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Quantitative measurement of motor and somatosensory impairments after mild (30 min) and severe (2 h) transient middle cerebral artery occlusion in rats
Journal of the Neurological Sciences 174 (2000) 141–146 www.elsevier.com / locate / jns
Quantitative measurement of motor and somatosensory impairments after mild (30 min) and severe (2 h) transient middle cerebral artery occlusion in rats a
a
a
a
Li Zhang , Jieli Chen , Yi Li , Zheng Gang Zhang , Michael Chopp a
a,b ,
*
Henry Ford Health Sciences Center, Department of Neurology, 2799 West Grand Boulevard, Detroit, MI 48202, USA b Oakland University, Department of Physics, Walton Boulevard & Squirrel Road, Rochester, MI 48309, USA Received 23 July 1999; received in revised form 7 January 2000; accepted 10 January 2000
Abstract We tested the hypothesis that mild and severe ischemic cell damage are reflected in neurological and functional recovery after stroke. Rats were subjected to either 30 min or 120 min of middle cerebral artery occlusion or sham operation. Neurological and functional tests including, gross neurological score, and rotarod and adhesive removal tests were performed at various time points up to 21 days after stroke. Significant differences between groups of animals were detected using the rotarod and adhesive removal test. A significant correlation between lesion volume and adhesive removal test was detected in rats subjected to 30 min of ischemia. Our data indicate that quantitative rotarod and adhesive removal tests measure different aspects of functional recovery after stroke, and both are useful in characterizing functional recovery from an ischemic insult. 2000 Elsevier Science B.V. All rights reserved. Keywords: Stroke; Rat; Motor impairment; Somatosensory impairment; Mild ischemia; Severe ischemia; Infarct volume; Neurological score
1. Introduction Several studies have examined correlations between neurological impairments and the histopathological features of ischemic brain after transient and permanent middle cerebral artery (MCA) occlusion [1–5]. Motor impairments have been directly correlated with duration of MCA occlusion and ischemic cell damage [6,7]. However, studies on long-term sensorimotor impairments are limited after transient MCA occlusion [7,8]. Significant neurological functional improvement occurs during the initial months after stroke [9–11]. Consequently, the assessment of long-term behavioral and functional impairments is critical for evaluating the efficacy of potential therapeutic agents in experimental stroke. The rotarod test is a sensitive index for assessing motor *Corresponding author. Tel.: 11-313-916-3936; fax: 11-313-9161318. E-mail address: [email protected] (M. Chopp)
impairment after traumatic brain injury and MCA occlusion [12]. The adhesive removal test is a reliable measure of sensorimotor impairment [13]. In the present study, we examined whether these tests are sufficiently sensitive to detect recovery of somatosensory and motor function after mild (30 min) and severe (120 min) transient MCA occlusion in rats over time (21 days) and whether impairments detected by these two tests correlate to the ischemic lesion measured on histopathology. In addition, the sensitivity of these two tests was compared with a neurological scale commonly used as a measure of neurological deficits after MCA occlusion [14].
2. Materials and methods All experimental procedures have been approved by the Care of Experimental Animals Committee of Henry Ford Hospital.
0022-510X / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0022-510X( 00 )00268-9
142
L. Zhang et al. / Journal of the Neurological Sciences 174 (2000) 141 – 146
2.1. Animal model Male Wistar rats (n548) weighing 270–300 g were employed in the present study. Rats were anesthetized with 3.5% halothane and maintained with 1.0–2.0% halothane in 70% N 2 O and 30% O 2 using a face mask. Rectal temperature was maintained at 378C throughout the surgical procedure using a feedback-regulated water heating system. Transient MCA occlusion was induced by a method of intraluminal vascular occlusion modified in our laboratory [15]. Briefly, the right common carotid artery, external carotid artery (ECA) and internal carotid artery (ICA) were exposed. A length of 4-0 monofilament nylon suture (18.5–19.5 mm), determined by the animal weight, with its tip rounded by heating near a flame, was advanced from the ECA into the lumen of the ICA until it blocked the origin of the MCA. Thirty minutes (n520) or 2 h (n520) after MCA occlusion, animals were reanesthetized with halothane and reperfusion was achieved by withdrawal of the suture until the tip cleared the lumen of the ICA. Sham operated animals (n58) underwent the same surgical procedure without inserting a suture.
2.2. Behavioral testing All animals with 30 min and 120 min of MCA occlusion were allowed to survive at 1, 2, 7, 14, 21 days (n54 / time point) after MCA occlusion, and all three behavioral tests were carried out at each time point until the end of each experiment. Sham animals (n58) were tested at 1 and 2 days after sham operation. 1. Accelerating rotarod test: An accelerating rotarod test was employed to measure motor function of rats [12]. In our preliminary experiments, naive rats with three to five training sessions per day for 3 days on the accelerating rotarod showed stable baseline values for remaining on the spindle. Therefore, in the present study, each animal received three to five training sessions per day for 3 consecutive days starting on day 3 prior to MCAo. The diameter of the rotarod spindle is 7 cm. The surface of the rotarod spindle is made of knurled perspex to provide an adequate grip which prevents animals from slipping down from the spindle. The speed of the spindle was increased from 4 to 40 rpm over a period of 5 min and training continued until the rat remained on the rotating spindle for approximately 300 s. Each rat received a test trial on the accelerating rotarod at all testing days after transient MCA occlusion and the duration the animal remained on the device was recorded. Data are presented as percentage of preischemic time which the rat remained on the rotating spindle. 2. Adhesive removal test: The adhesive removal test, which measures somatosensory deficit, was performed according to the method developed by Schallert and Whishaw [13]. Briefly, the rats were removed from their
home cages so that the adhesive paper dots could be firmly and accurately attached. Two small pieces of adhesivebacked paper dots (of equal size, 113.1 mm 2 ) were used as bilateral tactile stimuli occupying the distal–radial region on the wrist of each forelimb. The rats were then returned to their cages and they typically contacted and removed each stimulus one at a time using their teeth. The time required to remove both stimuli from each limb was recorded in 5 trials per day. Before surgery, the rats were trained 5 times a day for 3 days and all rats were able to remove the dots within 10 s at the end of training. The rats, therefore, were familiarized with the testing environment. Each animal received 5 trials at all testing days after transient MCA occlusion and the mean time required to remove both stimuli from limbs was recorded. 3. Neurologic score: Neurological deficits were examined at 1, 2, 7, 14, 21 days after transient MCA occlusion. A four point neurological score was employed [14]: 05no deficit, 15failure to extend left forepaw fully, 25circling to the left, 35falling to the left, 45no spontaneous walking with a depressed level of consciousness.
2.3. Histopathologic studies The animals were anesthetized with ketamine (44 mg / kg) and xylazine (13 mg / kg) and were sacrificed at 1, 2, 7, 14, 21 days (n54 / time point) after transient MCA occlusion. Sham animals (n58) were sacrificed at 2 days after sham operation. Each rat was transcardially perfused with heparinized saline followed by 10% formalin [16]. The brain was removed from the skull and cut into 7 coronal blocks, each with 2 mm thickness. The brain tissue was processed, embedded, and 6 mm thick paraffin sections from each block were cut and stained with hematoxylin and eosin (H&E) for evaluation of ischemic cell damage. Each H&E stained coronal section was digitized using a CCD camera (Hitachi RP-111 interfaced with Global Lab image analysis system; Data Translation, Marlboro, MA). The area of both hemispheres and the area containing the ischemic neuronal damage (mm 2 ) were calculated by tracing the area on the computer screen, and the volume (mm 3 ) was determined by multiplying the appropriate area by the section interval thickness [16,17]. To reduce errors associated with processing of tissue for histological analysis, the ischemic lesion volume is presented as the percentage of infarct volume of the contralateral hemisphere (indirect volume calculation) [18].
2.4. Statistics One way repeated ANOVA followed by Bonferroni correction was performed to analyze differences between 30 min and 120 min groups of the rotarod and the adhesive removal tests. Correlations between the lesion volumes and behavioral data at each time point were analyzed by Pearson correlation coefficients. A P-value of 0.05 or less
L. Zhang et al. / Journal of the Neurological Sciences 174 (2000) 141 – 146
was considered statistically significant. All values are presented as mean6S.E.M.
143
and ischemic lesion volume after 30 min and 120 min ischemia.
3.2. The adhesive removal test 3. Results
All sham operated animals did not exhibit a significant difference in the time spent on the accelerating rotarod before and after surgery. Animals subjected to 30 min transient ischemia showed a significant (P,0.05) reduction in the time spent on the accelerating rotarod at 1 and 2 days after transient ischemia compared with the preischemic values (Fig. 1). Motor function impairment was not detected at 14 days after 30 min transient ischemia (Fig. 1). However in animals subjected to 120 min of ischemia, a significant reduction of time spent on the accelerating rotarod persisted for 21 days (the end of the experiment) compared with the preischemic values (Fig. 1). Differences in time on the rotarod between 30 min and 120 min transient MCA occlusion were significant (P,0.05) at 7 days, 14 days, and 21 days after ischemia (Fig. 1). There was no correlation between the time spent on the rotarod
In sham-operated animals, there was no difference between before and after surgery on the time spent to remove the stimulus. In rats subjected to 30 min of ischemia, the mean times required to remove the stimulus were 35 s and 32 s at 1 and 2 days, respectively (Fig. 2). These times were significantly different (P,0.05) from the preischemia (6 s) and sham-operated animals (6 s). By day 7 after 30 min transient MCA occlusion, however, the mean time required to remove the stimulus returned to the preischemic level (7.3 s vs. 6.3 s; Fig. 2). In contrast, rats subjected to 120 min transient MCA occlusion failed to completely recover from sensorimotor impairment by the end of the experiment (21 days). A significant (P,0.05) difference in the time spent to remove the stimulus was detected at 7 days, 14 days and 21 days after ischemia between 30 min and 120 min transient MCA occlusion (Fig. 2). A significant linear relationship between the ischemic lesion volumes and the time required to remove the stimulus was detected at 1 day (r50.9, P50.0001) and 2 days (r50.9, P50.001) after 30 min transient MCA
Fig. 1. Shows the percentage of time that rats remain on the rotarod after ischemia compared to preischemia times as a function of time after stroke. Significant differences (P,0.05) between rats subjected to 30 min and 120 min of MCA occlusion for the percentage of the time on the rotarod were detected at 7, 14, 21 days after stroke. 1 30 min ischemia; j 120 min of ischemia; * P,0.05.
Fig. 2. Shows the time required to remove the adhesive paper dots from the forelimbs of animals subjected to 30 min and 120 min of MCA occlusion, as a function of time after onset of MCA occlusion. Significant differences (P,0.05) in time required to remove the dots were detected between groups at 7, 14 and 21 days after MCA occlusion. 1 30 min ischemia; j 120 min of ischemia; * P,0.05.
3.1. The rotarod test
L. Zhang et al. / Journal of the Neurological Sciences 174 (2000) 141 – 146
144 Table 1 Neurological deficit a
Sham 30 min 120 min a
1 day
2 days
7 days
14 days
21 days
0.060.0 0.560.3 1.860.1*1
0.060.0 0.360.3 1.660.2*1
0.060.0 1.060.2*1
0.060.0 0.860.3*1
0.060.0 0.360.3
Values are mean6S.E.M. * Significant difference from sham group, P,0.05. 1 Significant difference from 30 min ischemia group.
occlusion. In contrast, no significant correlation (1 day, r50.3, P50.29; 2 days, r50.2, P50.61) was detected between lesion volume and time to remove the adhesive stimulus in rats subjected to 120 min of MCA occlusion. No significant correlations between ischemia volume were detected for the 30 min and the 120 min ischemic groups and functional measurement (rotarod, adhesive removal, neurological score) at 7, 14 and 21 days after stroke.
3.3. Neurological score Sham operated animals did not exhibit any neurological deficits. Rats subjected to 30 min of transient MCA occlusion had neurological scores 0.5 and 0.3 at 1 day and 2 days after transient ischemia, respectively, which was not significantly different from sham operated rats (Table 1). However, a significant (P,0.05) neurological deficit was detected in rats subjected to 120 min of MCA occlusion at 1 days which persisted up to 14 days after ischemia (Table 1). Neurological deficit in rats subjected to 120 min of MCA occlusion was significantly different from sham and 30 min of ischemia at 1, 2, 7 and 14 days after ischemia.
3.4. Histopathology Ischemic neuronal damage was not detected in sham operated rats. Ischemic cell damage such as triangular shrunken, scalloped, and pyknotic neurons was detected in
rats subjected to 30 min and 120 min transient MCA occlusion, as previously described [19]. The ischemic lesion is primarily localized to the ipsilateral striatum in rats subjected to 30 min transient MCA occlusion (Fig. 3). Two rats from this group exhibited scattered ischemic neuronal damage within the ipsilateral parietal cortex. The ischemic lesion in the 120 min transient MCA occlusion group involved the ipsilateral territory supplied by the MCA (Fig. 3). The ischemic lesion volume was 8.864.4% and 35.865.6% of the contralateral hemisphere for 30 min and 120 min transient MCA occlusion, respectively.
4. Discussion In the present study, we observed a significant difference on recovery of motor and sensorimotor impairments between 30 min and 120 min of transient focal cerebral ischemia as assayed by the rotarod test and the adhesive removal test, respectively. No significant differences were detected on the neurological deficit scale between sham operated animals and animals subjected to 30 min of MCA occlusion. In contrast, 120 min of MCA occlusion induced significant neurological deficits compared to sham operated animals at 1 day after MCA occlusion which persisted up to 14 days after ischemia. These data suggest that the rotarod and the adhesive removal tests detect motor and sensorimotor deficits after mild and severe transient focal
Fig. 3. Representative distributions of ischemic damage (black areas) at 1, 2, 7, 14, 21 days after 30 min and 120 min of MCAo.
L. Zhang et al. / Journal of the Neurological Sciences 174 (2000) 141 – 146
cerebral ischemia, and provide a more sensitive indication of dysfunction than the standard neurological test. Our results are consistent with previous studies of Hamm et al. [12] which demonstrate that the accelerating rotarod test is a sensitive measure of motor impairment following both mild and moderate levels of brain trauma injury. The adhesive removal test measures forelimb somatosensorimotor asymmetries [13]. This test was originally developed to examine sensory function after damage to dopaminergic inputs to the neostriatum [20,21]. In the present study, we adapted this method to assess ischemic deficits. Our results show, for the first time, that there is a significant linear relationship between ischemic lesion volume and the time required to remove adhesive paper at 1 day (r50.9, P50.0001) and 2 days (r50.9, P50.001) after 30 min of ischemia, while a similar correlation between the adhesive removal test and the ischemic lesion after 120 min of MCA occlusion was not detected at 1 and 2 days after transient ischemia. One reason for this difference may be that, the ischemic lesion resulting from 30 min of MCA occlusion is primarily localized to the striatum, whereas the ischemic lesion from 120 min of MCA occlusion involves the striatum and the cortex. An ischemic lesion in the cortex can produce variable neurological deficits [22,23] related to the ischemic lesion location [24,25] rather than lesion volume alone. A lack of high correlation between the rotarod test and the ischemic volume for 30 min of ischemia indicates that the rotarod test and the adhesive removal test do not measure the same aspects of functional outcome after transient ischemia. Although rats subjected to 120 min of MCA occlusion exhibited more severe neurological deficits measured by rotarod and adhesive removal tests than rats subjected to 30 min of MCA occlusion, a significant correlation between lesion volume and these measurements was not achieved in the 120 min group, possibly due to large variations of deficits at day 1 and day 2 after transient MCA occlusion. In addition to the small sample size employed in the present study, transient reduction of CBF and disruption of blood brain barrier (BBB) may contribute to this variation, because occlusion of the origin of the MCA perturbs cerebral blood flow (CBF) and microvascular permeability in the frontal and sensorimotor cortex, striatum, and preoptic area [26]. Normal CBF and an intact BBB are necessary for normal neuronal function [27–29]. Assessment of functional recovery is important for evaluating efficacy of therapies designed to protect the ischemic brain. Our results indicate that although neurological deficits evaluated by the neurological scale disappeared at 2 days, complete recovery of motor and sensorimotor function assessed by the rotarod and adhesive removal tests required 7 days in rats subjected to 30 min of MCA occlusion. In rats subjected to 120 min of MCA occlusion, significant motor and sensorimotor deficits persisted at least 21 days, while neurological deficits recovered at 14 days in rats subjected to 120 min of MCA
145
occlusion. Spontaneous functional recovery may reflect tissue functional plasticity and circuit reorganization [30]. Our findings suggest that the rotarod test and the adhesive removal test are more sensitive than neurological testing by the Zea Longa scale [14] for detecting the degree of motor and sensorimotor impairments and recovery after transient MCA occlusion. Our data are consistent with others that motor impairment persists to 2 months after permanent MCA occlusion, and also extends previous studies by demonstrating persistence of sensorimotor impairment after 120 min transient MCA occlusion [31]. The rotarod test and the adhesive removal test are quantitative and objective compared with measurement of neurological deficit. Therefore, a battery of diverse functional tests should be used to characterize ischemic deficits and rates of behavioral recovery in experimental animals.
Acknowledgements The authors wish to thank Denice Bliesath for secretarial support. This work was supported in part by NINDS grants PO1 NS23393 and RO1 NS35504.
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Quantitative measurement of motor and somatosensory impairments after mild (30 min) and severe (2 h) transient middle cerebral artery occlusion in rats a
a
a
a
Li Zhang , Jieli Chen , Yi Li , Zheng Gang Zhang , Michael Chopp a
a,b ,
*
Henry Ford Health Sciences Center, Department of Neurology, 2799 West Grand Boulevard, Detroit, MI 48202, USA b Oakland University, Department of Physics, Walton Boulevard & Squirrel Road, Rochester, MI 48309, USA Received 23 July 1999; received in revised form 7 January 2000; accepted 10 January 2000
Abstract We tested the hypothesis that mild and severe ischemic cell damage are reflected in neurological and functional recovery after stroke. Rats were subjected to either 30 min or 120 min of middle cerebral artery occlusion or sham operation. Neurological and functional tests including, gross neurological score, and rotarod and adhesive removal tests were performed at various time points up to 21 days after stroke. Significant differences between groups of animals were detected using the rotarod and adhesive removal test. A significant correlation between lesion volume and adhesive removal test was detected in rats subjected to 30 min of ischemia. Our data indicate that quantitative rotarod and adhesive removal tests measure different aspects of functional recovery after stroke, and both are useful in characterizing functional recovery from an ischemic insult. 2000 Elsevier Science B.V. All rights reserved. Keywords: Stroke; Rat; Motor impairment; Somatosensory impairment; Mild ischemia; Severe ischemia; Infarct volume; Neurological score
1. Introduction Several studies have examined correlations between neurological impairments and the histopathological features of ischemic brain after transient and permanent middle cerebral artery (MCA) occlusion [1–5]. Motor impairments have been directly correlated with duration of MCA occlusion and ischemic cell damage [6,7]. However, studies on long-term sensorimotor impairments are limited after transient MCA occlusion [7,8]. Significant neurological functional improvement occurs during the initial months after stroke [9–11]. Consequently, the assessment of long-term behavioral and functional impairments is critical for evaluating the efficacy of potential therapeutic agents in experimental stroke. The rotarod test is a sensitive index for assessing motor *Corresponding author. Tel.: 11-313-916-3936; fax: 11-313-9161318. E-mail address: [email protected] (M. Chopp)
impairment after traumatic brain injury and MCA occlusion [12]. The adhesive removal test is a reliable measure of sensorimotor impairment [13]. In the present study, we examined whether these tests are sufficiently sensitive to detect recovery of somatosensory and motor function after mild (30 min) and severe (120 min) transient MCA occlusion in rats over time (21 days) and whether impairments detected by these two tests correlate to the ischemic lesion measured on histopathology. In addition, the sensitivity of these two tests was compared with a neurological scale commonly used as a measure of neurological deficits after MCA occlusion [14].
2. Materials and methods All experimental procedures have been approved by the Care of Experimental Animals Committee of Henry Ford Hospital.
0022-510X / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0022-510X( 00 )00268-9
142
L. Zhang et al. / Journal of the Neurological Sciences 174 (2000) 141 – 146
2.1. Animal model Male Wistar rats (n548) weighing 270–300 g were employed in the present study. Rats were anesthetized with 3.5% halothane and maintained with 1.0–2.0% halothane in 70% N 2 O and 30% O 2 using a face mask. Rectal temperature was maintained at 378C throughout the surgical procedure using a feedback-regulated water heating system. Transient MCA occlusion was induced by a method of intraluminal vascular occlusion modified in our laboratory [15]. Briefly, the right common carotid artery, external carotid artery (ECA) and internal carotid artery (ICA) were exposed. A length of 4-0 monofilament nylon suture (18.5–19.5 mm), determined by the animal weight, with its tip rounded by heating near a flame, was advanced from the ECA into the lumen of the ICA until it blocked the origin of the MCA. Thirty minutes (n520) or 2 h (n520) after MCA occlusion, animals were reanesthetized with halothane and reperfusion was achieved by withdrawal of the suture until the tip cleared the lumen of the ICA. Sham operated animals (n58) underwent the same surgical procedure without inserting a suture.
2.2. Behavioral testing All animals with 30 min and 120 min of MCA occlusion were allowed to survive at 1, 2, 7, 14, 21 days (n54 / time point) after MCA occlusion, and all three behavioral tests were carried out at each time point until the end of each experiment. Sham animals (n58) were tested at 1 and 2 days after sham operation. 1. Accelerating rotarod test: An accelerating rotarod test was employed to measure motor function of rats [12]. In our preliminary experiments, naive rats with three to five training sessions per day for 3 days on the accelerating rotarod showed stable baseline values for remaining on the spindle. Therefore, in the present study, each animal received three to five training sessions per day for 3 consecutive days starting on day 3 prior to MCAo. The diameter of the rotarod spindle is 7 cm. The surface of the rotarod spindle is made of knurled perspex to provide an adequate grip which prevents animals from slipping down from the spindle. The speed of the spindle was increased from 4 to 40 rpm over a period of 5 min and training continued until the rat remained on the rotating spindle for approximately 300 s. Each rat received a test trial on the accelerating rotarod at all testing days after transient MCA occlusion and the duration the animal remained on the device was recorded. Data are presented as percentage of preischemic time which the rat remained on the rotating spindle. 2. Adhesive removal test: The adhesive removal test, which measures somatosensory deficit, was performed according to the method developed by Schallert and Whishaw [13]. Briefly, the rats were removed from their
home cages so that the adhesive paper dots could be firmly and accurately attached. Two small pieces of adhesivebacked paper dots (of equal size, 113.1 mm 2 ) were used as bilateral tactile stimuli occupying the distal–radial region on the wrist of each forelimb. The rats were then returned to their cages and they typically contacted and removed each stimulus one at a time using their teeth. The time required to remove both stimuli from each limb was recorded in 5 trials per day. Before surgery, the rats were trained 5 times a day for 3 days and all rats were able to remove the dots within 10 s at the end of training. The rats, therefore, were familiarized with the testing environment. Each animal received 5 trials at all testing days after transient MCA occlusion and the mean time required to remove both stimuli from limbs was recorded. 3. Neurologic score: Neurological deficits were examined at 1, 2, 7, 14, 21 days after transient MCA occlusion. A four point neurological score was employed [14]: 05no deficit, 15failure to extend left forepaw fully, 25circling to the left, 35falling to the left, 45no spontaneous walking with a depressed level of consciousness.
2.3. Histopathologic studies The animals were anesthetized with ketamine (44 mg / kg) and xylazine (13 mg / kg) and were sacrificed at 1, 2, 7, 14, 21 days (n54 / time point) after transient MCA occlusion. Sham animals (n58) were sacrificed at 2 days after sham operation. Each rat was transcardially perfused with heparinized saline followed by 10% formalin [16]. The brain was removed from the skull and cut into 7 coronal blocks, each with 2 mm thickness. The brain tissue was processed, embedded, and 6 mm thick paraffin sections from each block were cut and stained with hematoxylin and eosin (H&E) for evaluation of ischemic cell damage. Each H&E stained coronal section was digitized using a CCD camera (Hitachi RP-111 interfaced with Global Lab image analysis system; Data Translation, Marlboro, MA). The area of both hemispheres and the area containing the ischemic neuronal damage (mm 2 ) were calculated by tracing the area on the computer screen, and the volume (mm 3 ) was determined by multiplying the appropriate area by the section interval thickness [16,17]. To reduce errors associated with processing of tissue for histological analysis, the ischemic lesion volume is presented as the percentage of infarct volume of the contralateral hemisphere (indirect volume calculation) [18].
2.4. Statistics One way repeated ANOVA followed by Bonferroni correction was performed to analyze differences between 30 min and 120 min groups of the rotarod and the adhesive removal tests. Correlations between the lesion volumes and behavioral data at each time point were analyzed by Pearson correlation coefficients. A P-value of 0.05 or less
L. Zhang et al. / Journal of the Neurological Sciences 174 (2000) 141 – 146
was considered statistically significant. All values are presented as mean6S.E.M.
143
and ischemic lesion volume after 30 min and 120 min ischemia.
3.2. The adhesive removal test 3. Results
All sham operated animals did not exhibit a significant difference in the time spent on the accelerating rotarod before and after surgery. Animals subjected to 30 min transient ischemia showed a significant (P,0.05) reduction in the time spent on the accelerating rotarod at 1 and 2 days after transient ischemia compared with the preischemic values (Fig. 1). Motor function impairment was not detected at 14 days after 30 min transient ischemia (Fig. 1). However in animals subjected to 120 min of ischemia, a significant reduction of time spent on the accelerating rotarod persisted for 21 days (the end of the experiment) compared with the preischemic values (Fig. 1). Differences in time on the rotarod between 30 min and 120 min transient MCA occlusion were significant (P,0.05) at 7 days, 14 days, and 21 days after ischemia (Fig. 1). There was no correlation between the time spent on the rotarod
In sham-operated animals, there was no difference between before and after surgery on the time spent to remove the stimulus. In rats subjected to 30 min of ischemia, the mean times required to remove the stimulus were 35 s and 32 s at 1 and 2 days, respectively (Fig. 2). These times were significantly different (P,0.05) from the preischemia (6 s) and sham-operated animals (6 s). By day 7 after 30 min transient MCA occlusion, however, the mean time required to remove the stimulus returned to the preischemic level (7.3 s vs. 6.3 s; Fig. 2). In contrast, rats subjected to 120 min transient MCA occlusion failed to completely recover from sensorimotor impairment by the end of the experiment (21 days). A significant (P,0.05) difference in the time spent to remove the stimulus was detected at 7 days, 14 days and 21 days after ischemia between 30 min and 120 min transient MCA occlusion (Fig. 2). A significant linear relationship between the ischemic lesion volumes and the time required to remove the stimulus was detected at 1 day (r50.9, P50.0001) and 2 days (r50.9, P50.001) after 30 min transient MCA
Fig. 1. Shows the percentage of time that rats remain on the rotarod after ischemia compared to preischemia times as a function of time after stroke. Significant differences (P,0.05) between rats subjected to 30 min and 120 min of MCA occlusion for the percentage of the time on the rotarod were detected at 7, 14, 21 days after stroke. 1 30 min ischemia; j 120 min of ischemia; * P,0.05.
Fig. 2. Shows the time required to remove the adhesive paper dots from the forelimbs of animals subjected to 30 min and 120 min of MCA occlusion, as a function of time after onset of MCA occlusion. Significant differences (P,0.05) in time required to remove the dots were detected between groups at 7, 14 and 21 days after MCA occlusion. 1 30 min ischemia; j 120 min of ischemia; * P,0.05.
3.1. The rotarod test
L. Zhang et al. / Journal of the Neurological Sciences 174 (2000) 141 – 146
144 Table 1 Neurological deficit a
Sham 30 min 120 min a
1 day
2 days
7 days
14 days
21 days
0.060.0 0.560.3 1.860.1*1
0.060.0 0.360.3 1.660.2*1
0.060.0 1.060.2*1
0.060.0 0.860.3*1
0.060.0 0.360.3
Values are mean6S.E.M. * Significant difference from sham group, P,0.05. 1 Significant difference from 30 min ischemia group.
occlusion. In contrast, no significant correlation (1 day, r50.3, P50.29; 2 days, r50.2, P50.61) was detected between lesion volume and time to remove the adhesive stimulus in rats subjected to 120 min of MCA occlusion. No significant correlations between ischemia volume were detected for the 30 min and the 120 min ischemic groups and functional measurement (rotarod, adhesive removal, neurological score) at 7, 14 and 21 days after stroke.
3.3. Neurological score Sham operated animals did not exhibit any neurological deficits. Rats subjected to 30 min of transient MCA occlusion had neurological scores 0.5 and 0.3 at 1 day and 2 days after transient ischemia, respectively, which was not significantly different from sham operated rats (Table 1). However, a significant (P,0.05) neurological deficit was detected in rats subjected to 120 min of MCA occlusion at 1 days which persisted up to 14 days after ischemia (Table 1). Neurological deficit in rats subjected to 120 min of MCA occlusion was significantly different from sham and 30 min of ischemia at 1, 2, 7 and 14 days after ischemia.
3.4. Histopathology Ischemic neuronal damage was not detected in sham operated rats. Ischemic cell damage such as triangular shrunken, scalloped, and pyknotic neurons was detected in
rats subjected to 30 min and 120 min transient MCA occlusion, as previously described [19]. The ischemic lesion is primarily localized to the ipsilateral striatum in rats subjected to 30 min transient MCA occlusion (Fig. 3). Two rats from this group exhibited scattered ischemic neuronal damage within the ipsilateral parietal cortex. The ischemic lesion in the 120 min transient MCA occlusion group involved the ipsilateral territory supplied by the MCA (Fig. 3). The ischemic lesion volume was 8.864.4% and 35.865.6% of the contralateral hemisphere for 30 min and 120 min transient MCA occlusion, respectively.
4. Discussion In the present study, we observed a significant difference on recovery of motor and sensorimotor impairments between 30 min and 120 min of transient focal cerebral ischemia as assayed by the rotarod test and the adhesive removal test, respectively. No significant differences were detected on the neurological deficit scale between sham operated animals and animals subjected to 30 min of MCA occlusion. In contrast, 120 min of MCA occlusion induced significant neurological deficits compared to sham operated animals at 1 day after MCA occlusion which persisted up to 14 days after ischemia. These data suggest that the rotarod and the adhesive removal tests detect motor and sensorimotor deficits after mild and severe transient focal
Fig. 3. Representative distributions of ischemic damage (black areas) at 1, 2, 7, 14, 21 days after 30 min and 120 min of MCAo.
L. Zhang et al. / Journal of the Neurological Sciences 174 (2000) 141 – 146
cerebral ischemia, and provide a more sensitive indication of dysfunction than the standard neurological test. Our results are consistent with previous studies of Hamm et al. [12] which demonstrate that the accelerating rotarod test is a sensitive measure of motor impairment following both mild and moderate levels of brain trauma injury. The adhesive removal test measures forelimb somatosensorimotor asymmetries [13]. This test was originally developed to examine sensory function after damage to dopaminergic inputs to the neostriatum [20,21]. In the present study, we adapted this method to assess ischemic deficits. Our results show, for the first time, that there is a significant linear relationship between ischemic lesion volume and the time required to remove adhesive paper at 1 day (r50.9, P50.0001) and 2 days (r50.9, P50.001) after 30 min of ischemia, while a similar correlation between the adhesive removal test and the ischemic lesion after 120 min of MCA occlusion was not detected at 1 and 2 days after transient ischemia. One reason for this difference may be that, the ischemic lesion resulting from 30 min of MCA occlusion is primarily localized to the striatum, whereas the ischemic lesion from 120 min of MCA occlusion involves the striatum and the cortex. An ischemic lesion in the cortex can produce variable neurological deficits [22,23] related to the ischemic lesion location [24,25] rather than lesion volume alone. A lack of high correlation between the rotarod test and the ischemic volume for 30 min of ischemia indicates that the rotarod test and the adhesive removal test do not measure the same aspects of functional outcome after transient ischemia. Although rats subjected to 120 min of MCA occlusion exhibited more severe neurological deficits measured by rotarod and adhesive removal tests than rats subjected to 30 min of MCA occlusion, a significant correlation between lesion volume and these measurements was not achieved in the 120 min group, possibly due to large variations of deficits at day 1 and day 2 after transient MCA occlusion. In addition to the small sample size employed in the present study, transient reduction of CBF and disruption of blood brain barrier (BBB) may contribute to this variation, because occlusion of the origin of the MCA perturbs cerebral blood flow (CBF) and microvascular permeability in the frontal and sensorimotor cortex, striatum, and preoptic area [26]. Normal CBF and an intact BBB are necessary for normal neuronal function [27–29]. Assessment of functional recovery is important for evaluating efficacy of therapies designed to protect the ischemic brain. Our results indicate that although neurological deficits evaluated by the neurological scale disappeared at 2 days, complete recovery of motor and sensorimotor function assessed by the rotarod and adhesive removal tests required 7 days in rats subjected to 30 min of MCA occlusion. In rats subjected to 120 min of MCA occlusion, significant motor and sensorimotor deficits persisted at least 21 days, while neurological deficits recovered at 14 days in rats subjected to 120 min of MCA
145
occlusion. Spontaneous functional recovery may reflect tissue functional plasticity and circuit reorganization [30]. Our findings suggest that the rotarod test and the adhesive removal test are more sensitive than neurological testing by the Zea Longa scale [14] for detecting the degree of motor and sensorimotor impairments and recovery after transient MCA occlusion. Our data are consistent with others that motor impairment persists to 2 months after permanent MCA occlusion, and also extends previous studies by demonstrating persistence of sensorimotor impairment after 120 min transient MCA occlusion [31]. The rotarod test and the adhesive removal test are quantitative and objective compared with measurement of neurological deficit. Therefore, a battery of diverse functional tests should be used to characterize ischemic deficits and rates of behavioral recovery in experimental animals.
Acknowledgements The authors wish to thank Denice Bliesath for secretarial support. This work was supported in part by NINDS grants PO1 NS23393 and RO1 NS35504.
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