Multifocal Electroretinograms in X-Linked Retinoschisis

Abstract

Purpose. To study local retinal cone function in patients with X-linked retinoschisis (XLRS) by multifocal ERGs (mfERGs).

Methods. mfERGs were recorded from seven eyes of seven patients with XLRS (mean age ± SD, 22.1 ± 3.2 years; range, 18 to 25 years). Five eyes had microcystic changes in the macula and two eyes had nonspecific macular degeneration. Two eyes had peripheral retinoschisis, and some of the stimuli fell on this area. The stimulus array consisted of 103 hexagons and the total recording time was set at approximately 4 minutes. The amplitudes and implicit times of both focal and summed responses for the first- and second-order kernels were analyzed.

Results. The amplitudes of the first-order kernel were markedly reduced in the central retina in all eyes. A large variation was observed in the amplitudes outside the fovea. The amplitudes of the focal cone ERGs at the peripheral retinoschisis did not differ from those recorded from adjacent retinal loci without the retinoschisis. The implicit times of the first-order kernel were significantly delayed, and the amplitudes of the second-order kernels were more affected than the first-order kernels across the whole field in all XLRS eyes.

Conclusions. The cone-mediated retinal responses were more impaired in the central than peripheral retina in eyes with XLRS. Delayed implicit times of the first-order kernel and reduced second-order kernel across the whole testing field in all XLRS eyes suggest that there is widespread cone-system dysfunction in XLRS.

X-linked retinoschisis (XLRS) is an inherited vitreoretinal dystrophy that is one of the most frequent causes of juvenile macular degeneration. ^{1 2} XLRS is characterized by microcystic changes within the macula in nearly all patients, and by peripheral retinoschisis in approximately 50% of the patients. The schisis occurs at the level of the nerve fiber and ganglion cell layers of the retina, ^{3 4 5} and it has long been proposed that defective degenerating Müller cells or inner retinal cells may be the primary cause of the pathologic changes. ⁵  

One of the typical functional characteristics in the retina of patients with XLRS is an abnormal scotopic ERG elicited by a bright-flash stimulus. The a-wave amplitude is normal or near normal, but the b-wave amplitude is substantially reduced, resulting in a negative-type ERG waveform. ^{6 7 8 9 10 11} The selective b-wave reduction in XLRS had been thought to be due to a generalized dysfunction of the Müller cells under the assumption that the b-wave originates from Müller cells. However, there has been growing evidence that the b-wave originates mainly from depolarizing ON bipolar cell directly. ^{12 13}  

Recent molecular genetic studies have provided new insights into the mechanism involved in the retinal dysfunction in the eyes of patients with XLRS. The gene causing XLRS encodes a retina-specific polypeptide, RS1, also called retinoschisin. ¹⁴ RS1 is expressed on the cell surface of rod and cone photoreceptor cells and also on bipolar cells, but not on Müller cells or ganglion cells. ¹⁵ This protein is thought to play an important role in cell adhesion or cell-cell interaction that maintains the integrity of the retinal neurons. ^{16 17} RS1-deficient mice show an overall disorganization of the retinal cell layers, a splitting of the inner nuclear layer, and a reduction of the ERG b-wave amplitude. ¹⁸  

Although there are many electrophysiological and psychophysical studies on the retinal function of eyes of patients with XLRS, very little is known about the retinal function in localized regions. ^{8 19 20} The multifocal ERGs (mfERGs) technique allows the simultaneous recordings of focal cone ERGs from multiple retinal loci in a single recording session of several minutes. ^{21 22 23 24 25} This technique is particularly useful for assessing cone-mediated function topographically in patients with retinal diseases. 

The purpose of this study was to investigate the functional status of the cone system at different loci in the posterior pole of the eye of seven young patients with XLRS by use of the mfERG technique. 

Subjects and Methods

Subjects

We recruited seven patients with XLRS from our clinic at the Department of Ophthalmology, Nagoya University School of Medicine. The clinical characteristics of these seven patients are summarized in Table 1 . Their mean age at the time of examination was 22.1 years with a range of 18 to 25 years. Each patient was diagnosed as having XLRS based on the ophthalmologic examination, standardized electrophysiological testing, and inheritance pattern. Molecular genetic examination of the RS1 gene was performed in two (P1 and P6) of the seven patients, and Ala101Pro ²⁶ and Glu72Lys mutations were identified in P1 and P6, respectively. 

All seven eyes had macular abnormalities. Five of seven eyes (P1–P5) had microcystic findings without any visible changes in the RPE. The other two eyes (P6 and P7) had nonspecific macular degeneration. In addition to the macular abnormalities, two of the seven eyes (P2 and P3) had peripheral retinoschisis, and in recording the mfERGs, some of the stimuli fell on the peripheral retinoschisis (Fig. 1) . 

For controls, 15 age-matched normal subjects (ages 19 to 38 years; mean, 27.1 years) were examined with the same recording protocol. None had known abnormalities of the visual system and their visual acuity was 1.0 (20/20) or better. 

Informed consent was obtained from all patients and volunteers after a full explanation of the procedures. All studies were conducted in accordance with the principles embodied in the Declaration of Helsinki. 

Multifocal ERGs

One randomly selected eye was examined with mfERG as previously reported in detail. ^{21 22 23 24 25} The mfERGs were recorded (VERIS; EDI, San Mateo, CA), and the visual stimuli consisted of 103 hexagonal elements scaled with eccentricity to give approximately equal responses. The stimulus array was displayed on a high-resolution monitor (CRT; Sony GDM, Tokyo, Japan) and the stimuli were driven at a 75-Hz frame rate. The diameter of the stimulus array subtended approximately 60° at a viewing distance of 27 cm. The luminance of each hexagon was independently modulated between black (3.5 cd/m²) and white (138.0 cd/m²) according to a binary m-sequence at 75 Hz. A small red fixation spot was placed at the center of the stimulus matrix. The luminance of the surround was set at 70.8 cd/m². 

Before the recordings, the subject’s pupil was fully dilated with a combination of 0.5% tropicamide and 0.5% phenylephrine hydrochloride, and the cornea was anesthetized with 0.4% proparacaine hydrochloride. ERGs were picked up with a bipolar contact lens electrode (Burian-Allen; Hansen Ophthalmic Laboratories, Iowa City, IA), and a grounded electrode was attached to the earlobe. After insertion of the contact lens electrode, the subjects were optically corrected for the viewing distance. The opposite eye was occluded. To estimate the overall fixation quality, we analyzed the region of the blind spot after the recordings. 

ERGs were amplified (×100,000), and band-pass filtered between 10 to 100 Hz (RPS-107; Grass, Quincy, MA). We used a 10-Hz cutoff because this is widely used in the clinical setting and is recommended by the International Society of Clinical Electrophysiology of Vision guideline. ²⁵ However, it has been suggested that a 10-Hz cutoff could alter the waveform of the response, and could be a problem if the waveform is negative. ^{27 28} The mfERGs recorded with a 10-Hz and a 3-Hz cutoff filter from one patient are shown in the Results. The sampling rate was 600 Hz (interval, 1.667 msec). 

The m-sequence had 2¹⁴ elements and required a total recording time of approximately 4 minutes. For the comfort of the subjects, the recording time was divided into eight segments. The first- and second-order kernels were extracted with software (VERIS 2.05; EDI). 

To improve the signal-to-noise ratio, an artifact reduction technique was used once. ²¹ Each individual response was also averaged with 6% of its six neighboring responses. ²¹  

Results

First-Order Kernel

The 103 first-order kernels of the focal responses from the right eye of a representative normal control (age, 25 years; refractive error, -2.00 D) and from seven eyes of the seven patients with XLRS are shown in Figure 2 . The averaged waveforms of the mfERGs for three eccentric rings (rings 1&2, 3&4, and 5&6) for 15 normal subjects and 7 XLRS patients are shown in Figure 3 . 

The results of a quantitative analysis of the amplitude and implicit times of the mfERG are shown in Figure 4 . The white areas represent values of the amplitudes and implicit times that were within 5 to 95 percentile limits of the normal range, and the black areas represent those with amplitudes or implicit times outside the 5 to 95 percentile limits of the normal range. The response densities and implicit times at five eccentric rings for seven patients are shown in Figures 5A and 5B , respectively. The gray region represents the 5 to 95 percentile range for the normal controls. 

In all patients, the responses in the central retina, which corresponded to the area of the foveal schisis, were severely attenuated (Figs. 2 3 4 5) . The amplitude of the central response in six of the eyes was lower than the lower limit of the normal range, and only one eye (P1), was within the lower limit (Fig. 5A) . 

In contrast, the amplitudes of the focal responses outside the foveal area varied considerably in the seven patients; the amplitudes were well-preserved and within normal range in one eye (P1), on the borderline in four eyes (P2 to P5), and nondetectable in two eyes (P6 and P7, Fig. 5A ). For two eyes with nonspecific macular degeneration (P6 and P7), the amplitudes of the local cone responses were lower than the 95 percentile at nearly all loci within the 30° field (Figs. 4 5A) . 

Interestingly, all seven XLRS patients had delayed implicit times at nearly all areas tested (Figs 3 4 5) . Significant delays were present even at the retinal areas where the amplitudes were within the normal range (e.g., P1, Figs. 4 5 ). Of the total 721 areas tested (103 locations in 7 patients), 707 areas (98.1%) had significantly delayed implicit times, whereas only 403 areas (55.9%) had reduced amplitudes. These findings suggest that the pathology of XLRS affects the implicit times more than the amplitude. 

Two of the seven eyes had peripheral retinoschisis. Nine and 11 hexagonal stimulus elements were included in the retinal area of the retinoschisis in P2 and P3, respectively (Fig. 1) . These two eyes had a balloon-like elevation of the inner retinal layer with large oval holes but did not have any visible change of the RPE. We plotted the stimulus area of the mfERG which corresponded to the peripheral retinoschisis (Fig. 2 , gray area of P2 and P3), and found that the mfERG amplitudes in the area of peripheral retinoschisis were within the normal range, and were not different from those at the adjacent retinal area without peripheral retinoschisis. 

To compare the waveforms of the XLRS patients with those of normal subjects, the 103 local responses were summed. The superimposed and averaged response waveforms for the 15 normal controls are shown on the left of Figure 6A , and the waveforms of the seven XLRS patients are shown on the right. The vertical dashed lines are drawn at 30 msec, and the results of statistical comparisons for the two groups are shown in Table 2 . The amplitudes of the initial negative (N1) and the following positive component (P1) were significantly reduced in the eyes of patients with XLRS (Table 2 and Fig. 6A ). The average amplitude of N1 and P1 in XLRS was 68% and 64% of the normal controls, respectively. The implicit times of N1 and P1 were also significantly delayed (Table 2 , P < 0.01). We also noted that the two or three oscillations followed by the N2 component (asterisks, Fig. 6A ) were essentially absent in all XLRS eyes. 

Second-Order Kernel

The 103 local responses of the first slice of the second-order kernel for the same control and seven XLRS patients shown in Figure 2 are shown in Figure 7 . Although the amplitude of local second-order kernel was very small for the normal control, positive and negative components were clearly visible. In contrast, the amplitude of the second-order kernel was substantially smaller and essentially absent at nearly all locations in the patients. This was true even at the retinal area with normal amplitude of first-order kernel (see peripheral retinal area of P1, Figs. 2 7 ). 

To compare the waveforms, all 103 second-order kernels were summed and are presented in Figure 7B . As in Figure 7A , the superimposed and averaged response waveforms for the 15 normal controls are shown on the left, and those for the seven XLRS patients on the right. The amplitude of the summed second-order kernel in XLRS patients was severely reduced when compared with normal controls. The amplitude of the second negative component (N2), the most prominent component of the second-order kernel, was measured as shown on the left of Figure 6C . The mean value was only 37% of the normal controls, which was significantly smaller (P < 0.01, Table 2 ). 

To compare the relative amplitude of the second-order kernels, we also calculated a ratio of the amplitudes of the second-order kernel to the first-order kernel. The ratio was significantly reduced (P < 0.01) in the XLRS patients, and a plot of these ratios showed that the two groups were clearly separated without any overlap (Fig. 6C) . 

Effect of Bandpass on Waveform of mfERGs

We used a bandpass setting of 10 to 100 Hz, but it has been suggested that a 10-Hz cutoff filter could alter the waveform of the response substantially, and could be a problem especially if the waveforms were negative. ^{27 28} One might expect that the waveform of the mfERGs can be negative if recorded with a lower cutoff. To determine the effect of the bandpass width on the mfERG waveforms, we recorded mfERGs using two bandpass settings, 10 to 100 Hz and 3 to 100 Hz, from a patient with XLRS (P5). The results of the summed mfERGs with a bandpass of 10 to 100 Hz, the summed mfERG with a bandpass of 3 to 100 Hz, as well as the conventional full-field ERG (bandpass, 0.3 to 300 Hz) are shown in Figure 8 . When compared with normal control, it was noted that the amplitude ratio of the positive to the negative component of the first-order kernel was slightly reduced in the XLRS for both 10-Hz and 3-Hz cutoff. This ratio was lower when the 3-Hz cutoff was used. However, the summed response of the mfERG still showed a positive peak even when a 3-Hz cutoff was used. The same results were also seen for the conventional full-field cone ERG results (bandpass, 0.3 to 300 Hz), where the amplitude ratio of the b-wave to the a-wave was lower than normal, but still larger than 1.0 even though the 0.3-Hz cutoff filter was used. These results indicated that, although a 10-Hz cutoff could alter the waveform, the waveform of the mfERGs did not show a negative waveform in patients with XLRS even when a lower cutoff filter is used. 

Discussion

Our results clearly demonstrate that the cone-mediated retinal function was most impaired in the central area that corresponded to the area of foveal schisis in all eyes (Figs. 2 3 4 5) . Considering recent evidence that the first-order kernel of the mfERG is mainly shaped by a combination of ON- and OFF-bipolar cell activities with smaller contribution from cone photoreceptors, ^{24 29 30 31} these results suggest that the retinal impairment in XLRS extends to the middle and outer retina in the central retina. 

These results are consistent with previous psychophysical and electrophysiological studies. ^{8 19 20} It is difficult to speculate from these results, however, whether the severe-middle and outer-retinal dysfunction in the central retina is primarily due to structural changes or to secondary damage associated with XLRS. 

Peachey et al. ⁸ investigated the cone and rod sensitivities at many points along the horizontal meridian in the eyes of patients with XLRS, and reported that the sensory neuronal pathways outside the fovea operates better than that within the foveal area. We have also previously studied the macular function of eyes with XLRS using the focal macular cone ERG technique, and found that the amplitude ratio of the b-wave to the a-wave gradually decreases toward the fovea. ¹⁹ This suggested that the retinal function in the middle and inner retina were more affected in the central retina. Muscut et al. recently also reported that the central response recorded by the mfERG technique was reduced in one patient with XLRS. ²⁰  

In contrast to the responses in the central retina, there was a large variation in the amplitude of the focal cone responses outside the fovea among the patients; one eye had nearly normal amplitudes, four had moderately reduced amplitudes, and two showed severely reduced amplitudes outside the fovea. Two eyes with severely reduced amplitude outside the fovea were those with nonspecific degenerative changes in the macula. These results are also in agreement with our previous finding that the focal macular cone ERGs were nearly nonrecordable in eyes with advanced macular changes in eyes with XLRS. ¹⁹ It is not known whether genetic or environmental factors are involved in such large phenotypic variations in the retina of XLRS. 

One interesting finding was that the amplitudes of the focal cone ERGs at the peripheral retinoschisis were not different from the responses from the adjacent retina without retinoschisis. These results suggest that the outer and middle retinal layers are still functioning relatively well in spite of the balloon-like peripheral retinoschisis without any visible RPE changes. It is not unreasonable to expect that the retinal function of middle and outer retina would be gradually impaired as the stage advances. 

In all XLRS patients, there was a delayed implicit time of the first-order kernel and a reduction of the amplitude of the second-order kernel across nearly the whole field. These changes were present in all eyes even at the retinal areas where the amplitudes of the first-order kernel were within the normal range (Figs. 2 7 ; peripheral responses of P1). These results suggest that in the retina of XLRS patients, the cone system is dysfunctional across the whole retina, which has been suggested by many authors. ^{8 9 11 32 33 34}  

Recently, Alexander and colleagues ^{32 33} recorded the photopic ERGs elicited by ON/OFF stimuli and sinusoidal stimuli in patients with XLRS, and suggested that the cone ON-pathway may be more impaired than the OFF-pathway. Naheed et al. ¹¹ also analyzed cone system dysfunction and reported that the function of the proximal retina was more affected than that of the cone photoreceptors. 

An implicit time delay of the first-order kernel has been reported in many other retinal diseases including retinitis pingentosa, ^{35 36 37} cone dystrophy, ^{38 39} macular dystrophy, ^{40 41} diabetic retinopathy, ^{42 43 44} retinal vascular disease, ⁴⁵ and congenital stationary night blindness. ⁴⁶ In addition, some of these diseases ^{24 42 45 46} also show a reduction of the second-order kernel as in XLRS. Thus, these mfERG findings are not specific for XLRS. Hood ²⁴ recently demonstrated that when the first-order kernel is large and abnormally slow, the second-order kernel is always nondetectable. Based on experimental studies and clinical observations, he also speculated that these mfERG changes can be a good marker for damage to structures proximal to the photoreceptor outer segment. The exact mechanism for the mfERG changes seen in the eyes of patients with XLRS is not entirely known, but one hypothesis is that the second-order kernel, which is thought to be involved in the short-term adaptive mechanism, is more affected than the first-order kernels, presumably due to widespread dysfunction of the proximal retina. The implicit time delay of the first-order kernel may be caused by the slowing of the synaptic transmission or inner nuclear layer response in the cone pathway. Alternatively, it is possible to speculate that the implicit time delay of the first-order kernel may be due to the reductions of the second-order kernel, because it is known that the second-order kernel affects the timing of the positive component of the first-order kernel. ²⁴  

In conclusion, our results demonstrated that the cone-system is more affected in the central retinal than the peripheral area in XLRS. There was considerable variation in cone function outside the fovea. The presence of peripheral retinoschisis itself did not reduce the focal cone ERGs at least at the stage without visible atrophic RPE changes. The delay in the implicit time of the first-order kernel and reduction of second-order kernel across whole field tested suggest that there are widespread cone-system dysfunctions, presumably due to the impairment of the proximal retina. We conclude that the mfERG technique is a valuable tool to evaluate local retinal cone function objectively in patients with XLRS. 

 Supported by Grant-in Aid 13307048 (YM), 14370557 (HT), 14770952 (MK) and 14901139 (C-HP) from the Ministry of Education, Science, Sports and Culture, Japan.

 Submitted for publication December 12, 2002; revised May 27, 2003; accepted June 5, 2003.

 Disclosure: C.-H. Piao, None; M. Kondo, None; M. Nakamura, None; H. Terasaki, None; Y. Miyake, None.

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Clinical Characteristics of Examined Patients

Patient No.	Age	Test Eye	Visual Acuity	Fundus Findings
1	22	L	0.7 (20/29)	FS
2	25	R	0.3 (20/66)	FS+ PRS
3	18	R	0.4 (20/50)	FS+ PRS
4	23	L	0.2 (20/100)	FS
5	26	L	0.2 (20/100)	FS
6	18	L	0.1 (20/200)	MD
7	25	L	0.2 (20/100)	MD

Figure 1.

 

Drawings of the fundus and Goldmann kinetic visual field in P2 and P3 who have peripheral retinoschisis with large inner retinal holes.

Figure 2.

 

The 103 focal first-order kernels of the mfERGs recorded from a normal subject and the seven patients with XLRS (P1–P7). Gray areas in P2 and P3 indicate the retinal areas corresponding to the peripheral retinoschisis.

Figure 3.

 

Averaged waveforms of the mfERGs for three eccentric rings (ring 1&2, 3&4, and 5&6). Results for 15 normal subjects and 7 XLRS patients are superimposed. Dotted line is drawn at 30 msec.

Figure 4.

 

Topographical maps of the amplitudes and implicit times for seven patients with XLRS. White areas represent amplitudes and implicit times within the 5 to 95 percentile range of control eyes; black areas represent reduced amplitudes or delayed implicit times greater than this range.

Figure 5.

 

(A) Response densities (amplitude/retinal area) for five eccentric rings in seven patients with XLRS. The gray region represents the 5 to 95 percentile range obtained from age-matched normal controls. (B) Implicit times at five eccentric rings in the seven patients with XLRS. The gray region represents the 5 to 95 percentile range obtained from age-matched normal controls.

Figure 6.

 

(A) Summed first-order kernels for 103 local responses for 15 normal controls (left) and seven XLRS patients (right). All these responses were superimposed in the upper traces and averaged waveforms are presented in the lower traces. (B) Summed second-order kernel responses for all 103 local responses for 15 normal controls (left) and seven XLRS patients (right). All these responses are superimposed in the upper traces and averaged waveforms are presented in the lower traces. (C) Amplitude ratio of the second- to first-order kernel for 15 normal subjects and seven XLRS patients.

Table 2.

 

Parameters of the mfERG of XLRS Group Compared with Normal Control Group

	n	First-Order Kernel							Second-Order Kernel			Amplitude Ratio of Second- to First-Order Kernel
				N1 Amplitude	P1 Amplitude	N1 Time	P1 Time	N2 Amplitude	N2 Time
Normal	15	13.9 ± 3.1	38.5 ± 9.1	15.2 ± 0.6	29.3 ± 0.9	9.3 ± 3.0	30.1 ± 0.8	0.24 ± 0.06
XLRS	7	9.5 ± 3.3*	24.6 ± 9.3*	17.6 ± 0.9*	33.6 ± 1.1*	3.4 ± 1.4*	36.2 ± 2.9*	0.14 ± 0.05*

Figure 7.

 

The 103 local second-order kernels of the mfERGs recorded from a normal subject and seven patients with XLRS (P1–P7).

Figure 8.

 

The results of the summed mfERG with a bandpass of 10 to 100 Hz, the summed mfERG with a bandpass of 3 to 100 Hz, and the conventional full-field ERGs (bandpass, 0.3 to 300 Hz) recorded from a patient with XLRS (P5).