Longstanding effects of continuous theta burst stimulation in adult amblyopes

ABSTRACT Clinical Relevance Continuous theta burst stimulation may be an important tool in the therapeutic management of amblyopia, when trying to correct the established neuronal imbalance. It is important to understand whether two sessions of continuous theta burst stimulation produce greater and longstanding changes in visual acuity and suppressive imbalance than one session of continuous theta burst stimulation Background We hypothesise that through the usage of continuous theta burst stimulation (cTBS) it is possible to change cortical excitability in a situation where visual impairment is present. Methods We selected 22 adult amblyopes, 18 females and 4 males, with an age range of 20–59 years. They were randomised into two groups: group A with 10 amblyopes was submitted to one session of cTBS and group B with 12 amblyopes submitted to two sessions of cTBS. Visual acuity (VA) and suppressive imbalance (SI) were evaluated immediately before and after stimulation in both groups A and B. A follow-up was done in both groups. Results For both group A and B, the VA improvements were significant after cTBS (p = 0.005 and p = 0.003, respectively). Regarding SI, both group A and B had significant improvements after cTBS (p = 0.03 and p = 0.005, respectively). Comparing groups, A and B no significant differences were found with regard to the results obtained both for VA (p = 0.72) and SI (p = 0.24). However, significant differences were found between group A and B with regard to the duration of stimulation effect for VA (p = 0.049) and SI (p = 0.03). Conclusion We conclude that two sessions of cTBS do not produce better results than one session of stimulation. However, it seems that two sessions of cTBS produce longstanding effects in VA and SI.


Introduction
Amblyopia is a neuronal visual disorder that occurs mostly in the first years of life. 1 It is characterised as a difference in visual acuity of at least 0.2 logMAR between the eyes when they are evaluated for the best corrected visual acuity (BCVA). 2,33][4] Although visual acuity is the visual parameter used to classify amblyopia, there are other visual parameters affected as well 3 : contrast sensitivity, 5 stereoacuity, 5,6 and sensorial ocular dominance 6,7 ; all are altered in amblyopic patients.
It is well established that each eye sends an input to the primary visual cortex, where information is then binocularly combined. 7,8][10] However, in an amblyopic patient these inhibitory interactions are unbalanced, which leads to an unbalanced visual system where the fellow eye has a greater strength over the amblyopic eye -suppressive imbalance. 8-10 For many years traditional therapies like refractive correction, occlusion and pharmacological penalisation were used to achieve a better balance between the eyes in infants.In recent years, a new technique -Transcranial Magnetic Stimulation (TMS) -has been used to improve this unbalanced equilibrium between the eyes in amblyopes.
TMS is a non-invasive and painless technique capable of neurostimulation and neuromodulation and was first used by Barker et al. in 1985, over the human motor cortex. 11TMS has been used as a therapeutic method to treat neurological pathologies or to recover from nervous disorders like stroke or trauma. 12There is scientific evidence that several TMS sessions have a positive effect on the rehabilitation of patients with depression and also that the number of applied sessions may influence the outcome of depressive patients. 13t has been reported before that an adult visual cortex still retains some plasticity. 14Based on this principle, Thompson et al. 14 applied one session of repetitive Magnetic Stimulation (rTMS) to a group of adult amblyopes and found improvements in contrast sensitivity. 14When rTMS is applied repetitively, it can create a process of cortical activation or inhibition, depending on the type of stimulation used. 15ost researchers believe that a homoeostatic plasticity mechanism, a long-term depression (LTD) and long-term potentiation (LTP), is responsible for the long-lasting effects of high-frequency stimulation, as they increase and decrease the strength of synapses, respectively. 12,16heta burst stimulation (TBS) is a patterned form of rTMS that presents shorter stimulation periods with similar neuronal effects.Several authors have used TMS/TBS in amblyopic patients, showing that through the use of TBS technique is possible to modify the responses of the amblyopic visual system. 6,14,17There are few research studies that used continuous TBS (cTBS) protocol, however with one session of cTBS it seems possible to improve several visual parameters, like visual acuity, suppressive imbalance, stereoacuity 6,18 and contrast sensitivity in amblyope patients. 17n our previous work, we applied one session of cTBS to a group of amblyopes and evaluated visual acuity and suppressive imbalance. 6,18The visual parameters were evaluated immediately before and after cTBS, and significant differences between before and after stimulation were found, but no follow-up was done. 6,18Previously, Clavagnier et al. 17 led a series of cTBS sessions in five adult amblyopes, measuring contrast sensitivity in every session before and after stimulation.They found improvements in contrast sensitivity in the volunteers, and they suggested that more stimulation sessions do not necessarily lead to better results but lead to a longstanding effect.
With this research study, we tried to evaluate the cumulative effects of TBS in amblyopia and answer the following question: Will two sessions of cTBS produce greater and longstanding changes in visual acuity and suppressive imbalance than one session of cTBS?

Methods
Adult participants with amblyopia were selected through community surveys and were submitted to an intensive eye examination.Furthermore, all refractive errors were corrected to achieve BCVA.
Inclusion criteria were as follows: absence of abnormalities in their ocular structures, with at least a difference of two lines of visual acuity between the eyes and the presence of an amblyogenic factor (strabismus, anisometropia, or both).Exclusion criteria were history of brain injury and/or head trauma, neurological and psychiatric disease, seizures, pregnancy, history of substance use, or the presence of metallic implants in the head or torso.

Participant characterisation
The study sample consisted of 22 adult amblyopes, 18 female and 4 male, with a mean age of 39.7 ± 12.6 SD years and an age range of 20-59 years.Of this group, 9 had amblyopia in the right eye, and the other 13 had amblyopia in the left eye.
Patients were randomised into two groups and submitted to stimulation.The allocation was done randomly with a fiftyfifty chance to be part of group A or B.
Group A was composed of 10 adult amblyopes, seven were female and three were male, with a mean age of 38.0 ± 12.5 SD.Five had amblyopia in the right eye, and the other five in the left eye.All 10 patients underwent one session of cTBS and were evaluated immediately before and after the stimulation sessions, re-evaluated at 15 days, and then monthly until they return to their baseline values.
Group B had 12 adult amblyopes, 11 female and 1 male, with a mean age of 41.9 ± 13.0 SD years.Five had amblyopia in the right eye and seven had amblyopia in the left eye.All 12 amblyopes were submitted to two sessions of stimulation which were delivered in consecutive days, and in the same period of the day.All patients were evaluated immediately before the first session of stimulation and immediately after the second session of stimulation, also re-evaluated at 15 days, and then monthly until they return to their baseline values.
All patients had given their written 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-Pp-2019-009).

Monitoring parameters
The outcomes evaluated were visual acuity (VA) and suppressive imbalance (SI).SI test is based on the 'The Modified Bagolini Striated Lens Test' and is an alternative test to traditional techniques used to measure sensory ocular dominance. 19,20Fundamentally, it allows not only to qualify but also to quantify the strength of the effect of one eye over the other. 19,20Methodology used for measuring both VA and SI was used in previous studies carried out by our research team and is presented in supplement 1. 6

Transcranial magnetic stimulation
The procedure took place in the Neurophysiology Laboratory of the Faculty of Health Sciences -University of Beira Interior.Group A underwent one session of cTBS, while group B was submitted to two sessions of cTBS.The equipment used was a MagVenture MagPro1G3 X100 5.0.1 with a butterfly coil MCF-B70, following standard safety recommendations. 21hosphenes threshold or transient visual phenomena were sought in order to find the more efficient location to deliver cTBS.Phosphenes induction indicated the exact point, in the occipital lobe, where stimulation should be delivered.An individualised intensity was used, consisting of 100% of the minimum intensity needed to evoke phosphenes (phosphene threshold intensity -PTI).The procedure was initiated by placing the coil in the mid-inion region with a lateral-medial orientation, starting with 50% of the maximum device output.Using a 1 cm right lateralisation (right-hemisphere stimulation), vertical steps of 1 cm (up to 3 cm) were used until unior bilateral phosphenes were described by each participant.Intensities over 80% of maximum device output were not used (but lower intensities could be used if the participant did not tolerate the higher intensities).In the cases where no phosphenes could be detected, this was also the maximum intensity used (if tolerated by the participant), and the coil localisation was in the mid-inion region with a lateral-medial orientation using a 1 cm right lateralisation.Regarding group B, all the patients had phosphenes in the first session and also showed phosphenes in the second session of stimulation.
After the stimulation, VA and SI follow-up were done with the patients being evaluated first at 15 days and then monthly, after the stimulation until they return to baseline values before stimulation, for both visual acuity and suppressive imbalance.The follow-up endpoint was when both visual acuity and suppressive imbalance return to the values measured before stimulation (baseline values).

Statistical analyses
Our data did not comply with the normality assumptions and Wilcoxon signed-rank test was performed, in order to compare the results between 'before' and 'after' cTBS.The comparison between the two different groups was done with the Mann-Whitney U test.Statistics were performed using IBM SPSS Statistics 25.0.P values ≤0.05 were considered as significant.
Table 1 shows the participant characterisation.Group A was formed by 10 adult amblyopes that were submitted to one session of cTBS.All of the participants showed improved visual acuity of the amblyopic eye with only one session of stimulation.Figure 1 presents these results.
Mean visual acuity values before cTBS were 0.35 logMAR ± 0.23 SD, and after cTBS was 0.22 logMAR ± 0.17 SD.There were statistically significant differences in visual acuity between 'before' and 'after' one session of stimulation (Wilcoxon = −2.81;p = 0.005), for group A.
The follow-up of the 10 patients was done.The reevaluation process was conducted until they returned to their baseline values before stimulation.In terms of visual acuity, the effects were sustained by an average of 114 ± 69 days.
Regarding suppressive imbalance, seven of the participants improved their suppressive imbalance after only one session of cTBS.Contrariwise, participants A15, A17 and A21 did not have any change in suppressive imbalance.These three patients had the amblyogenic factor (anisometropia) in common.Finally, patient A16 had a balanced system before cTBS and after cTBS was slightly unbalanced (Figure 2).
Mean value of suppressive imbalance before cTBS was 0.12 ± 0.10 SD, and after cTBS was 0.07 ± 0.06 SD.There were significant differences between before and after the cTBS technique for suppressive imbalance, in group A (Wilcoxon = −2.20;p = 0.03).
Regarding the duration of the stimulation effect for suppressive imbalance in group A, it lasted an average of 50 ± 53 SD days.After that, the patient returned to the suppressive imbalance values before the stimulation.
Almost all 12 amblyopes of group B subjected to two sessions of cTBS improved visual acuity, but patient A7 did not have any change.In Figure 3, it is possible to observe visual acuity improvements for this group.
Mean value of visual acuity before cTBS was 0.33 logMAR ± 0.18 SD, and after the two sessions of cTBS was 0.20 logMAR ± 0.15 SD.The differences between before cTBS and immediately after the two sessions of cTBS were significant (Wilcoxon = −2.94;p = 0.003).
As in group A, also for group B a follow-up was done.The effects of two sessions of magnetic stimulation were maintained for 183 days, after that, the patient return to visual acuity values before the stimulation.
In what concerns suppressive imbalance, there were no changes for participants A7 and A9In what concerns suppressive imbalance, there were no changes for participants A7 and A9 (Figure 4).Both patients had in common: being females, anisometropia, amblyopia detected early in life and having undergone some type of therapeutic intervention (patching and strabismus surgery, respectively).Ten of the amblyopes (A1, A2, A3, A4, A5, A6, A8, A10, A11 and A12), had a better suppressive imbalance (closer to 0) after the two sessions of cTBS.
Mean value of suppressive imbalance before cTBS was 0.10 logMAR ± 0.07 SD, and after the two sessions of cTBS was 0.05 logMAR ± 0.06 SD.The differences between before and after the two sessions of cTBS were significant (Wilcoxon = −2.82;p = 0.005).
In what concerns suppressive imbalance, the effect was sustained for 103 days, after that, the patient return to visual acuity values before the stimulation.
One of the visual parameters measured in group A and B was visual acuity.When comparing the results obtained with one (group A) and with two sessions (group B) of cTBS significant differences were found between groups (Mann-Whitney = −0.36;p = 0.75).There was a similar magnitude effect in both group A and B.
A follow-up was done in all subjects of groups A and B. With regard to the duration of the effect in days, significant differences were found between doing one or two sessions of cTBS (Mann Whitney = −1.97;p = 0.049).In Figure 5, there is a day's average of time effect of the participants submitted to one session (group A) and to two sessions (group B) of cTBS.There is a longer time effect in patients who underwent two sessions (group B) of cTBS, with a difference in the average of 69 days.
Suppressive imbalance is the other visual parameter evaluated in both groups A and B. No significant differences were found in the results achieved with one (group A) or two sessions (group B) of cTBS (Mann-Whitney = −1.19;p = 0.24).There was a similar magnitude effect in both group A and B.
Similarly, to what was done with visual acuity, patients were also followed-up for suppressive imbalance.Significant differences were found in time effect for one (group A) or two (group B) sessions of cTBS (Mann-Whitney = −2.24;p = 0.03, with a longer time effect for group B, with an average of more 53 days (Figure 6).

Discussion
In order to answer our question, we compared the differences between a group of amblyopic patients submitted to one session of cTBS (group A), and a group of amblyopic patients submitted to two sessions of cTBS (group B).Both group A and group B significantly improved visual acuity and suppressive imbalance for the amblyopic eye with one and two sessions of stimulation, respectively, in almost all of the amblyopes.Comparison between group A and B results after stimulation, both for visual acuity and suppressive imbalance, showed no significant differences between groups but effects were more long-lasting for the volunteers of group B (with two stimulation sessions).To sum up, it seems that there were no differences between groups in what concerns the magnitude of the immediate results but there was a significant difference in terms of time effectextended duration of the effects with the double session.
Clavagnier et al. applied seven sessions of cTBS to a group of five amblyopes.Contrast sensitivity for high spatial frequencies was evaluated, before, after and 30 min after the stimulation, achieving results that lasted for 78 days.These authors came to the conclusion that several sessions of stimulation will have the same effects in contrast sensitivity as one session but maintaining its effects for up to 78 days. 17We applied two sessions of stimulation to a group of 12 amblyopia on consecutive days, always in the same occipital cortex region.We evaluated visual acuity and suppressive imbalance immediately before the first session and immediately after the second session of stimulation, achieving results that lasted for 181 days, also suggesting that the difference between one versus two stimulation sessions remains in the time effect.Both group A and B were stimulated in the same hemisphere, with the same coil orientation, which allowed the uniformity and standardisation of the stimulation process.Both studies showed promising results regarding the longlasting effects of more than one session of stimulation.
Neuronal plasticity is the ability that neurons have to adapt and rearrange, resulting in the strengthening of some synaptic connections or in the impairment of others. 22During the first years of life, neuronal plasticity is increased and there is a critical period where development happens and it is possible to improve neuronal connections. 22,23This period is highly important for neuronal circuit maturation, however after a certain age the critical period ends, decreasing considerably plasticity levels. 22,23Recently, some authors suggested that the visual system can be manipulated beyond this critical period. 22,24he results achieved in group A (one session of cTBS), group B (two sessions of cTBS) and also in our previous work 6,18 suggest that the adult human brain may have enough neuronal capacity to adapt and be able to change the visual parameters in study.Several researchers believe that there are two mechanisms responsible for neuroplastic changes in synaptic structures after TMS, the long-term depression (LTD) and long-term potentiation (LTP). 12,25,26TBS may elicit a long-term increase in the strength of synapses (LTP) that can persevere for days, weeks, or even months. 12,25,26After a period of time, all the amblyopes returned to their baselines values of visual acuity and suppressive imbalance.
Intracortical inhibition has been proven to be an important factor in the beginning and at the end of the critical period, so an increase in cortical inhibition levels may lead to an early closing of the critical period. 22 Sale et al. (2010)  with animal studies suggested that, through the reduction of the inhibitory neurotransmitter GABA, it is possible to restore some plasticity in the visual cortex after the end of the critical period.Their findings support the theory that inhibition has a crucial role at the end of the critical period. 23  Previous results suggest that TBS can be used to induce ongoing-modulating activity in the visual cortex. 27ontinuous TBS may be able to promote the suppression of cortex excitability that may be responsible for intracortical inhibition, possibly facilitating occipital lobes to return to a different equilibrium. 28herefore, we believe that with cTBS, we may be able to reduce intracortical inhibition, thus promoting an improvement in visual acuity and suppressive imbalance as seen in our results.Curiously, this only appears to be effective in a disrupted system: published results from a normal control group previously evaluated did not show any change with one or two sessions either in visual acuity or in suppressive imbalance. 6,20 subject with normal binocular vision (NBV) has reciprocal interocular inhibition, but the same does not happen for an amblyopic eye. 9In our group of amblyopic patients, for both groups A and B, there was a strong dominance of the fellow fixing eye over the amblyopic eye.This interocular suppressive imbalance might be explained by the lack of balance in the process of reciprocal interocular inhibition which can be a cause or a consequence of the amblyopic process.However, these assumptions have to be made with caution and more research is needed.
The number of optimal stimulation sessions to obtain the maximum response remains unclear. 13,29When using rTMS to treat depression, it is known that a large number of sessions, usually over 10, are needed for optimal outcomes. 30In what concerns amblyopia, we observed that with two sessions of cTBS (group B), the visual acuity and suppressive imbalance results were not significantly better compared to one session (group A) of cTBS, but the effects were longer lasting, suggesting that there may be a cumulative effect leading to a stronger efficacy.More research is needed in order to understand the optimal number of stimulation sessions needed to achieve the best outcome for the maximum period of time.
Due to the sample size and its heterogenicity our results need to be interpreted with caution.They show, however, important progress in the therapeutic of the amblyopic patient.As future work, it would be important to have a larger sample of subjects to be able to subdivide it by amblyogenic factor, gender and age and to evaluate the effects of cTBS in each variable.

Figure 4 .
Figure 4. Suppressive Imbalance values before and immediately after the two sessions of cTBS.

Figure 5 .Figure 6 .
Figure 5.Time effect of one (group A) or two sessions (group B) of cTBS for the parameter visual acuity Figure 6.Time effect of one (group A) or two sessions (group B) of cTBS for the parameter suppressive imbalance