Evaluation of protective effects of trehalose on desiccation of epithelial cells in three dimensional reconstructed human corneal epithelium.
OBJECTIVE
Trehalose has been shown to protect epithelial cells from desiccation damage in cell culture and the murine dry eye model. The present study evaluates the protective role of trehalose in reconstructed human corneal epithelium (3D-HCE) during desiccation.


METHODS
The morphology of 3D-HCE was examined using in vivo an ex vivo confocal laser-scanning microscopy (CLSM). The 3D-HCE was desiccated with or without pre-treatment with trehalose. Evaluation of protective role of trehalose was conducted using different in vitro cell viability assays and CLSM. Tissue thickness for each condition was determined by optical coherence tomography (OCT).


RESULTS
3D-HCE tissue revealed similar features with human cornea at histological level. After desiccation the percentage of living cells was only 32% in 3D-HCE tissue without pre-incubation and 98% in trehalose-pre-incubated tissue, as shown by a cell viability assay. These findings were confirmed by using a Live-Dead assay. Also, the confocal immunofluorescence analysis revealed much better preservation of tight junctions in trehalose-pre-treated tissue.


CONCLUSIONS
CLSM and an in vitro cell viability assays could be successfully used for the characterization of 3D-HCE tissue. We demonstrated the protective role of trehalose using reconstructed corneal epithelium (3D-HCE), which mimics HCE and has the potential to become a valuable model in ophthalmic research.
Department of Ophthalmology, University of Rostock, Doberaner Strasse 140, Rostock, Germany. marine.hovakimyan@uni-rostock.de
Marine
M
Hovakimyan
Theresa
T
Ramoth
Marian
M
Löbler
Klaus-Peter
KP
Schmitz
Martin
M
Witt
Rudolf
R
Guthoff
Oliver
O
Stachs
D002452:Cell Count; D002470:Cell Survival / Q000187:drug effects; D003890:Desiccation / Q000379:methods*; D015352:Dry Eye Syndromes / Q000188:drug therapy / Q000473:pathology*; D019573:Epithelium, Corneal / Q000187:drug effects / Q000473:pathology*; D006801:Humans; D021621:Imaging, Three-Dimensional; D018613:Microscopy, Confocal; D009883:Ophthalmic Solutions; D019108:Tight Junctions; D046509:Tissue Culture Techniques; D041623:Tomography, Optical Coherence; D014199:Trehalose / Q000008:administration & dosage*
D009883:Ophthalmic Solutions; D014199:Trehalose