目的 建立臨床前毒理學(xué)研究中能充分涵蓋正常恒河猴眼球解剖組織學(xué)結(jié)構(gòu)和恒河猴眼球?qū)嵤┊惙N角膜內(nèi)皮植片移植后包含角膜及異種角膜內(nèi)皮植片的一致性組織制片方法。方法 成年恒河猴10只為對(duì)照組，10只為模型組，模型組右眼實(shí)施異種角膜內(nèi)皮植片移植手術(shù)。動(dòng)物剖檢時(shí)，摘取眼球，采用Davidson's液固定。左側(cè)正常眼球：在平行于顳側(cè)睫狀后長(zhǎng)動(dòng)脈及視神經(jīng)上方1 mm處，使用取材刀片沿視神經(jīng)、黃斑（中心凹）與角膜作一切面，切片經(jīng)常規(guī)脫水、包埋、切片、染色后進(jìn)行鏡檢。右側(cè)眼球：從睫狀體后方切取角膜及虹膜，摘除晶狀體，然后使角膜面向上，間隔2.5 mm切取角膜及虹膜，將切取的組織以切面向下按順序放入分格包埋盒中，剩余眼球組織再切3個(gè)橫切面并放入包埋盒，然后組織經(jīng)脫水、包埋、切片和HE染色及Masson染色，最后對(duì)正常眼球、角膜及異種角膜內(nèi)皮植片的解剖及組織學(xué)結(jié)構(gòu)特點(diǎn)進(jìn)行確認(rèn)。結(jié)果 所制備的正常眼球切片可清晰顯示眼球所有組織結(jié)構(gòu)，染色鮮艷，眼球形態(tài)真實(shí)。實(shí)施異種角膜內(nèi)皮植片移植后，眼球角膜及異種角膜內(nèi)皮植片結(jié)構(gòu)清晰，且能良好地展示異種角膜內(nèi)皮植片與角膜的貼合狀態(tài)。所制備的組織切片表面平整，未見人工假象。結(jié)論 通過自主建立的制片方法，可獲得包含所有眼球組織結(jié)構(gòu)特征的正常眼球切片，以及包含完整角膜、異種角膜內(nèi)皮植片及眼球其他組織的良好切片。所建立的眼球制片方法簡(jiǎn)單易行，并且一致性較高，可用于臨床前毒理學(xué)研究中眼部用藥及實(shí)施異種角膜內(nèi)皮植片移植后眼部的病理學(xué)評(píng)價(jià)。

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

國(guó)家“重大新藥創(chuàng)制”科技重大專項(xiàng)資助項(xiàng)目（2018ZX09201-017）