- Email: [email protected]
Early and late treadmill training after focal brain ischemia in rats
Neuroscience Letters 339 (2003) 91–94 www.elsevier.com/locate/neulet
Early and late treadmill training after focal brain ischemia in rats Yea-Ru Yanga, Ray-Yau Wanga,*, Paulus Shyi-Gang Wangb a
Faculty and Institute of Physical Therapy, National Yang-Ming University, 155, Sec 2, Li-Nong Street, Shih-Pai, Taipei, Taiwan b Department and Institute of Physiology, National Yang-Ming University, 155, Sec 2, Li-Nong Street, Shih-Pai, Taipei, Taiwan Received 11 March 2002; received in revised form 24 November 2002; accepted 9 December 2002
Abstract Treadmill training is increasingly recognized as an effective means to promote rhythmical vigorous walking and also as a useful method of task-related training. The present study endeavored to investigate the effects of early and late treadmill training after ligation of the middle cerebral artery. Forty male Sprague – Dawley rats were subjected to 60 min right middle cerebral artery occlusion (MCAO). All rats were randomly assigned to one of four groups: 24 h group, 2 week no training group, early training group (training started 24 h post MCAO), and late training group (training started 1 week post MCAO). Infarct volume was measured morphometrically. A five-point neurological evaluation scale was used to assess the neurological status of rats. Rats sacrificed 24 h post MCAO had the largest infarct volume (177.8 ^ 14.3 mm3) and the highest neurological score [2(1 – 4)]. Early treadmill training was found to have significant effects in reducing brain infarct volume and in improving neurologic function when compared with spontaneous recovery. However, the same effects cannot be found in late training. Based on the present findings, we would encourage early treadmill training for ischemic brain recovery. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Treadmill training; Middle cerebral artery occlusion; Stroke; Infarct volume; Neurologic function; Rats
It is a common practice among stroke centers to provide treatment and training programs to patients shortly after stroke onset. The findings of many recent studies suggest that the intensity of the manipulation, as well as the time point at which it is implemented, could have profound effects on outcome [15]. However, the optimal time window for therapeutic intervention is still not clear. Some experimental data have shown that general activation starting 24 h after an ischemic event promotes functional outcome without increasing tissue loss [6,12]. However, other reports indicated that intense early training might have a negative effect. Kozlowski et al. [7] immobilized the ipsilateral forelimbs of rats immediately after focal lesions of the forelimb sensorimotor cortex, forcing overuse of the affected limb for 14 days, resulting in retardation of functional improvement. Humm et al. [4] found that forced overuse of the impaired forelimb during the first 7 days after lesions of the forelimb representation area of the sensorimotor cortex in rats caused expansion of neural injury and greatly interfered with restoration of * Corresponding author. Tel.: þ886-2-2826-7210; fax: þ 886-2-28201841. E-mail address: [email protected] (R.-Y. Wang).
function. Risedal et al. [14] indicated that the specific training (1 h a day 5 days a week) initiated 24 h after focal brain ischemia can increase cortical tissue loss. Because of these conflicting reports, further studies are needed to determine the window for the critical period when training may add to promote functional outcome without increasing neuronal tissue loss. It has been postulated that neuroplasticity and the extent of recovery from forelimb motor asymmetries following brain injury depends in part on the degree of learned suppression of use of the impaired limb [11,17]. Animal studies have suggested that forced use engendering a repetitive motor task may best promote central neural plasticity [10]. Recently, treadmill walking has been shown to be effective in promoting rhythmical walking and also a useful method of training for chronic and acute individuals after a stroke [3,13]. The purpose of the present study was to investigate the effect of early and late treadmill training after brain ischemic lesions caused by middle cerebral artery occlusion (MCAO) in rats. Forty male Sprague– Dawley rats, between 2 and 3 months of age, were used as subjects. Animals were housed in groups of two and maintained under a 12/12 h light/dark cycle with food and water available ad libitum. Middle
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00010-7
92
Y.-R. Yang et al. / Neuroscience Letters 339 (2003) 91–94
cerebral artery occlusion procedure leading to focal cerebral ischemia was conducted under chloral hydrate anesthesia (with single 0.5 g/kg i.p. bolus in 1 ml of saline provided anesthesia lasting at least 2 h) for each rat. Rectal temperature was monitored throughout the surgical procedures and maintained at normothermic (37.0 ^ 0.5 8C) by a heating blanket controlled by an electronic temperature controller (HB 101/2, Debiomed). The right middle cerebral artery (MCA) was exposed using microsurgical techniques [18]. Briefly, a 2 mm burr hole was drilled at the junction of the zygomatic arch and the squamous portion of the temporal bone, following a 2 cm vertical skin incision midway between the right eye and ear and splitting of the temporalis muscle. The right MCA trunk was ligated above the rhinal fissure with 10 –0 suture. Complete interruption of blood flow was confirmed by using an operating microscope. Both common carotid arteries (CCAs) were immediately occluded following ligation of MCA with the use of nontraumatic aneurysm clips. After the predetermined duration of ischemia (60 min), the aneurysm clips and ligation were removed from both CCAs and MCA. Restoration of blood flow in all three arteries was observed directly under the microscope. A motor driven treadmill (Treadmill Simplex II, Columbus Instruments, USA) was used for the training protocol. Before MCAO procedure, all rats were placed on a moving belt facing away from the electrified grid and run in the direction opposite of the movement of the belt over a 3 day accommodation period. This allows rats to move forward in order to avoid foot shocks (with intensity in 1.0 mA, Stimulus Controller Model D48E, DRI Co., Taiwan). All rats learned how to run after 3 day accommodation period. After MCAO procedure, all rats were randomly assigned to one of four groups (n ¼ 10 for each group). Rats in Group I were sacrificed at 24 h post MCAO procedure. Rats in Group II remained relatively inactive for 2 weeks after MCAO and thereafter were sacrificed (spontaneous recovery). Rats in Group III were scheduled to run in the treadmill commenced 24 h after MCAO for a period of 1 week, and then remained relatively inactive for another 1 week before sacrifice (early training). Rats in Group IV remained relatively inactive for 1 week after MCAO and thereafter, scheduled to run in the treadmill for another 1 week, were sacrificed (late training). The treadmill training was 30 min per day, 5 days a week with a speed of 20 m/min and 08 slope. Neurological examination was performed before killing. A neurologic grading system with a five-point scale (0 – 4) described by Menzies et al. [9] was used: 0 ¼ no apparent deficits; 1 ¼ left forelimb flexion; 2 ¼ decreased grip of the left forelimb while tail pulled; 3 ¼ spontaneous movement in all directions; left circling only if pulled by tail; 4 ¼ spontaneous left circling. Rats were sacrificed under ketamine anesthesia by intracardiac perfusion with 200 ml of 0.9% NaCl. The brain was removed carefully and dissected into coronal 2
mm sections using a brain slicer. The fresh brain slices were immersed in a 2% solution of 2,3,5-triphenyltetrazolium chloride in normal saline at 37 8C for 30 min, and then fixed in 10% phosphate-buffered formalin at 4 8C [18]. Unstained areas on each brain slice, defined as infarction area, were then measured by an image analyzer (Image-Pro Plus). The damaged areas measured were mainly confined to cerebral cortex including its adjacent caudate nucleus, putamen, and hippocampus. Total measured infarct volume (MV) for each brain was calculated by summation of the infarcted area of all brain slices (area of infarct in square millimeters times thickness, 2 mm) from the same hemisphere. Both right hemisphere volume (RV) and left hemisphere volume (LV) were also measured and calculated. To compensate for the effect of brain edema on MV in the ischemic hemisphere, corrected infarct volume was calculated by the following formula, as previously described [16]: Corrected infarct volume ¼ LV 2 (RV 2 MV). Infarct volumes were expressed as mean ^ SEM. Comparison of infarct volumes was made by analysis of variance. The level of significance for a difference between two groups was further analyzed with post-hoc Tukey’s protected t test. Neurological scores were expressed as median (range) and were compared by Kruskal – Wallis analysis followed by the Mann – Whitney U test to compare medians. A probability value of less than 0.05 was considered to be significant. Rats sacrificed 24 h post MCAO had the largest infarct volume (177.8 ^ 14.3 mm3) and the highest neurological score [2(1 – 4)]. There was a significant effect of group on infarct volume [Fð3; 36Þ ¼ 7:2, P , 0:001] and neurological score (H ¼ 16:9, d:f: ¼ 3, P ¼ 0:001), as shown in Fig. 1 and Table 1. Post-hoc analysis revealed a significant difference in infarct volume (177.8 ^ 14.3 versus 102.1 ^ 6.5 mm3, P , 0:001) and neurological score [2(1 –4) versus 0(0 –1), P , 0:001] between 24 h group (Group I) and early training group (Group III). It also showed a significant difference for the infarct volume (177.8 ^ 14.3 versus 121.6 ^ 7.4 mm3, P , 0:01) and
Fig. 1. Infarct volume as mean ^ SEM at various conditions after middle cerebral artery occlusion in rats. ** Significantly different (P , 0:01) from the 24 h after middle cerebral artery occlusion. þ Significantly different (P , 0:05) from the 2 weeks without treadmill training after middle cerebral artery occlusion.
Y.-R. Yang et al. / Neuroscience Letters 339 (2003) 91–94 Table 1 Neurological score post middle cerebral artery occlusion in ratsa
24 h group 2 week no training group Early training group Late training group
Group
Number
Neurological Score
I II III IV
10 10 10 10
2 (1–4) 2 (0–3) 0 (0–1)***, 0 (0–3)*
þþ
a Neurological scores are expressed as median (range). *P , 0:05, ***P , 0:001 versus 24 h group. þ þ P , 0:01 versus 2 week no training group.
neurological score [2(1 – 4) versus 0(0 – 3), P , 0:05] between 24 h group and late training group (Group IV). There were no significant differences in the infarct volume and neurological score between 24 h group and 2 week no training group (Group II). Post-hoc analysis also revealed that the infarct volume (102.1 ^ 6.5 versus 143.6 ^ 15.0 mm3, P , 0:05) and neurological score [0(0 –1) versus 2(0 – 3), P , 0:005] of early training group were significantly lower than those of the 2 week no training group. In contrast, the infarct volume and neurological score of late training group showed no significant difference compared to those of rats in the 2 week no training group. The present investigation showed that treadmill training initiated 24 h post focal brain ischemia for 1 week can significantly reduce infarct volume in addition to improving neurologic function, but training initiated 1 week after the focal brain ischemia cannot significantly reduce infarct volume and improve neurologic function when compared to 2 week no training group. These findings suggest that early treadmill training can result in better recovery. The underlying mechanism for such effect remains unclear. Schallert et al. [15] have noted that residual impairments of function still exist following spontaneous recovery of stroke. Some of this residual impairment may be due to learned suppression of movement (learned nonuse) of the affected limb [15]. Learned nonuse can be overcome by specific training to use the affected limb in the postinjury situation. The better recovery of early treadmill training may be due in part to that these training may promote the reestablishment of normal motor patterns during the sensitive period soon after brain injury. Parallel to the present findings, forced exercise on a running wheel during the early postlesion period led to improvements in functional outcome after focal cortical lesions and no change in lesion volume [2]. Earlier experimental data have also shown that general activation starting 24 h after an ischemic event promoted functional outcome without increasing tissue loss [6,12]. Activation and training start early after stroke onset in most stroke centers today. It has been shown that early gaitrelated training is feasible and that it can be well tolerated by patients following a stroke [8]. A recent report by Johansson [5] has also indicated that clinical data strongly
93
favor early mobilization and training. The present study provides such evidence for clinical practice. However, there are findings that contradict to that mentioned above. Some evidences suggested that early training might exacerbate brain damage after focal brain ischemia in rats [4,7,14]. These studies were to constrain the intact forelimb immediately after the surgical procedure, thus forcing the animal to overuse the impaired forelimb for postural support and movements. Rather than enhancing recovery, forcing overuse of the affected limb resulted in retardation of functional improvement [7]. Bland et al. [1] have indicated that the intensity of training appears to be one of the important factors that contribute to early exclusive use-dependent exaggeration of injury. In the present study, the intensity of treadmill training for 30 min per day seems to be mild compared to forced use by casting procedures. Therefore, milder intensity of physical training after brain injury has not shown to be deleterious and in contrast, appears to promote reorganization of relevant cortical representation areas and led to functional improvements [11]. In summary, the present study has demonstrated that treadmill training, started 24 h after focal cerebral ischemia, significantly reduces infarct volume and improves neurologic function in MCAO rats. Based on the present findings, early treadmill training may be beneficial for ischemic brain recovery.
Acknowledgements This study was supported by NSC 91-2314-B-010-067 from the National Science Council of the Republic of China to R.Y.W.
References [1] S.T. Bland, T. Schallert, R. Strong, J. Aronowski, J.C. Grotta, Early exclusive use of the affected forelimb after moderate transient focal ischemia in rats: functional and anatomic outcome, Stroke 31 (2000) 1144– 1152. [2] C.L. Hart, G.W. Davis, T.M. Barth, Forced activity facilitates recovery of function following cortical lesions in rats, Natl. Neurotr. Soc. Abstr. (1997) 101. [3] S. Hesse, C. Bertelt, M.T. Jahnke, A. Schaffrin, P. Baake, M. Malezic, K.H. Mauritz, Treadmill training with partial weight support compared with physiotherapy in nonambulatory hemiparetic patients, Stroke 26 (1995) 976–981. [4] J.L. Humm, D.A. Kozlowski, D.C. James, J.E. Gotts, T. Schallert, Use-dependent exacerbation of brain damage occurs during an early post-lesion vulnerable period, Brain Res. 783 (1998) 286–292. [5] B.B. Johansson, Brain plasticity and stroke rehabilitation: the Willis Lecture, Stroke 31 (2000) 223–230. [6] B.B. Johansson, A.L. Ohlsson, Environment, social interaction and physical activity as determinants of functional outcome after cerebral infarction in the rat, Exp. Neurol. 139 (1996) 322– 327. [7] D.A. Kozlowski, D.C. James, T. Schallert, Use-dependent exagger-
94
[8]
[9]
[10]
[11]
[12] [13]
Y.-R. Yang et al. / Neuroscience Letters 339 (2003) 91–94 ation of neuronal injury after unilateral sensorimotor cortex lesions, J. Neurosci. 16 (1996) 4776–4786. F. Malouin, M. Potvin, J. Pre´vost, C.L. Richards, S. Wood-Dauphinee, Use of an intensive task-oriented gait training program in a series of patients with acute cerebrovascular accidents, Phys. Ther. 72 (1992) 781–793. S.A. Menzies, J.T. Hoff, A.L. Betz, Middle cerebral artery occlusion in rat: a neurological and pathological evaluation of a reproducible model, Neurosurgery 31 (1992) 100–107. R.J. Nudo, S. Barbay, J.A. Kleim, Role of neuroplasticity in functional recovery after stroke, in: H.S. Levin, J. Grafman (Eds.), Cerebral Reorganization of Function after Brain Damage, Oxford University Press, New York, 2000, pp. 168 –197. R.J. Nudo, B.M. Wise, F. SiFuentes, G.W. Milliken, Neural substrates for the effects of rehabilitative training on motor recovery after ischemic infarct, Science 272 (1996) 1791–1794. A.L. Ohlsson, B.B. Johansson, The environment influences functional outcome of cerebral infarction in rats, Stroke 26 (1995) 644–649. C.L. Richards, F. Malouin, S. Wood-Dauphinne, J.I. Williams, J.P. Bouchard, D. Brunet, Task-specific physical therapy for optimization
[14]
[15]
[16]
[17]
[18]
of gait recovery in acute stroke patients, Arch. Phys. Med. Rehab. 74 (1993) 612 –620. A. Risedal, J. Zeng, B.B. Johansson, Early training may exacerbate brain damage after focal brain ischemia in the rat, J. Cereb. Blood Flow Metab. 19 (1999) 997–1003. T. Schallert, S.T. Bland, J.L. Leasure, J. Tillerson, R. Gonzales, L. Williams, J. Aronowski, J. Grotta, Motor rehabilitation, use-related neural events, and reorganization of the brain after injury, in: H.S. Levin, J. Grafman (Eds.), Cerebral Reorganization of Function after Brain Damage, Oxford University Press, New York, 2000, pp. 145 –167. R.A. Swanson, M.T. Morton, G. Tsao-Wu, R.A. Savalos, C. Davidson, F.R. Sharp, A semiautomated method for measuring brain infarct volume, J. Cereb. Blood Flow Metab. 10 (1990) 290 –293. E. Taub, N.E. Miller, T.A. Novack, E.W. Cook, W.C. Fleming, C.S. Nepomuceno, J.S. Connell, J.E. Crago, Technique to improve chronic motor deficit after stroke, Arch. Phys. Med. Rehab. 74 (1993) 347 –354. R.Y. Wang, Y.R. Yang, S.M. Yu, Protective effects of treadmill training on infarction in rats, Brain Res. 922 (2001) 140–143.
Early and late treadmill training after focal brain ischemia in rats Yea-Ru Yanga, Ray-Yau Wanga,*, Paulus Shyi-Gang Wangb a
Faculty and Institute of Physical Therapy, National Yang-Ming University, 155, Sec 2, Li-Nong Street, Shih-Pai, Taipei, Taiwan b Department and Institute of Physiology, National Yang-Ming University, 155, Sec 2, Li-Nong Street, Shih-Pai, Taipei, Taiwan Received 11 March 2002; received in revised form 24 November 2002; accepted 9 December 2002
Abstract Treadmill training is increasingly recognized as an effective means to promote rhythmical vigorous walking and also as a useful method of task-related training. The present study endeavored to investigate the effects of early and late treadmill training after ligation of the middle cerebral artery. Forty male Sprague – Dawley rats were subjected to 60 min right middle cerebral artery occlusion (MCAO). All rats were randomly assigned to one of four groups: 24 h group, 2 week no training group, early training group (training started 24 h post MCAO), and late training group (training started 1 week post MCAO). Infarct volume was measured morphometrically. A five-point neurological evaluation scale was used to assess the neurological status of rats. Rats sacrificed 24 h post MCAO had the largest infarct volume (177.8 ^ 14.3 mm3) and the highest neurological score [2(1 – 4)]. Early treadmill training was found to have significant effects in reducing brain infarct volume and in improving neurologic function when compared with spontaneous recovery. However, the same effects cannot be found in late training. Based on the present findings, we would encourage early treadmill training for ischemic brain recovery. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Treadmill training; Middle cerebral artery occlusion; Stroke; Infarct volume; Neurologic function; Rats
It is a common practice among stroke centers to provide treatment and training programs to patients shortly after stroke onset. The findings of many recent studies suggest that the intensity of the manipulation, as well as the time point at which it is implemented, could have profound effects on outcome [15]. However, the optimal time window for therapeutic intervention is still not clear. Some experimental data have shown that general activation starting 24 h after an ischemic event promotes functional outcome without increasing tissue loss [6,12]. However, other reports indicated that intense early training might have a negative effect. Kozlowski et al. [7] immobilized the ipsilateral forelimbs of rats immediately after focal lesions of the forelimb sensorimotor cortex, forcing overuse of the affected limb for 14 days, resulting in retardation of functional improvement. Humm et al. [4] found that forced overuse of the impaired forelimb during the first 7 days after lesions of the forelimb representation area of the sensorimotor cortex in rats caused expansion of neural injury and greatly interfered with restoration of * Corresponding author. Tel.: þ886-2-2826-7210; fax: þ 886-2-28201841. E-mail address: [email protected] (R.-Y. Wang).
function. Risedal et al. [14] indicated that the specific training (1 h a day 5 days a week) initiated 24 h after focal brain ischemia can increase cortical tissue loss. Because of these conflicting reports, further studies are needed to determine the window for the critical period when training may add to promote functional outcome without increasing neuronal tissue loss. It has been postulated that neuroplasticity and the extent of recovery from forelimb motor asymmetries following brain injury depends in part on the degree of learned suppression of use of the impaired limb [11,17]. Animal studies have suggested that forced use engendering a repetitive motor task may best promote central neural plasticity [10]. Recently, treadmill walking has been shown to be effective in promoting rhythmical walking and also a useful method of training for chronic and acute individuals after a stroke [3,13]. The purpose of the present study was to investigate the effect of early and late treadmill training after brain ischemic lesions caused by middle cerebral artery occlusion (MCAO) in rats. Forty male Sprague– Dawley rats, between 2 and 3 months of age, were used as subjects. Animals were housed in groups of two and maintained under a 12/12 h light/dark cycle with food and water available ad libitum. Middle
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00010-7
92
Y.-R. Yang et al. / Neuroscience Letters 339 (2003) 91–94
cerebral artery occlusion procedure leading to focal cerebral ischemia was conducted under chloral hydrate anesthesia (with single 0.5 g/kg i.p. bolus in 1 ml of saline provided anesthesia lasting at least 2 h) for each rat. Rectal temperature was monitored throughout the surgical procedures and maintained at normothermic (37.0 ^ 0.5 8C) by a heating blanket controlled by an electronic temperature controller (HB 101/2, Debiomed). The right middle cerebral artery (MCA) was exposed using microsurgical techniques [18]. Briefly, a 2 mm burr hole was drilled at the junction of the zygomatic arch and the squamous portion of the temporal bone, following a 2 cm vertical skin incision midway between the right eye and ear and splitting of the temporalis muscle. The right MCA trunk was ligated above the rhinal fissure with 10 –0 suture. Complete interruption of blood flow was confirmed by using an operating microscope. Both common carotid arteries (CCAs) were immediately occluded following ligation of MCA with the use of nontraumatic aneurysm clips. After the predetermined duration of ischemia (60 min), the aneurysm clips and ligation were removed from both CCAs and MCA. Restoration of blood flow in all three arteries was observed directly under the microscope. A motor driven treadmill (Treadmill Simplex II, Columbus Instruments, USA) was used for the training protocol. Before MCAO procedure, all rats were placed on a moving belt facing away from the electrified grid and run in the direction opposite of the movement of the belt over a 3 day accommodation period. This allows rats to move forward in order to avoid foot shocks (with intensity in 1.0 mA, Stimulus Controller Model D48E, DRI Co., Taiwan). All rats learned how to run after 3 day accommodation period. After MCAO procedure, all rats were randomly assigned to one of four groups (n ¼ 10 for each group). Rats in Group I were sacrificed at 24 h post MCAO procedure. Rats in Group II remained relatively inactive for 2 weeks after MCAO and thereafter were sacrificed (spontaneous recovery). Rats in Group III were scheduled to run in the treadmill commenced 24 h after MCAO for a period of 1 week, and then remained relatively inactive for another 1 week before sacrifice (early training). Rats in Group IV remained relatively inactive for 1 week after MCAO and thereafter, scheduled to run in the treadmill for another 1 week, were sacrificed (late training). The treadmill training was 30 min per day, 5 days a week with a speed of 20 m/min and 08 slope. Neurological examination was performed before killing. A neurologic grading system with a five-point scale (0 – 4) described by Menzies et al. [9] was used: 0 ¼ no apparent deficits; 1 ¼ left forelimb flexion; 2 ¼ decreased grip of the left forelimb while tail pulled; 3 ¼ spontaneous movement in all directions; left circling only if pulled by tail; 4 ¼ spontaneous left circling. Rats were sacrificed under ketamine anesthesia by intracardiac perfusion with 200 ml of 0.9% NaCl. The brain was removed carefully and dissected into coronal 2
mm sections using a brain slicer. The fresh brain slices were immersed in a 2% solution of 2,3,5-triphenyltetrazolium chloride in normal saline at 37 8C for 30 min, and then fixed in 10% phosphate-buffered formalin at 4 8C [18]. Unstained areas on each brain slice, defined as infarction area, were then measured by an image analyzer (Image-Pro Plus). The damaged areas measured were mainly confined to cerebral cortex including its adjacent caudate nucleus, putamen, and hippocampus. Total measured infarct volume (MV) for each brain was calculated by summation of the infarcted area of all brain slices (area of infarct in square millimeters times thickness, 2 mm) from the same hemisphere. Both right hemisphere volume (RV) and left hemisphere volume (LV) were also measured and calculated. To compensate for the effect of brain edema on MV in the ischemic hemisphere, corrected infarct volume was calculated by the following formula, as previously described [16]: Corrected infarct volume ¼ LV 2 (RV 2 MV). Infarct volumes were expressed as mean ^ SEM. Comparison of infarct volumes was made by analysis of variance. The level of significance for a difference between two groups was further analyzed with post-hoc Tukey’s protected t test. Neurological scores were expressed as median (range) and were compared by Kruskal – Wallis analysis followed by the Mann – Whitney U test to compare medians. A probability value of less than 0.05 was considered to be significant. Rats sacrificed 24 h post MCAO had the largest infarct volume (177.8 ^ 14.3 mm3) and the highest neurological score [2(1 – 4)]. There was a significant effect of group on infarct volume [Fð3; 36Þ ¼ 7:2, P , 0:001] and neurological score (H ¼ 16:9, d:f: ¼ 3, P ¼ 0:001), as shown in Fig. 1 and Table 1. Post-hoc analysis revealed a significant difference in infarct volume (177.8 ^ 14.3 versus 102.1 ^ 6.5 mm3, P , 0:001) and neurological score [2(1 –4) versus 0(0 –1), P , 0:001] between 24 h group (Group I) and early training group (Group III). It also showed a significant difference for the infarct volume (177.8 ^ 14.3 versus 121.6 ^ 7.4 mm3, P , 0:01) and
Fig. 1. Infarct volume as mean ^ SEM at various conditions after middle cerebral artery occlusion in rats. ** Significantly different (P , 0:01) from the 24 h after middle cerebral artery occlusion. þ Significantly different (P , 0:05) from the 2 weeks without treadmill training after middle cerebral artery occlusion.
Y.-R. Yang et al. / Neuroscience Letters 339 (2003) 91–94 Table 1 Neurological score post middle cerebral artery occlusion in ratsa
24 h group 2 week no training group Early training group Late training group
Group
Number
Neurological Score
I II III IV
10 10 10 10
2 (1–4) 2 (0–3) 0 (0–1)***, 0 (0–3)*
þþ
a Neurological scores are expressed as median (range). *P , 0:05, ***P , 0:001 versus 24 h group. þ þ P , 0:01 versus 2 week no training group.
neurological score [2(1 – 4) versus 0(0 – 3), P , 0:05] between 24 h group and late training group (Group IV). There were no significant differences in the infarct volume and neurological score between 24 h group and 2 week no training group (Group II). Post-hoc analysis also revealed that the infarct volume (102.1 ^ 6.5 versus 143.6 ^ 15.0 mm3, P , 0:05) and neurological score [0(0 –1) versus 2(0 – 3), P , 0:005] of early training group were significantly lower than those of the 2 week no training group. In contrast, the infarct volume and neurological score of late training group showed no significant difference compared to those of rats in the 2 week no training group. The present investigation showed that treadmill training initiated 24 h post focal brain ischemia for 1 week can significantly reduce infarct volume in addition to improving neurologic function, but training initiated 1 week after the focal brain ischemia cannot significantly reduce infarct volume and improve neurologic function when compared to 2 week no training group. These findings suggest that early treadmill training can result in better recovery. The underlying mechanism for such effect remains unclear. Schallert et al. [15] have noted that residual impairments of function still exist following spontaneous recovery of stroke. Some of this residual impairment may be due to learned suppression of movement (learned nonuse) of the affected limb [15]. Learned nonuse can be overcome by specific training to use the affected limb in the postinjury situation. The better recovery of early treadmill training may be due in part to that these training may promote the reestablishment of normal motor patterns during the sensitive period soon after brain injury. Parallel to the present findings, forced exercise on a running wheel during the early postlesion period led to improvements in functional outcome after focal cortical lesions and no change in lesion volume [2]. Earlier experimental data have also shown that general activation starting 24 h after an ischemic event promoted functional outcome without increasing tissue loss [6,12]. Activation and training start early after stroke onset in most stroke centers today. It has been shown that early gaitrelated training is feasible and that it can be well tolerated by patients following a stroke [8]. A recent report by Johansson [5] has also indicated that clinical data strongly
93
favor early mobilization and training. The present study provides such evidence for clinical practice. However, there are findings that contradict to that mentioned above. Some evidences suggested that early training might exacerbate brain damage after focal brain ischemia in rats [4,7,14]. These studies were to constrain the intact forelimb immediately after the surgical procedure, thus forcing the animal to overuse the impaired forelimb for postural support and movements. Rather than enhancing recovery, forcing overuse of the affected limb resulted in retardation of functional improvement [7]. Bland et al. [1] have indicated that the intensity of training appears to be one of the important factors that contribute to early exclusive use-dependent exaggeration of injury. In the present study, the intensity of treadmill training for 30 min per day seems to be mild compared to forced use by casting procedures. Therefore, milder intensity of physical training after brain injury has not shown to be deleterious and in contrast, appears to promote reorganization of relevant cortical representation areas and led to functional improvements [11]. In summary, the present study has demonstrated that treadmill training, started 24 h after focal cerebral ischemia, significantly reduces infarct volume and improves neurologic function in MCAO rats. Based on the present findings, early treadmill training may be beneficial for ischemic brain recovery.
Acknowledgements This study was supported by NSC 91-2314-B-010-067 from the National Science Council of the Republic of China to R.Y.W.
References [1] S.T. Bland, T. Schallert, R. Strong, J. Aronowski, J.C. Grotta, Early exclusive use of the affected forelimb after moderate transient focal ischemia in rats: functional and anatomic outcome, Stroke 31 (2000) 1144– 1152. [2] C.L. Hart, G.W. Davis, T.M. Barth, Forced activity facilitates recovery of function following cortical lesions in rats, Natl. Neurotr. Soc. Abstr. (1997) 101. [3] S. Hesse, C. Bertelt, M.T. Jahnke, A. Schaffrin, P. Baake, M. Malezic, K.H. Mauritz, Treadmill training with partial weight support compared with physiotherapy in nonambulatory hemiparetic patients, Stroke 26 (1995) 976–981. [4] J.L. Humm, D.A. Kozlowski, D.C. James, J.E. Gotts, T. Schallert, Use-dependent exacerbation of brain damage occurs during an early post-lesion vulnerable period, Brain Res. 783 (1998) 286–292. [5] B.B. Johansson, Brain plasticity and stroke rehabilitation: the Willis Lecture, Stroke 31 (2000) 223–230. [6] B.B. Johansson, A.L. Ohlsson, Environment, social interaction and physical activity as determinants of functional outcome after cerebral infarction in the rat, Exp. Neurol. 139 (1996) 322– 327. [7] D.A. Kozlowski, D.C. James, T. Schallert, Use-dependent exagger-
94
[8]
[9]
[10]
[11]
[12] [13]
Y.-R. Yang et al. / Neuroscience Letters 339 (2003) 91–94 ation of neuronal injury after unilateral sensorimotor cortex lesions, J. Neurosci. 16 (1996) 4776–4786. F. Malouin, M. Potvin, J. Pre´vost, C.L. Richards, S. Wood-Dauphinee, Use of an intensive task-oriented gait training program in a series of patients with acute cerebrovascular accidents, Phys. Ther. 72 (1992) 781–793. S.A. Menzies, J.T. Hoff, A.L. Betz, Middle cerebral artery occlusion in rat: a neurological and pathological evaluation of a reproducible model, Neurosurgery 31 (1992) 100–107. R.J. Nudo, S. Barbay, J.A. Kleim, Role of neuroplasticity in functional recovery after stroke, in: H.S. Levin, J. Grafman (Eds.), Cerebral Reorganization of Function after Brain Damage, Oxford University Press, New York, 2000, pp. 168 –197. R.J. Nudo, B.M. Wise, F. SiFuentes, G.W. Milliken, Neural substrates for the effects of rehabilitative training on motor recovery after ischemic infarct, Science 272 (1996) 1791–1794. A.L. Ohlsson, B.B. Johansson, The environment influences functional outcome of cerebral infarction in rats, Stroke 26 (1995) 644–649. C.L. Richards, F. Malouin, S. Wood-Dauphinne, J.I. Williams, J.P. Bouchard, D. Brunet, Task-specific physical therapy for optimization
[14]
[15]
[16]
[17]
[18]
of gait recovery in acute stroke patients, Arch. Phys. Med. Rehab. 74 (1993) 612 –620. A. Risedal, J. Zeng, B.B. Johansson, Early training may exacerbate brain damage after focal brain ischemia in the rat, J. Cereb. Blood Flow Metab. 19 (1999) 997–1003. T. Schallert, S.T. Bland, J.L. Leasure, J. Tillerson, R. Gonzales, L. Williams, J. Aronowski, J. Grotta, Motor rehabilitation, use-related neural events, and reorganization of the brain after injury, in: H.S. Levin, J. Grafman (Eds.), Cerebral Reorganization of Function after Brain Damage, Oxford University Press, New York, 2000, pp. 145 –167. R.A. Swanson, M.T. Morton, G. Tsao-Wu, R.A. Savalos, C. Davidson, F.R. Sharp, A semiautomated method for measuring brain infarct volume, J. Cereb. Blood Flow Metab. 10 (1990) 290 –293. E. Taub, N.E. Miller, T.A. Novack, E.W. Cook, W.C. Fleming, C.S. Nepomuceno, J.S. Connell, J.E. Crago, Technique to improve chronic motor deficit after stroke, Arch. Phys. Med. Rehab. 74 (1993) 347 –354. R.Y. Wang, Y.R. Yang, S.M. Yu, Protective effects of treadmill training on infarction in rats, Brain Res. 922 (2001) 140–143.