Functional, structural, and molecular identification of lymphatic outflow from subconjunctival blebs
Functional, structural, and molecular identification of lymphatic outflow from subconjunctival blebs
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DOI：
10.1016/j.exer.2020.108049
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发表时间：
2020-07-01
影响因子：
3.4
通讯作者：
Huang, Alex S.
中科院分区：
文献类型：
--
作者：
Akiyama, Goichi;Saraswathy, Sindhu;Huang, Alex S.
本研究的目的是评价结膜下滤过泡的流出途径，并确定其身份。获取死后猪（n = 20）、人（n = 1）和牛（n = 1）的眼睛，并将示踪剂（荧光素、吲哚菁绿色或可固定/荧光葡聚糖）注射到结膜下空间中以产生凸起的滤过泡，其中定性和定量地观察流出途径。将具有荧光报告转基因的啮齿动物成像用于结构比较。同时获得光学相干断层扫描（OCT），以研究这些途径的结构性质。使用可固定/荧光葡聚糖，将示踪剂捕获到滤过泡流出通道管腔壁，用于组织学可视化和使用针对淋巴和血管标记物的免疫荧光进行分子鉴定。在所有物种中使用所有示踪剂均可观察到滤过泡流出途径。定量分析显示，鼻象限与颞象限相比具有更多的滤过泡相关流出通路（鼻：1.9 +/- 0.3通路vs.颞：0.7 +/- 0.2通路; p = 0.003）。然而，并非所有的滤过泡均导致流出道（0-通道= 18.2%; 1-通道= 36.4%; 2-通道= 38.6%; 3-通道= 6.8%）。使用光学相干断层扫描和组织学验证流出信号为真正的管腔通路。在猪眼的血流方向上识别出二尖瓣。标记途径的免疫荧光显示淋巴（Prox-1和podoplanin），但不是血管（CD 31）的身份。因此，结膜下滤过泡流出发生在离散的管腔通路中。通过瓣膜的结构鉴定和淋巴标志物的分子鉴定评估，它们是淋巴管。更好地了解淋巴外流可能会导致改善青光眼手术和眼部药物输送的眼部护理。
The purpose of this study is to evaluate outflow pathways from subconjunctival blebs and to identify their identity. Post-mortem porcine (n = 20), human (n = 1), and bovine (n = 1) eyes were acquired, and tracers (fluorescein, indocyanine green, or fixable/fluorescent dextrans) were injected into the subconjunctival space to create raised blebs where outflow pathways were visualized qualitatively and quantitatively. Rodents with fluorescent reporter transgenes were imaged for structural comparison. Concurrent optical coherence tomography (OCT) was obtained to study the structural nature of these pathways. Using fixable/fluorescent dextrans, tracers were trapped to the bleb outflow pathway lumen walls for histological visualization and molecular identification using immunofluorescence against lymphatic and blood vessel markers. Bleb outflow pathways could be observed using all tracers in all species. Quantitative analysis showed that the nasal quadrant had more bleb-related outflow pathways compared to the temporal quadrant (nasal: 1.9 +/- 0.3 pathways vs. temporal: 0.7 +/- 0.2 pathways; p = 0.003). However, not all blebs resulted in an outflow pathway (0-pathways = 18.2%; 1-pathway = 36.4%; 2-pathways = 38.6%; and 3-pathways = 6.8%). Outflow signal was validated as true luminal pathways using optical coherence tomography and histology. Bicuspid valves were identified in the direction of flow in porcine eyes. Immunofluorescence of labeled pathways demonstrated a lymphatic (Prox-1 and podoplanin) but not a blood vessel (CD31) identity. Therefore, subconjunctival bleb outflow occurs in discrete luminal pathways. They are lymphatic as assessed by structural identification of valves and molecular identification of lymphatic markers. Better understanding of lymphatic outflow may lead to improved eye care for glaucoma surgery and ocular drug delivery.