Different effects of running wheel exercise and skilled reaching training on corticofugal tract plasticity in hypertensive rats with cortical infarctions

Different effects of running wheel exercise and skilled reaching training on corticofugal tract plasticity in hypertensive rats with cortical infarctions

Behavioural Brain Research 336 (2018) 166–172 Contents lists available at ScienceDirect Behavioural Brain Research journal homepage: www.elsevier.co...

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Behavioural Brain Research 336 (2018) 166–172

Contents lists available at ScienceDirect

Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr

Research report

Different effects of running wheel exercise and skilled reaching training on corticofugal tract plasticity in hypertensive rats with cortical infarctions

MARK

ChanJuan Zhanga,b,1, Yan Zouc,1, Kui Lia, Chao Lia, YingPing Jiangb, Ju Suna, Ruifang Suna, ⁎ HongMei Wena, a b c

Department of Rehabilitation Medicine, The Third Affiliated Hospital, Sun Yat-sen University, 600 Tianhe Road, Guangzhou 510630, Guangdong Province, China Department of Rehabilitation Medicine, Guangdong Second Provincial General Hospital, 466 Xingang Middle Road, Guangzhou 510317, Guangdong Province, China Department of Radiology, The Third Affiliated Hospital, Sun Yat-sen University, 600 Tianhe Road, Guangzhou 510630, Guangdong Province, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Stroke Running wheel exercise Skilled reaching training Pyramidal tract Corticorubral tract Plasticity

The approaches that facilitate white matter plasticity may prompt functional recovery after a stroke. The effects of different exercise methods on motor recovery in stroke rats have been investigated. However, it is not clear whether their effects on axonal plasticity different. The aim of this study was to compare the effects of the forced running wheel exercise (RWE) and skilled reaching training (SRT) on axonal plasticity and motor recovery. Cortical infarctions were generated in stroke-prone renovascular hypertensive rats. The rats were randomly divided into the following three groups: the control (Con) group, the RWE group, and the SRT group. A sham group was also included. The mNSS and forelimb grip strength tests were performed on days 3, 7, 14, 21, 28, 35, and 42 after ischemia. The anterograde tract tracer biotinylated dextran amine (BDA) was injected into the rats to trace the axonal plasticity of the contralesional corticofugal tracts. Compared with the Con group, the mNSS scores in the SRT and RWE groups decreased on day 28 (P < 0.05) and on days 35 and 42 (P < 0.01). The grip strength in the SRT group increased relative to that in the RWE group at 42 day post-ischemia (P < 0.01). Both the RWE and SRT groups exhibited enhanced plasticity of the contralesional corticofugal tract axons at the level of the red nucleus (P < 0.01) and the cervical enlargement (P < 0.01). More contralateral corticorubral tract remodeling was observed at the red nucleus level in the SRT group than in the RWE group (P < 0.001). Taken together, these results suggest that SRT may enhance axon plasticity in the corticorubral tract more effectively than the forced RWE and is associated with better motor recovery after cerebral ischemia.

1. Introduction Stroke is the leading cause of adult disability worldwide, and the subsequent functional rehabilitation remains a major challenge for rehabilitation teams. As shown in recent studies, the infarct volume does not play a major role in motor dysfunction [1]. Instead, the pyramidal tract injury per se and the subsequent degeneration are closely related to the motor deficits after stroke [2]. Secondary anterograde degeneration occurs in remote non-ischemic brain areas, including the ipsilateral thalamus, the striatum, and the distal pyramidal tract, which are synaptically connected with the primary lesion [3,4]. Secondary degeneration is well characterized by diffusion tensor imaging (DTI) and is manifested as decreased fractional anisotropy (FA) in the pyramidal tract and other pathways [5]. As reported by an increasing number of studies, the more extensive the degeneration is, the poorer the grip strength and hand dexterity are, and the greater the level of physical ⁎

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impairment is [2,6,7]. Currently, the mechanism underlying motor recovery is not clear. Rong et al. [8] reported that the degree of degeneration and compensation by the spared peri-infarct corticospinal tract might be an important mechanism for motor recovery, whereas Liu et al. [9] showed that axons from the contralesional motor cortex sprouted into the denervated spinal cord after a stroke and might contribute to functional recovery after stroke. No matter which side is more important, since plasticity of the white matter tract occurs in all stages of stoke, including the acute, sub-acute, and chronic stages [3], therapies intended to alleviate axonal degeneration or promote axonal regeneration to improve functional recovery after stroke seem to have wide therapeutic time windows. Interventions that prompt corticofugal tract remodeling have been explored to enhance functional recovery following stroke, including erythropoietin, human stem cells, a monoclonal antibody against IN-1(a

Corresponding author. E-mail address: [email protected] (H. Wen). Zhang and Zou contributed equally

http://dx.doi.org/10.1016/j.bbr.2017.09.002 Received 25 May 2017; Received in revised form 30 August 2017; Accepted 1 September 2017 Available online 04 September 2017 0166-4328/ © 2017 Published by Elsevier B.V.

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h light/dark cycle throughout the experiment.

monoclonal antibody of recombinant Nogo-A), and constraint-induced movement therapy [10–13]. Because axonal reorganization responds to and is shaped by activity, physical exercise potentially has an influence on axonal rewiring post-stroke [11,14]. Improved behavioral recovery after constraint-induced movement therapy is partially associated with enhanced post-stroke axonal growth and synaptic plasticity [15]. Previous studies have indicated that the influences of different physical training approaches on neurological function recovery vary. Each type of physical training pattern has merits and limitations [16–18]. Physical exercise focuses on motion, particularly locomotor activity, whereas physical training consists of skilled motor tasks. Both methods exert different effects on structural and/or functional recovery in intact and brain-damaged animals [18]. In the study by Maldonado et al. [16], motor skill training was more effective than voluntary running exercise in promoting skilled reaching recovery in the sensorimotor cortices of ischemic rats, although the underlying mechanism was not explored. The hypothesis of this study was that physical training and physical exercise would promote different effects on the plasticity of the corticofugal tract after focal cerebral ischemia. The aims of this study were to compare the effects of forced running wheel exercise (RWE) and skilled reaching training (SRT) on motor function recovery and corticofugal tract remodeling in rats with cortical ischemia.

2.2.2. Pre-MCAO training Two to three weeks prior to the MCAO, all of the RHRSP rats received skilled reaching pre-training, which included group training and individual training. In group training, 3–4 rats were trained together in their feeding cages. A railing with 1-cm gaps was placed in each feeding cage. The rats were able to access food pellets (approximately 45 mg) through the gaps in the railing (Fig. 2A). During the last three days of pre-training, the rats underwent individual training in the single pelletreaching box (Fig. 4B) for 30 min per day to acquaint them with the reaching box used in post-operative training. Each rat was individually trained in a Plexiglas chamber (34cm × 14 cm × 29 cm) with a slit (10 mm) in the center of the front wall. A 5cm × 3.5 cm Plexiglas shelf was attached to the outer front wall in front of the slit at a height of 3 cm above the floor of the chamber, which was fixed to the left or right side of the slit according the preferred limb. One food pellet was placed on the shelf located contralateral to the preferred limb at a distance of 1 cm from the reaching window during each training session. The floor of the training box was composed of metal rods through which the lost pellets fell to prevent the rats from retrieving the pellets that fell inside the box. On the last day of pre-training, the preferred limb and the success rates were recorded. If the rat successfully reached the food and consumed it, the attempt was recorded as “successful”; otherwise, the attempt was recorded as “unsuccessful”. Only the rats with success rates on the reaching task of up to 60% were randomized into the MCAO or sham groups. Indeed, 46 rats that had success rates of more than 60% (approximately asymptotic performance) on the reaching task were selected for the MCAO or sham operations. The preferred-for-reaching forelimb, namely, the limb that was used to reach the food pellets 20 consecutive times over a 10-min session [16], was recorded. Among which 13 rats who failed to learn how to access food through the aperture at the end of pre-MCAO training were withdrawn from further study.

2. Material and methods 2.1. Experimental design The study timeline was illustrated in Fig. 1. All of the rats were pretrained on skilled reaching for 2–3 weeks prior to middle cerebral artery occlusion (MCAO) in the feeding cages (as shown in Fig. 2A). During the last three days of pre-training, the rats underwent individual training in the single pellet-reaching box (as shown in Fig. 2B) for 30 min per day to acquaint them with the reaching box used in the postoperative training. Those rats with success rates of more than 60% on the reaching task performed on the last day of pre-training were selected for MCAO surgery. The different physical training methods began 3 days after MCAO. The behavioral tests were assessed on days 3 and 7 and weekly thereafter until 42 days post-MCAO. The tract tracers were injected on day 42.The rats were sacrificed two weeks later.

2.3. MCAO surgical procedures 46 RHRSP rats (15–16 weeks) were assigned randomly to receive focal cortical infarction (n = 36) or sham MCAO surgery procedures (n = 10) as described previously [4,19]. Briefly, the rats were deeply anesthetized with 10% chloral hydrate (3.5 ml/kg body weight). The right or left (contralateral to the preferred-for-reaching limb) middle cerebral artery (MCA) was exposed and then occluded at the distal segment, which was approximately the origin of the striatal branches, using bipolar electrocoagulation, which resulted in a permanent focal infarction in the neocortex. In the sham-operated animals (n = 10), the MCA was only exposed but not occlusion. Neurological function was evaluated 6 h after MCAO using Bederson’s neurological function test [20]. The Bederson’s scores were evaluated according to the following criteria: no deficits, score of 0; unable to extend the contralateral forelimb, score of 1; flexion of the contra-lateral forelimb, score of 2; mild circling to the contra-lateral side, score of 3; and severe circling and allying to the contra-lateral side, score of 4. Two rats with scores of 4 according to the Bederson’s scores [20]

2.2. Animals 2.2.1. Stroke-prone renovascular hypertensive model 90 male Sprague Dawley rats (3–4 weeks) weighing 60–90 g were used to establish stroke-prone renovascular hypertensive model (RHRSP) as previously described [19]. All of the animals were deeply anesthetized with 10% chloral hydrate (3 ml/kg body weight). Systolic blood pressure was measured in preheated (60 °C, 15 min) conscious rats after bilateral renal artery constriction twice per week for 12 weeks using an indirect tail-cuff sphygmomanometer (China-Japan Friendship Hospital, Beijing, China). Next, 59 rats weighing 300–450 g with systolic blood pressures higher than 180 mm Hg and without stroke symptoms were selected for the pre-MCAO training. The rats were housed socially (3–4 rats/cage) in the same animal care facility on a 12-

Fig. 1. Experimental design. The arrows indicate the timing of pre-training, middle cerebral artery occlusion (MCAO), behavioral tests, different physical training methods (forced running wheel exercise and skilled reaching training), tract tracer injection, and sacrifice. The physical training began on day 3 after stroke and continued until day 42.

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Fig. 2. Pre- and post-operative skilled reaching training. Pre-MCAO group training (A): 3–4 rats in one group were trained in the feeding cage. The rats were allowed to retrieve the food pellets through the gaps (1 cm) in the railing. Individual pre- and postMCAO training (B). The rat reaches through the slit for a single food pellet located on the shelf using the preferred forelimb.

[21]. Neurological function was assessed on a scale of 0–18 points (normal, 0; maximal deficit, 18) by one blinded observer.

and the other 4 of the MCAO rats were excluded from the study because of subarachnoid hemorrhage or death during surgery. Thirty rats with scores of 1–3 were selected and were randomly divided into three groups: MCAO control (n = 10), SRT (n = 10) and RWE (n = 10). All of the procedures were performed during the animals’ dark phase. All of the experimental procedures and animal care were performed according to the guidelines of the Institutional Animal Ethical Committee of Sun Yat-sen University.

2.5.2. Grip strength test The forelimb grip strength was measured using a grip strength test instrument (YLS-13 grip strength test instrument, JiaShi Scientific Instruments Company, Shanghai, China) as previously described [19]. The rat was held by its tail and was pulled back gently until its front paw loosened its grip, and the rat’s maximum grip was recorded automatically. When the grip strength of the affected forelimb was evaluated, the unaffected forepaw was wrapped with tape. The grip strength was measured five times at each time point, and the mean value was calculated. The grip strength test was performed by an independent investigator who was blinded to the treatment.

2.4. Physical exercise and skilled reaching training 2.4.1. Post-MCAO skilled reaching training Beginning at day 3 after MCAO, the rats received skilled reaching training. Every rat with MCAO in the SRT group was placed into a single pellet reaching box with a slit in the middle of the front wall (Fig. 2B) through which the rat could retrieve a food pellet (approximately 45 mg) that had been placed on the shelf in front of the slit. The food pellet was placed contralateral to the preferred limb at a distance of 1 cm from the reaching window. The position of the food enabled the animal to reach the pellets with the preferred but affected limb. The rats were trained 30 min per day, 5 days per week for 40 days, until day 42 following MCAO.

2.6. Infarct volume measurement Five successive coronal sections at 2.0-mm intervals from bregma +4.7 to −5.2 mm were chosen for Nissl staining to quantify the infarct volume. Nissl staining was performed with 0.1% cresyl violet (Sigma) using a standard procedure. The images were captured with a camera and computer connected to the microscope (Olympus BX51). The infarct areas were referred to as zones of irreversible ischemic damage. The volumes of the ipsilateral and contralateral hemispheres were calculated by using ImageJ software (National Institutes of Health, Bethesda, USA), and the relative infarct volume was expressed as a percentage of the contralateral hemisphere.

2.4.2. Post-MCAO forced running wheel exercise Beginning at day 3 after MCAO, the rats received running wheel exercise. The rats were placed into a programmable, motorized wheel apparatus (21 cm diameter, 40 cm long, made at the South China University of Technology, Guangzhou, China) [19] and ran at a speed of 7 rev/min (approximately 5 m/min) for 10 min, which was then increased to 10 rev/min (approximately 7 m/min) for 10 min and finally to 13 rev/min (approximately 9 m/min) for 10 min [19]. The rats in the control group and sham group were housed in standard cages without exercise or training. The rats were trained 30 min per day, 5 days per week for 40 days, until day 42 following MCAO.

2.7. Delivery of anterograde tract tracers Biotinylated dextran amine (BDA) was injected into the forelimb motor cortex contralateral to the ischemic cortex to evaluate the corticofugal tract plasticity in the rats with focal cerebral ischemia [12]. Cranial burr holes were drilled 1.5 mm rostral, 0.5 mm caudal, and 3.5 mm lateral to the bregma, through which 10% BDA, which had a molecular weight of 10,000 Da and was diluted in 0.01 M phosphatebuffered saline, pH 7.2 (Molecular Probes Inc., Eugene, OR, United States), was injected into the cortex with a microsyringe 6 weeks after cerebral ischemia. A total volume of 5 μl of the tracer was administered at 5 sites in the forepaw motor cortex of each animal (stereotaxic coordinates: 1.5 mm rostral to the bregma, 2.5 mm lateral to the midline; 0.5 mm rostral to the bregma, 1.5, 2.5, and 3.5 mm lateral to the

2.5. Behavioral assessments 2.5.1. Modified neurological severity scores (mNSS) The mNSS is a comprehensive measurement of the motor tests (score 1–3). The rats were placed on the floor (score 0–3) and received sensory tests scores (scores of 1–2), balance beam tests scores (scores of 0–6), and lack of reflex and abnormal movements scores (scores of 1–4) 168

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Fig. 3. Location of the infarction area and the BDA injection sites. Gross appearance of the infarction area (A). The red dots illustrate the infarct border. The blue box depicts the cranial burr holes generated for the BDA injection, and the black dots indicate the BDA injection sites. The cresyl violet-stained sections show the typical infarct area (B). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

40 × objective, and then the peduncle was measured four times more in a systematic manner over the area of the peduncle using a 400 × objective. The number of BDA-positive axons was counted in four non-overlapping fields (425 μm × 320 μm), which corresponded to ∼22.51% of the total cerebral peduncle cross-section and was presented as the average number per field on each section. The four values were averaged, and the total number of BDA-positive fibers was extrapolated for each section. The number of BDA- positive fibers obtained from the three consecutive sections was averaged.

midline; and 0.5 mm caudal to the bregma, 2.5 mm lateral to the midline at depths of 1.0 mm, and 1.5 mm below the cortical surface; 1 μl per injection) through a finely drawn glass capillary. The tract tracer injections did not result in animal dropouts. No macroscopic bleeding or overt infections were observed around the tracer injection sites. After this procedure, the dura was covered with gelfoam, the skull was closed with dental cement, and the skin was sutured. Fourteen days after the tracer injection, the rats were sequentially transcardially perfused with saline and 4% paraformaldehyde. The entire brain and spinal cord were immersed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline and cryoprotected in increasing concentrations of sucrose (20% and 30%) until the tissues sank. The tissues were then frozen with isopentane and cut into 40-μm-thick coronal cryostat sections for immunohistochemistry.

2.10. Statistical analysis The statistical analyses were conducted using SPSS software version 17 for windows (IBM Inc., Chicago, IL, USA). All of the data are presented as means ± SEs and were analyzed using one-way analysis of variance followed by the least significant differences (LSD) test for multiple comparisons. The behavioral tests were analyzed by repeated measures ANOVAs at seven different time points. For the tests in which significant treatment or treatment by time interaction effects were noted (at the 0.05 level), one-way ANOVAs were performed for each time point using the LSD post hoc tests. Spearman’s correlation analyses were performed to explore the associations between the crossed contralesional fibers and grip strength.

2.8. Immunohistochemistry for BDA The brain sections were washed in 50 mM Tris-buffered saline (pH 8.0) containing 0.5% Triton X-100 before incubation with an avidinbiotin-peroxidase complex diluted in TBST-X (ABC Elite; Vector Laboratories, Burlingame, CA, USA) overnight at 4 °C, as previously described [12]. On the following day, the staining was revealed with 3,3′-diaminobenzidine (DAB) containing 0.4% ammonium nickel sulfate and 0.004% H2O2. The process was terminated with double-distilled water.

3. Results

2.9. Neuroanatomical analysis

3.1. Infarct volume

All of the brain areas under study were identified using the Paxinos and Watson atlas. Image-Pro Plus image analysis software (Media Cybernetics, Silver Spring, MD, USA) was applied to analyze all of the histological images in a blinded manner. The corticorubral projections were evaluated at the level of the parvocellular red nucleus (bregma −3.0 to −3.5 mm). A 500 μm long intersection line was superimposed on the brain midline [12]. The fibers crossing the midline into the red nucleus ipsilateral to the lesioned motor cortex were quantified using Image-Pro Plus image analysis software (Media Cybernetics, Silver Spring, MD, USA). Five consecutive sections were analyzed, and the mean values of the labeled fibers were determined. The corticospinal projections were evaluated in the denervated gray matter obtained from 40 adjacent cross-sections at the cervical enlargement (C5-7) [22]. The numbers of BDA-stained fibers in the corticospinal tract and the corticorubral tract were normalized to the total number of fibers at the level of the cerebral peduncle ipsilateral to the injection site and multiplied by 100 to control for possible variability in BDA uptake in different rats. The total number of labeled fibers in the cerebral peduncle in three consecutive sections from each animal was estimated [23]. First, the area of the cerebral peduncle was measured using a

The primary infarctions were consistently restricted to the somatosensory cortex (Fig. 3A, 3B). The relative infarct volumes were 13.10% ± 0.30%, 12.47% ± 0.40%, and 12.03% ± 0.43% in the control (Con, n = 10), running wheel exercise (RWE, n = 10), and skilled reaching training (SRT, n = 10) groups, respectively, at 56 days after MCAO. No significant differences in the infarct volumes were observed among the three groups (F = 1.994, P = 0.171). 3.2. Neural functional recovery and grip strength Both physical training and exercise reduced the mNSS scores, as illustrated in Fig. 4A. Significant differences were observed among the three groups at day 28 (F = 9.464, P = 0.001), day 35 (F = 46.098, P < 0.001), and day 42 (F = 43.991, P < 0.001). Compared with the Con group, the mNSS scores of the SRT and RWE groups decreased on day 28 (P < 0.05) and on days 35 and 42 (P < 0.01). Significant differences in the mNSS scores were not observed among the three groups on days 3, 7, 14, and 21 after MCAO (F = 0.091, P = 0.914; F = 0.871, P = 0.430; F = 2.353, P = 0.114, respectively; Fig. 3A). Two-way repeated measures ANOVAs revealed significant time by treatment condition interactions in the mNSS scores (F (5.688) 169

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Fig. 4. mNSS assessment of the different groups (A). *P < 0.05 and **P < 0.01 compared with the Con group. Grip strength tests of the affected forelimb (B). *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the Con group. △P < 0.05, △△P < 0.01, and △△△P < 0.001 compared to the sham group. #P < 0.01 compared with the RWE group. Grip strength tests of the unaffected forelimbs (C). mNSS, modified neurological severity scores; MCAO, middle cerebral artery occlusion; Con, control; RWE, running wheel exercise; SRT, skilled reaching training.

projection (Fig. 5A). In the Con group (n = 10), only a few BDA-labeled CRT fibers crossed the midline and terminated within the contralesional deafferentiated red nucleus area (Fig. 5B). The rats in the RWE (n = 10) and SRT (n = 10) groups exhibited increased number of BDA-positive fibers in the red nucleus ipsilateral to the infarction (Fig. 5C and 5D). The quantitative data revealed a significant increase in the number of fibers that crossed the midline in the RWE and SRT groups compared with the number of fibers that crossed the midline in the Con group (Fig. 5E, P < 0.01and P < 0.001, respectively). A significant difference was observed between the SRT and RWE groups (Fig. 5E, P < 0.001).

= 4.512, P < 0.001). The grip strengths of the affected and unaffected forelimbs are illustrated in Fig. 4B and C. The grip strengths of the affected forelimbs in the RWE and SRT groups were stronger than those in the control group on days 14, 21, 28, 35, and 42 (P < 0.05), but these strengths were weaker than those in the sham group beginning at 3 days post-MCAO (P < 0.05), as shown in Fig. 4B. The grip strength of the affected side was greater in the SRT group than in the RWE group on day 42 (P < 0.01). Two-way repeated measures ANOVAs revealed a significant time by treatment condition interaction on the grip strength of the affected forelimbs (F (11.424) = 3.353, P < 0.001). No significant differences were noted between the SRT and RWE groups on day 3, 7, 14, 21, 35 or 42 or in the unaffected forelimb among the three groups (F = 1.366, P = 0.265; F = 2.234, P = 0.101; F = 2.078, P = 0.116; F = 2.016, P = 0.129; F = 1.376, P = 0.263; F = 0.164, P = 0.920; and F = 1.019, P = 0.396, respectively) (Fig. 4B and C). The two-way repeated measures ANOVAs did not reveal a significant time by treatment condition interaction for the grip strength of the unaffected forelimbs (F (11.225) = 1.031, P = 0.424).

3.3.2. Contralesional corticospinal tract (CST) plasticity Few BDA-labeled CST crossing fibers projected into the cervical enlargement in the sham group (n = 10) and Con group (n = 10) (Fig. 6A, B). The number of BDA-labeled CST fibers that crossed the midline toward the denervated spinal cord was increased in the RWE (n = 10) (Fig. 6C) and SRT (n = 10) (Fig. 6D) groups compared to the number in the Con group (Fig. 6E, P < 0.01and P < 0.05, respectively). No significant difference was noted between the RWE and SRT groups.

3.3. Effects of the different interventions on corticofugal tract plasticity 3.3.1. Contralesional corticorubral tract (CRT) plasticity The CRT in the sham (n = 10) rats was primarily a contralesional

Fig. 5. Biotinylated dextran amine (BDA) staining of the contralesional corticorubral tract axons at the level of the red nucleus. Midline-crossing fibers at the red nucleus in the sham group (A), Con group (B), RWE group (C) and SRT group (D). Scale bars = 50 μm. The interventions increased the number of fibers crossing the midline at the level of red nucleus compared with the Con group. SRT enhanced the contralateral pyramidal tract remodeling in the red nucleus compared with the RWE group (E). *P < 0.01 and **P < 0.001 compared with the Con group, △P < 0.01, △△P < 0.001 compared with the sham group, #P < 0.001 compared with the RWE group. Con, control; RWE, running wheel exercise; SRT, skilled reaching training. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. Biotinylated dextran amine (BDA) staining of the contralesional pyramidal tract axons at the level of the cervical enlargement. The midline crossing fibers in the sham group (A), Con group (B), RWE group (C) and SRT group (D). Scale bar = 50 μm. The interventions increased the number of fibers crossing the midline at the level of the cervical enlargement (E). *P < 0.05 and **P < 0.01 compared with the Con group, △P < 0.01 compared with the sham group. Con, control; RWE, running wheel exercise; SRT, skilled reaching training.

3.3.3. Correlations between the crossed contralesional fibers and grip strength The numbers of contralesional midline-crossing fibers at the levels of the red nucleus (r = 0.892, P < 0.001) and the cervical enlargement (r = 0.553, P < 0.05) were significantly correlated with the grip strength of the affected limb on day 42 after cerebral ischemia.

erythropoietin [12] and human neural stem cells [10] improve contralateral corticofugal tract axon remodeling after ischemic stroke [6]. After the focal ischemic lesion was generated, axonal plasticity of the remaining motor system pathways occurred at several levels of the nervous system, including the red nucleus, cerebellum, and spinal cord, which are associated with skilled reaching behavior [6,12,15]. Axonal remodeling after damage to the motor cortex appears to be activitydependent. In the present study, both running wheel exercise and skilled reaching training enhanced the plasticity of the contralesional corticorubral tract and pyramidal tract axons at the level of the red nucleus and the cervical enlargement, the axonal plasticity was highly correlated with the improvement of forelimb strength after MCAO treated with physical exercise indicated that the axonal plasticity was playing an important role in behavioral recovery, which was in line with previous studies [12,15,22]. Furthermore, skilled reaching training more effectively enhanced contralateral corticorubral tract remodeling in the red nucleus than forced running wheel exercise. Although both the corticospinal tract and corticorubral tract control hand movements in the skilled reaching task, the red nucleus neurons, which are particularly activated during skilled reaching training, play an important role in controlling accurate distal movements [29], and mainly contribute to the aiming and reaching of hand movements [30]. Skilled reaching training might stimulate the function of the red nucleus to a greater extent than running wheel exercise and enhances the structural plasticity of the corticorubral tract. Brain plasticity depends on experience, but the experience of repetitive motor activity alone might not be sufficient to produce brain plasticity [14]. The stimulus, together with input from the limbic and paralimbic structures, is necessary for functional reorganization [31]. Skilled reaching training, also referred to as “task-specific training”, is complex behavioral training that consists of a meaningful task and not solely repetitive activity to train the paralyzed forelimb [14,17]. In addition, “successful” and “unsuccessful” outcomes facilitate the optimization of the motor pattern and functional improvement. This type of meaningful task may be more effective in improving the limb’s functional reorganization, including the forelimb strength, which is consistent with our study. In contrast, the running wheel exercise, which is a type of forced physical exercise, only consist of running repetition, without meaningful limb function, may be less efficacious to limb’s functional improvement [14]. As shown in previous studies, physical training that

4. Discussion In this study, we investigated the effects of physical exercise and training on corticofugal tract plasticity in hypertensive rats with MCAO. Both exercise and training promoted the plasticity of the contralateral pyramidal tract and corticorubral tract, in accordance with previous studies [12,15]. In addition, we found skilled reaching training could enhance the remodeling of the projections from the contralateral corticorubral tract to the red nucleus more effectively than forced running wheel exercise. The same amount of time of skilled reaching training improve the grip strength more than forced running wheel exercise at 42 days post-MCAO. To the best of our knowledge, this study is the first to examine the effects of different patterns of physical training on the axonal plasticity of the contralesional corticofugal tracts after cerebral ischemia in a hypertensive model. Hypertension is one of the most important risk factors for stroke and white matter lesions [24]. Moreover, the pathophysilology and the clinical outcome are quite different in stroke patients with hypertensive arteriosclerosis and normal cerebral blood vessels [24–26]. Spontaneously hypertensive rats are limited by its genetic factors [24,25,27], while RHRSP had a high incidence of spontaneous stroke without genetic susceptibility. Therefore, RHRSP rats share with stroke patients the same pathology basis of hypertension and are more clinically relevant than normotensive or spontaneously hypertensive rats. We found that both wheel running exercise and skilled reaching training enhanced the axonal remodeling of the contralateral corticofugal tract, indicating the beneficial role of contralateral pyramidal tract and corticorubral tract axon plasticity in ischemic stroke recovery, consistent with previous studies [12,15]. The unaffected hemisphere has been shown to compensate for the loss of motor function after stroke [28]. Furthermore, accumulating evidence suggests that the integrity of the contralesional pyramidal tract is uniquely and closely associated with multiple dimensions of motor recovery, particularly in the chronic phase of stroke [6,28]. Several other therapies, including 171

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involves training/learning is generally more effective in promoting the strength of the limb than running exercise, one type of repeated physical exercise [16,18]. Based on the results of the present study, taskoriented rehabilitation, such as skilled reaching training, might enhance pyramidal tract axon sprouting more effectively than forced repeated physical exercise, such as running wheel exercise. In this study, we did not investigate the potential molecular mechanisms of the different effects of the two types of interventions on axonal plasticity. The inhibition of the Nogo-A/NgR1 pathway might be involved in this process [19]. Further studies are needed to illustrate the underlying mechanisms. 5. Conclusions Skilled reaching training may increase the grip strength and enhance the contralateral corticorubral tract plasticity at the red nucleus to a greater extent than forced running wheel exercise in hypertensive rats with cerebral ischemia. Conflicts of interest The authors have no conflicts of interest to declare. Acknowledgments The experiments were performed in the Neurology and Stroke Center Laboratory in The First Affiliated Hospital of Sun Yat-sen University, which is the state’s key laboratory. This study was supported by the National Natural Science Foundation of China (No. 81101461; No.81472156; No. 81672259; and No.81260554). References [1] A. Sterr, S. Shen, A.J. Szameitat, K.A. Herron, The role of corticospinal tract damage in chronic motor recovery and neurorehabilitation: a pilot study, Neurorehabil. Neural Repair 24 (5) (2010) 413–419. [2] P.G. Lindberg, P.H. Skejo, E. Rounis, Z. Nagy, C. Schmitz, H. Wernegren, A. Bring, M. Engardt, H. Forssberg, J. Borg, Wallerian degeneration of the corticofugal tracts in chronic stroke: a pilot study relating diffusion tensor imaging, transcranial magnetic stimulation, and hand function, Neurorehabil. Neural Repair 21 (6) (2007) 551–560. [3] J. Zhang, Y. Zhang, S. Xing, Z. Liang, J. Zeng, Secondary neurodegeneration in remote regions after focal cerebral infarction: a new target for stroke management? Stroke 43 (6) (2012) 1700–1705. [4] S. Xing, Y. Zhang, J. Li, J. Zhang, Y. Li, C. Dang, C. Li, Y. Fan, J. Yu, Z. Pei, J. Zeng, Beclin 1 knockdown inhibits autophagic activation and prevents the secondary neurodegenerative damage in the ipsilateral thalamus following focal cerebral infarction, Autophagy 8 (1) (2012) 63–76. [5] Y. Takenobu, T. Hayashi, H. Moriwaki, K. Nagatsuka, H. Naritomi, H. Fukuyama, Motor recovery and microstructural change in rubro-spinal tract in subcortical stroke, NeuroImagen Clin. 4 (2014) 201–208. [6] M.R. Borich, C. Mang, L.A. Boyd, Both projection and commissural pathways are disrupted in individuals with chronic stroke: investigating microstructural white matter correlates of motor recovery, BMC Neurosci. 13 (2012) 107. [7] M.H. Tuszynski, O. Steward, Concepts and methods for the study of axonal regeneration in the CNS, Neuron 74 (5) (2012) 777–791. [8] D. Rong, M. Zhang, Q. Ma, J. Lu, K. Li, Corticospinal tract change during motor recovery in patients with medulla infarct: a diffusion tensor imaging study, BioMed. Res. Int. (2014) (2014) 524096. [9] Z. Liu, Y. Li, X. Zhang, S. Savant-Bhonsale, M. Chopp, Contralesional axonal remodeling of the corticospinal system in adult rats after stroke and bone marrow stromal cell treatment, Stroke 39 (9) (2008) 2571–2577.

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