J Neural Transm (2014) 121:307–313 DOI 10.1007/s00702-013-1102-1
PSYCHIATRY AND PRECLINICAL PSYCHIATRIC STUDIES - ORIGINAL ARTICLE
Serial repetitive transcranial magnetic stimulation (rTMS) decreases BDNF serum levels in healthy male volunteers Gerd Schaller • Wolfgang Sperling • Tanja Richter-Schmidinger • Christiane Mu¨hle • Annemarie Heberlein • Christian Maiho¨fner • Johannes Kornhuber • Bernd Lenz
Received: 9 July 2013 / Accepted: 9 October 2013 / Published online: 25 October 2013 Ó Springer-Verlag Wien 2013
Abstract Although repetitive transcranial magnetic stimulation (rTMS) is established in the treatment of depression, there is little knowledge about the underlying molecular mechanisms. In the last decade, the neurotrophic hypothesis of depression entailed a plethora of studies on the role of neurogenesis-associated factors in affective disorders and rTMS treatment. In the present study, we hypothesised a sham-controlled increase of peripheral brain-derived neurotrophic factor (BDNF) levels following serial rTMS stimulations in healthy individuals. We investigated the influence of a cycle of nine daily highfrequency (HF)-rTMS (25 Hz) stimulations over the left dorsolateral prefrontal cortex (DLPFC) on serum levels of BDNF in 44 young healthy male volunteers. BDNF serum concentrations were measured at baseline, on day 5 and on day 10. Overall, the statistical analyses showed that the active and sham group differed significantly regarding their responses of BDNF serum levels. Contrary to our expectations, there was a significant decrease of BDNF only during active treatment. Following the treatment period,
J. Kornhuber and B. Lenz contributed equally. G. Schaller (&) W. Sperling T. Richter-Schmidinger C. Mu¨hle J. Kornhuber B. Lenz Department of Psychiatry and Psychotherapy, FriedrichAlexander-University of Erlangen-Nuremberg, Schwabachanlage 6-10, 91054 Erlangen, Germany e-mail: [email protected] A. Heberlein Department of Psychiatry, Social Psychiatry and Psychotherapy, Hannover Medical School, Hannover, Germany C. Maiho¨fner Department of Neurology, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany
significantly lower BDNF serum levels were quantified in the active group on day 10, when compared to the sham group. The participants’ smoking status affected this effect. Our results suggest that serial HF-rTMS stimulations over the left DLPFC decrease serum BDNF levels in healthy male volunteers. This provides further evidence for an involvement of BDNF in clinical rTMS effects. Keywords RCT
rTMS BDNF Healthy volunteers
Introduction Although there is persuasive evidence for the antidepressant effect of repetitive transcranial magnetic stimulation (rTMS) (Hermann and Ebmeier 2006; Slotema et al. 2010), there is only limited knowledge about the underlying molecular mechanisms. Brain-derived neurotrophic factor (BDNF) is suggested to be involved in the pathogenesis of depression (Jacobs et al. 2000). Multiple studies have investigated the influence of rTMS on BDNF; however, the exact relationship still remains ambiguous. Hence, we here studied the impact of a cycle of high-frequency (HF)-rTMS over the left dorsolateral prefrontal cortex (DLPFC) on BDNF serum levels. To overcome the confounding factors, we recruited a very homogeneous sample of healthy young men and took alcohol drinking as well as smoking behaviour into account. The neurogenesis theory of depression (Jacobs et al. 2000) postulates that a stress-induced decrease in neurogenesis causes depression. Therefore, experimental and clinical studies were performed within the last decade to investigate the relationship between BDNF and depression. Correlations between BDNF, stress and depression have
123
308
been reported in several animal studies (Altar et al. 2003; Duman and Monteggia 2006; Shirayama et al. 2002). In humans, various investigations demonstrate decreased peripheral serum BDNF levels in depressed patients and positive effects of antidepressant treatment on serum BDNF levels. These observations indicate that neurotrophic factors are involved in affective disorders (Duman and Monteggia 2006; Gervasoni et al. 2005; Lee et al. 2007). Several clinical studies have already focused on the influence of rTMS on peripheral BDNF levels in patients with depression. Zanardini et al. (2006) reported a significant increase of serum BDNF following rTMS stimulations on five consecutive days in drug-resistant depressed patients. Yukimasa et al. (2006) found increased plasma levels of BDNF in patients with treatment-resistant major depression who responded to ten sessions of HF-rTMS. In contrast, Lang et al. (2006) reported no change of BDNF serum concentrations and no association of peripheral neurotrophin levels with clinical parameters following HFrTMS in a sample of treatment-resistant patients with major depression. In addition, Gedge et al. (2012) found no alteration of peripheral BDNF following treatment with HF-rTMS. In addition to these studies in depressed patients, Lang et al. (2007) investigated the acute effect of low-frequency (LF)-rTMS on BDNF in a sample of healthy volunteers. They found no change of serum BDNF concentrations. The authors discussed that the negative finding could possibly be due to a long-term expression effect of rTMS rather than an acute change of peripheral BDNF. In fact, Angelucci et al. (2004) reported a progressive reduction of BDNF plasma levels after 8 days of LF-rTMS over the motor cortex in healthy subjects and no effect on plasma BDNF in patients with amyotrophic lateral sclerosis (ALS). While there cannot be concluded a clear effect of rTMS on peripheral BDNF levels from these clinical investigations, in vitro studies are more unambiguous. rTMS increases BDNF mRNA in hippocampal areas, the granule cell layer, and in the parietal and the piriform cortex in rat brain (Mu¨ller et al. 2000) and in rat adrenal medulla PC-12 cells (Henkel et al. 2008). In addition, Ma et al. (2013) found an activating effect of LF-rTMS on BDNF in hippocampal neurons. Hence, in vitro studies clearly show that rTMS induces BDNF expression. We recently found a significant reduction of the Beck Depression Inventory (BDI) score in healthy young men during a 9-day series of HF-rTMS over the left DLPFC in a sham-controlled study design (Schaller et al. 2011). The present study investigated in the same sample whether serum levels of BDNF are influenced by serial rTMS stimulations. We administered HF-rTMS with typical antidepressive stimulation conditions to a very homogeneous sample of healthy young men and hypothesised that the
123
G. Schaller et al.
peripheral BDNF levels on day 5 and day 10 differed significantly between the active- and sham-treated study participants. The above-mentioned clinical studies show heterogeneous results regarding the influence of rTMS on peripheral BDNF. It was, thus, difficult to predict the direction of BDNF changes. However, with respect to the effects of a cycle of HF-rTMS stimulations on affective symptoms in our sample and the clear in vitro rTMS-effect, we hypothesised higher BDNF values in the active group as compared to the sham group.
Materials and methods Subjects, study design and rTMS treatment This study was part of a larger project, which aimed to investigate the effects of serial rTMS stimulations on affective symptoms (Schaller et al. 2011), on cognitive functions (Schaller et al. 2013) and on underlying molecular mechanisms in young healthy men. Forty-four male volunteers participated after written informed consent was obtained. The study protocol was approved by the local Ethics Committee. Exclusion criteria were severe organic or manifest psychiatric disorders, long-term medication, abnormal laboratory tests of clinical relevance and contraindications for rTMS (Wassermann 1998). A healthy medical condition was determined by a clinical interview, physical examination and laboratory data. We inquired the participants’ medical and psychiatric history, performed an internistic and neurological medical examination and analysed blood samples. None of the included volunteers had a physical or psychiatric illness in need of treatment except nicotine abuse or nicotine dependency and overweight. According to the Alcohol Use Disorder Identification Test (AUDIT) scores 18 of the 44 participants were at risk of hazardous alcohol consumption (AUDIT score C8, mean 6.2, SD 3.7, minimum 0, maximum 16). None of the participants fulfilled the diagnostic criteria for alcohol dependency. Besides, none of the participants reported another pre-existing psychiatric illness, nor complained about psychiatric symptoms, leading to a psychiatric diagnosis according to ICD-10 criteria. Especially, none of the participants reported symptoms leading to the diagnosis of depression or dysthymia. A BDI cut-off score was not implemented. In addition, analyses of blood samples were used to exclude acute and chronic infections (leucocytes, C-reactive protein), anaemia and polycythemia (erythrocytes, haemoglobin), thrombocytopenia and thrombocytosis (thrombocytes), electrolyte disorders (potassium, sodium, chloride), renal insufficiency (urea, creatinine), severe liver dysfunction (bilirubin, LDH, AST, ALT, GGT) and manifest hypo- and hyperthyroidism (TSH, T3, T4).
Serial rTMS decreases BDNF serum levels in healthy male volunteers
All participants were within the normal range of the following blood values: leucocytes (reference values: 4–10 9 103/ll), erythrocytes (4.2–5.9 9 106/ll), haemoglobin (13–17 g/dl), chloride (98–108 mmol/l), AST (\50 U/l), FT3 (3.5–8.1 pmol/l) and FT4 (10–25 pmol/l). We found slightly increased values for C-reactive protein (\5 mg/l) in two participants (maximum 7.3 mg/l), for thrombocytes (140–400 9 103/ll) in one participant (450 9 103/ll), for potassium (3.6–4.8 mmol/l) in one participant (5.1 mmol/l), for creatinine (\1.2 mg/dl) in two participants (maximum 1.35 mg/dl), for bilirubin (\1.1 mg/dl) in seven participants (maximum 2.8 mg/dl), for LDH (\250 U/l) in five participants (maximum 294 U/l), for ALT (\50 U/l) in four participants (maximum 122 U/l), for GGT (\60 U/l) in two participants (maximum 148 U/l) and for TSH (0.3–4.0 lU/ml) in three participants (maximum 4.43 lU/ml). One participant showed a slightly decreased value for sodium (134 mmol/l; 135–145 mmol/ l). Urea was, respectively, slightly decreased (12 mg/dl) and slightly increased (47 mg/dl) in one participant (17–43 mg/dl). RANCODE 3.6 professional (IDV, Gauting, Germany) was used for randomization to the active or sham group. The study was carried out in accordance with the Declaration of Helsinki and the ICH-GCP Guidelines. Stimulations were performed on nine consecutive days with the following stimulation parameters: 25 Hz, 100 % MT, 50 pulses/train, 15 trains/run, 8 s intertrain intervals, 750 pulses/session. For reasons of practicability, we restricted the cycle to a number of nine stimulations instead of the frequently implemented ten stimulations in studies with depressed patients. At the first session, the resting motor threshold of the right abductor pollicis brevis muscle was determined. We used the visual method to determine the motor threshold, since it has been shown to be equivalent to electroneuromyelography (Pridmore et al. 1998). Stimulations were administered over the left DLPFC, which was defined as the region 5-cm rostral to the point of optimal stimulation for the right abductor pollicis brevis muscle at a parasagittal plane in the left hemisphere (Rumi et al. 2005). Based on our hypothesis and related studies with depressed patients we decided to administer HF-rTMS over the left DLPFC, which is the best evaluated and most frequently employed therapeutic regimen in patients with depression. The investigator marked the point of stimulation and other points (bridge, ears) on a Lycra swim cap to facilitate location of the site of stimulation in subsequent sessions. For active stimulation (n = 22), the coil was placed flat against the scalp with its lower side centre directly over the previously determined point of stimulation (left DLPFC). With the participants’ consent, which was based on the lack of burdensome side effects, we increased the intensity of stimulation every session. We
309
assumed that higher stimulation intensities should cause greater effects. Using the described procedure, we were able to increase the applied dose to a mean of 124.3 % of MT [standard deviation (SD) 20] on day 5 and to a mean of 136.9 % of MT (SD 28) on day 9. The sham stimulation (n = 22) followed the same schedule, using a sham coil. Neuropsychological assessments and blood drawing were conducted prior to the first stimulation, immediately (within 5–30 min) after stimulation on day 5 and on day 10 (one day after the last stimulation). The participants and the persons who directly administered the stimulation and rated were blind to treatment allocation. The detailed stimulation procedures are described in Schaller et al. (2011). To assess affective symptoms we used the BDI (Beck and Steer 1987). In order to further characterise the study’s sample, we used the AUDIT (Babor et al. 1992), a valid screening instrument for pathological use of alcohol, as well as the Fagerstro¨m Test for Nicotine Dependence (FTND) (Bleich et al. 2002). Serum BDNF quantification Blood samples were centrifuged (4,000 g, 10 min, 4 °C) and serum samples were stored at -80 °C immediately after collection. BDNF serum levels of the two groups (active and sham) were investigated on day 1, day 5 and day 10. They were assessed using a commercial enzymelinked immunosorbent assay (Human BDNF ELISA, Catalog # LF-EK50005, AbFrontier, Young In Frontier Co., Ltd., Republic of Korea) which was performed according to the manufacturer’s instructions. The detection limit was defined as 2 pg/ml. The mean intra-assay and inter-assay coefficients of variation were 4.1 % and 5.4 %, respectively. The sample concentrations in each plate were calculated according to standard curves and dilution factors. Statistical analysis General linear models (repeated measures GLMs) and Student’s t tests were used to evaluate putative betweensubject differences (active vs. sham group) and withinsubject differences (before vs. after stimulations). Normal distribution was assumed with the Kolmogorov–Smirnov test. In the first step we performed a repeated measures GLM using BDNF serum levels (dependent variable), day 5 and 10 (within-subject factors), BDNF serum levels of day 1, the AUDIT scores (covariates), group (active vs. sham) and smoking status (between-subject factors). Alcohol drinking and smoking behaviour were included because of a recent study reporting the two factors as significant determinants of serum BDNF (Bus et al. 2011). Subsequently, we performed repeated measures GLMs independently for the active and sham groups with time (day 1, 5 and 10) as
123
310
within-subject factors and smoker status as between-subject factor. The AUDIT scores were not considered in these GLMs as we did not find a significant influence in the first GLM. Correlations of the BDI score reduction and BDNF serum level reduction (both from day 1 to day 10) as well as of the BDNF serum level reduction from day 1 to day 10 and the increase of stimulation intensities in the active group were evaluated with Pearson’s correlation tests. The results are presented as mean and standard deviation SD. A p value of \0.05 was considered significant. Data were analysed using IBM SPSS Statistics Version 20 for Windows (SPSS Inc., Chicago, IL).
Results Demographic characteristics The demographic characteristics of the 44 male participants were as follows. Age: mean 24.2 (±2.8) years; active mean 24.4 (±2.7) years, sham mean 24.1 (±2.9) years; body height: mean 183 (±7.5) cm; active mean 184 (±7.8) cm, sham mean 182 (±7.3) cm; body weight: mean 80.3 (±11.2) kg; active mean 81.0 (±11.7) kg, sham mean 79.7 (±10.9) kg; body mass index: mean 24.0 (±2.7) kg/ m2; active mean 24.0 (±3.0) kg/m2, sham mean 24.0 (±2.5) kg/m2; AUDIT: mean 6.2 (±3.7); active mean 6.8 (±3.2), sham mean 5.6 (±4.2). The group consisted of 13 smokers (n[active group] = 8, n[sham group] = 5) and 31 nonsmokers. The volunteers’ BDI scores (active and sham group) at baseline were as follows: mean: 4.18 (±3.44), minimum 0, maximum 14. One participant, in each group (active and sham), scored [10 points in the BDI at baseline, indicating mild depressive symptoms. BDNF serum levels General linear modelling [BDNF serum levels (dependent variable), day 5 and 10 (within-subject factors), the BDNF serum levels of day 1, the AUDIT scores (covariates), group (active vs. sham) and smoker status (between-subject factors)] revealed a significant difference of the BDNF serum levels between the active and sham group (main effect of group: F(1,38) = 5.339, p = 0.026, partial g2 = 0.123). There was a significant effect regarding BDNF serum levels of day 1 (F(1,38) = 6.588, p = 0.014, partial g2 = 0.148) and smoking status (F(1,38) = 6.622, p = 0.014, partial g2 = 0.148). Alcohol intake measured by the AUDIT showed no significant influence (F(1,38) = 2.754, p = 0.105, partial g2 = 0.068). Subsequently, we performed GLMs independently for the active and sham groups with BDNF serum levels as dependent variable, time (day 1, 5 and 10) as within-subject factor and smoker
123
G. Schaller et al.
Fig. 1 Change of BDNF serum level after five and nine sessions of rTMS (active vs. sham treatment). The y-axis shows BDNF serum levels (ng/ml). The graph shows means and 95 % confidence intervals. Starting with similar peripheral BDNF values at baseline, the two groups differed significantly following the treatment period on day 10 (Student’s t tests; day 1: active 3.47 [±2.63] ng/ml vs. sham 3.32 [±1.87] ng/ml, df = 37.886, T = 0.211, p = 0.834; day 10: active 1.99 [±1.50] ng/ml vs. sham 3.08 [±2.01] ng/ml, df = 42, T = -2.042, p = 0.047). General linear modelling revealed a significant difference of the BDNF serum levels between the active and sham group (F(1,38) = 5.339, p = 0.026, partial g2 = 0.123). Subsequent GLMs (independent for active and sham group) showed a significant reduction of BDNF serum levels during the treatment period exclusively in the active group (active: F(2,40) = 3.736, p = 0.033, partial g2 = 0.157; sham: F(2,40) = 0.883, p = 0.421, partial g2 = 0.042)
status as between-subject factor. These showed a significant reduction of BDNF serum levels during the treatment period exclusively in the active group (main effect of time: active: F(2,40) = 3.736, p = 0.033, partial g2 = 0.157; sham: F(2,40) = 0.883, p = 0.421, partial g2 = 0.042). Smoking behaviour showed a significant association only in the active group with a greater BDNF decrease from day 1 to day 10 in non-smoker than in current smoker (active: non-smoker -1.59 (±2.47) ng/ml vs. smoker -1.27 (±3.27) ng/ml, F(1,20) = 6.128, p = 0.022, partial g2 = 0.235; sham: F(1,20) = 0.991, p = 0.331, partial g2 = 0.047). Moreover, paired Student’s t tests revealed a significant reduction of BDNF serum levels following stimulations on day 10 only in the active group (main effect of time: active: df = 21, T = 2.549, p = 0.019; sham: df = 21, T = 0.495, p = 0.626). In addition, starting with comparable peripheral BDNF values at baseline, the two groups differed significantly following the treatment period on day 10 (main effect of group: Student’s t tests; day 1: active 3.47 [±2.63] ng/ml vs. sham 3.32 [±1.87] ng/ml, df = 37.886, T = 0.211, p = 0.834; day 10: active 1.99 [±1.50] ng/ml vs. sham 3.08 [±2.01] ng/ml, df = 42, T = -2.042, p = 0.047) (Fig. 1). The Pearson’s correlation between BDI reduction (Schaller et al. 2011) and BDNF serum level reduction (both from day 1 to day 10) was neither significant in the active group (r = 0.378, p = 0.083) nor in the sham group (r = -0.042, p = 0.854). In addition, the correlation of
Serial rTMS decreases BDNF serum levels in healthy male volunteers
the BDNF serum level reduction from day 1 to day 10 with the increase of stimulation intensities in the active group was not significant either (r = 0.163, p = 0.468).
Discussion To our knowledge, this study is the first to investigate the influence of a series of HF-rTMS (25 Hz) stimulations over the left DLPFC on BDNF serum levels in healthy volunteers. Contrary to our hypothesis of an rTMS-induced increase of peripheral BDNF levels, we found a significant sham-controlled reduction of peripheral BDNF levels during rTMS treatment. Whereas alcohol intake showed no significant relation to BDNF serum levels in our sample, smoking status was significantly related to the lowered BDNF serum levels in the active group in the sense that non-smokers showed a greater decrease than current smoker following the rTMS sessions. This is in line with the previously described positive correlation of smoking and BDNF levels (Bus et al. 2011). To date, investigations on the effects of rTMS stimulation on BDNF in depressed and healthy people report inconsistent results. Zanardini et al. (2006) and Yukimasa et al. (2006) revealed a significant increase of serum BDNF after rTMS treatment of several days’ duration in depressed patients [1 Hz or 17 Hz, 110 % MT, left DLPFC, five sessions, n = 16 (Zanardini et al. 2006); 20 Hz, 80 % MT, left prefrontal cortex, 10 sessions, n = 26 (Yukimasa et al. 2006)]. In contrast, Lang et al. (2006) found no changes of BDNF serum levels after serial HF-rTMS (20 Hz, 100 % MT, left DLPFC, 10 sessions, n = 14) in treatment-resistant depressive patients. Gedge et al. (2012) again found no significant alteration of the serum BDNF concentrations after serial HFrTMS stimulations (10 Hz, 80 % MT, left DLPFC, 10 sessions, n = 18) in patients with drug-resistant depression. In a study on the acute effect of rTMS on BDNF in healthy participants, Lang et al. (2007) reported no significant BDNF changes after one single LF-rTMS stimulation (1 Hz, 70–130 % MT, left and right motor cortex, one session, n = 42) over the motor cortex. As one possible reason for the negative finding, the authors discussed a long-term expression effect of rTMS rather than an acute change of BDNF. In fact, Angelucci et al. (2004) reported a progressive reduction of BDNF plasma levels after LF-rTMS (1 Hz, 110 % MT, left and right motor cortex, eight sessions, n = 10) over the motor cortex for 8 days in healthy subjects, whereas no effect on BDNF plasma levels was found in patients with ALS. The heterogeneous study designs with different samples (healthy individuals vs. depressive patients vs. patients with ALS), different stimulation parameters (HF-rTMS vs. LF-rTMS) and different sites of stimulation (motor cortex vs. left DLPFC) may account for these inconsistencies in
311
previous studies results which make the results’ comparison and their discussion difficult. However, the reported effect of rTMS on peripheral BDNF levels in our study is in line with findings of Angelucci et al. (2004). Although the two studies differed in sites of stimulation (motor cortex vs. left DLPFC) and applied frequencies (LF-rTMS vs. HF-rTMS), they consistently found a progressive reduction of BDNF plasma levels after serial rTMS stimulation in healthy subjects. In conclusion, this suggests that BDNF changes are neither specific for a particular site of stimulation, nor for the frequency of stimulation, and may be only caused by serial rTMS stimulation. In contrast to these findings in healthy participants, investigations on the rTMS-BDNF relationship in depressed patients indicated an increase of BDNF levels after serial rTMS stimulations (Zanardini et al. 2006; Yukimasa et al. 2006). This suggests that rTMS effects on peripheral BDNF levels depend on whether study participants are depressed or not. rTMS may entail both, a decrease of peripheral BDNF in healthy participants with physiological BDNF serum levels and an increase in depressed patients who show a` priori-reduced BDNF levels (Duman and Monteggia 2006; Gervasoni et al. 2005; Lee et al. 2007). This might indicate a role of BDNF in a negative feedback loop. It might also suggest that BDNF is not as hypothesised the primary causation factor of the antidepressant property of rTMS, but one important factor in the complex interaction between various factors. It is known that transcranial magnetic stimulation in addition to effects on BDNF induces other neurobiological effects [i.e. rTMS of the left DLPFC modulates dopamine release in the ipsilateral anterior cingulate cortex in healthy volunteers (Cho and Strafella 2009)]. We hypothesise that rTMS stimulation primarily influences neurotransmission [i.e. dopamine (Cho and Strafella 2009)]. These neural effects of rTMS are caused under the stimulation coil and might spread across neural networks. This might indicate a mediating role of BDNF. rTMS does not affect or even downregulate BDNF signalling in balanced neural networks with no need for changes such as in our study. As reported in studies on the influence of rTMS in depressed patients, BDNF, as a surrogate marker of neuroplasticity, increases in dysbalanced neural networks with demand for modification of pathological neurotransmitter concentrations. The strengths of our investigation include the shamcontrolled design, the highly standardised experimental procedures, the homogeneous cohort and the number of recruited participants which is higher or at least comparable to other studies in this field. Furthermore, we integrated alcohol intake and smoking status as determinants of serum BDNF into the analyses. Especially smoking seems to be a potential confounder that has been left out of consideration so far. This study has several limitations. The method to
123
312
G. Schaller et al.
target the left DLPFC used here (defined as the region 5-cm rostral to the point of optimal stimulation for the right abductor pollicis brevis muscle at a parasagittal plane in the left hemisphere) is imprecise. Recent studies on coil placement based on anatomical or functional MRI suggest the need for a TMS-coil positioning method, which incorporates individual anatomical information (Peleman et al. 2010; Sparing et al. 2008). We decided to use the above-mentioned standard procedure to detect the left DLPFC, since this is the method used in the majority of clinical trials evaluating the effect of rTMS on depressed people (Rumi et al. 2005) and because of reasons of practicability in daily clinical routine. Another limitation is that peripheral BDNF levels were quantified in this study. Since BDNF also originates from peripheral sources (Meyer et al. 1992), we cannot assure that the reported findings represent exclusively cerebral processes. Nevertheless, there are numerous studies which found a positive correlation between blood and brain BDNF levels (Karege et al. 2002; Klein et al. 2011) and, therefore, strongly support research on peripheral BDNF levels. Furthermore, aiming at a homogeneous sample we restricted our investigations to male participants. Thus, the findings cannot be transferred to women. In addition, the effects were small. Some statistical values showed marginal significance and might, therefore, represent a non-clinically significant observation, also given the numerous analyses performed and the post hoc nature of the study. In summary, our results show that serial HF-rTMS stimulations over the left DLPFC decrease serum BDNF levels in healthy male volunteers. This is in line with the one to date existing study on the effect of a LF-rTMS series on BDNF in healthy controls (Angelucci et al. 2004). Although our findings were contrary to the primary hypothesis, the study provides further evidence that BDNF is involved in the effects of rTMS. The underlying mechanisms still remain to be discovered. It is now interesting to investigate other neurotrophins, neurotransmitters and further putative causation factors for the clinical effect of transcranial magnetic stimulation. Acknowledgments We thank all volunteers for their participation. Funding for this study was provided by the Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University of ErlangenNuremberg, Erlangen, Germany. Conflict of interest conflict of interest.
None of the authors had a financial or personal
References Altar CA, Whitehead RE, Chen R, Wortwein G, Madsen TM (2003) Effects of electroconvulsive seizures and antidepressant drugs on
123
brain-derived neurotrophic factor protein in rat brain. Biol Psychiatry 54:703–709 Angelucci F, Oliviero A, Pilato F, Saturno E, Dileone M, Versace V, Musumeci G, Batocchi AP, Tonali PA, Di Lazzaro V (2004) Transcranial magnetic stimulation and BDNF plasma levels in amyotrophic lateral sclerosis. Neuroreport 15:717–720 Babor TF, de la Fuente JR, Saunders J, Grant M (1992) AUDIT. The alcohol use disorders identification test. Guidelines for use in primary health care, World Health Organization, Geneva, Switzerland Beck A, Steer R (1987) Beck Depression Inventory—Manual. The Psychological Corporation, San Antonio, TX, USA Bleich S, Havemann-Reinecke U, Kornhuber J (2002) Fagerstro¨mTest fu¨r Nikotinabha¨ngigkeit (FTNA) (Fagerstro¨m Test for Nicotine Dependence). Hogrefe-Verlag, Go¨ttingen, Germany Bus BA, Molendijk ML, Penninx BJ, Buitelaar JK, Kenis G, Prickaerts J, Elzinga BM, Voshaar RC (2011) Determinants of serum brain-derived neurotrophic factor. Psychoneuroendocrinology 36:228–239 Cho SS, Strafella AP (2009) rTMS of the left dorsolateral prefrontal cortex modulates dopamine release in the ipsilateral anterior cingulate cortex and orbitofrontal cortex. PLoS ONE 4:e6725 Duman RS, Monteggia LM (2006) A neurotrophic model for stressrelated mood disorders. Biol Psychiatry 59:1116–1127 Gedge L, Beaudoin A, Lazowski L, du Toit R, Jokic R, Milev R (2012) Effects of electroconvulsive therapy and repetitive transcranial magnetic stimulation on serum brain-derived neurotrophic factor levels in patients with depression. Front Psychiatry 3:12 Gervasoni N, Aubry JM, Bondolfi G, Osiek C, Schwald M, Bertschy G, Karege F (2005) Partial normalization of serum brain-derived neurotrophic factor in remitted patients after a major depressive episode. Neuropsychobiology 51:234–238 Henkel AW, Sperling W, Rotter A, Reulbach U, Reichardt C, Bo¨nsch D, Maler JM, Kornhuber J, Wiltfang J (2008) Antidepressant drugs modulate growth factors in cultured cells. BMC Pharmacol 4(8):6 Hermann LL, Ebmeier KP (2006) Factors modifying the efficacy of transcranial magnetic stimulation in the treatment of depression: a review. J Clin Psychiatry 67:1870–1876 Jacobs BL, van Praag H, Gage FH (2000) Adult brain neurogenesis and psychiatry: a novel theory of depression. Mol Psychiatry 5:262–269 Karege F, Schwald M, Cisse M (2002) Postnatal developmental profile of brain-derived neurotrophic factor in rat brain and platelets. Neurosci Lett 328:261–264 Klein AB, Williamson R, Santini MA, Clemmensen C, Ettrup A, Rios M, Knudsen GM, Aznar S (2011) Blood BDNF concentrations reflect brain-tissue BDNF levels across species. Int J Neuropsychopharmacol 14:347–353 Lang UE, Bajbouj M, Gallinat J, Hellweg R (2006) Brain-derived neurotrophic factor serum concentrations in depressive patients during vagus nerve stimulation and repetitive transcranial magnetic stimulation. Psychopharmacology 187:56–59 Lang UE, Hellweg R, Gallinat J, Bajbouj M (2007) Acute prefrontal cortex transcranial magnetic stimulation in healthy volunteers: no effects on brain-derived neurotrophic factor (BDNF) concentrations in serum. J Affect Disord 107:255–258 Lee BH, Kim H, Park SH, Kim YK (2007) Decreased plasma BDNF level in depressive patients. J Affect Disord 101:239–244 Ma J, Zhang Z, Su Y, Kang L, Geng D, Wang Y, Luan F, Wang M, Cui H (2013) Magnetic stimulation modulates structural synaptic plasticity and regulates BDNF-TrkB signal pathway in cultured hippocampal neurons. Neurochem Int 62:84–91 Meyer M, Matsuoka I, Wetmore C, Olson L, Thoenen H (1992) Enhanced synthesis of brain-derived neurotrophic factor in the
Serial rTMS decreases BDNF serum levels in healthy male volunteers lesioned peripheral nerve: different mechanisms are responsible for the regulation of BDNF and NGF mRNA. J Cell Biol 119:45–54 Mu¨ller M, Tosci N, Kresse AE, Poat A, Keck ME (2000) Long-term repetitive transcranial magnetic stimulation increases the expression of brain-derived neurotrophic factor and cholecystokinin mRNA, but not neuropeptide tyrosine mRNA in specific areas of rat brain. Neuropsychopharmacology 23:205–215 Peleman K, Van Schuerbeek P, Luypaert R, Stadnik T, De Raedt R, De Mey J, Bossuyt A, Baeken C (2010) Using 3D-MRI to localize the dorsolateral prefrontal cortex in TMS research. World J Biol Psychiatry 11:425–430 Pridmore S, Fernandes Filho JA, Nahas Z, Liberatos C, George MS (1998) Motor threshold in transcranial magnetic stimulation: a comparison of a neurophysiological method and a visualization of movement method. J ECT 14:25–27 Rumi DO, Gattaz WF, Rigonatti SP, Rosa MA, Fregni F, Rosa MO, Mansur C, Myczkowski ML, Moreno RA, Marcolin MA (2005) Transcranial magnetic stimulation accelerates the antidepressant effect of amitriptyline in severe depression: a double-blind placebo-controlled study. Biol Psychiatry 57:162–166 Schaller G, Lenz B, Friedrich K, Dygon D, Richter-Schmidinger T, Jacobi A, Mueller SE, Maiho¨fner C, Sperling W, Kornhuber J (2011) Repetitive transcranial magnetic stimulation influences mood in healthy male volunteers. J Psychiatr Res 45:1178–1183 Schaller G, Lenz B, Friedrich K, Dygon D, Richter-Schmidinger T, Sperling W, Kornhuber J (2013) No evidence for effects of a high-frequency repetitive transcranial magnetic stimulation (rTMS) series on verbal and figural fluency and TAP tasks
313 performance in healthy male volunteers. Neuropsychobiology 67:69–73 Shirayama Y, Chen AC, Nakagawa S, Russell DS, Duman RS (2002) Brain-derived neurotrophic factor produces antidepressant effects in behavioral models of depression. J Neurosci 22:3251–3261 Slotema CW, Blom JD, Hoek HW, Sommer IE (2010) Should we expand the toolbox of psychiatric treatment methods to include Repetitive Transcranial Magnetic Stimulation (rTMS)? A metaanalysis of the efficacy of rTMS in psychiatric disorders. J Clin Psychiatry 71:873–884 Sparing R, Buelte D, Meister IG, Paus T, Fink GR (2008) Transcranial magnetic stimulation and the challenge of coil placement: a comparison of conventional and stereotaxic neuronavigational strategies. Hum Brain Mapp 29:82–96 Wassermann EM (1998) Risk and safety of repetitive transcranial magnetic stimulation: report and suggested guidelines from the International Workshop on the Safety of Repetitive Transcranial Magnetic Stimulation, June 5-7, 1996. Electroencephalogr Clin Neurophysiol 108:1–16 Yukimasa T, Yoshimura R, Tamagawa A, Uozumi T, Shinkai K, Ueda N, Tsuji S, Nakamura J (2006) High-Frequency Repetitive Transcranial Magnetic Stimulation improves refractory depression by influencing catecholamine and Brain-Derived Neurotrophic Factors. Pharmacopsychiatry 39:52–59 Zanardini R, Gazzoli A, Ventriglia M, Perez J, Bignotti S, Rossini PM, Gennarelli M, Bocchio-Chiavetto L (2006) Effect of repetitive transcranial magnetic stimulation on serum brain derived neurotrophic factor in drug resistant depressed patients. J Affect Disord 91:83–86
123
PSYCHIATRY AND PRECLINICAL PSYCHIATRIC STUDIES - ORIGINAL ARTICLE
Serial repetitive transcranial magnetic stimulation (rTMS) decreases BDNF serum levels in healthy male volunteers Gerd Schaller • Wolfgang Sperling • Tanja Richter-Schmidinger • Christiane Mu¨hle • Annemarie Heberlein • Christian Maiho¨fner • Johannes Kornhuber • Bernd Lenz
Received: 9 July 2013 / Accepted: 9 October 2013 / Published online: 25 October 2013 Ó Springer-Verlag Wien 2013
Abstract Although repetitive transcranial magnetic stimulation (rTMS) is established in the treatment of depression, there is little knowledge about the underlying molecular mechanisms. In the last decade, the neurotrophic hypothesis of depression entailed a plethora of studies on the role of neurogenesis-associated factors in affective disorders and rTMS treatment. In the present study, we hypothesised a sham-controlled increase of peripheral brain-derived neurotrophic factor (BDNF) levels following serial rTMS stimulations in healthy individuals. We investigated the influence of a cycle of nine daily highfrequency (HF)-rTMS (25 Hz) stimulations over the left dorsolateral prefrontal cortex (DLPFC) on serum levels of BDNF in 44 young healthy male volunteers. BDNF serum concentrations were measured at baseline, on day 5 and on day 10. Overall, the statistical analyses showed that the active and sham group differed significantly regarding their responses of BDNF serum levels. Contrary to our expectations, there was a significant decrease of BDNF only during active treatment. Following the treatment period,
J. Kornhuber and B. Lenz contributed equally. G. Schaller (&) W. Sperling T. Richter-Schmidinger C. Mu¨hle J. Kornhuber B. Lenz Department of Psychiatry and Psychotherapy, FriedrichAlexander-University of Erlangen-Nuremberg, Schwabachanlage 6-10, 91054 Erlangen, Germany e-mail: [email protected] A. Heberlein Department of Psychiatry, Social Psychiatry and Psychotherapy, Hannover Medical School, Hannover, Germany C. Maiho¨fner Department of Neurology, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany
significantly lower BDNF serum levels were quantified in the active group on day 10, when compared to the sham group. The participants’ smoking status affected this effect. Our results suggest that serial HF-rTMS stimulations over the left DLPFC decrease serum BDNF levels in healthy male volunteers. This provides further evidence for an involvement of BDNF in clinical rTMS effects. Keywords RCT
rTMS BDNF Healthy volunteers
Introduction Although there is persuasive evidence for the antidepressant effect of repetitive transcranial magnetic stimulation (rTMS) (Hermann and Ebmeier 2006; Slotema et al. 2010), there is only limited knowledge about the underlying molecular mechanisms. Brain-derived neurotrophic factor (BDNF) is suggested to be involved in the pathogenesis of depression (Jacobs et al. 2000). Multiple studies have investigated the influence of rTMS on BDNF; however, the exact relationship still remains ambiguous. Hence, we here studied the impact of a cycle of high-frequency (HF)-rTMS over the left dorsolateral prefrontal cortex (DLPFC) on BDNF serum levels. To overcome the confounding factors, we recruited a very homogeneous sample of healthy young men and took alcohol drinking as well as smoking behaviour into account. The neurogenesis theory of depression (Jacobs et al. 2000) postulates that a stress-induced decrease in neurogenesis causes depression. Therefore, experimental and clinical studies were performed within the last decade to investigate the relationship between BDNF and depression. Correlations between BDNF, stress and depression have
123
308
been reported in several animal studies (Altar et al. 2003; Duman and Monteggia 2006; Shirayama et al. 2002). In humans, various investigations demonstrate decreased peripheral serum BDNF levels in depressed patients and positive effects of antidepressant treatment on serum BDNF levels. These observations indicate that neurotrophic factors are involved in affective disorders (Duman and Monteggia 2006; Gervasoni et al. 2005; Lee et al. 2007). Several clinical studies have already focused on the influence of rTMS on peripheral BDNF levels in patients with depression. Zanardini et al. (2006) reported a significant increase of serum BDNF following rTMS stimulations on five consecutive days in drug-resistant depressed patients. Yukimasa et al. (2006) found increased plasma levels of BDNF in patients with treatment-resistant major depression who responded to ten sessions of HF-rTMS. In contrast, Lang et al. (2006) reported no change of BDNF serum concentrations and no association of peripheral neurotrophin levels with clinical parameters following HFrTMS in a sample of treatment-resistant patients with major depression. In addition, Gedge et al. (2012) found no alteration of peripheral BDNF following treatment with HF-rTMS. In addition to these studies in depressed patients, Lang et al. (2007) investigated the acute effect of low-frequency (LF)-rTMS on BDNF in a sample of healthy volunteers. They found no change of serum BDNF concentrations. The authors discussed that the negative finding could possibly be due to a long-term expression effect of rTMS rather than an acute change of peripheral BDNF. In fact, Angelucci et al. (2004) reported a progressive reduction of BDNF plasma levels after 8 days of LF-rTMS over the motor cortex in healthy subjects and no effect on plasma BDNF in patients with amyotrophic lateral sclerosis (ALS). While there cannot be concluded a clear effect of rTMS on peripheral BDNF levels from these clinical investigations, in vitro studies are more unambiguous. rTMS increases BDNF mRNA in hippocampal areas, the granule cell layer, and in the parietal and the piriform cortex in rat brain (Mu¨ller et al. 2000) and in rat adrenal medulla PC-12 cells (Henkel et al. 2008). In addition, Ma et al. (2013) found an activating effect of LF-rTMS on BDNF in hippocampal neurons. Hence, in vitro studies clearly show that rTMS induces BDNF expression. We recently found a significant reduction of the Beck Depression Inventory (BDI) score in healthy young men during a 9-day series of HF-rTMS over the left DLPFC in a sham-controlled study design (Schaller et al. 2011). The present study investigated in the same sample whether serum levels of BDNF are influenced by serial rTMS stimulations. We administered HF-rTMS with typical antidepressive stimulation conditions to a very homogeneous sample of healthy young men and hypothesised that the
123
G. Schaller et al.
peripheral BDNF levels on day 5 and day 10 differed significantly between the active- and sham-treated study participants. The above-mentioned clinical studies show heterogeneous results regarding the influence of rTMS on peripheral BDNF. It was, thus, difficult to predict the direction of BDNF changes. However, with respect to the effects of a cycle of HF-rTMS stimulations on affective symptoms in our sample and the clear in vitro rTMS-effect, we hypothesised higher BDNF values in the active group as compared to the sham group.
Materials and methods Subjects, study design and rTMS treatment This study was part of a larger project, which aimed to investigate the effects of serial rTMS stimulations on affective symptoms (Schaller et al. 2011), on cognitive functions (Schaller et al. 2013) and on underlying molecular mechanisms in young healthy men. Forty-four male volunteers participated after written informed consent was obtained. The study protocol was approved by the local Ethics Committee. Exclusion criteria were severe organic or manifest psychiatric disorders, long-term medication, abnormal laboratory tests of clinical relevance and contraindications for rTMS (Wassermann 1998). A healthy medical condition was determined by a clinical interview, physical examination and laboratory data. We inquired the participants’ medical and psychiatric history, performed an internistic and neurological medical examination and analysed blood samples. None of the included volunteers had a physical or psychiatric illness in need of treatment except nicotine abuse or nicotine dependency and overweight. According to the Alcohol Use Disorder Identification Test (AUDIT) scores 18 of the 44 participants were at risk of hazardous alcohol consumption (AUDIT score C8, mean 6.2, SD 3.7, minimum 0, maximum 16). None of the participants fulfilled the diagnostic criteria for alcohol dependency. Besides, none of the participants reported another pre-existing psychiatric illness, nor complained about psychiatric symptoms, leading to a psychiatric diagnosis according to ICD-10 criteria. Especially, none of the participants reported symptoms leading to the diagnosis of depression or dysthymia. A BDI cut-off score was not implemented. In addition, analyses of blood samples were used to exclude acute and chronic infections (leucocytes, C-reactive protein), anaemia and polycythemia (erythrocytes, haemoglobin), thrombocytopenia and thrombocytosis (thrombocytes), electrolyte disorders (potassium, sodium, chloride), renal insufficiency (urea, creatinine), severe liver dysfunction (bilirubin, LDH, AST, ALT, GGT) and manifest hypo- and hyperthyroidism (TSH, T3, T4).
Serial rTMS decreases BDNF serum levels in healthy male volunteers
All participants were within the normal range of the following blood values: leucocytes (reference values: 4–10 9 103/ll), erythrocytes (4.2–5.9 9 106/ll), haemoglobin (13–17 g/dl), chloride (98–108 mmol/l), AST (\50 U/l), FT3 (3.5–8.1 pmol/l) and FT4 (10–25 pmol/l). We found slightly increased values for C-reactive protein (\5 mg/l) in two participants (maximum 7.3 mg/l), for thrombocytes (140–400 9 103/ll) in one participant (450 9 103/ll), for potassium (3.6–4.8 mmol/l) in one participant (5.1 mmol/l), for creatinine (\1.2 mg/dl) in two participants (maximum 1.35 mg/dl), for bilirubin (\1.1 mg/dl) in seven participants (maximum 2.8 mg/dl), for LDH (\250 U/l) in five participants (maximum 294 U/l), for ALT (\50 U/l) in four participants (maximum 122 U/l), for GGT (\60 U/l) in two participants (maximum 148 U/l) and for TSH (0.3–4.0 lU/ml) in three participants (maximum 4.43 lU/ml). One participant showed a slightly decreased value for sodium (134 mmol/l; 135–145 mmol/ l). Urea was, respectively, slightly decreased (12 mg/dl) and slightly increased (47 mg/dl) in one participant (17–43 mg/dl). RANCODE 3.6 professional (IDV, Gauting, Germany) was used for randomization to the active or sham group. The study was carried out in accordance with the Declaration of Helsinki and the ICH-GCP Guidelines. Stimulations were performed on nine consecutive days with the following stimulation parameters: 25 Hz, 100 % MT, 50 pulses/train, 15 trains/run, 8 s intertrain intervals, 750 pulses/session. For reasons of practicability, we restricted the cycle to a number of nine stimulations instead of the frequently implemented ten stimulations in studies with depressed patients. At the first session, the resting motor threshold of the right abductor pollicis brevis muscle was determined. We used the visual method to determine the motor threshold, since it has been shown to be equivalent to electroneuromyelography (Pridmore et al. 1998). Stimulations were administered over the left DLPFC, which was defined as the region 5-cm rostral to the point of optimal stimulation for the right abductor pollicis brevis muscle at a parasagittal plane in the left hemisphere (Rumi et al. 2005). Based on our hypothesis and related studies with depressed patients we decided to administer HF-rTMS over the left DLPFC, which is the best evaluated and most frequently employed therapeutic regimen in patients with depression. The investigator marked the point of stimulation and other points (bridge, ears) on a Lycra swim cap to facilitate location of the site of stimulation in subsequent sessions. For active stimulation (n = 22), the coil was placed flat against the scalp with its lower side centre directly over the previously determined point of stimulation (left DLPFC). With the participants’ consent, which was based on the lack of burdensome side effects, we increased the intensity of stimulation every session. We
309
assumed that higher stimulation intensities should cause greater effects. Using the described procedure, we were able to increase the applied dose to a mean of 124.3 % of MT [standard deviation (SD) 20] on day 5 and to a mean of 136.9 % of MT (SD 28) on day 9. The sham stimulation (n = 22) followed the same schedule, using a sham coil. Neuropsychological assessments and blood drawing were conducted prior to the first stimulation, immediately (within 5–30 min) after stimulation on day 5 and on day 10 (one day after the last stimulation). The participants and the persons who directly administered the stimulation and rated were blind to treatment allocation. The detailed stimulation procedures are described in Schaller et al. (2011). To assess affective symptoms we used the BDI (Beck and Steer 1987). In order to further characterise the study’s sample, we used the AUDIT (Babor et al. 1992), a valid screening instrument for pathological use of alcohol, as well as the Fagerstro¨m Test for Nicotine Dependence (FTND) (Bleich et al. 2002). Serum BDNF quantification Blood samples were centrifuged (4,000 g, 10 min, 4 °C) and serum samples were stored at -80 °C immediately after collection. BDNF serum levels of the two groups (active and sham) were investigated on day 1, day 5 and day 10. They were assessed using a commercial enzymelinked immunosorbent assay (Human BDNF ELISA, Catalog # LF-EK50005, AbFrontier, Young In Frontier Co., Ltd., Republic of Korea) which was performed according to the manufacturer’s instructions. The detection limit was defined as 2 pg/ml. The mean intra-assay and inter-assay coefficients of variation were 4.1 % and 5.4 %, respectively. The sample concentrations in each plate were calculated according to standard curves and dilution factors. Statistical analysis General linear models (repeated measures GLMs) and Student’s t tests were used to evaluate putative betweensubject differences (active vs. sham group) and withinsubject differences (before vs. after stimulations). Normal distribution was assumed with the Kolmogorov–Smirnov test. In the first step we performed a repeated measures GLM using BDNF serum levels (dependent variable), day 5 and 10 (within-subject factors), BDNF serum levels of day 1, the AUDIT scores (covariates), group (active vs. sham) and smoking status (between-subject factors). Alcohol drinking and smoking behaviour were included because of a recent study reporting the two factors as significant determinants of serum BDNF (Bus et al. 2011). Subsequently, we performed repeated measures GLMs independently for the active and sham groups with time (day 1, 5 and 10) as
123
310
within-subject factors and smoker status as between-subject factor. The AUDIT scores were not considered in these GLMs as we did not find a significant influence in the first GLM. Correlations of the BDI score reduction and BDNF serum level reduction (both from day 1 to day 10) as well as of the BDNF serum level reduction from day 1 to day 10 and the increase of stimulation intensities in the active group were evaluated with Pearson’s correlation tests. The results are presented as mean and standard deviation SD. A p value of \0.05 was considered significant. Data were analysed using IBM SPSS Statistics Version 20 for Windows (SPSS Inc., Chicago, IL).
Results Demographic characteristics The demographic characteristics of the 44 male participants were as follows. Age: mean 24.2 (±2.8) years; active mean 24.4 (±2.7) years, sham mean 24.1 (±2.9) years; body height: mean 183 (±7.5) cm; active mean 184 (±7.8) cm, sham mean 182 (±7.3) cm; body weight: mean 80.3 (±11.2) kg; active mean 81.0 (±11.7) kg, sham mean 79.7 (±10.9) kg; body mass index: mean 24.0 (±2.7) kg/ m2; active mean 24.0 (±3.0) kg/m2, sham mean 24.0 (±2.5) kg/m2; AUDIT: mean 6.2 (±3.7); active mean 6.8 (±3.2), sham mean 5.6 (±4.2). The group consisted of 13 smokers (n[active group] = 8, n[sham group] = 5) and 31 nonsmokers. The volunteers’ BDI scores (active and sham group) at baseline were as follows: mean: 4.18 (±3.44), minimum 0, maximum 14. One participant, in each group (active and sham), scored [10 points in the BDI at baseline, indicating mild depressive symptoms. BDNF serum levels General linear modelling [BDNF serum levels (dependent variable), day 5 and 10 (within-subject factors), the BDNF serum levels of day 1, the AUDIT scores (covariates), group (active vs. sham) and smoker status (between-subject factors)] revealed a significant difference of the BDNF serum levels between the active and sham group (main effect of group: F(1,38) = 5.339, p = 0.026, partial g2 = 0.123). There was a significant effect regarding BDNF serum levels of day 1 (F(1,38) = 6.588, p = 0.014, partial g2 = 0.148) and smoking status (F(1,38) = 6.622, p = 0.014, partial g2 = 0.148). Alcohol intake measured by the AUDIT showed no significant influence (F(1,38) = 2.754, p = 0.105, partial g2 = 0.068). Subsequently, we performed GLMs independently for the active and sham groups with BDNF serum levels as dependent variable, time (day 1, 5 and 10) as within-subject factor and smoker
123
G. Schaller et al.
Fig. 1 Change of BDNF serum level after five and nine sessions of rTMS (active vs. sham treatment). The y-axis shows BDNF serum levels (ng/ml). The graph shows means and 95 % confidence intervals. Starting with similar peripheral BDNF values at baseline, the two groups differed significantly following the treatment period on day 10 (Student’s t tests; day 1: active 3.47 [±2.63] ng/ml vs. sham 3.32 [±1.87] ng/ml, df = 37.886, T = 0.211, p = 0.834; day 10: active 1.99 [±1.50] ng/ml vs. sham 3.08 [±2.01] ng/ml, df = 42, T = -2.042, p = 0.047). General linear modelling revealed a significant difference of the BDNF serum levels between the active and sham group (F(1,38) = 5.339, p = 0.026, partial g2 = 0.123). Subsequent GLMs (independent for active and sham group) showed a significant reduction of BDNF serum levels during the treatment period exclusively in the active group (active: F(2,40) = 3.736, p = 0.033, partial g2 = 0.157; sham: F(2,40) = 0.883, p = 0.421, partial g2 = 0.042)
status as between-subject factor. These showed a significant reduction of BDNF serum levels during the treatment period exclusively in the active group (main effect of time: active: F(2,40) = 3.736, p = 0.033, partial g2 = 0.157; sham: F(2,40) = 0.883, p = 0.421, partial g2 = 0.042). Smoking behaviour showed a significant association only in the active group with a greater BDNF decrease from day 1 to day 10 in non-smoker than in current smoker (active: non-smoker -1.59 (±2.47) ng/ml vs. smoker -1.27 (±3.27) ng/ml, F(1,20) = 6.128, p = 0.022, partial g2 = 0.235; sham: F(1,20) = 0.991, p = 0.331, partial g2 = 0.047). Moreover, paired Student’s t tests revealed a significant reduction of BDNF serum levels following stimulations on day 10 only in the active group (main effect of time: active: df = 21, T = 2.549, p = 0.019; sham: df = 21, T = 0.495, p = 0.626). In addition, starting with comparable peripheral BDNF values at baseline, the two groups differed significantly following the treatment period on day 10 (main effect of group: Student’s t tests; day 1: active 3.47 [±2.63] ng/ml vs. sham 3.32 [±1.87] ng/ml, df = 37.886, T = 0.211, p = 0.834; day 10: active 1.99 [±1.50] ng/ml vs. sham 3.08 [±2.01] ng/ml, df = 42, T = -2.042, p = 0.047) (Fig. 1). The Pearson’s correlation between BDI reduction (Schaller et al. 2011) and BDNF serum level reduction (both from day 1 to day 10) was neither significant in the active group (r = 0.378, p = 0.083) nor in the sham group (r = -0.042, p = 0.854). In addition, the correlation of
Serial rTMS decreases BDNF serum levels in healthy male volunteers
the BDNF serum level reduction from day 1 to day 10 with the increase of stimulation intensities in the active group was not significant either (r = 0.163, p = 0.468).
Discussion To our knowledge, this study is the first to investigate the influence of a series of HF-rTMS (25 Hz) stimulations over the left DLPFC on BDNF serum levels in healthy volunteers. Contrary to our hypothesis of an rTMS-induced increase of peripheral BDNF levels, we found a significant sham-controlled reduction of peripheral BDNF levels during rTMS treatment. Whereas alcohol intake showed no significant relation to BDNF serum levels in our sample, smoking status was significantly related to the lowered BDNF serum levels in the active group in the sense that non-smokers showed a greater decrease than current smoker following the rTMS sessions. This is in line with the previously described positive correlation of smoking and BDNF levels (Bus et al. 2011). To date, investigations on the effects of rTMS stimulation on BDNF in depressed and healthy people report inconsistent results. Zanardini et al. (2006) and Yukimasa et al. (2006) revealed a significant increase of serum BDNF after rTMS treatment of several days’ duration in depressed patients [1 Hz or 17 Hz, 110 % MT, left DLPFC, five sessions, n = 16 (Zanardini et al. 2006); 20 Hz, 80 % MT, left prefrontal cortex, 10 sessions, n = 26 (Yukimasa et al. 2006)]. In contrast, Lang et al. (2006) found no changes of BDNF serum levels after serial HF-rTMS (20 Hz, 100 % MT, left DLPFC, 10 sessions, n = 14) in treatment-resistant depressive patients. Gedge et al. (2012) again found no significant alteration of the serum BDNF concentrations after serial HFrTMS stimulations (10 Hz, 80 % MT, left DLPFC, 10 sessions, n = 18) in patients with drug-resistant depression. In a study on the acute effect of rTMS on BDNF in healthy participants, Lang et al. (2007) reported no significant BDNF changes after one single LF-rTMS stimulation (1 Hz, 70–130 % MT, left and right motor cortex, one session, n = 42) over the motor cortex. As one possible reason for the negative finding, the authors discussed a long-term expression effect of rTMS rather than an acute change of BDNF. In fact, Angelucci et al. (2004) reported a progressive reduction of BDNF plasma levels after LF-rTMS (1 Hz, 110 % MT, left and right motor cortex, eight sessions, n = 10) over the motor cortex for 8 days in healthy subjects, whereas no effect on BDNF plasma levels was found in patients with ALS. The heterogeneous study designs with different samples (healthy individuals vs. depressive patients vs. patients with ALS), different stimulation parameters (HF-rTMS vs. LF-rTMS) and different sites of stimulation (motor cortex vs. left DLPFC) may account for these inconsistencies in
311
previous studies results which make the results’ comparison and their discussion difficult. However, the reported effect of rTMS on peripheral BDNF levels in our study is in line with findings of Angelucci et al. (2004). Although the two studies differed in sites of stimulation (motor cortex vs. left DLPFC) and applied frequencies (LF-rTMS vs. HF-rTMS), they consistently found a progressive reduction of BDNF plasma levels after serial rTMS stimulation in healthy subjects. In conclusion, this suggests that BDNF changes are neither specific for a particular site of stimulation, nor for the frequency of stimulation, and may be only caused by serial rTMS stimulation. In contrast to these findings in healthy participants, investigations on the rTMS-BDNF relationship in depressed patients indicated an increase of BDNF levels after serial rTMS stimulations (Zanardini et al. 2006; Yukimasa et al. 2006). This suggests that rTMS effects on peripheral BDNF levels depend on whether study participants are depressed or not. rTMS may entail both, a decrease of peripheral BDNF in healthy participants with physiological BDNF serum levels and an increase in depressed patients who show a` priori-reduced BDNF levels (Duman and Monteggia 2006; Gervasoni et al. 2005; Lee et al. 2007). This might indicate a role of BDNF in a negative feedback loop. It might also suggest that BDNF is not as hypothesised the primary causation factor of the antidepressant property of rTMS, but one important factor in the complex interaction between various factors. It is known that transcranial magnetic stimulation in addition to effects on BDNF induces other neurobiological effects [i.e. rTMS of the left DLPFC modulates dopamine release in the ipsilateral anterior cingulate cortex in healthy volunteers (Cho and Strafella 2009)]. We hypothesise that rTMS stimulation primarily influences neurotransmission [i.e. dopamine (Cho and Strafella 2009)]. These neural effects of rTMS are caused under the stimulation coil and might spread across neural networks. This might indicate a mediating role of BDNF. rTMS does not affect or even downregulate BDNF signalling in balanced neural networks with no need for changes such as in our study. As reported in studies on the influence of rTMS in depressed patients, BDNF, as a surrogate marker of neuroplasticity, increases in dysbalanced neural networks with demand for modification of pathological neurotransmitter concentrations. The strengths of our investigation include the shamcontrolled design, the highly standardised experimental procedures, the homogeneous cohort and the number of recruited participants which is higher or at least comparable to other studies in this field. Furthermore, we integrated alcohol intake and smoking status as determinants of serum BDNF into the analyses. Especially smoking seems to be a potential confounder that has been left out of consideration so far. This study has several limitations. The method to
123
312
G. Schaller et al.
target the left DLPFC used here (defined as the region 5-cm rostral to the point of optimal stimulation for the right abductor pollicis brevis muscle at a parasagittal plane in the left hemisphere) is imprecise. Recent studies on coil placement based on anatomical or functional MRI suggest the need for a TMS-coil positioning method, which incorporates individual anatomical information (Peleman et al. 2010; Sparing et al. 2008). We decided to use the above-mentioned standard procedure to detect the left DLPFC, since this is the method used in the majority of clinical trials evaluating the effect of rTMS on depressed people (Rumi et al. 2005) and because of reasons of practicability in daily clinical routine. Another limitation is that peripheral BDNF levels were quantified in this study. Since BDNF also originates from peripheral sources (Meyer et al. 1992), we cannot assure that the reported findings represent exclusively cerebral processes. Nevertheless, there are numerous studies which found a positive correlation between blood and brain BDNF levels (Karege et al. 2002; Klein et al. 2011) and, therefore, strongly support research on peripheral BDNF levels. Furthermore, aiming at a homogeneous sample we restricted our investigations to male participants. Thus, the findings cannot be transferred to women. In addition, the effects were small. Some statistical values showed marginal significance and might, therefore, represent a non-clinically significant observation, also given the numerous analyses performed and the post hoc nature of the study. In summary, our results show that serial HF-rTMS stimulations over the left DLPFC decrease serum BDNF levels in healthy male volunteers. This is in line with the one to date existing study on the effect of a LF-rTMS series on BDNF in healthy controls (Angelucci et al. 2004). Although our findings were contrary to the primary hypothesis, the study provides further evidence that BDNF is involved in the effects of rTMS. The underlying mechanisms still remain to be discovered. It is now interesting to investigate other neurotrophins, neurotransmitters and further putative causation factors for the clinical effect of transcranial magnetic stimulation. Acknowledgments We thank all volunteers for their participation. Funding for this study was provided by the Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University of ErlangenNuremberg, Erlangen, Germany. Conflict of interest conflict of interest.
None of the authors had a financial or personal
References Altar CA, Whitehead RE, Chen R, Wortwein G, Madsen TM (2003) Effects of electroconvulsive seizures and antidepressant drugs on
123
brain-derived neurotrophic factor protein in rat brain. Biol Psychiatry 54:703–709 Angelucci F, Oliviero A, Pilato F, Saturno E, Dileone M, Versace V, Musumeci G, Batocchi AP, Tonali PA, Di Lazzaro V (2004) Transcranial magnetic stimulation and BDNF plasma levels in amyotrophic lateral sclerosis. Neuroreport 15:717–720 Babor TF, de la Fuente JR, Saunders J, Grant M (1992) AUDIT. The alcohol use disorders identification test. Guidelines for use in primary health care, World Health Organization, Geneva, Switzerland Beck A, Steer R (1987) Beck Depression Inventory—Manual. The Psychological Corporation, San Antonio, TX, USA Bleich S, Havemann-Reinecke U, Kornhuber J (2002) Fagerstro¨mTest fu¨r Nikotinabha¨ngigkeit (FTNA) (Fagerstro¨m Test for Nicotine Dependence). Hogrefe-Verlag, Go¨ttingen, Germany Bus BA, Molendijk ML, Penninx BJ, Buitelaar JK, Kenis G, Prickaerts J, Elzinga BM, Voshaar RC (2011) Determinants of serum brain-derived neurotrophic factor. Psychoneuroendocrinology 36:228–239 Cho SS, Strafella AP (2009) rTMS of the left dorsolateral prefrontal cortex modulates dopamine release in the ipsilateral anterior cingulate cortex and orbitofrontal cortex. PLoS ONE 4:e6725 Duman RS, Monteggia LM (2006) A neurotrophic model for stressrelated mood disorders. Biol Psychiatry 59:1116–1127 Gedge L, Beaudoin A, Lazowski L, du Toit R, Jokic R, Milev R (2012) Effects of electroconvulsive therapy and repetitive transcranial magnetic stimulation on serum brain-derived neurotrophic factor levels in patients with depression. Front Psychiatry 3:12 Gervasoni N, Aubry JM, Bondolfi G, Osiek C, Schwald M, Bertschy G, Karege F (2005) Partial normalization of serum brain-derived neurotrophic factor in remitted patients after a major depressive episode. Neuropsychobiology 51:234–238 Henkel AW, Sperling W, Rotter A, Reulbach U, Reichardt C, Bo¨nsch D, Maler JM, Kornhuber J, Wiltfang J (2008) Antidepressant drugs modulate growth factors in cultured cells. BMC Pharmacol 4(8):6 Hermann LL, Ebmeier KP (2006) Factors modifying the efficacy of transcranial magnetic stimulation in the treatment of depression: a review. J Clin Psychiatry 67:1870–1876 Jacobs BL, van Praag H, Gage FH (2000) Adult brain neurogenesis and psychiatry: a novel theory of depression. Mol Psychiatry 5:262–269 Karege F, Schwald M, Cisse M (2002) Postnatal developmental profile of brain-derived neurotrophic factor in rat brain and platelets. Neurosci Lett 328:261–264 Klein AB, Williamson R, Santini MA, Clemmensen C, Ettrup A, Rios M, Knudsen GM, Aznar S (2011) Blood BDNF concentrations reflect brain-tissue BDNF levels across species. Int J Neuropsychopharmacol 14:347–353 Lang UE, Bajbouj M, Gallinat J, Hellweg R (2006) Brain-derived neurotrophic factor serum concentrations in depressive patients during vagus nerve stimulation and repetitive transcranial magnetic stimulation. Psychopharmacology 187:56–59 Lang UE, Hellweg R, Gallinat J, Bajbouj M (2007) Acute prefrontal cortex transcranial magnetic stimulation in healthy volunteers: no effects on brain-derived neurotrophic factor (BDNF) concentrations in serum. J Affect Disord 107:255–258 Lee BH, Kim H, Park SH, Kim YK (2007) Decreased plasma BDNF level in depressive patients. J Affect Disord 101:239–244 Ma J, Zhang Z, Su Y, Kang L, Geng D, Wang Y, Luan F, Wang M, Cui H (2013) Magnetic stimulation modulates structural synaptic plasticity and regulates BDNF-TrkB signal pathway in cultured hippocampal neurons. Neurochem Int 62:84–91 Meyer M, Matsuoka I, Wetmore C, Olson L, Thoenen H (1992) Enhanced synthesis of brain-derived neurotrophic factor in the
Serial rTMS decreases BDNF serum levels in healthy male volunteers lesioned peripheral nerve: different mechanisms are responsible for the regulation of BDNF and NGF mRNA. J Cell Biol 119:45–54 Mu¨ller M, Tosci N, Kresse AE, Poat A, Keck ME (2000) Long-term repetitive transcranial magnetic stimulation increases the expression of brain-derived neurotrophic factor and cholecystokinin mRNA, but not neuropeptide tyrosine mRNA in specific areas of rat brain. Neuropsychopharmacology 23:205–215 Peleman K, Van Schuerbeek P, Luypaert R, Stadnik T, De Raedt R, De Mey J, Bossuyt A, Baeken C (2010) Using 3D-MRI to localize the dorsolateral prefrontal cortex in TMS research. World J Biol Psychiatry 11:425–430 Pridmore S, Fernandes Filho JA, Nahas Z, Liberatos C, George MS (1998) Motor threshold in transcranial magnetic stimulation: a comparison of a neurophysiological method and a visualization of movement method. J ECT 14:25–27 Rumi DO, Gattaz WF, Rigonatti SP, Rosa MA, Fregni F, Rosa MO, Mansur C, Myczkowski ML, Moreno RA, Marcolin MA (2005) Transcranial magnetic stimulation accelerates the antidepressant effect of amitriptyline in severe depression: a double-blind placebo-controlled study. Biol Psychiatry 57:162–166 Schaller G, Lenz B, Friedrich K, Dygon D, Richter-Schmidinger T, Jacobi A, Mueller SE, Maiho¨fner C, Sperling W, Kornhuber J (2011) Repetitive transcranial magnetic stimulation influences mood in healthy male volunteers. J Psychiatr Res 45:1178–1183 Schaller G, Lenz B, Friedrich K, Dygon D, Richter-Schmidinger T, Sperling W, Kornhuber J (2013) No evidence for effects of a high-frequency repetitive transcranial magnetic stimulation (rTMS) series on verbal and figural fluency and TAP tasks
313 performance in healthy male volunteers. Neuropsychobiology 67:69–73 Shirayama Y, Chen AC, Nakagawa S, Russell DS, Duman RS (2002) Brain-derived neurotrophic factor produces antidepressant effects in behavioral models of depression. J Neurosci 22:3251–3261 Slotema CW, Blom JD, Hoek HW, Sommer IE (2010) Should we expand the toolbox of psychiatric treatment methods to include Repetitive Transcranial Magnetic Stimulation (rTMS)? A metaanalysis of the efficacy of rTMS in psychiatric disorders. J Clin Psychiatry 71:873–884 Sparing R, Buelte D, Meister IG, Paus T, Fink GR (2008) Transcranial magnetic stimulation and the challenge of coil placement: a comparison of conventional and stereotaxic neuronavigational strategies. Hum Brain Mapp 29:82–96 Wassermann EM (1998) Risk and safety of repetitive transcranial magnetic stimulation: report and suggested guidelines from the International Workshop on the Safety of Repetitive Transcranial Magnetic Stimulation, June 5-7, 1996. Electroencephalogr Clin Neurophysiol 108:1–16 Yukimasa T, Yoshimura R, Tamagawa A, Uozumi T, Shinkai K, Ueda N, Tsuji S, Nakamura J (2006) High-Frequency Repetitive Transcranial Magnetic Stimulation improves refractory depression by influencing catecholamine and Brain-Derived Neurotrophic Factors. Pharmacopsychiatry 39:52–59 Zanardini R, Gazzoli A, Ventriglia M, Perez J, Bignotti S, Rossini PM, Gennarelli M, Bocchio-Chiavetto L (2006) Effect of repetitive transcranial magnetic stimulation on serum brain derived neurotrophic factor in drug resistant depressed patients. J Affect Disord 91:83–86
123
