Effect of Direct Transcranial Stimulation by Continuous Current (Tdcs) in “Kiss Nightclub Fire” Patients with Post-Traumatic Stress Disorder: Phase II Controlled Clinical Trial

Kathy Aleixo dos Santos Marcolin Univeristy of Santa Maria (UFSM) Santa Maria City – Rio Grande do Sul State Ângelo Batista Miralha Cunha Univeristy of Santa Maria (UFSM) Santa Maria City – Rio Grande do Sul State Beatriz Capparros Yoneyama Univeristy of Santa Maria (UFSM) Santa Maria City – Rio Grande do Sul State Tiango Aguiar Ribeiro (  tiangoribeiro@gmail.com ) Univeristy of Santa Maria (UFSM) Santa Maria City – Rio Grande do Sul State

the event, avoidance of reminders, anger, hyper-vigilance, dysphoria, sleeplessness, increased arousal or anhedonia (1)(2)(3)(4). About 60.7% of men and 51.2% of women will be exposed to at least one potentially traumatic event during their lives (5). The prevalence of PTSD during life in the general population of the United States is 8% (6) but in direct victims of disasters these numbers can reach 30-40% (7). Most PTSD patients have severe distress and some impairment in their psychosocial functioning, such as family and work con icts, and/or the development of comorbidities (social phobia, panic syndrome, major depressive disorder, increased risk of suicide) (1,6,8).
Treatment includes psychotherapy and cognitive behavioral therapies such as prolonged exposure therapy, cognitive processing therapy, eye movement desensitization and reprocessing cognitive behavioral therapy (2,9). However, pharmacological therapy also has its place, the rst-line treatment being the use of antidepressants, particularly selective serotonin reuptake inhibitors (9); the best responses are seen with paroxetine and sertraline (10). However, despite the correct treatment, less than 60% of patients respond to treatment and only 30% have complete remission of symptoms (11).
A large re in a nightclub in Santa Maria, Rio Grande do Sul, Brazil, in January 2013, killed 242 young people, left hundreds more with physical and psychological consequences and was the biggest disaster to date in our country. The event, known in the media as "KISS nightclub re", caused an increase in the number of cases of several pathologies, among them PTSD. The impact of this disease, not only on KISS survivors but also on family members, rst responders and police, combined with the low effectiveness of standard treatment, made new forms of approach necessary.
In this context, transcranial direct current stimulation (tDCS), which is a neuromodulation technique, has shown promise in the treatment of neuropsychiatric disorders (12)(13)(14). tDCS consists of the passage of low-intensity constant electric current through the skull, thus modifying brain activity (12,14,15). The application of repetitive transcranial magnetic stimulation (rTMS), a technique similar to tDCS, has shown promising results in the treatment of patients with PTSD. Several studies with low-frequency rTMS in cerebral ow in the right dorsolateral prefrontal cortex (DLPFC) have found positive results (16)(17)(18)(19) and, given the similarity between these two techniques, it can be assumed that the results would be similar using tDCS.
The aim of this study is to present a clinical trial study conducted in patients with PTSD caused by the "KISS nightclub re" disaster who, being unresponsive to pharmacological therapy, underwent tDCS treatment. Symptoms of PTSD, depression and anxiety are expected to decrease and remain low after treatment and at follow-up for 60 days after intervention. Patients were recruited from the Integrated Center for the Attention of Accident Victims of the Psychiatry Department of Santa Maria University Hospital, which is responsible for the attendance of patients of the "KISS nightclub re" disaster. The inclusion criteria used for the study were: patients over 18 years of age diagnosed with PTSD without complete remission of symptoms; and maintenance of the pharmacological schedule unchanged in the three weeks preceding the beginning of the proposed treatment and during the proposed treatment. The study exclusion criteria were: patients with psychiatric indication for hospitalization; patients with severe personality disorder; presence of neurological diseases or neoplasms in activity; presence of neurodegenerative diseases; patients with metallic implants; and pregnant women and nursing mothers.

Study protocol
Neuroimaging studies suggest that the pathogenesis of PTSD may be related to changes in cortical excitability (20,21). Individuals exposed to trauma memories have increased cerebral ow in the right DLPFC, suggesting hyperexcitability in this region (22). Consequently, neuromodulation in this area would have the potential to rebalance brain function and return it to basal levels (17). tDCS sessions were performed with two electrodes positioned as follows: the cathode on the right DLPFC and the extracephalic anode on the contralateral deltoid muscle. The stimulations were performed once a day, each for 30 min; a total of 10 sessions were held over 10 continuous days. In each tDCS a current of 2 mA was used for an area of 25 cm² (current density of 0.08 mA/cm²). The electric current was administered through a direct current electrostimulator (Striat Ibramed). In order to minimize possible discomfort and increase the tolerability of the procedure, the current was maintained at 1 mA in the initial and nal 30 s of each session in order to scale the current increase and decrease, thus avoiding sudden changes in its intensity. The electrodes used were made of silicone and wrapped in sponges soaked in saline solution.

Assessment
All patients were assessed using four standardized scales applied at four distinct times: before tDCS (preintervention), after tDCS (post-intervention), 30 days' post-intervention and 90-days' post-intervention.
The Post-Traumatic Stress Disorder Checklist, Civilian version (PCL-C), is composed of 17 items based on the DSM-IV for the purpose of screening and to aid in diagnostic evaluation and monitoring. Each question has ve possible answers, ranging from 1 (nothing) to 5 (very). It is a self-applicable scale and its total score ranges from 17 to 85 points, classi ed as: 28-29, some PTSD symptoms; 30-44, moderate to moderately high severity of PTSD symptoms; 45-85, high severity of PTSD symptoms (23).
The Montreal Cognitive Assessment (MoCA) is a screening tool for mild cognitive impairment. It has a short application time, and its score varies in the range 0-30 points. Scores greater than 26 are considered normal (24). The Hamilton Depression Rating Scale (HAM-D) is widely used in research and clinical practice to assess patients with depressive symptoms. The version with 17 items was adopted for this trial, which is the most commonly used version (25). The scores are classi ed as: 0-7, normal; 8-16, mild depression; 17-23, moderate depression; and >24, severe depression (26).
The Hamilton Anxiety Rating Scale (HAM-A) consists of 14 items, with scores in the range 0-56 points (27). Each item is evaluated from 0 (none) to 4 (maximum) points. The intensity of anxious symptoms is classi ed as: <17, mild severity; 18-24, mild to moderate severity; and 25-30, moderate to severe severity (28).

Statistical analysis
Statistical analysis was performed using SPSS software, version 18.0 (SPSS Inc., IBM Corporation, Armonk, NY). The Kolmogorov-Smirnov test was applied to verify the distribution of variables. Normal quantitative variables were expressed as mean and standard deviation. Qualitative variables were expressed by their absolute and relative frequencies. To examine possible differences between repeated measures of the PCL-C, MoCA, HAM-D and HAM-A for the follow-up of subjects, generalized estimating equation (GEE) analysis with the Wald chi-square test was used. To verify the exact stratum where the difference was found, the Bonferroni post-hoc test was used. All statistical analyses performed were considered signi cant when the bicaudal p value was <0.05.

Results
Data from 145 subjects were initially screened. After applying the inclusion and exclusion criteria, eight subjects were analysed ( ow diagram -gure 1). The characteristics of the participants are shown in Table 1. Most of the subjects were female (87.5%) and the mean age of the studied population was 30.88 ± 7.74 years (mean ± standard deviation), ranging from 23 to 44 years.   Table 2. Subjects with moderate depression diagnoses on the HAM-D scale had an average reduction of 60% in scale values after tDCS, indicating a normal diagnosis on this scale. For symptoms of anxiety measured by the HAM-A scale, patients with a diagnosis of moderate to severe anxiety presented a reduction of 54.39% after tDCS, indicating mild symptoms of anxiety. There was a 20% decrease in the values of the PCL-C scale: patients with high severity symptoms of PTSD were assessed as having moderate to moderately high severity symptoms after tDCS. Evaluation of the MoCA scale showed that there was no cognitive impairment either before or after tDCS, with patients according to this scale assessment considered normal.  Although the scales showed an increase in their values from post-intervention to the 30-day' postintervention assessment (Table 3), these increases were not statistically signi cant, demonstrating that there was no signi cant involution of the improvements obtained with the treatment. Therefore, the effects of the treatment remained 30 days after the intervention.  Comparing pre-intervention and 30-day post-intervention values (Table 4), there was a statistically signi cant decrease in the PCL-C values and a 5.5% improvement of cognitive function in the MoCA scale.

Discussion
All patients undergoing experimental treatment with tDCS showed improvement in PTSD symptoms after the intervention and these positive effects were maintained after 30 days of intervention. Also, but in a lesser proportion, maintained improvement in relation to symptoms of depression and anxiety at the end of 90 days was observed.
Only minor and brief adverse effects have been reported: local hyperemia, pruritus, mild discomfort and headache. Local hyperemia ceased spontaneously within 1 hour after the end of the stimulus, itching and discomfort was reduced or ceased with extra application of saline solution on the sponges and headache ceased spontaneously within 24 hours on taking common analgesics. These data help to reinforce the evidence that tDCS is a safe technique for non-invasive neuromodulation (12,13).
In our study, a 20% reduction in the symptoms of refractory PTSD was observed after treatment. This improvement was seen immediately after the tenth neuromodulation session and was present throughout the rst month after treatment. Although the improvement continued, it was not stable over time and Corroborating the importance of DLPFC neuromodulation in the treatment of PTSD, Boggio et al. (31) demonstrated that stimulation with high frequencies (20 Hz) in both hemispheres caused an improvement in symptoms, with these effects being more evident in the right cortex. In addition, stimulus on the right DLPFC caused an improvement in anxiety symptoms and cognition, as in our study. Watts et al. (19) also evaluated the effects of rTMS on DLPFC and observed improvement in PTSD symptoms in the group submitted to the active stimulus as opposed to the simulated stimulus, but unlike the protocol used by Boggio et al. (31) the sessions were carried out with low-frequency stimuli (1 Hz). In a similar study there was also an improvement in PTSD (16). These ndings corroborate what we found in our research, due to the similarity between the cathodic stimuli of tDCS and the low frequencies in rTMS.
There are several examples in the literature demonstrating the bene ts of neuromodulation of the right prefrontal cortex and its repercussions both on the symptoms of PTSD and the frequent comorbidities of this disorder. The results found in our study corroborate those observed in other studies, reinforcing the importance of the right DLPFC. A peculiarity of our research was that, unlike most studies, which applied excitatory stimuli, we applied inhibitory stimuli and, even so, we obtained similar positive results. There is an increasing amount of evidence suggesting the relevance of DLPFC in PTSD and neuromodulation of this region proves to be a good alternative to be added to the treatment arsenal of this disorder. However, some aspects deserve attention and should be better clari ed, such as differentiating and quantifying the action of excitatory and inhibitory stimuli, as both seem to have bene cial effects. Not only should tDCS be explored as an isolated alternative but also as an adjunct treatment to psychotropic drugs or cognitive behavioral therapy. In addition, it should be analyzed whether improvement of the condition is due to improvement of PTSD itself or is an apparent improvement, a re ection of the important relief of concomitant depressive and anxious symptoms. All individuals undergoing experimental treatment in our study were refractory and had already used at least two different pharmacological regimens. They had been symptomatic patients for longer, symptoms were more severe and they responded less to conventional treatment. Therefore, the positive response to tDCS speci cally in this group would have been less than expected when compared to patients with less intense symptoms, which seems to be intuitive.
The main characteristic of depressive disorder is the presence of sadness and/or anhedonia, in addition to changes in sleep and appetite, tiredness, feelings of guilt and worthlessness, cognitive impairment and ideas of death. It is a highly prevalent disease and is a frequent comorbidity in patients with PTSD (32). In our study, after the tenth session of tDCS the depressive symptoms improved by 60%. This improvement was still noticeable after 1 month of treatment; however, the intensity of the improvement decreased over the period. Most studies that analyzed the effect of tDCS on depressive symptoms were carried out with unilateral assemblies applying the anodic stimulus in the left DLPFC because the hypoactivity of this area is one of the pathophysiological hypotheses of this disorder. Neuromodulation of this region has been promising, with a response comparable to the use of uoxetine (33), and the association of tDCS with sertraline proved to be superior to the use of tDCS or sertraline alone (34). Other studies have also shown encouraging results. Boggio et al. (31), in a randomized controlled study of 40 patients and with the anode on the left, found a 40% improvement in depressive symptoms. Similar studies have also found positive results (33,35), with a reduction of up to 60% in symptoms. However, due to the large functional differences between the cerebral hemispheres, it is impossible to compare these studies with this research. In contrast to these positive results, Loo et al. (36) randomized 64 depressed subjects resistant to treatment into active and simulated groups and found no signi cant difference between groups. One possible explanation for this may be the fact that fewer sessions were held ( ve in total). However, a clinical trial (37) that stimulated depressed patients resistant to escitalopram by taking 10 sessions also found no positive results. It is interesting to take into account that some studies that showed robust results in the reduction of depressive symptoms (18, 35) interrupted the use of antidepressants for up to 2 months before starting the procedure. For ethical reasons, we kept patients on standard treatment and achieved similar results. So far, there have been no unipolar assemblies used in the right prefrontal region in depressed patients. The closest have been bilateral assemblies in which the anode on the left is associated with the cathode on the right. Brunoni et al. (34), using an assembly similar to the one described, observed cognitive impairment in the right prefrontal cortex but it was the opposite of what we found in this study, in which there was an improvement in depressive and cognitive symptoms. The small number of patients in this study makes it impossible to generalize, yet all patients showed improvement in depression and none of them evolved with worsening cognition. In contrast to other studies, we observed a signi cant improvement in depressive symptoms without concomitant cognitive impairment. The role of the right DLPFC in depressive symptoms needs to be better understood, especially its role in the regulation of subcortical structures related to depressive symptoms. In addition, chronically ill patients are more likely to develop depressive disorder and the improvement in PTSD may have in uenced the improvement in depression.
Our study showed a 54% improvement in anxiety symptoms. This improvement was present 30 days after the end of treatment, despite some decrease in these effects. Even so, this improvement remained statistically signi cant when compared to before treatment. Although anxiety disorders are quite prevalent, there is still little research involving tDCS in patients with these symptoms and, so far, it is limited to case reports. There is only one case report of a patient with generalized anxiety disorder refractory to treatment undergoing tDCS sessions (38). As in our study, cathodic stimulus was applied in the right DLPFC with improvement of symptoms. There is also a case report of a patient with obsessivecompulsive disorder resistant to treatment who underwent cathodic stimulus in order to decrease cortical excitability. There was a 30% reduction in the severity of the condition but stimulation was performed in the motor cortex (39). Evidence of tDCS action on anxiety disorders is still incipient. This is partly due to the fact that anxiety disorders are very heterogeneous, involving different brain areas. It is expected that with the development of larger studies, the evidence supporting the use of this modality of neuromodulation as an alternative to standard treatment will increase.
There was a gradual improvement in cognition immediately after treatment. However, this improvement was only statistically signi cant when the patients were reassessed after 1 month of treatment, suggesting that the bene cial effects may have progressed even after the end of the stimulations. The sphere of cognition is very heterogeneous and composed of several aspects, such as attention, vigilance, perception (visual, auditory, somatosensory), working memory, learning, decision-making, etc., located in different brain circuits. The involvement of DLPFC in the various aspects of neurocognition has been the subject of investigation and numerous studies in the literature have shown positive effects on cognition after anodic stimuli, mainly in the left DLPFC (40)(41)(42)(43). Although patients undergoing experimental treatment have improved cognition, the literature shows that, unlike what happens with anodic stimuli, cathodic stimuli applied in the right and left DLPFC are related to worsening cognitive performance. Tanoue et al. (44) conducted a study to assess attention by dividing 24 patients into three groups: cathodic stimulation in the right parietal region; cathodic stimulation in the right DLPFC; and sham stimulus (simulated stimulation, performed with the device turned off). Patients undergoing cathodic stimulation showed worse performance when compared to the sham stimulus. In addition, this worsening of performance was more intense in patients stimulated in the right DLPFC. Elmer et al. (45) also observed a worsening of cognition after cathodic stimulation in the left DLPFC. Opposing these data, another study found that anodic stimulation in the right DLPFC improves learning and attention (40), corroborating the opposite effect that the cathode and anode exert. What would justify this apparent improvement in the cognitive performance of stimulated patients is the fact that the MoCA is a simple scale with a learning curve. There is a possibility that this improvement does not necessarily re ect an improvement in cognition but rather a memorization of the test by patients. Still, this scale alone is insu cient in view of the complexity of the cognitive domains, and for a more complete assessment it would be necessary to apply several neurocognitive tests together with tests of its different domains in a broader way. In addition, the MoCA is a screening scale for mild cognitive impairment, with some authors suggesting a cutoff point between 24 and 26 points. Of the eight patients studied, seven were above 26 points in all evaluations, that is, although they did not score the total 30 points they were outside the range considered for the presence of cognitive impairment. The study of cognitive processes is a complex area and the application of cathodic stimuli, which decrease cortical excitability, is not necessarily harmful, as demonstrated by Weiss and Lavidor (46) when they stimulated the parietal cortex of healthy individuals, which was corroborated by our work.
Important limiting factors of the study were the small number of participants, which makes generalizations di cult, and the absence of a control group. Individual characteristics also had a limiting effect as age, gender and shape and size of the skull can interfere in the amount of current reaching the brain and also in the correct positioning of the electrodes. In addition, all patients were using one or more psychotropic drugs and therefore were subject to interference from these drugs in cortical excitability. It has been shown that the use of psychotropic drugs in uences the rate of neuronal ring and may be related to longer lasting effects or decreased effects of tDCS (47). However, it is a unique study carried out in a population of young adults exposed to the same traumatic event, which was unique in our country and in the world, and the study used validated assessment tools for depression, anxiety, cognition and PTSD.

Conclusion
Our study found that patients with refractory PTSD, after adjuvant treatment with tDCS, showed improvement of 20% in the central symptoms of PTSD, in addition to an improvement of 60% in depressive symptoms and 54% in anxious symptoms. Despite a decrease in these effects over time, this improvement was maintained throughout the rst month after treatment. The improvement in cognition occurred slowly and progressively, being statistically signi cant at the end of the rst month of treatment.
There was no development of important side effects. Non-invasive neuromodulation techniques, especially tDCS, have been shown to be safe and promising in the treatment of neuropsychiatric pathologies and can be an alternative for the treatment of refractory disorders, either as a monotherapy or as a treatment enhancement strategy. They can also be an option for patients who do not want or do not tolerate pharmacological management.
There are multiple conditions that can in uence the outcome of the procedure and should be studied in <p>Study ow diagram</p>