Comparison of Macular and RNFL Thickness in Anisometropic Amblyopia as Compared to Normal Fellow Eyes

Purpose: To compare the macular and peripapillary RNFL thicknesses of the amblyopic and normal eye in patients with anisometropic amblyopia. Methods: This is a hospital based cross-sectional study for which we studied 36 amblyopic subjects aged between 5 to 16 years in which the amblyopic eye was taken as the case and the unaffected eye was taken as control eye. The inclusion criteria included children having unilateral strabismic or anisometropic amblyopia who underwent macular and RNFL thickness assessment by SD-OCT. Results: Central Macular thickness as well as the thickness of the other macular segments was signifi cantly greater in the affected eye (p<0.001) as compared to the normal eye. The average RNFL thickness was also found to be increased in the affected eye but the difference was not statistically signifi cant (p=0.106), however it was statistically signifi cant in quadrantic analysis (p<0.05). The Central Macular thickness showed a negative correlation with the BCVA (r=0.252), although it was not statistically signifi cant (p=0.139). Conclusion: A signifi cant increase was found in the macular as well as RNFL thickness in the amblyopic eyes as compared to the normal fellow eye which support the hypothesis of decreased apoptosis of the ganglion cells postnatally.


INTRODUCTION
Amblyopia is the most frequent cause of unilateral poor visual acuity (VA) in children, with an incidence of 0.20% to 6.2% in preschool and school-age children 1 . Amblyopia involves loss of Snellen and grating acuity, loss of contrast sensitivity and creation of distortions in the shape of a stimulus 2 . Amblyopia is thought to occur during the period of neuronal development of the retina and the cerebral cortex. Th erefore, it frequently arises during the fi rst 2-3 years of childhood. However, it may also occur in children up to 8-9 years and persists life-long.
Optical coherence tomography (OCT) is a non-invasive, noncontact technique that visualizes the retinal structure in vivo newer spectral domain machines may approach 3 micron resolution 3 , and can measure the thickness of both peripapillary retinal nerve fi ber layer (RNFL) and macula retinal layer which can be used as a helpful tool in assessing retinal changes in amblyopia During fetal development, there is a rapid decline in cell density in the retinal ganglion cell layer toward and had minimal segmentation errors. We excluded scans of signal strength <7 and those with centration errors. Th e proprietary Cirrus segmentation algorithm was used to produce retinal thickness maps, which were then averaged over nine retinal subfi elds within a 6-mm diameter circle centered at the true foveal location, as defi ned by the Age-Related Eye Disease Study. Macular thickness measurements were obtained in macular cube 512 x128 combination scan mode. Th e 6 x 6 mm circle corresponds to the Early Treatment Diabetic Retinopathy Study subfi eld, which is segmented by a concentric circle into central, inner, and outer circles (1, 3, and 6 mm). Th e inner and outer circles were both subdivided into four quadrants (superior, inferior, nasal, and temporal) to give nine areas in total. Th e thickness of each area was measured. Th e optic disk cube 200 x 200 scan mode was used to image the optic disk and RNFL over a 6 x 6 mm optic nerve head using 200 x 200 axial measurements.
Th e RNFL thickness of the four quadrants (superior, inferior, nasal, and temporal) was then measured.
Data was analyzed using IBM-SPSS Version 21.0 software. Data has been represented as numbers and percentages and mean±SD. Paired 't'-test was used for comparison. A 'p' value less than 0.05 indicates a signifi cant diff erence

OBSERVATION AND RESULTS
Th is is a hospital based cross-sectional study carried out at a tertiary centre of north India for which 36 subjects with unilateral amblyopia were enrolled.
A total of 36 patients (18 males and 18 females) aged between 5 to 16 years (mean age 11.58±4.96 years) were enrolled in the study. In 69.4% cases right eye was aff ected as opposed to only 30.6% subjects in whom left eye was aff ected. Mean best corrected visual acuity of aff ected eye was 1.21±0.32 LogMAR as compared to 0.56±0.21 LogMAR for the unaff ected eye (Table 1).
Total mean of total RNFL thickness of aff ected side (106.81) was greater than that of unaff ected side (98.58) but diff erence was not found to be signifi cant (p=0.106). An increase in the RNFL thickness was observed in the aff ected eye for superior (136.19), temporal (61.97) and inferior (100.89) quadrants as compared to the unaff ected side, where the thickness of the respective quadrants were found to be 96.31, 85.03 and 85.03 respectively but the diff erence was statistically signifi cant for superior and inferior quadrants the end of gestation. In humans, the total population of cells in the ganglion cell layer is highest (2.2-2.5 million cells) between approximately 18 and 30 weeks of gestation. After this, the cell population declines rapidly. If amblyopia aff ects the process of postnatal reduction of ganglion cells, RNFL thickness may be thicker than that in the normal eye 4 . Similarly, decreased foveal depression have been reported in amblyopic patients on ophthalmoscopic examination. Foveal depression and refl ex are absent in these eyes and slight discoloration (Wine coloration) of the fovea is sometimes observed, however, these are not consistent fi ndings in the amblyopic fundus 5 .
Th us, this study was taken up to compare the macular and peripapillary RNFL thicknesses of the amblyopic eye and the normal eye in patients with anisometropic amblyopia to fi nd the potential initial neural site of the visual defi cit in this condition.

MATERIALS AND METHODS
Th is is a hospital based cross-sectional study for which we enrolled 45 amblyopic subjects aged between 5 to 16 years in which the amblyopic eye was taken as the case and the unaff ected eye was taken as control eye. Th e inclusion criteria included children having unilateral strabismic or anisometropic amblyopia with the informed and written consent of their legal guardian. We excluded children having any systemic disorder aff ecting eyes, nystagmus, corneal opacities, glaucoma, retinal disorders, any infl ammatory condition of the eye, history of severe ocular trauma and the patients who refused to participate in the study. And fi nally we were left with 36 patients who underwent further evaluation.
All patients underwent a detailed eye examination including best corrected visual acuity (BCVA) determination using Snellen chart from 6m distance, manifest refraction and cycloplegic refraction after pupillary dilation with 1% cyclopentolate hydrochloride and 1% tropicamide, alternative cover test, duction and version testing, intraocular pressure (IOP) measurement, slitlamp biomicroscopy and fundus examination. In this study, anisometropia was defi ned as a cycloplegic, spherical equivalent diff erence greater than 2.00 diopter (D) between fellow eyes.
Further the patients underwent macular and RNFL thickness assessment by spectral domain OCT using Carl Zeiss Cirrus HD OCT. All scans were of high quality (signal strength ≥7), lacked centration errors, correlation came out to be positive when compared with the increased RNFL thickness (Table 4).

DISCUSSION
In the present hospital based observational study conducted at a tertiary care hospital of north India, we have attempted to study the eff ect of amblyopia on macular and RNFL thickness in subjects aged between 5 to 16 years.
We studied 36 patients with unilateral amblyopia with equal number of males and females (18 males and 18 females) which is in contrast with other studies which had greater number of male subjects as compared to the female subjects [5][6][7][8][9][10][11] . More number of subjects had right side aff ected as compared to the left eye [25 (69.4%) and 11 (30.6%) respectively]. No other study, to the best of our knowledge, has reported such comparison. Although we could explain this discrepancy but it may be the result of embryonal developmental behaviour On comparing the RNFL thickness between the amblyopic and unaff ected eye we found signifi cant diff erence in the values of both. Th e RNFL thickness was greater in the amblyopic eye as compared to the (p<0.001). In nasal segment average RNFL of aff ected side was signifi cantly lower (54.22) as compared to that of unaff ected side (68.00; p=0.001) (  Table 3).
In the study, comparison of BCVA, with the Macular and peripapillary RNFL thickness between normal and abnormal eye in children with unilateral amblyopia was done. It was observed that while assessing the relationship between the RNFL thickness and BCVA, there was a positive correlation in the average (r=0.262, P=0.123), superior (r=0.192, p=0.262), inferior (r=0.169, p=0.325) and nasal quadrants (r=0.118, p=0.492) except temporal, which showed a negative correlation (r= -0.065, p=0.707) although it was not statistically signifi cant. Since the worse vision is indicated by a higher LogMAR value, hence, the Pearson in central macular thickness between eyes with moderate to severe amblyopia and the external controls (P = 0.037). In a similar comparison drawn by Hyuhn et al. 10 amblyopic eyes had slightly greater foveal minimum thickness than the normal fellow eye (by 5.0 _m; 95% confi dence interval 0.1-9.9). Amblyopic eyes were found to have a slightly thicker central macula (1 mm diameter region) in both comparisons, although these diff erences were not statistically signifi cant. He also compared the RNFL thickness among the two, but did not fi nd any signifi cant diff erence between amblyopic and normal fellow eyes or normal eyes of non-amblyopic children. Chatterjee et al. 11 studied the diff erence in the macular thickness of the anisometropic and strabismic amblyopic eyes in comparison to the normal fellow eye and found that the macular thickness of the amblyopic eye was greater than that in the normal eye in the anisometropic amblyopia group,  11 and Yen et al. 7 , though the diff erence did not reach any statistical signifi cance in their studies. Th e macular thickness in our study was also found to be greater in the amblyopic eye as compared to the normal control eye. Th e Central macular thickness was signifi cantly higher in the amblyopic eye (p<0.001) as well as in all the quadrants (p<0.001) except in the inner superior quadrant where the diff erence did not reach statistical signifi cance. Agrawal et al. 12 also reported a similar increment in the macular thickness in the amblyopic eye with Mean macular thickness in the amblyopic eyes being 277.5 μ ± 15.3 and in the fellow normal eyes being 272.4 μ ± 13.1 (P < 0.05). Although Rajavi 5 et al. used external age matched controls to compare the macular thickness between the amblyopic and normal eyes, he also found a signifi cant diff erence  Histopathologic changes in the lateral geniculate nucleus and visual cortex have been reported. Histologic study of the lateral geniculate nucleus of monkeys with strabismic, anisometropic, and visual deprivation amblyopia reveals marked shrinkage of cells that receive input from the amblyopic eye. 5,8,9 Th ere are similar fi ndings in the lateral geniculate nucleus in human anisometropic amblyopia and strabismic amblyopia 11 . But we also think that at least in humans amblyopia may be associated with changes that aff ect retinal function at the level of production of Pattern ERG defi cit, which is assumed to be preganglionic, however other studies 16 have refuted this claim, especially those in which fi xation and focus were pre adjusted.

CONCLUSION
In our study we found a signifi cant increase in the macular as well as RNFL thickness in the amblyopic eyes as compared to the normal fellow eye which support the hypothesis of decreased apoptosis of the ganglion cells postnatally. Th is baseline evaluation can be used to monitor the occlusion therapy thereafter and hence, changes in the macular and RNFL thickness can be evaluated after a successful occlusion therapy.
Compliance with ethics requirements: Th e authors declare no confl ict of interest regarding this article. Th e authors declare that all the procedures and experiments of this study respect the ethical standards in the Helsinki Declaration of 1975, as revised in 2008(5), as well as the national law. Informed consent was obtained from all the patients included in the study.
Wang et al. 12 studied 14 subjects with hyperopic anisometropic amblyopia and reported no signifi cant diff erences in peripapillary retinal nerve fi ber layer, central macular thickness, and macular volume between amblyopic eyes and fellow eyes of the participants. Similarly, Dickmann et al. 13 reported no signifi cant diff erence between retinal nerve fi ber layer thickness, macular thickness, and foveal volume in amblyopic versus fellow eyes in patients with unilateral amblyopia. However, Pang et al. 14 reported that amblyopic children with unilateral high myopia tend to have a thicker fovea and thinner inner and outer macular ring in the amblyopic eye as compared to their normal fellow eye.
We also found an inverse correlation between the RNFL and Macular thickness and Best corrected visual acuity in the amblyopic eye. Th ere was a positive correlation between the BCVA (LogMar) and RNFL thickness, average as well as in all the quadrants except in the temporal quadrant. Similar positive correlation was found between the BCVA (LogMAR) and the macular thickness, except in the inner nasal, outer temporal and outer nasal quadrants. Th e higher the LogMAR value worse is the visual acuity, hence there was a positive correlation with the RNFL and macular thickness. To the best of our knowledge, no other study has derived the correlation between the RNFL and Macular thickness with the visual acuity.
Yen et al. 7 hypothesised that amblyopes have thicker retina and that the amblyopic process have eff ects at multiple levels in the visual pathway. Experiments have proved that retinal ganglion cells suff er changes with light deprivation from birth. Th ese changes may include some or all of these-cell loss or apoptosis 11 , mean nucleolar volume diminution in ganglion cell layers, and inner plexiform layer thinning in rats and cats 9 . Changes also include reduction in optic nerve head