Macular retinal and nerve fiber layer thickness in early glaucoma: clinical correlations.

PURPOSE: Previous studies have evaluated macular retinal thickness (RT) and nerve fiber layer thickness (RNFLT) changes in early glaucoma using elaborate optical coherence tomography (OCT) scanning protocols.
MATERIALS AND METHODS: This study examines RT and RNFLT using standard scanning protocols in early glaucoma. In this prospective, nonrandomized case series, 95 eyes of 95 patients were evaluated, including 29 nonglaucomatous subjects (control group), 34 glaucoma suspects, and 32 early manifest glaucoma patients. RT and RNFLT were measured using scanning fast macular thickness map and Fast RNFLT (3.4) protocols on a 1.70 mm radius around the macular center (respectively) in all four quadrants. The fast RNFLT (3.4) protocol was transposed on the macula from the peri-papillary area. Data were statistically analyzed for differences between groups, and for correlations between parameters. P<0.5 was statistically significant.
RESULTS: Both early manifest glaucoma patients and glaucoma suspects had significantly lower RT than controls in all quadrants. RNFLT differences in all quadrants were not statistically significant (P>0.05). RT was significantly inversely correlated with axial length in early manifest glaucoma patients and glaucoma suspects but not in controls.
CONCLUSIONS: The finding that RT was significantly lower in early manifest glaucoma patients and glaucoma suspects indicates that the transposition of the OCT fast RNFL thickness (3.4) protocol from the peri-papillary area to the peri-macular area can be used for early glaucoma diagnosis. Intraretinal changes in early glaucoma, likely precede nerve fiber changes.

INTRODUCTION

Historically, evaluation of early glaucomatous change has focused mostly on optic disk changes.1 Modalities such as optical coherence tomography (OCT), confocal scanning laser ophthalmoscopy (HRT) or scanning laser polarimetry (GDx) with specially developed software algorithms have been used to quantitatively assess such changes.2 However, glaucomatous damage is primarily focused on retinal ganglion cells, which are particularly abundant in the peri-macular region (the only retinal area with a ganglion cell layer more than 1 layer thick), constituting, together with the nerve fiber layer, up to 35% of retinal macular thickness.3 Therefore, glaucomatous changes causing ganglion cell death could potentially result in a reduction of retinal macular thickness.45 Indeed, by employing specially developed algorithms to analyze OCT scans, previous studies have reported that glaucoma, even during the early stage, results in the thinning of inner retinal layers at the macular region.67 This study employs an alternative OCT analysis, based on widely available OCT software, to comparatively evaluate retinal macular thickness (RT) and macular retinal nerve fiber layer thickness (RNFLT) in glaucoma suspects and early manifest glaucoma as well as in a control group and examines the correlation of RT and RNFLT with clinical parameters. The findings of this could help in understanding the importance of macular changes, determined by conventional and commercially available OCT algorithms, as a diagnostic tool for early glaucoma.

MATERIALS AND METHODS

This is a prospective, non-randomized case series. The protocol was approved by the ethics committee of the University Hospital of Heraklion, in Crete, Greece and all participants signed an informed consent form in accordance with the tenets of Declaration of Helsinki. Participants were classified into a control group, glaucoma suspects and early manifest glaucoma group, which included 29 subjects (12 males, 41.37%), 34 patients (16 males, 47.05%) and 32 patients (15 males, 46.87%), respectively. Control group subjects were recruited from cataract surgery candidates whereas glaucoma suspects and early manifest glaucoma group patients were recruited from the Glaucoma Service of the Department of Ophthalmology of the University Hospital of Heraklion, Crete, Greece. Patients with a history of ophthalmic surgery (including refractive surgery), ophthalmic trauma or inflammation, any form of macular pathology or any diagnosed neurological condition were excluded to rule out retinal nerve thickness changes or perimetric findings not directly related to glaucoma. Furthermore, only patients with Best Corrected Visual Acuity (BCVA) over 20/40 in both eyes were included, to maximize reliability for automated perimetry and target fixation in OCT.

Patient classification was based on previously published guidelines8 and was performed by two independent glaucoma specialists (ETD and IGP) who were masked to the other's decision. Only cases with a consensus in the classification by both specialists were included in the analyses. Furthermore, only patients in whom the glaucoma classification was the same in both eyes were included. In all patients only one eye (the right eye) was included in the analyses. The control group consisted of individuals with applanation tonometry readings under 21 mmHg in both eyes, no antiglaucomatous medications, no family history of glaucoma, a cup-to-disk ratio (CDR) under 0.5 with a uniform neuroretinal rim and automated perimetry with a pattern standard deviation within 95% limits of the normal reference, in both eyes. The early manifest glaucoma group consisted of patients diagnosed with glaucoma in both eyes, who had been administered antiglaucomatous medications achieving target IOP. The latter had been set according to previously proposed published studies.9 All early manifest glaucoma group patients had applanation tonometry readings above 21 mmHg in both eyes before the administration of antiglaucomatous medications but below 21 mmHg under treatment, CDR above 0.5 in both eyes whereas in automated perimetry, the pattern standard deviation was outside 95% limits of the normal reference in both eyes. Glaucoma suspects had applanation tonometry readings between 21 and 25 mmHg in both eyes without any antiglaucomatous medications, CDR over 0.5 in both eyes and pattern standard deviation within 95% limits of the normal reference, in both eyes.

All patients underwent ophthalmic examinations by an experienced examiner (VA) who was masked to the patient classification. Automated perimetry (with central 30-2 threshold test, Humphrey® Field Analyzer/HFA II-I, 30-2, Carl Zeiss-Meditec Inc., Dublin, CA, USA) was performed and the pattern standard deviation recorded. For dynamic contour tonometry (SMT Swiss Microtechnology AG, Port, Switzerland) three good quality readings (Q1-Q3, as recommended by the manufacturer) were recorded and the mean value calculated. Goldmann applanation tonometry was performed at least 10 minutes after DCT. Measurements of ocular axial length and central corneal thickness were performed with the Alcon OcuScan® RxP Ophthalmic Ultrasound System, with a 20 MHz probe for pachymetry and a 10 MHz probe for biometry (Alcon laboratories, Alcon, Fort Worth, TX, USA). The clinical parameters recorded for all groups, including age, axial length, central corneal thickness, applanation, and dynamic contour tonometry and pattern standard deviation are presented in Table 1.

Table 1

Clinical parameters and statistical differences of (mean±SD) the control group, glaucoma suspects, and early manifest glaucoma group

Subsequently, all participants underwent OCT testing of the macular area using the Stratus OCT 3, version 4 (Carl Zeiss, San Diego, CA, USA). Patients underwent two different scanning protocols, both applied to the macula:

The fast macular thickness map protocol with six linear scans with a length of 6 mm spaced 30° apart. Measurements of RT were taken on each axis at a distance of 1.70 mm from the macular center [Figure 1].

Figure 1

Fast macular thickness map protocol with six linear scans with a length of 6 mm spaced 30 with each other (a, b). Measurements of RT were taken on each axis 3.45 mm from the macular center along four quadrants (c, d)

The fast RNFL thickness (3.4) protocol focused on the macula, instead of the optic disk. The fast RNFL thickness (3.4) protocol is a glaucoma scanning protocol designed for the measurement of the peri-papillary RNFL thickness which compresses the three sequential circular scans of 3.40 mm diameter of the RNFL thickness protocol into one scan, performed in 1.92 seconds. In this study, the Fast RNFL thickness (3.4) protocol was transposed onto the macular area to provide an estimation of the thickness of the peri-macular RNFL [Figure 2].

Figure 2

Fast retinal nerve fiber layer thickness (3.4) protocol focused on the macula, instead of the optic disk. Peri-papillary scanning was transposed on the peri-macular area (a) resulting in a peri-macular scanning (b) and measurement of the retinal nerve fiber layer thickness along four quadrants on radius of 3.4 mm around the macular center (c). A B-scan image of the same eye showing the area of the posterior pole and the optic nerve (*) is also presented (d)

RNFL is differentiated from other retinal layers by employing a threshold algorithm in which RNFL is assumed to correspond to the highly reflective layer at the vitreoretinal interface. Borders are determined by searching for the first points on each scan where reflectivities exceed a preset threshold (e.g., when reflectivity reaches two-thirds of the maximum reflectivity in each smoothed axial scan evaluated on a logarithmic scale). The RNFL thickness is then defined as the number of pixels between the anterior and posterior boundaries of the RNFL.1011

The mean RT values in four peri-macular quadrants, defined as superior (46°-135°), inferior (226°-315°), nasal (136°-225°), and temporal (316°-45°) were extracted from the fast macular thickness map protocol. The generation of RT was based on the retinal thickness analysis report. The mean RNFLT was extracted from the fast RNFL thickness (3.4) protocol at a distance of 1.70 mm from the macular center, for each of the four quadrants (inferior, superior, nasal, temporal). The RNFLT measurements were performed after the RT measurements to make sure that the center of circular macular RNFLT scan was accurately centered on the fovea. An example of a cross-sectional retinal map with RT, RNFLT, and the distance from the macular center at which measurements were taken (1.70 mm) is shown in Figure 3.

Figure 3

A cross-sectional retinal map showing retinal thickness, retinal nerve fiber layer thickness, and the distance from the macular center (1.70 mm) at which measurements were taken

Scan quality was acceptable in all subjects, with a signal to noise ratio>35 dB. OCT scanning lasted less than 2 minutes in all cases. Data were statistically analyzed with SPSS software, ver. 14.0 (SPSS, Chicago, IL, USA). Statistical significance was set as 0.05. Differences in gender distribution between groups were examined with Pearson's Chi-square test. Differences in age, axial length, and central corneal thickness between groups were examined with one-way analysis of variance (ANOVA and subsequent post hoc analysis with Dunnett's T3 test). Differences in RT and RNFLT as well in their ratio between groups were also examined with one-way ANOVA and subsequent post hoc analysis with Dunnett's T3 test. Correlations between RT, RNFLT or their ratio and clinical parameters such as axial length, central corneal thickness, applanation and dynamic contour tonometry, and pattern standard deviation were also examined (Pearson's bivariate correlation coefficient).

RESULTS

Differences in gender and age distributions between groups were not statistically significant (Pearson's bivariate correlation coefficient and ANOVA, respectively) (P>0.05, all cases) [Table 1]. Differences in axial length between groups were also statistically not significant (ANOVA). On the contrary, the variation in central corneal thickness between groups was statistically significant, with the early manifest glaucoma group and glaucoma suspects having significantly lower central corneal thickness scores compared to that in the control group (P<0.05, all comparisons) [Table 1]. The differences in central corneal thickness between the early manifest glaucoma group and glaucoma suspects were not statistically significant (P <0.05, ANOVA, Dunnett's T3 test).

The variation in RT between groups was statistically significant for all peri-macular quadrants (P <0.05) [Table 2]. Differences in RT between the control group and the early manifest glaucoma group were statistically significant for all quadrants (P <0.05) [Table 2]. There were no statistical differences between the early manifest glaucoma group and glaucoma suspects for all quadrants (P >0.05) [Table 2]. The difference between the control group and glaucoma suspects was statistically significant for the superior quadrant (P <0.05) [Table 2]. Differences between the control group and glaucoma suspects approached (but did not exceed) the levels of statistical significance for inferior, nasal, and temporal quadrants [Table 2]. The statistical significance (post hoc analysis) for differences in RT between groups is presented in Table 3.

Table 2

Retinal thickness (mean±SD) in all four peri-macular quadrants with analysis of variance scores (F values) and statistical significance for the variance of the thickness in each quadrant between groups

Table 3

Post hoc analysis for the differences in retinal thickness quadrants between groups

The variation in RNFLT was not statistically significant for all quadrants (P>0.05, all comparisons) [Table 4]. The variation in the RNFLT:RT ratio was not statistically significant for all quadrants, although it approached (but did not exceed) statistical significance for the temporal quadrant (P>0.05. all comparison, Table 5).

Table 4

Retinal nerve fiber layer thickness (mean±SD) in the peri-macular quadrants and the variance in quadrant thickness between groups

Table 5

The ratio of retinal nerve fiber layer thickness to retinal thickness (mean±SD) in all four peri-macular quadrants and the variance of the ratio in each quadrant between groups

There was no statistically significant correlation between the control and the various parameters under investigation for all quadrants (Pearson's bivariate correlation coefficient, Table 6). For glaucoma suspects, statistically significant inverse correlations were detected in all quadrants between RT and applanation tonometry, dynamic contour tonometry as well as axial length (Pearson's bivariate correlation coefficient, Table 7). Correlations between RNFLT or RNFLT:RT ratio and all clinical parameters examined (age, axial length, central corneal thickness, applanation and dynamic contour tonometry, and pattern standard deviation) were statistically not significant in all quadrants for glaucoma suspects (Pearson's bivariate correlation coefficient).

Table 6

Pearson's bivariate correlation coefficients (r) and levels of statistical significance (P) for the correlations between retinal thickness and retinal nerve fiber layer thickness per quadrant and parameters recorded in the control group

Table 7

Pearson's bivariate correlation coefficients (r) and levels of statistical significance (P) for the correlations between retinal thickness and retinal nerve fiber layer thickness per quadrant and parameters recorded in glaucoma suspects

Statistically significant inverse correlations between RT and pattern standard deviation as well as between RT and axial length were detected in all quadrants for the early manifest glaucoma group (Pearson's bivariate correlation coefficient, Table 8). Correlations between RNFLT or RNFLT:RT ratio and all clinical parameters examined (age, axial length, central corneal thickness, applanation and dynamic contour tonometry and pattern standard deviation) were not statistically significant in all quadrants for the early manifest glaucoma group (Pearson's bivariate correlation coefficient).

Table 8

Pearson's bivariate correlation coefficients (r) and levels of statistical significance (P) for the correlations between retinal thickness and retinal nerve fiber layer thickness per quadrant and parameters recorded in the early manifest glaucoma group

DISCUSSION

Several studies have reported a decrease in macular retinal thickness in established glaucoma.12–19 As approximately 50% of ganglion cells are located in the peri-macular region (4-5 mm from the fovea with the peak density occurring 750-1100 μm from the foveal center, where the cell density may be 4-6 cell bodies thick), macular retinal thinning in glaucoma has been primarily attributed to loss of ganglion cell and nerve fiber layers.1920 By creating detailed OCT maps of the macular structures a recent study reported reduced thickness in all three inner retinal layers in both perimetric and preperimetric glaucoma.67 The authors of this previous study concluded that the combined evaluation of the thickness of the three innermost macular layers has the strongest discriminant power for glaucoma diagnosis. Nevertheless, detailed OCT mapping requires the use of proprietary automatic segmentation algorithms which may not be readily available in many glaucoma units. However, the present study employs software already installed in most OCT systems and thus may directly be applied for glaucoma diagnosis.

Normally performed around the optic disk, the RNFL thickness (3.4) protocol enables the quantification of the RNFLT.18 The observation that RNFLT scores of the control group extracted from that protocol after transposition on the peri-macular region in this study are similar to the normative data reported for the peri-macular area in several previous studies672122 using more elaborate scanning protocols especially designed for the macula supports the accuracy and feasibility of the methodology followed in this study. The RT reduction in all peri-macular quadrants for the early manifest glaucoma group, compared with the control group, is also in agreement with several previous studies2223 reporting retinal thinning at the macular area in glaucoma. RT was also reduced in glaucoma suspects in all peri-macular quadrants, compared with the control group, although differences exceeded statistical significance only for the superior quadrant. Another study6 evaluating segmentation macular maps reported reduced thickness in the inner retinal layer thickness in glaucomatous eyes was maximal for the inferior peri-macular quadrant, instead of the superior quadrant observed in the present study. Such disparities may be associated with differences in study populations since thickness changes in both inferior and superior peri-macular segments have been reported2425 in early glaucoma. Focal reduction in retinal thickness in the superior or inferior segments possibly reflects the retinotopic distribution of the affected ganglion cells. Interestingly, RNFLT was higher in both superior and inferior quadrants in CG, compared with the early manifest glaucoma group and glaucoma suspects, the differences did not reach statistical significance in this study. This finding may reflect the fact that in early glaucoma reversible ganglion cell malfunction, instead of death may occur; thus the axons of malfunctioning ganglion cells may also be spared. Furthermore, previous studies have also reported that RNFLT measurements obtained by Stratus OCT may display significant variability, especially in early or moderately advanced glaucomas.26 The fact that RT reduction in the early manifest glaucoma group and glaucoma suspects was much more pronounced than RNFLT reduction in all peri-macular quadrants imply that intraretinal changes may precede retinal nerve fiber changes in early glaucoma.

The potential association between RT and early glaucomatous changes is further supported by the statistically significant inverse correlation detected in this study between RT and applanation tonometry as well as between RT and dynamic contour tonometry in glaucoma suspects. Glaucoma suspects were not receiving any IOP-lowering medications, thus the IOP recorded, either by applanation or by dynamic contour tonometry, reflected true (pharmacologically unaltered) IOP values. Accordingly, the significant inverse correlation between IOP and RT in this group may possibly reflect a relationship between retinal structure and mechanical stress inflicted on the retina by IOP. A short-term sharp increase in IOP, such as that occurring during laser in situ keratomileusis (LASIK), has not been associated with a decrease in retinal thickness.27 However, studies based on animal glaucoma models evaluating changes associated with experimentally raised IOP have reported that a moderate but prolonged IOP increase may produce ongoing retinal strain.28 Alternately, in the case of the early manifest glaucoma group, the lack of a significant correlation between IOP (measured by either applanation or dynamic contour tonometry) and RT possibly reflects the fact that the IOP was pharmacologically modified (since target IOP had been achieved in all early manifest glaucoma group patients).

Interestingly, correlations between axial length and RT in all peri-macular quadrants were statistically significant in glaucoma suspects and early manifest glaucoma group but not in the control group. A decreased retinal thickness has been reported in eyes with increased axial length by previous studies2930 attributed to generalized thinning of posterior ocular walls in high myopia. Findings from the present study imply that eyes with early manifest glaucoma or even clinical suspicion for glaucoma development may be more prone to retinal thinning associated with an increased axial length. The fact that differences in axial length between groups were not statistically significant further supports this possibility. The significantly lower central corneal thickness in the early manifest glaucoma group and glaucoma suspects, compared with the control group, probably reflects the previously described inverse correlation between central corneal thickness and the predisposition to glaucoma development.31

The lack of significant correlations between RNFLT or RNFLT:RT ratio and all parameters examined, including axial length, central corneal thickness, applanation tonometry, dynamic contour tonometry or pattern standard deviation, in the control group, glaucoma suspects, and early manifest glaucoma group may possibly be attributed to a lack of glaucomatous changes (control group) or to the early stage of glaucoma (glaucoma suspects or early manifest glaucoma group) and further supports the concept that in early glaucoma intraretinal changes precede retinal nerve fiber changes. In the case of the early manifest glaucoma group, the significant correlation between pattern standard deviation and RT implies that retinal changes detected in this group have functional implications, corresponding to visual field defects, as previously suggested.32

Results of this study imply that peri-macular RT may be a more accurate indicator than peri-macular RNFLT for the course of the glaucomatous process in the early stages of glaucoma, in which intraretinal changes may predominate. We encourage further research in this area, to take advantage of such changes for earlier glaucoma diagnosis and timely treatment.