Silent Substitution Stimulation of S-cone Pathway and L- and M-cone Pathway in Glaucoma

Abstract

Purpose.: The study aimed for objective detection of primary open-angle glaucoma with selective color channel stimulation based on the silent substitution technique. In addition, an objective was analysis of the characteristics of individual color channels based on visual evoked potentials.

Methods.: Visual evoked potentials were recorded in 141 subjects (39 patients with glaucoma, 71 healthy subjects, and 31 age-matched healthy subjects) with two scalp electrodes after cone-specific flash stimulation. Silent substitution stimulation was presented with a 30-in. liquid crystal display. Separate responses were obtained for short-, medium-, and long-wavelength–sensitive cones. Age-matched subgroups were used to compare patients with glaucoma and healthy subjects.

Results.: The S-cone responses of age-matched healthy subjects had significantly different slopes for the first positive wave, compared with the responses of patients with moderate glaucoma. This difference was not observed in the L- and M-cone responses. Distinct changes in the S-cone response profiles were observed with increasing severity of glaucoma. Patients with severe glaucomatous damage were recognizable by the altered profiles of their visual evoked potentials. Healthy subjects showed significant differences between color channels.

Conclusions.: Glaucoma and its severity were objectively detected by using the silent substitution technique. The stimulation technique and signal analysis enabled assessment of the visual evoked potentials of individual color channels.

Primary open-angle glaucoma is a common disease throughout the world and is one of the most common causes of blindness. ¹ Early detection is essential in fighting its progression. ²  

Routine screening tests detect only a portion of glaucoma cases. Furthermore, these tests have limited sensitivities in the early stages of the disease. ^3,4 In recent years, significant advances have been made in imaging techniques. Their suitability for the early diagnosis of glaucoma and the effect on the techniques of influencing factors (e.g., cornea and anatomic variability) are currently being assessed. Imaging techniques such as retinal tomography and cornea pachymetry remain inadequate as single diagnostic tools. ^5,6 Standard diagnostic methods such as subjective perimetry are dependent on the patient's cooperation. Aside from their subjectivity, they have the disadvantage that significant retinal damage must be present for deficits in visual function to be diagnosed. ^4,7 Intensive research has been performed on developing objective vision diagnosis techniques. ^8–11 For example, the pattern electroretinogram (ERG) is a sensitive test for diagnosing glaucoma. ¹² Another objective examination method is the visual evoked potential (VEP), whereby vision deficiencies are objectively evaluated by analyzing the electrodiagnostic responses of the visual cortex. ^11,13–15  

Several studies have demonstrated that glaucoma is associated with disturbances in blue color perception. ^7,8,13,16 The photoreceptors (L-, M-, and S-cones) differ in some certain properties. In addition, differences are present in the phototransduction cascade. ^17–20 With glaucoma, the damage to intraocular neurons causes changes, even cell death, in higher levels of the visual pathway. Thus, changes in VEP may be of clinical use in diagnosing glaucoma. ^21,22 The resulting specific vulnerability to various clinical pictures could be useful in diagnosis if appropriate stimulation technology is applied. ^15,23–25  

For the specific stimulation of individual photoreceptor cells, tests have been performed to determine the characteristics of the color channels, functioning of the retina, and color perception. ^26–31  

In the present study, we sought to objectively analyze the cortical response signals after selective color channel stimulation, based on the silent substitution technique (SST). ^29,32 By this method, the characteristics of the color channels can be determined and parameters can be established. We applied our analysis to the VEP of healthy subjects and patients with glaucoma at different stages of the disease. 

Materials and Methods

Subjects

We studied 141 subjects (39 patients with glaucoma and 102 healthy subjects) at the AugenZentrum Siegburg and the Institute of Biomedical Engineering and Informatics, Ilmenau University of Technology (Fig. 1). All the patients were selected from AugenZentrum Siegburg. All subjects gave informed consent after the details and purpose of the study had been explained. The study was performed according to the tenets of the Declaration of Helsinki. 

Figure 1.

 

Summary of patient characteristics, including the number of examined subjects and subgroups for analysis, number of subjects in each subgroup, age in years, and sex. The group of 102 healthy subjects was used to examine several color channels. The group of 31 healthy subjects were age-matched and compared with the 29 patients with moderate glaucoma and 6 with severe glaucoma.

The group of healthy subjects consisted of 102 medically diagnosed volunteers without relevant diseases or defects in color perception. Optical deficits were corrected with existent visual aids. The subjects had visual acuities between 0.8 and 1.0, intraocular pressures less than 21 mm Hg, and normal visual fields. Sequential examinations were performed on the right and left eyes. Both eyes were subjected to S-cone stimulation and combined L-and M-cone stimulation. Thus, four recordings were collected for each subject. Because of the experimental setup, technical and biological artifacts occurred. Consequently, some of the recordings were excluded from further analysis. 

The measurements were repeated in eight subjects, to assess reproducibility. Only one of the measurements was used in the subsequent analysis (besides the reproducibility test). 

Furthermore, 39 patients with glaucoma were examined in an analogous manner. Four patients were excluded due to artifacts (Fig. 1). The patients were medically prediagnosed and divided into two groups: moderate and severe glaucomatous damage. All the patients with glaucoma had a history of intraocular pressure above 21 mm Hg. The intraocular pressures of all the patients were normalized by medical therapy. The group with moderate glaucoma consisted of 29 patients with visual acuities of between 0.6 and 1.0. Their visual fields showed areas of mild to moderate sensitivity loss but no absolute scotomata. The severe glaucoma group consisted of six patients who had absolute scotomata (Bjerrum scotoma [BS] and concentric constriction [CC]) in their visual fields (Table 1). 

Table 1.

 

Diagnosis of Patients with Severe Glaucoma

	Patient 110		Patient 129		Patient 140		Patient 144		Patient 175		Patient 176
	OS	OD	OS	OD	OS	OD	OS	OD	OS	OD	OS	OD
Diagnosis	BS	Artifact	BS	BS	BS	Artifact	CC	CC	BS	BS	BS	BS

An age-matched healthy subject group of approximately equal size (31 subjects from the 102 originally enrolled) was established (Fig. 1) to compare the control group of healthy subjects with the group of 29 patients with moderate glaucoma. 

Stimulation

A 30-in. liquid crystal display (Myrica V30–1; Fujitsu Siemens, Munich, Germany) was used with native display resolution (1280 × 768 pixels) and a frame rate of 60 Hz (Fig. 2D). The display was connected to a stimulation unit (THERA PRAX; neuroConn GmbH, Ilmenau, Germany). The frame rate was verified with a PIN-diode (BPX 65; Siemens, Munich, Germany) and a digital memory oscilloscope (TDS 3045; Tektronix, Beaverton, OR) by applying a 30 Hz black–white flicker sequence to the display, which was generated by the stimulation system. The display was adjusted to the chromatic coordinates x = 0.33 and y = 0.33 with a compact-array spectrometer (CAS 140B; Instrument Systems, Munich, Germany). The spectral distribution and temporal constancy of the emission spectra of the red, green, and blue color channels were measured with the spectrometer. Gamma correction was performed by determining the characteristic gamma curve for the stimulator and the graphic card (8-bit VGA). The color channel intensities were 117 (red), 282 (green), and 47 (blue) cd/m². 

Figure 2.

 

Schematic representation of the timing, configuration, and geometry of the stimuli, as well as the major color values. (A) The CIE coordinates (X, Y, Z) of the S-cone stimulation (S) and combined L- and M-cone stimulation (LM) were based on the 1964 standard colorimetric observer. (B) The ON and OFF terms corresponded to the two colors of each stimulation sequence and the stimulus size. (C) The timings and durations of the stimuli. A random ISI was chosen to reduce habituation effects, which can cause alpha spindles. (D) Additional information about the stimulator (L, luminance; R, red; G, green; B, blue). (E) The computed LMS values and resulting cone contrasts. S-cone stimulation (S) means the substitution (respectively no change in activation) of the L- and M-cones. Hence, the L and M values for the ON and OFF terms must be equal. For the combined L- and M-cone stimulation (LM), the S-cones are substituted, and the S values (activation of S-cones) are equal. Both stimulation conditions are optimized for approximately equal and maximum cone contrast (C).

The RGB color values were transformed into the standard color values of virtual XYZ space.³³Figure 2A shows the XYZ coordinates of the stimuli for the 10° CIE observer. Hunt fundamentals for 10° and larger viewing conditions were used for the transformation to LMS-space.³⁴ The transformation matrices for the applied stimulator system are as follows:  

 

 

 

A color combination for the S-cone stimulation (L1 = L2, M1 = M2, S1 ≠ S2) and the combined L-and M-cone stimulation (L1 ≠ L2, M1 ≠ M2, S1 = S2) was calculated for the display. The activation of several cone types and stimulation contrasts was determined (Fig. 2E), and they could be selectively changed by adjusting the color combinations. We verified the S-cone isolation with the help of an adaption and bleaching experiment, which is based on the isolation technique from Stiles.³⁵ The cone contrast (C) was determined by the following formula (according to the Michelson contrast)³⁶ 

 

where, E_ON and E_OFF represent the cone activation levels during the ON and OFF phase, respectively. E is the amount of light absorbed per unit of retinal area for the three cone types.³⁴  

The PIN-diode was also used to analyze the electro-optical transient behavior of the display caused by different color combinations of the stimulation sequences. The time between the starting and ending points of the presented color combination was measured by applying the S-cone and combined L- and M-cone sequence. The transient times were 6.2 ms for the S-cone stimulation and 5.4 ms for the L- and M-cone stimulation. To correct the frame delay in the electroencephalogram (EEG), we analyzed the photodiode signal and the synchronization trigger from the stimulation unit. The trigger coincided with the onset of stimulus generation by the unit, and the photodiode signal coincided with stimulus imaging by the display. The frame delay was 7.0 ms for all stimulations. 

During the examination, the subject's head was positioned on a chin–forehead rest. The left and right eyes were tested sequentially; the eye not being tested was covered with an eye patch. The eye being tested was positioned central to the stimulator at a distance of 0.5 m. This resulted in a maximum visual angle of 64° × 42° (Fig. 2B). 

Circular flash stimulation with a fixation point was selected. ³⁷ A balanced sequence was used for the stimulation order and for the order in which the eyes were tested. The stimulus size was ±11°. All volunteers were light adapted. Furthermore, the contribution of the rod system was suppressed with an alternating stimulus and by the ambient room luminance of 100 cd/m². There was an adaption area (∼375 cd/m², cold cathode fluorescent lamp spectrum with dominant wavelengths of λ₁ = 488 nm, λ₂ = 544 nm, λ₃ = 611 nm) surrounding the circular stimulus area to eliminate rod responses during stimulation of the S-cones. The luminance of the adaption area resulted in a retinal illuminance of 3.3 log td. 

The ON stimulus time was 17 ms and the OFF time was 767 ms. An additional random interstimulus interval (ISI) was chosen at 17 to 600 ms to prevent the influence of periodic disturbance signals and habituation effects (Fig. 2C). A total of 160 repetitive stimulations were performed per examination. 

Data Recording

The EEG signal was recorded simultaneously to the stimulation via the examination system (THERA PRAX; neuroConn GmbH). EEG signals were recorded with an electrode cap and Ag/AgCl ring electrodes (Easycap, Herrsching, Germany) placed over the visual cortex at Oz and the reference position Fz. The sample rate was 512 Hz. The hardware trigger signal from the stimulation unit was used and adjusted regarding the delay (see stimulation section), to precisely correlate the EEG signal with the excitation. 

Preprocessing

Signal processing and analysis of EEG and VEP signals were performed (MatLab software; The MathWorks, Natick, MA). A digital software filter was used to filter the electrode drift (0.8 Hz high-pass). In addition, a 30 Hz digital low-pass filter was used. The signal was filtered with elliptic infinite impulse response filters in the forward and backward directions to prevent phase shifts. In addition, trials that had physiological signal distortions (e.g., muscle activity, increased alpha-activity, and eye movement) were detected by using artifact detectors and excluded from the response signal analysis. ³⁸ A strong technical distortion in the area of the physiological response signal (17 Hz) also had to be eliminated by adapting and applying the matching pursuit method. ³⁹ A separate classification of the 160 recorded trials was performed. The correlation coefficient between the average signal of all valid trials and each single valid trial was determined. The 100 trials with the highest correlation coefficient were averaged. The constant number of averaged trials guaranteed comparability of the response signals. 

Analysis

As test parameters, we used the peak latency of the first negative wave (N1) and the first positive wave (P1), the peak-to-peak amplitude, the slope, and the area of the S-cone and combined L- and M-cone response signals (Fig. 3). These values were calculated by using automated analysis algorithms. The peak latency was determined by finding local latency minima and maxima. These latency periods and corresponding amplitudes were used to calculate the peak-to-peak amplitude and slope. The area was determined by integrating all the amplitudes in the range of the main response signal (between the first and second negative wave). A manual correction was performed in the case of false parameter detection. 

Figure 3.

 

Examples of S-cone responses (dashed curve) and combined L-and M-cone responses (solid curve) to SST stimulation and the analysis parameters (N1, P1, slope, area, and peak-to-peak amplitude).

The data were analyzed by multivariate analysis of variance (MANOVA) (SPSS software; SPSS Inc., Chicago, IL). 

Results

Healthy Subjects

Figure 3 shows two response signals for the stimulated color channels of a healthy subject; it also shows the parameters used to analyze the signals. The two typical response signals differ in the curve shape. 

A grand average response for the two stimulated color channels and the corresponding standard deviation (SD) was calculated from the recordings of healthy subjects (Fig. 4). 

Figure 4.

 

Grand averages (solid curves) and SD (dashed curves) of the combined L- and M-cone response (top) and the S-cone response (bottom) of the healthy subjects.

We found variations in the curve shapes of the stimulated color channels. The peak-to-peak amplitude of the S-cone response was 9 μV, which is markedly less than that of the L- and M-cone response (13 μV). The N1 latency of the S-cone response (111 ms) was greater than that of the L- and M-cone response (90 ms). The P1 latency was 182 ms for the S-cone response and 140 ms for the L- and M-cone response. The maximum variance of the response signals occurred during the main response (50–400 ms), with an average SD of 2.6 μV for the S-cone response and 3.8 μV for the L- and M-cone response. 

Parameter analysis was performed to investigate the differences between the color channels (Table 2). Statistical analysis with MANOVA revealed significant differences between the selective stimulated color channels for all parameters. 

Table 2.

 

Parameters for the S-cone Response and the Combined L-and M-cone Response of the Healthy Subjects

Parameter		S-cone Response (n = 178)	L- and M-cone Response (n = 183)	Δ	P
Nl (ms)	∅	110.0	95.0	15.0	<0.001
	SD	16.2	18.6
P1 (ms)	∅	183.0	150.0	33.0	<0.001
	SD	29.3	28.7
Slope (μV/ms)	∅	0.2	0.4	0.2	<0.001
	SD	0.1	0.2
Area (μVms)	∅	552.0	806.0	254.0	<0.001
	SD	278.3	402.2
Peak-to-peak amplitude (μV)	∅	14.0	19.0	5.0	<0.001
	SD	5.3	7.4

Repeated measurements showed no differences in the results. 

Given the large differences in the ages of the subject groups (healthy subjects and patients with moderate glaucoma), the relationship between the analysis parameters and age was evaluated in the healthy subject group. Examination of latency shifts revealed an average increase in the S-cone response in N1 latency of 4 ms per decade and in P1 latency of 3 ms per decade. The average L- and M-cone response values were calculated to be 5 ms per decade (N1 latency) and 4 ms per decade (P1 latency). 

Figure 5 shows the age structure of the test subjects. Based on this distribution, two groups were formed (age ranges, 10–44 and 47–85 years). As an example, Figure 6 shows the relationship between the calculated values and age for the N1 latency parameter. 

Figure 5.

 

Histogram of the age distribution in the healthy subject group.

Figure 6.

 

Example of age dependency: parameter N1 in the healthy subject group.

The statistical significance of the differences in analysis parameters for the two age groups was studied by using MANOVA. The resulting probabilities are P _N1 = 1E-13, P _P1 = 3E-4, P _slope = 0.13, P _area = 0.61, and P _{peak-to-peak amplitude} = 0.02. The peak-to-peak amplitude, N1 and P1 showed a significant difference between the two age groups (P < 0.05), whereas the slope and area showed no significant difference (P > 0.05). However, the statistical test results do not rule out an age dependency of the parameters. Therefore, the following observations were made when working with age-matched groups. 

Patients with Glaucoma

Table 3 lists the averages and SDs of the analysis parameters for age-matched healthy subjects and patients with moderate glaucoma, the differences between both groups (Δ), and the significance of the differences (P-value). Significant differences were observed in some parameters between patients with moderate glaucoma and healthy subjects. These differences were not observed in the L- and M-cone response. 

Table 3.

 

Parameters of Age-Matched Groups of Healthy Subjects and Patients with Moderate Glaucoma by Selective Cone Stimulation

Parameter		S-cone Response			L- and M-cone Response
		Patient (n = 45)	Volunteer (n = 62)	Δ (P)	Patient (n = 49)	Volunteer (n = 63)	Δ (P)
Nl (ms)	∅	121.0	118.0	3.0	108.0	106.0	2
	SD	18.1	13.1	(P = 0.32)	28.6	14.9	(P = 0.58)
Pl (ms)	∅	199.0	188.0	11.0	165.0	162.0	3
	SD	33.1	28.9	(P = 0.07)	39.9	30.6	(P = 0.66)
Slope (μV/ms)	∅	0.16	0.23	0.07	0.31	0.34	0.03
	SD	0.1	0.1	(P = 0.01)	0.2	0.2	(P = 0.37)
Area (μVms)	∅	486.0	573.0	87.0	707.0	715.0	8
	SD	408.6	307.9	(P = 0.23)	386.1	366.2	(P = 0.91)
Peak-to-peak amplitude (μV)	∅	12.0	14.0	2.0	14.0	16.0	2
	SD	10.2	6.3	(P = 0.21)	7.9	6.0	(P = 0.22)

 The difference between the two groups and the significance are expressed by Δ and the P-value. respectively. The patient group did not include the patients with severe glaucoma.

Distinct changes in the S-cone response profiles were observed in patients with severe glaucoma damage compared with that of a healthy subject after selective S-cone stimulation (Fig. 7). 

Figure 7.

 

S-cone response of a healthy subject and of patients with severe glaucomatous damage; clear changes are apparent in the S-cone response profiles of the patients.

The response signal of the healthy subject shows a typical, distinct VEP (Fig. 7, top). Six of the patients with glaucoma (10 S-cone response signals) were diagnosed with severe glaucoma damage. Six of the 10 response signals of these patients showed no VEP (Fig. 7, patient 176, left eye). Negativity of response (Fig. 7, patient 110, left eye) instead of the typical VEP was observed for three evaluations. One response showed a weak VEP despite diagnosis of concentric constriction. The second eye of this patient showed no VEP. In summary, it can be concluded that severe glaucoma damage is manifested in the S-cone response for 9 of the 10 eyes. 

A change in the S-cone response with disease severity can also be shown in the analysis of the grand averages of the respective group (Fig. 8). 

Figure 8.

 

Change in the S-cone response (grand averages: solid curves; SD: dashed curves) is dependent on glaucoma status. The S-cone response of an age-matched healthy subject (top), patients with moderate glaucoma (middle), and patients with severe glaucoma (bottom) are shown. Severity of glaucoma results in a latency shift and reduction of the response signal (middle) up to loss of VEP (bottom).

A strong VEP (Fig. 8, top) was found in the S-cone response grand average of the age-matched healthy subject group. A weaker signal and a change in latency were observed in the S-cone response grand average of patients with moderate glaucoma (Fig. 8, middle). The signal strength was distinctly weaker for patients with severe glaucoma (Fig. 8, bottom). The grand average shows that there is no response signal for this group. 

Discussion

To our knowledge, this study is the first in which optimal selective color channel stimulation has been performed based on the SST method, together with an objective validation with VEP, in a large number of healthy subjects and in patients with glaucoma. We observed damage to the S-cone pathways in patients with glaucoma and changes in the S-cone response with the severity of glaucoma. 

Cone isolation was demonstrated by an adaption and bleaching experiment. The absence of the S-cone response after the bleaching procedure in contrast to the unaffected L- and M-cone response was the evidence of the successful cone isolation. As shown in Figure 2E, both stimulation conditions were optimized for nearly equal and maximum cone contrast (≥90%). However, optimization for equal cone contrast was performed, since it is known that latency variations are much larger for different cone contrasts than for different luminance contrasts. ⁴⁰ Circular flash stimulation was selected because the study was designed to have as few restrictions as possible in terms of the health, age, and cooperation of the subjects. ³⁷ Onset–offset stimuli produce larger and more distinctive response signals to chromatic stimuli than do transient reversals or pattern-reversal stimuli. ^41,42 The light adaption of the volunteers and the adaption area (retinal illuminance of 3.3 log td) should prevent the rod contribution to the response signals. ⁴³  

It has been shown that glaucoma can be diagnosed based on deficits in the blue color channel, making a selective, objective examination of this color channel of great interest. ^{7,8,10,13,16,21,24,44–46} Many of these previous studies were performed by using perimetry, ^7,45,46 which has the considerable disadvantage of being a subjective method. In contrast, ERG (especially the pattern ERG) is a useful objective testing method. ^{24,44,47–49} However, disease-related changes in the layers of the visual pathways and the influence of cortical processing in the case of glaucoma could not be determined. ^21,22 VEP allows testing of the entire visual pathways. Some researchers, have performed VEP testing on patients with glaucoma. ^8,10,13,50 However, rather than the SST method, blue-on-yellow stimulus, ⁸ black-white pattern reversal stimulus, ^10,50 and heterochromatic flicker photometry (HFP) ¹³ were used to stimulate color channels. 

In the present study, we performed an objective examination of the visual pathways from the retina to the visual cortex by analyzing VEP. The results show that the S-cone response of patients with moderate glaucoma is different from that of age-matched healthy subjects (Table 3). Latency shifts of 3 (N1) and 11 (P1) ms were determined. We found a significant difference in the parameter slope (0.07 μV/ms) between the two groups. Therefore, the S-cone response revealed distinct differences between the two groups compared with the differences of the combined L-and M-cone response (Δ_N1 = 2 ms, Δ_P1 = 3 ms, Δ_slope = 0.03 μV/ms). A latency shift of the S-cone response has also been observed by other researchers. For example, with blue-on-yellow stimulation, Horn et al. ⁸ found a peak time difference of 7 to 19 ms between a glaucoma patient group and a control group. Rodarte et al. ¹⁰ found differences of approximately 3 to 6 ms. However, we found only a minor difference in the latency shift of the L- and M-cone response. This finding supports the assumption that glaucoma damage is initially manifested in the blue color channel. Note that the analyzed data originate from patients with moderate glaucoma and healthy subjects. 

In addition to the latency shift, distinct changes occurred in the configuration of the S-cone response toward the absence of response (Fig. 7), especially in patients with severe glaucoma damage. It was not possible to determine parameters when the response signals were highly deformed or absent. However, these abnormal S-cone response profiles were visually determined for 9 of 10 eyes in the group with severe glaucoma damage. Only one recording of an eye with severe glaucomatous damage had a detectable VEP; no VEP was recordable from the second eye of this patient. The remaining response in one eye could be explained by the different progression of the disease in each eye. 

Figure 8 shows a reduction in the response signal and a shift in the latency of the grand average response signal of the patients with moderate glaucoma (Fig. 8, middle) compared with the grand average response signal of the age-matched healthy subjects (Fig. 8, top). Furthermore, there is no defined VEP in the case of severe glaucoma damage (Fig 8, bottom). Therefore, the severity of the disease can be assessed by objective electrophysiological testing. Aldebasi et al., ¹³ using HFP stimulation, reported distinct changes in the configuration of the S-cone response in a comparison of a control group with a group of patients with primary open-angle. Using blue-on-yellow stimulation, Horn et al. ⁸ also found an increase in the latency of the S-cone response in the progression of glaucoma. These findings support the hypothesis of a change in S-cone response with glaucoma progression. 

In comparing the stimulated channels within the healthy group (Fig. 4), the grand average response signals exhibit larger amplitudes, a greater area, and an increased slope of the L- and M-cone response. The latency of N1 and P1 against the S-cone response was distinctly lower. It can be concluded that processing is faster for the red-green channel. Porciatti and Sartucci ⁴⁰ and Rabin et al. ⁴² reached the same finding by using blue-yellow stimulation in conjunction with sinusoidal gratings. The SDs of the calculated grand averages (Fig. 4) mirror the large variances of the interindividual response signals. This finding can be attributed to the fundamentally high variability of the EEG, in addition to the individually varying response signals of the stimulations. 

The extracted VEP parameter also showed significant differences (P < 0.001) between the two stimulated color channels (Table 2), as seen in the grand average curve shape (Fig. 4). The higher peak-to-peak amplitude, slope, and area of the L- and M-cone response represent a distinctly stronger response signal in the stimulation of L- and M-cones. The latency shift between the S-cone response and the combined L- and M-cone response was 15 to 33 ms. Other researchers have also determined differences between color channels. Latency shifts between color channels have been observed even in infants. ⁴¹ Rabin et al. ⁴² determined a difference of 25 to 30 ms in adults in blue-yellow stimulation with sinusoidal gratings, and Robson and Kulikowski ⁵¹ found a latency shift of 13 ms between color channels with blue-yellow stimulation. Porciatti and Sartucci ⁴⁰ found comparable waveforms with greater amplitudes and shorter latency for L- and M-cone response after onset VEP examinations for S-cone response and L- and M-cone response. Differences in reaction times during color channel examinations have been attributed to the visual processing system. ⁵² Processing and further transmission of color-selective stimulation up to the visual cortex occurs in different systems (parvocellular layers, red-green information; koniocellular layers, blue-yellow information) ^21,53,54 and probably causes differences in further signal processing. ^55,56  

The latency shifts of the selectively stimulated color channels observed in the present study correspond to the physiological characteristics of color-opponent neurons and processing in the parvocellular and koniocellular visual pathways. ⁴⁰ Possible reasons for latency shifts can be determined even in the very early processing stages of the visual system. ERG studies reveal that the response signals for different color channels have different latencies and amplitudes from the first processing steps on the retina. ^57,58 The latency is also dependent on the luminance of stimuli and the respective contrast of activation. 

Our analysis of the influence of age on VEP parameters showed significant differences between the two age groups in N1, P1, and peak-to-peak amplitude. The increase in latency was approximately 4 ms per decade. Similar age-related influences on VEP have been observed by other researchers. ^9,59,60 Latency shifts of approximately 8 ms per decade have been observed in onset and reversal studies, amplitude values dropped slightly, although a fundamental change in form could not be determined. ^9,60 In a multifocal black–white pattern reversal stimulation, Rodarte et al. ¹⁰ determined a slight age-related dependency of 1.3 ms per decade. In the present study the parameters slope and area did not show any significant differences between the two age groups; however, an age-related dependency could not be ruled out. Therefore, age-related dependencies should be taken into consideration when testing. Possible reasons for age-related dependencies of VEP are the effects of aging on the photoreceptors, a reduction in contrast sensitivity, or an increase in the opacity of the eye. ⁶⁰  

We presented the results of tests that involved optimal selective color channel stimulation based on SST methodology and an objective evaluation with VEP, as performed on a large number of healthy subjects and patients with glaucoma. The applied stimulation methods offered advantages in objectivity and flexibility of selective color channel stimulation. This approach could enable new diagnostic possibilities and improved early diagnosis. A receiver operating characteristic analysis of the classification, based on the developed parameters, should be performed to quantify the impact of the method for diagnosis. To further validate the significance of the methodology, progressive follow-up studies or studies with a large number of patients who are at various stages of the disease as well as the detailed observation of accompanying diseases are necessary. 

Footnotes

 Supported by Grants 13N8521 and 03IP605 from the German Federal Ministry of Education and Research.

Footnotes

 Disclosure: P. Bessler, None; S. Klee, None; U. Kellner, None; J. Haueisen, None

The authors thank Silke Weinitz and Sylvi Herzog for performing the examinations.