Peripapillary retinal nerve fiber layer thickness in different glaucoma stages measured by optical coherence tomography

Background/Aim. One of the most reliable methods for structural measurements of glaucomatous damage is spectral domain optical coherence tomography (SD-OCT). The aim of this study was to measure peripapillary retinal nerve fiber layer (RNFL) thickness with SD-OCT in eyes with different stages of glaucoma, as well as to determine which sector in the peripapillary circle is the most vulnerable to glaucomatous damage. Methods. The study included 153 eyes of 93 patients with confirmed primary open angle glaucoma (POAG). All the patients underwent a complete ophthalmic examination, including visual field testing and peripapillary RNFL thickness measured by SD-OCT. They were divided into three subgroups: early, moderate and severe stage of glaucoma based on the mean deviation (MD) index of visual field. The results were presented as mean RNFL thickness: total, in the four quadrants and 12 clock-hour RNFL thickness. Results. The overall mean peripapillary RNFL was 74.95  14.51 μm. The lower quadrant had the thickest RNFL (92.78  25.84 μm), followed by upper (88.82  22.04 μm), nasal (64.31  11.67 μm) and temporal ones (54.02  12.76 μm), showing a significant difference (χ2 = 273.36, DF = 3, p < 0.001). Comparison between RNFL thickness in early glaucoma and moderate and severe stages revealed that the most sensitive sectors were inferior and superior ones, as well as sectors at 5–7 clock hour position. The greatest decrease in RNFL thickness was observed in the 9 o'clock hour sector in all three glaucoma subgroups (46.99  13.28 μm), while the RNFL was the thickest in the 6 o'clock hour sector (102.63  34.12 μm). Conclusion. Peripapillary RNFL thickness is inversely proportional to the degree of glaucomatous damage: the greater the damage, the thinner peripapillary RNFL.


Introduction
Glaucoma is a progressive multifactorial optic neuropathy characterized by structural changes of the optic nerve head (ONH) and peripapillary retinal nerve fiber layer (RNFL) damage associated with functional visual field (VF) defects.An early detection and follow up of glaucoma require functional testing using standard automated perimetry (SAP) as gold standard, particularly the 24-2 Swedish Interactive Threshold Algorithm (SITA) strategy, as well as structural testing which can be based on ophthalmic findings and followed by stereoscopic photography of ONH.But, one of the most reliable methods for objective and precise structural measurements of glaucomatous damage is the optical coherence tomography (OCT) which provides both quantitative and qualitative measurements of the RNFL thickness.
OCT in diagnostics of the ONH structural changes became a part of standard procedure for diagnosis and monitoring of patients with retinal pathology.OCT is also highly sensitive in differentiating glaucomatous from nonglaucomatous ONH changes which include (non)arteritic anterior ischemic opticopathy, intracranial tumors, optical neuritis, dominant optic atrophy, methanol poisoning, Leber's opticopathy [1][2][3][4][5] where OCT finding shows more diffuse decrease in peripapillary RNFL thickness in comparison to glaucoma 6 .Also, the differences between healthy eyes and eyes with glaucoma are significant 7 .Due to its advantages in performing examinations, OCT method can significantly facilitate differentiation of ONH structural damages in suspected cases.
The aim of this study was to measure peripapillary RNFL thickness with spectral domain OCT (SD-OCT) in the eyes with different stages of glaucoma, as well as to determine which sector in the peripapillary circle is the most vulnerable one to glaucomatous damage.

Methods
This study included 153 eyes of 93 patients with confirmed primary open angle glaucoma (POAG).Patients with glaucoma were referred from the glaucoma department of the University Eye Clinic in Niš, Serbia.All the patients underwent complete ophthalmic examination, including best corrected visual acuity, intraocular pressure measurement by applanation tonometry, gonioscopy, visual field testing using the 24-2 SITA algorithm (Humphrey Field Analyzer II; Carl Zeiss Meditec, Inc., Dublin, CA), slit lamp examination of anterior segment and fundus examination with a plus 90diopter lens.Peripapillary, RNFL thickness was measured using a glaucoma analysis mode in Cirrus HD OCT device software version 6.0 (Carl Zeiss Meditec, Inc.).This study followed the tenets of the Declaration of Helsinki, the study protocol was approved by the Ethics Committee of the Medical faculty of Niš and informed consent was obtained from all the participants.They were classified into three subgroups on the basis of mean deviation (MD) index of VF: an early glaucoma (MD ≤ -6 dB), moderate glaucoma (MD between -6 dB up to -12 dB) and severe glaucoma with MD more than -12 dB.The patients with any other intraocular disease, opacification of ocular media, intraocular surgery, ocular trauma and secondary glaucoma were excluded from the study.
Inclusion criteria were: confirmed POAG (glaucomatous VF loss consistent with optic nerve damage), VF test reliability indices values, such as false positive, false negatives and fixation loss less than 20%; no ocular opacities or other ocular pathology, no other structural optic nerve abnormalities or secondary glaucoma; negative history of previous eye disease, trauma and/or eye surgery and no neurologic disease; axial length in referral values.
ONH imaging was automatically made over an area of 6×6 mm by a 200×200-pixel resolution axial scan.Each eye was dilated with tropicamide 1% drops before recording.Images with a signal power more than seven were used for analysis.In all subgroups overall mean of the whole circle circumference, linear maps at 12 o'clock hour positions and circular maps within 4 quadrants and mean peripapillary RNFL thickness were recorded for each patient.
For the statistical analysis, mean peripapillary RNFL and its segment in four quadrants, as well as at 12 o'clock hour positions were calculated.Comparison between the means of the groups for paired variables was evaluated by oneway ANOVA.Kolmogorov-Smirnov test was used to determine whole circle circumferences, 4 quadrants and 12 clockhour sectors RNFL thickness distribution in the observed group and subgroups.Data were analyzed using SPSS v. 20.0 for Windows (SPSS, Inc., Chicago, IL).The value of p < 0.05 was considered statistically significant.
There was a highly significant difference between the observed quadrants in different glaucoma stages (χ 2 = 273.36,DF = 3, p < 0.001).Temporal quadrant was highly significantly thinner than all the other quadrants, while the nasal one was significantly thinner than upper and lower ones (p < 0.001).A detailed comparison of the peripapillary RNFL thickness in all four sectors and among different stages of glaucoma showed the following results: in temporal and nasal sectors there was no difference in RNFL thickness regression between moderate and severe glaucoma (the loss of RNFL thickness was 17% vs 8%, respectively).But, when we calculated the RNFL thickness in moderate and severe glaucoma in comparison to early glaucoma measurements, we found the RNFL thickness  loss of by 18% in the superior sector compared to thickness in early glaucoma, as well as more thinning in severe glaucoma, up to 29%, compared to early glaucoma thickness.In inferior sector, results of peripapillary RNFL show thinning of 16% in moderate, up to 35% in severe glaucoma.The curves of RNFL thickness in three glaucomatous subgroups are presented in Figure 1.As it can be seen, the thinnest sector in all the three glaucoma subgroups (mean thickness was 46.99  13.28 μm) is at 9 o'clock hour position.The RNFL thicknesses among the different glaucoma subgroups was significantly different, but an overlapping was observed.At positions 3, 4, 8 and 9 o'clock, the thinnest peripapillary RNFL was in moderate glaucoma instead of severe glaucoma, as expected.It is interesting that at positions from 3 up to 5 o'clock, there was no difference in RNFL thickness with repeated overlapping in moderate glaucoma subgroup vs. severe glaucoma subgroup.
The results of this study show that the greatest thinning and linear regression of peripapillary RNFL from early to moderate and, finally, severe glaucoma, were in the following positions: 6 o'clock position -in moderate glaucoma 15% up to 38%, at 7 o'clock position -16% up to 37%, at 5 o'clock hour -18% up to 33%.

Discussion
OCT offers a possibility of quantitative and morphologic RNFL analysis.For the purposes of measuring peripapillary RNFL thickness in this study we used Cirrus HD-OCT, which was found to be a reliable diagnostic tool with excellent reproducibility regarding the monitoring of glaucoma progression, as demonstrated by many studies 14,15 .Also, SD-OCT demonstrates advantage over time domain OCT for two reasons.First, it shows an enlarged circle around the optic nerve head with sufficient detail to identify possible errors caused by influence of potential epiretinal membrane, vitreous detachments, etc.Second, high-quality scans allow detailed visualization 16 .
Basic facts about optic nerve fiber layer thickness are the following: average RNFL thickness represents the mean thickness of the entire nerve fiber that reaches the optic disc.Also, the majority of the nerve fibers converge on the optic disc either superiorly or inferiorly.In the present study, the peripapillary RNFL was thicker superiorly and inferiorly, which can be explained by normal peripapillary RNFL distribution.In early glaucoma, as reported in some histological studies, there is 25-35% loss of retinal ganglion cells before VF damage is confirmed, while many of these nerve fibers are still undamaged 17 .Taliantzis et al. 18 reported that deep structural alterations detected by OCT are an important indicator of early glaucomatous changes, even if they are not detected with SAP.Once the VF loss is established, smaller amounts of RNFL thickness are necessary for the reduction of mean deviation value 19 .
In our study, the inferior RNFL thickness shows the highest thinning in advanced glaucoma eyes, what is in correlation with the results of some previous studies 20 .Peripapillary, RNFL demonstrated higher sensitivity and specificity than macular RNFL for glaucomatous and suspect groups and even then, the inferior quadrant is the most sensitive parameter 21 .Mean RNFL thickness in inferior and superior quadrants, as well as 6, 7, 11 and 12 clock hour segment thickness have the highest sensitivity and specificity in distinguishing eyes with glaucomatous VF defects from normal eyes 22 .Hwang and Kim 23 reported that the eyes with more advanced glaucoma had thinner RNFL in global area, superior, inferior and temporal quadrants.All the mentioned studies report similar results, as we mentioned in this study.A significant difference in superior and inferior peripapillary quadrants between glaucomatous and normal eyes was detected by scanning laser polarimetry, as Galväo Filho et al. 19 reported as well.The authors found out a significant difference in RNFL thickness between healthy eyes and eyes of those with early glaucoma, but not between early and moderate glaucomatous eyes.Our study shows that the linear reduction of RNFL thickness as glaucomatous damage is more progressive.It should be mentioned that more glaucomatous eyes were included in the study (153 vs 68) and we used SD OCT, not scanning laser polarimetry for RNFL thickness measurement.However, even a complete loss of ganglion cells, as it is demonstrated in severe glaucoma, leaves some residual thickness which consists of glial cells and blood vessels 24 .In the end, even residual retinal thickness in the eyes with severe glaucoma is different in different peripapillary sectors, corresponding with papillomacular bundle.It supports the result of this study that in nasal and temporal sectors there is no difference between different glaucoma stages, while in superior and inferior quadrants the thinning of peripapillary RNFL is significant.
As previously mentioned, SD-OCT is useful for determining structural changes in peripapillary RNFL thickness, and also for evaluating the correlation between perimetric defects and corresponding nerve fiber loss 25 .New summary statistics combining VF and OCT results are being developed, as it has been reported in a study of Racette et al. 26 who integrated functional and structural measurements using Artificial Neural Networks.Very similar to these results, Bizios et al. 27 demonstrate relevance vector machine on a combined optimized confocal scanning laser ophthalmology (CSLO) device using the Heidelberg retina tomograph (HRT) and short-wavelength automated perimetry data for the purpose of improving glaucoma diagnostic accuracy.The multivariate and Moorfields algorithm of the HRT provide good ability to distinguish glaucoma tous from normal eyes 22 .
Most studies included patients with VF loss, as we did.But, when glaucoma suspects with normal VF were included in the study, the sensitivity and specificity of diagnostic tests were significantly lower when compared to cases with glaucomatous VF abnormalities 22 .In presented study we were focused on a SD OCT potential and power to detect the difference between RNFL thickness in different glaucoma stages without comparing with VF defects.Since the before mentioned approach could have limited utility, we considered that it is very important to define the peripapillary RNFL sectors that are most vulnerable to glaucomatous damage.
As it was reported, a simple OCT imaging report is sensitive to providing quantitative information for most cases, but for difficult ones interpreting physicians should have considerable experience in analyzing OCT scans 16 and they should always have on mind that OCT is only a part of the glaucoma diagnostics and the following up process which is of great importance when combined with SAP or CSLO results.

Conclusion
The sector of the thickest peripapillary RNFL is the temporal one, followed by nasal, superior and inferior sectors.The thinning of the RNFL at advanced stages of glaucoma is greater in the inferior and superior sectors.There is no decrease of peripapillary RNFL thickness in the nasal and temporal quadrants in comparison to early glaucoma RNFL thickness.The most valuable and sensitive parameters in assessing the degree of peripapillary RNFL glaucomatous damage are in the 5, 6 and 7 clock hour sectors.