Bruckner Daniela, Kaser-Eichberger Alexandra, Bogner Barbara, Runge Christian, Schrödl Falk, Strohmaier Clemens, Silva Maria Elena, Zaunmair Pia, Couillard-Despres Sebastien, Aigner Ludwig, Rivera Francisco J, Reitsamer Herbert A, Trost Andrea
a University Clinic of Ophthalmology and Optometry, Research Program for Experimental Ophthalmology and Glaucoma Research, Paracelsus Medical University/SALK , Salzburg , Austria.
b Department of Anatomy , Paracelsus Medical University Salzburg , Salzburg , Austria.
Curr Eye Res. 2018 Oct;43(10):1274-1285. doi: 10.1080/02713683.2018.1493130. Epub 2018 Jul 18.
Purpose/aim of the study: In the retina, defects in pericytes (PCs) function/loss are associated with various complications; however, the exact pathological mechanisms are still not fully elucidated. Following the behavior of retina-resident PCs during health and disease will reveal new insights for both the understanding of pathological mechanisms and the development of new regenerative therapies for the treatment of retinopathies. The main goal of this study is to determine whether the NG2-reporter mouse (NG2CreER-eGFP) is a suitable model to study the fate of retina-resident PCs.
Vascular development-dependent reporter induction in retinal PCs was evaluated at different time points [(a) > P21, (b) < P21, and (c) P1 to > P21)] and additionally four different modes of application were tested. Reporter expression was evaluated by enhanced green fluorescent protein (eGFP) immunofluorescence by confocal microscopy and induction efficiency was calculated by analyzing NG2-expressing PCs in comparison to eGFP-labeled PCs in the three capillary layers.
eGFP-positive PCs were detected in the three retinal capillary layers at all time points and administration routes tested. Multiple tamoxifen (TAM) applications in adult (> P21) NG2CreER-eGFP mice resulted in 3.59% eGFP-positive PCs. 2.37% eGFP-labeled PCs were detected after single intraperitoneal TAM injections at early postnatal days (P2/P5); however, just 1.61% PCs revealed reporter expression upon activation via the lactating mother (P4-P7). The highest number of eGFP-labeled PCs (7.09%) was detected following triple TAM administrations (P10-P12). The number of reporter-positive PCs doubled using homozygous animals.
Despite low recombination efficiency in the used PC-specific fate mapping mouse model, changes in NG2 promoter activity of PCs during vascular development are indicated by single and multiple TAM inductions at different developmental time points. Nevertheless, these findings need further confirmation in up-coming studies by using homozygous NG2CreER-eGFP mice and additionally by mating the NG2CreER with a different reporter mouse to increase the low recombination efficiency.
研究目的：在视网膜中，周细胞（PCs）功能缺陷/丧失与多种并发症相关；然而，确切的病理机制仍未完全阐明。追踪视网膜驻留PCs在健康和疾病状态下的行为，将为理解病理机制和开发治疗视网膜病变的新型再生疗法提供新的见解。本研究的主要目标是确定NG2报告基因小鼠（NG2CreER-eGFP）是否是研究视网膜驻留PCs命运的合适模型。
在不同时间点[（a）> P21，（b）< P21，以及（c）P1至> P21]评估视网膜PCs中血管发育依赖性报告基因的诱导情况，并另外测试了四种不同的应用模式。通过共聚焦显微镜用增强型绿色荧光蛋白（eGFP）免疫荧光评估报告基因表达，并通过分析三个毛细血管层中表达NG2的PCs与eGFP标记的PCs来计算诱导效率。
在所有测试的时间点和给药途径下，在三个视网膜毛细血管层中均检测到eGFP阳性PCs。在成年（> P21）NG2CreER-eGFP小鼠中多次给予他莫昔芬（TAM）后，3.59%的PCs为eGFP阳性。在出生后早期（P2/P5）单次腹腔注射TAM后，检测到2.37%的eGFP标记PCs；然而，通过哺乳期母亲激活（P4 - P7）时，只有1.61%的PCs显示报告基因表达。在三次给予TAM（P10 - P12）后，检测到的eGFP标记PCs数量最多（7.09%）。使用纯合动物时，报告基因阳性PCs的数量增加了一倍。
尽管在所使用的PC特异性命运图谱小鼠模型中重组效率较低，但在不同发育时间点进行单次和多次TAM诱导表明，PCs的NG2启动子活性在血管发育过程中发生了变化。然而，这些发现需要在未来的研究中通过使用纯合NG2CreER-eGFP小鼠，并另外将NG2CreER与不同的报告基因小鼠交配以提高低重组效率来进一步证实。
