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Title: Study comparing the regulation of epithelial homeostasis in transplantable human and porcine corneas
Author: Soltaninia, Jila
ISNI:       0000 0004 2748 1959
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2005
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PURPOSE To obtain a better understanding of corneal epithelial cell homeostasis during organ culture storage. METHODS Porcine corneas were used in this study as a model for organ culture storage of human cornea which is carried out routinely for storage of transplant corneas. Porcine corneas were maintained in immersion organ culture for varying periods of time. For proliferation studies BrdU at 1/500 dilution was added. Corneas were then stored in an incubator at 34 C for up to 14 days. At each time point the cornea was bisected: one half was fixed for wax sectioning and the other half snap frozen for cryosectioning. General morphology, epithelial stratification and cell density were assessed by light and fluorescence microscopy using Leica QFluoro image analysis software. Keratocyte number and density was quantified in the anterior, posterior and middle regions of the corneal stroma. The proliferative status of the epithelium was assessed as a function of BrdU incorporation and quantified in central, peripheral and limbal regions. The TUNEL assay was also performed to identify epithelial cell death. Changes in porcine corneas were compared to those in human organ cultured corneas. RESULTS A significant decrease in corneal epithelial thickness was demonstrated with increasing time of organ culture from a maximum value of 45um 3.6 at 0 day to a minimum of 7.7pm 0.6 at 14 days (PO.0001). Stromal thickness increased from 691 um 53 at 0 day to a maximum of 1569pm 126 at 14 days organ culture storage (PO.0001). There was also an apparent decrease in keratocyte density in anterior, posterior and middle stromal (PO.0001). The BrdU labelling index as a measure of the proliferation rate was not equally distributed across the cornea. Proliferation was greatest in the peripheral epithelium at 12 days (27.7% 8.6%) and the labelling index appeared to increase in all regions: peripheral (r=0.79), central (r=0.62) and limbal (r=0.67) as a function of organ culture storage time (PO.05). The cytoskeletal properties of epithelial cornea seemed to remain stable during storage. Lack of ZO-1, Cx43 and 50 expression were observed in cornea at some time points of culture (especially day 6). An increase in percentage of TUNEL-positive corneal epithelial cells was also demonstrated in the central, peripheral and limbal regions (58% at day 12 in peripheral region) with organ culture storage time (p<0.05). The lack of positive activated caspase 9 staining with the high intensity of immunorelated caspase-8 detected in the corneal organ storage model cornea suggests that the epithelial apoptotic pathway during storage is initiated by an extrinsic death receptor-mediated pathway CONCLUSIONS Porcine cornea is a good model for organ culture storage. Epithelial loss occurs during organ culture and is likely to cause barrier dysfunction, therefore contribute to stromal swelling and thus indirectly endothelial cell death. The lack of ZO-1, Cx43 and 50 expression in corneal epithelium is likely to contribute towards deregulation of the corneal epithelium. Epithelial cell loss during organ culture storage appears to be a consequence of accelerated cell death during storage. It is important for the injured epithelium to be replaced with new cells rapidly. However the proliferative activity of corneal epithelial cells during organ culture does not appear to be sufficient to replace the shed cells. This may result in barrier dysfunction and therefore indirectly endothelial cell death that is responsible for the 30% discard rate of corneas stored for transplantation. Changes in porcine corneas on the whole correlated with those in human corneas. Thus the porcine corneas provide a good model by which to study corneal integrity and cell homeostasis during organ culture.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available