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Nishal Kishinchand Primalani, Gary S L Peh, Khadijah Adnan, Jodhbir S Mehta; Evaluation of the migratory capacity of the human corneal endothelium across bare Descemet’s Membrane and exposed corneal stroma. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2069.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the migratory potential and cellular morphology of human corneal endothelial cells across bare Descemet’s membrane (DM) and peeled DM with exposed corneal stroma.
Donor human corneas unsuitable for corneal transplantation were used in this study. Corneas were washed in a saline buffered solution containing antibiotics and antimycotics. Under direct visualisation using a stereomicroscope, distinct regions within each corneal endothelial surface were exposed to insult. Up to 50% of a central 5mm area within each cornea was peeled to expose the underlying stroma whilst a further 15-25% was scratched with a sterile pipette tip (DM-intact). Each cornea was subsequently cultured in a 12-well plate containing F99 culture medium with rho-kinase inhibitor, Y-27632. The rate of recovery of each endothelial surface was compared across two groups of donors: young (ages 2, 23 and 40) and old (ages 64, 65 and 73). Over a 3-month period, the scratched and peeled areas of each cornea were exposed to Trypan Blue (TB). Positively stained areas were subsequently visualised and quantitatively assessed using pixel counts on Adobe Photoshop CS4. Morphological analysis along the scratched and peeled regions were performed at a single time point for each cornea using Alizharin Red under high power magnification and further assessed using Scanning Electron Microscopy.
Over the initial 2-week period, human cornea endothelium (ex-vivo) within the young group displayed up to 1.5 times the rate of recovery across bare (scratched) DM, compared to the old group (p<0.05). Over the initial 4-week period, cornea endothelium from all ages displayed up to a three-fold increased rate of recovery across bare DM, compared to exposed stroma (peeled DM) (p<0.05). Images obtained at all time points across this period showed progressively reduced TB staining of the bare DM with no significant change over exposed stroma. Over the 3 month period, gradually reduced TB staining of exposed corneal stroma across all donors suggested a recovering endothelial cell layer. This was confirmed on morphological analysis using Alizharin Red.
Our study shows that intact DM allows for a more rapid and complete corneal endothelial recovery through cell migration. The age of each cornea donor played a significant contributing factor in determining the rate of recovery.
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