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Dr. Nimrata Bajaj Dhami, Dr. Rohit Shetty

AIM:

To determine biomechanical alterations associated with the zonal tissue changes between ectatic cone and non-ectatic peripheral regions in keratoconus (KC)

INTRODUCTION:

Keratoconus is an ectatic disorder of cornea which is characterized by progressive focal thinning and irregular astigmatism resulting in reduced visual acuity.1,2The clinical features depend on the stage of disease and range from myopia, irregular astigmatism, stromal thinning (centrally or paracentrally), Fleischer’s ring, prominent corneal nerves to Vogt’s striae.3Despite the well-described clinical signs, the pathogenesis of KC is not so well understood.Various studies have attempted to associate the proteomic and cytokine expression profile of various molecules like IL-1, IL-6, IL 17, TNF-α, TGF-β and collagen homeostasis to KC etiology and progression.4,5 According to previous studies done by our institute, we have also shown the association of increased levels of MMP 9, IL-6 expression in KC tears, corneal epithelial cells and reductionin levels of Lysyl oxidase (LOX), collagen I (COL IA1), collagen IV (COL IVA1) in KC cones.7 The role of studying corneal biomechanics in keratoconus diagnosis and management strategies also been highlighted by a few studies.8,9With the emerging understanding of KC as a focal biomechanical weakness of cornea10, rather than a generalised disease, our study fills the knowledge lacunae in correlating the molecular and biomechanical profiles to better understand and treat the disease.

MATERIALS AND METHODS:

INCLUSION CRITERIA

This study was approved by the Narayana Nethralaya institutional review board (Bangalore, India) and carried outas per Indian Council of Medical Research and institutional human ethics guidelines in accordance to ARVO’s policies and the tenets of the Declaration of Helsinki. All samples were collected after informed written consent. The study group was selected from patients who reported to the cornea clinic at Narayana Nethralaya. A total of 50 Keratoconus eyes undergoing corneal cross-linking procedure and 25 eyes with normal corneal topography, undergoing photorefractive keratectomy (PRK) for correction of refractive errors were included in the study.

EXCLUSION CRITERIA: H/O recent infection, ocular trauma, H/O ocular surgery (cataract, collagen cross linking), pregnant/ lactating females, contact lens wearers, systemic medications (esp. HRT, steroids, anti-inflammatory medications)

Pre-operatively, workup included -refraction and retinoscopy, slit-lamp biomicroscopy, corneal topography was done on Pentacam, zonal corneal deformation amplitude was collected from prototype next-gen Corvis- ST in the superior, inferior and central gaze giving corneal biomechanics in the downward, central and upward gaze respectively.

KC patients were further graded based on the Amsler– Krumeich classification based on the clinical and topographic imaging (Grade 1=24, Grade 2=20, and Grade 3=6). The tangential map from topography analysis on the Pentacam was used to determine the cone location.

Intra-operatively, the epithelium was derived from two zones – CONE & PERIPHERY, using a manual epithelium scrapper. Using a 4.5 mm trephine centred on the steepest area, the “cone” in KC patients, while the pupil centre in the subjects undergoing PRK was considered as the “cone”, which was debrided and collected followed by separate scraping over the remaining corneal surface upto 9mm cornea which was designated as the ‘‘periphery’’. Debrided epithelium was stored separately in Eppendorf vials in normal saline and immediately transferred to a biorepository at -80 degree Celsius and subsequently subjected for molecular expression and analysis using qPCR for RNA extraction and analysis.

The regional biomechanics was then correlated with the focal molecular expression of various extracellular matrix proteins and inflammatory genes profile already correlated with the immunopathogenesis driving keratoconus in the previous studies done by our institute.

STATISTICAL ANALYSIS:

All statistical analysis was performed using MedCalc v16.2.0 and GraphPad Prizm 6.0 Software. The values of the individual groups were reported as average±SEM. Linear regression analyses were performed for analysing gene expression ratios for each gene as well as clinical parameters in this study.

RESULTS:

The clinical parameters between the two study groups – 25 controls and 50 keratoconic eyes were as follows- the mean keratometry values (Km) of grade1, grade 2, grade 3 and controls were 53.4±0.2, 58.67±2.1, 63.4±2.9 and 44.9±0.9 D respectively. The mean refractive spherical equivalent MRSE was -2.05±0.5, -3.9±0.1, -8.1±0.2 and -3.2±0.5 D in the four groups respectively and the thinnest corneal thickness TCT is 468.2±6.1, 457.5±6.3, 426±9.5 and 505.2±6.5 µm respectively. All the four groups showed statistically significant difference in their clinical presentation.

The corvis deformation data was analysed using analytical viscoelastic biomechanical model to derive the corneal stiffness. The corneal stiffness was then correlated with the cone location derived from the keratometric information on the topographic imaging.

Comparison of corneal stiffness among various corneal zones in controls versus KC patients

CORNEAL ZONES CONTROLS (N/m) KC (N/m)
SUPERIOR 105±4 104±3
CENTRAL 106±3.5 90±2.7
INFERIOR 109±4.3 93±3

 

Superior zones (non-ectatic) showed statistically significant difference with the remaining zones central or inferior (ectatic) in KC (p=0.01) but not controls(p=0.4).

The results of the gene expression characteristics of the keratoconus cone relative to its periphery were reported as the ratio of gene expression from the cone to periphery for each subject in order use an internal control to reduce the inter-individual variations that arise due to epidemiological factors like age, gender, environmental stimuli and genetic makeup and hence get a deeper understanding of the focal nature of the disease process in keratoconus.

The molecular expression of LOX and Collagen IVA1 mRNA expression from cone epithelium showed significant differences from the periphery in keratoconus patients (p=0.04) and (p=0.023) respectively, whereas no such differences were seen in controls with healthy corneas. The cone area also showed elevated levels of inflammatory and matrix modulatory proteins like MMP9, TNF α and IL6 (p=0.038), (p=0.008) and (p=0.005) as compared to periphery in keratoconus patients but not in controls. The molecular profile could further be correlated with the grades of keratoconus. Corneal stiffness values were correlated with ectatic zone specific TNF-α and LOX levels.

DISCUSSION:

Independent studies including previous studies done by our institute have suggested that inflammation is an important factor driving keratoconus, which is in coherence with our results which show that keratoconus cones have increased inflammatory and matrix modulatory protein gene expression profile like MMP 9, IL 6, TNF α as compared to the surrounding peripheral cornea while the controls who have no such statistically significant differences from centre to periphery and much lower expression of such factors in their cornea as compared to keratoconus. Also, the lowered levels of LOX, which is the endogenous collagen crosslinker and Collagen IVA1 showed inherent biomechanical weakness of the KC cones, also elucidating the focal nature of the disease.

This observation provides the foundation for customisation and zone selectivity of our treatment approaches to KC, which show more regularisation of corneal surface, translating into better refractive outcomes and reduced aberrations. 11,12

Since such extensive molecular studies requires elaborate infrastructure, we tested the novel idea of correlating the molecular expression levels of KC cones with the multizonal biomechanics which can be derived from the new multi-dimensional corvis. Our data suggests a strong correlation of biomechanical indices with the disease areas of the cornea as shown, which can be an easy surrogate marker for understanding gene expression and focal nature of this disease.

Hence, this study elucidates that KC corneas are non-uniform at biomechanical and molecular levels, which could help plan future differential customized cross-linking treatment protocols and monitor disease progression beyond topographic indices.

References:

  1. Gordon-Shaag A, Millodot M, Shneor E, Liu Y. The genetic and environmental factors for keratoconus. Bio Med Res Int. 2015; 2015:795738.
  2. Zadnik K, Barr JT, Edrington TB, et al. Baseline findings in the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study. Invest Ophthalmol Vis Sci. 1998;39:2537–2546
  3. Rabinowitz YS. Keratoconus. Survey of ophthalmology. 1998 Jan 1;42(4):297-319.
  4. Wisse RP, Kuiper JJ, Gans R, Imhof S, Radstake TR, Van der Lelij A. Cytokine expression in keratoconus and its corneal microenvironment: a systematic review. Ocular Surf. 2015; 13:272–283.
  5. Ghosh A, Zhou L, Ghosh A, Shetty R, Beuerman R. Proteomic and gene expression patterns of keratoconus. Indian J Ophthalmol. 2013;61:389–391.
  6. Shetty R, Ghosh A, Lim RR, et al. Elevated expression of matrix metalloproteinase-9 and inflammatory cytokines in keratoconus patients is inhibited by cyclosporine A. Invest Ophthalmol Vis Sci. 2015;56:738–750.
  7. Shetty R, Sathyanarayanamoorthy A, Ramachandra RA, et al. Attenuation of lysyl oxidase and collagen gene expression in keratoconus patient corneal epithelium corresponds to disease severity. Mol Vis. 2015;21:12–25.
  8. Roy AS, Shetty R, Kummelil MK. Keratoconus: a biomechanical perspective on loss of corneal stiffness. Indian journal of ophthalmology. 2013 Aug;61(8):392.
  9. Bao F, Geraghty B, Wang Q, Elsheikh A. Consideration of corneal biomechanics in the diagnosis and management of keratoconus: is it important?. Eye and Vision. 2016 Dec;3(1):18.
  10. Pahuja N, Kumar NR, Shroff R, Shetty R, Nuijts RM, Ghosh A, Sinha-Roy A, Chaurasia SS, Mohan RR, Ghosh A. Differential molecular expression of extracellular matrix and inflammatory genes at the corneal cone apex drives focal weakening in keratoconus. Investigative ophthalmology & visual science. 2016 Oct 1;57(13):5372-82.
  11. Seiler TG, Fischinger I, Koller T, et al. Customized corneal cross-linking: one-year results. Am J Ophthalmol. 2016;166:14–21
  12. Roberts CJ, Dupps WJ, Jr. Biomechanics of corneal ectasia and biomechanical treatments. J Cataract Refract Surg. 2014;40:991–998.

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