FP453 : Analysis of Confocal Microscopy Using A Novel Software In Healthy And Diseased Cornea

Dr.Shreya Shah, Dr.Rohit Shetty,Dr.NEHA SHILPY 

Presenting author: Dr. Shreya Shah

Co-Authors: Dr. Rohit Shetty , Dr. Arkasubhra Ghosh, Dr. Swaminathan Sethu, Dr. Neha Shilpy

Introduction

In vivo confocal microscopy (IVCM) is becoming an indispensable tool for studying corneal physiology and disease. This technique enables the imaging of the corneal architecture at a cellular level and offers a fast and non-invasive in vivo imaging of the cornea comparable to ex vivo histochemical techniques. IVCM is used to study corneal diseases such as dry eye disease (DED), ectasia, keratitis and dystrophies[1]

Alternations in corneal nerves, epithelial cells, keratocytes, endothelial cells, and immune cells have been demonstrated on IVCM in different ocular and systemic diseases [1-4]. The hallmarks of DED include discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface. There is a lack of substantial understanding of the etiopathology of the symptoms and their association with other in vivo clinical findings. The source of ocular discomfort or pain in DED cannot solely be explained by tear film metrics suggesting the role of other factors in causation of symptoms. IVCM studies provide valuable insights into the etiology of DED and allow longitudinal imaging and quantification of cellular changes such as dendritic cells and subbasal nerve plexus morphology in the cornea of patients over time.

Keratoconus (KC) is characterized by stromal thinning and protrusion of the cornea. Bilateral disease is more common, however, the frequency of unilateral disease has been reported to range from 14.3% to 41% [5]. Unilateral KC provides insight into disease pathogenesis as the unaffected fellow eye acts as an ideal control for the affected one with other contributing factors constant for both. Corneal nerves are known to regulate multiple pathways, which play a role in KC disease pathogenesis [6]. In-vivo confocal microscopy (IVCM) studies have demonstrated changes in the Sub-basal nerve plexus (SBNP) of patients suffering from KC, however it’s role in Unilateral KC has not been described so far [7]. 

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. Some studies suggest the role of inflammatory mediators in the pathogenesis of PLE. 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. [8-10] 

Purpose:

Using a novel software to quantify corneal nerve morphological changes in healthy and diseased corneas by in vivo confocal microscopy(IVCM)

Materials and methods:

A cross-sectional study consisting of three arms was undertaken after prior ethics committee approval. The first arm had 52 evaporative dry eye (EDE) patients and 43 normal individuals. The second arm consisted of 30 eyes of healthy patients and 33 patients with unilateral keratoconus with 33 normal eyes and 33 affected eyes. The third arm had 12 eyes of post LASIK ectasia (PLE) subjects and 14 eyes of post-LASIK controls (PLCs) After obtaining prior informed consent, all participating study patients underwent detailed ophthalmic examination followed by in vivo confocal microscopy (IVCM) to study the corneal nerve characteristics in each of the cohorts.

In vivo confocal microscopy

IVCM imaging was performed using Rostock Corneal Module/Heidelberg Retina Tomograph ll (RCM/HRT ll; Heidelberg Engineering GmBH, Dossenheim, Germany). [5] The device uses a diode laser of 670 nm wavelength. 0.5% proparacaine drops were used to anaesthetize the cornea before the procedure. Study subjects were asked to fixate on a distant target such as to enable examination of the central cornea. The central cornea was scanned in a single area at a desired depth. A drop of 0.5% moxifloxacin was instilled after the procedure. 

Corneal sub-basal nerve plexus and dendritic cell density assessments 

Five representative IVCM frames (400×400 microns 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 dendritiform structures with cell bodies in each image at the level of basal epithelium or at subbasal nerve plexus [11].

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 nerve features quantified included corneal nerve fiber density (CNFD), the total number of major nerves per square millimeter; nerve fiber length (CNFL), the total length of all nerve fibers and branches (millimeters per square millimeter); nerve branch density (CNBD), number of branches emanating from major nerve trunks per square millimeter, total branch density (CTBD) the total number of branch points per square millimeter; the nerve fiber area (CNFA) and the total nerve fiber area per square millimeter and the nerve fiber width (CNFW) the average nerve fiber width per square millimeter [3,7].

Results:

IVCM investigations revealed the presence of corneal dendritic cells (DCs) in EDE. Image based analyses revealed a significant increase in corneal dendritic cell (DC) density and subsets (DCs with and without dendritic processes) in the eyes of EDE patients compared to controls. SBNP features such as .

Keratoconus eyes had significantly lower CNFD compared to controls (?< 0.001) and contralateral unaffected eye (? = 0.01). The CNFL also followed a similar pattern being lower in keratoconic eye compared to controls (p =0.001) and contralateral unaffected eye (p = 0.02). CNBD was significantly higher in eyes with decentered cones (>2mm outside the optical zone).

Significantly higher numbers of dendritic cells, including dendritic cells with and without dendritic process were observed in the patients with PLE compared with post-LASIK controls. (p<0.05) In addition, sub basal nerve plexus morphology analysis 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.

Conclusion:

IVCM analysis using Automated CCmetrics software offers a fast, unique and non-invasive method to study SBNP in a myriad of conditions, and may be used as an imaging marker for early diagnosis of disease.

Discussion:

The persistence of ocular pain and discomfort in a subset of patients with DED following standard therapeutic strategies as well as the lack of tear film metrics to predict this population poses a major challenge in the management of DED. It is therefore imperative to identify diagnostic modalities that can accurately predict patients whose symptoms may not resolve with conventional therapy or may require additional dietary or environmental interventions along with topical therapy to ensure a favourable prognosis. IVCM used to study architecture of the cornea in dry eye and other ocular conditions can provide additional predictive information such as corneal DCD and SBNP features which are altered in DED. In our study, changes in sub-basal nerve plexus, revealed decrease in CNFL, CNBD and CNFD particularly in patients with higher discomfort or exaggerated symptoms. Presence of DCD’s has been established in various inflammatory corneal pathologies as well as their role in mediating a nociceptive response to stimuli [20]. In our study,

A significant increase in dendritic cell density was observed in the EDE cohort. We propose that an increase in inflammatory cells and the associated changes in sub-basal nerve plexus may be responsible for ocular discomfort experienced by patients in our cohort. Furthermore, an increase in the number of dendritic cells in close proximity to the sub-basal nerves was observed in patients with severe symptoms. Whether DC- mediated inflammatory or physical irritation of the nerve or changes in nerve physiology are responsible for pain in these patients needs to be determined.

Light and electron microscopy studies have demonstrated involvement of all layers of the cornea in KC [12,13] in vivo studies suffer from a limitation of low resolution. IVCM makes it possible to dissect the corneal layers to the resolution of a few microns at the cellular level [6]. The sub-basal nerve plexus has been mapped using the IVCM in KC with gross abnormal morphological changes even in patients with subclinical KC [14].

The plexus of nerves in KC shows a reduced nerve fiber density and increased tortuosity as compared to controls [15]. In our study, the quantification of the subbasal nerves was done by an automated software which extracts the nerve fiber data from a raw image thereby giving a “response” image which provides automated quantitative data regarding the CNFD, CNFL, CNBD, CTBD, CNFA, and CNFW. The analysis is objective, quick, and more reliable with negligible inter/intra-observer variability [16]. In our study, the quantitative analysis between the affected eye of unilateral KC and controls revealed a significant reduction in CNFD and CNFL.

The sub-basal nerve fiber quantitative changes might help in establishing a diagnosis of KC in these eyes on follow- up even before it is manifested clinically. Besides serving as a disease marker it can also aid in monitoring disease progression.

Corneal ectasia, which was previously considered to be a non-inflammatory condition has been proven to be otherwise. [17,18] Current knowledge thus emphasises the need to investigate the inflammatory status of keratectasia following LASIK.  Post-LASIK dry eye is another common postoperative complication that is inflammatory in nature. [19] Altered DCD has been observed in dry eye disease. [20] 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 preoperative evaluation for LASIK. It would be beneficial to

Study corneal sub-basal nerve plexus morphology both preoperatively and post refractive 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. [20,21] 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. 

References:

1. Alhatem, B. Cavalcanti, and P. Hamrah, “In vivo confocal 
microscopy in dry eye disease and related conditions,” Seminars 
in Ophthalmology, vol. 27, no. 5-6, pp. 138–148, 2012.
2. L. Niederer and C. N. J. McGhee, “Clinical in vivo confocal microscopy of the human cornea in health and disease,” Progress 
in Retinal and Eye Research, vol. 29, no. 1, pp. 30–58, 2010.
3. Pritchard, K. Edwards, A. M. Shahidi, G. P. Sampson, A. W. Russell, R. A. Malik and N. Efron, “Corneal markers of diabetic neuropathy,” Ocul Surf, vol. 9, no. 1, pp. 17-28, 2011.
4. V. Patel and C. N. J. McGhee, “In vivo laser scanning 
confocal microscopy confirms that the human corneal sub- basal nerve plexus is a highly dynamic structure,” Investigative Ophthalmology & Visual Science, vol. 49, no. 8, pp. 3409–3412, 2008.
5. H. Krachmer, R. S. Feder, and M. W. Belin, “Keratoconus and related noninflammatory corneal thinning disorders,” Survey of Ophthalmology, vol. 28, no. 4, pp. 293–322, 1984.
6. S. Shaheen, M. Bakir, and S. Jain, “Corneal nerves in health and disease,” Survey of Ophthalmology, vol. 59, no. 3, pp. 263– 285, 2014.
7. V. Patel and C. N. J. McGhee, “In vivo confocal microscopy of human corneal nerves in health, in ocular and systemic disease, and following corneal surgery: a review,” British Journal of Ophthalmology, vol. 93, no. 7, pp. 853–860, 2009.
8. Fan Gaskin JC, Loh IP, McGhee CN, et al. An immunohistochemical study of inflammatory cell changes and Matrix remodeling with and without acute Hydrops in Keratoconus. Invest Ophthalmol Vis Sci 2015;56:5831–7.
9. Mayer WJ, Mackert MJ, Kranebitter N, et al. Distribution of antigen presenting cells in the human cornea: correlation of in vivo confocal microscopy
and immunohistochemistry in different pathologic entities. Curr Eye Res 2012;37:1012–8.
10. Mastropasqua L, Nubile M, Lanzini M, et al. Epithelial dendritic cell distribution in normal and inflamed human cornea: in vivo confocal microscopy study. Am J Ophthalmol 2006;142:736–44.
11. Kheirkhah, R. Muller, J. Mikolajczak et al., “Comparison of standard versus wide-field composite images of the corneal subbasal layer by in vivo confocal microscopy,” Investigative Opthalmology & Visual Science, vol. 56, no. 10, pp. 5801–5807, 2015.
12. Sykakis,F.Carley,L.Irion,J.Denton,andM.C.Hillarby,“An in depth analysis of histopathological characteristics found in keratoconus,” Pathology, vol. 44, no. 3, pp. 234–239, 2012.
13. Sawaguchi,T.Fukuchi,H.Abe,T.Kaiya,J.Sugar,andB.V.J.T. Yue, “Three-dimensional scanning electron microscopic study of keratoconus corneas,” Archives of Ophthalmology, vol. 116, no. 1, pp. 62–68, 1998.
14. Bitirgen, A. Ozkagnici, B. Bozkurt, and R. A. Malik, “In vivo corneal confocal microscopic analysis in patients with keratoconus,” International Journal of Ophthalmology, vol. 8, no. 3, pp. 534–539, 2015.
15. S. Mannion, C. Tromans, and C. O’Donnell, “An evaluation of corneal nerve morphology and function in moderate kerato- conus,” Contact Lens and Anterior Eye, vol. 28, no. 4, pp. 185–192, 2005
16. A.Dabbah, J.Graham, I.N.Petropoulos, M.Tavakoli,and R. A. Malik, “Automatic analysis of diabetic peripheral neuropathy using multi-scale quantitative morphology of nerve fibres in corneal confocal microscopy imaging,” Medical Image Analysis, vol. 15, no. 5, pp. 738–747, 2011.
17. Jhanji V, Sharma N, Vajpayee RB. Management of keratoconus: current scenario. Br J Ophthalmol 2011;95:1044–50.
18. Galvis V, Sherwin T, Tello A, et al. Keratoconus: an inflammatory disorder? Eye 2015;29:843–59.
19. Shtein RM. Post-LASIK dry eye. Expert Rev Ophthalmol 2011;6:575–82.
20. Shetty R, Sethu S, Deshmukh R, et al. Corneal dendritic cell density is associated with subbasal nerve plexus features, ocular surface Disease Index, and serum vitamin D in evaporative Dry Eye Disease. Biomed Res Int 2016;2016:1–10.
21. Lum E, Golebiowski B, Swarbrick HA. Mapping the corneal sub-basal nerve plexus in Orthokeratology Lens wear using in vivo laser scanning confocal microscopy. Invest Ophthalmol Vis Sci 2012;53:1803–9.