University of Houston College of Optometry, 4901 Calhoun Rd, Houston, TX 77204, USA.
University of Houston College of Optometry, 4901 Calhoun Rd, Houston, TX 77204, USA.
Exp Eye Res. 2018 Sep;174:40-50. doi: 10.1016/j.exer.2018.05.020. Epub 2018 May 21.
Adenosine receptor (ADOR) antagonists, such as 7-methylxanthine (7-MX), have been shown to slow myopia progression in humans and animal models. Adenosine receptors are found throughout the body, and regulate the release of neurotransmitters such as dopamine and glutamate. However, the role of adenosine in eye growth is unclear. Evidence suggests that 7-MX increases scleral collagen fibril diameter, hence preventing axial elongation. This study used immunohistochemistry (IHC) and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) to examine the distribution of the four ADORs in the normal monkey eye to help elucidate potential mechanisms of action. Eyes were enucleated from six Rhesus monkeys. Anterior segments and eyecups were separated into components and flash-frozen for RNA extraction or fixed in 4% paraformaldehyde and processed for immunohistochemistry against ADORA1, ADORA2a, ADORA2b, and ADORA3. RNA was reverse-transcribed, and qPCR was performed using custom primers. Relative gene expression was calculated using the ΔΔCt method normalizing to liver expression, and statistical analysis was performed using Relative Expression Software Tool. ADORA1 immunostaining was highest in the iris sphincter muscle, trabecular meshwork, ciliary epithelium, and retinal nerve fiber layer. ADORA2a immunostaining was highest in the corneal epithelium, trabecular meshwork, ciliary epithelium, retinal nerve fiber layer, and scleral fibroblasts. ADORA2b immunostaining was highest in corneal basal epithelium, limbal stem cells, iris sphincter, ciliary muscle, ciliary epithelium, choroid, isolated retinal ganglion cells and scattered scleral fibroblasts. ADORA3 immunostaining was highest in the iris sphincter, ciliary muscle, ciliary epithelium, choroid, isolated retinal ganglion cells, and scleral fibroblasts. Compared to liver mRNA, ADORA1 mRNA was significantly higher in the brain, retina and choroid, and significantly lower in the iris/ciliary body. ADORA2a expression was higher in brain and retina, ADORA2b expression was higher in retina, and ADORA3 was higher in the choroid. In conclusion, immunohistochemistry and RT-qPCR indicated differential patterns of expression of the four adenosine receptors in the ocular tissues of the normal non-human primate. The presence of ADORs in scleral fibroblasts and the choroid may support mechanisms by which ADOR antagonists prevent myopia. The potential effects of ADOR inhibition on both anterior and posterior ocular structures warrant investigation.
腺苷受体 (ADOR) 拮抗剂，如 7-甲基黄嘌呤 (7-MX)，已被证明可减缓人类和动物模型的近视进展。腺苷受体存在于全身，调节多巴胺和谷氨酸等神经递质的释放。然而，腺苷在眼球生长中的作用尚不清楚。有证据表明，7-MX 增加巩膜胶原纤维直径，从而防止轴向伸长。本研究使用免疫组织化学 (IHC) 和逆转录定量聚合酶链反应 (RT-qPCR) 检查正常猴眼中的四种 ADOR 的分布，以帮助阐明潜在的作用机制。从六只恒河猴中取出眼球。前节和眼杯被分离成成分，并进行快速冷冻以提取 RNA 或固定在 4%多聚甲醛中，用于针对 ADORA1、ADORA2a、ADORA2b 和 ADORA3 的免疫组织化学检测。RNA 逆转录，使用定制引物进行 qPCR。使用相对表达软件工具，通过将肝脏表达归一化来计算相对基因表达。使用相对表达软件工具进行统计分析。ADORA1 免疫染色在虹膜括约肌、小梁网、睫状上皮和视网膜神经纤维层中最高。ADORA2a 免疫染色在角膜上皮、小梁网、睫状上皮、视网膜神经纤维层和巩膜成纤维细胞中最高。ADORA2b 免疫染色在角膜基底上皮、角膜缘干细胞、虹膜括约肌、睫状肌、睫状上皮、脉络膜、孤立的视网膜神经节细胞和分散的巩膜成纤维细胞中最高。ADORA3 免疫染色在虹膜括约肌、睫状肌、睫状上皮、脉络膜、孤立的视网膜神经节细胞和巩膜成纤维细胞中最高。与肝 mRNA 相比，ADORA1 mRNA 在大脑、视网膜和脉络膜中显著升高，在虹膜/睫状体中显著降低。ADORA2a 在大脑和视网膜中的表达更高，ADORA2b 在视网膜中的表达更高，ADORA3 在脉络膜中的表达更高。总之，免疫组织化学和 RT-qPCR 表明，在正常非人类灵长类动物的眼部组织中，四种腺苷受体的表达模式存在差异。ADOR 存在于巩膜成纤维细胞和脉络膜中，可能支持 ADOR 拮抗剂预防近视的机制。ADOR 抑制对前节和后节眼部结构的潜在影响值得进一步研究。
