Transcranial direct current stimulation (tDCS) has been suggested as an adjuvant tool to promote recovery of function after stroke, but the mechanisms of its action to date remain poorly understood. Moreover, studies aimed at unraveling those mechanisms have essentially been limited to the rat, where tDCS activates resident microglia as well as endogenous neural stem cells. Here we studied the effects of tDCS on microglia activation and neurogenesis in the mouse brain. Male wild-type mice were subjected to multisession tDCS of either anodal or cathodal polarity; sham-stimulated mice served as control. Activated microglia in the cerebral cortex and neuroblasts generated in the subventricular zone as the major neural stem cell niche were assessed immunohistochemically. Multisession tDCS at a sublesional charge density led to a polarity-dependent downregulation of the constitutive expression of Iba1 by microglia in the mouse cortex. In contrast, both anodal and, to an even greater extent, cathodal tDCS induced neurogenesis from the subventricular zone. Data suggest that tDCS elicits its action through multifacetted mechanisms, including immunomodulation and neurogenesis, and thus support the idea of using tDCS to induce regeneration and to promote recovery of function. Furthermore, data suggest that the effects of tDCS may be animal- and polarity-specific.
Transcranial direct current stimulation (tDCS) can be used to induce alterations of cortical excitability in a polarity-specific way, in both animals and humans [
Cerebral ischemia induces various processes at the cellular level, including the activation of brain-resident microglia (“neuroinflammation”) as well as the mobilization of neural stem cells from their niches [
All animal procedures were approved by the local animal care and use committee and governmental authorities (LANUV, # 84-02.04.2013.A068). Surgery was performed on twenty 10–12-week-old male C57BL/6JRj mice (Janvier Labs, France), weighing 28–35 g, under light isoflurane anesthesia, and additional local anesthesia with bupivacaine. To ensure identical electrode placement for tDCS, custom-made polycarbonate tubes with a contact area of 2.27 mm2 (Medres Medical Research, Cologne, Germany) were stereotactically placed on the skull of the mice prior to tDCS, as described previously [
Experimental setup for tDCS. (a) The anesthetized mouse was connected to the direct current stimulator (apparatus in the back) via a silver-coated electrode cable attached to the polycarbonate tube mounted on its skull. The cable protruding from under the mouse’ abdomen originates from the counter electrode. (b) The epicranial electrode (dashed circular line) was mounted on the intact skull using dental cement at the coordinates AP +0.5 mm and ML +1.5 mm from bregma.
Animals were randomized to receive 10 days of tDCS with either cathodal or anodal polarity; a third group of mice was not stimulated for control (sham group). Additionally, mice were randomized to receive different currents of tDCS, 250
Overview over the experimental groups.
Current (C) |
Multisession cathodal tDCS | Multisession anodal tDCS | Control (no tDCS/sham) |
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C: 250 |
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C: 500 |
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TDCS was repeated daily for 5 consecutive days, followed by a tDCS-free interval of 2 days; then animals were subjected to tDCS for 5 more days, resulting in 10 days of tDCS in total. This experimental design was adapted from clinical studies with stroke patients. For each tDCS session, the polycarbonate tube was filled with saline, a silver-coated tDCS electrode (Medres Medical Research, Cologne, Germany) was inserted, and a silver-coated sensor electrode (Spes Medica, Italy; #DENIS01526) was placed under the shaved thorax as counter electrode. TDCS was applied continuously for 15 minutes using a constant current stimulator (CX-6650, Schneider-Electronics, Gleichen, Germany) under light isoflurane anesthesia. In the control group, mice were anesthetized for 15 minutes without connection to the stimulator (“sham”). After each tDCS session, animals were allowed to recover in their home cages with access to food and water ad libitum.
On every second day of tDCS or sham stimulation, respectively, animals received intraperitoneal injections of bromodeoxyuridine (BrdU; Sigma-Aldrich, Munich, Germany) at a concentration of 100 mg/kg, in order to label proliferating cells.
Mice were euthanized by decapitation two days after the last tDCS session. Brains were removed rapidly and frozen at −80°C until used. Frozen brains were cut in coronal sections of 10
Image analysis and data quantification was performed by an independent observer blinded to the treatment conditions. To quantify DCX-positive neuroblasts or BrdU-positive proliferating cells in the SVZ, the area covered by immunoreactive cells was measured in
For all quantifications, mean values were established among equally treated mice.
Descriptive statistics, calculating means and standard errors, were performed with Microsoft Excel 2010 (Microsoft Corp.). All other statistical analyses were performed with the software Prism (Version 6.01, GraphPad, USA). For comparison of multiple groups, multifactor Analysis of Variance (ANOVA) was performed, followed by Tukey’s Honest Significant Difference (HSD) test. If data were not normally distributed, an ANOVA on ranks was performed, followed by Dunn’s multiple comparisons test as post hoc analysis. Statistical significance was set at the less than 5% level (
TDCS was applied repetitively for 10 consecutive days at two different charge densities (Table
Multisession transcranial direct current stimulation (tDCS) at a high charge density causes cortical lesions. (a) After multisession tDCS with 198 kC/m2, several animals presented with a disruption of neuronal integrity on NeuN neuronal staining. The lower image depicts the magnified region from the upper image. The scale bars represent 1 mm (upper image) and 200
Since at the low charge density of 99 kC/m2, no impairment of neuronal integrity or any other sign of cortical lesions were observed in any of the experimental animals. Thus, further immunohistochemical analyses were exclusively conducted on mouse brains stimulated with 99 kC/m2.
Mice stimulated at the low charge density of 99 kC/m2 were stained for Iba1 to assess and quantify activated microglia. Iba1+ microglia were found equally distributed throughout the cortex, without any focal areas of microglia accumulation (Figure
Multisession anodal tDCS at low charge density downregulates the constitutive activation of microglia. (a) Multisession tDCS with 99 kC/m2 did not cause focal cortical lesions (scale bar represents 1 mm). (b) Microglia expressing Iba1 displayed an amoeboid morphology irrespective of stimulation polarity (scale bar represents 20
Mouse brains stimulated at the low current density of 99 kC/m2 were stained for DCX to assess and quantify the effects of tDCS on neuroblasts in the SVZ (Figures
Multisession tDCS induces neurogenesis in the subventricular zone (SVZ). (a), (a′) Neuroblasts in the SVZ were identified by their expression of DCX under control conditions (sham). The scale bar represents 100
To assess the effect of tDCS on the proliferation of undifferentiated stem cells in the SVZ, animals were repetitively injected with BrdU during multisession tDCS. Staining for BrdU incorporation revealed that tDCS with 99 kC/m2 did not affect the amount of the BrdU+ stem cells in the SVZ (Figures
Multisession tDCS does not affect proliferation in the SVZ. (a) Proliferating cells in the SVZ were labeled with BrdU during anodal, cathodal, or sham tDCS. Immunohistochemistry revealed the size of the SVZ labeling positive for BrdU; the scale bar represents 100
In a previous study, we methodologically established tDCS in the mouse and found that a single stimulation with a charge density of 198 kC/m2 or below did not cause lesions to the cortex. We here performed multisession tDCS in healthy mice for a total of 10 sessions, simulating a clinical rehabilitation paradigm. In this multisession setting, 50% of the mice did develop lesions to the cerebral cortex at a charge density of 198 kC/m2, but not at the lower charge density of 99 kC/m2. This indicates that the lesion threshold is lower for multisession tDCS than for single tDCS, suggesting a cumulative effect of the stimulation. Since not all mice develop lesions even at 198 kC/m2, we suggest stochastic effects within this dose range. Other effects of tDCS, such as sequence learning or pain perception, are known to be affected by the number of sessions as well, corroborating this cumulative response to stimulation [
Until now, research on tDCS in experimental animals has mostly been limited to the rat [
Microglia activation goes along with increased expression of Iba1 antigen as detected by immunocytochemistry [
Brain-resident neural stem cells in the neurogenic niche of the SVZ were affected by both cathodal and anodal tDCS, leading to an increase in young neuroblasts. Similarly, tDCS mobilizes neural stem cells in the rat, leading to proliferation [
TDCS elicits its actions through multifacetted mechanisms, far exceeding its primary effects on resting membrane potential. We here show that anodal tDCS downregulates constitutive expression of Iba1 on microglia in the cortex of the mouse, suggesting immunomodulatory effects. Moreover, cathodal, more than anodal, tDCS induced neurogenesis, supporting the use of tDCS in facilitating regeneration and recovery of function after stroke.
The authors declare that they have no competing interests.
This work was supported by the “Marga und Walter Boll-Stiftung” (no. 210-12-12 and no. 210-10-15) and the Köln-Fortune-Program/Faculty of Medicine, University of Cologne, Germany (106/2012). The authors thank Ms. Claudia Drapatz for excellent technical assistance.