Research article Received: 21 December 2013,
Revised: 9 November 2014,
Accepted: 14 November 2014,
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/nbm.3244
Transcranial direct current stimulation promotes the mobility of engrafted NSCs in the rat brain Meike Hedwig Keutersa,b, Markus Aswendtb, Annette Tennstaedtb, Dirk Wiedermannb, Anton Pikhovycha,b, Steffen Rotthuesa, Gereon Rudolf Finka,b, Michael Schroetera,b, Mathias Hoehnb,c and Maria Adele Ruegera,b,d* Transcranial direct current stimulation (tDCS) is used in numerous clinical studies and considered an effective and versatile add-on therapy in neurorehabilitation. To date, however, the underlying neurobiological mechanisms remain elusive. In a rat model of tDCS, we recently observed a polarity-dependent accumulation of endogenous neural stem cells (NSCs) in the stimulated cortex. Based upon these findings, we hypothesized that tDCS may exert a direct migratory effect on endogenous NSCs towards the stimulated cortex. Using noninvasive imaging, we here investigated whether tDCS may also cause a directed migration of engrafted NSCs. Murine NSCs were labeled with superparamagnetic particles of iron oxide (SPIOs) and implanted into rat striatum and corpus callosum. MRI was performed (i) immediately after implantation and (ii) after 10 tDCS sessions of anodal or cathodal polarity. Sham-stimulated rats served as control. Imaging results were validated ex vivo using immunohistochemistry. Overall migratory activity of NSCs almost doubled after anodal tDCS. However, no directed migration within the electric field (i.e. towards or away from the electrode) could be observed. Rather, an undirected outward migration from the center of the graft was detected. Xenograft transplantation induced a neuroinflammatory response that was significantly enhanced following cathodal tDCS. This inflammatory response did not impact negatively on the survival of implanted NSCs. Data suggest that anodal tDCS increases the undirected migratory activity of implanted NSCs. Since the electric field did not guide implanted NSCs over large distances, previously observed polarity-dependent accumulation of endogenous NSCs in the cortex might have originated from local proliferation. Results enhance our understanding of the neurobiological mechanisms underlying tDCS, and may thereby help to develop a targeted and sustainable application of tDCS in clinical practice. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: migration; galvanotaxis; MRI; superparamagnetic particles of iron oxide; neuroinflammation; phagocytes; microglia
INTRODUCTION Transcranial direct current stimulation (tDCS) as a non-invasive neuromodulatory technique induces long-lasting alterations of cortical excitability in both animals (1) and humans (2). tDCS has been used to (i) enhance the effects of motor learning in
* Correspondence to: Maria Adele Rueger, Department of Neurology, University Hospital of Cologne, Cologne, Germany. E-mail: [email protected] a M. H. Keuters, A. Pikhovych, S. Rotthues, G. R. Fink, M. Schroeter, M. A. Rueger Department of Neurology, University Hospital of Cologne, Cologne, Germany b M. H. Keuters, M. Aswendt, A. Tennstaedt, D. Wiedermann, A. Pikhovych, G. R. Fink, M. Schroeter, M. Hoehn, M. A. Rueger In-vivo-NMR Laboratory, Max Planck Institute for Neurological Research, Cologne, Germany c M. Hoehn Department of Radiology, Leiden University Medical School, Leiden, The Netherlands
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healthy volunteers (3) and (ii) ameliorate functional deficits following stroke, including motor deficits (4), aphasia (5), and neglect (6). Despite the current use of tDCS in numerous clinical studies, to date the basic mechanisms and details underlying the neurobiological effects of tDCS remain unknown (for comprehensive reviews, see (7) and (8)). Recent reports provide evidence that the electrical stimulation exceeds primary
d M. A. Rueger Cognitive Neuroscience, Institute of Neuroscience and Medicine (INM3), Research Centre Juelich, Juelich, Germany Abbreviations used: ANOVA, analysis of variance; DAB, diaminobenzidine; ES cells, embryonic stem cells; FMRIB, Oxford Centre for Functional Magnetic Resonance Imaging of the Brain; FOV, field of view; FSL, FMRIB’s Software Library; GE-EPI, gradient echo–echo planar imaging; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; mAb, monoclonal antibody; MHC, major histocompatibility complex; NSC, neural stem cell; n.s., not significant; PBS, phosphate-buffered saline; ROI, region of interest; SPIO, superparamagnetic particle of iron oxide; SVZ, subventricular zone; tDCS, transcranial direct current stimulation; TE, echo time; TR, repetition time.
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M. H. KEUTERS ET AL. electrophysiological effects on neurons, e.g., it additionally modulates responses of non-neuronal cells in the brain (9–11). In a rat model of tDCS, we recently demonstrated a pro-inflammatory effect of both cathodal and anodal tDCS as well as a polarity-dependent accumulation of endogenous neural stem cells (NSCs) in the stimulated cortex (12). Data suggest that beneficial effects of tDCS in stroke may at least in part result from NSC activation and the modulation of neuroinflammation. Regarding a putative polarity-dependent NSC accumulation in the stimulated cortex, we hypothesized that cathodal tDCS might induce a directed migration of endogenous NSCs from the subventricular zone (SVZ, i.e. the major NSC niche) towards the cortex. Migration of cells in culture can be induced by electric fields, a phenomenon referred to as galvanotaxis (13). Among many other cell types, rodent neural progenitor cells (14,15), human embryonic stem (ES) cells (16), and human ES-cell derived NSCs (17) have all been demonstrated to migrate in an electric field in vitro. However, whether such effects can also be observed in vivo has not yet been investigated. Accordingly, we here explored the migration of transplanted contrast-agent-labeled NSC towards the cortex upon tDCS stimulation, using MRI in vivo as well as immunohistochemistry ex vivo.
Stereotactic implantation of NSCs Each rat received two grafts of labeled NSCs: one in the right striatum, the other one in the left corpus callosum (Fig. 1(B)). For implantation, anesthetized rats were placed in a stereotactic rat frame equipped with a digital positioning system (Digital Lab Standard, Stoelting, Wood Dale, IL, USA). Carprofen (4 mg ml 1; Pfizer/Zoetis, Berlin, Germany) was injected subcutaneously as analgetic. After incising the skin and removing the periosteum, a burr hole was drilled over the first implantation site in the right striatum, using a dental drill (Bien-Air 810 Technobox, Bien-Air Dental, Bienne, Switzerland). A microsyringe (5 μl volume; Hamilton Messtechnik, Hoechst, Germany) was slowly inserted to the stereotactic coordinates bregma +1.0 mm AP, 2.5 mm ML, 5.0 mm VD. After positioning the needle, a 2 μl suspension of 300 000 labeled NSCs were injected at 0.5 μl min 1 using an automatic microinjection control system (UltraMicroPump, World Precision Instruments, Sarasota, FL, USA). After injection, the needle was left in place for 5 min to allow the cell suspension to distribute evenly within the tissue. Then, the needle was slowly withdrawn at 1 mm min 1. The same injection procedure was repeated for the second implantation site, i.e. the left corpus callosum, at the coordinates +1.0 mm AP, +2 mm ML, 3.0 mm VD. After the procedure, the skin was closed with single sutures.
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EXPERIMENTAL METHODS Cell culture and labeling The NSC line D3WT_N2Euro was established from the murine ES cell line D3 and retrovirally transduced to stably express green fluorescent protein (GFP) cloned from the copepod Pontellina plumata as described previously (18). Adherent NSCs were grown in N2Euro medium (Biozol, Siziano, Italy) in the presence of 5 ng ml 1 EGF and 10 ng ml 1 FGF-2 (both PeproTech, Hamburg, Germany) to keep them in an undifferentiated state. Prior to transplantation, cells were labeled with superparamagnetic particles of iron oxide (SPIOs) with a mean diameter of 150 nm, using the clinically established contrast agent Endorem (9 μl ml ml 1; Guerbet, France) and the transfection reagent Metafectene (1 μl ml 1; Biontex, Martinsried, Germany) as described previously (19). In brief, NSCs were incubated with Endorem plus Metafectene for 24 hours, then washed with phosphate-buffered saline (PBS; Invitrogen, Darmstadt, Germany), detached from plates with Accutase (PAA, Pasching, Austria), and re-suspended in Hank’s Balanced Salt Solution (Invitrogen) at 150 000 cells μl 1 for immediate transplantation.
Immediately after the implantation of labeled NSCs, MRI was performed to determine the exact position of each graft. The rat was placed in a special rat holder designed for MRI (medres, Cologne, Germany), including an in-house feedback control system for
Animals and general aspects of surgery All animal procedures were carried out in accordance with the German Laws for Animal Protection and were approved by the local animal care committee as well as local governmental authorities (LANUV NRW, no 87-51.04.2010.A332). Spontaneously breathing male adult Wistar rats weighing 290–310 g were anesthetized with 5% isoflurane, and maintained with 2.5% isoflurane in a 65%/35% nitrous oxide/oxygen atmosphere. Throughout all surgical procedures, body temperature was maintained at 37 °C with a feedback-controlled heating pad. For a timeline of experiments see Figure 1(A).
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Figure 1. Experimental setup to monitor the migration of NSCs. (A) Timeline of experiments. (B) GFP-positive NSCs labeled with SPIOs were stereotactically implanted into two distinct sites, the right striatum (a) and the left corpus callosum (b), of adult rats. (C) For tDCS, an epicranial electrode was mounted on the rat skull over the striatal implantation site, as indicated by the red dot.
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TDCS PROMOTES THE MOBILITY OF ENGRAFTED NEURAL STEM CELLS body temperature. MRI was performed on a BioSpec 11.7 T dedicated animal scanner system (Bruker BioSpin, Ettlingen, Germany) with a 16 cm horizontal bore magnet, equipped with actively shielded gradient coils (BGA9S, 750 mT m 1; Bruker). RF transmission was achieved with a birdcage quadrature resonator coil (72 mm diameter), and the signal was detected with quadrature rat brain surface coils (Bruker). A single-shot gradient echo–echo planar imaging (GE-EPI) pilot was used for shimming, as described previously (20). Two scans were then obtained for each animal. The first was a T2-weighted image as rapid acquisition with relaxation enhancement, using the following protocol: field of view (FOV) 3.2 cm, matrix 256 × 256, slice thickness 0.5 mm, echo time (TE) 32.5 ms, repetition time (TR) 6500 ms. Second, a T2 map was generated using a multislice multiecho spin echo sequence with the parameters FOV 3.2 cm, matrix 160 × 160, slice thickness 0.5 mm, TE1 = 10 ms, TE2 = 20 ms, TR = 4000 ms. MRI was repeated two days after the last tDCS stimulation to determine the exact location of the transplanted NSC after tDCS, and thus to assess a potential tDCS-induced migration. tDCS Starting one day after NSC implantation, multi-session tDCS was performed as described previously (12). In brief, 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 stereotactic coordinates bregma AP +2.0 mm, ML +2.0 mm; the electrode was left in place for the entire experiment (Fig. 1(C)). Animals were randomized to receive 10 days of tDCS with either cathodal or anodal polarity; a third group of rats was sham-stimulated for control. tDCS was repeated daily using the same parameters for five consecutive days, followed by a tDCS-free interval of two days; then animals were subjected to tDCS for five more days, resulting in 10 days tDCS in total. tDCS was applied continuously for 15 min at 500 μA using a constant current stimulator (CX-6650, Schneider Electronics, Gleichen, Germany) under isoflurane anesthesia. For sham stimulation, rats were also anesthetized for 15 min, albeit without connecting them to the stimulator. After each tDCS session, animals were allowed to recover in their home cages with access to food and water ad libitum. MRI post-processing and data analysis In order to assess the migration of implanted NSCs, the T2 maps acquired before and after tDCS were accurately co-registered for each animal. First, images were converted into NIFTI format (Neuroimaging Informatics Technology Initiative; http://nifty. nimh.nih.gov), scaling up the voxel size by a factor of 10 to reach a human-like voxel size for correct further processing. Next, skin and skull were removed from the images using the Brain Extraction Tool in the software FSL (Oxford Centre for Functional Magnetic Resonance Imaging of the Brain, FMRIB, Software Library, Oxford, UK), followed by visual inspection and manual correction, if necessary (21). The resulting brain-only images were segmented using the FMRIB’s Automated Segmentation Tool of FSL, also correcting for spatial intensity variations (22). Next, a selected T2-weighted and manually brain-extracted dataset with ideal positioning and minimal artefacts was selected as the template brain. Each segmented image was co-registered to this
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template brain in order to ensure identical positioning: for all animals, the T2-weighted image from time point 1 was first coregistered to this template; in a second step, time point 2 was co-registered on a subject basis to imaging time point 1, providing an alignment with the template brain, using the FMRIB’s Linear Image Registration Tool of FSL (23,24). The transformation matrices were applied to the T2 maps accordingly. Migration of labeled cells was quantified on a region of interest (ROI)-based analysis using the ImageJ software (http://imagej.nih. gov/ij). Circular ROIs with a diameter of 500 μm were manually drawn on each slice to cover the area of the graft as well as the adjacent tissue three dimensionally. The number of ROIs for each individual implantation site was adapted to its spatial extent, varying slightly between animals. This enabled the distinction between ROIs within the graft, adjacent to the graft, and remote from the graft (Fig. 2(A), (A′)). ROIs on T2 maps were used to determine T2 values, which correlate with the number of labeled cells, since the iron label generates a quantifiable decrease in transverse relaxation time T2 (19). ΔT2 was calculated between the two scans according to ΔT2 = T2 no 1 (before tDCS) – T2 no 2 (after tDCS). Thus, a negative ΔT2 indicated a loss of iron oxide-labeled NSCs at this position, whereas a positive ΔT2 was associated with a higher number of NSCs following tDCS. We determined T2 stability between different animals and imaging sessions in order to correct for methodological data variability. A standard deviation was determined for each animal by comparing T2 values from reference ROIs for scans no 1 and no 2 (before and after tDCS). These reference ROIs were defined in regions remote from the cell graft location, namely in the striatum and the cortex on five consecutive T2 map slices, and had the same diameter (500 μm) as data ROIs. The standard deviation σ for each animal was calculated from these 10 reference ROIs. Twice the standard deviation (2σ) was then used to evaluate statistical significances. Thus, ΔT2 < 2σ indicated a significant migration of labeled NSCs away from the ROI, while ΔT2 > 2σ suggested that NSCs had migrated into the ROI (Fig. 2(B)). The number of ROIs with ΔT2 > 2σ was determined for each graft. Additionally, a mean ΔT2 was established for each graft, including only those ΔT2 values > 2σ. For both measures of migration, means were calculated for each group of grafts subjected to the same stimulation (anodal n = 4, cathodal n = 6, or sham n = 6). Immunohistochemistry Rats were perfused with PBS under deep anesthesia, followed by 4% paraformaldehyde for tissue fixation. The brains were removed, frozen in 2-methylbutane, and stored at 80 °C. Coronal brain sections with a slice thickness of 10 μm were cut at 500 μm intervals and stained with the following primary antibodies: monoclonal antibody (mAb) against rat CD68 (dilution 1:2000; MCA341, AbD Serotec, Oxford, UK) to identify phagocytic microglia/macrophages, mAb against rat major histocompatibility complex (MHC) class II (dilution 1:400; MCA46R, AbD Serotec, Oxford, UK) to assess activated microglia and macrophages, polyclonal antibody against turbo-GFP (dilution: 1: 5000; AB511, Evrogen, Moscow, Russia) to detect transplanted NSCs, or mAb against glial fibrillary acidic protein (GFAP, clone GA-5; dilution 1:1000; MAB360, Millipore, Schwalbach, Germany) to visualize astrogliosis. For visualization using light microscopy, the ABC Elite kit (Vector Laboratories, Burlingame, CA, USA), with diaminobenzidine (DAB; Sigma-Aldrich, Munich, Germany) as the final reaction product, was used. For co-staining of iron particles for
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M. H. KEUTERS ET AL.
Figure 2. MRI detects subtle migratory behavior of transplanted NSCs into the periphery. (A) Based on co-registered T2 maps acquired before and after tDCS, multiple ROIs were defined within the graft, exemplarily depicted for the implantation site in the corpus callosum (within the red ellipse, compare excerpt for higher magnification), the adjacent tissue (blue), and more remote areas (green), to assess the migration of implanted NSCs into the periphery; scale bars represent 1 mm (overview image) and 500 μm (magnified excerpt). (A′) Magnification of the left (striatal) excerpt defined in (A), depicting a representative example for ROI definition within the striatal graft, color-coded in analogy to (A); the scale bar represents 500 μm. (B) Changes in T2 after anodal tDCS in one representative graft. Each data point represents ΔT2 within a specific ROI; ROIs are color-coded by their proximity to the graft according to (A). Values within 2σ of the data are underlaid in grey. Values above 2σ indicate a significant increase in T2 negativity after tDCS, suggesting that NSCs had migrated into the ROI. Regions from which NSCs had migrated away are characterized by values below 2σ. (C) Immunohistochemical staining of the striatal graft from the animal depicted in (A). GFP-positive (brown) NSCs are co-labeled with SPIOs (blue) in the entire graft; the scale bar represents 250 μm.
light microscopy, Prussian blue solution was applied for 20 min following DAB visualization (16 ml 2% hydrochloric acid plus 8 g potassium hexacyanoferrate II 3-hydrate, both Roth, Karlsruhe, Germany, in 400 ml dH2O) as described previously (25). For double immunostaining and visualization via fluorescence microscopy, fluorescein-labeled secondary antibodies were used (Alexa Fluor 488 anti-mouse IgG, dilution 1:200, and Alexa Fluor 568 anti-rabbit IgG, dilution 1:200; both Invitrogen, Karlsruhe, Germany); all cells were additionally counterstained with Hoechst 33342 (Life Technologies, Darmstadt, Germany). Sections were co-stained for GFP and iron particles in order to verify that transplanted NSCs co-expressed the two labels (Fig. 2 (C)), and for CD68 and iron particles to identify phagocytes that took up the label (Fig. 5(F) later). NSC migration was quantified in consecutive slices by counting GFP-positive cells lying adjacent to the graft, i.e. that had migrated towards the periphery. Sums were calculated for each implantation site. To assess the extent of reactive neuroinflammation towards the implanted NSCs, CD68-positive phagocytes were counted for each section and summed for each implantation site. In parallel, the extent of MHCII immunoreactivity as a surrogate for the quantity of MHCII-expressing microglia and infiltrating macrophages was determined volumetrically using ImageJ. To assess the survival of transplanted NSCs throughout the observation period of 16 days, GFP-positive cells, irrespective of their localization – within the graft or adjacent to it – were counted for each section and summed for each implantation site.
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For all immunohistochemical measures described above, means were calculated for each group of grafts subjected to the same stimulation (anodal, cathodal, or sham). Statistical analysis Descriptive statistics were performed with Microsoft Excel 2003 (Microsoft, Redmond, WA, USA). One-way analysis of variance (ANOVA) (followed by Holm–Šidák post hoc test) and Kruskal– Wallis ANOVA on ranks (with multiple comparison procedure following Dunn’s method) were performed with SigmaPlot 11.0 for Windows (Systat Software, San Jose, CA, USA). Statistical significance was set at p < 0.05.
RESULTS We observed no difference between NSC grafts localized in the striatum and those implanted in the corpus callosum (cf. Fig. 1 (B)) with regard to all parameters assessed. In particular, the tissue volume covered by grafts did not differ between injection sites (p = 0.24), indicating that the cell solution distributed equally in both areas. As a consequence, data from the two implantation sites were merged, and further subgroup analyses were based on stimulation polarity only. Anodal tDCS increases the mobility of NSCs In the ROI-based MRI-data analysis, we did not observe any directed migration either towards or away from the stimulation electrode after tDCS of either polarity. Rather, labeled NSCs
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TDCS PROMOTES THE MOBILITY OF ENGRAFTED NEURAL STEM CELLS migrated from the center of the graft towards the periphery (cf. Fig. 2(A), (B)). However, the number of ROIs displaying such an outward migration was significantly increased after anodal tDCS compared with sham stimulation (Fig. 3(A); p < 0.05). Most ROIs that NSCs had migrated to were observed closely adjacent to the graft site (visualized in blue in Fig. 2(A), (B)) as opposed to remote from the graft (visualized in green in Figure 2(A), (B)). Undirected migratory activity was additionally quantified as ΔT2, based on the quantitative T2 values assessed by MRI before and after tDCS. Anodal tDCS caused an almost twofold increase in the migratory activity of NSCs compared with cathodal or sham tDCS (Fig. 3(B); both p < 0.05). Transplanted NSCs expressed GFP in addition to the SPIO label. We found all GFP-positive NSC to contain the iron label, irrespective of their localization within or remote from their graft, indicating an efficient in vitro SPIO labeling procedure (Figures 2 (C), 4(F)). The number of iron-labelled GFP-positive NSCs did not vary between treatment groups (Fig. 4(E)). Thus, MRI results could be verified by immunohistochemistry. After 10 days of tDCS, numerous GFP-positive NSCs were observed in the vicinity
Figure 4. Immunohistochemical validation of MRI data on NSC migration. (A) Representative image of transplanted GFP-positive NSCs in the corpus callosum after 10 days of anodal tDCS. The graft is depicted in the upper left corner. Several single cells have migrated away from the site of grafting (arrows on migrating cells). (B) GFP-positive NSCs in the striatum after 10 days of cathodal tDCS. Single cells (arrow) are found at short distances from the site of grafting. (C) GFP-positive NSCs in the striatum after 10 days of sham tDCS. Single cells (arrows) have migrated away from the site of grafting. (D) The number of GFP-positive NSCs migrating from the site of grafting towards the periphery (thus found adjacent to the graft) was significantly increased after anodal tDCS, suggesting a polarity-dependent effect of tDCS on the mobility of NSCs (mean ± SEM; *p < 0.05). (E) The number of GFP-positive NSCs labeled with SPIOs (thus detected by MRI) was similar between groups treated with different tDCS parameters (mean ± SEM; n.s.). (F) GFP-positive (brown) NSCs both within and adjacent to the graft co-labeled with SPIOs (blue).
Figure 3. Anodal tDCS increases the mobility of NSCs as quantified by MRI. (A) The number of ROIs presenting with a positive ΔT2, i.e. regions that NSCs had migrated to, was determined for each experimental group. Anodal tDCS was associated with a higher number of these regions than sham stimulation (mean ± SEM; *p < 0.05). (B) The average ΔT2 of all regions revealing migration was markedly increased by almost twofold after anodal tDCS compared with cathodal and sham stimulation (mean ± SEM; *p < 0.05).
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of the cell grafts, consistent with an undirected outward migration as demonstrated by MRI (Fig. 4(A)–(C)). NSCs migrated for up to 1.5 mm following anodal stimulation. The number of GFP-positive NSCs that had migrated from the grafts was significantly increased after anodal tDCS compared with cathodal or sham tDCS, corroborating the MRI findings (Fig. 4(D); both p < 0.05). In anodally stimulated grafts, 16% of all labeled NSCs migrated, compared with 6% after cathodal and 9% after sham tDCS (not significant, n.s.).
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M. H. KEUTERS ET AL. tDCS enhances the reactive immune response Implanted mouse NSCs caused a circumscribed reactive neuroinflammatory response in the rat brain. CD68-positive phagocytes were localized in close proximity to the cellular grafts (Fig. 5
Figure 5. Phagocytosis of iron particles by macrophages. (A) Representative image of CD68-positive phagocytes (brown), co-stained against iron with Prussian blue to mark SPIO-labeled transplanted NSCs (blue) in the corpus callosum after 10 days of anodal tDCS (scale bar valid for images (A)– (C)). (B) CD68-positive phagocytes (brown), co-stained against iron with Prussian blue to mark SPIO-labeled transplanted NSCs (blue) in the striatum after 10 days of cathodal tDCS. (C) CD68-positive phagocytes (brown), costained against iron with Prussian blue to mark SPIO-labeled transplanted NSCs (blue) in the striatum after 10 days of sham tDCS. (D) The increase in CD68-positive phagocytes after both anodal and cathodal tDCS compared with sham stimulation did not reach statistical significance (mean ± SEM; n. s.). (E) The number of SPIO-labeled CD68-positive phagocytes was similar between groups treated with different tDCS parameters (mean ± SEM; n. s.). (F) Adjacent to the graft, single CD68-positive phagocytes (brown) coexpressed the iron label (blue; asterisk), while others did not stain positive for iron (white arrow). SPIO-labeled cells negative for CD68 represent labeled NSCs (black arrow). (G) Co-staining of GFP-expressing NSCs (red) and CD68-positive phagocytes (green) shows no co-localization of the markers, indicating that inflammatory cells did not take up the GFP label by phagocytosis (exemplary image of a striatal graft from a cathodally stimulated animal; blue: nuclear counterstain with Hoechst).
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(A)–(C)). Phagocyte numbers were increased after both anodal and cathodal tDCS compared with sham stimulation; these increases did not, however, reach significance (Fig. 5(D)). Single phagocytes were detected that had internalized the iron label (Fig. 5(F), asterisk), but most of the iron label was confined to NSCs (Figures 4(F); 5(F), black arrow), and most CD68+ cells remained without iron label (Fig. 5(F), white arrow). The number of CD68-positive phagocytes with internalized iron label was not different between treatment groups (Fig. 5(E)). Importantly, we did not observe any co-expression of GFP and CD68, either in the striatal or in the callosal injection sites, or in either region of the grafts, indicating that inflammatory cells did not take up the GFP label by phagocytosis (Fig. 5(G)). Furthermore, activated microglia and macrophages expressing MCH II were observed in the area of the graft and along the injection trajectory (Fig. 6(A)–(C)). Activation of microglia and macrophages was quantified by measuring the volume of
Figure 6. Reactive activation of microglia and astroglia. (A) Representative image of activated MHCII-expressing microglia and macrophages after 10 days of anodal tDCS (scale bar valid for images (A)–(C)). (B) MHCII-expressing activated microglia and macrophages after 10 days of cathodal tDCS. (C) MHCII-expressing activated microglia and macrophages after 10 days of sham tDCS. (D) The tissue volume covered by MHCII-expressing activated microglia and macrophages was expanded after tDCS; after cathodal tDCS this increase was statistically significant (mean ± SEM; *p < 0.05). (E) Reactive astrogliosis to NSC grafting in a striatal graft after 10 days of anodal tDCS, as assessed by GFAP immunoreactivity (scale bar valid for images (E)–(G)). (F) Reactive astrogliosis in a striatal graft after 10 days of cathodal tDCS. (G) Reactive astrogliosis in a graft in the corpus callosum after 10 days of sham tDCS.
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TDCS PROMOTES THE MOBILITY OF ENGRAFTED NEURAL STEM CELLS
Figure 7. Effects of tDCS on the survival of transplanted NSCs. (A) Representative image of viable transplanted GFP+ NSCs in the corpus callosum after 10 days of anodal tDCS. The cells appear morphologically intact. (B) The increased numbers of viable GFP+ NSCs after both anodal and cathodal tDCS compared with sham stimulation was not statistically significant (mean ± SEM).
tissue covered by MHC II immunoreactivity. Activated microglia and macrophages expanded after tDCS of either polarity; a significant difference was reached for cathodal tDCS compared with sham stimulation (Fig. 6(D); p < 0.05). There was no relevant effect of tDCS on astrogliosis after cell grafting as assessed by GFAP staining (Fig. 6(E)–(G)). Since reactive neuroinflammation might lead to decreased survival of transplanted cells, viable and morphologically intact NSCs expressing GFP were counted in the entire graft plus its vicinity (Fig. 7(A)), and compared amongst stimulation groups. The number of NSCs remaining after 10 days of tDCS was higher after both anodal and cathodal tDCS compared with sham stimulation; however, this increased number was not statistically significant, falling short of proving a positive effect of tDCS on cell survival (Fig. 7(B)).
DISCUSSION MRI-based tracking of cells labeled in vitro with iron oxide particles is a well-established method to monitor in vivo the migration of NSCs in the rat brain. Iron oxide particles decrease the T2 and IT2* relaxation times, causing signal void on T2/T2*weighted MRI (for comprehensive reviews, see (26) and (27)). This technique possesses an excellent sensitivity and promotes the detection of clusters of a few hundred cells or less in vivo (19,28,29). To ensure an appropriate labeling efficiency, cells are exposed to SPIOs in combination with a transfection reagent,
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a procedure with only minimal effects on survival and differentiation potential of NSCs (19). The label is stable for over a month, although some dilution of SPIOs due to cell migration, cell division, and/or metabolism may occur (30). Note, however, that MRI cannot differentiate live from dead cells containing the iron label, and that dead cells carrying SPIOs may be taken up by phagocytes, which then incorporate the label (for review, see (31)). Thus, the hypointense contrast detected by T2-weighted MRI may also originate in part from inflammatory cells that have taken up the particles by phagocytosis. As a consequence, validation of imaging findings by immunohistochemistry is of utmost importance. We here used two complementary approaches to verify that the iron label was localized in the NSCs. First, we used NSCs stably expressing GFP as an additional label for immunohistochemistry. GFP staining independently corroborated our imaging results on NSC migration, and furthermore confirmed NSC viability over the 16 day observation period. Second, we co-stained for (i) SPIOs in NSCs, showing that particles were still present in the cells of interest, and (ii) GFP and phagocytes, showing that they did not adopt the GFP label, as well as for (iii) SPIOs in phagocytes, quantifying how many of them had taken up the MRI label. Our results demonstrate that most SPIOs remained in NSCs over the observation period. NSCs are capable of migrating over long distances towards their target if they are engaged by strong chemotactic cues. Proinflammatory cytokines expressed in the peri-infarct zone of focal cortical ischemia attract labeled NSCs from the contralateral hemisphere (29,32,33). In vitro, galvanotaxis induces a comparable attraction, directing cultured NSCs towards the cathodal pole (14,15,17). We here assessed whether galvanotaxis mediates a similar effect on NSC migration in vivo. To this end we used the same tDCS parameters as previously shown to elicit an accumulation of endogenous NSCs in the stimulated cortex (12). Current findings indicate that multi-session tDCS does not evoke the migration of engrafted NSCs over similarly long distances as chemotactic clues. However, NSCs did migrate for ~1.5 mm following anodal tDCS. This distance is comparable to stroke-directed NSC migration upon transplantation directly into the peri-infarct zone (34). Of note, not all cells left the graft’s core during anodal tDCS, some also migrated into – most likely more peripheral – ROIs of the core region (cf. Fig. 2(B)). This phenomenon of (apparent) short-range migration within the graft’s core might have been confounded by background tissue effects, such as changes in T2 due to needle-induced hemorrhage resolving over time. Overall, we observed an undirected migration of NSCs from the center of the graft towards the periphery, but no directed migration within the electric field, i.e. towards or away from the electrode. These results suggest that – with the stimulation parameters used in this study – galvanotaxis elicited by multisession tDCS is not strong enough to attract engrafted NSCs over long distances in vivo. Possibly, the surrounding tissue constrains NSC migration in vivo, while cultured cells retain their full motility and are capable of following galvanotactic cues. Moreover, we here investigated the migration of engrafted NSCs derived from a cell line in vitro. It is conceivable that endogenous NSCs might be more mobile in vivo than engrafted cells. Furthermore, a recent report suggests that iron oxide labeling might mitigate the motility of NSCs in the brain (35). Thus, further studies on the migration of NSCs are needed to address putative differences between endogenous and engrafted cells, as well as the galvanotactic effects of tDCS on iron oxidelabeled cells in culture.
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M. H. KEUTERS ET AL. Interestingly, in vitro studies have demonstrated galvanotactic effects directing cultured NSCs towards the cathode (14,15,17), while we here found that tDCS in vivo only enhanced NSC migration when the anodal pole was placed on the rat’s skull. However, this polarity-dependent migration we observed was undirected and possibly resembled a general activation of migratory disposition. Polarity-dependent effects of tDCS are well known (11,36,37), but only recent studies have started to unravel how this polarity dependence is mediated. Sun et al. suggested that different signal transduction pathways are involved for cells to respond to anodal versus cathodal stimulation (38). Tanaka et al. reported a polarity-dependent release of neurotransmitters such as dopamine (39). Further studies will need to clarify the mechanisms through which the disposition to migrate is affected in NSCs by anodal tDCS. If the previously described accumulation of NSCs in the cathodally stimulated cortex did not emanate from NSC migration from the SVZ, the most parsimonious interpretation of the data is that cathodal tDCS may elicit proliferation of NSCs residing in the cortex. Several recent reports have provided evidence that endogenous NSCs do not only reside in the major stem cell niches, but are diffusely distributed throughout the mammalian brain (40–43). These disseminated NSCs may be more difficult to detect than NSCs in the established niches, since they tend to remain quiescent and thus do not express markers associated with proliferation or neurogenesis, as is typical of NSCs in the SVZ and hippocampus (reviewed in (44)). Further studies are warranted to investigate the specific effects of tDCS on the activation and proliferation of cortical NSCs. As expected, xenograft transplantation of mouse NSCs into the rat brain evoked a reactive neuroinflammatory response involving microglia activation and phagocytic activity. In line with our previous finding that tDCS elicits an innate immune response in the naïve rat brain independent of its polarity (12), we here show that, while tDCS of either polarity enhanced the reactive neuroinflammatory response towards cellular xenografts, this effect was statistically significant after cathodal tDCS. No systemic immunosuppression was used in the current study, allowing for the detection of tDCS-induced effects of reactive neuroinflammation. Likewise, experimental studies focusing on effects other than the long-term survival of transplanted NSCs have successfully omitted a systemic immunosuppressive treatment, especially since immunosuppression interferes with any putative immunomodulatory effects of the transplanted cells and causes additional side effects (45,46). However, long-term survival of transplanted stem cells for more than ~4 weeks may only be achieved with at least a short course of systemic immunosuppression (47,48). Interestingly, our data suggest that the enhanced inflammatory response evoked by tDCS does not impact negatively on the survival of the implanted NSCs. Recent findings of Wang and colleagues even describe an anti-apoptotic effect of electric stimulation on NSCs mediated through PI3K/Akt signaling (49). Moreover, tDCS was recently shown to act in a neuroprotective way in acute stroke (50). We observed a higher number of viable NSCs after 10 days of tDCS of either polarity compared with sham stimulation; however, this effect was not statistically significant, falling short of proving a positive effect of tDCS on cell survival. Further studies are required to investigate a potential role for tDCS in the protection of transplanted (stem) cells. We implanted NSCs into two different sites in the rat brain, offering a differential environment. The striatal injection site
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was chosen close to the SVZ as a major NSC niche; the corpus callosum was chosen to potentially support cell migration along its fiber tracts. Our data suggest that, at least under our experimental conditions, transplanted NSCs did not preferentially migrate along the corpus callosal fiber tracts. Another possibility, however, could be that differences in NSC migration between the implantation sites were not detected within the chosen observation period. In summary, we observed that the migratory activity of labeled NSCs was affected by tDCS in a polarity-dependent manner. However, galvanotaxis elicited by multi-session tDCS was not strong enough to attract engrafted NSCs over long distances, suggesting that the previously observed polarity-dependent accumulation of endogenous NSCs in the cortex might originate from a local proliferation. Cathodal tDCS enhanced the reactive neuroinflammatory response towards cellular xenografts, but this did not impact negatively on the survival of implanted NSCs. The results obtained here add to our understanding of the neurobiological mechanisms underlying tDCS, which may help to develop a targeted and sustainable application of tDCS in patients suffering from neurological disease, e.g. stroke.
Acknowledgements We thank Claudia Drapatz, Andreas Beyrau, Michael Diedenhofen and Karina Peters for excellent technical support. This work was supported by the Marga und Walter Boll-Stiftung (no 210-1212), by the Koeln Fortune Program/Faculty of Medicine, University of Cologne (no 106/2012), and by the EU-FP7 programs TargetBraIn (no HEALTH-F2-2012-279017) and BrainPath (no 612360).
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Revised: 9 November 2014,
Accepted: 14 November 2014,
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/nbm.3244
Transcranial direct current stimulation promotes the mobility of engrafted NSCs in the rat brain Meike Hedwig Keutersa,b, Markus Aswendtb, Annette Tennstaedtb, Dirk Wiedermannb, Anton Pikhovycha,b, Steffen Rotthuesa, Gereon Rudolf Finka,b, Michael Schroetera,b, Mathias Hoehnb,c and Maria Adele Ruegera,b,d* Transcranial direct current stimulation (tDCS) is used in numerous clinical studies and considered an effective and versatile add-on therapy in neurorehabilitation. To date, however, the underlying neurobiological mechanisms remain elusive. In a rat model of tDCS, we recently observed a polarity-dependent accumulation of endogenous neural stem cells (NSCs) in the stimulated cortex. Based upon these findings, we hypothesized that tDCS may exert a direct migratory effect on endogenous NSCs towards the stimulated cortex. Using noninvasive imaging, we here investigated whether tDCS may also cause a directed migration of engrafted NSCs. Murine NSCs were labeled with superparamagnetic particles of iron oxide (SPIOs) and implanted into rat striatum and corpus callosum. MRI was performed (i) immediately after implantation and (ii) after 10 tDCS sessions of anodal or cathodal polarity. Sham-stimulated rats served as control. Imaging results were validated ex vivo using immunohistochemistry. Overall migratory activity of NSCs almost doubled after anodal tDCS. However, no directed migration within the electric field (i.e. towards or away from the electrode) could be observed. Rather, an undirected outward migration from the center of the graft was detected. Xenograft transplantation induced a neuroinflammatory response that was significantly enhanced following cathodal tDCS. This inflammatory response did not impact negatively on the survival of implanted NSCs. Data suggest that anodal tDCS increases the undirected migratory activity of implanted NSCs. Since the electric field did not guide implanted NSCs over large distances, previously observed polarity-dependent accumulation of endogenous NSCs in the cortex might have originated from local proliferation. Results enhance our understanding of the neurobiological mechanisms underlying tDCS, and may thereby help to develop a targeted and sustainable application of tDCS in clinical practice. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: migration; galvanotaxis; MRI; superparamagnetic particles of iron oxide; neuroinflammation; phagocytes; microglia
INTRODUCTION Transcranial direct current stimulation (tDCS) as a non-invasive neuromodulatory technique induces long-lasting alterations of cortical excitability in both animals (1) and humans (2). tDCS has been used to (i) enhance the effects of motor learning in
* Correspondence to: Maria Adele Rueger, Department of Neurology, University Hospital of Cologne, Cologne, Germany. E-mail: [email protected] a M. H. Keuters, A. Pikhovych, S. Rotthues, G. R. Fink, M. Schroeter, M. A. Rueger Department of Neurology, University Hospital of Cologne, Cologne, Germany b M. H. Keuters, M. Aswendt, A. Tennstaedt, D. Wiedermann, A. Pikhovych, G. R. Fink, M. Schroeter, M. Hoehn, M. A. Rueger In-vivo-NMR Laboratory, Max Planck Institute for Neurological Research, Cologne, Germany c M. Hoehn Department of Radiology, Leiden University Medical School, Leiden, The Netherlands
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healthy volunteers (3) and (ii) ameliorate functional deficits following stroke, including motor deficits (4), aphasia (5), and neglect (6). Despite the current use of tDCS in numerous clinical studies, to date the basic mechanisms and details underlying the neurobiological effects of tDCS remain unknown (for comprehensive reviews, see (7) and (8)). Recent reports provide evidence that the electrical stimulation exceeds primary
d M. A. Rueger Cognitive Neuroscience, Institute of Neuroscience and Medicine (INM3), Research Centre Juelich, Juelich, Germany Abbreviations used: ANOVA, analysis of variance; DAB, diaminobenzidine; ES cells, embryonic stem cells; FMRIB, Oxford Centre for Functional Magnetic Resonance Imaging of the Brain; FOV, field of view; FSL, FMRIB’s Software Library; GE-EPI, gradient echo–echo planar imaging; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; mAb, monoclonal antibody; MHC, major histocompatibility complex; NSC, neural stem cell; n.s., not significant; PBS, phosphate-buffered saline; ROI, region of interest; SPIO, superparamagnetic particle of iron oxide; SVZ, subventricular zone; tDCS, transcranial direct current stimulation; TE, echo time; TR, repetition time.
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M. H. KEUTERS ET AL. electrophysiological effects on neurons, e.g., it additionally modulates responses of non-neuronal cells in the brain (9–11). In a rat model of tDCS, we recently demonstrated a pro-inflammatory effect of both cathodal and anodal tDCS as well as a polarity-dependent accumulation of endogenous neural stem cells (NSCs) in the stimulated cortex (12). Data suggest that beneficial effects of tDCS in stroke may at least in part result from NSC activation and the modulation of neuroinflammation. Regarding a putative polarity-dependent NSC accumulation in the stimulated cortex, we hypothesized that cathodal tDCS might induce a directed migration of endogenous NSCs from the subventricular zone (SVZ, i.e. the major NSC niche) towards the cortex. Migration of cells in culture can be induced by electric fields, a phenomenon referred to as galvanotaxis (13). Among many other cell types, rodent neural progenitor cells (14,15), human embryonic stem (ES) cells (16), and human ES-cell derived NSCs (17) have all been demonstrated to migrate in an electric field in vitro. However, whether such effects can also be observed in vivo has not yet been investigated. Accordingly, we here explored the migration of transplanted contrast-agent-labeled NSC towards the cortex upon tDCS stimulation, using MRI in vivo as well as immunohistochemistry ex vivo.
Stereotactic implantation of NSCs Each rat received two grafts of labeled NSCs: one in the right striatum, the other one in the left corpus callosum (Fig. 1(B)). For implantation, anesthetized rats were placed in a stereotactic rat frame equipped with a digital positioning system (Digital Lab Standard, Stoelting, Wood Dale, IL, USA). Carprofen (4 mg ml 1; Pfizer/Zoetis, Berlin, Germany) was injected subcutaneously as analgetic. After incising the skin and removing the periosteum, a burr hole was drilled over the first implantation site in the right striatum, using a dental drill (Bien-Air 810 Technobox, Bien-Air Dental, Bienne, Switzerland). A microsyringe (5 μl volume; Hamilton Messtechnik, Hoechst, Germany) was slowly inserted to the stereotactic coordinates bregma +1.0 mm AP, 2.5 mm ML, 5.0 mm VD. After positioning the needle, a 2 μl suspension of 300 000 labeled NSCs were injected at 0.5 μl min 1 using an automatic microinjection control system (UltraMicroPump, World Precision Instruments, Sarasota, FL, USA). After injection, the needle was left in place for 5 min to allow the cell suspension to distribute evenly within the tissue. Then, the needle was slowly withdrawn at 1 mm min 1. The same injection procedure was repeated for the second implantation site, i.e. the left corpus callosum, at the coordinates +1.0 mm AP, +2 mm ML, 3.0 mm VD. After the procedure, the skin was closed with single sutures.
MRI
EXPERIMENTAL METHODS Cell culture and labeling The NSC line D3WT_N2Euro was established from the murine ES cell line D3 and retrovirally transduced to stably express green fluorescent protein (GFP) cloned from the copepod Pontellina plumata as described previously (18). Adherent NSCs were grown in N2Euro medium (Biozol, Siziano, Italy) in the presence of 5 ng ml 1 EGF and 10 ng ml 1 FGF-2 (both PeproTech, Hamburg, Germany) to keep them in an undifferentiated state. Prior to transplantation, cells were labeled with superparamagnetic particles of iron oxide (SPIOs) with a mean diameter of 150 nm, using the clinically established contrast agent Endorem (9 μl ml ml 1; Guerbet, France) and the transfection reagent Metafectene (1 μl ml 1; Biontex, Martinsried, Germany) as described previously (19). In brief, NSCs were incubated with Endorem plus Metafectene for 24 hours, then washed with phosphate-buffered saline (PBS; Invitrogen, Darmstadt, Germany), detached from plates with Accutase (PAA, Pasching, Austria), and re-suspended in Hank’s Balanced Salt Solution (Invitrogen) at 150 000 cells μl 1 for immediate transplantation.
Immediately after the implantation of labeled NSCs, MRI was performed to determine the exact position of each graft. The rat was placed in a special rat holder designed for MRI (medres, Cologne, Germany), including an in-house feedback control system for
Animals and general aspects of surgery All animal procedures were carried out in accordance with the German Laws for Animal Protection and were approved by the local animal care committee as well as local governmental authorities (LANUV NRW, no 87-51.04.2010.A332). Spontaneously breathing male adult Wistar rats weighing 290–310 g were anesthetized with 5% isoflurane, and maintained with 2.5% isoflurane in a 65%/35% nitrous oxide/oxygen atmosphere. Throughout all surgical procedures, body temperature was maintained at 37 °C with a feedback-controlled heating pad. For a timeline of experiments see Figure 1(A).
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Figure 1. Experimental setup to monitor the migration of NSCs. (A) Timeline of experiments. (B) GFP-positive NSCs labeled with SPIOs were stereotactically implanted into two distinct sites, the right striatum (a) and the left corpus callosum (b), of adult rats. (C) For tDCS, an epicranial electrode was mounted on the rat skull over the striatal implantation site, as indicated by the red dot.
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TDCS PROMOTES THE MOBILITY OF ENGRAFTED NEURAL STEM CELLS body temperature. MRI was performed on a BioSpec 11.7 T dedicated animal scanner system (Bruker BioSpin, Ettlingen, Germany) with a 16 cm horizontal bore magnet, equipped with actively shielded gradient coils (BGA9S, 750 mT m 1; Bruker). RF transmission was achieved with a birdcage quadrature resonator coil (72 mm diameter), and the signal was detected with quadrature rat brain surface coils (Bruker). A single-shot gradient echo–echo planar imaging (GE-EPI) pilot was used for shimming, as described previously (20). Two scans were then obtained for each animal. The first was a T2-weighted image as rapid acquisition with relaxation enhancement, using the following protocol: field of view (FOV) 3.2 cm, matrix 256 × 256, slice thickness 0.5 mm, echo time (TE) 32.5 ms, repetition time (TR) 6500 ms. Second, a T2 map was generated using a multislice multiecho spin echo sequence with the parameters FOV 3.2 cm, matrix 160 × 160, slice thickness 0.5 mm, TE1 = 10 ms, TE2 = 20 ms, TR = 4000 ms. MRI was repeated two days after the last tDCS stimulation to determine the exact location of the transplanted NSC after tDCS, and thus to assess a potential tDCS-induced migration. tDCS Starting one day after NSC implantation, multi-session tDCS was performed as described previously (12). In brief, 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 stereotactic coordinates bregma AP +2.0 mm, ML +2.0 mm; the electrode was left in place for the entire experiment (Fig. 1(C)). Animals were randomized to receive 10 days of tDCS with either cathodal or anodal polarity; a third group of rats was sham-stimulated for control. tDCS was repeated daily using the same parameters for five consecutive days, followed by a tDCS-free interval of two days; then animals were subjected to tDCS for five more days, resulting in 10 days tDCS in total. tDCS was applied continuously for 15 min at 500 μA using a constant current stimulator (CX-6650, Schneider Electronics, Gleichen, Germany) under isoflurane anesthesia. For sham stimulation, rats were also anesthetized for 15 min, albeit without connecting them to the stimulator. After each tDCS session, animals were allowed to recover in their home cages with access to food and water ad libitum. MRI post-processing and data analysis In order to assess the migration of implanted NSCs, the T2 maps acquired before and after tDCS were accurately co-registered for each animal. First, images were converted into NIFTI format (Neuroimaging Informatics Technology Initiative; http://nifty. nimh.nih.gov), scaling up the voxel size by a factor of 10 to reach a human-like voxel size for correct further processing. Next, skin and skull were removed from the images using the Brain Extraction Tool in the software FSL (Oxford Centre for Functional Magnetic Resonance Imaging of the Brain, FMRIB, Software Library, Oxford, UK), followed by visual inspection and manual correction, if necessary (21). The resulting brain-only images were segmented using the FMRIB’s Automated Segmentation Tool of FSL, also correcting for spatial intensity variations (22). Next, a selected T2-weighted and manually brain-extracted dataset with ideal positioning and minimal artefacts was selected as the template brain. Each segmented image was co-registered to this
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template brain in order to ensure identical positioning: for all animals, the T2-weighted image from time point 1 was first coregistered to this template; in a second step, time point 2 was co-registered on a subject basis to imaging time point 1, providing an alignment with the template brain, using the FMRIB’s Linear Image Registration Tool of FSL (23,24). The transformation matrices were applied to the T2 maps accordingly. Migration of labeled cells was quantified on a region of interest (ROI)-based analysis using the ImageJ software (http://imagej.nih. gov/ij). Circular ROIs with a diameter of 500 μm were manually drawn on each slice to cover the area of the graft as well as the adjacent tissue three dimensionally. The number of ROIs for each individual implantation site was adapted to its spatial extent, varying slightly between animals. This enabled the distinction between ROIs within the graft, adjacent to the graft, and remote from the graft (Fig. 2(A), (A′)). ROIs on T2 maps were used to determine T2 values, which correlate with the number of labeled cells, since the iron label generates a quantifiable decrease in transverse relaxation time T2 (19). ΔT2 was calculated between the two scans according to ΔT2 = T2 no 1 (before tDCS) – T2 no 2 (after tDCS). Thus, a negative ΔT2 indicated a loss of iron oxide-labeled NSCs at this position, whereas a positive ΔT2 was associated with a higher number of NSCs following tDCS. We determined T2 stability between different animals and imaging sessions in order to correct for methodological data variability. A standard deviation was determined for each animal by comparing T2 values from reference ROIs for scans no 1 and no 2 (before and after tDCS). These reference ROIs were defined in regions remote from the cell graft location, namely in the striatum and the cortex on five consecutive T2 map slices, and had the same diameter (500 μm) as data ROIs. The standard deviation σ for each animal was calculated from these 10 reference ROIs. Twice the standard deviation (2σ) was then used to evaluate statistical significances. Thus, ΔT2 < 2σ indicated a significant migration of labeled NSCs away from the ROI, while ΔT2 > 2σ suggested that NSCs had migrated into the ROI (Fig. 2(B)). The number of ROIs with ΔT2 > 2σ was determined for each graft. Additionally, a mean ΔT2 was established for each graft, including only those ΔT2 values > 2σ. For both measures of migration, means were calculated for each group of grafts subjected to the same stimulation (anodal n = 4, cathodal n = 6, or sham n = 6). Immunohistochemistry Rats were perfused with PBS under deep anesthesia, followed by 4% paraformaldehyde for tissue fixation. The brains were removed, frozen in 2-methylbutane, and stored at 80 °C. Coronal brain sections with a slice thickness of 10 μm were cut at 500 μm intervals and stained with the following primary antibodies: monoclonal antibody (mAb) against rat CD68 (dilution 1:2000; MCA341, AbD Serotec, Oxford, UK) to identify phagocytic microglia/macrophages, mAb against rat major histocompatibility complex (MHC) class II (dilution 1:400; MCA46R, AbD Serotec, Oxford, UK) to assess activated microglia and macrophages, polyclonal antibody against turbo-GFP (dilution: 1: 5000; AB511, Evrogen, Moscow, Russia) to detect transplanted NSCs, or mAb against glial fibrillary acidic protein (GFAP, clone GA-5; dilution 1:1000; MAB360, Millipore, Schwalbach, Germany) to visualize astrogliosis. For visualization using light microscopy, the ABC Elite kit (Vector Laboratories, Burlingame, CA, USA), with diaminobenzidine (DAB; Sigma-Aldrich, Munich, Germany) as the final reaction product, was used. For co-staining of iron particles for
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Figure 2. MRI detects subtle migratory behavior of transplanted NSCs into the periphery. (A) Based on co-registered T2 maps acquired before and after tDCS, multiple ROIs were defined within the graft, exemplarily depicted for the implantation site in the corpus callosum (within the red ellipse, compare excerpt for higher magnification), the adjacent tissue (blue), and more remote areas (green), to assess the migration of implanted NSCs into the periphery; scale bars represent 1 mm (overview image) and 500 μm (magnified excerpt). (A′) Magnification of the left (striatal) excerpt defined in (A), depicting a representative example for ROI definition within the striatal graft, color-coded in analogy to (A); the scale bar represents 500 μm. (B) Changes in T2 after anodal tDCS in one representative graft. Each data point represents ΔT2 within a specific ROI; ROIs are color-coded by their proximity to the graft according to (A). Values within 2σ of the data are underlaid in grey. Values above 2σ indicate a significant increase in T2 negativity after tDCS, suggesting that NSCs had migrated into the ROI. Regions from which NSCs had migrated away are characterized by values below 2σ. (C) Immunohistochemical staining of the striatal graft from the animal depicted in (A). GFP-positive (brown) NSCs are co-labeled with SPIOs (blue) in the entire graft; the scale bar represents 250 μm.
light microscopy, Prussian blue solution was applied for 20 min following DAB visualization (16 ml 2% hydrochloric acid plus 8 g potassium hexacyanoferrate II 3-hydrate, both Roth, Karlsruhe, Germany, in 400 ml dH2O) as described previously (25). For double immunostaining and visualization via fluorescence microscopy, fluorescein-labeled secondary antibodies were used (Alexa Fluor 488 anti-mouse IgG, dilution 1:200, and Alexa Fluor 568 anti-rabbit IgG, dilution 1:200; both Invitrogen, Karlsruhe, Germany); all cells were additionally counterstained with Hoechst 33342 (Life Technologies, Darmstadt, Germany). Sections were co-stained for GFP and iron particles in order to verify that transplanted NSCs co-expressed the two labels (Fig. 2 (C)), and for CD68 and iron particles to identify phagocytes that took up the label (Fig. 5(F) later). NSC migration was quantified in consecutive slices by counting GFP-positive cells lying adjacent to the graft, i.e. that had migrated towards the periphery. Sums were calculated for each implantation site. To assess the extent of reactive neuroinflammation towards the implanted NSCs, CD68-positive phagocytes were counted for each section and summed for each implantation site. In parallel, the extent of MHCII immunoreactivity as a surrogate for the quantity of MHCII-expressing microglia and infiltrating macrophages was determined volumetrically using ImageJ. To assess the survival of transplanted NSCs throughout the observation period of 16 days, GFP-positive cells, irrespective of their localization – within the graft or adjacent to it – were counted for each section and summed for each implantation site.
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For all immunohistochemical measures described above, means were calculated for each group of grafts subjected to the same stimulation (anodal, cathodal, or sham). Statistical analysis Descriptive statistics were performed with Microsoft Excel 2003 (Microsoft, Redmond, WA, USA). One-way analysis of variance (ANOVA) (followed by Holm–Šidák post hoc test) and Kruskal– Wallis ANOVA on ranks (with multiple comparison procedure following Dunn’s method) were performed with SigmaPlot 11.0 for Windows (Systat Software, San Jose, CA, USA). Statistical significance was set at p < 0.05.
RESULTS We observed no difference between NSC grafts localized in the striatum and those implanted in the corpus callosum (cf. Fig. 1 (B)) with regard to all parameters assessed. In particular, the tissue volume covered by grafts did not differ between injection sites (p = 0.24), indicating that the cell solution distributed equally in both areas. As a consequence, data from the two implantation sites were merged, and further subgroup analyses were based on stimulation polarity only. Anodal tDCS increases the mobility of NSCs In the ROI-based MRI-data analysis, we did not observe any directed migration either towards or away from the stimulation electrode after tDCS of either polarity. Rather, labeled NSCs
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TDCS PROMOTES THE MOBILITY OF ENGRAFTED NEURAL STEM CELLS migrated from the center of the graft towards the periphery (cf. Fig. 2(A), (B)). However, the number of ROIs displaying such an outward migration was significantly increased after anodal tDCS compared with sham stimulation (Fig. 3(A); p < 0.05). Most ROIs that NSCs had migrated to were observed closely adjacent to the graft site (visualized in blue in Fig. 2(A), (B)) as opposed to remote from the graft (visualized in green in Figure 2(A), (B)). Undirected migratory activity was additionally quantified as ΔT2, based on the quantitative T2 values assessed by MRI before and after tDCS. Anodal tDCS caused an almost twofold increase in the migratory activity of NSCs compared with cathodal or sham tDCS (Fig. 3(B); both p < 0.05). Transplanted NSCs expressed GFP in addition to the SPIO label. We found all GFP-positive NSC to contain the iron label, irrespective of their localization within or remote from their graft, indicating an efficient in vitro SPIO labeling procedure (Figures 2 (C), 4(F)). The number of iron-labelled GFP-positive NSCs did not vary between treatment groups (Fig. 4(E)). Thus, MRI results could be verified by immunohistochemistry. After 10 days of tDCS, numerous GFP-positive NSCs were observed in the vicinity
Figure 4. Immunohistochemical validation of MRI data on NSC migration. (A) Representative image of transplanted GFP-positive NSCs in the corpus callosum after 10 days of anodal tDCS. The graft is depicted in the upper left corner. Several single cells have migrated away from the site of grafting (arrows on migrating cells). (B) GFP-positive NSCs in the striatum after 10 days of cathodal tDCS. Single cells (arrow) are found at short distances from the site of grafting. (C) GFP-positive NSCs in the striatum after 10 days of sham tDCS. Single cells (arrows) have migrated away from the site of grafting. (D) The number of GFP-positive NSCs migrating from the site of grafting towards the periphery (thus found adjacent to the graft) was significantly increased after anodal tDCS, suggesting a polarity-dependent effect of tDCS on the mobility of NSCs (mean ± SEM; *p < 0.05). (E) The number of GFP-positive NSCs labeled with SPIOs (thus detected by MRI) was similar between groups treated with different tDCS parameters (mean ± SEM; n.s.). (F) GFP-positive (brown) NSCs both within and adjacent to the graft co-labeled with SPIOs (blue).
Figure 3. Anodal tDCS increases the mobility of NSCs as quantified by MRI. (A) The number of ROIs presenting with a positive ΔT2, i.e. regions that NSCs had migrated to, was determined for each experimental group. Anodal tDCS was associated with a higher number of these regions than sham stimulation (mean ± SEM; *p < 0.05). (B) The average ΔT2 of all regions revealing migration was markedly increased by almost twofold after anodal tDCS compared with cathodal and sham stimulation (mean ± SEM; *p < 0.05).
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of the cell grafts, consistent with an undirected outward migration as demonstrated by MRI (Fig. 4(A)–(C)). NSCs migrated for up to 1.5 mm following anodal stimulation. The number of GFP-positive NSCs that had migrated from the grafts was significantly increased after anodal tDCS compared with cathodal or sham tDCS, corroborating the MRI findings (Fig. 4(D); both p < 0.05). In anodally stimulated grafts, 16% of all labeled NSCs migrated, compared with 6% after cathodal and 9% after sham tDCS (not significant, n.s.).
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M. H. KEUTERS ET AL. tDCS enhances the reactive immune response Implanted mouse NSCs caused a circumscribed reactive neuroinflammatory response in the rat brain. CD68-positive phagocytes were localized in close proximity to the cellular grafts (Fig. 5
Figure 5. Phagocytosis of iron particles by macrophages. (A) Representative image of CD68-positive phagocytes (brown), co-stained against iron with Prussian blue to mark SPIO-labeled transplanted NSCs (blue) in the corpus callosum after 10 days of anodal tDCS (scale bar valid for images (A)– (C)). (B) CD68-positive phagocytes (brown), co-stained against iron with Prussian blue to mark SPIO-labeled transplanted NSCs (blue) in the striatum after 10 days of cathodal tDCS. (C) CD68-positive phagocytes (brown), costained against iron with Prussian blue to mark SPIO-labeled transplanted NSCs (blue) in the striatum after 10 days of sham tDCS. (D) The increase in CD68-positive phagocytes after both anodal and cathodal tDCS compared with sham stimulation did not reach statistical significance (mean ± SEM; n. s.). (E) The number of SPIO-labeled CD68-positive phagocytes was similar between groups treated with different tDCS parameters (mean ± SEM; n. s.). (F) Adjacent to the graft, single CD68-positive phagocytes (brown) coexpressed the iron label (blue; asterisk), while others did not stain positive for iron (white arrow). SPIO-labeled cells negative for CD68 represent labeled NSCs (black arrow). (G) Co-staining of GFP-expressing NSCs (red) and CD68-positive phagocytes (green) shows no co-localization of the markers, indicating that inflammatory cells did not take up the GFP label by phagocytosis (exemplary image of a striatal graft from a cathodally stimulated animal; blue: nuclear counterstain with Hoechst).
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(A)–(C)). Phagocyte numbers were increased after both anodal and cathodal tDCS compared with sham stimulation; these increases did not, however, reach significance (Fig. 5(D)). Single phagocytes were detected that had internalized the iron label (Fig. 5(F), asterisk), but most of the iron label was confined to NSCs (Figures 4(F); 5(F), black arrow), and most CD68+ cells remained without iron label (Fig. 5(F), white arrow). The number of CD68-positive phagocytes with internalized iron label was not different between treatment groups (Fig. 5(E)). Importantly, we did not observe any co-expression of GFP and CD68, either in the striatal or in the callosal injection sites, or in either region of the grafts, indicating that inflammatory cells did not take up the GFP label by phagocytosis (Fig. 5(G)). Furthermore, activated microglia and macrophages expressing MCH II were observed in the area of the graft and along the injection trajectory (Fig. 6(A)–(C)). Activation of microglia and macrophages was quantified by measuring the volume of
Figure 6. Reactive activation of microglia and astroglia. (A) Representative image of activated MHCII-expressing microglia and macrophages after 10 days of anodal tDCS (scale bar valid for images (A)–(C)). (B) MHCII-expressing activated microglia and macrophages after 10 days of cathodal tDCS. (C) MHCII-expressing activated microglia and macrophages after 10 days of sham tDCS. (D) The tissue volume covered by MHCII-expressing activated microglia and macrophages was expanded after tDCS; after cathodal tDCS this increase was statistically significant (mean ± SEM; *p < 0.05). (E) Reactive astrogliosis to NSC grafting in a striatal graft after 10 days of anodal tDCS, as assessed by GFAP immunoreactivity (scale bar valid for images (E)–(G)). (F) Reactive astrogliosis in a striatal graft after 10 days of cathodal tDCS. (G) Reactive astrogliosis in a graft in the corpus callosum after 10 days of sham tDCS.
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TDCS PROMOTES THE MOBILITY OF ENGRAFTED NEURAL STEM CELLS
Figure 7. Effects of tDCS on the survival of transplanted NSCs. (A) Representative image of viable transplanted GFP+ NSCs in the corpus callosum after 10 days of anodal tDCS. The cells appear morphologically intact. (B) The increased numbers of viable GFP+ NSCs after both anodal and cathodal tDCS compared with sham stimulation was not statistically significant (mean ± SEM).
tissue covered by MHC II immunoreactivity. Activated microglia and macrophages expanded after tDCS of either polarity; a significant difference was reached for cathodal tDCS compared with sham stimulation (Fig. 6(D); p < 0.05). There was no relevant effect of tDCS on astrogliosis after cell grafting as assessed by GFAP staining (Fig. 6(E)–(G)). Since reactive neuroinflammation might lead to decreased survival of transplanted cells, viable and morphologically intact NSCs expressing GFP were counted in the entire graft plus its vicinity (Fig. 7(A)), and compared amongst stimulation groups. The number of NSCs remaining after 10 days of tDCS was higher after both anodal and cathodal tDCS compared with sham stimulation; however, this increased number was not statistically significant, falling short of proving a positive effect of tDCS on cell survival (Fig. 7(B)).
DISCUSSION MRI-based tracking of cells labeled in vitro with iron oxide particles is a well-established method to monitor in vivo the migration of NSCs in the rat brain. Iron oxide particles decrease the T2 and IT2* relaxation times, causing signal void on T2/T2*weighted MRI (for comprehensive reviews, see (26) and (27)). This technique possesses an excellent sensitivity and promotes the detection of clusters of a few hundred cells or less in vivo (19,28,29). To ensure an appropriate labeling efficiency, cells are exposed to SPIOs in combination with a transfection reagent,
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a procedure with only minimal effects on survival and differentiation potential of NSCs (19). The label is stable for over a month, although some dilution of SPIOs due to cell migration, cell division, and/or metabolism may occur (30). Note, however, that MRI cannot differentiate live from dead cells containing the iron label, and that dead cells carrying SPIOs may be taken up by phagocytes, which then incorporate the label (for review, see (31)). Thus, the hypointense contrast detected by T2-weighted MRI may also originate in part from inflammatory cells that have taken up the particles by phagocytosis. As a consequence, validation of imaging findings by immunohistochemistry is of utmost importance. We here used two complementary approaches to verify that the iron label was localized in the NSCs. First, we used NSCs stably expressing GFP as an additional label for immunohistochemistry. GFP staining independently corroborated our imaging results on NSC migration, and furthermore confirmed NSC viability over the 16 day observation period. Second, we co-stained for (i) SPIOs in NSCs, showing that particles were still present in the cells of interest, and (ii) GFP and phagocytes, showing that they did not adopt the GFP label, as well as for (iii) SPIOs in phagocytes, quantifying how many of them had taken up the MRI label. Our results demonstrate that most SPIOs remained in NSCs over the observation period. NSCs are capable of migrating over long distances towards their target if they are engaged by strong chemotactic cues. Proinflammatory cytokines expressed in the peri-infarct zone of focal cortical ischemia attract labeled NSCs from the contralateral hemisphere (29,32,33). In vitro, galvanotaxis induces a comparable attraction, directing cultured NSCs towards the cathodal pole (14,15,17). We here assessed whether galvanotaxis mediates a similar effect on NSC migration in vivo. To this end we used the same tDCS parameters as previously shown to elicit an accumulation of endogenous NSCs in the stimulated cortex (12). Current findings indicate that multi-session tDCS does not evoke the migration of engrafted NSCs over similarly long distances as chemotactic clues. However, NSCs did migrate for ~1.5 mm following anodal tDCS. This distance is comparable to stroke-directed NSC migration upon transplantation directly into the peri-infarct zone (34). Of note, not all cells left the graft’s core during anodal tDCS, some also migrated into – most likely more peripheral – ROIs of the core region (cf. Fig. 2(B)). This phenomenon of (apparent) short-range migration within the graft’s core might have been confounded by background tissue effects, such as changes in T2 due to needle-induced hemorrhage resolving over time. Overall, we observed an undirected migration of NSCs from the center of the graft towards the periphery, but no directed migration within the electric field, i.e. towards or away from the electrode. These results suggest that – with the stimulation parameters used in this study – galvanotaxis elicited by multisession tDCS is not strong enough to attract engrafted NSCs over long distances in vivo. Possibly, the surrounding tissue constrains NSC migration in vivo, while cultured cells retain their full motility and are capable of following galvanotactic cues. Moreover, we here investigated the migration of engrafted NSCs derived from a cell line in vitro. It is conceivable that endogenous NSCs might be more mobile in vivo than engrafted cells. Furthermore, a recent report suggests that iron oxide labeling might mitigate the motility of NSCs in the brain (35). Thus, further studies on the migration of NSCs are needed to address putative differences between endogenous and engrafted cells, as well as the galvanotactic effects of tDCS on iron oxidelabeled cells in culture.
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M. H. KEUTERS ET AL. Interestingly, in vitro studies have demonstrated galvanotactic effects directing cultured NSCs towards the cathode (14,15,17), while we here found that tDCS in vivo only enhanced NSC migration when the anodal pole was placed on the rat’s skull. However, this polarity-dependent migration we observed was undirected and possibly resembled a general activation of migratory disposition. Polarity-dependent effects of tDCS are well known (11,36,37), but only recent studies have started to unravel how this polarity dependence is mediated. Sun et al. suggested that different signal transduction pathways are involved for cells to respond to anodal versus cathodal stimulation (38). Tanaka et al. reported a polarity-dependent release of neurotransmitters such as dopamine (39). Further studies will need to clarify the mechanisms through which the disposition to migrate is affected in NSCs by anodal tDCS. If the previously described accumulation of NSCs in the cathodally stimulated cortex did not emanate from NSC migration from the SVZ, the most parsimonious interpretation of the data is that cathodal tDCS may elicit proliferation of NSCs residing in the cortex. Several recent reports have provided evidence that endogenous NSCs do not only reside in the major stem cell niches, but are diffusely distributed throughout the mammalian brain (40–43). These disseminated NSCs may be more difficult to detect than NSCs in the established niches, since they tend to remain quiescent and thus do not express markers associated with proliferation or neurogenesis, as is typical of NSCs in the SVZ and hippocampus (reviewed in (44)). Further studies are warranted to investigate the specific effects of tDCS on the activation and proliferation of cortical NSCs. As expected, xenograft transplantation of mouse NSCs into the rat brain evoked a reactive neuroinflammatory response involving microglia activation and phagocytic activity. In line with our previous finding that tDCS elicits an innate immune response in the naïve rat brain independent of its polarity (12), we here show that, while tDCS of either polarity enhanced the reactive neuroinflammatory response towards cellular xenografts, this effect was statistically significant after cathodal tDCS. No systemic immunosuppression was used in the current study, allowing for the detection of tDCS-induced effects of reactive neuroinflammation. Likewise, experimental studies focusing on effects other than the long-term survival of transplanted NSCs have successfully omitted a systemic immunosuppressive treatment, especially since immunosuppression interferes with any putative immunomodulatory effects of the transplanted cells and causes additional side effects (45,46). However, long-term survival of transplanted stem cells for more than ~4 weeks may only be achieved with at least a short course of systemic immunosuppression (47,48). Interestingly, our data suggest that the enhanced inflammatory response evoked by tDCS does not impact negatively on the survival of the implanted NSCs. Recent findings of Wang and colleagues even describe an anti-apoptotic effect of electric stimulation on NSCs mediated through PI3K/Akt signaling (49). Moreover, tDCS was recently shown to act in a neuroprotective way in acute stroke (50). We observed a higher number of viable NSCs after 10 days of tDCS of either polarity compared with sham stimulation; however, this effect was not statistically significant, falling short of proving a positive effect of tDCS on cell survival. Further studies are required to investigate a potential role for tDCS in the protection of transplanted (stem) cells. We implanted NSCs into two different sites in the rat brain, offering a differential environment. The striatal injection site
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was chosen close to the SVZ as a major NSC niche; the corpus callosum was chosen to potentially support cell migration along its fiber tracts. Our data suggest that, at least under our experimental conditions, transplanted NSCs did not preferentially migrate along the corpus callosal fiber tracts. Another possibility, however, could be that differences in NSC migration between the implantation sites were not detected within the chosen observation period. In summary, we observed that the migratory activity of labeled NSCs was affected by tDCS in a polarity-dependent manner. However, galvanotaxis elicited by multi-session tDCS was not strong enough to attract engrafted NSCs over long distances, suggesting that the previously observed polarity-dependent accumulation of endogenous NSCs in the cortex might originate from a local proliferation. Cathodal tDCS enhanced the reactive neuroinflammatory response towards cellular xenografts, but this did not impact negatively on the survival of implanted NSCs. The results obtained here add to our understanding of the neurobiological mechanisms underlying tDCS, which may help to develop a targeted and sustainable application of tDCS in patients suffering from neurological disease, e.g. stroke.
Acknowledgements We thank Claudia Drapatz, Andreas Beyrau, Michael Diedenhofen and Karina Peters for excellent technical support. This work was supported by the Marga und Walter Boll-Stiftung (no 210-1212), by the Koeln Fortune Program/Faculty of Medicine, University of Cologne (no 106/2012), and by the EU-FP7 programs TargetBraIn (no HEALTH-F2-2012-279017) and BrainPath (no 612360).
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