International Journal of Neuroscience, 2015; 00(00): 1–6 Copyright © 2015 Informa Healthcare USA, Inc. ISSN: 0020-7454 print / 1543-5245 online DOI: 10.3109/00207454.2014.998759
ORIGINAL ARTICLE
Combination of early constraint-induced movement therapy and fasudil enhances motor recovery after ischemic stroke in rats Yun-hai Liu,1 Yan Zhao,1,3 Feng-zhen Huang,4 Yun-hong Chen,1 Hong-xing Wang,2 Emmanuel Bonney,2 and Bao-qiong Liu2 Int J Neurosci Downloaded from informahealthcare.com by Gazi Univ. on 05/03/15 For personal use only.
1
Department of Neurology; 2 Rehabilitation, Xiangya Hospital, Central South University, Changsha, P. R. China; Department of Neurology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, P. R. China; 4 Department of Neurology & Institute of Translational Medicine at University of South China, the First People’s Hospital of Chenzhou, Chenzhou, Hunan, P. R. China 3
Purpose: Constraint-induced movement therapy (CIMT) is a promising technique for the recovery of upper extremity movement in chronic stroke patients. However, the effectiveness of its use in acute ischemia has not been confirmed. Myelin-associated inhibitors, which have upregulated functions in tissues affected by acute focal infarction, limit axonal regeneration via activation of the Rho–Rho-associated protein kinase (ROCK) pathway. The present study examined whether early CIMT combined with the ROCK inhibitor fasudil promotes motor recovery after acute ischemic stroke. Materials and Methods: Rats were trained to perform the skilled-reach test and then subjected to middle cerebral artery occlusion (MCAO), producing a stroke affecting the preferred forelimb. Rats were assigned to one of four groups (N = 6/group): (nontreated) Control, CIMT, Fasudil, or CIMT+fasudil. CIMT and/or intraperitoneal infusion of fasudil were initiated 1 day postMCAO. Skilled reach and foot fault test data were collected once before and repeatedly over 4 weeks after the operation. Infarct volumes were calculated. Results: All four groups showed similar forelimb impairment before treatment. The performance of CIMT alone group was similar to that of controls on both tests. Fasudil alone facilitated recovery in the foot-fault test, but not in the skilled-reach test. Rats in the CIMT+fasudil group demonstrated enhanced recovery in both tests, including better performance over time than the Fasudil group on the foot-fault test. Infarct size did not differ significantly between the groups. Conclusions: Early CIMT promotes motor recovery after acute ischemic stroke when it is administered with fasudil pharmacotherapy, but not without it. KEYWORDS: Constraint-induced movement therapy (CIMT), ischemic stroke, fasudil
Introduction Stroke is the leading cause of disability in adults. Although some degree of spontaneous neurological recovery occurs, there is an urgent need to identify therapeutic strategies that can enhance poststroke recovery. It is widely recognized that restorative treatments, including rehabilitative training and pharmacologic augmentation, can support motor recovery after a stroke. In constraint-induced movement therapy (CIMT), which Received 27 April 2014; accepted 11 December 2014 Yun-hai Liu and Yan Zhao contributed equally to this work. Correspondence: Bao-qiong Liu, M.D., Department of Rehabilitation, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China. Tel: +86-13739070224. E-mail: [email protected]
is considered a highly promising rehabilitative training technique, the affected arm is subjected to intensive training by immobilizing the intact arm. Several studies have demonstrated beneficial effects of CIMT on the restoration of impaired upper extremity movement, especially in chronic stroke patients [1, 2]. Meanwhile, there is some evidence from “forced overuse” experiments that suggests that very early CIMT, that is CIMT within 2 weeks of a stroke, may not be beneficial [3, 4]. Myelin-derived molecules inhibit axonal regeneration in the adult central nervous system. In particular, the protein Nogo is a major myelin-derived inhibitor of neural growth. It limits axonal regeneration by activating the Rho–Rho-associated protein kinase (ROCK) pathway in acute focal infarction brain tissue [5, 6]. The presence of Nogo could be limiting the effectiveness of 1
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Y.-h. Liu et al.
CIMT in acute ischemia. The ROCK inhibitor fasudil has been reported to increase cerebral blood flow in the ischemic brain and protect against neuronal cell death, and it has shown promise in the treatment of acute ischemic stroke in animals [7, 8]. We hypothesized that combining early CIMT with fasudil pharmacotherapy could attenuate blockade of neural growth after acute cerebral ischemia, enabling CIMT with fasudil to produce greater benefit than either CIMT or fasudil as monotherapy. In the present study, early CIMT was combined with administration of the ROCK inhibitor fasudil in rats, and behavioral and infarct volumes measurements were compared between treatment groups to determine whether the CIMT plus fasudil combination therapy yields a greater therapeutic benefit after ischemic stroke than either intervention alone.
Materials and Methods Subjects, shaping period, and group assignment Ethical approval of the experimental protocol was obtained from the ethics committee for animal research at our institution. Experiments were conducted on adult male Sprague-Dawley rats (250–300 g body weight). All rats were group housed in standard cages (≥4 per cage) and kept in a controlled temperature with a 12-h light/dark cycle and easy access to food and water. Access to food was removed for 12 h before each skilledreach training and assessment session. Forelimb preference was determined during a preinfarct shaping period in the skilled-reach task apparatus (see description in behavioral assessments section). Animals meeting the training criterion (described below) were assigned randomly to four groups (N = 6/group): (1) Control (ischemia, saline without CIMT); (2) CIMT (ischemia, saline with early CIMT); (3) Fasudil (ischemia, fasudil without CIMT); and (4) CIMT+fasudil (ischemia, fasudil with early CIMT).
Manipulations Surgery. The middle cerebral artery ischemicreperfusion model was applied to impair the preferred forelimb. Rats were anesthetized with chloral hydrate (400 mg/kg, i.p). Transient focal cerebral ischemia was induced by intraluminal middle cerebral artery occlusion (MCAO) with a monafilament [9]. After a 90-min period of MCAO, the filament was withdrawn to allow reperfusion. Treatments. Therapeutic treatments consistent with group assignments were initiated 1 day postMCAO,
and continued for 2 weeks. Twenty-four hours after induction of ischemia, each rat in the CIMT and CIMT+fasudil groups was fitted with a one-sleeve plaster cast to restrain use of the unaffected forelimb by holding it in a slightly retracted position. Thus, CIMT forced animals to rely on their impaired upper limb. Rats only persist in the reaching behavior for 1 h, at which point they may be tired or full. Therefore, the skilled reach task described below was also used for rehabilitative training for 1 h/day [10]. Fasudil was diluted in saline (0.5 ml final volume) and administered intraperitoneally (10 mg/kg daily) from postMCAO day 1 to postMCAO day 14.
Behavioral assessments Behavioral assessments were conducted before MCAO, on post-MCAO days 1 and 4, and weekly during postinfarct weeks 1 to 4 by an investigator who was blinded to the experimental group assignments of the rats. Forelimb motor performance was assessed by a skilled-reach test and a foot fault test. Skilled-reach test. Each animal was placed in a chamber (38.5-cm long × 45-cm high × 12.5 cm wide) designed to limit the use of either the right or left forelimb. The external shelf used to present food pellets was 5 cm from the bottom of the chamber. A single food pellet (45 mg) was delivered during each trial. PreMCAO training sessions were carried out until baseline scores reached a plateau (>65% success rate) with the preferred limb [11]. Rats that could not meet the inclusion criteria were excluded from the experiment. In the skilled reach test sessions, the number of times that the rat was able to reach the food pellet out of 20 trials was calculated. Foot fault test. Each animal was placed on an elevated grid (110 cm × 30 cm with 3.5 cm × 3.5-cm grid openings) and allowed to explore the grid for 2 min. The number of times each forelimb was used and the number of slips made with each forelimb were video-recorded. Foot fault score was determined according to the following formula: Foot f aul t s cor e = noi mpair ed f or elimb sl i ps − no.intact f or el i mb sl i ps total no.movements wi th both f or e limbs
Infarct volume measurement Rats were sacrificed by decapitation at day 28 postMCAO. Their brains were removed immediately and then frozen by being placed in a freezer (−20◦ C) for 15 min. Consecutive 2-mm coronal slices were cut and International Journal of Neuroscience
Early CIMT combines with fasudil enhances recovery Table 1.
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Percentage of successful food pellet retrievals (mean ± SEM). Day(s) postMCAO
Group Control CIMT CIMT+fasudil Fasudil
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∗
Baseline
1
4
7
14
21
28
72.5 ± 7.6 74.2 ± 5.9 75.0 ± 8.4 76.7 ± 6.1
2.5 ± 4.2 4.2 ± 6.7 2.5 ± 4.2 4.2 ± 6.7
11.7 ± 6.8 12.5 ± 6.9 12.5 ± 5.2 8.3 ± 6.8
19.2 ± 7.4 20.8 ± 9.2 27.5 ± 7.6 20.0 ± 8.9
31.7 ± 17.8 45.0 ± 8.4 67.5 ± 17.3∗ 32.5 ± 16.4
33.3 ± 9.8 40.0 ± 5.5 52.5 ± 5.3∗ 38.3 ± 18.6
37.5 ± 5.3 45.0 ± 8.4 58.3 ± 13.7∗ 40.0 ± 11.8
p < 0.05 vs. Control group, post hoc comparisons.
immersed in 10 ml of 2% triphenyltetrazolium chloride (TTC) phosphate buffer (0.2 mmol/L, pH 8.7) at 37◦ C for 30 min. The slices were then postfixed in 4% paraformaldehyde overnight. Infarct volume was calculated with a computerized image analyzer (ImageJ, NIH) based on TTC visualization of the infarct lesion.
Statistical analysis Data were analyzed and compared among the groups using SPSS (version 17.0). Percentage data were normalized with an arcsine square root transformation. The normality of the distribution of each dataset was verified with the Shapiro-Wilk test (normal distribution accepted at p > 0.05). Behavioral data were analyzed with analyses of variance (ANOVAs). Comparisons between the groups were made using the least significant difference post hoc test (similar data variation in compared groups) or Tamhane’s test (equality of variances assumption not suitable). Infarct volume was analyzed across groups with a one-way ANOVA. Statistical significance level was set at p < 0.05.
Results Skilled reach test Before MCAO, there were no significant differences among the groups in terms of their pellet retrieval performance (F3, 20 = 0.337; p = 0.799; Table 1). All groups showed marked deficits in obtaining pellets 1 day
Table 2.
postMCAO, with no significant differences between the groups (F3, 20 = 0.059; p = 0.981; Table 1). All groups showed improvement in their performance over time. The performance of the CIMT and fasudil groups remained similar to that of the nontreated Control group throughout the experimental period. By 2 weeks postMCAO, rats in the CIMT+fasudil group demonstrated better forelimb function on the skilled reach test than the Control group (p < 0.05; Table 1). Thus, CIMT in combination with fasudil facilitated recovery after 2 weeks of treatment, whereas CIMT alone failed to significantly enhance recovery (Figure 1).
Foot fault test There were no significant differences in foot faults among the groups preMCAO (F3, 20 = 1.097; p = 0.373; Table 2). All groups exhibited impaired locomotion to a similar extent on the foot fault test on day 1 postMCAO (F3, 20 = 0.243; p = 0.865; Table 2). Rats in the Fasudil and CIMT+fasudil groups exhibited a more favorable motor recovery on the foot fault test than did rats in the Control group (Figure 2). This divergence from the Control group reached significant levels for both groups on day 14 postMCAO and persisted in its significance until the end of the experiment (day 28). Early CIMT alone did not enhance motor recovery significantly compared with the Control group. Moreover, rats in the combined treatment group demonstrated better forelimb function than fasudil group on the foot-fault test at weeks 2 (p = 0.002) and weeks 4 (p = 0.039).
Foot fault scores (mean ± SEM). Day(s) postMCAO
Group
Baseline
1
4
7
14
21
28
Control CIMT CIMT+fasudil Fasudil
2.2 ± 1.7 1.7 ± 2.8 3.3 ± 3.0 1.1 ± 2.7
33.9 ± 3.9 34.4 ± 5.0 32.2 ± 4.0 33.3 ± 5.6
31.1 ± 3.4 32.2 ± 6.2 30.0 ± 4.2 30.6 ± 7.1
21.1 ± 3.4 20.6 ± 5.3 19.4 ± 4.9 17.2 ± 3.9
13.9 ± 2.5 11.7 ± 1.8 3.9 ± 2.5∗# 8.3 ± 1.8∗
11.7 ± 1.8 10.0 ± 2.1 2.2 ± 1.7∗ 5.6 ± 4.0
10.6 ± 4.4 7.8 ± 1.7 1.7 ± 1.8∗# 5.0 ± 3.5∗
∗
p < 0.05 vs. Control group, post hoc comparisons; # p < 0.05 vs. Fasudil group, post hoc comparisons.
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Skilled-reach test performance. All groups showed marked deficits in obtaining pellets with the strokeimpaired limb after MCAO, with no significant differences between the groups. The performance of all infarct groups improved over time. Rats in the CIMT + fasudil group, but not in the CIMT or Fasudil (monotherapy) groups, demonstrated improved forelimb function after 2 weeks of CIMT, relative to the control group. ∗ differs significantly from controls.
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Figure 1.
Infarct volume As shown in Figure 3, infarct size did not differ significantly between the groups.
Discussion The results of the present study support our hypothesis that combining CIMT with fasudil treatment would improve neurological outcome after an acute ischemic stroke compared with CIMT alone or fasudil alone. The addition of the ROCK inhibitor fasudil during the early recovery phase appears to have improved the neural response to motor training through CIMT [12]. These effects could not be attributed to group differences in infarct size. Our finding that CIMT alone did not significantly improve functional outcome of rats in the acute stroke phase is consistent with previous studies indicating that
Foot-fault test performance. All groups committed more missteps after MCAO versus before, with no significant differences between the groups before or 1 day after the operation. Fasudil monotherapy facilitated recovery of foot placement over 2 weeks of treatment. Furthermore, rats in the CIMT + fasudil combined therapy group exhibited significantly greater improvement than the Fasudil group at 2 weeks postMCAO, and this divergence was sustained through the end of the study. ∗ differs significantly from controls, #differs significantly from Fasudil alone. Figure 2.
Figure 3. Infarct sizes did not differ between the groups.
CIMT during the early poststroke period (e.g. from day 2 to day 11) seems to be safe, but may not yield any significant benefits [4, 13]. Interestingly, rats in the Fasudil (monotherapy) group exhibited significant improvement in the foot fault test, but not in the skilledreach test, perhaps because the latter is more complex. In the skilled-reach test, rats need to complete a series of motions to obtain a pellet, including advancing the forelimb, opening the digits, pronating the wrist, grasping with the paw, supinating the wrist, and releasing the digits. Because of this complexity, rats were pretrained in the skilled-reach test to a criterion level before being subjected to the MCAO operation. Conversely, rats were able to perform the foot-fault task well the first time they were faced with it (single preMCAO trial). Our findings are consistent with the prevalent notion that axonal regeneration, sprouting, and new network formation are blocked by neurite outgrowth inhibitory proteins in the adult central nervous system [14]. Furthermore, this work provides additional support for the model that myelin-derived axonal growth inhibitor acts to limit axonal regeneration in brain tissue affected by acute focal infarction through activation of the Rho-ROCK pathway [15–17]. The ability of CIMT to enhance functional recovery later in rehabilitation has been attributed to neuroplasticity, including changes in neuronal networks mediated by axonal outgrowth and synaptic plasticity as well as changes in long-distance connectivity [18]. Thus, it appears to be the time-limited upregulation of neurite outgrowth inhibitors and their receptors in the acute ischemic brain that constrain axonal sprouting and thereby limit the influence of CIMT in the early phase of stroke recovery. It has been suggested that ROCK inhibition may be a promising therapeutic strategy to enhance recovery after ischemic stroke [19]. Several potential indirect mechanisms for such enhancement have been suggested including limiting inflammatory response [20], increasing regional cerebral blood flow [21], reducing hyperviscosity [22], and reversing down-regulation of eNOS activity in endothelial cells under hypoxic conditions [23]. Furthermore, administration of fasudil has International Journal of Neuroscience
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Early CIMT combines with fasudil enhances recovery
the potential to support neural recovery directly through its molecular blockade of Rho-ROCK pathway signaling; this blockade would be expected to interfere with the anti-sprouting and anti-neural pathfinding actions of Rho-ROCK pathway activation after stroke [24]. Given that sprouting and pathfinding actions are an integral prerequisite for adaptive rewiring of neuronal networks during functional recovery, it seems likely that fasudil’s recovery-promoting effects can be attributed, in large part, to its inhibition of the Rho-ROCK pathway. There are some limitations of the present study to be noted. First, we did not obtain a detailed accounting of histological changes over the recovery period nor did we relate such changes to treatment. In future work, we will conduct anterograde tracing experiments to examine the interacting effects of ROCK inhibition and CIMT on axonal growth in acute ischemic stroke. Second, on postMCAO days 2 and 3, the rats were quite sedentary and showed little motivation to reach for pellets even after 12 h of fasting. Thus, we suspect that there may have been negligible benefit to the first couple days of postMCAO CIMT. It should be determined whether the same benefit could be achieved when commencing CIMT following a 3-day poststroke delay.
Conclusion Although CIMT alone does not enhance recovery in rats in the early stroke recovery phase, at least not as evidenced by the skilled-reach and foot-fault tests, significant improvement in functional outcome can be obtained when early CIMT is combined with fasudil administration. The present results indicate that RhoROCK pathway inhibition with fasudil during early CIMT promotes motor recovery after acute ischemic stroke. Indeed, delivery of fasudil alone in the acute phase of the ischemia recovery can yield a positive impact on the recovery of upper limb use in rats, though the effect is less pronounced than that observed with a combination of CIMT and fasudil.
Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by a grant from Hunan Provincial Natural Science Foundation of China (13JJ3013) and Fundamental Research Funds for the Central Universities (Grant Number: 2012QNZT094). C
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References 1. Taub E, Miller NE, Novack TA, et al. Technique to improve chronic motor deficit after stroke. Arch Phys Med Rehabil 1993; 74(4):347–54. 2. Wolf SL, Winstein CJ, Miller JP et al; EXCITE Investigators. Effect of constraint-induced movement therapy on upper extremity function 3 to 9 months after stroke: the EXCITE randomized clinical trial. JAMA 2006; 296(17):2095–104. 3. Dromerick AW, Lang CE, Birkenmeier RL, et al. Very Early Constraint-Induced Movement during Stroke Rehabilitation (VECTORS): A single-center RCT. Neurology 2009;73(3):195–201. 4. Diederich K, Quennet V, Bauer H, et al. Successful regeneration after experimental stroke by granulocyte-colony stimulating factor is not further enhanced by constraint-induced movement therapy either in concurrent or in sequential combination therapy. Stroke 2012;43(1):185–92. 5. Wang F, Xing S, He M, et al. Nogo-A is associated with secondary degeneration of substantia nigra in hypertensive rats with focal cortical infarction. Brain Res 2012;1469:153–63. 6. Cheatwood JL, Emerick AJ, Schwab ME, Kartje GL. Nogo-A expression after focal ischemic stroke in the adult rat. Stroke. 2008 Jul;39(7):2091–8. 7. Yamashita K, Kotani Y, Nakajima Y, et al. Fasudil, a Rho kinase (ROCK) inhibitor, protects against ischemic neuronal damage in vitro and in vivo by acting directly on neurons. Brain Res 2007;1154:215–24. 8. Satoh S, Toshima Y, Hitomi A, Ikegaki I, Seto M, Asano T. Wide therapeutic time window for Rho-kinase inhibition therapy in ischemic brain damage in a rat cerebral thrombosis model. Brain Res 2008;1193:102–8. 9. Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 1989;20(1):84–91. 10. DeBow SB, Davies ML, Clarke HL, Colbourne F. Constraintinduced movement therapy and rehabilitation exercises lessen motor deficits and volume of brain injury after striatal hemorrhagic stroke in rats. Stroke 2003;34(4):1021–6. 11. Girgis J, Merrett D, Kirkland S, et al. Reaching training in rats with spinal cord injury promotes plasticity and task specific recovery. Brain 2007;130(11):2993–3003. 12. Satoh S, Toshima Y, Hitomi A, et al. Wide therapeutic time window for Rho-kinase inhibition therapy in ischemic brain damage in a rat cerebral thrombosis model. Brain Res 2008;1193:102–8. 13. Bland ST, Schallert T, Strong R, et al. Early exclusive use of the affected forelimb after moderate transient focal ischemia in rats: functional and anatomic outcome. Stroke; a journal of cerebral circulation. 2000;31(5):1144–52. 14. Gonzenbach RR, Schwab ME. Disinhibition of neurite growth to repair the injured adult CNS: focusing on Nogo. Cellular Mol Life Sci: CMLS 2008;65(1):161–76. 15. Craveiro LM, Weinmann O, Roschitzki B, et al. Infusion of antiNogo-A antibodies in adult rats increases growth and synapse related proteins in the absence of behavioral alterations. Exp Neurol 2013;250:52–68. 16. Adelson JD, Barreto GE, Xu L, et al. Neuroprotection from stroke in the absence of MHCI or PirB. Neuron 2012;73(6):1100–7. 17. Wang H, Xiong Y, Mu D. PirB restricts neuronal regeneration in developing rat brain following hypoxia-ischemia. Mol Med Rep 2012 Aug;6(2):339–44. 18. Zhao S, Zhao M, Xiao T, et al. Constraint-induced movement therapy overcomes the intrinsic axonal growth-inhibitory signals in strokerats. Stroke 2013;44(6):1698–705.
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Y.-h. Liu et al. 22. Hitomi A, Satoh S, Ikegaki I, et al. Hemorheological abnormalities in experimental cerebral ischemia and effects of protein kinase inhibitor on blood fluidity. Life Sci 2000;67(16): 1929–39. 23. Takemoto M, Sun J, Hiroki J, et al. Rho-kinase mediates hypoxia-induced downregulation of endothelial nitric oxide synthase. Circulation 2002;106(1): 57–62. 24. Fournier AE, Takizawa BT, Strittmatter SM. Rho kinase inhibition enhances axonal regeneration in the injured CNS. J Neurosci 2003;23(4):1416–23.
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19. Lemmens R1, Jaspers T, Robberecht W, Thijs VN. Modifying expression of EphA4 and its downstream targets improves functional recovery after stroke. Hum Mol Genet 2013;22(11):2214–20. 20. Higashi M, Shimokawa H, Hattori T, et al. Long-term inhibition of Rho-kinase suppresses angiotensin II-induced cardiovascular hypertrophy in rats in vivo: effect on endothelial NAD(P)H oxidase system. Circulation Res 2003;93(8):767–75. 21. Nagata K, Kondoh Y, Satoh Y, et al. Effects of fasudil hydrochloride on cerebral blood flow in patients with chronic cerebral infarction. Clinical Neuropharmacol 1993;16(6):501–10.
International Journal of Neuroscience
ORIGINAL ARTICLE
Combination of early constraint-induced movement therapy and fasudil enhances motor recovery after ischemic stroke in rats Yun-hai Liu,1 Yan Zhao,1,3 Feng-zhen Huang,4 Yun-hong Chen,1 Hong-xing Wang,2 Emmanuel Bonney,2 and Bao-qiong Liu2 Int J Neurosci Downloaded from informahealthcare.com by Gazi Univ. on 05/03/15 For personal use only.
1
Department of Neurology; 2 Rehabilitation, Xiangya Hospital, Central South University, Changsha, P. R. China; Department of Neurology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, P. R. China; 4 Department of Neurology & Institute of Translational Medicine at University of South China, the First People’s Hospital of Chenzhou, Chenzhou, Hunan, P. R. China 3
Purpose: Constraint-induced movement therapy (CIMT) is a promising technique for the recovery of upper extremity movement in chronic stroke patients. However, the effectiveness of its use in acute ischemia has not been confirmed. Myelin-associated inhibitors, which have upregulated functions in tissues affected by acute focal infarction, limit axonal regeneration via activation of the Rho–Rho-associated protein kinase (ROCK) pathway. The present study examined whether early CIMT combined with the ROCK inhibitor fasudil promotes motor recovery after acute ischemic stroke. Materials and Methods: Rats were trained to perform the skilled-reach test and then subjected to middle cerebral artery occlusion (MCAO), producing a stroke affecting the preferred forelimb. Rats were assigned to one of four groups (N = 6/group): (nontreated) Control, CIMT, Fasudil, or CIMT+fasudil. CIMT and/or intraperitoneal infusion of fasudil were initiated 1 day postMCAO. Skilled reach and foot fault test data were collected once before and repeatedly over 4 weeks after the operation. Infarct volumes were calculated. Results: All four groups showed similar forelimb impairment before treatment. The performance of CIMT alone group was similar to that of controls on both tests. Fasudil alone facilitated recovery in the foot-fault test, but not in the skilled-reach test. Rats in the CIMT+fasudil group demonstrated enhanced recovery in both tests, including better performance over time than the Fasudil group on the foot-fault test. Infarct size did not differ significantly between the groups. Conclusions: Early CIMT promotes motor recovery after acute ischemic stroke when it is administered with fasudil pharmacotherapy, but not without it. KEYWORDS: Constraint-induced movement therapy (CIMT), ischemic stroke, fasudil
Introduction Stroke is the leading cause of disability in adults. Although some degree of spontaneous neurological recovery occurs, there is an urgent need to identify therapeutic strategies that can enhance poststroke recovery. It is widely recognized that restorative treatments, including rehabilitative training and pharmacologic augmentation, can support motor recovery after a stroke. In constraint-induced movement therapy (CIMT), which Received 27 April 2014; accepted 11 December 2014 Yun-hai Liu and Yan Zhao contributed equally to this work. Correspondence: Bao-qiong Liu, M.D., Department of Rehabilitation, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China. Tel: +86-13739070224. E-mail: [email protected]
is considered a highly promising rehabilitative training technique, the affected arm is subjected to intensive training by immobilizing the intact arm. Several studies have demonstrated beneficial effects of CIMT on the restoration of impaired upper extremity movement, especially in chronic stroke patients [1, 2]. Meanwhile, there is some evidence from “forced overuse” experiments that suggests that very early CIMT, that is CIMT within 2 weeks of a stroke, may not be beneficial [3, 4]. Myelin-derived molecules inhibit axonal regeneration in the adult central nervous system. In particular, the protein Nogo is a major myelin-derived inhibitor of neural growth. It limits axonal regeneration by activating the Rho–Rho-associated protein kinase (ROCK) pathway in acute focal infarction brain tissue [5, 6]. The presence of Nogo could be limiting the effectiveness of 1
Int J Neurosci Downloaded from informahealthcare.com by Gazi Univ. on 05/03/15 For personal use only.
2
Y.-h. Liu et al.
CIMT in acute ischemia. The ROCK inhibitor fasudil has been reported to increase cerebral blood flow in the ischemic brain and protect against neuronal cell death, and it has shown promise in the treatment of acute ischemic stroke in animals [7, 8]. We hypothesized that combining early CIMT with fasudil pharmacotherapy could attenuate blockade of neural growth after acute cerebral ischemia, enabling CIMT with fasudil to produce greater benefit than either CIMT or fasudil as monotherapy. In the present study, early CIMT was combined with administration of the ROCK inhibitor fasudil in rats, and behavioral and infarct volumes measurements were compared between treatment groups to determine whether the CIMT plus fasudil combination therapy yields a greater therapeutic benefit after ischemic stroke than either intervention alone.
Materials and Methods Subjects, shaping period, and group assignment Ethical approval of the experimental protocol was obtained from the ethics committee for animal research at our institution. Experiments were conducted on adult male Sprague-Dawley rats (250–300 g body weight). All rats were group housed in standard cages (≥4 per cage) and kept in a controlled temperature with a 12-h light/dark cycle and easy access to food and water. Access to food was removed for 12 h before each skilledreach training and assessment session. Forelimb preference was determined during a preinfarct shaping period in the skilled-reach task apparatus (see description in behavioral assessments section). Animals meeting the training criterion (described below) were assigned randomly to four groups (N = 6/group): (1) Control (ischemia, saline without CIMT); (2) CIMT (ischemia, saline with early CIMT); (3) Fasudil (ischemia, fasudil without CIMT); and (4) CIMT+fasudil (ischemia, fasudil with early CIMT).
Manipulations Surgery. The middle cerebral artery ischemicreperfusion model was applied to impair the preferred forelimb. Rats were anesthetized with chloral hydrate (400 mg/kg, i.p). Transient focal cerebral ischemia was induced by intraluminal middle cerebral artery occlusion (MCAO) with a monafilament [9]. After a 90-min period of MCAO, the filament was withdrawn to allow reperfusion. Treatments. Therapeutic treatments consistent with group assignments were initiated 1 day postMCAO,
and continued for 2 weeks. Twenty-four hours after induction of ischemia, each rat in the CIMT and CIMT+fasudil groups was fitted with a one-sleeve plaster cast to restrain use of the unaffected forelimb by holding it in a slightly retracted position. Thus, CIMT forced animals to rely on their impaired upper limb. Rats only persist in the reaching behavior for 1 h, at which point they may be tired or full. Therefore, the skilled reach task described below was also used for rehabilitative training for 1 h/day [10]. Fasudil was diluted in saline (0.5 ml final volume) and administered intraperitoneally (10 mg/kg daily) from postMCAO day 1 to postMCAO day 14.
Behavioral assessments Behavioral assessments were conducted before MCAO, on post-MCAO days 1 and 4, and weekly during postinfarct weeks 1 to 4 by an investigator who was blinded to the experimental group assignments of the rats. Forelimb motor performance was assessed by a skilled-reach test and a foot fault test. Skilled-reach test. Each animal was placed in a chamber (38.5-cm long × 45-cm high × 12.5 cm wide) designed to limit the use of either the right or left forelimb. The external shelf used to present food pellets was 5 cm from the bottom of the chamber. A single food pellet (45 mg) was delivered during each trial. PreMCAO training sessions were carried out until baseline scores reached a plateau (>65% success rate) with the preferred limb [11]. Rats that could not meet the inclusion criteria were excluded from the experiment. In the skilled reach test sessions, the number of times that the rat was able to reach the food pellet out of 20 trials was calculated. Foot fault test. Each animal was placed on an elevated grid (110 cm × 30 cm with 3.5 cm × 3.5-cm grid openings) and allowed to explore the grid for 2 min. The number of times each forelimb was used and the number of slips made with each forelimb were video-recorded. Foot fault score was determined according to the following formula: Foot f aul t s cor e = noi mpair ed f or elimb sl i ps − no.intact f or el i mb sl i ps total no.movements wi th both f or e limbs
Infarct volume measurement Rats were sacrificed by decapitation at day 28 postMCAO. Their brains were removed immediately and then frozen by being placed in a freezer (−20◦ C) for 15 min. Consecutive 2-mm coronal slices were cut and International Journal of Neuroscience
Early CIMT combines with fasudil enhances recovery Table 1.
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Percentage of successful food pellet retrievals (mean ± SEM). Day(s) postMCAO
Group Control CIMT CIMT+fasudil Fasudil
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∗
Baseline
1
4
7
14
21
28
72.5 ± 7.6 74.2 ± 5.9 75.0 ± 8.4 76.7 ± 6.1
2.5 ± 4.2 4.2 ± 6.7 2.5 ± 4.2 4.2 ± 6.7
11.7 ± 6.8 12.5 ± 6.9 12.5 ± 5.2 8.3 ± 6.8
19.2 ± 7.4 20.8 ± 9.2 27.5 ± 7.6 20.0 ± 8.9
31.7 ± 17.8 45.0 ± 8.4 67.5 ± 17.3∗ 32.5 ± 16.4
33.3 ± 9.8 40.0 ± 5.5 52.5 ± 5.3∗ 38.3 ± 18.6
37.5 ± 5.3 45.0 ± 8.4 58.3 ± 13.7∗ 40.0 ± 11.8
p < 0.05 vs. Control group, post hoc comparisons.
immersed in 10 ml of 2% triphenyltetrazolium chloride (TTC) phosphate buffer (0.2 mmol/L, pH 8.7) at 37◦ C for 30 min. The slices were then postfixed in 4% paraformaldehyde overnight. Infarct volume was calculated with a computerized image analyzer (ImageJ, NIH) based on TTC visualization of the infarct lesion.
Statistical analysis Data were analyzed and compared among the groups using SPSS (version 17.0). Percentage data were normalized with an arcsine square root transformation. The normality of the distribution of each dataset was verified with the Shapiro-Wilk test (normal distribution accepted at p > 0.05). Behavioral data were analyzed with analyses of variance (ANOVAs). Comparisons between the groups were made using the least significant difference post hoc test (similar data variation in compared groups) or Tamhane’s test (equality of variances assumption not suitable). Infarct volume was analyzed across groups with a one-way ANOVA. Statistical significance level was set at p < 0.05.
Results Skilled reach test Before MCAO, there were no significant differences among the groups in terms of their pellet retrieval performance (F3, 20 = 0.337; p = 0.799; Table 1). All groups showed marked deficits in obtaining pellets 1 day
Table 2.
postMCAO, with no significant differences between the groups (F3, 20 = 0.059; p = 0.981; Table 1). All groups showed improvement in their performance over time. The performance of the CIMT and fasudil groups remained similar to that of the nontreated Control group throughout the experimental period. By 2 weeks postMCAO, rats in the CIMT+fasudil group demonstrated better forelimb function on the skilled reach test than the Control group (p < 0.05; Table 1). Thus, CIMT in combination with fasudil facilitated recovery after 2 weeks of treatment, whereas CIMT alone failed to significantly enhance recovery (Figure 1).
Foot fault test There were no significant differences in foot faults among the groups preMCAO (F3, 20 = 1.097; p = 0.373; Table 2). All groups exhibited impaired locomotion to a similar extent on the foot fault test on day 1 postMCAO (F3, 20 = 0.243; p = 0.865; Table 2). Rats in the Fasudil and CIMT+fasudil groups exhibited a more favorable motor recovery on the foot fault test than did rats in the Control group (Figure 2). This divergence from the Control group reached significant levels for both groups on day 14 postMCAO and persisted in its significance until the end of the experiment (day 28). Early CIMT alone did not enhance motor recovery significantly compared with the Control group. Moreover, rats in the combined treatment group demonstrated better forelimb function than fasudil group on the foot-fault test at weeks 2 (p = 0.002) and weeks 4 (p = 0.039).
Foot fault scores (mean ± SEM). Day(s) postMCAO
Group
Baseline
1
4
7
14
21
28
Control CIMT CIMT+fasudil Fasudil
2.2 ± 1.7 1.7 ± 2.8 3.3 ± 3.0 1.1 ± 2.7
33.9 ± 3.9 34.4 ± 5.0 32.2 ± 4.0 33.3 ± 5.6
31.1 ± 3.4 32.2 ± 6.2 30.0 ± 4.2 30.6 ± 7.1
21.1 ± 3.4 20.6 ± 5.3 19.4 ± 4.9 17.2 ± 3.9
13.9 ± 2.5 11.7 ± 1.8 3.9 ± 2.5∗# 8.3 ± 1.8∗
11.7 ± 1.8 10.0 ± 2.1 2.2 ± 1.7∗ 5.6 ± 4.0
10.6 ± 4.4 7.8 ± 1.7 1.7 ± 1.8∗# 5.0 ± 3.5∗
∗
p < 0.05 vs. Control group, post hoc comparisons; # p < 0.05 vs. Fasudil group, post hoc comparisons.
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Y.-h. Liu et al.
Skilled-reach test performance. All groups showed marked deficits in obtaining pellets with the strokeimpaired limb after MCAO, with no significant differences between the groups. The performance of all infarct groups improved over time. Rats in the CIMT + fasudil group, but not in the CIMT or Fasudil (monotherapy) groups, demonstrated improved forelimb function after 2 weeks of CIMT, relative to the control group. ∗ differs significantly from controls.
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Figure 1.
Infarct volume As shown in Figure 3, infarct size did not differ significantly between the groups.
Discussion The results of the present study support our hypothesis that combining CIMT with fasudil treatment would improve neurological outcome after an acute ischemic stroke compared with CIMT alone or fasudil alone. The addition of the ROCK inhibitor fasudil during the early recovery phase appears to have improved the neural response to motor training through CIMT [12]. These effects could not be attributed to group differences in infarct size. Our finding that CIMT alone did not significantly improve functional outcome of rats in the acute stroke phase is consistent with previous studies indicating that
Foot-fault test performance. All groups committed more missteps after MCAO versus before, with no significant differences between the groups before or 1 day after the operation. Fasudil monotherapy facilitated recovery of foot placement over 2 weeks of treatment. Furthermore, rats in the CIMT + fasudil combined therapy group exhibited significantly greater improvement than the Fasudil group at 2 weeks postMCAO, and this divergence was sustained through the end of the study. ∗ differs significantly from controls, #differs significantly from Fasudil alone. Figure 2.
Figure 3. Infarct sizes did not differ between the groups.
CIMT during the early poststroke period (e.g. from day 2 to day 11) seems to be safe, but may not yield any significant benefits [4, 13]. Interestingly, rats in the Fasudil (monotherapy) group exhibited significant improvement in the foot fault test, but not in the skilledreach test, perhaps because the latter is more complex. In the skilled-reach test, rats need to complete a series of motions to obtain a pellet, including advancing the forelimb, opening the digits, pronating the wrist, grasping with the paw, supinating the wrist, and releasing the digits. Because of this complexity, rats were pretrained in the skilled-reach test to a criterion level before being subjected to the MCAO operation. Conversely, rats were able to perform the foot-fault task well the first time they were faced with it (single preMCAO trial). Our findings are consistent with the prevalent notion that axonal regeneration, sprouting, and new network formation are blocked by neurite outgrowth inhibitory proteins in the adult central nervous system [14]. Furthermore, this work provides additional support for the model that myelin-derived axonal growth inhibitor acts to limit axonal regeneration in brain tissue affected by acute focal infarction through activation of the Rho-ROCK pathway [15–17]. The ability of CIMT to enhance functional recovery later in rehabilitation has been attributed to neuroplasticity, including changes in neuronal networks mediated by axonal outgrowth and synaptic plasticity as well as changes in long-distance connectivity [18]. Thus, it appears to be the time-limited upregulation of neurite outgrowth inhibitors and their receptors in the acute ischemic brain that constrain axonal sprouting and thereby limit the influence of CIMT in the early phase of stroke recovery. It has been suggested that ROCK inhibition may be a promising therapeutic strategy to enhance recovery after ischemic stroke [19]. Several potential indirect mechanisms for such enhancement have been suggested including limiting inflammatory response [20], increasing regional cerebral blood flow [21], reducing hyperviscosity [22], and reversing down-regulation of eNOS activity in endothelial cells under hypoxic conditions [23]. Furthermore, administration of fasudil has International Journal of Neuroscience
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Early CIMT combines with fasudil enhances recovery
the potential to support neural recovery directly through its molecular blockade of Rho-ROCK pathway signaling; this blockade would be expected to interfere with the anti-sprouting and anti-neural pathfinding actions of Rho-ROCK pathway activation after stroke [24]. Given that sprouting and pathfinding actions are an integral prerequisite for adaptive rewiring of neuronal networks during functional recovery, it seems likely that fasudil’s recovery-promoting effects can be attributed, in large part, to its inhibition of the Rho-ROCK pathway. There are some limitations of the present study to be noted. First, we did not obtain a detailed accounting of histological changes over the recovery period nor did we relate such changes to treatment. In future work, we will conduct anterograde tracing experiments to examine the interacting effects of ROCK inhibition and CIMT on axonal growth in acute ischemic stroke. Second, on postMCAO days 2 and 3, the rats were quite sedentary and showed little motivation to reach for pellets even after 12 h of fasting. Thus, we suspect that there may have been negligible benefit to the first couple days of postMCAO CIMT. It should be determined whether the same benefit could be achieved when commencing CIMT following a 3-day poststroke delay.
Conclusion Although CIMT alone does not enhance recovery in rats in the early stroke recovery phase, at least not as evidenced by the skilled-reach and foot-fault tests, significant improvement in functional outcome can be obtained when early CIMT is combined with fasudil administration. The present results indicate that RhoROCK pathway inhibition with fasudil during early CIMT promotes motor recovery after acute ischemic stroke. Indeed, delivery of fasudil alone in the acute phase of the ischemia recovery can yield a positive impact on the recovery of upper limb use in rats, though the effect is less pronounced than that observed with a combination of CIMT and fasudil.
Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by a grant from Hunan Provincial Natural Science Foundation of China (13JJ3013) and Fundamental Research Funds for the Central Universities (Grant Number: 2012QNZT094). C
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