Transcranial Magnetic Stimulation in Adults With Amblyopia

Background: Through transcranial magnetic stimulation (TMS) it is possible to change cortical excitability of the visual cortex, and to influence binocular balance. The main goal of our study is to assess the effect of transcranial magnetic stimulation, specifically theta burst stimulation (TBS), in a group of amblyopic volunteers measuring several visual parameters: visual acuity, suppressive imbalance, and stereoacuity. Methods: Thirteen volunteers aged 19 to 24 years, randomly split in 2 groups, underwent 1 session of continuous TBS, stimulating the right occipital lobe. The first group with 8 volunteers was exposed to active stimulation with cTBS, and the other group with 5 volunteers was exposed to placebo stimulation. Results: Significant improvements in visual acuity, suppressive imbalance, and stereoacuity were found in the amblyopic eye after cTBS. The average value of amblyopia in visual acuity before stimulation was 0.32 ± 0.20 logMar and after cTBS was 0.19 ± 0.17 logMar. The mean value for the control group before placebo stimulation was 0.28 ± 0.17 and after placebo stimulation was 0.28 ± 0.16. The suppressive imbalance in the group of amblyope subjects stimulated before cTBS was 0.26 ± 0.18 and after was 0.12 ± 0.12; the suppressive imbalance of the control group before the placebo stimulation was 0.34 ± 0.37 and after was 0.32 ± 0.40. Conclusions: Visual acuity, suppressive imbalance, and stereoacuity had significant enhancements compared with baseline after cTBS over the right occipital lobe in an ambliopic population.

Original Contribution Transcranial Magnetic Stimulation in Adults With Amblyopia Ana Rita Tuna, MSc, Nuno Pinto, Lc, Francisco Miguel Brardo, PhD, Andresa Fernandes, MSc, Amélia Fernandes Nunes, PhD, Maria Vaz Pato, PhD Background: Through transcranial magnetic stimulation (TMS) it is possible to change cortical excitability of the visual cortex, and to influence binocular balance. The main goal of our study is to assess the effect of transcranial magnetic stimulation, specifically theta burst stimulation (TBS), in a group of amblyopic volunteers measuring several visual parameters: visual acuity, suppressive imbalance, and stereoacuity. Methods: Thirteen volunteers aged 19 to 24 years, randomly split in 2 groups, underwent 1 session of continuous TBS, stimulating the right occipital lobe. The first group with 8 volunteers was exposed to active stimulation with cTBS, and the other group with 5 volunteers was exposed to placebo stimulation. Results: Significant improvements in visual acuity, suppressive imbalance, and stereoacuity were found in the amblyopic eye after cTBS. The average value of amblyopia in visual acuity before stimulation was 0.32 ± 0.20 logMar and after cTBS was 0.19 ± 0.17 logMar. The mean value for the control group before placebo stimulation was 0.28 ± 0.17 and after placebo stimulation was 0.28 ± 0.16. The suppressive imbalance in the group of amblyope subjects stimulated before cTBS was 0.26 ± 0.18 and after was 0.12 ± 0.12; the suppressive imbalance of the control group before the placebo stimulation was 0.34 ± 0.37 and after was 0.32 ± 0.40. Conclusions: Visual acuity, suppressive imbalance, and stereoacuity had significant enhancements compared with baseline after cTBS over the right occipital lobe in an ambliopic population. Journal of Neuro-Ophthalmology 2020;40:185-192 doi: 10.1097/WNO.0000000000000828 © 2019 by North American Neuro-Ophthalmology Society CICS-Health Sciences Research Centre (ART, NP, FMB, AF, AFN, MVP), Faculty of Health Sciences, University of Beira Interior, Covilhã, Portugal; Dr. Lopes Dias School of Health (NP), Polytechnic Institute of Castelo Branco, Castelo Branco, Portugal; Department of Physics (FMB, AFN), University of Beira Interior, Covilhã, Portugal; and CICS- Health Sciences Research Centre (MVP), Faculty of Health Sciences, University of Beira Interior; ULS Guarda, Guarda, Portugal. All relevant data are within the article and its Supporting Information files. Our data are not ethically or legally restricted. Address correspondence to Ana Rita Tuna, MSc, CICS-Health Sciences Research Centre, Faculty of Health Sciences, University of Beira Interior, Covilhã, Urbanização Quinta dos Negreiros Lt5, 2E 6200-506, Covilhã, Portugal; E-mail: [e-mail] Tuna et al: J Neuro-Ophthalmol 2020; 40: 185-192 A mblyopia is a visual disorder that is due to an abnormal binocular interaction or visual deprivation occurring in the first several years of life. It is estimated that the prevalence of this disorder is around 1.6%-3.6% of the world's population (1). Clinically, amblyopia is a loss of unilateral visual acuity of an apparently normal eye. Less frequently, there is bilateral loss of visual acuity. Amblyopia is characterized by the existence of a 2 or more line difference in visual acuity between the 2 eyes, despite best optical correction (2). Reduced visual acuity is not sufficient to diagnose amblyopia. In fact, it is required that an underlying amblyogenic factor be identified (1) because such factors may interfere with the development of the visual pathways during the growth process (3). The most common amblyogenic factors are strabismus, anisometropia, or even a combination of these; less frequently, amblyopia can be caused by visual deprivation as may occur in patients with congenital cataract or ptosis (4). Ocular dominance can exist in motor and sensorial domains. The first domain is known as "sighting," and involves a preference for using 1 eye when performing a certain activity, such as using a telescope. The second domain can be defined as preference for using the eye in which the image on the retina is clearer and brighter. With these criteria in mind, the visual system "chooses" one of the eyes (5). In the amblyopic patient, there is a greater tendency for the nonamblyopic eye to dominating over the other (6). New paradigms consider amblyopia a binocular disorder (2), and it is believed that it reflects binocular dysfunction (7). Li et al (8), for instance, suggested that the disruption of binocular vision is caused by a suppression of the image received by the brain from a malfunctioning eye; this can subsequently lead to chronic suppression, thereby inducing amblyopia. To reestablish binocular visual function, it is necessary to reduce suppression of the amblyopic eye while decreasing the degree of sensorial dominance of the nonamblyopic eye (6). The most widely used treatments for amblyopia include optical treatments (lenses), occlusion, and pharmacological 185 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Original Contribution therapy with atropine. Optical treatments include refractive adaptation; by correcting the refractive error, it is expected that progressive improvements in visual acuity will occur (1). Occlusion is the second type of treatment; this consists of externally blocking visual input to the nonamblyopic eye (patching), thereby increasing the use of the amblyopic eye (9). The third type of treatment is the pharmacological paralysis of accommodation of the nonamblyopic eye using atropine (Atropisol; 1 drop of 0.5%-1% solution) (4). This blocks the parasympathetic innervation of the pupil, inducing blurring of images (9,10). These treatments have been used successfully in younger patients. However, when applied in adults, their potential effectiveness decreases considerably (4,11). Transcranial magnetic stimulation (TMS), which was introduced in 1985 by Anthony Barker, has been used in the treatment of amblyopia. TMS is a technique involving neurostimulation and neuromodulation that is based on Faraday's principles of electromagnetic induction (12,13). It involves inducing electrical currents in the cortex through a coil positioned adjacent to the scalp. Magnetic fields created by the current on the coil go through the skull with almost no resistance and painlessly generate new electrical currents in the cerebral cortex (6,14). When applied in a repetitive way, magnetic fields allow for the modulation of central nervous system circuits; this is accomplished by increasing synaptic connections and allowing for activation or suppression of motor, sensory, or cognitive functions (7,12). In the past decade, studies have shown the potential of magnetic transcranial stimulation in the recovery of amblyopia, suggesting that the brain has enough neuronal plasticity to recover the normal function amblyopic eyes both in childhood and during adulthood (6,15). Until the present study, investigations of TMS only explored the recovery of visual contrast (15,16). The purpose of our pilot study was to evaluate the effects of TMS visual acuity, suppressive imbalance, and stereoacuity in a group of amblyopic adult patients. METHODS Study participants included 13 adult volunteers, aged 19-24 years, with amblyopia. The participants were randomized to active stimulation with cTBS (cTBS group) or placebo stimulation (placebo group). Participants were recruited using community surveys. All participants in this study were evaluated for visual function. Refractive error was corrected to ensure that best-corrected visual acuity could be measured (BCVA). Based on inclusion criteria, there was a minimum of 2 lines difference in BCVA between the 2 eyes. An amblyogenic factor (microstrabismus/isometropy/strabismus or/ and anisometropia) was also required as was the absence of physical anomalies in the ocular structures. Exclusion criteria included history of brain injury and/or head trauma, neurological and psychiatric disease, seizures, pregnancy, history of 186 substance use, or the presence of metallic implants in the head or torso (17). All participants provided written informed consent in accordance with the Helsinki declaration, and data protection legislation was followed in terms of anonymity. The study was approved by the Ethics Committee of the University of Beira Interior (Study nr. CE-UBI-Pj-2017-019). OUTCOME MEASURES Suppressive Imbalance Outcome measures used were as follows: suppressive imbalance (SI), visual acuity, and stereoacuity. SI represents the relative depth of the amblyopic eye suppression vs the nonamblyopic eye; this represents the degree of dominance of the nonamblyopic eye (18). Determination of the SI is performed with both eyes open. Bagolini lenses are placed over the best optical prescription. For the right eye, the lens is placed at 45° and, for the left eye, the orientation of the lens is 135°. The participant is instructed to view a source of light at a distance of 4 m and to note the white lines that are produced by the Bagolini lenses. Participants with normal binocular function perceive an "X"; this represents the combination of what is seen by the right eye (\) and what is seen by the left eye (/). To quantify the relative suppression of the amblyopic eye, a neutral density filter (NDF) was used. The NDF is placed in front of the eye; the density is of the filter is progressively increased until the patient does not perceive the line generated by the Bagolini lens. This measurement is then repeated for the contralateral eye (8,18). The degree suppressive imbalance, or SI value, is calculated using the following equation: SI ¼ NDF ðRight eyeÞ 2 NDF ðLeft eyeÞ NDF ðRight eyeÞ þ NDF ðLeft eyeÞ SI can vary between 21 and 1, where 0 means there is no ocular dominance. Values of 1 (or 21) correspond to complete suppression in one of the eyes. The closer the value is to 0, the less pronounced is the dominance of the nonamblyopic eye. A negative value signifies dominance of the left eye, whereas a positive value indicates dominance of the right eye (19). Visual Acuity Distance visual acuity was measured for each eye using the Early Treatment Diabetic Retinopathy Study charts at a distance of 4 m. Visual acuity was scored letter-by-letter instead, and logarithm of the minimum angle of resolution (LogMAR) scores were recorded (20). Stereoacuity Stereoacuity was measured using the Randot acuity test at a distance of 33 cm. It is scored from 400 to 20 arcseconds. The participant is instructed to choose among 3 circles in Tuna et al: J Neuro-Ophthalmol 2020; 40: 185-192 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Tuna et al: J Neuro-Ophthalmol 2020; 40: 185-192 Age Gender History of Treatment S1 19 Female No previous treatment S2 20 Female No previous treatment S3 21 Female No previous treatment S4 20 Female No previous treatment S5 24 Female S6 22 Male First detected age 5 Patching for 1 yr No previous treatment S7 24 Female S8 24 Male First detected age 6 Patching for 1 yr First detected age 3 Patching for 2 yr P9 21 Female No previous treatment P10 22 Female No previous treatment P11 24 Male No previous treatment P12 20 Female No previous treatment P13 21 Male No previous treatment Refractive Error (OD/OS) 210/21.75 · 90 OD 24.50/20.25 · 10 OS +1.00/21.25 · 85 OD 20.50/20.75 · 30 OS Plano OD Plano OS +0.75/23.75 · 5 OD Plano OS 27.00 OD 27.00 OS +2.25/22.00 · 140 +3.00/22.50 · 60 Plano OD Plano OS 21.00/21.25 · 105 OD 20.75/21.00 · 15 OS Plano OD Plano OS 211 OD 29.50 OS Plano OS Plano OD 23.00/20.75 · 180 25.25/20.75 · 180 Plano OD Plano OS LogMar Visual Acuity (OD) LogMar Visual Acuity (OS) 0.3 20.1 A 80 0.14 20.12 AST (18 ET) 70 20.04 0.26 M 60 A 55 0.5 20.1 Ambliogenic Factor Intensity (MDO), % 0.0 0.48 I 80 0.06 0.64 ST (6 ET) 80 ST (14 ET) 55 0.1 20.1 20.1 0.1 ST (6 ET) 50 20.1 0.2 M 30 0.28 0.0 A 25 0.58 0.1 ST (20 XT) 25 0.0 0.2 A 30 0.16 ST (10 XT) 30 20.1 Original Contribution Id A, anisometropy; AS, anisometropy and strabismus; ET, esotropia; I, isometropy; M, microstrabismus; MDO, maximum device output; OD, right eye; OS, left eye; P, placebo group; S, cTBS group; ST, strabismus; XT, exotropia. 187 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. TABLE 1. Characteristics of the volunteers and stimulation details Original Contribution each frame, the one which appears closest. Groups of circles are presented with progressively decreasing levels of disparity (appearance of closeness) (21). Transcranial Magnetic Stimulation All participants underwent 1 session of magnetic stimulation with theta burst stimulation (TBS); this allows shorter stimulation periods and produces longer effects using a continuous protocol (cTBS) (15,22). The procedure includes determining the specific occipital point at which place the stimulation coil; this corresponds to the area where magnetic stimulation is most capable of inducing phosphenes or transient visual phenomena (16,23). The phosphenes are directly induced by stimulation of the visual cortex and are thus induced even in patients with amblyopia; their presence indicates that the magnetic stimulation is in the correct location. The intensity used in cTBS stimulation varied and was determined for each study participant and was calculated as the minimum intensity needed to evoke phosphenes (phosphene threshold intensity-PTI) (24). The stimulation coil was placed with a lateromedial orientation over the right occipital region. Using as a spatial reference the medium inion area, the center of the coil was then moved 1 cm to the right. This was the initial stimulation point, with 50% of the maximum device output (MDO). We then proceeded upward on the occipital region, from the initial point to a maximum of 3 cm, in steps of 1-cm length, until phosphenes were reported by the subject. To ensure participant safety, intensities higher than 80% of MDO were not used, even when phosphenes were not induced. This intensity was used in cases in which the volunteer did not see phosphenes (23-26). After locating the point where phosphenes were induced, magnetic stimulation was applied following the cTBS protocol. For this procedure, 600 stimuli were continuously given in bursts of 3 pulses at 50 Hz repeated in the 5-Hz range (at 200 milliseconds intervals), with a total duration of 40 seconds. (15,22). The placebo group underwent the initial portion of the protocol in which the location of stimulation that induced phosphenes was located. This group was then administered a placebo stimulation, performed through the change of the coil's position (with a 90° tilt), with the intensity lowered to 50% of PTI. Independently of the affected eye, the protocol was similarly applied to all participants using right hemisphere stimulation. Before and immediately after the stimulation sessions, measurements of visual acuity, suppressive imbalance, and stereoacuity were evaluated. Stimulation procedures were performed by a researcher masked to outcome measures. Similarly, the technicians measuring the visual acuity, suppressive imbalance, and stereoacuity were masked to treatment assignment of active TMS or placebo. longitudinal data in factorial experiments (RStudio) as an alternative to repeated-measures analysis of variance. The R Project for Statistical Computing (Ri386 3.5.2) was the software used for data analysis. P values ,0.05 were regarded as significant. RESULTS The cTBS treatment group in this pilot study included 8 participants (6 women and 2 men with a median age of 21.5 [20.03-23.47]). Four of these participants had amblyopia of the right eye; the others had left eye amblyopia. The placebo group included 5 participants (3 women and 2 men with a median age of 21 [19.72-23.48]). Two had amblyopia of the right eye, whereas the others had amblyopia of the left eye (Table 1). Visual acuity was the first outcome that was studied before and after stimulation in both treatment groups (Figs. 1, 2). After 1 treatment session in the cTBS group, there was improvement of visual acuity in all but 1 participant, who maintained stable visual acuity (Fig. 1). The median value of visual acuity before the stimulation was 0.28 (0.14-0.49) logMAR (20/38 Snellen), whereas the median value after stimulation was 0.18 (0.05-0.34) logMAR (20/30 Snellen). A review of the results showed that participants who had fewer changes with the cTBS session were Participants 2 and 3; both of these individuals had strabismus as the cause of their amblyopia. Participants 1, 4, and 6 showed the greatest improvements in visual acuity, and in 2 of them, the amblyogenic factor was anisometropy. None of the cTBS group participants had had any previous treatments for amblyopia. By contrast, there were no significant changes in visual acuity for the placebo group. The median value was 0.2 (0.07-0.5) logMAR (20/32 Snellen) before placebo stimulation and 0.2 (0.08-0.49) logMAR (20/32 Snellen) after placebo stimulation (Fig. 2). Significant differences (P = 0.01; nonparametric analysis of longitudinal data in factorial experiments) were observed Statistical Analyses Because our data did not fit assumptions for a normal distribution, we used the nonparametric test for analysis of 188 FIG. 1. Visual acuity measurements before and after cTBS treatment. AE, amblyopic eye. Tuna et al: J Neuro-Ophthalmol 2020; 40: 185-192 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Original Contribution FIG. 2. Visual acuity measurements before and after cTBS placebo stimulation (control group). AE, amblyopic eye. for visual acuity before and after stimulation in the cTBS group. Results of this statistical test are in agreement with the Wilcoxon signed-rank test with a P = 0.017. This was observed independently of the lateralization of the amblyopic eye. By contrast, in the placebo group, there were no significant differences for prestimulation vs poststimulation visual acuity (P = 0.81; nonparametric analysis of longitudinal data in factorial experiments). This was confirmed once again with the Wilcoxon signed-rank test with a P = 1. SI was then determined for both the cTBS group and in the placebo group (Figs. 3, 4). Six of the 8 participants in the cTBS group showed a reduction SI from prestimulation to poststimulation, suggesting that most had an improved balance between the 2 eyes in terms of ocular dominance. These improvements were reflected in a value of SI that was closer to zero after TBS treatment 0.3 (0.1-0.4) before vs 0.15 (0.03-0.22) after treatment (Fig. 3). In the placebo group, there were no changes in the suppressive imbalance before vs after the placebo session; 1 exception was participant number 2, who showed a slight improvement in the FIG. 4. Suppressive imbalance measurements before and after cTBS placebo stimulation. A negative value indicates dominance of the left eye, whereas a positive value signifies dominance of the right eye. SI. SI before the stimulation was 0.17 (20.12 to 0.81), and afterward, the median was 0.17 (20.17 to 0.82) (Fig. 4). When results were analyzed using nonparametric analysis of longitudinal data in factorial experiments, the cTBS group showed a significant difference in suppressive imbalance (P = 0.013) for measurements before vs after stimulation. On the other hand, the placebo group showed a nonsignificant result (P = 0.81). The Wilcoxon signed-rank test is in agreement with the statistical test that was performed, with a result of P = 0.028 and P = 0.317, respectively. Stereoacuity was then studied. In the cTBS group after the TBS session, most participants showed improvements. This was with exception of participant numbers 3 and 8 who did not demonstrate improvement (Table 2). The median value for stereoacuity in arcseconds before the stimulation was 100 (19.4-432.6) compared with 63 (13.6- 288.4) after stimulation. In the placebo group, there were no changes in the stereoacuity from before vs after stimulation. The median value was the same 80 (228.8 to 192.8) both before and after the stimulation (Table 3). Nonparametric analysis of longitudinal data in factorial experiments confirmed a significant difference between TABLE 2. Values of stereoacuity for participants in the cTBS group Stereoacuity FIG. 3. Suppressive imbalance measurements before and after cTBS treatment. A negative value indicates dominance of the left eye, whereas a positive value signifies dominance of the right eye. Tuna et al: J Neuro-Ophthalmol 2020; 40: 185-192 ID Before After 1 2 3 4 5 6 7 8 6899 10099 40099 10099 40099 70099 4099 No stereo 5099 6399 40099 6399 20099 40099 3299 No stereo 189 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Original Contribution TABLE 3. Values of stereoacuity for participants in the control group Stereoacuity ID Before After 9 10 11 12 13 20099 14099 No stereo 8099 No stereo 20099 14099 No stereo 8099 No stereo before and after TBS in the cTBS group (P = 0.0005), whereas there were no significant differences in the placebo group (P = 0.49). The Wilcoxon signed-rank test was performed for the cTBS group and the placebo group, and the results are in agreement with the test that was conducted before (P = 0.027 vs P = 1). DISCUSSION In this pilot study, transcranial magnetic stimulation was associated with improved visual acuity in the amblyopic eyes of most participants in the active treatment (cTBS) group. Improvements of SI were also seen. Visual acuity improvements were most notable in the amblyopic eyes; visual acuities for the contralateral eyes did not change. Regarding stereoacuity, there was also improvement noted in almost all of the participants in the cTBS group. With just 1 treatment session, there were notable improvements in all measures. The placebo group participants did not significantly change with respect to visual outcomes before vs after treatment. Larger studies with greater standardization of the treatment duration for cTBS and placebo groups will further define the potential role for neuroplasticity in patients with amblyopia. Neuroplasticity is defined as the capacity for neurons to adapt and reorganize circuits in both structural and functional capacities (27). Plasticity enables the brain to learn and remember as well as to recover from injury (28). Brain mechanisms of neuroplasticity are described as being able to change existing synapses and neurons and to rewire the cortical circuits of the brain, with the creation of new synapses (28). Through TMS, it is possible to induce and manipulate neuroplastic changes, changing neuronal activation (29). Our preliminary results suggest that the visual cortex of an amblyopic adult may have enough neuronal plasticity to change apparently stable processes using TMS. With 1 session of cTBS, it was possible to improve several visual outcome measures in the stimulated amblyopic group, possibly due to neuronal plasticity. In our group of participants, there was a tendency for amblyopic subjects with anisometropy as the amblyogenic factor to show more evident improvements than noted for participants with a history of strabismus. A larger cohort 190 should be studied to confirm this finding. Nevertheless, the amblyogenic mechanism may be an important factor in recovery and in the capacity for TMS to modulate improvement of visual acuity. Other authors have investigated the potential role for TMS in patients with amblyopia and have suggested that the visual systems of amblyopic adults have enough plasticity to undergo change. Those studies used contrast sensitivity as the visual outcome, showing that TBS produces more stable improvements in the amblyopic eye for a period of 78 days. However, these studies may have also been limited by the fact that the control groups consisted of healthy volunteers without amblyopia (15,16). One limitation of our study, however, is that the duration of the effect of 1 session of stimulation is not yet known. Other studies have suggested a long duration of effect (15); larger investigations with longitudinal follow-up will be important to address this question. Cortical inhibition may be an important factor defining the limits of neuronal plasticity. A reduction of GABAergic interneuron activity may be a crucial factor in restoring neuronal plasticity among adult patients. During the critical period of growth in childhood, neuronal plasticity is robust and gamma‐aminobutyric acid (GABA) neurotransmitter levels are low. However, as this period ends, the levels of neuronal plasticity decrease as a result of the increase in inhibitory transmission (26). When there is an excess of GABA production, it may result in the anticipated end of the development period, leading to, or failing to prevent, amblyopia (30). In adults, it becomes more difficult to switch ocular dominance from the contralateral eye to the amblyopic eye. Previous studies have demonstrated that inhibition of the system does not allow cortical cells to have enough plasticity to be able to make this change in ocular dominance (30,31). The usage of Food and Drug Administration-approved drugs such as fluoxetine or diazepam (used in conditions such as depression) can have an inhibitory effect, thus reducing a possible GABAergic inhibition and allowing to re-establish ocular dominance plasticity (30,32). These drugs may reduce cortical inhibition, thereby allowing for some synaptic plasticity and inducing recovery in the amblyopic eye (30). Although it is controversial, some authors argue that inhibitory magnetic stimulation exerts a negative effect on GABAergic activity (33). That assumption, associated with the results reported by Sale et al (30) showing an excess in GABA transmission in the visual cortex of amblyopic patients, suggests that cTBS may promote an improvement in visual outcomes of acuity, SI, and stereopsis through downregulation of GABAergic activity (30). Measurements of visual acuity are susceptible to small amounts of random variability, potentially resulting from lighting, scaling of optotypes based on distance, or patientrelated performance factors. Rosser et al and Peter have noted that test-retest variability may be a factor (20,34). As Tuna et al: J Neuro-Ophthalmol 2020; 40: 185-192 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Original Contribution such, and as noted in our study, such variability should be interpreted in the context of sample size and results of statistical testing, which takes into account the role of chance in the significance of observed differences. SI is a very useful tool to quantify the dominance of 1 eye. It is an easy test to apply and with low cost. However, measurement of SI is a subjective test for which it is necessary for the patient to be very attentive to small differences induced by the NDF (19). We believe that the improvements observed in participant number 2 of the placebo group may be due to misinterpretation of the test when it was applied the first time. When a second measurement was made after stimulation, it was better understood, and there was, in fact, a slight difference, showing a trend toward having a better balance in these patients. Importantly, the need for participant focus and understanding of the test applies to both the cTBS group and placebo group in this study. It should be emphasized that, given the nature of this pilot study to apply an innovative TMS protocol in adults with amblyopia, only the right side of the brain was stimulated. For this pilot investigation, we chose the right hemisphere following the results of previous studies indicating that there may be some predominance of right hemisphere over the left (35,36). In our group's ongoing studies, we have already performed bilateral stimulation for the next cohort. Our results suggest that TMS may be associated with improvement of visual function in adults with amblyopia. The TMS methods tested in this pilot study were rapid and painless as per the participants' reports. This technique may also be combined with other treatments, such as the antisuppressive visual therapy. TMS has been investigated by other authors in amblyopic adults (15,16) with positive results. However, in our study, we used TBS instead of its classical form (TMS) because it is faster and may have longer lasting effects (15). We also created a specific protocol, in the form of the visual "hot spot" identification (participant notes phosphenes), in the intensity applied and in coil orientation (16,23-26). One potential limitation of our pilot study is the fact that although the participants and evaluators were masked to the treatment assignment, there may have been some subtle differences in the treatment protocols noted by participants themselves. Nonetheless, the outcome measure assessments were largely objective in nature in terms of the uniformity of instructions. It will be important for our results to be reproduced in larger groups with greater uniformity of the durations of testing protocols and with randomization by using random-number generators or other methods used in clinical trials. Our results, independently of being very promising, need to be interpreted with caution given the small sample sizes. Statistical tests demonstrating differences beyond those expected based on chance alone are encouraging, and indicates that future studies should proceed to investigate the potential role for TMS and related technolTuna et al: J Neuro-Ophthalmol 2020; 40: 185-192 ogies in adults with amblyopia. This pilot study represents an important step toward our understanding cortical mechanisms involved in the visual system in amblyopia. STATEMENT OF AUTHORSHIP Category 1: a. Conception and design: A. R. Tuna, N. Pinto, F. M. Brardo, M. V. Pato, and A. Nunes; b. Acquisition of data: A. R. Tuna, N. Pinto, M. V. Pato, and A. Nunes; c. Analysis and interpretation of data: A. R. Tuna, N. Pinto, M. V. Pato, F. M. Brardo, and A. Fernandes. Category 2: a. Drafting the manuscript: A. R. Tuna, N. Pinto, and M. V. Pato; b. Revising it for intellectual content: A. R. Tuna, N. Pinto, F. M. Brardo, M. V. Pato, and A. Nunes. Category 3: a. Final approval of the completed manuscript: A. R. Tuna, N. Pinto, F. M. Brardo, A. Fernandes, M. V. Pato, and A. Nunes. REFERENCES 1. Barrett BT, Bradley A, Candy TR. The relationship between anisometropia and amblyopia. Prog Retin Eye Res. 2013;36:120-158. 2. Joly O, Frankó E. Neuroimaging of amblyopia and binocular vision: a review. Front Integr Neurosci. 2014;8:62. 3. Wong AMF. New concepts concerning the neural mechanisms of amblyopia and their clinical implications. Can J Ophthalmol. 2012;47:399-409. 4. Doshi NR, Rodriguez ML. Amblyopia. Am Fam Physician. 2007;75:361-367. 5. 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