Ashbery Drew, Baez Hector C, Kanarr Rye E, Kunala Karteek, Power Derek, Chu Colin J, Schallek Jesse, McGregor Juliette E
University of Rochester School of Medicine and Dentistry, Rochester, New York, United States.
Center for Visual Science, University of Rochester, Rochester, New York, United States.
Invest Ophthalmol Vis Sci. 2024 Sep 3;65(11):23. doi: 10.1167/iovs.65.11.23.
Insight into the immune status of the living eye is essential as we seek to understand ocular disease and develop new treatments. The nonhuman primate (NHP) is the gold standard preclinical model for therapeutic development in ophthalmology, owing to the similar visual system and immune landscape in the NHP relative to the human. Here, we demonstrate the utility of phase-contrast adaptive optics scanning light ophthalmoscope (AOSLO) to visualize immune cell dynamics on the cellular scale, label-free in the NHP.
Phase-contrast AOSLO was used to image preselected areas of retinal vasculature in five NHP eyes. Images were registered to correct for eye motion, temporally averaged, and analyzed for immune cell activity. Cell counts, dimensions, velocities, and frequency per vessel were determined manually and compared between retinal arterioles and venules. Based on cell appearance and circularity index, cells were divided into three morphologies: ovoid, semicircular, and flattened.
Immune cells were observed migrating along vascular endothelium with and against blood flow. Cell velocity did not significantly differ between morphology or vessel type and was independent of blow flood. Venules had a significantly higher cell frequency than arterioles. A higher proportion of cells resembled "flattened" morphology in arterioles. Based on cell speeds, morphologies, and behaviors, we identified these cells as nonclassical patrolling monocytes (NCPMs).
Phase-contrast AOSLO has the potential to reveal the once hidden behaviors of single immune cells in retinal circulation and can do so without the requirement of added contrast agents that may disrupt immune cell behavior.
随着我们致力于理解眼部疾病并开发新的治疗方法，深入了解活体眼睛的免疫状态至关重要。由于非人灵长类动物（NHP）与人类具有相似的视觉系统和免疫格局，因此它是眼科治疗开发的金标准临床前模型。在此，我们展示了相衬自适应光学扫描激光检眼镜（AOSLO）在细胞水平上可视化NHP中免疫细胞动态的效用，且无需标记。
使用相衬AOSLO对5只NHP眼睛中预先选定的视网膜血管区域进行成像。对图像进行配准以校正眼球运动，进行时间平均，并分析免疫细胞活性。手动确定每条血管的细胞计数、尺寸、速度和频率，并比较视网膜小动脉和小静脉之间的差异。根据细胞外观和圆形度指数，将细胞分为三种形态：卵形、半圆形和平坦形。
观察到免疫细胞沿着血管内皮随血流和逆血流迁移。细胞速度在形态或血管类型之间没有显著差异，并且与血流量无关。小静脉的细胞频率明显高于小动脉。小动脉中较高比例的细胞呈现“扁平”形态。根据细胞速度、形态和行为，我们将这些细胞鉴定为非经典巡逻单核细胞（NCPM）。
相衬AOSLO有潜力揭示视网膜循环中单个免疫细胞曾经隐藏的行为，并且无需添加可能干扰免疫细胞行为的造影剂即可做到这一点。
