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Repetitive transcranial magnetic stimulation increases serum brain-derived neurotrophic factor and decreases interleukin-1β and tumor necrosis factor-α in elderly patients with refractory depression

Objective To investigate the effects of repetitive transcranial magnetic stimulation (rTMS) on serum levels of brain-derived neurotrophic factor (BDNF), interleukin (IL)-1β, and tumor necrosis factor (TNF)-α in elderly patients with refractory depression. Methods A total of 58 elderly patients diagnosed with refractory depression between January 2015 and December 2016 were divided randomly into two groups: 29 patients who received rTMS and 29 controls without rTMS. Thirty healthy individuals were also enrolled and all received rTMS. Serum levels of BDNF, IL-1β, and TNF-α were measured before the study (0 days), and at 48 hours and 1, 2, 3, and 4 weeks after the first TMS treatment. Results BDNF levels gradually increased with treatment duration in the rTMS group and were significantly higher compared with the control group. In contrast, IL-1β and TNF-α levels gradually decreased and were significantly lower than in the control group. None of the serum factors were affected by rTMS in the healthy individuals. BDNF levels were negatively correlated and IL-1β and TNF-α levels were positively correlated with Hamilton Depression Rating Scale-24 scores. Conclusion These results suggest that rTMS may increase BDNF and decrease IL-1β and TNF-α serum levels in elderly patients with refractory depression.


Introduction
Depression is predicted to become the second most prevalent disease after cardiovascular disease by 2020, and thus represents a major public health problem, especially in elderly individuals. 1,2 One study showed that almost 20% of adults in the US experienced at least one episode of major depression in their lifetime, 3 and approximately 10% to 30% of patients with depression will develop treatmentrefractory depression unresponsive to antidepressants. 1,4 Furthermore, depression is often missed, ignored, or inadequately managed in elderly patients. This is partly a result of the misunderstanding that depression is an inevitable result of aging, and the treatment for refractory depression in elderly patients may thus be particularly challenging. 5,6 Various treatment methods and medications have been developed for depression, but the mechanisms responsible for depression remain unclear. 7,8 Inflammatory factors such as interleukin (IL)-1, 9 tumor necrosis factor (TNF)-a, 10 and nuclear factor-kappaB (NF-jB) 11 have been shown to be increased in patients with depression. Brain-derived neurotrophic factor (BDNF) has also attracted recent attention in relation to depression, 12 and was shown to be associated with the pathophysiology of major depressive disorder and the therapeutic mechanisms of antidepressant treatment. 13 BDNF levels are generally decreased in depressive patients and can be increased by antidepressant therapies. 14,15 Among the available therapeutic strategies for depression, electrostimulation, including deep-brain stimulation, 16 electroconvulsive treatment, 17 and transcranial magnetic stimulation (TMS), 18 have proven effective and are widely adopted in clinical practice. Repeat TMS (rTMS) treatment can improve depression in patients with bipolar affective disorder 19 and medication-resistant depression. 20 Some studies showed that electroconvulsive treatment could increase BDNF levels and thus improve the condition of patients with depression, 21 while other studies of TMS in healthy volunteers showed different results for BDNF. 22,23 However, studies focusing on the effects of TMS on BDNF, IL-1, and TNF-a levels in elderly patients with refractory depression are lacking.
In the present study, we measured serum levels of BDNF, IL-1b, and TNF-a in elderly patients with refractory depression. The results of this study may provide deeper insights into the mechanisms of TMS treatment and identify new therapeutic targets in patients with depression.

Patients
The present study enrolled 58 elderly patients (!60 years old) diagnosed with refractory depression who were admitted to our hospital between January 2015 and December 2016. All patients were diagnosed with depression according to DMS-IV criteria and continued to experience depressive symptoms despite adequatedosage antidepressant treatments with at least two different medications for more than 6 weeks (Hamilton Depression Rating Scale-17 (HAMD) score >17 and <50% reduction in HAMD score compared with baseline values). 24 The following patients were excluded: those with a history of manic episodes, cerebral organic disorders, psychoactive substance use, drug dependence, other mental disorders, severe physical diseases (such as liver or kidney dysfunction or serious cardiovascular diseases); or with a cardiac pacemaker or other metal implant.
The patients were divided randomly into two groups of 29 patients each, treated with or without rTMS, respectively. Patients in the rTMS group were stimulated in the left dorsolateral prefrontal cortex at a frequency of 10 Hz and intensity of stimulation of 80% of the motion threshold using a YRDCCY-I TMR apparatus (Yiruide Medical Equipment New Technology Co., Ltd., Wuhan, China). Stimulation lasted for 1 s with 11 s intervals, with a total of 1200 pulses per session. 25 The stimulation point in the left dorsolateral prefrontal cortex was determined according to the anatomical location and coils were placed parallel to the skull. All patients were treated with TMS five times a week for 1 month, for 20 minutes each time. Patients in the control group received no rTMS. All other antidepressants were stopped during the study, and all patients only received venlafaxine sustained release tablets 75 mg/day, gradually increasing to 150 to 300 mg/day according to the patient's condition. Alprazolam 0.4 to 0.8 mg was administered in patients with a sleep disorder. Thirty healthy individuals were enrolled and received rTMS as described above. Informed consent was obtained from all participants. The present study was approved by the ethics committee of Shanghai Jiading District Mental Health Center.

Measurement and data collection
Demographic data including age, sex, education level, and family history, and clinical variables including disease course and HAMD-24 scores were recorded. Blood samples were collected before the study (0 days), and at 48 hours and 1, 2, 3, and 4 weeks after the first TMS treatment. Serum levels of BDNF, IL-1b, and TNF-a were determined by enzyme-linked immunosorbent assay (ELISA) using commercially available ELISA kits (R&D Systems, China) according to manufacturer's instructions. HAMD-24 scores were determined at the same time as blood sampling.

Statistical analysis
The data were expressed as mean AE standard deviation. Non-continuous data and ratios were compared by v 2 tests. Comparisons between two groups of continuous data were performed using Student's t-test and comparisons among three or more groups were conducted by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Correlations were analyzed by Pearson's correlation analysis. Results were considered to be significant when the P-value was < 0.05. All calculations were performed using PASW Statistics for Windows, Version 18.0 (SPSS Inc., Chicago, IL, USA).

Baseline clinical characteristics of all participants
The baseline clinical information for all participants is shown in Table 1. There was no significant difference in age, sex, education level, family history, medication condition, or other clinical variables, including disease course and HAMD-24 scores, among the treated and control patients and the healthy volunteers. However, initial BDNF levels were significantly lower and IL-1b and TNF-a levels were significantly higher in both patient groups compared with the healthy individuals (P < 0.001 for all comparisons). No patients dropped out of the study during the study period and no adverse effects were observed in any patients.

Changes in serum levels of BDNF, IL-1b, and TNF-a
We measured serum levels of BDNF, IL-1b, and TNF-a in all groups at 48 hours and 1, 2, 3, and 4 weeks after the first TMS using ELISA. Serum BDNF levels were significantly lower in both patient groups compared with the healthy group before the study (all P < 0.001) ( Figure 1). However, BDNF levels gradually increased with treatment duration in the rTMS group, and were significantly higher than in the control group (all P < 0.001). Meanwhile, serum levels of IL-1b and TNF-a were significantly higher in the patient groups compared with the healthy group before the study (all P < 0.001), but gradually decreased with rTMS treatment duration, and were significantly lower than in the control group from 1 week (all P < 0.001). Meanwhile, levels of BDNF, IL-1b, and TNF-a were unaffected by rTMS in the healthy individuals. These results indicated that rTMS could increase BDNF levels and Relationships of serum BDNF with HAMD score, IL-1b, and TNF-a We further examined the relationships among serum levels of BDNF, IL-1b, TNF-a, and clinical outcomes. HAMD-24 scores were evaluated at the same time points as serum factors. HAMD-24 scores were significantly decreased in the rTMS group compared with the control group (P < 0.001) ( Figure  2). Meanwhile, Pearson's correlation analysis showed that BDNF levels were negatively correlated and IL-1b and TNF-a levels were positively correlated with HAMD-24 scores. Serum BDNF levels were also negatively correlated with IL-1b and TNF-a levels (P < 0.001) ( Table 2). These results suggested that Figure 1. Changes in serum BDNF, IL-1b, and TNF-a levels in all participants. *P < 0.05, compared with controls; # P < 0.05, compared with healthy volunteers. BDNF, brain-derived neurotrophic factor; IL-1, interleukin-1; TNF-a, tumor necrosis factor-a; rTMS, repetitive transcranial magnetic stimulation.  rTMS might improve depression by influencing these serum factors.

Discussion
It is estimated that around 3% of the general population have depression, and approximately 10% to 30% of these patients will develop treatment-refractory depression that fails to respond to an adequate trial of an antidepressant. 26 These patients thus require other treatment strategies such as TMS. However, although TMS has proven effective in many studies, 19,20 its mechanisms remain unclear. BDNF is considered to be a key factor in the development and treatment of depression, 13 though studies of the effects of TMS on BDNF in patients with depression have shown conflicting results. Yukimasa et al. 27 demonstrated that high-frequency rTMS could improve refractory depression by influencing BDNF, while Lang et al. 28 found no effect of brain stimulation on serum BDNF concentrations. The effects of TMS in healthy individuals have also shown different results. Lang et al. 22 found that acute prefrontal cortex TMS had no effect on serum BDNF levels in healthy volunteers, but Schaller et al. 23 showed that serial rTMS could decrease BDNF serum levels in healthy male volunteers. These contradictory results encouraged us to confirm the mechanisms of rTMS. The results of the present study demonstrated that rTMS could increase serum BDNF levels and decrease IL-1b and TNF-a levels in patients with refractory depression, but that it had no effect on levels of these factors in healthy individuals.
We confirmed that serum BDNF levels were decreased and IL-1b and TNF-a were increased in patients with refractory depression. These results were in accordance with previous studies, including Li et al.,29 who showed that patients with major depression had lower levels of serum BDNF at admission. Furthermore, Maes et al. 30 showed that serum IL-6 and IL-1 receptor antagonist concentrations were increased in major depression, and Howren et al. 31 demonstrated that IL-1 was positively associated with depression. Karson et al. also found that a TNF-a inhibitor could decrease depression and anxiety-like behaviors in a rat model. 10 We also confirmed that rTMS could enhance serum BDNF levels and decrease serum IL-1b and TNF-a levels in patients with depression, but that it had no effect on serum levels of these factors in healthy individuals. As discussed above, previous studies have shown contradictory results regarding the effect of TMS on BDNF levels. These discrepancies may be due to different study populations, and further studies are needed to confirm the results. However, few studies have examined the effects of rTMS on levels of other factors and, to the best of our knowledge, the current study provides the first evidence showing that rTMS can decrease IL-1b and TNF-a levels in patients with depression. We also demonstrated that BDNF levels were negatively correlated while IL-1b and TNF-a levels were positively correlated with HAMD-24 scores.
The present study had some limitations. First, we only analyzed the short-term effects of rTMS and did not investigate the long-term effects. Second, the control group comprised patients without rTMS, rather than patients with sham rTMS. Third, we examined the effects of rTMS on BDNF, IL-1b, and TNF-a, but did not analyze levels of other inflammatory factors, such as NF-jB and IL-10. Further studies are therefore needed to address these limitations.
In conclusion, we conducted a clinical study to investigate the effect of rTMS on serum BDNF, IL-1b, and TNF-a levels in elderly patients with refractory depression. rTMS increased serum BDNF levels and decreased serum IL-1b and TNF-a levels in patients with depression, but had no effect on any of these factors in healthy individuals. These results provide deeper insights into the mechanism of TMS and identify potential new therapeutic targets in patients with depression.

Availability of data and materials
The data and materials used in this study are available upon reasonable request from the corresponding author once the paper has been published.

Consent for publication
All personal information has been allowed for publication by all participants.