目的研究快速、精确获取小鼠角膜神经三维(3D)图像及其参数的技术方案。
方法选取SPF级雌性C57BL/6小鼠4只，吸入过量乙醚麻醉后使小鼠安乐死，立即在解剖显微镜下获取具有完整角膜缘的角膜4个，经过常规固定、透膜和抗β-Ⅲ微管蛋白荧光抗体标记后整铺片处理。在高分辨率去卷积显微镜下采用科学互补性金属氧化物半导体探测器捕获图像，通过显微镜系统自携带图像处理软件对图像进行3D去卷积运算，Z轴数据平面投影以及自动拼接处理得到完整的角膜神经纤维3D图像。采用交互式显微图像分析软件Imaris的丝状追踪模块中的自动检测模式获得不同区域的角膜神经密度，采用自动路径模式手动指定计算起始点到终止点的神经纤维长度。
结果在去卷积显微镜60倍油镜下，可以观察到角膜缘处呈密集网络状的基质层神经纤维在角膜缘附近进入前弹力层，并发出密集的分枝，形成基底下神经丛。这些神经丛向角膜中心伸展形成密集的神经网络样结构，在角膜顶点汇聚成漩涡状结构。少部分神经纤维丛垂直进入上皮层，并发出许多微小的神经末梢分枝。采用Imaris软件丝状物追踪模块中的自动检测模式自动统计，发现角膜神经末梢密度从角膜缘的(2 488.88±282.84)μm/μm 2逐渐增多至角膜中央的(5 766.66±298.55)μm/μm 2；角膜基质神经纤维密度从角膜缘的(40.99±0.99)μm/μm 2递减至角膜中央的(34.57±1.28)μm/μm 2。通过自动路径模式手动测量发现，角膜缘处基质层神经纤维进入前弹力层约151 μm处开始分枝形成基底下神经丛。
结论去卷积显微镜系统可以获得整个角膜神经纤维的3D分布，Imaris图像分析软件可以自动、快速统计待测区域角膜神经纤维的不同参数。
ObjectiveTo investigate a rapid protocol for the acquisition of a three-dimensional (3D) image of corneal nerve distribution and its various parameters.
MethodsFour SPF female C57BL/6 mice were selected and four corneal samples with complete limbi were obtained using a dissecting microscope after the sacrifice of mice euthanized by ether.After conventional fixation, permeabilization, and immunostaining by an anti-β-Ⅲ tubulin fluorescent-conjugated antibody, a whole-nerve image of the whole-mount cornea was captured under a 60X oil lens using a scientific complementary metal-oxide-semiconductor detector and a high-resolution deconvolution microscope.The 3D image of the corneal nerve fiber was obtained after 3D deconvolution processing, Z-axis data projection, and automatic stitching using the self-contained image processing software of the microscope system.The corneal nerve density in different areas was analyzed using the automatic detection mode in the Filament Tracer module and the manual Autopath module of the interactive microscopic image analysis software Imaris.The use and care of animals complied with the statement of the Association for Research in Visual and Ophthalmology, and the study protocol was approved by an Experimental Animal Welfare Committee of Jinan University (No.JN-A-2002-01).
ResultsIt was found that stromal nerve fibers in a dense network entered the Bowman membrane near the limbus, and branches of stromal nerve fibers formed subbasal nerve plexus, which stretched toward the center of the cornea to form a dense neural network-like structure and converged into a vortex-like structure at the apex of the cornea.Some subbasal nerves entered the epithelial layer vertically and some branches of nerve endings were found.Through the automatic detection mode of Imaris software, a gradual increase of the density from (2 488.88±282.84)μm/μm 2 at the limbus to (5 766.66±298.55)μm/μm 2 at the center of the cornea of the subbasal nerve branches, and a decrease of the density from (40.99±0.99)μm/μm 2 at the limbus to (34.57±1.28)μm/μm 2 at the center of the stromal nerves were found.It was also found that the stromal nerves at the limbus entered the Bowman membrane for about 151 μm and then began to branch to form subbasal nerves.
ConclusionsThe high-resolution deconvolution microscope system can be used to study the 3D distribution of the whole corneal nerve.Additionally, Imaris can be used for obtaining various parameters of the corneal nerves automatically and quickly.
傅婷,黄奕嘉,刘俊,等. 快速精确获取小鼠角膜神经三维图像及其参数的技术方案研究[J]. 中华实验眼科杂志,2022,40(02)：104-109.
DOI:10.3760/cma.j.cn115989-20200817-00595版权归中华医学会所有。
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傅婷：文章选题及撰写、实验研究、分析数据；黄奕嘉：实验研究、分析数据；刘俊：论文修改；李志杰：文章选题及撰写
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