Objective: To determine the viability and microbiological safety of canine [c], feline [f] and equine [e] corneal and scleral tissue cryopreserved at -20°C, comparing tissues cryopreserved for less than 1 year (G≤1a) with those cryopreserved for long periods (G≥6a). Materials and Methods: Thirty-six eyeballs from dogs, 20 from cats, and 34 from horses were obtained from the Fundació Hospital Clinic Veterinari between 2003 and 2013. All animals were free of neoplasia or infectious diseases and had no ocular abnormalities. After a decontamination protocol, the eyes were enucleated under sterile conditions within 10 hours post-mortem and stored at -20°C under atmospheric conditions with a broad spectrum antibiotic until analysis. Microbiological, histological and ultrastructural studies of both, the scleral and corneal tissue were performed. Microbiology consisted on cultures of corneal and scleral samples on blood, McConkey and Sabouraud agar and brain heart broth. Cryopreservationartefacts were evaluatedby histology. Transmission electron microscopy (TEM) was used to establish collagen integrity, as well asthe number and characteristics of keratocytes, classifying those in normal, apoptotic and necrotic. Cell death by apoptosis was assessed by TUNEL in selected canine samples Results.Microbiology: Direct cultures were positive in 27.5% [c] 15.0% [f] and 0% [e] of G≤1aand in none of the G≥6a.Enriched cultures were positive in 47.5% [c] 30.0% [f] and 27.5% [e] of G≤1aand in 16.7% [c] 0% [f] and 25.0% [e] ofG≥6a.Histopathology: In all three species, cryopreservation artefacts were more frequent in G≤1a samples than in G≥6a. TEM: The predominant keratocyte was apoptotic in all samples of the 3 species. Collagen structure is retained over time, classified as organized or semi-organized in all cases, except in one sample of canine cornea. Conclusion. The canine, feline and equine corneal and scleral tissues cryopreserved at -20°C can be used for at least 8, 10 and 9 years respectively without microbiological or structural impediments. Bacterial contamination in these tissues seems to be reduced over time. Apoptosis is the most important cell death pathway for cryopreserved (-20º)keratocytes.
Crioconservación de córnea y esclera a -‐20°C en diferentes especies animales
Costa Moya, D. (Author). 10 Jul 2015
Student thesis: Doctoral thesis
Student thesis: Doctoral thesis