A cross-sectional study was performed on consecutive patients presenting to the Glaucoma Service at the Fondazione Policlinico Universitario A. Gemelli IRCCS - Università Cattolica del Sacro Cuore of Roma, Italy, from April to June 2021. The study protocol (ID 3934) was approved by Policlinico Gemelli's Ethics Committee and carried out in accordance with the tenets of the Declaration of Helsinki. Written informed consent was obtained from all subjects following an explanation of the nature and intent of the study.
Subjects
The study population included a group of 28 glaucoma patients consisting of pre-perimetric, early and moderate stage patients (64.3% women and 35.7% men; mean age ± SD: 58.64 ± 14.04 years, range: 40–80). Seventeen normal subjects, whose sex and age distribution were comparable with those of patients, were also enrolled into the study as a control group.
Both patients and controls underwent a full ophthalmologic examination, including best-corrected Snellen visual acuity measurement, slit-lamp biomicroscopy of the ocular anterior segment and fundus, Goldmann applanation tonometry, and gonioscopy. Moreover, central corneal pachymetry by the digital ultrasonic pachymeter Pachmate DGH55 (DGH Technology, Inc., Exton PA, USA), computerized white-on-white 30 − 2 visual field testing by Humphrey Field Analyzer 750i (HFA; Carl Zeiss Meditec, Inc., Dublin CA, USA), SS-PERG with adaptation paradigm recording by Retimax (CSO, Florence, Italy), and measurement of both peripapillary retinal nerve fiber layer (RNFL) and macular ganglion cell/inner plexiform layer (GCIPL) thicknesses by spectral-domain Cirrus HD-OCT (model 5000, sw. version 10.0, Carl Zeiss Meditech, Inc., Dublin CA, USA) were also performed. Visual field, PERG, and OCT analyses were obtained for each subject within 1 week of each other.
Inclusion criteria were as follows: age between 40 and 80 years, normal range central corneal thickness values (520–570 µm), and fulfillment of current clinical practice criteria used to diagnose glaucoma patients: (1) elevated intraocular pressure (IOP) at diagnosis (> 21 mmHg on two separate occasions); (2) open anterior chamber angle assessed by gonioscopy; (3) abnormal optic disc as defined on routine stereoscopic examination with slit-lamp biomicroscopy and 78-diopter (D) lens by vertical cup/disc (C/D) diameter ratio > 0.6 in relation to optic disc size, an interocular C/D diameter ratio asymmetry ≥ 0.2 unexplained by side differences in disc size, diffuse or focal rim thinning, notching; (4) reliable and reproducible visual field abnormalities (see below) accomplishing the Hodapp–Parrish–Anderson criteria for early to moderate glaucoma stages, as well as normal visual field for pre-perimetric glaucoma.
All patients were under treatment with one or more topical hypotensive drugs (β-blockers, prostaglandin analogues, carbonic anhydrase inhibitors, and 𝛼2-agonists) providing a stable IOP lower than 21 mmHg, and sometimes neuroprotectants.
Exclusion criteria were as follows: corrected Snellen visual acuity < 20/25, refractive errors equal to or more than 2 diopters (D) of myopia or hyperopia and 1 D of astigmatism, optic disc pallor exceeding cupping, cataract surgery or changes in the IOP-lowering and/or neuroprotectant therapies within the 3 months before patient recruitment and morpho-functional assessment, low perimetric reliability, and ophthalmologic or neurologic diseases which may affect visual function and exam execution.
Perimetry
Visual field sensitivity was determined for each eye using the HFA central 30 − 2 SITA-standard test. Only visual field exams with good reliability indices (fixation losses, false positive and negative errors < 20%)20 were evaluated. Abnormal perimetry was defined as a typical reproducible defect (arcuate and/or paracentral scotoma or nasal step) in three consecutive exams,21 with one or more of the following alterations: Glaucoma Hemifield Test outside normal limits, pattern standard deviation (PSD) with p < 5%, and a cluster of ≥ 3 adjacent points, not contiguous with the field borders nor the blind spot, in the upper and/or lower hemifield of the total and pattern deviation plots with p < 5%, one of which reached p < 1%. For data analysis, the two global indices of field sensitivity, mean deviation (MD) and PSD, were collected.
OCT Recording
OCT imaging was performed using the Cirrus HD-OCT on both peripapillary RNFL and macular GCIPL. The OCT lens was adjusted for the refractive error. The subject was instructed to stare at the internal fixation target with the eye under examination, to enable the optic disc and the macula to subsequently come into the appropriate windows and to be centered. The scan protocols were the Optic Disc Cube 200x200 and Macular Cube 512x128 for the study of peripapillary RNFL and macular GCIPL, respectively. After optimizing the reflective signal, three separate scans were obtained per eye by each protocol during the same session, and the best one with optimal signal strength (> 6/10) and scan image centering, no movements during scans or anomalous internal/external boundary definition was used for the analysis. Average RNFL and GCIPL thicknesses were collected.
Technique of PERGx Recording and Analysis
The PERG was acquired simultaneously from both eyes with standard skin surface electrodes (Grass gold, 10 mm diameter) taped on the lower eyelids (active), ipsilateral temples (reference), and central forehead (ground) using the Retimax system. Subjects fixated at the center of the stimulating field (size, 60° width x 50° height) with natural pupils, whose size was measured (mean value, 3.5 ± 1.0 mm) at a viewing distance of 57 cm wearing full refractive correction. No statistically significant differences in pupil size were observed between patients and normal subjects. Fixation was monitored by a trained observer. Signals were amplified (gain of 100 k, 1–250 Hz bandwidth, 6 dB/octave slope), digitized (12 bit resolution, 2 kHz sampling rate, 100 µV AC range) and averaged in synchronism with stimulus onset. Artifacts, mainly from blinks or large eye movements, were automatically rejected to minimize amplitude bias.
PERGx was recorded similarly to a published protocol.19 In particular, SS-PERG was elicited by black-white horizontal gratings of 0.8 cycles/degree spatial frequency and 95% contrast (mean luminance: 35 cd/m2), modulated in counterphase at 7.5 Hz (15 reversals/s). Stimulus was electronically generated on a high-resolution organic light-emitting diode television (OLED TV) monitor and administered continuously over nearly 2 minutes. The response was recorded as a sequence of 10 partial averages (packets), each one (10 seconds average duration) obtained summing up to 60 cycles.18 The first packet was obtained with the patient exposed to a uniform gray stimulus equiluminant with the pattern stimulus, to obtain a “noise” response. Two replications of the entire adaptation paradigm, including noise level assessment, were recorded, and an appropriate time interval between replications was chosen to avoid residual adaptive effects.
A discrete Fourier analysis was performed on the recordings in order to isolate the PERG second harmonic (2P, the significant outcome of PERG experiments)22. The resulting waveforms (9 for each patient, excluding the first noise waveform) were further analyzed by plotting 2P amplitude and phase as a function of time for each subject. At the end of the procedure each response consisted of 9 second harmonic packets. Furthermore, PERGx amplitude and phase values were averaged across group subjects both for single packets (average scalar amplitude and phase) as well as for each response over the 9 packets (grand-average vector amplitude and phase). We assumed that the grand-average vector parameters represented an index of non-adapted RGC activity and corresponded to the ordinary SS-PERG.15
In addition to the grand-average measurements, we studied the PERGx habituation in terms of amplitude slope and phase angular dispersion. With regard to the former, a linear regression analysis was automatically applied to the PERGx vector amplitude plotted as a function of packets’ order number from each patient, to determine the slope (angular coefficient of linear function) of the adaptive amplitude changes. The residuals of this regression provided an estimate of the “noise”, that is the component of variance not attributable to the adaptive modifications. Angular dispersion23 was considered as an index of PERGx phase variability during the habituation process.
Statistical analysis
Only the right eyes were considered. The following PERGx parameters were chosen as the primary outcome measures of the study: average scalar amplitude and phase for each packet; amplitude slope and phase angular dispersion, as measures of adaptive PERGx changes; and grand-average vector amplitude and phase, as surrogates of ordinary non-adapted SS-PERG. The secondary outcomes were the OCT morphometric parameters of peripapillary and macular retina, namely the RNFL and GCIPL thicknesses.
Statistical analysis was performed using SPSS 17.0 for Windows (IBM SPSS, Armonk NY, USA) and Origin 6.0 (Microcal Origin, Microcal Software Inc., Northampton MA, USA). Alpha and beta error were established at 5% and 20%, respectively. The following variables were considered as continuous quantitative variables: age; IOP measurement; perimetric MD and PSD indices; OCT RNFL and GCIPL thicknesses; PERGx amplitude slope, grand-average vector amplitude, angular dispersion and grand-average vector phase. Assimilability to normal distribution was evaluated using the Kolmogorov-Smirnov test.
The electrophysiological 9 signal packets were separately analyzed and PERGx amplitude and phase were initially studied as individual temporal series, and then averaged across subjects and plotted as a function of the single sequential packets. Linear regression analyses were applied to the amplitude and phase data in order to evaluate the presence of an adaptive behavior.
Univariate comparison between the two groups’ parameters was performed using the two-tailed Student’s t-test for independent groups. A Bonferroni corrected p value < 0.05 was considered to establish the statistical significance of the results. Receiver operating characteristic (ROC) curves were used to study the diagnostic accuracy of PERGx parameters (i.e. their ability to differentiate between unhealthy and healthy eyes) by evaluating the area under the curve (AUC), with an AUC of 0.5 indicating no discrimination ability and an AUC of 1.0 indicating maximal discrimination ability. The optimal cutoff points for glaucoma diagnosis were estimated as the values that warranted the best combination of true positive rate (sensitivity) and false positive rate (1-specificity).