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Transcranial direct current stimulation induces polarity-specific changes of cortical blood perfusion in the rat
Experimental Neurology 227 (2011) 322–327
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Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r
Transcranial direct current stimulation induces polarity-specific changes of cortical blood perfusion in the rat Dorothee Wachter a,⁎, Arne Wrede c, Walter Schulz-Schaeffer c, Ali Taghizadeh-Waghefi a, Michael A. Nitsche b, Anna Kutschenko b, Veit Rohde a, David Liebetanz b a b c
Department of Neurosurgery, University Medical Center Göttingen, Germany Department of Clinical Neurophysiology, University Medical Center Göttingen, Germany Department of Neuropathology, University Medical Center Göttingen, Germany
a r t i c l e
i n f o
Article history: Received 2 September 2010 Revised 30 November 2010 Accepted 3 December 2010 Available online 11 December 2010 Keywords: Cerebral blood flow CBF Laser Doppler flowmetry tDCS Stroke
a b s t r a c t Objective: Transcranial direct current stimulation (tDCS) induces changes in cortical excitability and improves hand-motor function in chronic stroke. These effects depend on polarity, duration of stimulation and current intensity applied. Towards evaluating the therapeutic potential of tDCS in acute stroke, we investigated tDCSeffects on cerebral blood flow (CBF) in a tDCS rat model adapted for this purpose. Methods: In a randomised crossover design eight Sprague–Dawley rats received three single cathodal and anodal tDCS for 15 min every other day. At each polarity, current intensities of 25, 50 and 100 μA were applied. CBF was measured prior and after tDCS for at least 30 min with laser Doppler flowmetry (LDF). Results: At higher intensities (50 and 100 μA) anodal tDCS increased CBF up to 30 min. At 100 μA CBF was increased by about 25%, at 50 μA by about 18%. In contrast, cathodal tDCS led to a decrease of CBF, likewise depending on the current intensity applied. At 100 μA the effects were about 25% of baseline levels and persisted for at least 30 min. At 25 and 50 μA, baseline-levels were mostly re-established within 30 min. Conclusions: tDCS modulates CBF in a polarity specific way, the extent of modulation depending on the stimulation parameters applied. Because of its polarity-specificity, we assume that CBF-alterations are causally related to tDCS-induced alterations in cortical excitability via neuro-vascular coupling. tDCS may constitute a therapeutic option in acute stroke patients or in patients at risk for vasospasm-induced ischemia after subarachnoid hemorrhage. © 2010 Elsevier Inc. All rights reserved.
Introduction Transcranial direct current stimulation (tDCS) induces lasting changes in cortical excitability in the human brain (Nitsche and Paulus, 2000). The after-effects are based on an alteration in membrane potentials and NMDA-receptor dependent synaptic plasticity (Liebetanz et al., 2002). The stability is controlled by the duration as well as by the current intensity of the stimulation, and the direction of the induced excitability changes is controlled by the polarity of the stimulation (Nitsche and Paulus, 2001; Nitsche et al., 2003). Clinical pilot studies suggest that there are beneficial effects from tDCS for various neurological and psychiatric diseases (Brown et al., 2006; Fregni et al., 2005; Hummel et al., 2005; Liebetanz et al., 2006; Nitsche et al., 2009). In chronic stroke patients, motor performance can be improved by cathodal stimulation of the unaffected hemisphere that suppresses transcallosal inhibition of the affected ⁎ Corresponding author. Neurochirurgische Klinik, Georg-August-Universität Göttingen, Robert-Koch-Strasse 40, D-37075 Göttingen, Germany. Fax: +49 641 551 398794. E-mail address: [email protected] (D. Wachter). 0014-4886/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2010.12.005
hemisphere (Hummel et al., 2005) or by anodal stimulation of the affected hemisphere, which directly increases cortical excitability of the affected hemisphere (Fregni et al., 2005). In contrast to chronic stroke, there are so far no experimental data concerning the influence of tDCS on acute stroke. As tDCS-induced excitability changes are supposed to be associated with modulations in cerebral blood perfusion (CBF) and blood oxygenation levels (Baudewig et al., 2001; Jang et al., 2009; Lang et al., 2005; Merzagora et al., 2010), tDCS may theoretically also be of value in controlling regional CBF parameters with the purpose of increasing the cortical ischemic tolerance. But also on the other hand, a direct influence of tDCS on the musculature of the blood vessel wall has been proposed by Fox and Yasargil (1974) who induced a focal vasodilatation of the basilar artery by the application of DC stimulation directly to the artery in an experimental dog model. Presuming that the application of tDCS might lead to relevant changes in CBF, this technique could be of therapeutic importance in acute stroke patients or in patients at risk of ischemic damage from subarachnoid hemorrhage associated vasospasms. Being able to alter cerebral perfusion would have a tremendous effect on treatment strategies in these patients. To explore such possible therapeutic
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options of tDCS, the present study systematically explores the effects of tDCS on regional CBF in a rat model.
Materials and methods The experiments were performed on eight male Sprague–Dawley rats (Charles River, Germany; mean body weight 310 g). The animals were housed in a temperature- and humidity-controlled room on a 12-hour light/12-hour dark cycle in single cages under standard laboratory conditions, with food and water ad libitum. All experiments were conducted in accordance with the “Guide for the Care and Use of Laboratory Animals of the NIH” and were ethically approved by the Government of Lower Saxony.
tDCS For the transcranial application of cathodal and anodal tDCS we fixed an epicranial electrode with a defined contact area (3.5 mm2) onto the skull in a surgical procedure using a non-toxic glass ionomer cement (Ketac™ Cem Maxicap™, ESPE Dental AG, Seefeld, Germany) as described before (Liebetanz et al., 2006). The electrode was placed approximately 2 mm behind the coronal suture and 4 mm lateral to the sagittal suture (middle cerebral artery territory). This surgical procedure was performed 2 days prior to the first tDCS under anaesthesia with isoflurane. The body temperature was maintained between 36.5 and 37.5 °C by a heating blanket, controlled by a rectal temperature probe (Homeothermic Monitor, Harvard Apparatus, Hugo Sachs Elektronik, Germany). The counter electrode, which is composed of a large conventional rubber plate electrode (10.5 cm2, Physiomed Elektromedizin AG, Schnaittach, Germany), was placed onto the ventral thorax of the anaesthetised rat (Fig. 1). tDCS was applied continuously for 15 min with 25, 50 and 100 μA using a constant current stimulator (CX-6650, Schneider Electronics, Gleichen, Germany). To avoid stimulation break effects, the current intensity was automatically ramped for 10 s instead of being switching on and off abruptly (Liebetanz et al., 2009). Sequences of six 15-minute long stimulations were randomised to each animal (three cathodal and three anodal tDCSs of 25, 50 and 100 μA). Prior to stimulation the electrode was filled with saline solution (0.9% NaCl) to ensure the defined contact area. During tDCS and CBF measurements, the animals were continuously anaesthetised with isoflurane (1.0–1.5%; flow of 1.8–2.0 l/min) at a low level in order to reduce the influence of the narcotic agent on stimulation as well as CBF effects. The animals had 48 h of rest after each tDCS.
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Laser Doppler flowmetry (LDF) The aim of this study is to determine the degree of tDCS-induced cerebral blood flow (CBF) changes as influenced by the stimulation parameters applied (current intensity and current polarity). To determine the duration and extent of changes, cerebral blood flow (CBF) was measured before and after each tDCS by LDF (PerfiFlux System 5000, Perimed, Järfälla, Stockholm, Sweden). To guarantee stable baseline values not affected by anaesthesia-induced changes in CBF, CBF was monitored for 30 min until baseline measures were obtained. We obtained one single baseline recording with duration of 15 min, after an initial monitoring of 30 min that was not part of the recording used for analysis. After tDCS, laser Doppler flowmetry was continued for at least 30 min. LDF was measured over the electrode holder using a probe with the same diameter as the electrode for tDCS (Probe 407-1, Perimed). This procedure enabled us to place the probe and the electrode onto the skull through the same probe holder, which ensured that the laser Doppler flowmetry was recorded in the same cortical area to which tDCS was applied. CBF was measured in perfusion units (PU) with a frequency of 1 Hz and analysed using PeriSoft® for Windows (Software version 2.50, Perimed AB). Histological analysis The brains of all tDCS-treated animals were histologically evaluated with light microscopy for pathological changes such as edema, necrosis, hematoma and for cellular changes induced by tDCS. Twenty-four hours after the final DC stimulation, the animals were deeply anaesthetised with an overdose of isoflurane (3.5%) and perfused transcardially with a saline solution and heparine (10 000 I. E. heparine in 1000 ml NaCl 0.9%). After perfusion the brains were carefully removed from the skull and fixated in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). On the following day, the brains were cut in 3 mm-thick coronal slices and embedded in low-melting paraffin. For histological analysis, slices of 1–3 μm were cut on a microtome. H&E staining and Bielschowsky's silver impregnation were performed according to standard procedures. Immunohistochemical staining was carried out using antibodies against GFAP (rabbit polyclonal, 1:1000, Dako, Glostrup, Denmark), APP (mouse monoclonal, 1:100, Millipore, Billerica, USA), and ED1 (mouse monoclonal, 1:500, Serotec, Oxford, UK). Detection was performed with a biotinylated secondary antibody against mouse IgG (sheep, 1:200, GE Healthcare/Amersham) or rabbit IgG (donkey, 1:200, GE/ Amersham). Extravidine coupled to horseradish peroxidase (SigmaAldrich) was used to visualize antibody binding by enzymatic conversion of the diaminobenzidine chromogen (DAB). ED1 and APP required a heat-induced epitope retrieval using a citrate buffer at pH 6. Statistical analysis
Fig. 1. Experimental set-up: position of the cephalic and thoracic electrode. LDF was measured over the electrode holder using a probe with the same diameter as the electrode for tDCS.
All CBF measurements were recorded in perfusion units (PU). For statistical calculation, an average baseline value of CBF was calculated prior to tDCS (duration of 15 min). CBF measurements after tDCS were averaged over 1–10 min, 10–20 min and 20–30 min after tDCS and expressed as baseline quotients. To assess the tDCS-induced changes in CBF depending on current intensity and current polarity over a defined time course, we performed a three-way factorial ANOVA analysis with the withinsubject-factor time course (baseline, 1–10 min, 10–20 min and 20– 30 min after tDCS), the between-subject-factors of tDCS polarity (anodal, cathodal) and current intensity (either 25, 50 or 100 μA), and the dependent variable CBF. Conditional on significant effect in the ANOVA, we performed post hoc Student's t-tests to compare differences of CBF over time, and between the above-mentioned
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Table 1 Results of the three-way factorial repeated measurements ANOVA. Variables
Degrees of freedom
F values
P values
Polarity of current stimulation Current intensity Time course Time course × polarity Time course × intensity Polarity × intensity Time course × polarity × intensity
2 3 3 6 9 6 18
51.789 0.870 1.189 29.658 1.452 5.460 4.557
b 0.001 0.427 0.327 b 0.001 0.242 0.008 0.008
factors for a specific time bin, including baseline values. Statistical Product and Service Solutions (SPSS for MAC) was used for statistical analysis. A P-value b 0.05 was considered significant. Results All animals coped well with the surgical and stimulation procedures. The data of one tDCS session (50 μA anodal, rat number 6) was excluded due to leakage of the epicranial electrode. In total, 47 measurements were available for analysis. Changes in cerebral perfusion by tDCS
baseline perfusion
600 550 500 450 400 350 0
5
10
time course in min
15
perfusion in perfusion units
perfusion in perfusion units
The results of the ANOVA revealed a significant main effect of the polarity on CBF (P = b0.001; Table 1). Moreover, the interactions polarity × intensity (P = 0.008) as well as time course × polarity (P = b0.001), and polarity × intensity × time course were significant (P = 0.008). The interaction of time course, polarity and intensity demonstrates that the duration of tDCS-induced CBF changes and its direction can be altered depending on the current intensity as well as the current polarity applied. As shown by the post hoc tests, cathodal tDCS with an intensity of 25 μA leads to a significant decrease in CBF during the first 10 min after the current application, whereas the application with 25 μA of anodal polarity did not produce a significant increase in CBF. Similar to the course of CBF changes after cathodal tDCS with 25 μA, the application of 50 μA induced a significant decrease in CBF within the first 10 min after tDCS, before the baseline level was re-established. Significant changes in CBF were induced by 50 and 100 μA of anodal (increase in CBF, Fig. 2) as well as 100 μA of cathodal polarity (decrease in CBF) for at least 30 min. In summary, at low intensities cathodal tDCS seems to be more effective in producing CBF changes than anodal tDCS. At medium intensities (50 μA) anodal tDCS effects were more pronounced than cathodal effects, i.e. baseline-levels were re-established within 10 min after cathodal tDCS but persisted for at least 30 min after anodal tDCS. tDCS at 100 μA induced comparable effects. Both stimulation techniques led to significant changes in CBF that continued to be significant for at least 30 min. Depending on the polarity applied, we
CBF Perfusion Units after tDCS /Baseline
324
200
*
100
*
*
*
150
*
*
25uA anodal
50uA anodal
50
100uA anodal
0
*
-50
*
25uA cathodal
*
*
-100
*
50uA cathodal 100uA cathodal
-150 10min -200
20min
30min
Time Course (min after tDCS)
Fig. 3. Cerebral blood flow (CBF) changes measured as baseline ratios in perfusion units (PU) with laser Doppler flowmetry after tDCS with anodal and cathodal polarity of 100 μA, 50 μA and 25 μA for 15 min. The data are presented as SD +/- SEM. The applications of anodal tDCS lead to an increase, whereas the applications of cathodal tDCS lead to a decrease in CBF. The magnitude and duration of CBF changes depended on the current intensity applied. *P b 0.05.
were either able to decrease (cathodal tDCS) or increase (anodal tDCS) CBF (Fig. 3). Duration of after-effects was significantly altered according to the current intensity applied. A selecting bias by varying baseline values was ruled out by Student's t-test. Microscopic results In general, no astrocytic reaction, edema, hemorrhage or other detectable cytological changes were seen. In a single case, a unilateral lesion in the parieto-occipital cortex was observed adjacent to the electrode. The lesion was characterized by a loss of the cortical layer I and a proliferation of the leptomeningeal tissue. An enhancement of vasculature was observed in the leptomeninges and in the cortical layers below the defect. Within the leptomeninges and in proximity to the defect, pigmented macrophages were detectable. With antibodies against the glial fibrillary acidic protein and the myeloid cell marker ED1, an astrocytic gliosis and microglial reaction adjacent to the defect was seen. Silver impregnation staining according to Bielschowsky no longer revealed signs of axonal degeneration. These results correspond to a colliquative necrosis that was induced a few days before (Fig. 4). Discussion Combining tDCS and transcranial LDF in a rat model, we demonstrated that tDCS induces sustained changes on CBF. These changes are polarity-specific, i.e. anodal tDCS leads to an increase, whereas the application of cathodal tDCS leads to a decrease in CBF.
post-anodal tDCS perfusion
600 550 500 450 400 350 0
5
10
15
20
25
30
time course in min
Fig. 2. Increase of CBF in perfusion units (PU) in rat number after tDCS with 100 μA for 15 min as compared to baseline perfusion. Mean values were 437.5 PU prior to and 527.8 PU after tDCS.
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Fig. 4. Superficial cortical defect in a single stimulated animal. (a) The H&E staining shows a circumscribed loss of the cortical layer I (indicated by arrows in unlesioned tissue) and a proliferation of blood vessels in the overlaying leptomeninges. (b) The immunohistochemical reaction against the glial fibrillary acid protein (brown colour) identifies astrogliosis in the surrounding tissues as a reaction to the lesion. (c) With the silver staining according to Bielschowsky, a reactive proliferation of connective tissue (arrow) is seen in the lesion, but no axonal degeneration. (d) The immunohistochemical reaction against the macrophage/microglia marker ED1 (brown colour) shows macrophages in the leptomeninges and microglial activation in the area of the lesion. In summary, the changes are consistent with residua of a superficial cortical necrosis.
The duration and degree of CBF changes depend on the intensity of the current applied. Thus a current intensity of 100 μA for 15 min induced CBF changes that lasted for at least 30 min after the cessation of stimulation. Possible mechanisms of tDCS induced CBF changes In chronic stroke patients, motor performance can be significantly improved by cathodal stimulation of the unaffected hemisphere or by anodal stimulation of the affected hemisphere (Fregni et al., 2005). It is assumed that cathodal stimulation decreases cortical excitability, which suppresses transcallosal inhibition of the affected hemisphere, whereas anodal stimulation of the lesioned hemisphere directly increases cortical excitability (Baker et al., 2010; Brown et al., 2006; Fregni et al., 2005; Hummel et al., 2005; Schlaug et al., 2008). Regarding the modulation of cortical excitability, pharmacological studies suggest that they depend on membrane polarisation and a related alteration of spontaneous discharge rates during tDCS (Islam et al., 1995; Liebetanz et al., 2002; Nitsche et al., 2003). If tDCS is applied long enough, i.e. a few minutes, these polarity-driven alterations in membrane potentials lead to a change of synaptic strength via a modulation of NMDA-receptor activity (Liebetanz et al., 2002). As suggested by fNIRS, fMRI and PET studies, these lasting tDCS effects are associated with a change in BOLD signal and cerebral perfusion (Baudewig et al., 2001; Jang et al., 2009; Lang et al., 2005; Merzagora et al., 2010). This fact could lead to the conclusion that the polarity-specific changes of LDF signal as demonstrated in the present rat study are also accountable for the effects of CBF in terms of neuro-vascular coupling by alternating the activation of astrocytes. Neuro-vascular coupling is
the ability of neurons to modulate cerebral blood flow in regions of activation. It is thought that microvessels, glia, and neurons cooperate in terms of a ‘neuro-vascular unit’ (del Zoppo, 2010). Up to date, the precise mechanism of neuro-vascular coupling is still unclear. But, there is evidence that the activation of Ca(2+) elevations in astrocyte endfeet is one essential step in the control. Upon activation, astrocytes can release both vasodilating and vasoconstrictive agents. The type of vasomotor response is thought to depend on the resting state of the cerebral arterioles (Carmignoto and Gómez-Gonzalo, 2010). Regarding these facts, tDCS might modulate CBF via a change of Ca(2+) concentration in astrocyte endfeet that can either result in vasodilatation or vasoconstriction depending on the polarity applied. However, a direct action of tDCS on the muscles of the vessel walls could be an alternative explanation for the tDCS-induced CBF alterations in our study. First hints that underline this option are found by Fox and Yasargil (1974), who induced a focal vasodilatation of the basilar artery by applying either anodal or cathodal DC stimulation directly to the artery in an experimental dog model. This dilatation was more rapid and more pronounced after cathodal DC application and lasted for at least 4 days after stimulation. The authors hypothesised that muscle protein bonds of the vascular wall were affected by the local accumulation of ions produced during electrolysis, which resulted in vasodilatation. In contrast, in the present study, we observed a polarity-specific change of CBF that cannot be explained by focal vasodilatation irrespective of the polarity applied. Since we did not apply the current directly to the artery, this might explain why our results differ from those described by Fox and Yasargil. It might be possible that the transcranial application of either anodal or cathodal tDCS could produce different ions during electrolysis that either result in vasodilatation or vasoconstriction.
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Recently, Vernieri et al. showed that tDCS even has a polarityspecific bilateral effect on cerebral vasomotor reactivity (VMR) (Vernieri et al., 2010). Anodal tDCS decreased, whereas cathodal tDCS increased VMR bilaterally. Consensual changes were demonstrated in heart rate variability (HRV). Based on the facts that tDCSeffects on cortical excitability are unilateral, whereas Vernieri et al. (2010) demonstrated bihemispheric polarity-specific changes on VMR and HRV, they concluded that these effects were the result of modulations by the sympathetic nervous system, but did not exclude a possible influence of tDCS on the myogenic and the metabolic control of cerebral circulation. So far, it is not known whether tDCSinduced CBF changes represent an indirect result of the induced modulation of neurovascular-coupling or whether tDCS acts directly on the blood vessels or even on the general sympathetic nervous system as described by Vernieri et al. (2010). However, since the effects presented here are symmetrically polarity-specific, in contrast to the direct blood vessel effects described by Fox and Yasargil (1974), we assume that neuro-vascular coupling is probably the underlying mechanism for tDCS-induced CBF changes. Further studies should explore these mechanisms in more detail. Pharmacological approaches could help to further differentiate between these possible mechanisms by selectively interfering with the neuronal effects of tDCS. Experimental set-ups that include the VMR assessment by transcranial Doppler could serve as additional devices in differentiating the possible mechanisms for the results herein described. Technical aspects We utilized the established method of epicranial electrode placement in the rat that was recently introduced by our laboratory (Liebetanz et al., 2006, 2009). This enabled us to induce changes in cortical excitability by tDCS in a polarity-specific manner. To investigate tDCS-induced CBF changes, we successfully extended this method by combining it with LDF. This method of LDF is a well-established method for CBF measurements and for the documentation of ischemia in stroke animal models (Skarphedinsson et al., 1989; Zwagerman et al., 2010). However, since it only provides a punctiform point of contact, we cannot define the real expanse of CBF changes induced by tDCS in our experiments. Therefore, further studies with enhanced technologies like laser speckle are needed to determine the local or global extent of tDCS effects on CBF. In this study, we used a large conventional rubber plate electrode as a counter electrode that was placed onto the ventral thorax of the anaesthetised rat, opposed to the classical “bicephalic” tDCS montages used in humans. Recently, Vandermeeren et al. (2010) demonstrated in healthy human volunteers that this technique did not modulate the activity of brainstem autonomic centres and concluded that the method does not produce significant side effects and that the method seems to be safe under similar experimental conditions. However, Vernieri et al. (2010), who placed the counter electrode above the ipsilateral arm, did demonstrate a modulation of the sympathetic nervous system by tDCS. As we have not constantly monitored heart rate and global blood pressure, there is a theoretical possibility that the observed effects could have been a result of modulating brainstem regulatory centres or due to a systemic sympatho-vagal reaction. In view of these ambiguous results, we cannot definitely exclude a possible influence on autonomic centres. Further studies are needed to clearly define the safety aspects and possible influences on brainstem regulatory centres under continuous monitoring of vital parameters before transferring our experimental set-up to human subjects, since a change of sympathetic activation could also be harmful to the patient. Like in our previous animal studies on the effects of tDCS on cortical spreading depression or focal seizure threshold, all animals
underwent histopathological examination, which excluded morphological changes related to tDCS. However, in the present study, one of the 8 animals developed a necrosis in the parieto-occipital region of the left hemisphere, dorso-lateral to the stimulated region. This is an unexpected finding, since we clearly respected the safety limits which have been established for cathodal tDCS in rats (Liebetanz et al., 2009). In the rat study referred to, which was based on lesion experiments, a threshold for deleterious effects from cathodal tDCS was estimated to begin at a charge density of 52 400 C/m2. In the present study, the maximum charge density applied in a single session was 25 714 C/m2 in each rat. However, all animals received at least two stimulations at this charge density, one stimulation session being with anodal polarity, for which no safety criteria are available so far. Therefore, although we respected the safety criteria for cathodal stimulation, the combination with the anodal stimulation may have contributed to the tissue damage in one animal. Furthermore, Fox and Yasargil (1974) mentioned coagulation effects and micro-emboli after either anodal or cathodal DC stimulation to basal intracranial arteries. Since we have found a necrosis dorso-lateral to the stimulated region in one animal, the side effects described by Fox and Yasargil might be an alternative explanation. Another factor contributing to a different safety margin could be that in the current study the electrode was placed more caudally and laterally, i.e. above the region of the middle cerebral artery, as compared to the foregoing safety study (Liebetanz et al., 2009). It is possible that different brain regions may be unequally susceptible to DC stimulation. Applying the same stimulation over different cortical areas may possibly also lead to a different distribution in the local flow of current, resulting in different local maxima of current density. Besides, it should be taken into account that the rat brain size is about 500–1000 fold smaller than the human brain (Nieuwenhuys et al., 1998). However, due to the small sized electrode, the current density is 25 to 100 fold higher than applied in humans. Since there are huge differences in the size and shape of the rat brain, the applied current might spread deeper in the rat than in humans. Moreover, the current distribution can vary and might induce different changes. This case illustrates that the experimental basis for the definition of safety criteria for DC stimulation is still insufficient. Further safety studies are warranted, especially before intensified tDCS protocols are transferred to clinical application. Conclusions The results demonstrate that CBF can be modulated in a polarityspecific way by the non-invasive technique of tDCS effects. Because of its effects on CBF, tDCS may have potential with regard to ischemic tolerance and to reducing ischemic neuronal damage, acute stroke or vasospasm. A possible approach could be the application of repeated tDCS immediately after stroke or in patients at high risk from vasospasm-induced ischemic lesions after SAH. References Baker, J.M., Rorden, C., Fridriksson, J., 2010. Using transcranial direct-current stimulation to treat stroke patients with aphasia. Stroke 41, 1229–1236. Baudewig, J., Nitsche, M.A., Paulus, W., et al., 2001. Regional modulation of BOLD MRI responses to human sensorimotor activation by transcranial direct current stimulation. Magn. Reson. Med. 45, 196–201. Brown, J.A., Lutsep, H.L., Weinand, M., 2006. Mortor cortex stimulation for the enhancement of recovery from stroke: a prospective, multicenter safety study. Neurosurgery 58, 464–473. Carmignoto, G., Gómez-Gonzalo, M., 2010. The contribution of astrocyte signalling to neurovascular coupling. Brain Res. Rev. 63, 138–148. del Zoppo, G.J., 2010. The neurovascular unit in the setting of stroke. J. Intern. Med. 267, 156–171. Fox, J.L., Yasargil, M.G., 1974. The experimental effect of direct electrical current on intracranial arteries and the blood-brain barrier. J. Neurosurg. 41, 582–589. Fregni, F., Boggio, P.S., Mansur, C.G., 2005. Transcranial direct current stimulation of the unaffected hemisphere in stroke patients. NeuroReport 16, 1551–1555.
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Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r
Transcranial direct current stimulation induces polarity-specific changes of cortical blood perfusion in the rat Dorothee Wachter a,⁎, Arne Wrede c, Walter Schulz-Schaeffer c, Ali Taghizadeh-Waghefi a, Michael A. Nitsche b, Anna Kutschenko b, Veit Rohde a, David Liebetanz b a b c
Department of Neurosurgery, University Medical Center Göttingen, Germany Department of Clinical Neurophysiology, University Medical Center Göttingen, Germany Department of Neuropathology, University Medical Center Göttingen, Germany
a r t i c l e
i n f o
Article history: Received 2 September 2010 Revised 30 November 2010 Accepted 3 December 2010 Available online 11 December 2010 Keywords: Cerebral blood flow CBF Laser Doppler flowmetry tDCS Stroke
a b s t r a c t Objective: Transcranial direct current stimulation (tDCS) induces changes in cortical excitability and improves hand-motor function in chronic stroke. These effects depend on polarity, duration of stimulation and current intensity applied. Towards evaluating the therapeutic potential of tDCS in acute stroke, we investigated tDCSeffects on cerebral blood flow (CBF) in a tDCS rat model adapted for this purpose. Methods: In a randomised crossover design eight Sprague–Dawley rats received three single cathodal and anodal tDCS for 15 min every other day. At each polarity, current intensities of 25, 50 and 100 μA were applied. CBF was measured prior and after tDCS for at least 30 min with laser Doppler flowmetry (LDF). Results: At higher intensities (50 and 100 μA) anodal tDCS increased CBF up to 30 min. At 100 μA CBF was increased by about 25%, at 50 μA by about 18%. In contrast, cathodal tDCS led to a decrease of CBF, likewise depending on the current intensity applied. At 100 μA the effects were about 25% of baseline levels and persisted for at least 30 min. At 25 and 50 μA, baseline-levels were mostly re-established within 30 min. Conclusions: tDCS modulates CBF in a polarity specific way, the extent of modulation depending on the stimulation parameters applied. Because of its polarity-specificity, we assume that CBF-alterations are causally related to tDCS-induced alterations in cortical excitability via neuro-vascular coupling. tDCS may constitute a therapeutic option in acute stroke patients or in patients at risk for vasospasm-induced ischemia after subarachnoid hemorrhage. © 2010 Elsevier Inc. All rights reserved.
Introduction Transcranial direct current stimulation (tDCS) induces lasting changes in cortical excitability in the human brain (Nitsche and Paulus, 2000). The after-effects are based on an alteration in membrane potentials and NMDA-receptor dependent synaptic plasticity (Liebetanz et al., 2002). The stability is controlled by the duration as well as by the current intensity of the stimulation, and the direction of the induced excitability changes is controlled by the polarity of the stimulation (Nitsche and Paulus, 2001; Nitsche et al., 2003). Clinical pilot studies suggest that there are beneficial effects from tDCS for various neurological and psychiatric diseases (Brown et al., 2006; Fregni et al., 2005; Hummel et al., 2005; Liebetanz et al., 2006; Nitsche et al., 2009). In chronic stroke patients, motor performance can be improved by cathodal stimulation of the unaffected hemisphere that suppresses transcallosal inhibition of the affected ⁎ Corresponding author. Neurochirurgische Klinik, Georg-August-Universität Göttingen, Robert-Koch-Strasse 40, D-37075 Göttingen, Germany. Fax: +49 641 551 398794. E-mail address: [email protected] (D. Wachter). 0014-4886/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2010.12.005
hemisphere (Hummel et al., 2005) or by anodal stimulation of the affected hemisphere, which directly increases cortical excitability of the affected hemisphere (Fregni et al., 2005). In contrast to chronic stroke, there are so far no experimental data concerning the influence of tDCS on acute stroke. As tDCS-induced excitability changes are supposed to be associated with modulations in cerebral blood perfusion (CBF) and blood oxygenation levels (Baudewig et al., 2001; Jang et al., 2009; Lang et al., 2005; Merzagora et al., 2010), tDCS may theoretically also be of value in controlling regional CBF parameters with the purpose of increasing the cortical ischemic tolerance. But also on the other hand, a direct influence of tDCS on the musculature of the blood vessel wall has been proposed by Fox and Yasargil (1974) who induced a focal vasodilatation of the basilar artery by the application of DC stimulation directly to the artery in an experimental dog model. Presuming that the application of tDCS might lead to relevant changes in CBF, this technique could be of therapeutic importance in acute stroke patients or in patients at risk of ischemic damage from subarachnoid hemorrhage associated vasospasms. Being able to alter cerebral perfusion would have a tremendous effect on treatment strategies in these patients. To explore such possible therapeutic
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options of tDCS, the present study systematically explores the effects of tDCS on regional CBF in a rat model.
Materials and methods The experiments were performed on eight male Sprague–Dawley rats (Charles River, Germany; mean body weight 310 g). The animals were housed in a temperature- and humidity-controlled room on a 12-hour light/12-hour dark cycle in single cages under standard laboratory conditions, with food and water ad libitum. All experiments were conducted in accordance with the “Guide for the Care and Use of Laboratory Animals of the NIH” and were ethically approved by the Government of Lower Saxony.
tDCS For the transcranial application of cathodal and anodal tDCS we fixed an epicranial electrode with a defined contact area (3.5 mm2) onto the skull in a surgical procedure using a non-toxic glass ionomer cement (Ketac™ Cem Maxicap™, ESPE Dental AG, Seefeld, Germany) as described before (Liebetanz et al., 2006). The electrode was placed approximately 2 mm behind the coronal suture and 4 mm lateral to the sagittal suture (middle cerebral artery territory). This surgical procedure was performed 2 days prior to the first tDCS under anaesthesia with isoflurane. The body temperature was maintained between 36.5 and 37.5 °C by a heating blanket, controlled by a rectal temperature probe (Homeothermic Monitor, Harvard Apparatus, Hugo Sachs Elektronik, Germany). The counter electrode, which is composed of a large conventional rubber plate electrode (10.5 cm2, Physiomed Elektromedizin AG, Schnaittach, Germany), was placed onto the ventral thorax of the anaesthetised rat (Fig. 1). tDCS was applied continuously for 15 min with 25, 50 and 100 μA using a constant current stimulator (CX-6650, Schneider Electronics, Gleichen, Germany). To avoid stimulation break effects, the current intensity was automatically ramped for 10 s instead of being switching on and off abruptly (Liebetanz et al., 2009). Sequences of six 15-minute long stimulations were randomised to each animal (three cathodal and three anodal tDCSs of 25, 50 and 100 μA). Prior to stimulation the electrode was filled with saline solution (0.9% NaCl) to ensure the defined contact area. During tDCS and CBF measurements, the animals were continuously anaesthetised with isoflurane (1.0–1.5%; flow of 1.8–2.0 l/min) at a low level in order to reduce the influence of the narcotic agent on stimulation as well as CBF effects. The animals had 48 h of rest after each tDCS.
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Laser Doppler flowmetry (LDF) The aim of this study is to determine the degree of tDCS-induced cerebral blood flow (CBF) changes as influenced by the stimulation parameters applied (current intensity and current polarity). To determine the duration and extent of changes, cerebral blood flow (CBF) was measured before and after each tDCS by LDF (PerfiFlux System 5000, Perimed, Järfälla, Stockholm, Sweden). To guarantee stable baseline values not affected by anaesthesia-induced changes in CBF, CBF was monitored for 30 min until baseline measures were obtained. We obtained one single baseline recording with duration of 15 min, after an initial monitoring of 30 min that was not part of the recording used for analysis. After tDCS, laser Doppler flowmetry was continued for at least 30 min. LDF was measured over the electrode holder using a probe with the same diameter as the electrode for tDCS (Probe 407-1, Perimed). This procedure enabled us to place the probe and the electrode onto the skull through the same probe holder, which ensured that the laser Doppler flowmetry was recorded in the same cortical area to which tDCS was applied. CBF was measured in perfusion units (PU) with a frequency of 1 Hz and analysed using PeriSoft® for Windows (Software version 2.50, Perimed AB). Histological analysis The brains of all tDCS-treated animals were histologically evaluated with light microscopy for pathological changes such as edema, necrosis, hematoma and for cellular changes induced by tDCS. Twenty-four hours after the final DC stimulation, the animals were deeply anaesthetised with an overdose of isoflurane (3.5%) and perfused transcardially with a saline solution and heparine (10 000 I. E. heparine in 1000 ml NaCl 0.9%). After perfusion the brains were carefully removed from the skull and fixated in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). On the following day, the brains were cut in 3 mm-thick coronal slices and embedded in low-melting paraffin. For histological analysis, slices of 1–3 μm were cut on a microtome. H&E staining and Bielschowsky's silver impregnation were performed according to standard procedures. Immunohistochemical staining was carried out using antibodies against GFAP (rabbit polyclonal, 1:1000, Dako, Glostrup, Denmark), APP (mouse monoclonal, 1:100, Millipore, Billerica, USA), and ED1 (mouse monoclonal, 1:500, Serotec, Oxford, UK). Detection was performed with a biotinylated secondary antibody against mouse IgG (sheep, 1:200, GE Healthcare/Amersham) or rabbit IgG (donkey, 1:200, GE/ Amersham). Extravidine coupled to horseradish peroxidase (SigmaAldrich) was used to visualize antibody binding by enzymatic conversion of the diaminobenzidine chromogen (DAB). ED1 and APP required a heat-induced epitope retrieval using a citrate buffer at pH 6. Statistical analysis
Fig. 1. Experimental set-up: position of the cephalic and thoracic electrode. LDF was measured over the electrode holder using a probe with the same diameter as the electrode for tDCS.
All CBF measurements were recorded in perfusion units (PU). For statistical calculation, an average baseline value of CBF was calculated prior to tDCS (duration of 15 min). CBF measurements after tDCS were averaged over 1–10 min, 10–20 min and 20–30 min after tDCS and expressed as baseline quotients. To assess the tDCS-induced changes in CBF depending on current intensity and current polarity over a defined time course, we performed a three-way factorial ANOVA analysis with the withinsubject-factor time course (baseline, 1–10 min, 10–20 min and 20– 30 min after tDCS), the between-subject-factors of tDCS polarity (anodal, cathodal) and current intensity (either 25, 50 or 100 μA), and the dependent variable CBF. Conditional on significant effect in the ANOVA, we performed post hoc Student's t-tests to compare differences of CBF over time, and between the above-mentioned
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Table 1 Results of the three-way factorial repeated measurements ANOVA. Variables
Degrees of freedom
F values
P values
Polarity of current stimulation Current intensity Time course Time course × polarity Time course × intensity Polarity × intensity Time course × polarity × intensity
2 3 3 6 9 6 18
51.789 0.870 1.189 29.658 1.452 5.460 4.557
b 0.001 0.427 0.327 b 0.001 0.242 0.008 0.008
factors for a specific time bin, including baseline values. Statistical Product and Service Solutions (SPSS for MAC) was used for statistical analysis. A P-value b 0.05 was considered significant. Results All animals coped well with the surgical and stimulation procedures. The data of one tDCS session (50 μA anodal, rat number 6) was excluded due to leakage of the epicranial electrode. In total, 47 measurements were available for analysis. Changes in cerebral perfusion by tDCS
baseline perfusion
600 550 500 450 400 350 0
5
10
time course in min
15
perfusion in perfusion units
perfusion in perfusion units
The results of the ANOVA revealed a significant main effect of the polarity on CBF (P = b0.001; Table 1). Moreover, the interactions polarity × intensity (P = 0.008) as well as time course × polarity (P = b0.001), and polarity × intensity × time course were significant (P = 0.008). The interaction of time course, polarity and intensity demonstrates that the duration of tDCS-induced CBF changes and its direction can be altered depending on the current intensity as well as the current polarity applied. As shown by the post hoc tests, cathodal tDCS with an intensity of 25 μA leads to a significant decrease in CBF during the first 10 min after the current application, whereas the application with 25 μA of anodal polarity did not produce a significant increase in CBF. Similar to the course of CBF changes after cathodal tDCS with 25 μA, the application of 50 μA induced a significant decrease in CBF within the first 10 min after tDCS, before the baseline level was re-established. Significant changes in CBF were induced by 50 and 100 μA of anodal (increase in CBF, Fig. 2) as well as 100 μA of cathodal polarity (decrease in CBF) for at least 30 min. In summary, at low intensities cathodal tDCS seems to be more effective in producing CBF changes than anodal tDCS. At medium intensities (50 μA) anodal tDCS effects were more pronounced than cathodal effects, i.e. baseline-levels were re-established within 10 min after cathodal tDCS but persisted for at least 30 min after anodal tDCS. tDCS at 100 μA induced comparable effects. Both stimulation techniques led to significant changes in CBF that continued to be significant for at least 30 min. Depending on the polarity applied, we
CBF Perfusion Units after tDCS /Baseline
324
200
*
100
*
*
*
150
*
*
25uA anodal
50uA anodal
50
100uA anodal
0
*
-50
*
25uA cathodal
*
*
-100
*
50uA cathodal 100uA cathodal
-150 10min -200
20min
30min
Time Course (min after tDCS)
Fig. 3. Cerebral blood flow (CBF) changes measured as baseline ratios in perfusion units (PU) with laser Doppler flowmetry after tDCS with anodal and cathodal polarity of 100 μA, 50 μA and 25 μA for 15 min. The data are presented as SD +/- SEM. The applications of anodal tDCS lead to an increase, whereas the applications of cathodal tDCS lead to a decrease in CBF. The magnitude and duration of CBF changes depended on the current intensity applied. *P b 0.05.
were either able to decrease (cathodal tDCS) or increase (anodal tDCS) CBF (Fig. 3). Duration of after-effects was significantly altered according to the current intensity applied. A selecting bias by varying baseline values was ruled out by Student's t-test. Microscopic results In general, no astrocytic reaction, edema, hemorrhage or other detectable cytological changes were seen. In a single case, a unilateral lesion in the parieto-occipital cortex was observed adjacent to the electrode. The lesion was characterized by a loss of the cortical layer I and a proliferation of the leptomeningeal tissue. An enhancement of vasculature was observed in the leptomeninges and in the cortical layers below the defect. Within the leptomeninges and in proximity to the defect, pigmented macrophages were detectable. With antibodies against the glial fibrillary acidic protein and the myeloid cell marker ED1, an astrocytic gliosis and microglial reaction adjacent to the defect was seen. Silver impregnation staining according to Bielschowsky no longer revealed signs of axonal degeneration. These results correspond to a colliquative necrosis that was induced a few days before (Fig. 4). Discussion Combining tDCS and transcranial LDF in a rat model, we demonstrated that tDCS induces sustained changes on CBF. These changes are polarity-specific, i.e. anodal tDCS leads to an increase, whereas the application of cathodal tDCS leads to a decrease in CBF.
post-anodal tDCS perfusion
600 550 500 450 400 350 0
5
10
15
20
25
30
time course in min
Fig. 2. Increase of CBF in perfusion units (PU) in rat number after tDCS with 100 μA for 15 min as compared to baseline perfusion. Mean values were 437.5 PU prior to and 527.8 PU after tDCS.
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Fig. 4. Superficial cortical defect in a single stimulated animal. (a) The H&E staining shows a circumscribed loss of the cortical layer I (indicated by arrows in unlesioned tissue) and a proliferation of blood vessels in the overlaying leptomeninges. (b) The immunohistochemical reaction against the glial fibrillary acid protein (brown colour) identifies astrogliosis in the surrounding tissues as a reaction to the lesion. (c) With the silver staining according to Bielschowsky, a reactive proliferation of connective tissue (arrow) is seen in the lesion, but no axonal degeneration. (d) The immunohistochemical reaction against the macrophage/microglia marker ED1 (brown colour) shows macrophages in the leptomeninges and microglial activation in the area of the lesion. In summary, the changes are consistent with residua of a superficial cortical necrosis.
The duration and degree of CBF changes depend on the intensity of the current applied. Thus a current intensity of 100 μA for 15 min induced CBF changes that lasted for at least 30 min after the cessation of stimulation. Possible mechanisms of tDCS induced CBF changes In chronic stroke patients, motor performance can be significantly improved by cathodal stimulation of the unaffected hemisphere or by anodal stimulation of the affected hemisphere (Fregni et al., 2005). It is assumed that cathodal stimulation decreases cortical excitability, which suppresses transcallosal inhibition of the affected hemisphere, whereas anodal stimulation of the lesioned hemisphere directly increases cortical excitability (Baker et al., 2010; Brown et al., 2006; Fregni et al., 2005; Hummel et al., 2005; Schlaug et al., 2008). Regarding the modulation of cortical excitability, pharmacological studies suggest that they depend on membrane polarisation and a related alteration of spontaneous discharge rates during tDCS (Islam et al., 1995; Liebetanz et al., 2002; Nitsche et al., 2003). If tDCS is applied long enough, i.e. a few minutes, these polarity-driven alterations in membrane potentials lead to a change of synaptic strength via a modulation of NMDA-receptor activity (Liebetanz et al., 2002). As suggested by fNIRS, fMRI and PET studies, these lasting tDCS effects are associated with a change in BOLD signal and cerebral perfusion (Baudewig et al., 2001; Jang et al., 2009; Lang et al., 2005; Merzagora et al., 2010). This fact could lead to the conclusion that the polarity-specific changes of LDF signal as demonstrated in the present rat study are also accountable for the effects of CBF in terms of neuro-vascular coupling by alternating the activation of astrocytes. Neuro-vascular coupling is
the ability of neurons to modulate cerebral blood flow in regions of activation. It is thought that microvessels, glia, and neurons cooperate in terms of a ‘neuro-vascular unit’ (del Zoppo, 2010). Up to date, the precise mechanism of neuro-vascular coupling is still unclear. But, there is evidence that the activation of Ca(2+) elevations in astrocyte endfeet is one essential step in the control. Upon activation, astrocytes can release both vasodilating and vasoconstrictive agents. The type of vasomotor response is thought to depend on the resting state of the cerebral arterioles (Carmignoto and Gómez-Gonzalo, 2010). Regarding these facts, tDCS might modulate CBF via a change of Ca(2+) concentration in astrocyte endfeet that can either result in vasodilatation or vasoconstriction depending on the polarity applied. However, a direct action of tDCS on the muscles of the vessel walls could be an alternative explanation for the tDCS-induced CBF alterations in our study. First hints that underline this option are found by Fox and Yasargil (1974), who induced a focal vasodilatation of the basilar artery by applying either anodal or cathodal DC stimulation directly to the artery in an experimental dog model. This dilatation was more rapid and more pronounced after cathodal DC application and lasted for at least 4 days after stimulation. The authors hypothesised that muscle protein bonds of the vascular wall were affected by the local accumulation of ions produced during electrolysis, which resulted in vasodilatation. In contrast, in the present study, we observed a polarity-specific change of CBF that cannot be explained by focal vasodilatation irrespective of the polarity applied. Since we did not apply the current directly to the artery, this might explain why our results differ from those described by Fox and Yasargil. It might be possible that the transcranial application of either anodal or cathodal tDCS could produce different ions during electrolysis that either result in vasodilatation or vasoconstriction.
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Recently, Vernieri et al. showed that tDCS even has a polarityspecific bilateral effect on cerebral vasomotor reactivity (VMR) (Vernieri et al., 2010). Anodal tDCS decreased, whereas cathodal tDCS increased VMR bilaterally. Consensual changes were demonstrated in heart rate variability (HRV). Based on the facts that tDCSeffects on cortical excitability are unilateral, whereas Vernieri et al. (2010) demonstrated bihemispheric polarity-specific changes on VMR and HRV, they concluded that these effects were the result of modulations by the sympathetic nervous system, but did not exclude a possible influence of tDCS on the myogenic and the metabolic control of cerebral circulation. So far, it is not known whether tDCSinduced CBF changes represent an indirect result of the induced modulation of neurovascular-coupling or whether tDCS acts directly on the blood vessels or even on the general sympathetic nervous system as described by Vernieri et al. (2010). However, since the effects presented here are symmetrically polarity-specific, in contrast to the direct blood vessel effects described by Fox and Yasargil (1974), we assume that neuro-vascular coupling is probably the underlying mechanism for tDCS-induced CBF changes. Further studies should explore these mechanisms in more detail. Pharmacological approaches could help to further differentiate between these possible mechanisms by selectively interfering with the neuronal effects of tDCS. Experimental set-ups that include the VMR assessment by transcranial Doppler could serve as additional devices in differentiating the possible mechanisms for the results herein described. Technical aspects We utilized the established method of epicranial electrode placement in the rat that was recently introduced by our laboratory (Liebetanz et al., 2006, 2009). This enabled us to induce changes in cortical excitability by tDCS in a polarity-specific manner. To investigate tDCS-induced CBF changes, we successfully extended this method by combining it with LDF. This method of LDF is a well-established method for CBF measurements and for the documentation of ischemia in stroke animal models (Skarphedinsson et al., 1989; Zwagerman et al., 2010). However, since it only provides a punctiform point of contact, we cannot define the real expanse of CBF changes induced by tDCS in our experiments. Therefore, further studies with enhanced technologies like laser speckle are needed to determine the local or global extent of tDCS effects on CBF. In this study, we used a large conventional rubber plate electrode as a counter electrode that was placed onto the ventral thorax of the anaesthetised rat, opposed to the classical “bicephalic” tDCS montages used in humans. Recently, Vandermeeren et al. (2010) demonstrated in healthy human volunteers that this technique did not modulate the activity of brainstem autonomic centres and concluded that the method does not produce significant side effects and that the method seems to be safe under similar experimental conditions. However, Vernieri et al. (2010), who placed the counter electrode above the ipsilateral arm, did demonstrate a modulation of the sympathetic nervous system by tDCS. As we have not constantly monitored heart rate and global blood pressure, there is a theoretical possibility that the observed effects could have been a result of modulating brainstem regulatory centres or due to a systemic sympatho-vagal reaction. In view of these ambiguous results, we cannot definitely exclude a possible influence on autonomic centres. Further studies are needed to clearly define the safety aspects and possible influences on brainstem regulatory centres under continuous monitoring of vital parameters before transferring our experimental set-up to human subjects, since a change of sympathetic activation could also be harmful to the patient. Like in our previous animal studies on the effects of tDCS on cortical spreading depression or focal seizure threshold, all animals
underwent histopathological examination, which excluded morphological changes related to tDCS. However, in the present study, one of the 8 animals developed a necrosis in the parieto-occipital region of the left hemisphere, dorso-lateral to the stimulated region. This is an unexpected finding, since we clearly respected the safety limits which have been established for cathodal tDCS in rats (Liebetanz et al., 2009). In the rat study referred to, which was based on lesion experiments, a threshold for deleterious effects from cathodal tDCS was estimated to begin at a charge density of 52 400 C/m2. In the present study, the maximum charge density applied in a single session was 25 714 C/m2 in each rat. However, all animals received at least two stimulations at this charge density, one stimulation session being with anodal polarity, for which no safety criteria are available so far. Therefore, although we respected the safety criteria for cathodal stimulation, the combination with the anodal stimulation may have contributed to the tissue damage in one animal. Furthermore, Fox and Yasargil (1974) mentioned coagulation effects and micro-emboli after either anodal or cathodal DC stimulation to basal intracranial arteries. Since we have found a necrosis dorso-lateral to the stimulated region in one animal, the side effects described by Fox and Yasargil might be an alternative explanation. Another factor contributing to a different safety margin could be that in the current study the electrode was placed more caudally and laterally, i.e. above the region of the middle cerebral artery, as compared to the foregoing safety study (Liebetanz et al., 2009). It is possible that different brain regions may be unequally susceptible to DC stimulation. Applying the same stimulation over different cortical areas may possibly also lead to a different distribution in the local flow of current, resulting in different local maxima of current density. Besides, it should be taken into account that the rat brain size is about 500–1000 fold smaller than the human brain (Nieuwenhuys et al., 1998). However, due to the small sized electrode, the current density is 25 to 100 fold higher than applied in humans. Since there are huge differences in the size and shape of the rat brain, the applied current might spread deeper in the rat than in humans. Moreover, the current distribution can vary and might induce different changes. This case illustrates that the experimental basis for the definition of safety criteria for DC stimulation is still insufficient. Further safety studies are warranted, especially before intensified tDCS protocols are transferred to clinical application. Conclusions The results demonstrate that CBF can be modulated in a polarityspecific way by the non-invasive technique of tDCS effects. Because of its effects on CBF, tDCS may have potential with regard to ischemic tolerance and to reducing ischemic neuronal damage, acute stroke or vasospasm. A possible approach could be the application of repeated tDCS immediately after stroke or in patients at high risk from vasospasm-induced ischemic lesions after SAH. References Baker, J.M., Rorden, C., Fridriksson, J., 2010. Using transcranial direct-current stimulation to treat stroke patients with aphasia. Stroke 41, 1229–1236. Baudewig, J., Nitsche, M.A., Paulus, W., et al., 2001. Regional modulation of BOLD MRI responses to human sensorimotor activation by transcranial direct current stimulation. Magn. Reson. Med. 45, 196–201. Brown, J.A., Lutsep, H.L., Weinand, M., 2006. Mortor cortex stimulation for the enhancement of recovery from stroke: a prospective, multicenter safety study. Neurosurgery 58, 464–473. Carmignoto, G., Gómez-Gonzalo, M., 2010. The contribution of astrocyte signalling to neurovascular coupling. Brain Res. Rev. 63, 138–148. del Zoppo, G.J., 2010. The neurovascular unit in the setting of stroke. J. Intern. Med. 267, 156–171. Fox, J.L., Yasargil, M.G., 1974. The experimental effect of direct electrical current on intracranial arteries and the blood-brain barrier. J. Neurosurg. 41, 582–589. Fregni, F., Boggio, P.S., Mansur, C.G., 2005. Transcranial direct current stimulation of the unaffected hemisphere in stroke patients. NeuroReport 16, 1551–1555.
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