1. Corneal morphology in Epithelial Basement Membrane Dystrophy: A large cross-sectional study with in vivo confocal microscopy Johan Germundsson M.D, Per Fagerholm M.D Ph.D, Neil Lagali Ph.D Department of Clinical and Experimental Medicine Faculty of Health Sciences, Linköping University Linköping, Sweden Authors have no financial interest

2. Purpose To document corneal morphology in a large group of patients with Epithelial Basement Membrane Dystrophy (EBMD), in both surgically-treated and non-treated eyes To document corneal morphology in a large group of patients with Epithelial Basement Membrane Dystrophy (EBMD), in both surgically-treated and non-treated eyes

3. Methods We examined, under a 6 month period in 2009, 76 eyes of 39 EBMD patients treated with phototherapeutic keratectomy (PTK) between the years 2001-2008. There were 52 operated and 24 fellow eyes. We examined, under a 6 month period in 2009, 76 eyes of 39 EBMD patients treated with phototherapeutic keratectomy (PTK) between the years 2001-2008. There were 52 operated and 24 fellow eyes. Slit lamp examinations and in vivo confocal microscopy (IVCM) were performed Slit lamp examinations and in vivo confocal microscopy (IVCM) were performed By IVCM, we: By IVCM, we: Determine the presence of map, dot, or fingerprint features Determine the presence of map, dot, or fingerprint features Grade the severity with a numerical score in a blinded fashion Grade the severity with a numerical score in a blinded fashion Quantify subbasal nerve density Quantify subbasal nerve density Assess stromal morphology Assess stromal morphology

4. Grading 1. Mild 1. Mild  Presence of isolated, cell-sized dot opacities and/or small (< 250  m) isolated regions with folding of basement membrane 2. Intermediate 2. Intermediate  Larger dots or confluent dot features, map-like regions, and inhomogeneous epithelial cells 3. Severe 3. Severe  Extensive basement membrane folds, large map regions (with dimensions exceeding 1mm), and large, extensive dot opacities Three-point grading scale was used to classify the severity of EBMD morphology as observed by IVCM Two observers independently graded 76 images in a blinded fashion. Grading differed by 1 grade in 7 cases (p = 0.89, Mann-Whitney Rank Sum Test)

5. Results - EBMD morphology Recurrence of EBMD morphology was seen in 33% of operated eyes by IVCM, with a mild to moderate grade (mean score 1.5) Recurrence of EBMD morphology was seen in 33% of operated eyes by IVCM, with a mild to moderate grade (mean score 1.5) EBMD morphology was noted in 78% of fellow eyes, with a grade of moderate to severe (mean score 2.5) EBMD morphology was noted in 78% of fellow eyes, with a grade of moderate to severe (mean score 2.5) EBMD grade was significantly more severe in unoperated eyes (p<0.001, Mann-Whitney test) EBMD grade was significantly more severe in unoperated eyes (p<0.001, Mann-Whitney test)

6. Results - subbasal nerve density Trend towards regeneration with postoperative time Trend towards regeneration with postoperative time Similar density between eyes operated in 2001 and unoperated eyes Similar density between eyes operated in 2001 and unoperated eyes Nerve density was measured by two independent observers Nerve density was measured by two independent observers 95% LOA for difference in nerve density: -3260 to 3098  m/mm 2 (76 measurements) 95% LOA for difference in nerve density: -3260 to 3098  m/mm 2 (76 measurements) Average interobserver difference in nerve density was ± 9% Average interobserver difference in nerve density was ± 9%

7. Subbasal nerve density: EBMD vs Normal In EBMD eyes operated in 2001 (n=4, 60.8±20y) In EBMD eyes operated in 2001 (n=4, 60.8±20y) 17191±8598 µm/mm 2 (mean ± SD) 17191±8598 µm/mm 2 (mean ± SD) In unoperated EBMD eyes (n=24, age 57.5±14y) In unoperated EBMD eyes (n=24, age 57.5±14y) 14370±6931 µm/mm 2 14370±6931 µm/mm 2 In a normal population (n=20, age 60.7±7y) In a normal population (n=20, age 60.7±7y) 10552±6688 µm/mm 2 10552±6688 µm/mm 2 Patel DV et al., Cornea 2009; 28:735-40. Patel DV et al., Cornea 2009; 28:735-40. ~17000 µm/mm 2 ~17000 µm/mm 2 Niederer RL et al., Br JOphthalmol 2007; 91:1165-69. Niederer RL et al., Br JOphthalmol 2007; 91:1165-69. Large inter-patient variations in subbasal nerve density Large inter-patient variations in subbasal nerve density No clear differences between EBMD (unoperated or 8y postop) and a normal age-matched population No clear differences between EBMD (unoperated or 8y postop) and a normal age-matched population

8.  IVCM images from the stroma of 6 different patients with EBMD  In 29% of all eyes examined, abnormal stromal nerve morphology was observed with nerves exhibiting a tortuous path, being intimately associated with stromal cells  Abnormally tortuous stromal nerves, with multiple nerve-keratocyte linkages.  Seen in 9 operated and 14 unoperated eyes Results - nerve morphology

9. Conclusions EBMD grade was significantly reduced in eyes operated by PTK (p<0.001) EBMD grade was significantly reduced in eyes operated by PTK (p<0.001) Subbasal nerves regenerate over time after PTK Subbasal nerves regenerate over time after PTK Subbasal nerve density in EBMD does not appear to be pathologically low Subbasal nerve density in EBMD does not appear to be pathologically low Examining corneas with EBMD using IVCM may give new insight into the disorder and the efficacy of PTK treatment Examining corneas with EBMD using IVCM may give new insight into the disorder and the efficacy of PTK treatment We believe this to be the first report of stromal abnormalities in EBMD We believe this to be the first report of stromal abnormalities in EBMD