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Cryopreservation (−20 °C) of canine corneoscleral tissue: histological, microbiological, and ultrastructural study
Author(s) -
Costa Daniel,
Leiva Marta,
Naranjo Carolina,
Ríos José,
Peña Maria T.
Publication year - 2018
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.12544
Subject(s) - cryopreservation , tunel assay , andrology , ultrastructure , biology , pathology , h&e stain , staining , immunohistochemistry , anatomy , medicine , embryo , microbiology and biotechnology
Objective To evaluate microbiological, histological, and ultrastructural characteristics of short‐term cryopreserved ( STC ) canine corneoscleral tissue (<1 year) and to compare it with long‐term cryopreserved ( LTC ) tissue (>6 years). Animals studied Thirty‐six healthy canine globes. Procedure After a decontamination protocol, globes were enucleated and stored at −20 °C. Corneoscleral tissue was evaluated at different periods: <1 year (20 eyes) and >6 years (12 eyes). Four 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 (TEM). Terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL ) was also used for keratocyte characterization. Results Corneal microbial growth was observed in 25% of the direct STC cultures, and in 47.4% and 16.7% of the enriched STC and LTC cultures, respectively. Scleral STC direct cultures were 30% positive, while enrichment cultures were positive in 66.7% and 16.7% of the STC and LTC , respectively ( P = 0.011). Cryopreservation artifacts were higher in LTC tissues ( P < 0.001). Apoptotic keratocytes were predominant by TEM and TUNEL , in both STC and LTC . Minimal structural differences were detected in collagen organization between STC and LTC . Conclusions Cryopreservation of canine corneoscleral tissue seems to reduce bacterial contamination over time. Apoptosis is the main way of death of cryopreserved canine 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 8 years without structural or microbiological impediment.