FP33 : Corneal Neural Imaging Using in Vivo Confocal Microscopy in Cases of Congenital Corneal Anesthesia

Dr. Madhumita Gopal, M21204, Dr. Sunita Chaurasia, Dr. Muralidhar Ramappa

Abstract

Purpose: Quantitative estimation and comparison of the corneal neural architecture using real-time, in vivo confocal microscopy (IVCM) in patients with congenital corneal anesthesia (CCA) as against a control population.

Design: Prospective, non-consecutive, comparative interventional clinical case series.

Participants:Co-operative children (n=8) diagnosed to have CCA, without excessive scarring of the cornea, as cases, and a consenting blood relative each (n=8) without any corneal pathology as controls underwent IVCM, using the Nidek Confoscan-4, of the superficial corneal nerves.

Main Outcome Measures:Primary outcome was measure corneal neural architecture in children diagnosed with an anesthetic cornea and compare with the age matched controls. Correlate structural changes and functional loss as secondary outcomes.

Method: The clearest 3-5 images from each eye were selected, coded and the nerves analyzed for length, thickness and density, dichotomous pattern and beading, using ImageJ software. The images were then decoded and the values statistically analyzed.

Result: 12 eyes of 8 cases and 16 eyes of 8 controls were imaged. Measurements on corneal nerve density showed a significant difference (p= 0.007), cases having a lower mean (4.11 ± 1.03 mm per mm²) compared with the controls (6.52 ± 1.87 mm per mm²). Measurements on corneal nerve length (p= 0.22), thickness (p=0.24) and presence of beading (p=0.87) did not reveal a significant difference between cases and controls. In dichotomous patterns, the medians were 45% and 82% for cases and controls respectively (p = 0.11).

Conclusion:There appears to be no relationship between the functional loss (absent corneal sensation) and anatomical decrease (reduced sub-basal nerve density) of corneal nerves in CCA.

Introduction

Congenital corneal anesthesia (CCA) is a rare complex neurological condition that is frequently overlooked or under diagnosed or often the diagnosis is delayed, leading to irreversible damage of the visual axis. Typically,the presentation is bilateral though unilateral presentation is not uncommon. This clinical entity often presents with a lusterless ocular surface, poor tearing, painless sterile erosions through early childhood and, at times, may result in the deterioration of optical quality of the cornea if not intervened on time¹.CCA is characterized by neurosensory deficits that encompass not only ocular surface but often other divisions of the trigeminal nerve are involved. This sensory deficit may be an isolated finding or occur as part of a complex neurological syndrome and or in association with multiple somatic abnormalities and congenital insensitivity to pain (CIPA).^1-8

Rosenberg classified the disorder into three distinct groups:³ Group I is associated with isolated trigeminal anesthesia, probably due to primary hypoplasia of the hindbrain. Group II is associated with mesenchymal anomalies, which include Goldenhar syndrome, Mobius syndrome and Riley-Day syndrome or familial dysautonomia (FD).Group III is associated with focal brain stem signs without evidence of mesenchymal dysplasia. Reduced or absent corneal sensation, irrespective of the causal origin, can adversely affect corneal integrity.¹ An anaesthetized cornea has poor epithelial turnover and a defective epithelial repair mechanism due to depleted cAMP levels, acetylcholine and acetylcholine transferases at nerve terminals.⁸ In addition, there is decrease in reflex tearing, reduced blink rates and increased tear mucus secretion, which are aggravated by progressive abnormalities of corneal epithelial microvilli, making the cornea increasingly vulnerable to self-inflicted injury, infection, and poor self-repair.^10-13

Children with this disorder are prone to frequent epithelial erosions which, unless recognized and managed in a timely manner, can progress rapidly, leading to corneal ulceration and perforation or opacities, described as neurotrophic keratitis¹.Because of challenges involved in evaluating these children, the diagnosis of CCA is often missed or delayed leading to permanent visual disability. Perhaps the only effective treatment strategy at present is performing a 2/3^rdwidth tarsorrhaphy.Therefore, timely recognition is the key in minimizing the morbidity.To the best of our knowledge, there are no previous studies describing in vivo, real-time architectural anatomy of the corneal nerves particularly in settings of CCA.

Our prior understanding of corneal nerve architecture is limited to either light microscopic and or electron microscopic observations. However, ultrastructural information of human corneal nerves is a matter of debate due to scarcity of data in the literature owing to the autolysis of these nerves immediately after death. Hence, fresh human corneas are required for optimal ultrastructural analyses.However, these are difficult to obtain because the majority of these specimens are targeted for corneal transplantations.In vivo confocal microscopy (IVCM) offers numerous advantages over the traditional techniques. It is a non-invasive, real-time, in vivo imaging of corneal neural architecture at their physiological state. In addition, confocal imaging minimizes the artifacts and enables us to study the corneal neural architecture in its entirety.⁵

In this context, we intended to study the application of real-time, in vivo confocal corneal neural imaging exclusively in children with anesthetic corneas. The current study aims to explore the pattern of corneal neural architecture in comparison with normal age-matched controls and clinical utility in this setting carried out at a tertiary eye care center in Southern India.

Methods:

All diagnosed cases of congenital corneal anesthesia who presented to Tej Kohli Cornea Institute, L V Prasad Eye Institute, Hyderabad, India from Jun 2015 to Dec2015 and who fulfilled the inclusion criteria were included as cases for this study. Their relatives were included as controls.

Inclusion criteria:

Cases

1. Clinical history of recurrent redness and photophobia with or without associated absence of tearing and/or lack of response to touch sensations either localized to face or generalized to the whole body.
2. Parents noted signs of self-mutilation and or failure in responding to intramuscular injection at the time of immunization.
3. Absent/reduced corneal sensations and lusterless ocular surface noted on clinical examination when no other etiological cause could explain the clinical picture.
4. Ancillary clues towards corneal anesthesia in patients such as: an indifferent  response on instillation of topical anesthetic agents and absence of resistance to examination under microscope without general anesthesia and while performing corneal scrapings.

Controls

1. Unaffected blood relatives of patients.
2. No other corneal pathologies.

Exclusion Criteria:

Cases

1. Dense corneal scarring that is precluding the assessment of corneal nerves.
2. Corneal epithelial defect at the time of examination.
3. Non-cooperative children.

Controls

1. Individuals not co-operative for IVCM.

Patients who came to the out-patient department of the Tej Kohli Cornea Institute were evaluated and determined to meet eligibility criteria by a pediatric cornea specialist as per previously defined inclusion and exclusion criteria. Informed written consent was then taken from subjects or their legal guardian for those under 18 yrs of age. The relatives were also recruited in a similar manner to serve as controls. Each of the study subject then assigned a study number.

The cases and controls underwent a comprehensive eye examination including the following in the given order

1. Best Corrected Visual Acuity (BCVA): Measured using a LogMAR chart.
2. Slit lamp microscopic examination: To look for uptake of stain, areas of scarring and presence of other ocular pathologies.
3. Corneal sensation using a sterile wisp of cotton.
4. Schirmer’s Test: Done without a topical pharmacological anesthetic in cases. In controls, a drop of proparacaine hydrochloride 0.5% was instilled before measuring the Schirmer’s values.
5. Intraocular pressure measurement using Goldman’s Applanation Tonometer.

Confocal Microscopy

Following the above-mentioned examination, the study recruits underwent corneal neural imaging using the Nidek Confoscan-4(Nidek Technologies, Chiyoda-ku,Tokyo, Japan) which is a white-light, slit-scanning confocal microscope (SSCM). A 40x immersion lens was used using an optically clear gel as a medium.

The capturing of images was performed by the same investigator for all cases and controls. The procedure was explained prior to imaging. Imaging was then done in a topically anesthetized cornea in the control population and lubricant used for the congenital anesthetic cornea. The subject was made to sit comfortably on a stool and rest their chin and forehead on the rest provided. Depending on which eye was to be imaged, the left or right chin rest was used. The subject was asked to fixate on the yellow light. An optically clear gel was placed on the tip of the lens and the microscope advanced with the help of the joystick so that the gel is just in contact with the subject’s eye (working distance of 2mm). With the subject keeping their eye completely still, the desired region of the cornea was scanned and images captured on the display and saved onto a connected computer containing the Navis software.

Images were obtained from the central cornea or, in the presence of scarring, the nasal paracentral region. Attempt was made to obtain images from the same area of the cornea in both cases and controls. Images were obtained at the level of the sub-basal plexus approximately at a depth of 50-150 µm. Before and after imaging every subject, the lens was disinfected using 70% isopropanol solution (isopropyl alcohol). Subjects were then examined at the slit lamp once again to look for any epithelial abrasions.

Once the test was done for all the recruits, the images with the best detailing of the subbasal plexus were chosen (3-5 per eye) and coded such that in further analysis the analyzer is masked to the origin of each image. Each image was then analyzed using the Image J software for the following parameters: length of the corneal nerves, presence of dichotomous pattern, presence of beads, thickness and density.The images were then decoded and the values were first averaged for each eye and then averaged for each recruit. These values were then statistically analyzed.

Statistical Analysis:

Statistical analysis was done using Origin v7.0 (OriginLab Corporation, Northampton, MA, USA). The continuous data from right and left eyes of the same patient were averaged. The continuous data were checked for normality using Shapiro-Wilk test and equality of variance by Levene test. The normally distributed data were described by mean and standard deviation and analyzed by Student t-test while non-parametric data were described using median and interquartile range (IQR) and analyzed by Mann-Whitney test. Categorical data were compared using Fisher Exact Probability test. A p-value of <0.05 was considered statistically significant.

Results

Baseline characteristics: The study was conducted on eight patients of CCA and eight controls as defined by the inclusion and exclusion criteria. While 12 eyes matched criteria amongst the cases, all sixteen eyes were examined in the control group. In terms of the male to female split, the respondents were better represented in the cases group (5:3 compared with 7:1 in controls). The mean age amongst the cases was 11.75 ± 4.5years while it was 27.4 ± 8 in the control group (Table1).

Subjects were evaluated on corneal sensation, BCVA and Schirmer’s value. Corneal sensation was present on all patients from the control group, while being absent in the cases group. Schirmer’s values were higher in the control group (mean of 25.7 ± 4mm) compared with the test cases (mean of 15± 7mm). There was no uptake of stain before or after imaging in all 16 subjects.The BCVA was 0.00 LogMAR for controls compared with 0.83 ± 0.76 LogMAR for the cases.

Neural imaging

Measurements on corneal nerve density using confocal microscopy showed a significant difference (p = 0.007) between the cases and the control group (Table 2). The cases had a lower mean of 4.11 ± 1.03 mm/mm² compared with the control group witha mean of 6.52 ± 1.87mm/mm².(Figure 1).

As CCA is a very rare disease, only eight cases were obtained during the study period. Measurements on corneal nerve length and thickness did not reveal a significant difference between cases and controls. The cases had a mean corneal nerve length of 242.60 ± 79.99mm and controls had a mean of 283.86 ± 42.29mm, while the mean corneal thickness of the cases and controls were 3.10 ± 0.35 mm and 2.88 ± 0.38mm respectively (Table2).

The beading and dichotomous patterns (Table3)did not significant difference between the two groups.In measuring, corneal nerve beading, both the test and control groups had a median of 100% presencein dichotomous patterns, the medians were 45% and 82% for cases and controls respectively (p = 0.11).

Discussion

Congenital corneal anesthesia is a rare disorder of which little is known regarding its etiopathogenesis. In this study, we aim to look at the architecture of the sub-basal corneal plexus of nerves in patients and compare them to those of unaffected individuals. Even though there have been several reports indicating an autosomal dominant mode of inheritance for CCA, the controls that we chose for this study had functionally normal corneal nerves.

Confocal microscopy is a useful tool for not only imaging corneal nerves but also for their qualitative and quantitative analysis. Numerous studies have been done on the analysis of the corneal neural architecture in both healthy and diseased cornea. These include the structure, density, length, width and beading as done in our study. Of these, the change in density of corneal nerves has been the most common parameter analyzed. There have been varying results for the normal density of the sub-basal plexus of nerves which have been put down to differences in the type of confocal microscopy used that is tandem, slit or laser scanning confocal microscopy. As our study was done on the same instrument and images obtained by the same examiner in similar locations of the cornea in both cases and controls, our values are reliable.

A few studies have been done using IVCM for acquired causes of corneal anesthesia and have shown varying results. Dhillon et al¹⁴ described a case of acquired trigeminal anesthesia of probable viral etiology where the corneal nerves were intact. However, a study by Rosenberg et al¹⁵ on 16 patients with unilateral keratitis showed absent corneal nerves in the affected eye in two cases and a reduction in density in three cases. Reduced density has been shown in diabetic patients as compared to controls and this has shown to correlate with the presence of reduced corneal sensation and peripheral neuropathy¹⁶. No studies were found that have used IVCM for congenital corneal anesthesia.

In our study, we found a significant decrease in the density of corneal nerves in CCA. Clarke et al¹⁷ in their study of six family members having CCA also found a decrease in density of corneal nerves in all their subjects. In a case described by Anseth⁷, no corneal nerves could be visualized. However, these cases were described before confocal microscopy was commonly used for the analysis of corneal nerves.

An observation made during the conduct of this study was the absence of reflex blinking in cases when an object is brought close to the eye. This appears to be a conditioned reflex requiring intact corneal sensations during early childhood. It is thus important to provide protection in the form of protective glasses to these patients.

To conclude, there appears to be no relationship between the functional loss (absent corneal sensation) and anatomical decrease (reduced subbasal nerve density) of corneal nerves in cases of CCA. Although the sample size of cases is small, considering the rarity of CCA, the findings of this study can be considered relevant.

 

References:

1. Ramappa M, Chaurasia S, Chakrabarti S, Kaur I. Congenital corneal anesthesia. Journal of AAPOS: the official publication of the American Association for Pediatric Ophthalmology and Strabismus / American Association for Pediatric Ophthalmology and Strabismus. 2014;18(5):427-32.
2. Ramaesh K, Stokes J, Henry E, Dutton G, Dhillon B. Congenital corneal anesthesia. Survey of ophthalmology. 2007;52(1):50-60.
3. Rosenberg ML. Congenital trigeminal anaesthesia. Brain. 1984;107(4):1073-82.
4. Stewart HL, Wind CA, Kaufman HE. Unilateral congenital corneal anesthesia. American journal of ophthalmology. 1972;74(2):334-5.
5. Baxter D, Olszewski J. Congenital universal insensitivity to pain. Brain: a journal of neurology. 1960.
6. Hewson EG. CONGENITAL TRIGEMINAL anaesthesia. The British journal of ophthalmology. 1963;47:308-11.
7. Anseth A. Congenital bilateral corneal anesthesia. Acta ophthalmologica. 1968;46(5):909-11.
8. Carpel EF. Congenital corneal anesthesia. American journal of ophthalmology. 1978;85(3):357-9.
9. Cavanagh HD, Colley AM. The molecular basis of neurotrophic keratitis. Acta ophthalmologica. 1989;67(S192):115-34.
10. Baum JL, Feingold M. Ocular aspects of Goldenhar’s syndrome. American journal of ophthalmology. 1973;75(2):250-7.
11. .Mackie I. Role of the corneal nerves in destructive disease of the cornea. Transactions of the ophthalmological societies of the United Kingdom. 1978;98(3):343.
12. Mohandessan MM, Romano PE. Neuroparalytic keratitis in Goldenhar-Gorlin syndrome. American journal of ophthalmology. 1978;85(1):111-3.
13. van Bijsterveld OP. Unilateral corneal anesthesia in oculoauriculovertebral dysplasia. Archives of ophthalmology. 1969;82(2):189-90.
14. Dhillon VK, Elalfy MS, Al-Aqaba M, Dua HS. Anaesthetic corneas with intact sub-basal nerve plexus. British Journal of Ophthalmology. 2014 Jan 3:bjophthalmol-2013.
15. Rosenberg ME, Tervo TM, Müller LJ, Moilanen JA, Vesaluoma MH. In vivo confocal microscopy after herpes keratitis. Cornea. 2002 Apr 1;21(3):265-9.
16. Malik RA, Kallinikos P, Abbott CA, van Schie CH, Morgan P, Efron N, Boulton AJ. Corneal confocal microscopy: a non-invasive surrogate of nerve fibre damage and repair in diabetic patients. Diabetologia. 2003 May 1;46(5):683-8.
17. Clarke MP, Sullivan TJ, Kobayashi J, Rootman DS, Cherry PM. Familial congenital corneal anaesthesia. Australian and New Zealand journal of ophthalmology. 1992;20(3):207-10.