Cryopreservation (-20 °C) of feline corneoscleral tissue: histologic, microbiologic, and ultrastructural study.
OBJECTIVE
To evaluate microbiological, histologic, and ultrastructural characteristics of short-term cryopreserved (STC) feline corneoscleral tissue (<1 year) and to compare it with long-term cryopreserved (LTC) tissue (>7 years).


METHODS
Twenty healthy feline globes were obtained from 2003 to 2013.


METHODS
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 year (10 eyes) and >7 years (8 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
Although microbiologic cultures were positive only in STC [direct (20.8%); enrichment (37.5%)], significant differences between periods were only found in enrichment cultures (P = 0.006). Cryopreservation artifacts were most commonly observed in LTC tissues (P < 0.001). Normal keratocytes were predominant in STC corneas (STC 58.3%, LTC 12.5%) and apoptotic ones in LTC (STC 41.7%, LTC 75%), whereas necrotic keratocytes were only seen in LTC (LTC 12.5%) (P = 0.046). No structural differences were detected in collagen organization between STC and LTC (Pcornea = 0.147; Psclera = 0.362).


CONCLUSIONS
Cryopreservation of feline corneoscleral tissue seems to reduce bacterial contamination over time. Apoptosis is the main cause of death of cryopreserved feline 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 10 years without structural or microbiological impediment.
Facultat de Veterinària, Servei d'Oftalmologia de la Fundació Hospital Clínic Veterinari, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain.
Daniel
D
Costa
Facultat de Veterinària, Servei d'Oftalmologia de la Fundació Hospital Clínic Veterinari, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain.|Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain.
Marta
M
Leiva
Departamento de Medicina y Cirugía Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain.
Carolina
C
Naranjo
Laboratory of Biostatistics & Epidemiology, Facultat de Medicina, Bellaterra and Biostatistics and Medical Statistics Core Facility, Hospital Clínic, IDIBAPS, Universitat Autònoma de Barcelona, Barcelona, Spain.
José
J
Ríos
Facultat de Veterinària, Servei d'Oftalmologia de la Fundació Hospital Clínic Veterinari, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain.|Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain.
Maria Teresa
MT
Peña
D000818:Animals; D017209:Apoptosis; D002415:Cats; D003315:Cornea / Q000166:cytology* / Q000382:microbiology / Q000648:ultrastructure; D060527:Corneal Keratocytes / Q000166:cytology / Q000648:ultrastructure; D015925:Cryopreservation / Q000662:veterinary*; D046529:Microscopy, Electron, Transmission / Q000662:veterinary; D012590:Sclera / Q000166:cytology* / Q000382:microbiology / Q000648:ultrastructure
cat; collagen; cornea; keratocyte; sclera; storage method