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Omer Iqbal; Keratoconus-An inflammatory Primary Ectasia?. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2993.
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© ARVO (1962-2015); The Authors (2016-present)
Keratoconus is designated as a non-inflammatory, bilateral, often asymmetric primary ectasia associated with irregular astigmatism and one of the common indications for corneal transplantation. There is a growing debate that Keratoconus may be an inflammatory condition. This study aims to determine the role of TNF-α, TGF-β2 and NF-κB in the pathogenesis of Keratoconus.
Under an Institutional Review Board approved protocol, surgically discarded and de-identified samples, normal and keratoconic corneas, were obtained from the Department of Ophthalmology at Loyola University Health System. A total of 12 normal and 12 keratoconic corneas were selected for this study. The tissue samples were fixed in formalin/PIPES buffer and stored at 4ᴼC until paraffin embedding and sectioning at 4 microns thickness. Deconvolution immunofluorescence (IF) was performed on unstained slides. After imaging, the relative expression (normal vs Keratoconus) of TNF-α, TGF-β2 and NF-κB was quantified in Imaris®. For TNF-α, TGF-β2 and NF-κB, Center channel measurements were obtained per 10µm cubes in the epithelial and stromal layers of both keratoconic and normal corneas. Samples devoid of intact epithelium were not quantified. Statistical analysis was performed using Graph Pad Prism one way ANOVA with multiple comparisons. To determine the intensity of NF-κB in the nucleus, surfaces were created around Imaris® in the 435 nm channel (Dapi stained nuclei) and the mean of Cy5 intensities was quantified within those volumes. Student’s unpaired t-test with Welch’s correction was performed in Graph Pad prism.
TGF-β2 and TNF-α, both demonstrated higher expression in the epithelial layer of keratoconic corneas when compared to the normal corneas (p=<0.0001). NF-κB had similar expression levels in the epithelial and stromal layers of keratoconic corneas when compared to normal corneas (p=0.64 and p=0.99 respectively). However, there was higher intensity staining of NF-κB in the nuclei of keratoconic corneal samples when compared to normal corneal samples (p=0.002).
Higher expression of TGF-β2, TNF-α and NF-κB in the epithelial and stromal layers and higher intensity staining of NF-κB in the nuclei of keratoconic corneas when compared to the normal corneas suggest the involvement of inflammatory processes in the pathogenesis of keratoconus.
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