Neuroprotective effect of cathodal transcranial direct current stimulation in a rat stroke model Francesca Notturno, Marta Pace, Filippo Zappasodi, Etrugul Cam, Claudio L. Bassetti, Antonino Uncini PII: DOI: Reference:
S0022-510X(14)00307-4 doi: 10.1016/j.jns.2014.05.017 JNS 13194
To appear in:
Journal of the Neurological Sciences
Received date: Accepted date:
28 April 2014 6 May 2014
Please cite this article as: Notturno Francesca, Pace Marta, Zappasodi Filippo, Cam Etrugul, Bassetti Claudio L., Uncini Antonino, Neuroprotective effect of cathodal transcranial direct current stimulation in a rat stroke model, Journal of the Neurological Sciences (2014), doi: 10.1016/j.jns.2014.05.017
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ACCEPTED MANUSCRIPT Neuroprotective effect of cathodal transcranial direct current stimulation
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in a rat stroke model
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Francesca Notturno* a,b, Marta Pace* b, Filippo Zappasodia,c, Etrugul Camd, Claudio L. Bassettib,d,
Department of Neuroscience and Imaging, University “G. d’Annunzio”, via dei Vestini 31, 66100
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a
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Antonino Uncinia,b
Chieti, Italy
Neurocenter of Southern Switzerland, Via Tesserete 46, 6903 Lugano, Switzerland
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Institute of Advanced Biomedical Technologies, University “G. d’Annunzio”, via dei Vestini 31,
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b
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Universitätsklinik für Neurologie, Inselspital, Bern, Switzerland
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d
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66100 Chieti, Italy
*These authors contributed equally to this work and are shared first authors.
Corresponding author: Prof. Antonino Uncini
Department of Neuroscience and Imaging University "G. d'Annunzio", Chieti-Pescara, Via dei Vestini, 66100 Chieti, Italy [email protected] Tel : +39 0871 3556942
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Abstract
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Experimental focal brain ischemia generates in the penumbra recurrent depolarizations which spread
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across the injured cortex inducing infarct growth. Transcranial direct current stimulation can induce a lasting, polarity-specific, modulation of cortical excitability. To verify whether cathodal transcranial
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direct current stimulation could reduce the infarct size and the number of depolarizations, focal
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ischemia was induced in the rat by the 3 vessels occlusion technique. In the first experiment 12 ischemic rats received cathodal stimulation (alternating 15 minutes on and 15 minutes off) starting 45
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minutes after middle cerebral artery occlusion and lasting 4 hours. In the second experiment 12 ischemic rats received cathodal transcranial direct current stimulation with the same protocol but
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starting soon after middle cerebral artery occlusion and lasting 6 hours. In both experiments controls
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were 12 ischemic rats not receiving stimulation. Cathodal stimulation reduced the infarct volume in the
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first experiment by 20% (p=0.002) and in the second by 30% (p=0.003). The area of cerebral infarction was smaller in animals receiving cathodal stimulation in both experiments (p = 0.005). Cathodal stimulation reduced the number of depolarizations (p=0.023) and infarct volume correlated with the number of depolarizations (p=0.048).
Our findings indicate that cathodal transcranial direct current stimulation exert a neuroprotective effect in the acute phase of stroke possibly decreasing the number of spreading depolarizations. These findings may have translational relevance and open a new avenue in neuroprotection of stroke in humans.
Keywords: peri-infarction depolarizations, cortical spreading depression, non-invasive brain stimulation, brain infarct volume, rat stroke model, neuroprotection.
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ACCEPTED MANUSCRIPT 1. Introduction Experimental ischemia generates electrical instability in the penumbra manifesting with recurrent
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spontaneous waves of depolarization denominated peri-infarction depolarizations (PIDs) (Nedergaard and Hansen 1993; Hossmann 1996). These depolarizations spread across the injured cortex into the
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normally perfused tissue, where they take the characteristics of cortical spreading depression (CSD),
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inducing transient depression of electrocorticogram (Hossmann, 1996). Infarct size correlates, in the
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stroke experimental model, with the numbers of PIDs or duration of PID aggregates (Mies et al., 1993; Dijkhuizen et al., 1999), and the PID number is the independent, determining variable in this
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relationship (Busch et al., 1996).
Transcranial direct current stimulation (tDCS) is a form of non-invasive modulation of brain
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excitability currently employed to modulate brain excitability in many neurological disorders (Fregni
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and Pascual-Leone, 2007). TDCS induces lasting alterations of cortical excitability and changes in
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neural cell membrane potential by manipulating ion channels or by shifting electrical gradients which influence the electrical balance of ions inside and outside the neural membrane. The direction of the excitability shifts is determined by the current polarity (Priori et al., 1998; Nitsche et al., 2000; Nitsche and Paulus, 2000). Experimental and human studies suggested that the after-effects of tDCS might originate from persistent modifications of synaptic efficacy similar to those underlying long-term potentiation and long-term depression of synaptic activity studies (Islam et al., 1995; Liebetanz et al., 2002). In vivo cathodal polarization directly applied to cortical surface in rats is able to completely block CSD and it has been demonstrated that tDCS interferes with the propagation velocity of CSD in a focal and polarity-specific way that lasts beyond the end of tDCS (Richter et al., 1996; Liebetanz et al., 2006a).
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ACCEPTED MANUSCRIPT A very recent study showed that cathodal tDCS applied intermittently for 40 minutes in the acute phase of stroke due transitory middle cerebral artery (MCA) occlusion in the mouse reduced infarct
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volume and promote a better clinical recovery (Peruzzotti-Jametti et al. 2013).
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The aim of this study was to verify the safety and efficacy of long duration cathodal tDCS in the acute phase of stroke induced the rat by the permanent 3 vessels occlusion technique. TDCS was
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discontinuously applied, with cycles of 15 minutes of stimulation alternating with15 minutes of no
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stimulation up to 180 minutes. Moreover we investigated whether cathodal tDCS could inhibits
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recurrent depolarizations.
2. Materials and Methods
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2.1 Animals
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Fifty-three male Sprague Dawley rats (Harlan Laboratories, Udine, Italy) 8-9 weeks old and weighting
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165-265 g at the time of surgery were employed in the study. The rats were housed under 12 hours light/dark cycle and room temperature of 22 °C. Animals were provided with food and water ad libitum. All experiments were approved by the Cantonal Veterinary Authority of Ticino, Switzerland and conducted according to guidelines for the care and use of laboratory animals (Kantonales Veterinäramt Zürich, Switzerland). Effort was made to minimize the number of animals used. The experiments were carried out in the laboratory of the Neurocenter of Southern Switzerland in Bellinzona.
2.2 Induction of focal cerebral ischemia Cerebral ischemia in the left hemisphere was induced by permanent occlusion of the MCA, permanent occlusion of the ipsilateral distal common carotid artery, and temporary occlusion of the contralateral common carotid artery (Chen et al., 1986; Zunzunegui et al., 2011). The rats were anesthetized with 4
ACCEPTED MANUSCRIPT 2% isoflurane in 2 liters/min of O2. A 2 cm vertical skin incision was made midway between the ear and the eye. The skull was exposed where the frontal bone joins the temporal bone and a small area
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(5×5 mm) of the bone overlying the MCA was removed (Fig. 1). After removing the dura mater, the
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MCA and its three main branches dorsal to the rhinal fissure were occluded by bipolar electrocoagulation. The common carotid artery ipsilateral to the occluded MCA was ligated
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permanently using a 4-0 silk suture thread. Finally, the contralateral common carotid artery was
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temporarily occluded for 60 minutes by an aneurism clip. Body temperature was maintained using a heating pad servo-controlled by a rectal temperature probe. The left femoral artery was cannulated for
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continuous blood pressure monitoring by a blood pressure transducer (Harvard, 220 VAC/50 Hz) and
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for serial measurements of pO2, pCO2 and glucose concentration (Radiometer ABL 5).
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2.3 Transcranial direct current stimulation
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TDCS was applied using a constant current stimulator (DC-stimulator Eldith, Germany). The epicranial electrode was a home-made plastic jacket with an inner diameter of 3 mm fixed on the skull 2 mm left and 1 mm posterior to the bregma (Fig. 1). Fixation was achieved by an ionomeric cement (Ketac Cem, ESPE Dental AG, Seefeld, Germany). The jacket was filled with cotton wool soaked in saline solution (0.9% NaCl) resulting in a contact surface of 7 mm2. To prevent the bypassing of currents that would occur in the case of two juxtaposed epicranial electrodes and to induce polarityspecific tDCS effects, a rubber electrode (10.5 cm2) was positioned on the ventral side of the thorax (Liebetanz et al., 2006b). The electrode on the skull was connected to the cathode and the electrode on the chest to the anode of the stimulator. A constant current intensity of 0.2 mA (current density of 2.86 mA/cm2) was applied, within the reported limits of safety and tolerability (Liebetanz et al., 2009). The stimulation protocol consisted of cycles of 15 minutes of stimulation alternating with15 minutes of no stimulation. This protocol was chosen because in rats PIDs are frequent and separated by an interval of 5
ACCEPTED MANUSCRIPT 5-10 minutes and in mice a reduction of the amplitude of the motor potentials, evoked by a transcranial electrical stimulation, is observed for 10 minutes after a cathodal tDCS lasting for 10 minutes
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(Nakamura et al., 2010; Cambiaghi et al., 2010). In the not stimulated animals the electrodes were
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positioned on the skull and the chest without delivering the stimulation. The rats were maintained under slight anesthesia by 2% isoflurane in 1 liter/min of O2 and analgesia by buprenorphine 0.03 mg/kg
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during real and sham tDCS. At the end of the experiment the rats, once awake, were placed back into
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their home cages.
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2.4 Brain damage analysis
Fourthy-eight hours after the induction of the ischemia, the rats were decapitated under isoflurane
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anesthesia. The brain was rapidly removed, frozen in dry ice and stored at -80 °C. For each brain,
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sections of 20 m corresponding to six levels [A = + 2.7 (L1), + 1.7 (L2), + 0.7 (L3), -0.3 (L4), -1.3
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(L5) , -2.3 (L6) mm from bregma] were cut on a cryostat (Paxinos et al., 2005). Twelve to 15 slices for each level were mounted on SuperFrost Plus (Menzel GmbH, Braunschweig, Germany) for histological evaluation. To assess the infarct volume, all the sections were first fixed by means of PBS containing 4% paraformaldehyde for 20 minutes at room temperature and then stained with cresyl violet. On digitized cresyl violet sections the infarct area was delineated and measured using the NIH Image J software (NIH, Bethesda, MD, USA). The infarct volume was estimated knowing the distance between each chosen level and corrected for the edema by multiplying the ratio of the contralateral to ipsilateral volume and taking also into account the volume of the contralateral hemisphere (Gao et al., 2010). Moreover, the brain swelling was calculated as follows: [(ipsilateral hemisphere volume - contralateral hemisphere volume)/contralateral hemisphere volume] x 100 (Junge et al., 2003).
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ACCEPTED MANUSCRIPT To assess the potential damage induced by tDCS coronal brain sections of 20 m thickness were cut serially from the block on a microtome, mounted on a glass slide, and stained with haematoxylin and
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eosin.
2.5 Recording and analysis of slow potential changes
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A slow potential change (SPC) is the hallmark of both peri-infarction depolarizations and cortical
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spreading depression (Leao, 1951; Koroleva and Bures, 1996; Fabricius et al., 2006). We recorded SPCs in direct current modality by using a gold coated miniature screw (0.9 mm diameter) inserted in
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the skull overlying the infarcted hemisphere 1.0 mm left and 1.5 mm anterior to bregma (Fig. 1 ). Care was taken not to pass the dura and this was verified by histology. The head of the screw was soldered
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to a multipolar connector and the assembly was fixed to the skull with cement. The reference was an Ag/AgCl electrode placed in the neck musculature and the animal was grounded with a steel needle
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electrode inserted into the hind limb and connected to the Faraday cage. The signals were amplified in direct current modality (AC/DC Differential Amplifier Model 3000), digitized at 1000 Hz (CED 14101 mKII) and after analog-to-digital conversion stored into a computer for subsequent analyses offline by Spike2 software (CED, UK). SPCs were recorded right after MCA occlusion for the following 6 hours. For quantitative analysis a SPC was defined as a shift in direct current potential of amplitude (negative to positive peak) 0.5 mV. The duration of SPCs was calculated as the interval between the starting point and the return to the baseline. The number, the frequency (number of SPCs in one hour), the mean amplitude and the mean duration in each animal were calculated.
2.6 Experiments and experimental groups Two experiments were performed (Fig. 2). In the first experiment 12 rats, 45 minutes after MCA occlusion, received cathodal tDCS for 4 hours with a cumulative duration of stimulation of 120 minutes 7
ACCEPTED MANUSCRIPT (group 1); 12 rats were not stimulated after ischemia (group 2). Since PIDs start in rats soon after MCA occlusion (Hartings et al., 2003), to anticipate the onset and increase the duration of tDCS without
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prolonging excessively the anesthesia, the time for the surgical procedure to MCA occlusion was
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shortened of about one hour and tDCS was started immediately after the MCA occlusion (Fig. 2). In this second experiment 12 rats received cathodal tDCS for 6 hours with a cumulative duration of
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stimulation of 180 minutes (group 3); 12 rats were not stimulated after ischemia (group 4). Five rats
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(group 5) went through the same surgical procedure of the other animals except for the occlusion of MCA and carotid cerebral arteries (sham ischemia) and received cathodal tDCS for 6 hours.
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Since we thought important to maintain comparable in all animals the effect of isoflurane, a N-methylD-aspartate receptor antagonist, in the possible inhibition of depolarizations the duration of anesthesia
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was approximately 9 hours for all experimental groups (Fig. 2). In 16 animals of the experiment 2 (8
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as described above.
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animals of group 3, treated with tDCS, and 8 animals of group 4, not stimulated) SPCs were recorded
2.7 Statistical analysis
Gaussian distribution of values was verified by Kolmogorov-Smirnov test. To identify the effect of tDCS on infarct volume, independent sample t-test was applied for each experiment to compare the volume values of the rats stimulated with real tDCS with those of the not stimulated rats. Moreover, to check for possible specific effects of stimulation on predefined brain levels, repeated measures ANOVA was applied to the different infarct area values of brain levels, with Stimulation type (real stimulation, no stimulation) as between-subject factors and Level (L1-L6) as within subject factor. In the rat of experiment 2, in which SPCs were recorded (8 stimulated rats of group 3 and 8 not stimulated rats of group 4), repeated measures ANOVA was applied to the number of SPCs, with Stimulation type as between subject factor and Time (hour 1-6) as within subject factor. For all these 16 8
ACCEPTED MANUSCRIPT rats Pearson correlation was performed in order to assess a link between the number and duration of
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SPCs and the infarct volume.
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3. Results 3.1 Physiological variables
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Rectal temperature was 36.5 ± 0.5 °C before MCA occlusion, increased to an average of 37.5 ± 0.5 °C
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within 2 hours and returned to previous values at the end of the experiment. Baseline blood glucose levels were 160 ± 20 mg/dl before the surgery and raised to 200 ± 30 mg/dl
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within 4-6 hours. An increase of blood glucose has been described in the rodent models of cerebral ischemia (Ginsberg and Busto, 1989). Blood pressure and blood gases remained in the normal range
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3.2 Infarct area and volume
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during the experiment.
We did not find macroscopic or microscopic lesions in the brains of group 5 rats with sham ischemia and cathodal tDCS for 6 hours.
The volume of cerebral infarction was significantly smaller in animals receiving cathodal tDCS (group 1 and 3) than no stimulation (group 2 and 4), both in experiment 1 and 2. The volume reduction between groups of stimulated and not stimulated rats was 20% in experiment 1 (62 ± 6 mm3 vs 78 ± 14 mm3, independent t-test t (22) = 3.650, p=0.001), and 30% in experiment 2 (38 ± 9 mm3 vs 55 ± 15 mm3 , independet t-test t (22) = 3.381, p=0.003), (Fig. 3A). The ANOVA design on the area of cerebral infarction evidenced main effects of Stimulation Type both in experiment 1 (F(1,22) = 9.648; p = 0.005) and experiment 2 (F(1,22)=9.648; p=0.005). The lack of the interaction Stimulation Type × Level (p>0.05 in both experiments) suggests that this reduction was not specific for some levels. In Fig. 3B are reported representative coronal brain sections showing the 9
ACCEPTED MANUSCRIPT infarct area at the 6 predefined levels in a rat receiving cathodal tDCS and in a not stimulated rat of experiment 2.
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In the second experiment the infarct area and volumes resulted smaller both in the stimulated (group 3)
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and not stimulated (group 4) animals compared to the first experiment (Fig. 3A). Anyway the comparisons were always made between groups inside the same experiment.
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We found no significant effects in brain swelling between stimulated and not stimulated rats in both
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experiment 1 and 2 (16 ± 7 % and 19 ± 9 % respectively, independent t-test p>0.1 consistently).
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3.3 SPCs
SPCs were recorded in 8 rats of group 3 treated with tDCS for 6 hours and in 8 rats of group 4
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receiving no stimulation. Within few minutes after MCA occlusion, SPCs appeared and recurred
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periodically during the 6 hours of recordings (Fig. 4A). The analysed SPCs had amplitudes ranging
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from 0.5 to 4.9 mV and duration up to 50 sec. The ANOVA design returned a Stimulation Type effect (F(1,14) = 8.210; p = 0.012) and a lack of significance of the interaction Time × Stimulation Type (p>0.05). Indeed, the stimulated rats displayed a total number of SPCs lower than the non stimulated rats (19.8 ± 11.7 vs 112.3 ± 90.5; independent sample t-test: t(7.2) = -2.865, p= 0.023). The difference in the number of SPCs between group 3 and group 4 rats was already significant during the first hour after the MCA occlusion and remained significant for the following 5 hours (lack of the significance Time × Stimulation Type, Fig. 4B). The inter-rat variability of the number of SPCs was much greater in the not stimulated rats (SD = 90.5) than in the rats receiving cathodal tDCS (SD = 11.9) as indicated by the Levene's Test for equality of variances (p < 0.001). No difference in the mean amplitude and mean duration of SPCs was found between stimulated and not stimulated rats (independent t-test p > 0.200, consistently).
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ACCEPTED MANUSCRIPT In the 16 animals in which SPCs were recorded, a positive correlation was found between the total number of SPCs and the infarct volume (Pearson’s r = 0.501, p = 0.048, Fig. 4C). No correlation was
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found between the total duration of SPCs and the infarct volume (p > 0.200).
4. Discussion
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In this study we applied cathodal tDCS during the very early phase of stroke induction employing a
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current density approximately half of the current density used in studying the anticonvulsant effect in a rat model of focal epilepsy (Liebetanz et al., 2006b). Although we cannot exclude that because of the
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operculum in the skull the actual current reaching the cerebral cortex was higher, we did not find macroscopic nor microscopic lesions in the brains of rats with sham ischemia which received tDCS for
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a cumulative time of 180 minutes confirming that the stimulation technique was safe even with a
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duration of stimulation four and half times longer that in the mouse (Peruzzotti-Jametti et al., 2013).
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Cathodal tDCS was able to reduce the infarct size, compared to controls, by 20% in the animals stimulated (15 minutes on and 15 minutes off) for 4 hours. In the animals in which the onset of stimulation was anticipated soon after MCA occlusion and lasted 6 hours the infarct size was reduced , compared to controls by 30%.
TDCS significantly reduced not only the number but interestingly also the inter-rat variability of SPCs in the whole period of stimulation. The link between number of PIDs and infarct size is thought to be causal since eliciting PIDs by potassium application or electrical stimulation results in larger infarct volumes (Busch et al., 1996; Back et al., 1996). On the other hand, administration of glutamate receptor blockers or hypothermia, which reduce PIDs, have neuroprotective effect (Chen et al., 1993; Mies et al., 1993; Tatlisumak et al., 2000; Hartings et al., 2003). The basis for the relationship between PIDs and infarct growth may be an abnormal microvascular vasoconstriction in response to the depolarizations as well as an imbalance between increased metabolic workload, induced by the 11
ACCEPTED MANUSCRIPT depolarizations, and delivery of oxygen and glucose to cerebral cells in the penumbra (Shin et al., 2006; Hashemi et al., 2009).
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The relationships among PIDs, glutamate and growth of the infarcted territory are debated.
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Glutamate excitotoxicity is prominent in the early phase of ischemic stroke and glutamate in the ischemic core may triggers PIDs that propagates in the penumbra causing an expansion of infarction
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(Somjen, 2001). On the other hand it is possible that cell death in the penumbra occurs primarily as a
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consequence of metabolic stress and vasoconstriction induced by PIDs whereas glutamate release and excitotoxicity are secondary to the depolarizations (Hartings et al., 2003). Moreover the
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neuroprotective effect of N-methyl-D-aspartate receptor antagonist may be attributable to block of the spread of PIDs, which depends in part on glutamate receptors, and not to a direct effect on glutamate-
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induced currents per se (Hartings et al., 2003). Whatever is the first trigger of the complex cascade of
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events leading to infarct expansion our findings indicate that cathodal tDCS blocks the origin and the
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repeatedly spontaneous cycling of PIDs which are thought to enlarge ischemic lesions (Nakamura et al., 2010). In the present, as in other experimental studies, infarct size was correlated with the number of SPCs (Mies et al., 1993; Busch et al., 1996; Back et al., 1996; Hartings et al., 2003). Therefore we think that cathodal-tDCS exerts, at least in part, its effect by reducing recurrent spreading depolarizations. Nevertheless we cannot exclude that cathodal tDCS reduces infarct size by other mechanisms. Cortical, continuous, low-frequency electrical stimulation in in a transitory MCA occlusion model had antiapoptotic, anti-inflammatory and angiogenic effects (Baba et al.. 2009). Cathodal tDCS reduces cortical glutamate, and downregulates the N-methyl-D-aspartate receptor NR2B in healthy mice (Petruzzoli-Jannetti et al. 2013). In the transitory MCA occlusion model in the mouse cathodal tDCs, applied in the early phase of stroke, reduced number of apoptotic neuronal cells and postischemic inflammatory reaction (Petruzzoli-Jannetti et al.2013).
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PIDs start in rats soon after MCA occlusion and recur through 40 hours in a biphasic pattern: an initial
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phase lasting about 3 hours and a secondary phase predominating at 8-18 hours which was
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demonstrated to be even more important for the recruitment of penumbral tissue into the infarct core. (Hartings et al., 2003). Peruzzotti-Jametti and colleagues. (2013) showed that delaying of 4.5 hours the
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onset of tDCS after the induction of ischemia induced only a slight, not significant, reduction of the
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infarct volume. Therefore it seems crucial to start cathodal tDCS as soon as possible after the induction of the ischemia. Moreover to explore the full extent of neuroprotective effect a 24 hour experiment of
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stimulation in freely behaving animals should be performed (Hartings et al., 2003). Moving from bench to bedside, CSDs and PIDs have been recorded also in patients with malignant
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middle cerebral artery infarction and has been suggested that halting spreading depolarizations may
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help to avoid the deterioration in these patients due secondary ischemia and fatal brain edema.
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(Fabricius et al., 2006; Nakamura et al., 2010; Dohmen et al., 2008). In conclusion, although its mechanisms are far from being fully elucidated, cathodal tDCS a noninvasive, easy to delivery, inexpensive, method of brain excitability modulation, already employed in the experimental treatment of various neurological and psychiatric disorders, seems to be a promising neuroprotective strategy in the very acute phase of stroke treatment.
Acknowledgments This work was partially founded by “Ticino foundation for neurodegenerative diseases”.
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Tergau, F., 2006b. Anticonvulsant effects of transcranial direct-current stimulation (tDCS) in the rat cortical ramp model of focal epilepsy. Epilepsia 47, 1216-1224. Liebetanz, D., Koch, R., Mayenfels, S., König, F., Paulus, W., Nitsche, M.A., 2009. Safety limits of cathodal transcranial direct current stimulation in rats. Clin. Neurophysiol. 120, 1161-1167. Mies, G., Iijima, T., Hossmann, K., 1993. Correlation between peri-infarct DC shifts and ischaemic neuronal damage in rat. Neuroreport 4, 709-711. Nakamura, H., Strong, A.J., Dohmen, C., Sakowitz, O.W., Vollmar, S., Sué, M., Kracht, L., Hashemi, P., Bhatia, R., Yoshimine, T., Dreier, J.P., Dunn, A.K., Graf, R., 2010. Spreading depolarizations cycle around and enlarge focal ischaemic brain lesions. Brain 133, 1994-2006. Nedergaard, M., Hansen, A.J., 1993. Characterization of cortical depolarizations evoked in focal cerebral ischemia. J. Cereb. Blood Flow Metab. 13, 568-574.
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ACCEPTED MANUSCRIPT Nitsche, M.A., Doemkes, S., Karakose, T., Antal, A., Liebetanz, D., Lang, N., Tergau, F., Paulus, W., 2000. Shaping the effects of transcranial direct current stimulation of the human motor cortex. J.
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Neurophysiol. 797, 3109-3117.
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Nitsche, M.A., Paulus, W., 2000. Excitability changes induced in the human motor cortex by weak transcranial direct current stimulation. J. Physiol. 527, 633–639.
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Paxinos, G., Watson, C., 2005. The rat brain in stereotaxic coordinates. Sixth edition. Elsevier London,
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Amsterdam, Burlington.
Priori, A., Berardelli, A., Rona, S., Accornero, N., Manfredi, M., 1998. Polarization of the human
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motor cortex through the scalp. Neuroreport 9, 2257–2260. Peruzzotti-Jannetti, L., Cambiaghi, M., Bacigalupi, M., Gallizioli, M., Gaude, E., Mari, S., Sandrone,
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S., Cursi, M., Teneud, L., Comi, G., Musco, G., Martino, G., Leocani, L., 2013. Safety and efficacy
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of trancranial direct current stimulation in acute experimental ischemic stroke. Stroke, 44, 3166-
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3174.
Richter, F., Fechner, R., Haschke, W., 1996. Initiation of spreading depression can be blocked by transcortical polarization of rat cerebral cortex. Int. J. Neurosci. 86, 111-118. Shin, H.K., Dunn, A.K., Jones, P.B., Boas, D.A., Moskowitz, M.A., Ayata, C., 2006. Vasoconstrictive neurovascular coupling during focal ischemic depolarizations. J. Cereb. Blood Flow Metab. 26,1018-1030. Schlaug, G., Renga, V., 2008. Transcranial direct current stimulation: a non invasive tool to facilitate stroke recovery. Expert Rev. Med. Devices 5,759-768. Somjen, G.G. 2001. Mechanisms of spreading depression and hypoxic spreading depression-like depolarization. Physiol. Rev. 81, 1065–1096.
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ACCEPTED MANUSCRIPT Tatlisumak, T., Takano, K., Meiler, M.R., Fisher, M., 2000. A glycine site antagonist ZD9379 reduces number of spreading depressions and infarct size in rats with permanent middle cerebral artery
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occlusion. Acta Neurochir. Suppl. 76, 331-333.
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Zunzunegui, C., Gao, B., Cam, E., Hodor, A., Bassetti, C.L., 2011. Sleep disturbance impairs stroke
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recovery in the rat. 34, 1261-1269.
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ACCEPTED MANUSCRIPT Figure legends
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Figure 1 Schematic illustration on the rat’s skull of the localization of surgical operculum, of the electrode for
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cathodal transcranial direct current stimulation (tDCS), and of the recording direct current (DC)-
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coupled electrode in relation to bregma (adapted from Paxinos and Watson 2007). Further details are
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given in the methods section.
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Figure 2
Experiments (EXP) and experimental groups with indication of onset of anaesthesia, of surgery
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duration to occlusion of middle cerebral artery (MCA), onset and duration of cathodal transcranial
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direct current stimulation (tDCS) and no stimulation. Note that the duration of surgery in the animals
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belonging to groups 3 and 4 was about 1 hour shorter than in the animals belonging to groups 1 and 2. TDCS was started about 45 minutes after MCA occlusion in groups 1 and 2 and immediately after the MCA occlusion in groups 3 and 4.
Figure 3
A) Results of experiments 1 and 2. Left: histograms representing mean ± standard deviation of the total infarct area at the 6 predefinite levels (L1-L6) in rats receiving cathodal transcranial direct current stimulation (tDCS) and no stimulation. Right: histograms representing mean ± standard deviation of the total infarct volume of rats receiving cathodal tDCS and no stimulation. Circles represent the infarct volume values of individual rats. B) Representative coronal brain sections showing the infarct area at 6 predefined levels in a non stimulated rat of group 4 and in a rat of group 3 treated with cathodal tDCS for 6 hours. The 19
ACCEPTED MANUSCRIPT infarct areas are delimited. L1 is at 2.7 mm anterior to bregma and the interval between each
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level is 1 mm.
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Figure 4
A) Representative recordings of slow potential changes (SPCs) in rats of experiment 2 treated with
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cathodal transcranial direct current stimulation (tDCS) and no stimulation, 3-4 hours after MCA
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occlusion. The insert shows a high-resolution image of a SPC event. B) Histograms representing mean number of SPCs per hour in rats of experiment 2 treated with
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cathodal tDCS (8 animals belonging to group 3) and no stimulation (8 animals belonging to group 4). Bars are standard deviations.
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C) Scatter-plot of number of SPCs and cortical infarct volume and fitting line. Values refer to
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the16 rats of experiment 2 (8 rats treated with cathodal tDCS and 8 rats not stimulated).
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S0022-510X(14)00307-4 doi: 10.1016/j.jns.2014.05.017 JNS 13194
To appear in:
Journal of the Neurological Sciences
Received date: Accepted date:
28 April 2014 6 May 2014
Please cite this article as: Notturno Francesca, Pace Marta, Zappasodi Filippo, Cam Etrugul, Bassetti Claudio L., Uncini Antonino, Neuroprotective effect of cathodal transcranial direct current stimulation in a rat stroke model, Journal of the Neurological Sciences (2014), doi: 10.1016/j.jns.2014.05.017
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Neuroprotective effect of cathodal transcranial direct current stimulation
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in a rat stroke model
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Francesca Notturno* a,b, Marta Pace* b, Filippo Zappasodia,c, Etrugul Camd, Claudio L. Bassettib,d,
Department of Neuroscience and Imaging, University “G. d’Annunzio”, via dei Vestini 31, 66100
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a
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Antonino Uncinia,b
Chieti, Italy
Neurocenter of Southern Switzerland, Via Tesserete 46, 6903 Lugano, Switzerland
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Institute of Advanced Biomedical Technologies, University “G. d’Annunzio”, via dei Vestini 31,
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b
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Universitätsklinik für Neurologie, Inselspital, Bern, Switzerland
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d
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66100 Chieti, Italy
*These authors contributed equally to this work and are shared first authors.
Corresponding author: Prof. Antonino Uncini
Department of Neuroscience and Imaging University "G. d'Annunzio", Chieti-Pescara, Via dei Vestini, 66100 Chieti, Italy [email protected] Tel : +39 0871 3556942
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Abstract
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Experimental focal brain ischemia generates in the penumbra recurrent depolarizations which spread
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across the injured cortex inducing infarct growth. Transcranial direct current stimulation can induce a lasting, polarity-specific, modulation of cortical excitability. To verify whether cathodal transcranial
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direct current stimulation could reduce the infarct size and the number of depolarizations, focal
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ischemia was induced in the rat by the 3 vessels occlusion technique. In the first experiment 12 ischemic rats received cathodal stimulation (alternating 15 minutes on and 15 minutes off) starting 45
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minutes after middle cerebral artery occlusion and lasting 4 hours. In the second experiment 12 ischemic rats received cathodal transcranial direct current stimulation with the same protocol but
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starting soon after middle cerebral artery occlusion and lasting 6 hours. In both experiments controls
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were 12 ischemic rats not receiving stimulation. Cathodal stimulation reduced the infarct volume in the
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first experiment by 20% (p=0.002) and in the second by 30% (p=0.003). The area of cerebral infarction was smaller in animals receiving cathodal stimulation in both experiments (p = 0.005). Cathodal stimulation reduced the number of depolarizations (p=0.023) and infarct volume correlated with the number of depolarizations (p=0.048).
Our findings indicate that cathodal transcranial direct current stimulation exert a neuroprotective effect in the acute phase of stroke possibly decreasing the number of spreading depolarizations. These findings may have translational relevance and open a new avenue in neuroprotection of stroke in humans.
Keywords: peri-infarction depolarizations, cortical spreading depression, non-invasive brain stimulation, brain infarct volume, rat stroke model, neuroprotection.
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ACCEPTED MANUSCRIPT 1. Introduction Experimental ischemia generates electrical instability in the penumbra manifesting with recurrent
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spontaneous waves of depolarization denominated peri-infarction depolarizations (PIDs) (Nedergaard and Hansen 1993; Hossmann 1996). These depolarizations spread across the injured cortex into the
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normally perfused tissue, where they take the characteristics of cortical spreading depression (CSD),
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inducing transient depression of electrocorticogram (Hossmann, 1996). Infarct size correlates, in the
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stroke experimental model, with the numbers of PIDs or duration of PID aggregates (Mies et al., 1993; Dijkhuizen et al., 1999), and the PID number is the independent, determining variable in this
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relationship (Busch et al., 1996).
Transcranial direct current stimulation (tDCS) is a form of non-invasive modulation of brain
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excitability currently employed to modulate brain excitability in many neurological disorders (Fregni
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and Pascual-Leone, 2007). TDCS induces lasting alterations of cortical excitability and changes in
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neural cell membrane potential by manipulating ion channels or by shifting electrical gradients which influence the electrical balance of ions inside and outside the neural membrane. The direction of the excitability shifts is determined by the current polarity (Priori et al., 1998; Nitsche et al., 2000; Nitsche and Paulus, 2000). Experimental and human studies suggested that the after-effects of tDCS might originate from persistent modifications of synaptic efficacy similar to those underlying long-term potentiation and long-term depression of synaptic activity studies (Islam et al., 1995; Liebetanz et al., 2002). In vivo cathodal polarization directly applied to cortical surface in rats is able to completely block CSD and it has been demonstrated that tDCS interferes with the propagation velocity of CSD in a focal and polarity-specific way that lasts beyond the end of tDCS (Richter et al., 1996; Liebetanz et al., 2006a).
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ACCEPTED MANUSCRIPT A very recent study showed that cathodal tDCS applied intermittently for 40 minutes in the acute phase of stroke due transitory middle cerebral artery (MCA) occlusion in the mouse reduced infarct
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volume and promote a better clinical recovery (Peruzzotti-Jametti et al. 2013).
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The aim of this study was to verify the safety and efficacy of long duration cathodal tDCS in the acute phase of stroke induced the rat by the permanent 3 vessels occlusion technique. TDCS was
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discontinuously applied, with cycles of 15 minutes of stimulation alternating with15 minutes of no
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stimulation up to 180 minutes. Moreover we investigated whether cathodal tDCS could inhibits
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recurrent depolarizations.
2. Materials and Methods
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2.1 Animals
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Fifty-three male Sprague Dawley rats (Harlan Laboratories, Udine, Italy) 8-9 weeks old and weighting
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165-265 g at the time of surgery were employed in the study. The rats were housed under 12 hours light/dark cycle and room temperature of 22 °C. Animals were provided with food and water ad libitum. All experiments were approved by the Cantonal Veterinary Authority of Ticino, Switzerland and conducted according to guidelines for the care and use of laboratory animals (Kantonales Veterinäramt Zürich, Switzerland). Effort was made to minimize the number of animals used. The experiments were carried out in the laboratory of the Neurocenter of Southern Switzerland in Bellinzona.
2.2 Induction of focal cerebral ischemia Cerebral ischemia in the left hemisphere was induced by permanent occlusion of the MCA, permanent occlusion of the ipsilateral distal common carotid artery, and temporary occlusion of the contralateral common carotid artery (Chen et al., 1986; Zunzunegui et al., 2011). The rats were anesthetized with 4
ACCEPTED MANUSCRIPT 2% isoflurane in 2 liters/min of O2. A 2 cm vertical skin incision was made midway between the ear and the eye. The skull was exposed where the frontal bone joins the temporal bone and a small area
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(5×5 mm) of the bone overlying the MCA was removed (Fig. 1). After removing the dura mater, the
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MCA and its three main branches dorsal to the rhinal fissure were occluded by bipolar electrocoagulation. The common carotid artery ipsilateral to the occluded MCA was ligated
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permanently using a 4-0 silk suture thread. Finally, the contralateral common carotid artery was
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temporarily occluded for 60 minutes by an aneurism clip. Body temperature was maintained using a heating pad servo-controlled by a rectal temperature probe. The left femoral artery was cannulated for
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continuous blood pressure monitoring by a blood pressure transducer (Harvard, 220 VAC/50 Hz) and
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for serial measurements of pO2, pCO2 and glucose concentration (Radiometer ABL 5).
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2.3 Transcranial direct current stimulation
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TDCS was applied using a constant current stimulator (DC-stimulator Eldith, Germany). The epicranial electrode was a home-made plastic jacket with an inner diameter of 3 mm fixed on the skull 2 mm left and 1 mm posterior to the bregma (Fig. 1). Fixation was achieved by an ionomeric cement (Ketac Cem, ESPE Dental AG, Seefeld, Germany). The jacket was filled with cotton wool soaked in saline solution (0.9% NaCl) resulting in a contact surface of 7 mm2. To prevent the bypassing of currents that would occur in the case of two juxtaposed epicranial electrodes and to induce polarityspecific tDCS effects, a rubber electrode (10.5 cm2) was positioned on the ventral side of the thorax (Liebetanz et al., 2006b). The electrode on the skull was connected to the cathode and the electrode on the chest to the anode of the stimulator. A constant current intensity of 0.2 mA (current density of 2.86 mA/cm2) was applied, within the reported limits of safety and tolerability (Liebetanz et al., 2009). The stimulation protocol consisted of cycles of 15 minutes of stimulation alternating with15 minutes of no stimulation. This protocol was chosen because in rats PIDs are frequent and separated by an interval of 5
ACCEPTED MANUSCRIPT 5-10 minutes and in mice a reduction of the amplitude of the motor potentials, evoked by a transcranial electrical stimulation, is observed for 10 minutes after a cathodal tDCS lasting for 10 minutes
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(Nakamura et al., 2010; Cambiaghi et al., 2010). In the not stimulated animals the electrodes were
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positioned on the skull and the chest without delivering the stimulation. The rats were maintained under slight anesthesia by 2% isoflurane in 1 liter/min of O2 and analgesia by buprenorphine 0.03 mg/kg
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during real and sham tDCS. At the end of the experiment the rats, once awake, were placed back into
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their home cages.
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2.4 Brain damage analysis
Fourthy-eight hours after the induction of the ischemia, the rats were decapitated under isoflurane
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anesthesia. The brain was rapidly removed, frozen in dry ice and stored at -80 °C. For each brain,
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sections of 20 m corresponding to six levels [A = + 2.7 (L1), + 1.7 (L2), + 0.7 (L3), -0.3 (L4), -1.3
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(L5) , -2.3 (L6) mm from bregma] were cut on a cryostat (Paxinos et al., 2005). Twelve to 15 slices for each level were mounted on SuperFrost Plus (Menzel GmbH, Braunschweig, Germany) for histological evaluation. To assess the infarct volume, all the sections were first fixed by means of PBS containing 4% paraformaldehyde for 20 minutes at room temperature and then stained with cresyl violet. On digitized cresyl violet sections the infarct area was delineated and measured using the NIH Image J software (NIH, Bethesda, MD, USA). The infarct volume was estimated knowing the distance between each chosen level and corrected for the edema by multiplying the ratio of the contralateral to ipsilateral volume and taking also into account the volume of the contralateral hemisphere (Gao et al., 2010). Moreover, the brain swelling was calculated as follows: [(ipsilateral hemisphere volume - contralateral hemisphere volume)/contralateral hemisphere volume] x 100 (Junge et al., 2003).
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ACCEPTED MANUSCRIPT To assess the potential damage induced by tDCS coronal brain sections of 20 m thickness were cut serially from the block on a microtome, mounted on a glass slide, and stained with haematoxylin and
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eosin.
2.5 Recording and analysis of slow potential changes
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A slow potential change (SPC) is the hallmark of both peri-infarction depolarizations and cortical
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spreading depression (Leao, 1951; Koroleva and Bures, 1996; Fabricius et al., 2006). We recorded SPCs in direct current modality by using a gold coated miniature screw (0.9 mm diameter) inserted in
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the skull overlying the infarcted hemisphere 1.0 mm left and 1.5 mm anterior to bregma (Fig. 1 ). Care was taken not to pass the dura and this was verified by histology. The head of the screw was soldered
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to a multipolar connector and the assembly was fixed to the skull with cement. The reference was an Ag/AgCl electrode placed in the neck musculature and the animal was grounded with a steel needle
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electrode inserted into the hind limb and connected to the Faraday cage. The signals were amplified in direct current modality (AC/DC Differential Amplifier Model 3000), digitized at 1000 Hz (CED 14101 mKII) and after analog-to-digital conversion stored into a computer for subsequent analyses offline by Spike2 software (CED, UK). SPCs were recorded right after MCA occlusion for the following 6 hours. For quantitative analysis a SPC was defined as a shift in direct current potential of amplitude (negative to positive peak) 0.5 mV. The duration of SPCs was calculated as the interval between the starting point and the return to the baseline. The number, the frequency (number of SPCs in one hour), the mean amplitude and the mean duration in each animal were calculated.
2.6 Experiments and experimental groups Two experiments were performed (Fig. 2). In the first experiment 12 rats, 45 minutes after MCA occlusion, received cathodal tDCS for 4 hours with a cumulative duration of stimulation of 120 minutes 7
ACCEPTED MANUSCRIPT (group 1); 12 rats were not stimulated after ischemia (group 2). Since PIDs start in rats soon after MCA occlusion (Hartings et al., 2003), to anticipate the onset and increase the duration of tDCS without
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prolonging excessively the anesthesia, the time for the surgical procedure to MCA occlusion was
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shortened of about one hour and tDCS was started immediately after the MCA occlusion (Fig. 2). In this second experiment 12 rats received cathodal tDCS for 6 hours with a cumulative duration of
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stimulation of 180 minutes (group 3); 12 rats were not stimulated after ischemia (group 4). Five rats
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(group 5) went through the same surgical procedure of the other animals except for the occlusion of MCA and carotid cerebral arteries (sham ischemia) and received cathodal tDCS for 6 hours.
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Since we thought important to maintain comparable in all animals the effect of isoflurane, a N-methylD-aspartate receptor antagonist, in the possible inhibition of depolarizations the duration of anesthesia
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was approximately 9 hours for all experimental groups (Fig. 2). In 16 animals of the experiment 2 (8
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as described above.
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animals of group 3, treated with tDCS, and 8 animals of group 4, not stimulated) SPCs were recorded
2.7 Statistical analysis
Gaussian distribution of values was verified by Kolmogorov-Smirnov test. To identify the effect of tDCS on infarct volume, independent sample t-test was applied for each experiment to compare the volume values of the rats stimulated with real tDCS with those of the not stimulated rats. Moreover, to check for possible specific effects of stimulation on predefined brain levels, repeated measures ANOVA was applied to the different infarct area values of brain levels, with Stimulation type (real stimulation, no stimulation) as between-subject factors and Level (L1-L6) as within subject factor. In the rat of experiment 2, in which SPCs were recorded (8 stimulated rats of group 3 and 8 not stimulated rats of group 4), repeated measures ANOVA was applied to the number of SPCs, with Stimulation type as between subject factor and Time (hour 1-6) as within subject factor. For all these 16 8
ACCEPTED MANUSCRIPT rats Pearson correlation was performed in order to assess a link between the number and duration of
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SPCs and the infarct volume.
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3. Results 3.1 Physiological variables
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Rectal temperature was 36.5 ± 0.5 °C before MCA occlusion, increased to an average of 37.5 ± 0.5 °C
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within 2 hours and returned to previous values at the end of the experiment. Baseline blood glucose levels were 160 ± 20 mg/dl before the surgery and raised to 200 ± 30 mg/dl
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within 4-6 hours. An increase of blood glucose has been described in the rodent models of cerebral ischemia (Ginsberg and Busto, 1989). Blood pressure and blood gases remained in the normal range
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3.2 Infarct area and volume
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during the experiment.
We did not find macroscopic or microscopic lesions in the brains of group 5 rats with sham ischemia and cathodal tDCS for 6 hours.
The volume of cerebral infarction was significantly smaller in animals receiving cathodal tDCS (group 1 and 3) than no stimulation (group 2 and 4), both in experiment 1 and 2. The volume reduction between groups of stimulated and not stimulated rats was 20% in experiment 1 (62 ± 6 mm3 vs 78 ± 14 mm3, independent t-test t (22) = 3.650, p=0.001), and 30% in experiment 2 (38 ± 9 mm3 vs 55 ± 15 mm3 , independet t-test t (22) = 3.381, p=0.003), (Fig. 3A). The ANOVA design on the area of cerebral infarction evidenced main effects of Stimulation Type both in experiment 1 (F(1,22) = 9.648; p = 0.005) and experiment 2 (F(1,22)=9.648; p=0.005). The lack of the interaction Stimulation Type × Level (p>0.05 in both experiments) suggests that this reduction was not specific for some levels. In Fig. 3B are reported representative coronal brain sections showing the 9
ACCEPTED MANUSCRIPT infarct area at the 6 predefined levels in a rat receiving cathodal tDCS and in a not stimulated rat of experiment 2.
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In the second experiment the infarct area and volumes resulted smaller both in the stimulated (group 3)
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and not stimulated (group 4) animals compared to the first experiment (Fig. 3A). Anyway the comparisons were always made between groups inside the same experiment.
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We found no significant effects in brain swelling between stimulated and not stimulated rats in both
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experiment 1 and 2 (16 ± 7 % and 19 ± 9 % respectively, independent t-test p>0.1 consistently).
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3.3 SPCs
SPCs were recorded in 8 rats of group 3 treated with tDCS for 6 hours and in 8 rats of group 4
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receiving no stimulation. Within few minutes after MCA occlusion, SPCs appeared and recurred
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periodically during the 6 hours of recordings (Fig. 4A). The analysed SPCs had amplitudes ranging
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from 0.5 to 4.9 mV and duration up to 50 sec. The ANOVA design returned a Stimulation Type effect (F(1,14) = 8.210; p = 0.012) and a lack of significance of the interaction Time × Stimulation Type (p>0.05). Indeed, the stimulated rats displayed a total number of SPCs lower than the non stimulated rats (19.8 ± 11.7 vs 112.3 ± 90.5; independent sample t-test: t(7.2) = -2.865, p= 0.023). The difference in the number of SPCs between group 3 and group 4 rats was already significant during the first hour after the MCA occlusion and remained significant for the following 5 hours (lack of the significance Time × Stimulation Type, Fig. 4B). The inter-rat variability of the number of SPCs was much greater in the not stimulated rats (SD = 90.5) than in the rats receiving cathodal tDCS (SD = 11.9) as indicated by the Levene's Test for equality of variances (p < 0.001). No difference in the mean amplitude and mean duration of SPCs was found between stimulated and not stimulated rats (independent t-test p > 0.200, consistently).
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ACCEPTED MANUSCRIPT In the 16 animals in which SPCs were recorded, a positive correlation was found between the total number of SPCs and the infarct volume (Pearson’s r = 0.501, p = 0.048, Fig. 4C). No correlation was
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found between the total duration of SPCs and the infarct volume (p > 0.200).
4. Discussion
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In this study we applied cathodal tDCS during the very early phase of stroke induction employing a
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current density approximately half of the current density used in studying the anticonvulsant effect in a rat model of focal epilepsy (Liebetanz et al., 2006b). Although we cannot exclude that because of the
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operculum in the skull the actual current reaching the cerebral cortex was higher, we did not find macroscopic nor microscopic lesions in the brains of rats with sham ischemia which received tDCS for
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a cumulative time of 180 minutes confirming that the stimulation technique was safe even with a
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duration of stimulation four and half times longer that in the mouse (Peruzzotti-Jametti et al., 2013).
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Cathodal tDCS was able to reduce the infarct size, compared to controls, by 20% in the animals stimulated (15 minutes on and 15 minutes off) for 4 hours. In the animals in which the onset of stimulation was anticipated soon after MCA occlusion and lasted 6 hours the infarct size was reduced , compared to controls by 30%.
TDCS significantly reduced not only the number but interestingly also the inter-rat variability of SPCs in the whole period of stimulation. The link between number of PIDs and infarct size is thought to be causal since eliciting PIDs by potassium application or electrical stimulation results in larger infarct volumes (Busch et al., 1996; Back et al., 1996). On the other hand, administration of glutamate receptor blockers or hypothermia, which reduce PIDs, have neuroprotective effect (Chen et al., 1993; Mies et al., 1993; Tatlisumak et al., 2000; Hartings et al., 2003). The basis for the relationship between PIDs and infarct growth may be an abnormal microvascular vasoconstriction in response to the depolarizations as well as an imbalance between increased metabolic workload, induced by the 11
ACCEPTED MANUSCRIPT depolarizations, and delivery of oxygen and glucose to cerebral cells in the penumbra (Shin et al., 2006; Hashemi et al., 2009).
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The relationships among PIDs, glutamate and growth of the infarcted territory are debated.
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Glutamate excitotoxicity is prominent in the early phase of ischemic stroke and glutamate in the ischemic core may triggers PIDs that propagates in the penumbra causing an expansion of infarction
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(Somjen, 2001). On the other hand it is possible that cell death in the penumbra occurs primarily as a
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consequence of metabolic stress and vasoconstriction induced by PIDs whereas glutamate release and excitotoxicity are secondary to the depolarizations (Hartings et al., 2003). Moreover the
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neuroprotective effect of N-methyl-D-aspartate receptor antagonist may be attributable to block of the spread of PIDs, which depends in part on glutamate receptors, and not to a direct effect on glutamate-
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induced currents per se (Hartings et al., 2003). Whatever is the first trigger of the complex cascade of
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events leading to infarct expansion our findings indicate that cathodal tDCS blocks the origin and the
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repeatedly spontaneous cycling of PIDs which are thought to enlarge ischemic lesions (Nakamura et al., 2010). In the present, as in other experimental studies, infarct size was correlated with the number of SPCs (Mies et al., 1993; Busch et al., 1996; Back et al., 1996; Hartings et al., 2003). Therefore we think that cathodal-tDCS exerts, at least in part, its effect by reducing recurrent spreading depolarizations. Nevertheless we cannot exclude that cathodal tDCS reduces infarct size by other mechanisms. Cortical, continuous, low-frequency electrical stimulation in in a transitory MCA occlusion model had antiapoptotic, anti-inflammatory and angiogenic effects (Baba et al.. 2009). Cathodal tDCS reduces cortical glutamate, and downregulates the N-methyl-D-aspartate receptor NR2B in healthy mice (Petruzzoli-Jannetti et al. 2013). In the transitory MCA occlusion model in the mouse cathodal tDCs, applied in the early phase of stroke, reduced number of apoptotic neuronal cells and postischemic inflammatory reaction (Petruzzoli-Jannetti et al.2013).
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PIDs start in rats soon after MCA occlusion and recur through 40 hours in a biphasic pattern: an initial
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phase lasting about 3 hours and a secondary phase predominating at 8-18 hours which was
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demonstrated to be even more important for the recruitment of penumbral tissue into the infarct core. (Hartings et al., 2003). Peruzzotti-Jametti and colleagues. (2013) showed that delaying of 4.5 hours the
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onset of tDCS after the induction of ischemia induced only a slight, not significant, reduction of the
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infarct volume. Therefore it seems crucial to start cathodal tDCS as soon as possible after the induction of the ischemia. Moreover to explore the full extent of neuroprotective effect a 24 hour experiment of
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stimulation in freely behaving animals should be performed (Hartings et al., 2003). Moving from bench to bedside, CSDs and PIDs have been recorded also in patients with malignant
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middle cerebral artery infarction and has been suggested that halting spreading depolarizations may
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help to avoid the deterioration in these patients due secondary ischemia and fatal brain edema.
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(Fabricius et al., 2006; Nakamura et al., 2010; Dohmen et al., 2008). In conclusion, although its mechanisms are far from being fully elucidated, cathodal tDCS a noninvasive, easy to delivery, inexpensive, method of brain excitability modulation, already employed in the experimental treatment of various neurological and psychiatric disorders, seems to be a promising neuroprotective strategy in the very acute phase of stroke treatment.
Acknowledgments This work was partially founded by “Ticino foundation for neurodegenerative diseases”.
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ACCEPTED MANUSCRIPT Figure legends
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Figure 1 Schematic illustration on the rat’s skull of the localization of surgical operculum, of the electrode for
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cathodal transcranial direct current stimulation (tDCS), and of the recording direct current (DC)-
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coupled electrode in relation to bregma (adapted from Paxinos and Watson 2007). Further details are
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given in the methods section.
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Figure 2
Experiments (EXP) and experimental groups with indication of onset of anaesthesia, of surgery
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duration to occlusion of middle cerebral artery (MCA), onset and duration of cathodal transcranial
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direct current stimulation (tDCS) and no stimulation. Note that the duration of surgery in the animals
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belonging to groups 3 and 4 was about 1 hour shorter than in the animals belonging to groups 1 and 2. TDCS was started about 45 minutes after MCA occlusion in groups 1 and 2 and immediately after the MCA occlusion in groups 3 and 4.
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A) Results of experiments 1 and 2. Left: histograms representing mean ± standard deviation of the total infarct area at the 6 predefinite levels (L1-L6) in rats receiving cathodal transcranial direct current stimulation (tDCS) and no stimulation. Right: histograms representing mean ± standard deviation of the total infarct volume of rats receiving cathodal tDCS and no stimulation. Circles represent the infarct volume values of individual rats. B) Representative coronal brain sections showing the infarct area at 6 predefined levels in a non stimulated rat of group 4 and in a rat of group 3 treated with cathodal tDCS for 6 hours. The 19
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level is 1 mm.
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Figure 4
A) Representative recordings of slow potential changes (SPCs) in rats of experiment 2 treated with
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cathodal transcranial direct current stimulation (tDCS) and no stimulation, 3-4 hours after MCA
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occlusion. The insert shows a high-resolution image of a SPC event. B) Histograms representing mean number of SPCs per hour in rats of experiment 2 treated with
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cathodal tDCS (8 animals belonging to group 3) and no stimulation (8 animals belonging to group 4). Bars are standard deviations.
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C) Scatter-plot of number of SPCs and cortical infarct volume and fitting line. Values refer to
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the16 rats of experiment 2 (8 rats treated with cathodal tDCS and 8 rats not stimulated).
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