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Clinical science
In vivo confocal microscopy and tear cytokine analysis in post-LASIK ectasia
  1. Natasha Kishore Pahuja1,
  2. Rohit Shetty1,
  3. Rashmi Deshmukh1,
  4. Anupam Sharma2,
  5. Rudy M M A Nuijts3,
  6. Vishal Jhanji4,5,
  7. Swaminathan Sethu2,
  8. Arkasubhra Ghosh2,6
  1. 1 Cornea and Refractive Services, Narayana Nethralaya, Bangalore, India
  2. 2 Narayana Nethralaya Foundation, GROW Research Laboratory, Bangalore, India
  3. 3 Cornea Clinic, Department of Ophthalmology, Maastricht University Medical Centre, Maastricht, The Netherlands
  4. 4 Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong, China
  5. 5 UPMC Eye Center, University of Pittsburgh School of Medicine, Pittsburgh, USA
  6. 6 Singapore Eye Research Institute, Singapore
  1. Correspondence to Dr Swaminathan Sethu, Narayana Nethralaya Foundation, GROW Research Laboratory, #258/A Hosur Road, Narayana Health City, Bangalore, KA 560099, India; swaminathansethu{at}narayananethralaya.com

Abstract

Aim Corneal keratectasia is one of the complications associated with laser in situ keratomileusis (LASIK) that results in vision impairment. The pathogenesis of post-LASIK ectasia (PLE) remains underexplored. We report the tear cytokine profile and in vivo confocal microscopy (IVCM) findings in eyes with PLE.

Methods 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. Ocular Surface Disease Index (OSDI) scores obtained were based on standard questionnaire and IVCM images were used to determine corneal dendritic cells density (DCD) and sub-basal nerve plexus morphology. Inflammatory cytokines/chemokines in the tears were quantified using flow cytometry based cytometric bead array.

Results Pentacam-based scores, OSDI scores and corneal DCD were significantly (p<0.05) higher in patients with PLE compared with PLC. Discomfort-related subscale of OSDI score exhibited a positive correlation with total corneal DCD in the PLE cohort. The fold difference of chemokine (C-C motif) ligand/monocyte chemotactic protein-1 (CCL2/MCP1) (3.4±0.6) was found to be significantly (p<0.05) higher in the PLE cohorts and a positive correlation between CCL2/MCP1 levels and total corneal DCD was also observed in the PLE cohort.

Conclusion The current study found a significant difference in the tear film cytokine profile between normal and PLE eyes. Presence of increased corneal dendritic cells and altered tear cytokines suggests an ongoing inflammatory response in PLE.

  • Post-LASIK ectasia
  • corneal dendritic cell density
  • OSDI
  • inflammation

https://doi.org/10.1136/bjophthalmol-2016-309142

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    Introduction

    Laser in situ keratomileusis (LASIK), a refractive surgical procedure has gained popularity for correction of myopia, hyperopia and astigmatism, with proven safety and efficacy.1 However, it entails both intraoperative and postoperative complications. Post-LASIK ectasia (PLE) is one of the postoperative complications which is of major concern.2 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 steepening.3 The incidence of PLE ranges from 0.04% to 0.6%.4 5 A variety of risk factors have been proposed for PLE including high myopia, lower preoperative corneal thickness, residual stromal bed less than 250 µm and abnormal topography.3 Randleman et al have devised a scoring system, which is a quantitative method to screen for patients at risk to develop ectasia.6 However, some patients develop ectasia without having the proposed risk factors.7 8 The role of inflammatory mediators in the mechanism of ectasia in keratoconus and possibly post-LASIK has also been suggested.9 We have previously reported the pivotal role of inflammatory mediators in the pathogenesis of keratoconus.10 Presence of inflammatory mediators and dendritic cells has been demonstrated using immunohistochemistry and in vivo confocal microscopy (IVCM) in various inflammatory corneal conditions, including advanced ectatic cornea.11–13 Improved understanding of the cellular and molecular pathobiology in PLE would provide alternate management strategies. We hypothesise that an altered inflammatory response may drive the pathobiological process in PLE. Therefore, we investigated the corneal dendritic cell density (DCD) and tear cytokine profile in patients with PLE.

    Methods

    Study design

    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).14

    Patients

    The study cohort included subjects suffering from corneal ectasia with history of LASIK (post-LASIK ectasia (PLE)) and those without corneal ectasia but with history of LASIK (post-LASIK non-ectatic controls–PLC). The subjects in these two groups were age, gender and post-LASIK duration matched. The subjects were recruited from our refractive clinics. Six patients with PLE with LASIK surgery performed elsewhere were included. Patients who had presented with complaint of increasing refractive error (myopia with or without astigmatism), loss of uncorrected visual acuity with history of good vision for a certain period after LASIK procedure, were included. In addition, there was topographic evidence of asymmetrical keratometric steepening, with or without central or paracentral corneal thinning with corresponding increase of surface elevation on the topography maps.3 Seven patients who had successful LASIK procedures during the same period at our centre and had not developed any ocular symptoms were included as PLC. Refractive indices and ectasia risk scores (see online supplementary table 1) suggested comparable preoperative parameters in both the study groups. Patients with a history or active allergic eye disease, dry eye disease, abnormal meibomian glands, history of eye rubbing, systemic autoimmune disease and contact lens users were excluded from the study.

    Supplemental material

    Clinical evaluation and investigations

    At the time of presentation, all patients underwent detailed routine clinical eye examination after obtaining a thorough medical history. Patients were subjected to corneal topography evaluations (Pentacam HR, Oculus, Wetzlar, Germany) as shown in figure 1. BAD-D-based evaluation of corneal ectasia was performed.14 Dry eye evaluation as described earlier15 was performed to rule out dry eye disease related symptoms. Meibomian gland status was evaluated on Keratograph 5M (OCULUS Optikgeräte GmbH, Germany). Laser scanning IVCM was performed to assess corneal DCD using the Rostock Corneal Module/ Heidelberg Retina Tomograph ll (RCM/HRT ll, Heidelberg Engineering GmBH, Dossenheim, Germany). Furthermore, subjective symptoms were graded using Ocular Surface Disease Index (OSDI) questionnaire. Total OSDI scores were subclassified into discomfort-related and vision-related subscales for further evaluation. OSDI scores were used to evaluate ocular surface symptoms in general, if any, and was not used as an assessment tool for inflammation-related symptoms.

    Figure 1
    Figure 1

    Four maps refractive of study subjects. (A) Representative refractive maps of a post-LASIK control subject. Axial/sagittal curvature map shows symmetrical keratometry with relative central flattening attributed to myopic ablation with normal corneal thickness map and elevation (front and back) maps. (B) Representative refractive maps of a post-LASIK ectasia subject. Axial/sagittal curvature map shows asymmetrical inferior steepening; corneal thickness map shows paracentral thinning; Elevation (front) map shows paracentral surface elevation corresponding to the area of thinning on thickness map; elevation (back) maps shows paracentral surface elevation corresponding to the area of elevation on elevation (front) map and thinning on thickness map. Topography for all subjects was obtained using the Pentacam HR, Oculus, Wetzlar, Germany. Similar observations were made in the subjects from the respective study groups. LASIK, laser in situ keratomileusis.

    In vivo confocal microscopy

    IVCM has been a valuable, minimally invasive tool in assessing the status of antigen presenting cells (dendritic cells) in the cornea.16 Corneal dendritic cells density (DCD) at the level of sub basal nerve plexus in the cornea was analysed using IVCM as described earlier.15 Briefly, five representative IVCM frames (400×400 microns2 each) taken from the centre of the cornea for each subject were analysed by a blinded experienced observer. Dendritic cells (cells/mm2) were quantified using Cell Count software (Heidelberg Engineering GmbH) by identifying bright individual cell bodies with or without dendritic process as shown in figure 2. 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) as described earlier.15 The nerve features quantified include corneal nerve fibre density (CNFD), the total number of major nerves per square millimetre; nerve fibre length (CNFL), the total length of all nerve fibres and branches (millimetres per square millimetre); nerve branch density, number of branches emanating from major nerve trunks per square millimetre; total branch density the total number of branch points per square millimetre; the nerve fibre area and the total nerve fibre area per square millimetre and the average nerve fibre width per square millimetre. Two blinded observers analysed the images and average of the values were used for statistical analysis.

    Figure 2
    Figure 2

    Increased corneal dendritic cells density in post-LASIK ectasia. Panels exhibit laser-scanning in vivo confocal microscopy (IVCM) images exhibiting corneal sub basal nerve plexus region with or without dendritic cells (DCs) from post-LASIK control (A and B) and post-LASIK ectasia (C and D) subjects. DCs with dendritic process are indicated with blue arrow and those without dendritic process are indicated with yellow arrows. (Please see online version for colour image.) Panels shown are representative IVCM images with frame size 400×400 microns2 at a depth of 45 microns. LASIK, laser in situ keratomileusis.

    Tear collection

    Tear samples were collected using Schirmer’s strips (Whatman filter paper, 5×35 mm2, ContaCare Ophthalmics and Diagnostics, India) by following Schirmer’s test I protocol and stored in a sterile microcentrifuge tube at −80°C until further use. The wetting length of the Schirmer’s strip at the time of tear collection was recorded. Tear analytes were extracted from Schirmer’s strips by cutting the latter into small pieces and incubating in sterile 1 x phosphate buffered saline (PBS) for 2 hours at 4°C with agitation. The tear elute was separated from the pieces of the Schirmer’s strip by centrifugation at 4°C.

    Cytometric bead array

    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).

    Statistical analysis

    Based on the distribution of data (Shapiro-Wilk normality test), statistical tests such as t-test and Mann-Whitney test were performed. A p value <0.05 was considered to be statistically significant. Statistical dependence between two variables was measured by Spearman’s rank correlation analysis. Statistical analyses were performed using MedCalc V.12.5 (MedCalc Software bvba, Belgium) and GraphPad Prism V.6.0 (GraphPad Software, La Jolla, California, USA). Data are represented as both mean±SEM and median with range.

    Results

    Visual acuity and refractive parameters of the study cohort are listed in table 1. Corneal ectasia was determined on the basis of corneal topography (refractive maps) as shown in figure 1. The PLC group did not show any clinical or topographic signs of ectasia. Parameters such as BAD-D, OSDI, corneal DCD and tear inflammatory factors were measured and analysed in seven PLC subjects and six patients with PLE. Significantly higher BAD-D values were observed in patients with ectasia (PLE) compared with controls (PLC; table 1). We observed bilaterally asymmetrical BAD-D values in the study cohort. Therefore, BAD-D ratio was used to evaluate the differences between two eyes of each individual. BAD-D ratio was obtained by dividing the BAD-D values of the eye with higher BAD-D score by the BAD-D values of the contralateral eye in the same subject. We observed significantly higher BAD-D ratio in the PLE group compared with the controls (table 1). Patients with PLE recorded significantly higher total OSDI scores including discomfort-related and vision-related OSDI subscales compared with PLC (table 1). Laser-scanning IVCM-based investigations showed the presence of corneal dendritic cells in the sub basal nerve plexus region in the study cohort (figure 2). However, the significantly higher numbers of dendritic cells, including dendritic cells with and without dendritic process were observed in the patients with PLE compared with PLC (table 1). In addition, we observed ~threefold increase in corneal DCD (dendritic cells with and without dendritic process) in eyes with higher BAD-D score compared with the contralateral eye in the same subject in the PLE group, whereas, no such difference was observed in PLC (table 1). Total OSDI score and discomfort-related OSDI subscale exhibited a positive correlation with the density of dendritic cells with and without dendritic process in the PLE cohort (table 2). In addition, 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 (see online supplementary table 2), whereas, no differences were observed in any of the morphological features in the post-LASIK control group (see online supplementary table 2). 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 preop evaluation for LASIK. To circumvent this caveat of the nerve morphology analysis, we have performed the contralateral eye comparison.

    View this table:
    Table 1

    Study parameters in post-LASIK control and ectasia cohorts

    View this table:
    Table 2

    Correlation between OSDI scores and corneal dendritic cell density in post-LASIK control and ectasia cohorts

    Tear inflammatory cytokines, chemokines and secreted cell adhesion molecules levels were measured in both patients with PLC and PLE (absolute concentration shown in online supplementary table 3). Increased tear inflammatory factors correlated with higher BAD-D scores in PLE subjects compared with PLC (table 3A and online supplementary figure 1). Specifically, IL-2, CCL2/MCP1 and CXCL10/IP10 were found to be significantly higher in the PLE cohort (table 3A). 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. Furthermore, 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 (table 3B and online supplementary table 4). The current observations strongly indicate the association between aberrant inflammatory status and LASIK-elated corneal ectasia.

    Supplemental material

    View this table:
    Table 3A

    Tear inflammatory mediators in post-LASIK control and ectasia cohorts

    View this table:
    Table 3B

    Correlation between CCL2/MCP1 and corneal dendritic cell density in post-LASIK control and ectasia cohorts

    Discussion

    An effective strategy to manage PLE is to arrest the ectatic process, in addition to correcting corneal curvature irregularity and residual refractive error.17 Corneal ectasia has previously been considered to be a non-inflammatory condition.18 However, recent evidence on the pathobiology of keratoconus, an ectatic corneal disease has proven it otherwise.19 Furthermore, we have reported reduction in progression of ectasia in keratoconus by the use cyclosporine A, an immunomodulatory agent.10 Current knowledge thus emphasises the need to investigate the inflammatory status of corneal ectasia following LASIK. Post-LASIK dry eye is another common postoperative complication that is inflammatory in nature.20 Altered DCD has been observed in dry eye disease.15 Interestingly, we observed an increase in the density of corneal dendritic cells in PLE and threefold higher DCD in eyes with a higher degree of ectasia (table 1). DCs are potent antigen presenting cells well known to play a critical role in the mediation of inflammatory and immune responses. They bring about their actions either by direct interaction with other cell types or by the secretion of proinflammatory mediators. Although dry eye is one of the postoperative complications of LASIK, it is important to note that subjects in the current study were not suffering from either evaporative or aqueous deficient dry eye. Patients with PLE did not present with any other ocular infectious or inflammatory conditions which could possibly contribute towards the increased DCD. Markedly higher OSDI scores in the PLE subjects compared with controls (table 1) with both discomfort-related and vision-related scales contributing to it indicate vision-related and pain-related symptoms in PLE subjects at the time of presentation. Increased corneal DCD exhibited significant correlation with OSDI scores (table 2). This was similar to that observed in our earlier reports on evaporative dry eye15 which reiterates the association between increased corneal DCD and ocular surface disease symptoms. Corneal sub basal nerve density was observed to have increased in 3 months after LASIK in patients without ectatic changes.21 However, a decrease in CNFL and CNFD was measured in the eyes with higher degree of ectasia in the PLE group. However, the observation lacks robustness due to the non-existence of pre-LASIK sub basal nerve plexus morphology information, as IVCM-based imaging is currently not a standard of care for preop evaluation for LASIK. It would be beneficial to study corneal sub basal nerve plexus morphology both preoperatively and postrefractive surgery to understand its role in PLE. This would be of particular relevance in those who opt for LASIK but have been suffering from dry eye or are long-term contact lens users, since these have been associated with changes in the corneal sub basal plexus nerve morphology.15 22 Such altered corneal sub basal plexus nerve morphology along with changing molecular signature can predispose to events leading to corneal weakening and ectasia. Hence, long-term prospective studies are essential to confirm this hypothesis and render IVCM imaging a worthwhile tool in determining additional ectasia risk markers.

    The current study for the first time shows an increase in the levels of various tear inflammatory cytokines/chemokines in PLE. Furthermore, the levels of inflammatory factors were found to be few folds higher in the tears from eyes of PLE with greater degree of ectasia. The ultrastructure studies have revealed altered lamellar collagen organisation, collagen fibril thinning, decreased epithelial hypoplasia, Bowman’s layer breaks and stromal thinning in the ectatic region of the cornea.23 24 Functional association between collagen degradation process (characteristic of ectasia) and inflammation has been reported.25 Other factors such as pregnancy and eye rubbing that serve as triggers for PLE also suggest a plausible inflammatory component to the pathogenesis of PLE. Of the various inflammatory mediators measured in the current study, IL-2, MCP-1/CCL2 and CXCL10/IP10 levels were found to be significantly higher in PLE (table 3A). These inflammatory factors regulate corneal immune cell infiltration and trafficking in ocular surface conditions such as dry eye disease as well as infections and injury. IL-2, which regulates T cell function, is elevated in dry eye disease and its targeting results in alleviation of the disease.26 CXCL10/IP10, a chemokine that mediates trafficking of effector T cells and natural killer cells is expressed by activated immune cells, keratinocytes, fibroblasts and endothelial cells. It is reported to be increased in the tear fluid of patients with dry eye associated with Sjögren’s syndrome.27 MCP-1, a chemokine produced by a wide variety of immune cells and non-immune cells including keratocytes, epithelial cells and fibroblasts attracts monocytes, dendritic cells and T cells to the site of disease, injury or infection.28 In vitro and human studies have reported an increase in the levels of MCP-1 following LASIK.29 In the current study we observed a positive correlation between the MCP-1 levels and corneal DCD in PLE (table 3B). Migratory, tissue homing, maturation and T cell interaction properties of dendritic cells, necessary for mounting an immune response, are influenced by MCP-1.30 Similar, associations and functional interactions between dendritic cells and MCP-1 has been reported in amyotrophic lateral sclerosis spinal cord tissue.31

    The limitations of the study include small sample size and lack of comprehensive preoperative information, mainly because the PLE cases underwent LASIK in other centres. Nevertheless, our preliminary observations suggesting an aberrant inflammatory state is rather compelling in PLE, as evidenced by increased corneal DCD and tear cytokine/chemokine levels. Hence, managing the underlying subclinical inflammation could be a plausible additional strategy to corneal collagen cross-linking, intrastromal corneal ring segments and deep anterior lamellar keratoplasty in the treatment of PLE.

    Acknowledgments

    The authors thank Dr Abhijit Sinha-Roy, Narayana Nethralaya Foundation, Bangalore for his valuable inputs towards corneal topography and statistical analysis. The authors also thank Dr Rayaz Malik, University of Manchester, UK for CCMetrics Corneal Nerve Fibre Quantification software.

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    Footnotes

    • Contributors Study concept and design (RS, AG, VJ, SS, RMMAN). Data collection (NKP, RD, AS). Analysis and interpretation of data (RS, AG, SS). Writing the manuscript (AG, SS). Critical revision of the manuscript (RS, AG, SS). All of the authors were involved in the finalisation of the manuscript.

    • Funding This work was supported by Narayana Nethralaya Foundation, Bangalore, India.

    • Competing interests None declared.

    • Ethics approval Ethics Committee of Narayana Nethralaya Eye Hospital.

    • Provenance and peer review Not commissioned; externally peer reviewed.