Day/night difference in extradural cortical stimulation for motor relearning in a subacute stroke rat model

Abstract

Purpose: The aim of this study was to assess the proper timing of extradural cortical stimulation (ECS) on the motor relearning in a rat model of subacute photothrombotic stroke.

Methods: Photothrombotic infarction was induced on the dominant sensorimotor cortex in male Sprague-Dawley rats after training in a single-pellet reaching task (SPRT). Rats were randomly divided into three groups after stroke: ECS during the inactive period (Day-ECS group), ECS during the active period (Night-ECS group) and no ECS (Non-stimulated group). Six sham-operated rats were assigned to the control group. The Day- and Night-ECS group received continuous ECS for 12 hours during the day or night for 2 weeks from day 4 after the stroke. Behavioral assessment with SPRT was performed daily.

Results: SPRT showed a significantly faster and greater improvement in the Day and Night-ECS groups than in the Non-stimulated group. In the Day- and Night-ECS groups, the success rate of SPRT differed significantly from Non-stimulated group on day 11 and day 8, respectively. In addition, the Night-ECS group showed a significantly higher SPRT success rate than the Day-ECS group from days 10 to 13.

Conclusion: ECS during the active period might be more effective for motor relearning in the subacute stroke rat model.

Keywords

Day/night extradural cortical stimulation electrical stimulation motor learning motor recovery stroke

1 Introduction

Stroke is a major cause of disability in the adult population (Langhorne et al., 2011). Although stroke can induce many different impairments, motor impairment after stroke is a leading cause of disability and inability to perform activities of daily living(Braddom, 2007). Many rehabilitation strategies based on motor learning have been used to enhance motor recovery in stroke patients (Langhorne et al., 2011). Motor learning consists of skill acquisition through training and consolidation that involves stabilization or enhancement of the motor memory (Cohen et al., 2005; Reis et al., 2015). Consolidation depends on the passage of time, and implicit motor learning exhibits specific gains after a posttraining period that includes sleep (Debas et al., 2010). From this perspective, the passage of time could be considered an endogenous learning process. Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that directly enhances cortical excitability and motor function in both healthy individuals and stroke patients (Bastani & Jaberzadeh, 2012). Motor learning through tDCS may help to consolidate motor skills (Reis et al., 2009; Tecchio et al., 2010). In this respect, tDCS could be considered an augmenting exogenous tool for motor learning in humans. Therefore, the interaction between exogenously applied brain stimulation with tDCS and the endogenous learning process induced by the passage of time may be relevant to the consolidation process itself. In healthy subjects, the simultaneous application of tDCS and training was needed to obtain offline skill gains (Reis et al., 2015). However, the appropriate timing of tDCS has been poorly investigated and further information is needed to maximize the effect of tDCS in motor recovery after stroke (Kang et al., 2015).

Some researchers have suggested that direct cortical stimulation with epidural electrodes may produce a similar effect, while at the same time eliminating variations caused by individual differences in skin, hair, and skull thickness (Schlaug et al., 2008). Animal studies of extradural cortical stimulation (ECS) have reported improvements in motor function in acute and subacute photothrombotic infarction in rats (Adkins-Muir & Jones, 2003; Kleim et al., 2003; Machado et al., 2009; Moon et al., 2009; Chang et al., 2015). One previous study showed mechanisms of continuous ECS in the subacute stroke phase that encouraged axonal sprouting from mature neurons or from adult-generated immature neurons in the peri-infarct area (Chang et al., 2015). ECS in subacute photothrombotic infarction in rats could be used as a basic study of tDCS and its relation to motor recovery in human stroke patients. However, motor relearning after ECS during the subacute phase has not been well studied in even rat stroke model. The aim of this study was to evaluate the proper timing of ECS on the improvement of motor function in a rat model of the subacute photothrombotic stroke. This study seeks to provide basic evidence for the appropriate timing of tDCS in human subacute stroke patients.

2 Materials and methods

2.1 Subjects

This experiment was conducted according to the guidelines of the Committee on Animal Research at Samsung Medical Center. Thirty-six 250– 300 g male Sprague– Dawley rats were housed in a controlled animal husbandry unit at 21±1°C with food and water ad libitum. The 30 rats were randomly divided into three groups after stroke into the Day-ECS group, Night-ECS group, and Non-stimulated group. Six other rats were assigned to the control group. The rats in the Non-stimulated group underwent the same procedures as the rats in the treatment group, except for electrical stimulation (sham stimulation). The rats in the control group underwent a sham operation with normal saline instead of a photosensitive dye and no electrical stimulation.

2.2 Behavioral assessment

The single-pellet reaching task (SPRT) was used as the motor learning test for all rats (Gharbawie et al., 2005). From day 4 after stroke onset, the rats were trained to reach for pellets on a shelf using one of their forelimbs. The schedule was 20 pellets or 20 min in one session at 8:50 a.m. each day for 14 consecutive days. A successful reach was one in which an animal grasped a food pellet and brought it into the cage and to its mouth without dropping it. Reaching performance was scored as a percentage of successful reaches. The Non-stimulated group received an extradural cortical electrode implant but no stimulation. The reaching success rate was reported as a percentage. Preoperative reaching performance was an average of scores from the last 5 days of training (Fig. 1).

2.3 Photothrombotic infarction model and implantation of electrodes

Photothrombotic infarction was induced in the dominant cortex as previously described (Watson et al., 1985). The rats were anesthetized with a mixture of ketamine hydrochloride (100 mg/kg) and xylazine (10 mg/kg), their heads were fixed in a small animal stereotaxic frame, and a skin incision was made in the frontoparietal area. A fiber optic cold illumination light source with a 5 mm aperture (Fiberoptic Korea Co., Cheonan, Korea) was stereotaxically positioned 1.5 mm anterior to the bregma and 3.5 mm lateral to the midline of the skull, which is the area controlling forelimb motor activity and part of the sensory cortex.The skull was irradiated with cold light for 2 min, and then a photosensitive dye (Rose Bengal, 20 mg/kg) was injected through a femoral vein. This was followed by additional illumination for 18 min. This infarction model was used to investigate forelimb motor recovery after stroke in previous studies (Moon et al., 2006, 2009; Chang et al., 2015).

A stimulating electrode was implanted immediately after the induction of cortical lesions in all of the rats. A small craniotomy was made at the anterior border of the illumination area to expose the peri-infarct cortex. A 2×4 mm rectangular stimulating electrode (Oscor, Tampa, Florida, USA) was placed on the exposed dura and a 1-mm diameter stainless steel reference electrode was placed on the ipsilateral posterior parietal bone (1.5 cm posterior to the stimulating electrode), such that the electrical current would be delivered around the infarction lesion. The stimulating electrode cable was connected to a swivel adaptor attached to the top of the behavior-testing chamber to prevent the connecting cables from kinking. The electrodes were secured to the skull using additional fixing screws and dental acrylic resin.

2.4 Electrical stimulation

Pulsed electrical stimulation was delivered via a programmable stimulator (Cybermedic, Iksan, Korea) 12 h/day for two weeks after the fourth postoperative day. Any rats that scored more than 10 on Garcia’s neurological examination (Garcia et al., 1995) on the second day after the operation were excluded because of insufficient infarction. Movement thresholds were measured three days after the operation to determine the level of stimulation required to evoke movement of the dominant forelimb and/or dominant face. The stimulation intensity was set to half the movement threshold for individual animals, but a frequency of 50 Hz and a 194-μs pulse duration were uniformly used (Yang et al., 2008; Chang et al., 2015).

The Day-ECS group received continuous cortical stimulation throughout the inactive period from 9 a.m. to 9 p.m. for 14 days starting on day 4 after stroke onset. The Night-ECS group received continuous cortical stimulation throughout the active period from 9 p.m. to 9 a.m. for 14 days starting on day 4 after stroke onset. The Non-stimulated and control groups received no cortical stimulation.

2.5 Immunohistochemical analysis

For immunohistochemical analysis, the brains of 5 rats in each group were cryoprotected in 30% sucrose, sectioned (40 μm), and processed by free-floating methods (Kim et al., 2012). Primary antibodies were applied overnight. We used primary antibodies against rat anti-GFAP (1:1000, Invitrogen), mouse anti-NeuN (1:1000, Millipore) and rabbit anti-Tuj1 (1:1000, Sigma-Aldrich, St. Louis, MO USA). After 3 time washes with PBS, Alexa Fluor 488-conjugated (1:500, Jackson ImmunoResearch Laboratories), Alexa 647-conjugated or Cy3-conjugated (1:500, Jackson ImmunoResearch Laboratories) secondary antibodies and Hoechst 33342 were applied for 30 min at RT. Subsequently, sections were washed, mounted, and observed with a fluorescence microscope (Zeiss LSM700, Goettingen, Germany). For quantitative comparison, the distance of zone, which were located by NeuN-labeled cells from the glial scar border, and thickness of glial scar were analyzed in each section. The intensity of Tuj1-labeled neuritis in the injury core was measured. The average value was used for comparison between the each group.

2.6 Data analysis

We performed separate repeated-measures analysis of variance (ANOVA) with an intervention that was considered a within-subject factor to evaluate the influence of ECS on motor learning, as evaluated by SPRT in each group, including the control group. An additional ANOVA was performed to analyze the differences among the four groups. The criterion for significance was p < 0.05.

3 Results

Two rats dropped out due to a score >10 on Garcia’sneurological examination on the second day post-operation. One rat in the Night-ECS group was excluded for analysis because the electrode was removed spontaneously on day 3 after stroke. We analyzed data from 33 rats in the Day-ECS group (n = 10), Night-ECS group (n = 10), Non-stimulated group (n = 7), and control group (n = 6).

3.1 Behavioral assessment

Figure 2 shows the success rate of SPRT in each group. The analysis of the SPRT success rate shows a significant interaction between the group and time variables (F_39,377 = 6.113, p < 0.001). All groups except the control group improved gradually over 17 days after stroke. SPRT from D4 to D17 in the control group was significantly higher than it was in the Day-ECS, Night-ECS, and Non-stimulated groups, respectively (p < 0.05). The Night-ECS group showed a significantly higher SPRT success rate than the Non-stimulated group from days 8 to 17 (p < 0.05). The Day-ECS group showed a significantly higher SPRT success rate than the Non-stimulated group from days 11 to 17 (p < 0.05). In addition, the Night-ECS group showed a significantly higher SPRT success rate than the Day-ECS group (p < 0.05) from days 10 to 13.Collectively, these results suggest that ECS for 12 h/day significantly improved motor function, and that ECS during the active period was more effective for accelerating motor recovery in rats with subacute stroke.

3.2 Immunohistochemical analysis: Effect of ECS on the infarction lesion

To examine whether ECS affect the improvement of regeneration on rat brain, we explored immunohistological analysis. The thickness of GFAP-labeled glial scar seems to be similar between the Non-stimulated and ECS groups. NeuN-labeled neurons were frequently found below the glial scar in the Non-stimulated group (– 11.36±13.90). Interestingly, NeuN-labeled neurons in the ECS groups were observed above the glial scar (Day-ECS: 49.42±14.10, Night-ECS: 53.19±11.28, Fig. 3C, D). Distance of the NeuN-labeled zone from the border of injury scar was significantly high in the Day- and Night-ECS groups (Fig. 3F). Although the Night-ECS group exhibited higher distance from the injury scar border, there was no significant difference between the Day-ECS and Night-ECS groups. These results suggest that ECS could protect massive cell death near the injury core.

Next, we also examined whether ECS encourages axonal sprouting into the injury core. Tuj1-labeled neurites were frequently found in the glial scar or even in the core region in the Day-ECS and Night-ECS groups. In contrast, Tuj1-labeled fibers were less localized in the glial scar and penumbra region of the Non-stimulated group (Fig. 4). In addition, there was no significant difference between the Day-ECS and Night-ECS groups. These results suggest that ECS for 12 hours per day could encourage axonal sprouting into the injury core from the injury penumbra through the glial scar.

4 Discussion

This study demonstrated that 12 hours of continuous ECS per day for 2 weeks during the subacute stage significantly improved motor relearning in a rat stroke model. ECS may promote both the re-mapping of preexisting neurons and the recruitment of neuroblasts in peri-infarct regions. In addition, ECS during the active period could induce earlier motor relearning than the ECS during the inactive period. Therefore, in the rat model of the subacute photothrombotic stroke, ECS during the active period is recommended to improve motor function.

Motor relearning after a stroke involves a combination of spontaneous and learning-dependent processes (Langhorne et al., 2011). This post-stroke relearning can be the result of plasticity mechanisms (Song et al., 2000; Carmichael & Chesselet, 2002). In adult brain injury such as stroke, plasticity may be induced by activity-, experience-, and injury-dependent paradigms (Overman & Carmichael, 2014). These paradigms can produce axonal sprouting, dendritic morphology changes, and alterations in synaptic connectivity (Overman & Carmichael, 2014). In addition, these mechanisms in cortical region can be determined by NeuN density (Unal-Cevik et al., 2004; Collombet et al., 2006). Potential mechanisms underlying the recovery-promoting effect of electrical stimulation include partial regeneration of neural tissue due to the migration of newborn neural cells toward the stimulation site (Jahanshahi et al., 2014). A previous study showed that ECS encouraged axonal sprouting from mature neurons or from adult-generated immature neurons in the peri-infarct area to improve motor learning in the subacute stroke rat model (Chang et al., 2015). Our results from 12 hours of daily ECS for two weeks are consistent with earlier findings which suggest that electrical stimulation such as ECS may induce brain plasticity. In this study, ECS during the active period led to earlier motor relearning than the ECS during the inactive period. There may have been a synergistic effect between activity- and ECS-dependent paradigms to improve plasticity in this study.

We used ECS to apply electrical current to the cortex instead of tDCS, in order to eliminate confounding factors for tDCS effect such as skin, hair, and skull thickness (Schlaug et al., 2008; Filmer et al., 2014). Anodal tDCS over the primary motor cortex can improve the consolidation of motor skills, which may be the physiologic mechanism by which tDCS improves motor function in humans (Reis et al., 2009; Tecchio et al., 2010; Saucedo Marquez et al., 2013; Reis et al., 2015). Therefore, electrical simulation over primary motor cortex is an exogenous method to promote motor learning consolidation. Also, sleep can be considered as the endogenous process for motor learning consolidation, because consolidation of motor skills after training can occur in a time- or sleep-dependent fashion (Cohen et al., 2005; Reis et al., 2015). The interaction of exogenous and endogenous inductions could produce a synergistic effect on motor learning consolidation. The results of this study indicate that the exogenous induction of ECS may have a stronger synergistic effect during activity than during the inactive period. Coapplication of tDCS and training is required to induce offline skill gains, whereas anodal tDCS applied after the training did not induce skill gains in healthy individuals (Reis et al., 2015). These results indicate that tDCS interacts directly with the physiological consolidation process that already exists during training. This finding is consistent with the results of this study with subacute stroke rat models. Therefore, in both molecular and physiologic concerns, electrical stimulation during the active period can improve motor function.

A previous study of chronological changes in photothrombotic infarction in rats showed that the acute stage lasted for less than 2 days after stroke and that the subacute stage started between the third and seventh day (Moon et al., 2006). Therefore, ECS was delivered from day 4 after stroke to investigate the effect of ECS from the subacute phase onwards. However, the duration of ECS in that study (2 weeks) in a rat stroke model was much longer than any existing tDCS or rTMS protocol for improvement of motor recovery in human stroke patients (Pollock et al., 2014). The duration of ECS in this study was determined based on our previous studies investigating the effect of ECS on motor recovery in a rat stroke model (Moon et al., 2009; Chang et al., 2015). The application of the electrical stimulation protocol in this study is not recommended for human studies. Based on this study, we cannot suggest the appropriate duration of electrical stimulation for improving motor recovery after stroke in human patients nor in a rat model. Further investigation with variable ECS duration will be needed. In addition, the direct application of this method should be carefully considered in stroke rehabilitation because of the lack of information on the timing through which tDCS affects motor recovery after stroke.

There are some limitations to this study. We could not perform immunohistochemical assessment throughout ECS during the active period, during which significantly higher motor relearning occurred. The immunohistochemical analysis was done once at day 17, which is one of the limitations of this study. Another limitation of this study is that a behavioral assessment was only performed once in the morning. tDCS over M1 for 20 mins has a long lasting effect on cortical excitability for 30 to 60 min in healthy individuals (Filmer et al., 2014). According to a previous study with healthy individuals, after-effects of ECS may have influenced the Night-ECS more strongly than the Day-ECS group. Therefore, another behavioral assessment in the evening might yield more accurate results. In addition, we did not assess changes in infarct size after ECS, although no change in infarct size was observed after continuous ECS for 2 weeks in a previous study (Chang et al., 2015). Further experimentation will be needed to address these limitations.

In spite of these limitations, this study showed that 12 hours of ECS a day for 2 weeks could significantly improve motor learning in a rat stroke model that included continuous ECS for 2 weeks (Chang et al., 2015). ECS may encourage axonal sprouting from mature neurons or from adult-generatedimmature neurons in the peri-infarct area. There was a synergistic effect of activity and electrical stimulation on motor improvement. This study could be used to design the proper timing of electrical stimulation to improve motor function in stroke patients.

Conflict of interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Footnotes

Acknowledgments

This study was supported by Samsung Biomedical Research Institute grant [#SMX1131661], the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NRF-2014R1A2A1A01005128), the Brain Research Program through the National Research Foundation (NRF) funded by the Korean Ministry of Science, ICT & Future Planning (NRF-2012M3A9C6049933 and NRF-2013R1A1A3011896).

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