Abstract：
BACKGROUND: This study aimed to investigate the characteristics of retinal vascular degeneration and the expression of vessel-related claudin (CLD) proteins in retinal degeneration mouse (Pde6βrd1/rd1 rd1 mouse).
METHODS: Retinas from wild-type (WT) mice and rd1 mice at postnatal day 3 (P3), P5, P8, P11, P13, P15, P18, and P21 were collected. Immunofluorescence staining was used to assess the retinal vascular plexus, cell proliferation, CLD expression, and retinal ganglion cells (RGCs). The distribution of retinal superficial and deep vessels was determined by isolectin B4 fluorescence staining of retinal flat mounts and frozen sections. Hematoxylin and eosin staining and terminal deoxynucleotidyl transferase-mediated dNTP nick-end labeling were used to investigate retinal histological degeneration and apoptosis in rd1 mice, respectively. Quantitative real-time PCR and Western blot were used to measure the expression of vessel-related CLD-1, -2, -3, and -5, vascular endothelial growth factor A (VEGFA), and vascular endothelial growth factor receptor 2 (VEGFR2) in the retinas.
RESULTS: Compared to the WT mice, the rd1 mice displayed delayed but completed progressive development in the retinal superficial vascular plexuses (SVPs) and deep vascular plexuses (DVPs). In the rd1 mice, the thickness of retinal layers gradually decreased and the retinas underwent progressive atrophy and degeneration. The deterioration got worse at the late developmental stage. The declined vessel density of SVP and DVP correlated with the decreased thickness of the full and inner parts of the retina and the reduced number of RGCs. DVP degeneration and the thinning of the outer nuclear layer exhibited an obvious reduction at P15. The expression levels of CLD-1, CLD-2, CLD-3, CLD-5, VEGFA, and VEGFR2 decreased and were consistently lower in the rd1 mice than in WT mice since P15.
CONCLUSIONS: Rd1 mice exhibited progressive vascular degeneration of retinal SVP and DVP, the thinning and atrophy of retinal ONL and RGC, and the downregulation of vessel-related CLD proteins during the late developmental period. Thus, the rd1 mouse is a useful model of not only retinal neuro-degeneration but also retinal vascular degeneration.
METHODS: Retinas from wild-type (WT) mice and rd1 mice at postnatal day 3 (P3), P5, P8, P11, P13, P15, P18, and P21 were collected. Immunofluorescence staining was used to assess the retinal vascular plexus, cell proliferation, CLD expression, and retinal ganglion cells (RGCs). The distribution of retinal superficial and deep vessels was determined by isolectin B4 fluorescence staining of retinal flat mounts and frozen sections. Hematoxylin and eosin staining and terminal deoxynucleotidyl transferase-mediated dNTP nick-end labeling were used to investigate retinal histological degeneration and apoptosis in rd1 mice, respectively. Quantitative real-time PCR and Western blot were used to measure the expression of vessel-related CLD-1, -2, -3, and -5, vascular endothelial growth factor A (VEGFA), and vascular endothelial growth factor receptor 2 (VEGFR2) in the retinas.
RESULTS: Compared to the WT mice, the rd1 mice displayed delayed but completed progressive development in the retinal superficial vascular plexuses (SVPs) and deep vascular plexuses (DVPs). In the rd1 mice, the thickness of retinal layers gradually decreased and the retinas underwent progressive atrophy and degeneration. The deterioration got worse at the late developmental stage. The declined vessel density of SVP and DVP correlated with the decreased thickness of the full and inner parts of the retina and the reduced number of RGCs. DVP degeneration and the thinning of the outer nuclear layer exhibited an obvious reduction at P15. The expression levels of CLD-1, CLD-2, CLD-3, CLD-5, VEGFA, and VEGFR2 decreased and were consistently lower in the rd1 mice than in WT mice since P15.
CONCLUSIONS: Rd1 mice exhibited progressive vascular degeneration of retinal SVP and DVP, the thinning and atrophy of retinal ONL and RGC, and the downregulation of vessel-related CLD proteins during the late developmental period. Thus, the rd1 mouse is a useful model of not only retinal neuro-degeneration but also retinal vascular degeneration.
摘要：
背景：本研究旨在探讨视网膜血管变性小鼠(Pde6βrd1/rd1rd1小鼠)视网膜血管变性的特点及血管相关Claudin(CLD)蛋白的表达。
方法：来自野生型（WT）小鼠和rd1小鼠的视网膜在出生后第3天（P3），收集P5、P8、P11、P13、P15、P18和P21。免疫荧光染色用于评估视网膜血管丛，细胞增殖，CLD表达式,和视网膜神经节细胞（RGC）。通过视网膜扁平支架和冷冻切片的IsolectinB4荧光染色确定视网膜浅层和深层血管的分布。苏木精染色和伊红染色以及末端脱氧核苷酸转移酶介导的dNTP缺口末端标记分别用于研究rd1小鼠的视网膜组织学变性和凋亡。实时定量PCR和免疫印迹检测血管相关CLD-1、2、3和CLD-5、血管内皮生长因子A(VEGFA)的表达,和视网膜中的血管内皮生长因子受体2（VEGFR2）。
结果：与WT小鼠相比，rd1小鼠在视网膜浅层血管丛(SVP)和深层血管丛(DVP)中显示出延迟但完成的进行性发育.在rd1小鼠中，视网膜层厚度逐渐减少，视网膜进行性萎缩和变性。在晚期发育阶段恶化。SVP和DVP的血管密度下降与视网膜完整和内部的厚度下降以及RGC数量减少有关。在P15时，DVP的变性和外核层的变薄发生了明显的减少。CLD-1、CLD-2、CLD-3、CLD-5、VEGFA、和VEGFR2降低,并且在rd1小鼠中始终低于WT小鼠,因为P15。
结论：Rd1小鼠表现出视网膜SVP和DVP的进行性血管变性，视网膜ONL和RGC的变薄和萎缩，以及发育后期血管相关CLD蛋白的下调。因此，rd1小鼠不仅是视网膜神经变性而且是视网膜血管变性的有用模型。
方法：来自野生型（WT）小鼠和rd1小鼠的视网膜在出生后第3天（P3），收集P5、P8、P11、P13、P15、P18和P21。免疫荧光染色用于评估视网膜血管丛，细胞增殖，CLD表达式,和视网膜神经节细胞（RGC）。通过视网膜扁平支架和冷冻切片的IsolectinB4荧光染色确定视网膜浅层和深层血管的分布。苏木精染色和伊红染色以及末端脱氧核苷酸转移酶介导的dNTP缺口末端标记分别用于研究rd1小鼠的视网膜组织学变性和凋亡。实时定量PCR和免疫印迹检测血管相关CLD-1、2、3和CLD-5、血管内皮生长因子A(VEGFA)的表达,和视网膜中的血管内皮生长因子受体2（VEGFR2）。
结果：与WT小鼠相比，rd1小鼠在视网膜浅层血管丛(SVP)和深层血管丛(DVP)中显示出延迟但完成的进行性发育.在rd1小鼠中，视网膜层厚度逐渐减少，视网膜进行性萎缩和变性。在晚期发育阶段恶化。SVP和DVP的血管密度下降与视网膜完整和内部的厚度下降以及RGC数量减少有关。在P15时，DVP的变性和外核层的变薄发生了明显的减少。CLD-1、CLD-2、CLD-3、CLD-5、VEGFA、和VEGFR2降低,并且在rd1小鼠中始终低于WT小鼠,因为P15。
结论：Rd1小鼠表现出视网膜SVP和DVP的进行性血管变性，视网膜ONL和RGC的变薄和萎缩，以及发育后期血管相关CLD蛋白的下调。因此，rd1小鼠不仅是视网膜神经变性而且是视网膜血管变性的有用模型。
