Characteristics of KC Patients.

a<p>Grade 4 KC: Severe; VA &gt;6/7.5 with contact lens correction; severe corneal thinning and Munson’s sign.</p>b<p>DALK: Deep anterior lamellar keratoplasty.</p>c<p>Ectasia developed post-LASIK.</p

Graphs summarising semi-quantitative grading for SFRP immunolabelling of KC and control specimens.

<p>The graphs show the range of grading for SFRP1 to 5 immunostaining in the basal epithelium adjacent to the cone region, and a similar region in control corneas. SFRP1 to 4 immunostaining is significantly increased compared to controls (p&lt;0.05, Mann-Whitney test).</p

SFRP Immunolabelling: Semi-Quantitative Grading^a.

a<p>Semi-quantitative grading = Intensity+% immunolabelling (minimum = 0 and maximum = 6):</p><p><i>Intensity of Immunoreactivity.</i></p><p>0 = no staining.</p><p>0.5 = very weak.</p><p>1 = weak.</p><p>2 = moderate.</p><p>3 = strong.</p><p><i>% Immunolabelling.</i></p><p>0 = 0%.</p><p>1 = 1% to 10%.</p><p>2 = 11% to 50%.</p><p>3 = &gt;50%.</p>b<p>Significance.</p>c<p>SFRP1, 2 and 3 expressed in keratocytes only; SFRP5 expressed in stroma including matrix and cells.</p>d<p>Mann-Whitney test, p&lt;0.05 significant; NS - not significant.</p>e<p>ND - not detected.</p>f<p>KC full thickness graft n = 7/15; deep anterior lamellar keratoplasty (DALK) n = 8/15.</p

Characteristics of Control Corneas.

<p>Characteristics of Control Corneas.</p

Expression of SFRP Family Proteins in Human Keratoconus Corneas

<div><p>We investigated the expression of the secreted frizzled-related proteins (SFRPs) in keratoconus (KC) and control corneas. KC buttons (∼8 mm diameter) (n = 15) and whole control corneas (n = 7) were fixed in 10% formalin or 2% paraformaldehyde and subsequently paraffin embedded and sectioned. Sections for histopathology were stained with hematoxylin and eosin, or Periodic Acid Schiff’s reagent. A series of sections was also immunolabelled with SFRP 1 to 5 antibodies, visualised using immunofluorescence, and examined with a Zeiss LSM700 scanning laser confocal microscope. Semi-quantitative grading was used to compare SFRP immunostaining in KC and control corneas. Overall, KC corneas showed increased immunostaining for SFRP1 to 5, compared to controls. Corneal epithelium in all KC corneas displayed heterogeneous moderate to strong immunoreactivity for SFRP1 to 4, particularly in the basal epithelium adjacent to cone area. SFRP3 and 5 were localised to epithelial cell membranes in KC and control corneas, with increased SFRP3 cytoplasmic expression observed in KC. Strong stromal expression of SFRP5, including extracellular matrix, was seen in both KC and control corneas. In control corneas we observed differential expression of SFRP family proteins in the limbus compared to more central cornea. Taken together, our results support a role for SFRPs in maintaining a healthy cornea and in the pathogenesis of epithelial and anterior stromal disruption observed in KC.</p></div

Antibodies used for Immunohistochemistry.

<p>Antibodies used for Immunohistochemistry.</p

Light microscopy of control and KC corneas stained with PAS and Mayer's hematoxylin.

<p><b>a.</b> Control corneas showed uniform thickness multilayered epithelium in the central region, with increased stromal thickness towards the limbus. Blood vessels and undulating basal epithelium (asterisks) are only seen in the limbus region. <b>b.</b> In KC buttons, obvious epithelial thinning associated with stromal thinning is seen more centrally. <b>c to h.</b> Representative images at higher magnification show (c) control corneal epithelium and anterior stroma, (d) obvious epithelial thinning in the KC cone compared to the adjacent epithelium. (e, f) Enlarged basal epithelial cells and irregular Bowman’s layer (thinning and breaks, asterisks) are common features in KC specimens. In some KC specimens (g) evidence of incursion of stromal tissue between Bowman’s layer and the epithelium is seen (arrows). (h) A leukocyte is also seen in the KC basal epithelium, where stroma invaded between epithelium and Bowman’s layer (arrows). Epi: epithelium; bv: blood vessel; Adj: adjacent region; BL: Bowman’s layer.</p

Groups for TGF-β1 signaling pathway experiments.

<p>Groups for TGF-β1 signaling pathway experiments.</p

Groups for exogenous TGF-β1 treatment.

<p>Groups for exogenous TGF-β1 treatment.</p

Groups for NF-κB pathway.

<p>DMSO - Dimethyl sulfoxide (Research Organics, OH USA).</p