The authors have declared that no competing interests exist.
Conceived and designed the experiments: MAR MW RS MS. Performed the experiments: MAR MHK MW RB RK. Analyzed the data: MAR MHK RG MS. Contributed reagents/materials/analysis tools: RS RG. Wrote the paper: MAR GRF MS.
Transcranial direct current stimulation (tDCS) is increasingly being used in human studies as an adjuvant tool to promote recovery of function after stroke. However, its neurobiological effects are still largely unknown. Electric fields are known to influence the migration of various cell types
Transcranial direct current stimulation (tDCS) has long been known to induce long-lasting alterations of cortical excitability both in experimental animals
(A) For multi-session tDCS, an epicranial electrode was mounted as schematically depicted (red circle). (B) H&E staining confirmed an intact cortex after multi-session focal tDCS, and (C) no astrogliotic scarring was detected by GFAP staining (scale bar = 1 mm).
All animal procedures were in accordance with the German Laws for Animal Protection and were approved by the local animal care committee and local governmental authorities. Spontaneously breathing male Wistar rats weighing 290–330 g were anesthetized with 5% isoflurane and maintained with 2.5% isoflurane in 65% / 35% nitrous oxide / oxygen. Throughout surgical procedures, body temperature was maintained at 37.0°C with a thermostatically controlled heating pad.
(A) The density of activated microglia identified by Iba1 immunoreactivity was increased in the cortex ipsilateral to multi-session tDCS (scale bar = 0.05 mm). (B) This increase in Iba1+ cells compared to the respective contralateral hemisphere was most pronounced early after cathodal tDCS, but also present after anodal stimulation; effects decreased with time (means ± SEM; *p = 0.05, **p<0.01).
An epicranial electrode with a defined contact area of 3.5 mm2 was mounted onto the intact skull using non-toxic glass ionomer luting cement (Ketac Cem Plus, 3 M ESPE, Seefeld, Germany) at the following stereotaxic coordinates: bregma AP +2.0 mm, ML +2.0 mm. After electrode placement, the skin around the electrode was closed with sutures, and the electrode left in place for the entire experiment. The counter electrode was placed on the rat's ventral thorax. tDCS was applied continuously for 15 min at 500 µA using a constant current stimulator (CX-6650, Schneider Electronics, Gleichen, Germany), according to the protocol by Liebetanz et al
(A) ICAM1 immunoreactivity was increased in the ipsilateral hemisphere after multi-session cathodal or anodal tDCS, (scale bar = 0.2 mm). (B) ICAM1+ vessels trended to increase with time upon cathodal tDCS, while anodal tDCS caused only a transient increase (note that differences were not statistically significant; means ± SEM).
In all animals, the tracer bromodeoxyuridine (BrdU) was injected intraperitoneally for the duration of the experiment, starting on the first day of tDCS, at a concentration of 50 mg/kg per injection, as described previously
(A) After multi-session cathodal tDCS, the number of proliferating cells identified by BrdU-incorporation was increased in the ipsilateral hemisphere (scale bar = 0.5 mm). (B) This effect was only observed with cathodal stimulation. Anodal tDCS had no effect on proliferation in the cortex (means ± SEM; **p<0.01, *p<0.05).
Three days after the last tDCS session, rats were deeply anesthetized and decapitated. The brains were rapidly removed, frozen in isopentane at −40°C, and stored at −80°C prior to further histological and immunohistochemical processing. Ten µm thick adjacent serial coronal brain sections were cut at 500 µm intervals. H&E staining was performed according to standard protocols to detect any cortical lesion due to tDCS. For immunohistochemistry, sections were fixed with either 4% paraformaldehyde (for Iba1, BrdU, and Hes3-stainings), or with 100% acetone (for ICAM1-stainings). For antigen-retrieval prior to BrdU-staining, sections were microwave-heated in 0.01 M citrate buffer, pH 6.0, for 5 min, followed by 2N HCl at 37°C for 30 min. The following antibodies were used: anti-Iba1 (rabbit polyclonal, cat# 019-19741, dilution 1∶1000, Wako, Neuss, Germany); anti-ICAM-1 (mouse monoclonal, anti-rat CD54, cat-# MCA773GA, dilution 1∶500, AbD Serotec, Puchheim, Germany); anti-BrdU (mouse monoclonal, cat-# B2531, dilution 1∶100, Sigma, Munich, Germany); anti-Hes3 (rabbit polyclonal, cat-# sc-25393, dilution 1∶1000, Santa Cruz Biotechnology, Heidelberg, Germany). For visualization of all antibodies, the ABC Elite kit (Vector Laboratories, Burlingame, CA, USA) with diaminobenzidine (Sigma, Munich, Germany) as the final reaction product was used.
(A) The density of NSC identified by Hes3 immunoreactivity was increased in the cortex ipsilateral to multi-session tDCS (scale bar = 0.05 mm). (B) The number of Hes3-positive NSC increased in the ipsilateral hemisphere with the duration of cathodal tDCS (means ± SEM; *p<0.05).
To determine the number of Iba1-positive, BrdU-positive and Hes3-positive cells and of ICAM1-positive vessels in the cortex, 20 coronal sections at 500 µm intervals were stained with the respective antibody. Using a Zeiss Axiophot microscope with a 40x objective, 7 images of adjacent fields of view (FOV) of the cortex were taken of each hemisphere, and of each section. The number of positive cells in the ipsilateral, stimulated hemisphere was divided by the corresponding number in the contralateral, control hemisphere. Mean values and standard errors of the mean of these ratios were calculated for each group of animals that had received the same treatment.
Descriptive statistics and Student's t-tests were performed with Microsoft Excel 2003 (Microsoft Corp.); statistical significance was set at the less than 5% level (p<0.05).
Rats were subjected to 15 min sessions of tDCS using a 3.5 mm2 epicranial electrode and a current of 500 µA (
After 5 or 10 sessions of anodal or cathodal tDCS, histological analyses revealed that none of the rats had suffered a cortical lesion (
To assess the effects of tDCS on innate neuroinflammatory processes, rat brains were stained for Iba1, labelling activated microglia. In the ipsilateral (stimulated) cortex, the number of Iba1-positive cells was increased after both cathodal and anodal stimulation (
To explore a secondary involvement of adaptive immunity, we investigated the endothelial intercellular adhesion molecule-1 (ICAM1), involved in leukocyte transmigration through the blood-brain barrier. After 5 days, ICAM-1 was increased by trend in the ipsilateral (stimulated) cortex, irrespective of the polarity (
During the 5 or 10 days of multi-session tDCS, animals were repeatedly injected i.p. with BrdU to label proliferating cells. The protocol ensured that all animals received the same cumulative dose of BrdU, regardless of the duration of the experiment. BrdU-positive cells were counted in the ipsilateral cortex and compared to the contralateral side (
To further investigate what subpopulations of potentially proliferating cells were expanded by cathodal tDCS, brains were stained for Hes3 as a marker of endogenous NSC (
In this study, we investigated the effects of multi-session tDCS on the adult rat brain
In our experimental paradigm, we took great care to ensure that tDCS would not cause any lesion to the brain tissue. In a recent pioneer study, Liebetanz et al. described the relationship between charge density and the occurrence and size of cortical lesions
We found the numbers of Iba1-positive cells to be increased early after cathodal tDCS, decreasing again with time. Similar findings were obtained after anodal tDCS, albeit to a lesser extent. This rapid and transient activation of resident microglia is typical for an innate neuroinflammatory response to tDCS. ICAM1 expression is a prerequisite of recruitment of haematogenous cells in an antigen specific manner, categorized as adaptive immunity. ICAM-1 was used as an indicator of upcoming adaptive immune responses. It showed a typical later time-course with a maximum at 10 days following cathodal stimulation, while anodal stimulation caused a more transient, unspecific effect. However, those neuroinflammatory processes observed after both cathodal and anodal stimulation could – at least in part – also be caused by thermal effects that were sub-threshold to induce a lesion to the brain tissue.
Based upon work in focal cerebral ischemia, neuroinflammation has already been characterized as a ‘double-edged sword’ with both beneficial and detrimental effects on the prevention of secondary tissue damage, regeneration and recovery
The transcription factor Hes3 was recently identified as a biomarker for a widespread population of quiescent endogenous NSC that is dynamically upregulated upon their activation
Although BrdU-positive proliferating cells could at least in part constitute microglia, the extent of their upregulation and their exclusive appearance after cathodal tDCS makes it likely that at least the majority of those proliferating cells are not microglia. Furthermore, we found the number of Hes3-positive cells to increase with the number of cathodal tDCS session, further confirming an effect on NSC. In addition to the direct effect of the electric field on NSC migration, a second mechanism for the activation of cortical NSC is conceivable: tDCS was recently shown to modulate cerebral blood flow in a polarity-specific way, with an increase after anodal and decrease after cathodal stimulation, lasting for at least 30 min after tDCS
This study provides evidence that electric stimulation modulates responses of non-neuronal cells in the brain, and relevantly contributes to our scarce knowledge about the neurobiological effects of tDCS. tDCS both attracts endogenous NSC and activates innate immune responses. From the clinical point of view, applying tDCS after stroke may help to locally augment endogenous NSC known to promote neuroprotection and repair.