Dr Xu Yan Shan Eye Center and College of Optometry Tianjin Medical University 64 Tong-An Road Heping District Tianjin 300070 CHINA E-mail: firstname.lastname@example.org
Purpose: The purpose of this project was to investigate the changes in macular function and macular morphology of severe non-proliferative diabetic retinopathy (NPDR), due to photocoagulation, using the multifocal electroretinogram (mfERG) and optical coherence tomography (OCT).
Methods: Thirty-five volunteers were in the control group, with one eye per person examined with the mfERG. Both the mfERG and OCT were conducted on 30 patients with diabetes who had severe NPDR before, and two, seven and 14 days after, treatment with photocoagulation.
Results: Compared with the control group, the P1 and N1 response densities in the patients with NPDR appeared to decrease significantly at rings 2–3 and rings 3–4, respectively, whereas no difference was seen in the implicit times. At two days after photocoagulation, the P1 and N1 response densities decreased significantly in ring 1 and they were still lower than the pre-photocoagulation values at 14 days after photocoagulation. In addition, no change was found in the implicit times before and after photocoagulation. There was no obvious difference in the macular thickness after treatment. At two days after treatment, the P1 response density in ring 1 negatively correlated with the corresponding macular thickness.
Conclusion: The para-macular function was significantly impaired in those patients with severe NPDR and photocoagulation reduced the central macular function. Even after 14 days, the central macular function had not returned to pre-photocoagulation levels.
The increase in the incidence of diabetes has made diabetic retinopathy the leading cause of vision impairment and blindness.1,2 Retinal photocoagulation plays an important role in therapy for proliferative and non-proliferative diabetic retinopathy (NPDR);3 however, photocoagulation can induce macular oedema, resulting in temporary or persistent impairment of visual function.4,5
Many studies have focused on the multifocal electroretinogram (mfERG) at different stages of diabetic retinopathy, but studies on photocoagulation are rare. Laser photocoagulation has become a major method of intervention in diabetic retinopathy, but there are few studies of retinal functional changes after laser treatment. As laser spots at the posterior pole can affect macular function, which is the area of mfERG testing, the mfERG is well suited to research and clinical after-care.6 The mfERG can examine retinal responses from many localised regions and can provide a functional assessment, solving the limitation of full-field ERGs in detecting localised lesions, as well as the limited test field of focal ERGs.7,8
To study the macular function and macular morphology of severe NPDR, as well as the changes that are influenced by photocoagulation, especially in the early stage after laser treatment, the mfERG, combined with optical coherence tomography (OCT), was applied on normal participants and those with diabetes who had severe NPDR.
MATERIALS AND METHODS
Thirty-five normal participants (20 male and 15 female), aged from 43 to 76 years (mean: 60.3 ± 10.2 years), were recruited for the control group. One eye for each participant was randomly selected. The visual acuity of each eye was 6/6 or better, as measured by the Snellen letter chart, with refractive errors of ±3.00 D or less and no more than -1.00 D. There was no history of ophthalmic surgery or evidence of either eye or systemic disease. One eye each of 30 patients with type 2 diabetes who had severe NPDR were recruited, aged 39 to 74 years (mean: 54.9 ± 9.7 years ), and the duration of the diabetes was 7.9 ± 3.8 years. The diagnostic criteria of severe NPDR were based on the new international staging of diabetic retinopathy.9 The selected criteria for the patients were:
1without severe media opacity
2visual acuity of 6/12 or better
3refractive errors ≤±3.00 DS and -1.00 DC
5no retinal disease, except diabetic retinopathy
6no current or previous intraocular surgery, trauma, serious renal insufficiency or cerebral infarction.
The normal eyes of the control group were tested by a mfERG and OCT. The same procedures were conducted on the patients with diabetes on four occasions: before, and two, seven and 14 days after, photocoagulation.
All the research procedures adhered to the tenets of the Declaration of Helsinki and were approved by the Ethics Committee of the Tianjin Medical University's Eye Center, Tianjin, China.
MULTIFOCAL ELECTRORETINOGRAM STIMULI
The mfERG results were recorded by using a RETIscan System (3.20.26; ROLAND CONSULT Stasche & Finger, Bandenberg, Germany) with a 21-inch cathode ray tube monitor. The working distance from the screen to the participant was 30 cm and the diameter of the tested field was 50°. The stimulus matrix consisted of 61 scaled hexagonal elements with five concentric rings (the size of the hexagons increase with the eccentricity and the eccentricities of the five rings were 2.3°, 7.4°, 10°, 16° and 25°) (Figure 1). Each hexagon alternated between white and black at a frame rate of 75 Hz. The bright hexagons were ∼120 cd/m2, while the dark presentations were <1.0 cd/m2. The average luminance of the stimulus matrix was ∼60 cd/m2, the same as the background luminance. The room's luminance was ∼60 lux.
The fixation target was a red cross of <1 mm width with a 5° visual angle, placed at the centre of the stimulus matrix. The stimulus presentation was followed by a pseudo-random binary m-sequence (m = 9) and the total duration of the stimulation was 6 min and 16 s, consisting of eight sections of 47 s each.
MULTIFOCAL ELECTRORETINOGRAM RECORDING
The pupil of the participants was dilated with 0.5% compound tropicamide (Santen, Osaka, Japan) before testing. For the patients with NPDR, the pupil was dilated to ∼7 mm. For the control group, the pupil was dilated to 7–8 mm. A monopolar contact lens electrode (JET; FABRINAL SA, La Chaux-de-Fords, Switzerland) was used for the recording. The reference and ground electrodes were silver chloride and were attached to the outer canthus and central forehead separately. The first-order response density and implicit times of the P1 and N1 waves in all the rings were analysed. Not all the participants were fully corrected for working distance because of low-level myopia. Those who complained of blurred vision could wear glasses for the mfERG measurement. During the recording, the untested eye was occluded. The participants were asked to fixate on the target, which was located at the centre of the matrix. If the waveforms were unsteady, contaminated with blinks and eye movements or fixation shifts to the periphery, the recording segment would be rejected and repeated.
OPTICAL COHERENCE TOMOGRAPHY RECORDING
All the participants were examined by OCT (OCT2000; Zeiss-Humphrey, Jena, Germany) after the mfERG test. A radial linear scan was used in the OCT measurement and for a retinal topographic map. Computerised image analysis software was used to measure the macular neurosensory retinal thickness automatically. Each participant had six OCT measurements to obtain an average value.
Before photocoagulation, all the patients underwent examinations of visual acuity and intraocular pressure, anterior and posterior segment assessment and routine fundus photography and fundus fluorescein angiography. The severity of the retinopathy was determined by the findings of the fluorescein angiography.
The patients were treated by pan-retinal photocoagulation three times at 14-day intervals. For the first treatment, the laser mainly focused on the posterior pole of the retina (outside the vascular arcade), which has great influence on the macular function. All the mfERG and OCT measurements were carried out between the first and second treatments.
The initial photocoagulation burns were placed in a double arc at about two-disc diameters, temporal to the centre of the macula, with the burns spaced about one-half burn apart, and grey–white burns of gentle intensity were produced. The area outside the temporal vascular arcades and the area temporal to the macula were treated (Figure 2). The laser burns of the second and third treatments were given outside the former treatment area and were covered as anteriorly as possible. The krypton yellow and green multi-wavelength laser was operated with a spot size that ranged from 300 to 500 µm, with a power of 200 mW and a constant exposure time of 150–200 ms. Nearly 1200 burns were applied in the entire treatment process, ∼500 of which were given in the first treatment and were located outside the vascular arc, the closest to the posterior pole of the retina.
No eye examination was conducted immediately after the treatment. On days two, seven and 14 after the treatment, routine eye examinations were carried out, including visual acuity, intraocular pressure and anterior and posterior segment assessments before the mfERG and OCT.
The statistical analysis was carried out with SPSS for Windows (v. 12.0; SPSS, Chicago, IL, USA). The control group and group with NPDR were compared by using a two-way ANOVA and unpaired t-test with Bonferroni correction. A two-way repeated-measures ANOVA and Bonferroni test were used as post-hoc tests for the photocoagulation. A correlation analysis between the macular thickness and the mfERG response was tested by Pearson's correlation, with p < 0.050 as the statistically significant difference.
Comparison between the control group and the group with non-proliferative diabetic retinopathy
Between the control group and the group with NPDR, the interactions of the mfERG response densities and the rings are as follows: P1 response density (F = 3.500, p = 0.008); N1 response density (F = 6.928, p < 0.001); P1 implicit time (F = 1.038, p = 0.338); and N1 implicit time (F = 2.357, p = 0.054). Both the P1 and N1 response densities of the group with NPDR had a significant decrease at rings 2–3 and rings 3–4, respectively, compared with those of the control group (p < 0.010) (unpaired t-test, shown in Figures 3A and 3B).
For the P1 implicit time, although there were statistically significant effects between the groups (F = 6.900, p = 0.009), there was no significant difference between the groups in the individual rings. An unpaired t-test also was carried out (Figure 3C). The main effects between the groups in relation to the N1 implicit time showed no significant difference (F = 0.195, p = 0.659) (Figure 3D).
Comparison of the multifocal electroretinogram of the patients with non-proliferative diabetic retinopathy in different periods before and after photocoagulation
For the response density, the interaction between the rings and the period mainly affected the P1 response density (F = 3.412, p < 0.001) and the N1 response density (F = 12.531, p < 0.001). The one-way ANOVA showed that the P1 and N1 response densities had the largest degree of decrease in ring 1 (p < 0.001). At seven and 14 days after photocoagulation, the response density of ring 1 seemed to recover to some extent, but was still much lower than before photocoagulation (p < 0.010) (Figures 4A and 4B).
No interaction between the rings and period was found during the P1 implicit time (F = 0.844, p = 0.544) and the N1 implicit time (F = 1.327, p = 0.239). The P1 implicit time showed a significant main effect of the period (F = 3.365, p = 0.022) (Figure 4C), whereas the N1 implicit time had no significant main effect of the period (F = 2.072, p = 0.110) (Figure 4D).
Correlation between the macular thickness and the multifocal electroretinogram response
The macular thickness level, from the OCT measurement, seemed to increase two days after treatment and then it declined at seven and 14 days after photocoagulation (Figure 5). No significant difference was observed (F = 1.007, p = 0.405) (Table 1). The correlation analysis for the mfERG and OCT demonstrated that, two days after the photocoagulation treatment, the P1 response density in ring 1 had a negative correlation with the corresponding macular thickness (r = -0.389, p = 0.034) (Table 2).
Table 1. Macular thickness before and after the photocoagulation treatment
Macular thickness (µm) (mean ± SD)
248.10 ± 87.63
2 days after treatment
269.07 ± 89.22
7 days after treatment
253.87 ± 69.60
14 days after treatment
253.30 ± 66.91
Table 2. Correlation between the P1 response density and the optical coherence tomography (OCT)
211.03 ± 53.54
248.10 ± 87.63
2 days after treatment
120.78 ± 45.25
269.07 ± 89.22
7 days after treatment
148.44 ± 42.68
253.87 ± 69.60
14 days after treatment
176.23 ± 49.19
253.30 ± 66.91
For the patients with severe NPDR, the retinal function of the para-macular region showed a significant decline, despite the patients' vision having been maintained at a good level. Compared with the implicit times, the response densities had a significant decrease in the larger areas, indicating that the response densities were more susceptible. Yu and colleagues argued that all the response densities considerably decreased in their patients with NPDR and that the implicit times were delayed significantly in the 10.5–22.5° region.10 There were decreased response densities in rings 2–3 (P1) and rings 3–4 (N1), but no significant difference was found in the implicit times, although they had a prolonged tendency in the 0–25° region (rings 3–5) of the tested field. These results show that, compared with the implicit time, the response density was more easily influenced by diabetic retinopathy.10,11 A previous study on mild-to-moderate NPDR found that the implicit times increased in most retinal areas, while the amplitude was insensitive to retinopathy in these stages because of its larger inter-subjective variation.6
Why does the sensitivity of the response density change in different stages of NPDR? An explanation might be that the amplitude reflects the strength of the summed responses that are generated by the retinal cells and might be significantly affected only when the generators are severely damaged or cell loss occurs.12–14 Therefore, when the lesion becomes severe, the mfERG's amplitude begins to show obvious change. Regarding the implicit time, the variable data might be the main reason why the implicit time is very difficult to extract.
In this study, a large area of the patients' retina was affected by NPDR, as indicated by the impaired mfERG results. These findings are consistent with the clinical observation that the central vision of patients with NPDR is often unaffected despite the presence of microaneurysms and/or exudates elsewhere in the retina. This might explain why patients are unaware of the severity of the disease, which delays its prevention and early treatment. No obvious change in the mfERG response was found among the four quadrants of the retina, which might indicate no close correlation between the pathological changes of NPDR and the quadrant area. The observed changes were only part of the individual differences.
A marked decrease in the mfERG response density in ring 1 was observed after photocoagulation, indicating that the macula was a susceptible area. It is more likely that the increased macular thickness that was shown by OCT suggests that the photocoagulation induced some macular oedema. In addition, Maeshima and co-workers suggested that the disturbance of the microcirculation and the negative after-effect of the laser treatment had greater effects at the macula and could reduce the signal transmission in adjacent areas.15 These may support the possibility that photocoagulation induces macular oedema. Furthermore, Leibu and colleagues demonstrated that photocoagulation could lead to oedema and the destruction of retinal barriers, causing inflammation and toxic effects due to free radicals.16 Also, they reported that both diabetic retinopathy and laser-induced oedema could cause a disturbance of the retinal microcirculation. Thus, all these effects result in significant changes in the sensitive macular region.
Some patients with diabetic retinopathy who did not have macular oedema also reported a decline in colour contrast vision and contrast sensitivity after scatter treatment, indicating that their foveal function was still affected.17 Previous research that examined the direct influence of laser burns, using focal laser treatment, showed increased amplitudes in areas with oedema and hard exudates; however, the results demonstrated decreased amplitudes in newly pan-retinal photocoagulated areas and in untreated adjacent areas. The main reason that was considered was that the laser burns that are applied in pan-retinal photocoagulation are much larger and more powerful than those that are used in focal laser treatment;18 however, the exact mechanism for the changes in the macular function after laser treatment is not clear. Additionally, using the response density to evaluate the macular function of those with severe NPDR is more meaningful.
Before the photocoagulation treatment, the patients with severe NPDR had mild oedema, indicating that macular oedema was prevalent in this group. No significant change in the macular thickness was observed. This agrees with a study in which no change in the macular thickness and reduced response densities after photocoagulation were found.17 There was a negative correlation between the P1 response density in ring 1 and the corresponding macular thickness two days after the photocoagulation treatment. Diabetic macular oedema and laser-induced macular oedema might influence the mfERG response.
Many studies have indicated that the corresponding macular function of macular lesions changes, even if there is no significant change in histomorphology. This further demonstrates that the mfERG is sensitive to detecting lesions, especially at the macula. Thus, only a histological examination, accompanied by functional inspection, can reflect fully the real status of the retinal condition in different stages.
As viewed from electrophysiological and morphological changes, the mfERG, combined with OCT, could be more comprehensive in quantitatively evaluating macular function and morphology, as well as providing meaningful clinical data of disease progression, treatment and follow-up assessment.19–21 In this study, only a short-term observation was carried out after the first photocoagulation; hence, further investigation in a long-term study is necessary to have a better understanding of NPDR.