Supplementary Material for: Evaluation of Retinal Pigment Epithelium and Choroidal Neovascularization in Rats Using Laser-Scanning Optical-Resolution Photoacoustic Microscopy

<b><i>Purpose:</i></b> To demonstrate the value of the laser-scanning optical-resolution (LSOR)-photoacoustic (PA) microscopy (PAM) system and the conventional multimodal imaging techniques in the evaluation of laser-induced retinal injury and choroidal neovascularization (CNV) in rats. <b><i>Methods:</i></b> Different degrees of retinal injury were induced using laser photocoagulation. We compared the LSOR-PAM system with conventional imaging techniques in evaluating retinal injury with or without CNV. Six additional rats, treated with an anti-VEGF antibody or immunoglobulin G immediately after photocoagulation, were imaged 7 and 14 days after injection, and CNV lesion areas were compared. <b><i>Results:</i></b> In the retinal injury model, fundus autofluorescence showed well-defined hyperreflection, while the lesion displayed abundant PA signals demonstrating nonuniform melanin distribution in retinal pigment epithelium (RPE). RPE was detected with higher contrast in the PAM B-scan image than optical coherence tomography (OCT). Additionally, the CNV lesion was present with multiple PA signal intensities which distinctly characterized the location and area of CNV as found in fundus fluorescein angiography. Furthermore, the decreased PA signals extending from the CNV lesion were similar to those of the vascular bud in ex vivo imaging, which was invisible in other in vivo images. When treated with anti-VEGF agents, statistically significant differences can be demonstrated by PAM similar to other modalities. <b><i>Conclusions:</i></b> LSOR-PAM can detect the melanin distribution of RPE in laser-induced retinal injury and CNV in rats. PAM imaging provides a potential new tool to evaluate the vitality and functionality of RPE in vivo as well as to monitor the development and treatment of CNV.

<b><i>研究目的：</i></b> 本研究旨在验证激光扫描光学分辨率（laser-scanning optical-resolution, LSOR）光声（photoacoustic, PA）显微镜（PAM）系统及传统多模态成像技术，在评估大鼠激光诱导视网膜损伤与脉络膜新生血管（choroidal neovascularization, CNV）中的应用价值。<b><i>研究方法：</i></b> 采用激光光凝术构建不同程度的视网膜损伤模型。将LSOR-PAM系统与传统成像技术进行对比，用于评估伴或不伴CNV的视网膜损伤情况。另选取6只大鼠，在激光光凝术后即刻分别给予抗血管内皮生长因子（anti-VEGF）抗体或免疫球蛋白G（immunoglobulin G）治疗，并于给药后第7天、第14天进行成像检测，对比两组的CNV病灶面积。<b><i>研究结果：</i></b> 在视网膜损伤模型中，眼底自发荧光（fundus autofluorescence）可呈现边界清晰的高反射信号，而病灶区域则显示出丰富的PA信号，提示视网膜色素上皮（retinal pigment epithelium, RPE）内黑色素分布不均。相较于光学相干断层扫描（optical coherence tomography, OCT），PAM断层扫描（B-scan）图像可对RPE实现更高对比度的成像。此外，CNV病灶呈现出多样的PA信号强度，可如荧光素眼底血管造影（fundus fluorescein angiography）一般，清晰表征CNV的位置与病灶面积。进一步观察发现，从CNV病灶延伸出的PA信号衰减区域，与离体成像中的血管芽结构特征一致，而该结构在其他在体成像中无法被观测到。当大鼠接受抗VEGF药物治疗后，PAM可与其他成像模态一样，检测到具有统计学意义的差异。<b><i>研究结论：</i></b> LSOR-PAM可检测大鼠激光诱导视网膜损伤及CNV模型中RPE的黑色素分布情况。PAM成像为在体评估RPE的活性与功能，以及监测CNV的发生发展与治疗效果提供了一种潜在的新型检测工具。