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K. Mireskandari, A. Khalili, S. Tuft, M. Sleeman, I.K. Anderson, P.T. Khaw; A New Model of Investigating Corneal Stromal Fibrosis in vitro Using the Heidelberg HRT–II Confocal Microscope With the Rostock Cornea Module . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2746.
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© ARVO (1962-2015); The Authors (2016-present)
We investigate the potential of the Heidelberg HRT–II confocal microscope with Rostock cornea module as a new tool for the investigation of cellular behaviour and extracellular matrix modulation in the corneal stroma. In the laboratory, the technique may provide an alternative to sacrificing animals and specimens to obtain time point histology. The reproducibility and quantification of the images have not been previously defined; hence the implications of this technique currently remains uncertain.
We devised a novel corneal organ culture model to investigate and validate the Heidelberg HRT–II in–vivo confocal microscope with Rostock cornea module in controlled experiments. Thirty rabbit corneo–scleral segments were cultured for one week after wounding. The intra–observer variability of confocal images at various stromal depths in the short term was investigated. We also demonstrate how quantification is possible by controlling the settings on the machine and how these correlate with the histological grading of the samples.
The organ culture model was successfully adapted for imaging by the vertically mounted Heidelberg confocal microscope. Imaging was successfully performed and quantified in 90% of samples. The imaging failure rate of 10% was due to collapse of the gel under the organ culture and was related to the learning curve for the technique. The intra–observer reproducibility within and between imaging sessions was very good (r=0.975 and r=0.773 respectively; p=0.01). Histological comparison of sample demonstrates the contribution of the extracellular matrix and cellular elements to the image.
This technique can be utilized to enhance quantification during organ culture studies in the laboratory and may be useful in clinical trials of treatment for corneal disease.
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