Improved protocol for histological and histopathological preparation of large eyes

The development of new treatments for ocular diseases often requires investigating eyes similar in size and structure to human eyes. Such studies are challenging because analyzing the histopathology of large, human-sized eyes can be technically difficult. In particular, obtaining high-quality frozen sections is almost impossible due to the formation of ice crystals in the vitreous, which causes crush artifacts during the procedures of section and post sectioning manipulations. Herein, we describe a new method that provides high-quality frozen sections for large eyes and demonstrate its usefulness in the eyes of rabbits, pigs, minipigs, monkeys, and humans. We observed that artifactual separation of the photoreceptors from the retinal pigment epithelium is minimized and photoreceptor morphology is preserved. This method can be highly beneficial for investigators seeking to translate new treatments for ocular disease into the clinic. RESEARCH HIGHLIGHTS: Histopathological analysis of large and human-sized eyes presents significant challenges, particularly in obtaining high-quality frozen sections. A multistep fixation followed by vitreous removal and replacement ensures better cryopreservation and embedding of large eyes, minimizing the morphological and structural retinal loss found in many studies. Our results demonstrate that a multistep fixation and cryopreservation method for large eyes in histopathology consistently minimizes the artifactual separation of photoreceptors from the retinal pigment epithelium, thereby preserving photoreceptor morphology and providing high-quality frozen sections. A new method providing high-quality sections is necessary and will be highly useful for investigators aiming to translate new treatments for ocular diseases into clinical applications.