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Ocular Toxicity Studies: Specialized Necropsy, Histology, and Pathology Methods

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The eye and the ocular adnexa are common target organs in toxicology studies, but the appropriate evaluation of ocular tissues is a specialized endeavor.  Compared to routine toxicity studies, ocular toxicity studies require methodological modifications at almost every stage from study design to the final pathology report.

For example, at necropsy, the fragile nature of many ocular tissues requires particularly careful handling, very rapid exenteration, and specialized fixatives such as modified Davidson’s solution.  The left and right eyes and their adnexa should be identified and stored in separate labeled containers.  The globes should be marked (with sutures, indelible ink, etc.) in such a manner that the superior-inferior and nasal-temporal aspects remain identifiable.

Many anatomic subsites in the eye are small and localized but must be present on the glass slides so they can be examined by the pathologist (e.g., the approximately 1.5 mm fovea centralis of the macaque retina).  Many toxicologically relevant microscopic eye lesions can also be highly localized and small.  Some iatrogenic findings are also restricted to specific and often small areas of the globe (e.g., intravitreal injection sites at the pars plana).

For these reasons, many refinements of routine histology techniques are needed in ocular toxicity studies.  To ensure that the various eye regions (i.e., central, nasal, and temporal) are adequately sampled, multiple sections (calottes) of each globe should be taken, and in many cases, multiple step-sections of each calotte are appropriate.  In addition, the various eye sections should be placed on the glass slide in such a manner that the superior-inferior and nasal -temporal orientation is uniformly identifiable.  EPL has developed special histology methods to trim, embed, and section eyes to ensure that this critically important orientation is maintained.

Additional customized microscopic sections may also be required depending on the route of administration (e.g., topical, intravitreal, intracameral, juxtascleral, sub-Tenon, topical, or systemic).  The pathologist for ocular toxicity studies should have detailed knowledge of eye and adnexal anatomy and physiology in various laboratory animal species such as rabbits, monkeys, dogs, rats, mice and others, including appreciation of the numerous species-specific differences.

The pathologist should also modify the standard approach as needed for the specialized ocular toxicity study.  For example, in routine studies, the pathologist correlates microscopic lesions with gross necropsy observations in various tissues.  However, gross observations are rarely visible in eyes at necropsy.  Instead, many gross ocular changes (e.g., many cataracts) are noted only during in-life slit-lamp, indirect, or other ophthalmological examinations.  Thus, in ocular toxicity studies, the appropriate correlation for a microscopic ocular lesion is often a clinical in-life ophthalmological finding rather than a gross necropsy observation.

In many ocular studies, one eye is treated and the other left untreated as a contralateral control.  In these studies, the pathologist must record pathology findings separately for the left and right eyes, something which is very rarely if ever done in routine studies for other paired organs such as kidney, thyroid, etc.

In routine studies, the anatomic hierarchical level used for recording pathology findings is generally the organ as a whole, such as “liver”, “kidney”, etc.  The total number of organs examined per dose group serves as the denominator for statistical analyses of the microscopic pathology findings.  This approach must be modified for the specialized ocular toxicity study.  In these studies, the organ “eye” is not an appropriate anatomic level for recording microscopic findings or for serving as the dose group denominator.  Instead, tissue accountability and microscopic findings should be recorded at the level of major anatomic subsites of the eye, such as “cornea”, “lens”, “retina”, etc.

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