目的 建立临床前毒理学研究中能充分涵盖正常恒河猴眼球解剖组织学结构和恒河猴眼球实施异种角膜内皮植片移植后包含角膜及异种角膜内皮植片的一致性组织制片方法。方法 成年恒河猴10只为对照组，10只为模型组，模型组右眼实施异种角膜内皮植片移植手术。动物剖检时，摘取眼球，采用Davidson's液固定。左侧正常眼球：在平行于颞侧睫状后长动脉及视神经上方1 mm处，使用取材刀片沿视神经、黄斑（中心凹）与角膜作一切面，切片经常规脱水、包埋、切片、染色后进行镜检。右侧眼球：从睫状体后方切取角膜及虹膜，摘除晶状体，然后使角膜面向上，间隔2.5 mm切取角膜及虹膜，将切取的组织以切面向下按顺序放入分格包埋盒中，剩余眼球组织再切3个横切面并放入包埋盒，然后组织经脱水、包埋、切片和HE染色及Masson染色，最后对正常眼球、角膜及异种角膜内皮植片的解剖及组织学结构特点进行确认。结果 所制备的正常眼球切片可清晰显示眼球所有组织结构，染色鲜艳，眼球形态真实。实施异种角膜内皮植片移植后，眼球角膜及异种角膜内皮植片结构清晰，且能良好地展示异种角膜内皮植片与角膜的贴合状态。所制备的组织切片表面平整，未见人工假象。结论 通过自主建立的制片方法，可获得包含所有眼球组织结构特征的正常眼球切片，以及包含完整角膜、异种角膜内皮植片及眼球其他组织的良好切片。所建立的眼球制片方法简单易行，并且一致性较高，可用于临床前毒理学研究中眼部用药及实施异种角膜内皮植片移植后眼部的病理学评价。

Objective To establish a consistent section preparation method that can fully cover anatomical and histological structures of normal eyes, and cornea and xeno-corneal endothelium grafts after implementing xeno-corneal grafts transplantation in Rhesus monkey for preclinical toxicology research. Methods There were ten adult rhesus monkeys in control group and ten adult rhesus monkeys in model group. Xenogenic corneal endothelial graft was performed on the right eye of the model group. Normal eyes and eyes that underwent xenograft transplantation of xeno-corneal endothelial grafts were sampled and fixed with Davidson's fluid. Normal eyes: Use a slicing blade to make a section along the optic nerve, the macula (fovea centralis), and the cornea at a distance of 1mm parallel to the temporal posterior long ciliary artery (LPCA) and above the optic nerve. The samples were routinely dehydrated, embedded, sectioned and stained before undergoing microscopic observation. Eyes that underwent xenograft transplantation of the xeno-corneal endothelial grafts: Cut the cornea and iris from behind the ciliary body, remove the lens, and then cut the corneal and iris with the corneal facing upwards at an interval of 2.5 mm. Sampling surface of the tissue were placed downward and sequentially into a grid embedding box. Three transverse plane sections of the remaining ocular tissues were cut and placed in an embedding box. After dehydration and embedding, the wax block was sectioned and stained with HE and Masson, respectively. Finally, the anatomical and histological characteristics of the normal eyes, the cornea, and the xeno-corneal endothelial grafts were confirmed. Results The prepared eyes sections can clearly display all tissue structures of the normal eyes, with bright staining and realistic morphology. After implementation of the xeno-corneal endothelial grafts transplantation, the structures of the cornea and the xenocorneal endothelial grafts were clear, and the adhesion between the xeno-corneal endothelium and the cornea can be well displayed. The surface of the prepared tissue sections was flat and no artificial artifacts were observed. Conclusion By using a self-developed preparation method, we can obtain good eye section containing all structural features of the normal eye tissues, intact cornea and xeno-corneal endothelial grafts, and the other eye tissues. The method is easy, and has high consistency. It can be used for histopathological examination of ocular medication and pathological evaluation of the eyes after performing xeno-corneal endothelial grafts transplantation. We hoped that the section preparation method presented in this study will provide technical support for scientific assessment of the safety and efficacy of preclinical ophthalmic drugs/xeno-corneal endothelial grafts transplantation.

R965.2

国家“重大新药创制”科技重大专项资助项目（2018ZX09201-017）