Augenklinik, Universitätsklinikum Freiburg, Killianstr. 5, 79106 Freiburg, Germany.
Mol Cell Neurosci. 2013 Sep;56:225-33. doi: 10.1016/j.mcn.2013.06.001. Epub 2013 Jun 10.
The mouse model of oxygen-induced retinopathy (OIR) is commonly used to investigate various aspects of the pathogenesis of the retinopathy of prematurity (ROP) as well as angiogenesis in general. Retinal astrocytes were suggested to be involved in retinal angiogenesis. This study aimed to describe their localization and cell density during the course of physiological vascularization and pathological revascularization. Mice expressing H2B-GFP (green fluorescent protein fused to histone 2B) from the endogenous Pdgfra promoter were kept in 75% oxygen from P7 (post natal day 7) to P12 (mouse model of OIR). Retinal flatmounts or cryosections were immunostained for glial fibrillary acidic protein (Gfap), glutamine synthetase (Glul), collagen IV (Col IV), desmin (Des), caspase 3 (Casp3), paired box 2 (Pax2), or Ki67. Astrocytic nuclei were counted with the ImageJ macro AuTOCellQuant. The hypoxic state of the retina was investigated by Hypoxyprobe. The GFP signal of the Pdgfra reporter mice co-localized with Pax2, a nuclear marker for retinal astrocytes. This bright label was much easier to quantify than Gfap or Pax2 staining. Quantification of the cell density of astrocytes during physiological development specified the spreading of astrocytes in a concentrical wave from the optic nerve head towards the periphery. Astrocyte density was reduced during the remodelling of the primary vascular plexus into a hierarchical vascular tree (maximal astrocyte density at P1: 2800 astrocytes/mm2, final astrocyte density: 800 astrocytes/mm2). In the OIR model, cell density of astrocytes was elevated in the peripheral vascularized zone. In contrast, astrocyte density dropped to a half (400 astrocytes/mm2) of the normal value in the central avascular zone during the hyperoxic phase between P8 and P10 by apoptosis and rose only after P17 as the retinal network normalized. An additional drop of astrocyte density was observed within the angles between the large vessels of the central avascular zone during hypoxia between P12 and P17. Astrocyte density was not altered at vascular tufts. The hyperoxia effect on astrocytes including the reduced astrocyte density is not the reason for vascular tuft formation. Hypoxia-affected astrocytes in combination with a reduced astrocytic network in the central avascular zone during the hypoxic phase are important determinants in the formation of pathological features during retinal revascularization.
氧诱导视网膜病变（OIR）的小鼠模型常用于研究早产儿视网膜病变（ROP）的发病机制以及血管生成的各个方面。视网膜星形胶质细胞被认为参与了视网膜血管生成。本研究旨在描述其在生理血管生成和病理性再血管化过程中的定位和细胞密度。从内源性 Pdgfra 启动子表达 H2B-GFP（融合组蛋白 2B 的绿色荧光蛋白）的小鼠从 P7（出生后第 7 天）到 P12（OIR 小鼠模型）保持在 75%的氧气中。对视网膜平片或冷冻切片进行神经胶质纤维酸性蛋白（Gfap）、谷氨酰胺合成酶（Glul）、胶原 IV（Col IV）、结蛋白（Des）、半胱天冬酶 3（Casp3）、配对盒 2（Pax2）或 Ki67 的免疫染色。使用 ImageJ 宏 AuTOCellQuant 对星形胶质细胞核进行计数。通过 Hypoxyprobe 研究视网膜的缺氧状态。Pdgfra 报告小鼠的 GFP 信号与 Pax2 共定位，Pax2 是视网膜星形胶质细胞的核标记物。与 Gfap 或 Pax2 染色相比，这种明亮的标记物更容易定量。在生理发育过程中对星形胶质细胞密度的定量表明，星形胶质细胞从视神经头部向周围呈同心波状扩散。在初级血管丛重塑为分级血管树的过程中（P1 时最大星形胶质细胞密度为 2800 个/平方毫米，最终星形胶质细胞密度为 800 个/平方毫米），星形胶质细胞密度降低。在 OIR 模型中，在周边血管化区域，星形胶质细胞的密度升高。相反，在 P8 和 P10 之间的高氧阶段，由于细胞凋亡，星形胶质细胞密度降至正常水平的一半（400 个/平方毫米），仅在 P17 之后，当视网膜网络正常化后才升高。在 P12 和 P17 之间缺氧期间，在中央无血管区的大血管之间的角度处观察到星形胶质细胞密度的进一步下降。在血管丛中星形胶质细胞密度没有改变。高氧对星形胶质细胞的影响，包括星形胶质细胞密度的降低，并不是血管丛形成的原因。在缺氧阶段，受缺氧影响的星形胶质细胞与中央无血管区星形胶质细胞网络的减少相结合，是视网膜再血管化过程中病理性特征形成的重要决定因素。
