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Cryopreservation (−20°C) of equine corneoscleral tissue: Microbiological, histological, and ultrastructural study
Author(s) -
Costa Daniel,
Leiva Marta,
Naranjo Carolina,
Ríos José,
Peña Maria Teresa
Publication year - 2019
Publication title -
veterinary ophthalmology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.594
H-Index - 50
eISSN - 1463-5224
pISSN - 1463-5216
DOI - 10.1111/vop.12618
Subject(s) - cryopreservation , sclera , cornea , biology , andrology , ultrastructure , pathology , ophthalmology , medicine , anatomy , embryo , microbiology and biotechnology
Abstract Objective To evaluate microbiological, histological, and ultrastructural characteristics of short‐term cryopreserved ( STC ) equine corneoscleral tissue (<1 year), and to compare it with long‐term cryopreserved ( LTC ) tissue (>7 years). Animals studied Thirty‐four healthy equine globes. Procedure After a decontamination protocol, globes were enucleated and stored at −20°C in broad‐spectrum antibiotics. Corneoscleral tissue was evaluated at different storage periods: 1 month‐1 year (20 eyes) and 7‐9 years (12 eyes). Two eyes were used as controls. Microbiologic study included direct (blood, McConkey, and Sabouraud agars) and enrichment (brain‐heart infusion broth) cultures. Cryopreservation artifacts were evaluated by hematoxylin‐eosin. Corneoscleral collagen organization and number of normal and dead keratocytes were established by transmission electron microscopy. Results All microbiologic direct cultures were negative. Enrichment cultures were positive in 12.5% of corneal and 59.4% of scleral tissues (p cornea = 0.136; p sclera = 1.000). Cryopreservation artifacts were most commonly observed in LTC tissues ( P = 0.002). Normal keratocytes were predominant in STC corneas ( STC 60% and LTC 0%) and apoptotic ones in LTC ( STC 40% and LTC 90%), whereas necrotic keratocytes were only seen in LTC ( LTC 10%) ( P = 0.001). No structural differences were detected in collagen organization between STC and LTC (p cornea = 1.000; p sclera = 0.703). Conclusions Cryopreservation of equine corneoscleral tissue did not yield direct bacterial contamination. Apoptosis is the main cause of death of cryopreserved equine keratocytes. Based on the lack of significant structural differences between STC and LTC samples, these cryopreserved tissues could potentially be used for tectonic support for at least 9 years without structural or microbiological impediment.