Nuffield Laboratory of Ophthalmology and Oxford Eye Hospital Biomedical Research Centre, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.
Invest Ophthalmol Vis Sci. 2011 Sep 21;52(10):7340-6. doi: 10.1167/iovs.11-7996.
To develop a robust ex vivo model for evaluating cone survival in end-stage retinitis pigmentosa (RP) and apply this to quantify the effects of putative neuroprotective compounds.
Rhodopsin knockout mice were crossed with OPN1-GFP reporter mice so that GFP-positive cones could be identified against the background of a rod-specific degeneration. Retinal explants were harvested from 10-week-old mice and maintained in organotypic culture. Ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), or vascular endothelial growth factor 165b (VEGF(165b)) was administered daily to treatment groups at three doses (200 ng/mL, 100 ng/mL, or 50 ng/mL; n = 5 explants per group). Fluorescence microscopy was performed on days 1, 3, 5, 7, 9, and 12 to document the number of GFP-expressing cones.
Cone survival could be assessed reliably and reproducibly in this model, and cone degeneration was significantly greater in the absence of rods, in keeping with clinical observations of RP. Daily administration of 200 ng/mL CNTF led to significantly increased cone survival compared with sham-treated controls. The effect was dose dependent; 100 ng/mL CNTF reduced cone loss but to a lesser extent, and 200 ng/mL GDNF showed significant protection against cone loss at later time points (day 9-12) but was much less effective than CNTF at all doses. VEGF(165b) showed no neuroprotective effect in this model at any dose.
This model allows precise quantification of the neuroprotective effects of various compounds on cone survival and may therefore provide a robust method of screening neuroprotective compounds before application in vivo.
开发一种稳健的离体模型，用于评估晚期色素性视网膜炎（RP）中的锥体存活，并应用该模型来量化潜在神经保护化合物的作用。
将视紫红质敲除小鼠与 OPN1-GFP 报告小鼠杂交，以便在杆状特异性变性的背景下识别 GFP 阳性锥体。从 10 周龄的小鼠中采集视网膜外植体，并在器官型培养中维持。睫状神经营养因子（CNTF）、胶质细胞源性神经营养因子（GDNF）或血管内皮生长因子 165b（VEGF(165b)）以三种剂量（200ng/ml、100ng/ml 或 50ng/ml；每组 5 个外植体）每天给予治疗组。在第 1、3、5、7、9 和 12 天进行荧光显微镜检查，以记录 GFP 表达锥体的数量。
在该模型中可以可靠且可重复地评估锥体存活，并且在没有杆的情况下，锥体变性明显更大，与 RP 的临床观察一致。每天给予 200ng/ml CNTF 可显著增加锥体存活，与假处理对照相比。该作用呈剂量依赖性；100ng/ml CNTF 减少锥体丢失，但程度较小，而 200ng/ml GDNF 在稍后时间点（第 9-12 天）显示出对锥体丢失的显著保护作用，但在所有剂量下均不如 CNTF 有效。VEGF(165b)在该模型中任何剂量均无神经保护作用。
该模型允许对各种化合物对锥体存活的神经保护作用进行精确量化，因此在体内应用之前可能提供一种筛选神经保护化合物的稳健方法。
