Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States.
Center for Biologic Imaging and the Department of Cellular Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States.
Invest Ophthalmol Vis Sci. 2018 May 1;59(6):2371-2380. doi: 10.1167/iovs.17-23075.
The rate of conventional aqueous humor outflow is the highest nasally. We hypothesized that this is reflected in regionally different outflow structures and analyzed the entire limbus by high-resolution, full-thickness ribbon-scanning confocal microscopy (RSCM).
We perfused pig eyes by anterior chamber cannulation with eight lectin-fluorophore conjugates, followed by optical clearance with benzyl alcohol benzyl benzoate (BABB). RSCM and advanced analysis software (Imaris) were used to reconstruct a three-dimensional (3D), whole-specimen rendering of the perilimbal outflow structures. We performed morphometric analyses of the outflow tract from the level of the trabecular meshwork (TM) to the scleral vascular plexus (SVP).
Except for pigmented structures, BABB cleared the entire eye. Rhodamine-conjugated Glycine max agglutinin (soybean [SBA]) labeled the outflow tract evenly and retained fluorescence for months. RSCM produced terabyte-sized files allowing for in silico dissection of outflow tract vessels at a high resolution and in 3D. Networks of interconnected lumens were traced from the TM to downstream drainage structures. The collector channel (CC) volumes were 10 times smaller than the receiving SVP vessels, the largest of which were in the inferior limbus. Proximal CC diameters were up to four times the size of distal diameters and more elliptical at their proximal ends. The largest CCs were found in the superonasal and inferonasal quadrants where the highest outflow occurs.
RSCM of cleared eyes enabled high-resolution, volumetric analysis of the outflow tract. The proximal structures had greater diameters nasally, whereas the SVP was larger in the inferior limbus.
常规房水流出率在鼻侧最高。我们假设这反映在区域间不同的流出结构上，并通过高分辨率、全层带状扫描共聚焦显微镜（RSCM）分析整个角膜缘。
我们通过前房套管用八种凝集素-荧光染料缀合物对猪眼进行灌流，然后用苯甲醇苯甲酸苄酯（BABB）进行光学清除。RSCM 和高级分析软件（Imaris）用于重建角膜缘周围流出结构的三维（3D）全标本渲染。我们对从小梁网（TM）到巩膜血管丛（SVP）水平的流出道进行形态计量分析。
除了色素结构外，BABB 清除了整个眼睛。罗丹明缀合的 Glycine max agglutinin（大豆[SBA]）均匀标记流出道，并保持数月的荧光。RSCM 生成了兆字节大小的文件，允许以高分辨率和 3D 在计算机上对流出道血管进行解剖。从 TM 到下游引流结构追踪到相互连接的腔网络。收集管（CC）的体积比接收 SVP 血管小 10 倍，其中最大的血管位于下角膜缘。近端 CC 直径可达远端直径的 4 倍，在近端更呈椭圆形。最大的 CC 位于鼻上和鼻下象限，此处流出量最大。
清除后的眼睛的 RSCM 实现了流出道的高分辨率、体积分析。鼻侧的近端结构直径较大，而下部角膜缘的 SVP 较大。
