Imaging techniques have revolutionised the assessment of retinal disease in humans and animal models. Here we describe a novel technique for the in vivo visualisation of rod photoreceptors which permits semiquantitative assessment of outer retinal degeneration, and validate this approach in two mouse models of retinitis pigmentosa (RP). Transgenic mice carrying an Nrl-EGFP allele and homozygous for either knock-out of rhodopsin (Nrl-EGFP, Rho-/-) or heterozygous for knock-in of P23H mutant rhodopsin (Nrl-EGFP, RhoP23H/+) were used in this study. These novel strains have green fluorescent rods which undergo a progressive degeneration. Fundus imaging was performed at three-weekly intervals by near infrared reflectance (NIR) and blue light autofluorescence (BAF) confocal scanning laser ophthalmoscopy (cSLO). Mean grey values (mGV), which quantify fluorescence levels within such images, were compared for degenerate and age-matched non-degenerate (Nrl-EGFP, Rho+/+) controls. Mean grey value significantly decreased over time in the Rho-/- and RhoP23H/+ groups but was maintained in Rho+/+ mice (P < 0.001, two-way ANOVA). This corresponded to outer nuclear layer (ONL) thinning as observed by histology. The mGV of superior retina was significantly greater than that of inferior retina in RhoP23H/+ (P = 0.0024) but not in age-matched Rho+/+ (P = 0.45) or Rho-/- (P = 0.65) mice reflecting histological findings. Focal loss of rods could be visualised and mapped in vivo with this technique following a toxic insult, with thinning of the ONL being confirmed in hypofluorescent regions by spectral domain ocular coherence tomography (OCT). Fluorescence labelling of rods permits in vivo characterisation of models of RP and may provide new insights into patterns of degeneration, or rescue effect after treatment. mGV can be used in such cases as a semiquantitative metric of ONL degeneration, and can be used to identify regional variations in photoreceptor loss.

中文翻译：

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使用色素性视网膜炎的荧光报告小鼠模型对杆感光细胞变性进行动态体内定量分析。

成像技术彻底改变了人类和动物模型中视网膜疾病的评估方法。在这里，我们描述了一种用于杆状光感受器的体内可视化的新技术，该技术允许对外部视网膜变性进行半定量评估，并在两种视网膜色素变性（RP）小鼠模型中验证该方法。在此使用了带有Nrl-EGFP等位基因且纯合子用于剔除视紫红质的转基因小鼠（Nrl-EGFP，Rho-/-）或杂合了用于剔除P23H突变体视紫红质的纯合子（Nrl-EGFP，RhoP23H / +）学习。这些新菌株具有绿色荧光棒，该荧光棒经历了渐进的变性。通过近红外反射（NIR）和蓝光自发荧光（BAF）共聚焦扫描激光检眼镜（cSLO）每三周进行一次眼底成像。平均灰度值（mGV），比较了在这些图像中量化荧光水平的荧光蛋白，对简并和年龄匹配的未简（Nrl-EGFP，Rho + / +）对照进行了比较。在Rho-/-和RhoP23H / +组中，平均灰度值随时间显着降低，但在Rho + / +小鼠中保持不变（P <0.001，双向ANOVA）。通过组织学观察，这对应于外核层（ONL）变薄。RhoP23H / +（P = 0.0024）的视网膜上层的mGV显着大于下视网膜的mGV，但在年龄匹配的Rho + / +（P = 0.45）或Rho-/-（P = 0.65）的小鼠中，其mGV不能反映组织学结果。棒的局灶性损失可以在毒性损伤后通过该技术进行可视化并在体内绘制，通过光谱域眼相干断层扫描（OCT）在荧光不足的区域确认了ONL的变薄。棒的荧光标记允许对RP模型进行体内表征，并可能为变性模式或治疗后的抢救效果提供新的见解。mGV可用于ONL变性的半定量指标，并可用于识别感光细胞损失的区域变化。