were classified according to previous researches and the Cellmarker database.Pseudo-time analysis was performed in EC, revealing the gene expression matrix of different states.CNV-EC were further selected with preliminary analysis of the expression characteristics.Another 6 mice were selected to establish the CNV model and eyeball frozen sections were prepared 7 days after modeling.Expression and distribution as well as the area percentage of EC marker Pecam1, mitochondrial outer membrane proteins Tomm20 and mt-Co1, and capillary markers Kdr and Plvap were observed by immunofluorescence staining, and the vascular diameter was calculated.The use and care of animals followed the ARVO statement.This study protocol was approved by the Experimental Animal Welfare and Ethics Committee of Air Force Military Medical University (No.20200181).Results:The cell viability of the single-cell suspension prepared from choroidal-scleral fragments and choroidal scrapings was 99.4% and 99.1%, respectively, both of which met the sequencing requirements.The percentage of EC detected by flow cytometry was approximately 1.58%.The scRNA-seq result revealed that both the normal control and CNV groups contained 13 choroidal cell clusters.Compared with the normal control group, the proportions of rod/cone photoreceptor cells, EC and hematopoietic cells all increased, while the retinal pigment epithelium (RPE) and Schwan cells reduced in the CNV group.Among all clusters, EC constituted 18.4%.The pseudo-time analysis demonstrated that EC could be further divided into 4 states.The percentage of state 2 EC was 29.1% in the CNV group, which was significantly higher than 9.5% in the normal control group.Differentially expressed gene analysis showed that the expression of mitochondrion-related genes, including mt-Nd4 and mt-Atp6, were upregulated in state 2 EC, while capillary-related genes, including Kdr and Esm1, were downregulated.Immunofluorescent staining revealed that the area of Tomm20 and mt-Co1 in Pecam1-positive EC in the CNV area was (19.50±4.68)% and (4.64±2.82)%, respectively, which were both higher than (3.00±2.09)% and (0.18±0.34)% in normal area ( t=7.88, 3.84; both at P<0.01). The area of Kdr and Plvap in Pecam1-positive EC in the CNV area was (1.50±0.29)% and (0.79±0.97)%, respectively, which were both lower than (31.30±5.44)% and (10.43±2.28)% in the normal area ( t=13.40, 9.48; both at P<0.01). The vascular diameter in the CNV area was (5.52±1.85)μm, which was larger than (4.21±1.84)μm in the normal area ( t=9.57, P<0.001). Conclusions:When CNV occurs, the proportion of EC in choroid increases, and CNV-EC shows pathologic features of mitochondrial metabolic activation and loss of capillary properties, suggesting the mitochondrial activation of EC may play a role in the formation of CNV.
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Objective:To investigate the fundus fluorescein angiography (FFA) characteristics of spontaneous regression in retinopathy of prematurity (ROP) and the range of retinal vascularization.Methods:A clinical retrospective study. A total of 82 eyes of 41 infants with ROP, who underwent FFA from January 2019 to December 2021 in Department of Ophthalmology of Xijing Hospital after completion of ROP regression, were included. There were 25 males (50 eyes) and 16 females (32 eyes). ROP was diagnosed in Zone Ⅱ in 44 eyes, with 38 eyes in stage 2 and 6 eyes in stage 3, and in zone Ⅲ in 38 eyes of stage 2. All patients underwent FFA examination under general anesthesia, at postmenstrual age of 70.70±12.25 weeks, after the natural regression of ROP was completed. Focus on the retinal vascular development, as well as choroid circulation and macular abnormalities, and compare and observe the differences between zone Ⅱ and Ⅲ after spontaneous regression. The extent of retinal vascularization was determined by the ratio between the distance of the center of the disc to the border of the vascularized zone (DB) and the center of the disc to the fovea distance (DF). The width of avascular area, recorded as the distance from the ora serrata to the vascular termination, was counted by disc diameters (DD). The measurement data between zone Ⅱ and zone Ⅲ ROP were compared by the independent sample t-test, and the count data were compared by χ2 test or Fisher exact probability test. Results:The linear choroidal pattern was present in 9 eyes (21.95%, 9/41), and the tortuous arteries in the posterior retina were detected in 32 eyes (39.02%, 32/82). It was noted that increased branching of vessels presented in 45 eyes (54.88%, 45/82), straight shape of vessels shown in 27 eyes (32.93%, 27/82), circumferential vessels arisen in 45 eyes (54.88%, 45/82), arteriovenous shunt appeared in 18 eyes (21.95%, 18/82), and capillary bed lost in 46 eyes (56.10%, 46/82) in areas from initial ridge to vascular termini. Punctate or linear dye leakage was observed in 23 eyes (28.05%, 23/82) during the late phase of FFA. Macular abnormalities, such as the absence of foveal avascular zone and hypoperfusion, were observed in 28 eyes (34.15%, 28/82), of which the macular ectopia presented in 1 eye. The mean DB/DF ratio of all the 82 eyes on the temporal side was 4.63±0.29 and 3.77±0.23 in the nasal. The mean avascular area on the temporal retina was 1.74±1.00 DD. Compared with ROP in zone Ⅲ, increased branching of vessels and dye leakage were more common ( χ2=9.303, 10.774; P=0.002, 0.001), the extent of temporal retinal vascularization was smaller ( t=-2.285, P=0.026), and the avascular area of the retina was more significant ( t=5.491, P<0.001) in zone Ⅱ ROP. Conclusions:Even after completion of spontaneous regression in ROP, incomplete retinal vascularization and vascular abnormalities may exist permanently in FFA, including those such as the tortuous arteries in the posterior retina, increased branching and straight shape of vessels, circumferential vessels, capillary bed lost and macular abnormality. Further appropriate follow-up is needed for long-term safety.
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Objective:To investigate the ocular clinical manifestations in pediatric patients with incontinentia pigmenti (IP).Methods:A case series study was carried out and a retrospective analysis was performed.Clinical data of 13 pediatric patients with IP treated from January 2013 to December 2019 in Xijing Hospital were collected.All the patients underwent regular ophthalmologic examination.Three patients accepted fundus fluorescein angiography and six eyes of five patients were treated with retinal photocoagulation or anti-vascular endothelial growth factor (VEGF) intravitreal injection according to severity of the condition.The follow-up period ranged from 6 months to 6 years.The medical history, family history, systemic manifestations, ocular characteristics, diagnosis, treatment as well as ocular and systemic changes during follow-up were recorded and analyzed.This study followed the Declaration of Helsinki and the study protocol was approved by the Ethics Committee of Xijing Hospital, Fourth Military Medical University (No.KY20203287-1).Results:All the 13 patients were female aged from 5 days to 42 months at first visit, with the average age of 2.0 (1.0, 8.5) months.As for the main skin lesions at first visit, there were 4 cases in erythematous vesicle stage, 3 cases in verrucous exanthema stage, and 6 cases in hyperpigmented stage.There were 7 cases in shrinkage stage during follow-up.Among the 26 eyes of 13 patients, 18 eyes of 10 patients showed ocular anomalies, accounting for 76.9% of total patients (69.2% of total eyes). Among the 13 patients, 8 patients presented bilateral ocular involvement, 2 patients showed unilateral anomalies, and 3 patients had no ocular lesions.The retina was involved in all patients with ocular manifestations.The typical retinal lesions included avascular zone of peripheral retina in 13 eyes, tortuous and dilated retinal vessels in 10 eyes, increased vascular branch in 7 eyes, white linear retinal arteries and partial vascular occlusion in 4 eyes, retinal neovascularization in 3 eyes, total retinal detachment in 2 eyes, and retinal fold with macular lamellar hole in 1 eye.In addition, there was retinal hemorrhage in 11 eyes, retinal pigment changes in 4 eyes, grey ridge lesions in 3 eyes, macular dysplasia in 2 eyes, choroidal atrophy in 1 eye, optic gliosis in 1 eye and yellowish-white retinal exudate in 1 eye.There were also 4 patients with other ocular manifestations, such as strabismus and eyeball atrophy.Retinal photocoagulation was performed in 4 eyes of 3 patients and anti-VEGF intravitreal injection in 2 eyes of 2 patients.The retinal lesions regressed and the condition of patients kept stable during follow-up.Conclusions:The ocular clinical manifestations in patients with IP are usually typical and diverse, and the retinal vascular lesion is the main type.Early diagnosis and timely treatment are of great significance.