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Dr.Pooja Khamar,Dr.Rohit Shetty 

Introduction

Laser in situ keratomileusis (LASIK), a refractive surgical procedure has gained popularity for correction of myopia, hyperopia and astigmatism, with proven safety and efficacy1.Post-LASIK ectasia (PLE) is one of the postoperative complications which is of major concern2. It is characterised by loss of uncorrected visual acuity, progressive thinning and protrusion usually in the central or paracentral cornea and topographical evidence of asymmetrical corneal steepening3. SMILE (Small-incision lenticule extraction) is relatively new refractive technique using flap-free intrastromal laser for correcting myopia and astigmatism4.

The molecular signature of the diseased tissue adds a new dimension to the existing knowledge of disease pathogenesis and its mechanism. We have reported increased corneal dendritic cell density and an aberrant inflammatory state in the tear cytokine profile of post-LASIK ectasia patients when compared with normal5. In another study, we observed that the local, tissue specific molecularfactors (LOX, MMP9, IL-6, IL-10, TNFα, TIMP1) at the ectatic region of corneal epithelium and stroma are responsible for the focal corneal weakening in keratoconus6.

Methods

The patients were divided into two groups

In Group A:The cross-sectional study was approved by the Ethics Committee of Narayana Nethralaya Eye Hospital (E.C. Ref No.: C/2015/05/05) and was conducted in accordance with guidelines of the Declaration of Helsinki. Written informed consent was obtained from all subjects. The study was designed to evaluate differences in levels of tear cytokines/chemokines and corneal DCD in patients with and without corneal ectasia following LASIK. The measurements were also normalised to the contralateral eye to reduce basal variation among the subjects and to determine the effect of corneal ectasia severity on the measured parameters. The eye with a higher degree of corneal ectasia (eye of interest) in a subject was determined based on Belin-Ambrósio Deviation Index (BAD-D).This retrospective study included age- matched 7 (14 eyes) post-LASIK controls (PLCs) and 6 (12 eyes) PLE subjects. Corneal topography was used to categorise the subjects into PLC and PLE groups.

IVCM has been a valuable, minimally invasive tool in assessing the status of antigen presenting cells (dendritic cells) in the cornea7. Cells were included after assessment of two sides of the image for cells that overlapped with the edge of the frame. Quantitative analyses of the sub basal nerve plexus features from IVCM images were performed using Automatic CCMetrics software, V.1.0 (University of Manchester, UK).The levels of inflammatory cytokines, chemokines and secreted cell adhesion molecules in the tears were measured using cytometric bead array, CBA (BD cytometric bead array (CBA) Human Soluble Protein Flex Set System, BD Biosciences) on a flow cytometer (BD FACSCalibur, BD Biosciences). The CBA was performed for the quantification of interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, chemokine (C-C motif) ligand (CCL)2 (monocyte chemotactic protein-1 (MCP1), CCL4 (macrophage inflammatory protein 1β (MIP1β)), CCL5 (regulated on activation, normal T cell expressed and secreted), CXCL10 (IP10), E-selectin and intercellular adhesion molecule 1 (ICAM1). The assay was a performed as per manufacturer’s instruction using BD Human Soluble Protein Master buffer kit. The capture beads and analyte signals were acquired by a flow cytometer using BD Cell Quest Pro Software (V.6.0). Computation of analyte signal intensities with reference to the respective standards to determine absolute concentrations of individual analytes were performed by BD FCAP Array Software (V.3). The absolute concentration of the analytes were later adjusted according to the tear elute volume (wetting length of Schirmer’s strip during tear collection and volume of tear elution buffer).

Group B: gene expression profile was done in stored lenticules of a single patient developing ectasia post SMILE(PSE) with normal topography and in controls (n=25) (did not develop ectasia) by qPCR. SMILE (small incision lenticule extraction) surgery was performed using VisuMax femtosecond laser system (Carl ZiessMeditec AG) with a 500 kHz repetition rate. Spot spacing was 2µm for creation of lenticule side cut and 4.5 µm for creation of lenticule with preset laser-cut energy of 170nJ and track distance of 3 mm. Cap thicknesses was kept as 110µm with lenticule diameter of 6.0mm and cap diameter of 7.7mm (optical zone). After creation of refractive lenticule, it was dissected and separated through the 3mm side-cut located superiorly and removed manually. Cornea was remoistened with a wet merocel sponge at the end of the procedure followed by one drop of moxifloxacin hydro- chloride 0.5% (Vigamox®, Alcon Laboratories, Inc., Fort Worth, TX, USA) was instilled in both eyes. Routine post-operative regimen was given in both eyes, which included moxifloxacin hydrochloride 0.5% eye drops (Vigamox®, Alcon Laboratories, Inc., Fort Worth, TX, USA) four times a day for 1 week, tapering doses of topical 1% Fluorometholone eye drops (Flarex®, Alcon Laboratories, Inc., Fort Worth, TX, USA) and topical lubricants (Optive, Allergan, Inc.) four times a day for three months.

Intra-operatively collected lenticules were immediately transferred to -80˚C for storage and hence used later for RNA extraction.Stromal lenticule was finely chopped using sterile surgical blade (ListerTM, Sterilized by Gamma Radiation, ISO 9001:2000 Co.) and total RNA was extracted using TRIZOL reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA) followed by quantification and quality assessment. Briefly, the quantitative real-time PCR cycle includes pre-incubation at 95˚C for 5min, 40 amplification cycles at 95˚C for 10s, 58˚C for 15s and 72˚C for 30s using a CFX ConnectTM real-time PCR detection system (Bio-Rad, Philadelphia, PA, USA). Total levels of LOX, MMP9, IL-6, IL-10, BMP7, TNFα, TIMP1, COL IA1, COL IVA1, CD68, CTSK, ITGβ1, STAT3, TGFβ and TGFβR2 were estimated after normalization to actin. Total RNA was also isolated from primary HCFs transduced for LOX over expression and matching controls for subsequent measurement of LOX (lysyl oxidase) expression. Primer sequences are available on request.

Results

Group A: Significantly higher inflammatory factors, corneal dendritic cell density and reduced corneal nerve features were observed in PLE compared to controls. sub basal nerve plexus morphology analysis based on IVCM images revealed CNFL and CNFD to be significantly lower in the eyes with higher degree of ectasia in the PLE group whereas, no differences were observed in any of the morphological features in the post-LASIK control group. It is important to note that the pre-LASIK sub basal nerve plexus morphology information of the patients with PLE were not available, since IVCM-based imaging is not a standard of care for pre-op evaluation for LASIK. To circumvent this caveat of the nerve morphology analysis, we have performed the contralateral eye comparison. Tear inflammatory cytokines, chemokines and secreted cell adhesion molecules levels were measured in both patients with PLC and PLE. Specif- ically, IL-2, CCL2/MCP1 and CXCL10/IP10 were found to be significantly higher in the PLE cohort . CCL2/MCP1 was found to show the highest fold increase (3.4±0.6; p=0.009) in the eyes with higher BAD-D in the PLE cohort compared with other inflammatory factors investigated in this study. Further- more, a positive correlation was also observed to exist between CCL2/MCP1 levels and total corneal DCD, as well as DCDs with dendritic process in the PLE cohort . The current observations strongly indi- cate the association between aberrant inflammatory status and LASIK-elated corneal ectasia.

Group B: The ectatic eye lenticule had reduced expression (fold change) of LOX (0.57), COL IA1 (0.063), compared to controls without ectasia. Increased mRNA fold change expression of TGFβ (1.5), BMP7 (1.4), IL-6 (1.5), CTSK (1.37), Integrin β1 (1.4) was noted in severely ectatic eye compared to controls. However, MMP9 and TIMP1 levels were not altered in the ectatic eye compared to controls. Ectopic LOX expression in HCF induced significantly more collagen gel contraction indicating its role in strengthening the corneal stroma.

Discussion

Lysyl oxidase (LOX) is a natural, collagen cross linking enzyme produced in the extra cellular matrix in cornea and known to be reduced in ectatic eye diseases8. LOX is a copper amine oxidase that catalyzes the formation of crosslinks between collagen bundles in the cornea providing them with strength and stiffness. Reduced LOX levels have been associated with disease severity of corneal ectatic disease, keratoconus9. Ectopic expression of LOX in primary corneal fibroblasts led to a dose dependent increase in tensile strength of the collagen matrix observed in the form of increased contraction of collagen gel. These data and the known literature of LOX supports a scenario where inherently lower levels of LOX could be a critical factor in predisposing corneas to ectasia post surgical insult. Blum et al,10 have reported in a study on post-operative outcomes of SMILE suggesting that it is an effective, stable and safe procedure currently in use for treating myopia and myopic astigmatism. According to a recent review by Moshirfar et al,11 out of 750,000 cases of SMILE only four cases of post-SMILE ectasia have been reported till date worldwide. Sachdev, et al reported a patient who developed post-SMILE ectasia despite presenting with normal pre-surgical clinical indices. However, no explanation of the possible underlying mechanism was offered. Our study provides a plausible reason to the development of post-SMILE ectasia by uncovering an altered, inherent tissue specific profile, in particular, reduced LOX levels, which could have predisposed an otherwise clinically healthy eye to develop ectasia post-SMILE surgery.

Conclusion

Aberrant inflammation and altered stromal molecular factors observed in ectasia are novel finds. They can be potential targets in future diagnostics & therapeutics. Our study is the first evidence to associate molecular profile of ectasia post-refractive surgery beyond topography. Tissue specific molecular factors and their response to healing post-surgery are shown to be important determinants of subsequent outcomes. Hence, determining inflammatory markers and LOX status in tears might aid in screening risk to post-refractive surgery ectasia.

Refrences

  1. Sugar A, Rapuano CJ, Culbertson WW, et al. Laser in situ keratomileusis for myopia and astigmatism: safety and efficacy: a report by the American Academy of Ophthalmology. Ophthalmology 2002;109:175–87.
  2. Binder PS. Ectasia after laser in situ keratomileusis. J Cataract Refract Surg2003;29:2419–29.
  3. 3 Binder PS, Lindstrom RL, Stulting RD, et al. Keratoconus and corneal ectasia afterLASIK. J Cataract Refract Surg2005;31:2035–8.
  4. Alio Del Barrio, J. L., Vargas, V., Al-Shymali, O. &Alio, J. L. Small incision lenticule extraction (SMILE) in the correction of myopic astigmatism: outcomes and limitations – an update. Eye and vision 4, 26, doi:10.1186/s40662-017-0091-9 (2017).
  5. Pahuja, N. K. et al. In vivo confocal microscopy and tear cytokine analysis in post-LASIK ectasia. The British journal of ophthalmology 101, 1604-1610, doi:10.1136/bjophthalmol-2016-309142 (2017)
  6. Pahuja, N. et al. Correlation of Clinical and Biomechanical Outcomes of Accelerated Crosslinking (9 mW/cm2 in 10 minutes) in Keratoconus with Molecular Expression of Ectasia-Related Genes. Current eye research, 1-5, doi:10.3109/02713683.2015.1133831 (2016).
  7. Villani E, Baudouin C, Efron N, et al. In vivo confocal microscopy of the ocular surface: from bench to bedside. Curr Eye Res 2014;39:213–31.
  8. Pahuja, N. et al. Correlation of Clinical and Biomechanical Outcomes of Accelerated Crosslinking
  9.  mW/cm2 in 10 minutes) in Keratoconus with Molecular Expression of Ectasia-Related Genes.Current eye research, 1-5, doi:10.3109/02713683.2015.1133831 (2016).
  10. Shetty, R. et al. Attenuation of lysyl oxidase and collagen gene expression in keratoconus patient corneal           epithelium corresponds to disease severity. Molecular vision 21, 12-25 (2015).
  11. Blum, M., Taubig, K., Gruhn, C., Sekundo, W. &Kunert, K. S. Five-year results of Small Incision Lenticule Extraction (ReLEx SMILE). The British journal of ophthalmology 100, 1192-1195, doi:10.1136/bjophthalmol-2015-306822 (2016).
  12. Moshirfar, M. et al. Ectasia following small-incision lenticule extraction (SMILE): a review of the literature. Clinical ophthalmology 11, 1683-1688, doi:10.2147/OPTH.S147011 (2017).

 

 

 

 

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