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Quantitative measurement of neurological deficit after mild (30 min) transient middle cerebral artery occlusion in rats
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Quantitative measurement of neurological deficit after mild (30 min) transient middle cerebral artery occlusion in rats Kouji Wakayama a,b , Munehisa Shimamura a , Masataka Sata a , Naoyuki Sato c , Koji Kawakami a , Hirotsugu Fukuda d , Takuji Tomimatsu d , Toshio Ogihara b , Ryuichi Morishita c,⁎ a
Department of Advanced Clinical Science and Therapeutics, Graduate School of Medicine, The University of Tokyo, Japan Department of Geriatric Medicine, Graduate School of Medicine, Osaka University, Japan c Department of Clinical Gene Therapy, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita 565-0871, Japan d Department of Obstetrics and Gynecology, Graduate School of Medicine, Osaka University, Japan b
A R T I C LE I N FO
AB S T R A C T
Article history:
Although 30-min transient middle cerebral artery occlusion (30-min tMCAo) causes
Accepted 4 October 2006
reproducible subcortical infarction in rats, it is difficult to evaluate the resulting
Available online 14 December 2006
neurological deficit using common behavioral tests such as the rota-rod test, adhesiveremoval test, or narrow beam test. Establishment of a method of quantitative evaluation
Keywords:
would help to develop a novel therapeutic approach to treat cerebral infarction. To solve this
Transient middle cerebral
problem, we examined whether the neurological deficit could be detected by the Montoya
artery occlusion
staircase test or methamphetamine-induced rotation, which are commonly used in a
Subcortical infarction
Parkinson disease model induced by intrastriatal injection of 6-hydroxydopamine (6-
Behavior test
OHDA). From 10 to 14 days after tMCAo, the Montoya staircase test showed significant
Methamphetamine
clumsiness in forelimb tasks contralateral to the lesion side, whereas sham-operated rats
Montoya staircase test
showed no significant clumsiness in both forelimbs. The number of ipsilateral rotations induced by methamphetamine was also increased in tMCAo-rats at 21 days after tMCAo. Although Pearson's correlations coefficient showed that the results of these tests were correlated with the infarction volume, there was no significant correlation between the results of these two tests. These findings imply that the neurological deficit detected by both tests might reflect the severity of ischemic injury, but each test might evaluate different aspects of neurological deficit. Thus, the Montoya staircase test and methamphetamineinduced rotation are useful to evaluate neurological deficit in the chronic stage of subcortical infarction induced by 30-min tMCAo. © 2006 Published by Elsevier B.V.
1.
Introduction
Although 30-min transient middle cerebral artery occlusion (30-min tMCAo) in rats, which causes subcortical infarction without widespread neocortical ischemic damage (Goto et al., ⁎ Corresponding author. Fax: +81 6 6879 3409. E-mail address: [email protected] (R. Morishita). 0006-8993/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.brainres.2006.10.088
1994; Zhang et al., 2000), is a reliable model for research, it is difficult to evaluate neurological deficit in this model, since the sensori-motor deficit assessed by the commonly used neurological severity score, rota-rod test, grid walking test, cylinder test, ledged beam walking, or adhesive-removal test
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quickly recovers to the level in sham-operated rats (Rogers et al., 1997; Susanne et al., 2005; Zhang et al., 2000). Therefore, the development of a novel therapeutic approach to treat cerebral infarction is limited by methodological issues in evaluation. From this perspective, the development of other behavioral tests is necessary to evaluate neurological deficit quantitatively. In the present study, we focused on the Montoya staircase test and methamphetamine-induced rotation, which are commonly used in a Parkinson disease model in rats induced by intrastriatal injection of 6-hydroxydopamine (6-OHDA) (Barneoud et al., 2000; Jeyasingham et al., 2001; Kirik et al., 1998), for the evaluation of neurological deficit in a cerebral infarction model. The Montoya staircase test evaluates independent forelimb reaching tasks and grasping ability in rats (Montoya et al., 1990, 1991), and reflects the extent of striatal damage (Montoya et al., 1990). It is applied not only in Parkinson disease models, but also in focal cerebral ischemic rats such as 60, 90, and 120-min tMCAo models and neonatal hypoxic– ischemic brain injury model (Colbourne et al., 2000; Hudzik et al., 2000; Palmer et al., 2001; Tomimatsu et al., 2002; Virley et al., 2000), which exhibit widespread infarction of the neocortex and basal ganglia. However, this test has not been evaluated in subcortical infarction induced by 30-min tMCAo. Alternatively, methamphetamine-induced rotation examines postural instability and reflects subcortical damage (Pycock, 1980). Theoretically, methamphetamine stimulates the release of dopamine from neurons in the intact side of the brain and induces rotation ipsilateral to the lesion (Deumens et al., 2002). Similarly to the Montoya staircase test, this test has been applied in permanent MCAo (Grabowski et al., 1993) and 60–120 min tMCAo models (Hudzik et al., 2000; Veizovic et al., 2001), but it has not been examined in the 30-min tMCAo
model. From these viewpoints, the present study examined whether these two tests could detect the neurological deficit in the chronic stage of subcortical infarction in rats induced by 30-min tMCAo. Here, we demonstrated that the Montoya staircase test and methamphetamine-induced rotation were useful tests for the quantitative assessment of neurological deficit after 30-min tMCAo, and that these two tests reflected the severity of ischemic damage.
2.
Results
2.1.
Behavioral tests
After training sessions (day − 8 to −1, Fig. 1a), rats were exposed to 30-min tMCAo (day 0, sham: n = 4, tMCAo: n = 7). The staircase test was performed from day 8 to 14 (testing sessions). The commonly used neurological severity score (Table 1) was also determined on day 14. Then, rats were subjected to methamphetamine-induced rotation test on day 21. To confirm the severity of cerebral infarction, infarction volume was assessed by examining immunoreactivity for glial fibrillary acid protein (GFAP) as described previously (Hoehn et al., 2005). Rats exposed to 30-min tMCAo showed strong immunoreactivity for GFAP in subcortical regions, including the striatum, ventral pallidum, lateral globus pallidus, and internal capsule (Fig. 1b). Although small areas of cortical infarction could also be detected, most of them were restricted to a part of the granular insular cortex, dysgranular cortex, or agranular insular cortex. The sensory or motor cortex was not damaged. The modified neurological severity score (Table 1) was 0.0± 0.0 on day 14 in rats subjected to tMCAo, which corresponded to a previous report (Zhang et al., 2000). Sham rats also exhibited no
Fig. 1 – (a) Timing of behavioral tests and surgery. MCAo: middle cerebral artery occlusion, MP: methamphetamine, mNSS: modified neurological severity score. (b) Immunohistochemical staining for glial fibrillary acid protein (GFAP). Ischemic lesions showed strong GFAP immunoreactivity. In the 30-min tMCAo rat, the striatum was widely injured throughout the rostrocaudal axis accompanied by damage in the ventral pallidum, lateral globus pallidus, and internal capsule. Although a small cortical lesion was observed, no obvious GFAP immunoreactivity could be detected in the motor and somatosensory cortex. Bar= 2 mm.
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Table 1 – Modified neurological severity score Points Motor tests 1 Flexion of forelimb 1 Flexion of hindlimb Placing rat on the floor (normal = 0; maximum = 3) 0 Normal walk 1 Inability to walk straight 2 Circling toward the paretic side 3 Fall down to the paretic side Sensory test 1 Placing test (visual tactile test) 2 Proprioceptive test (deep sensation, pushing the paw against the table edge) Beam balance test (normal = 0; maximum = 6) 0 Balance with steady posture 1 Grasps side of beam 2 Hugs the beam and one limb falls down from the beam 3 Hugs the beam and two limbs fall down from the beam, or spins on beam (> 60 s) 4 Attempts to balance on the beam but falls off (> 40 s) 5 Attempts to balance on the beam but falls off (> 20 s) 6 Falls off: No attempt to balance or hang on the beam (< 20 s) Reflexes absent and abnormal movements 1 Corneal reflex (eye blink when lightly touching the cornea with cotton) 1 Startle reflex (motor response to a brief noise) 1 Seizures, myoclonus, myodystony Maximum points
2
3
2
6
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deficit in the tMCAo model, we further focused on the relation between the two tests. Thus, we examined the correlations between infarction volume and the results of behavioral tests as assessed by Pearson's correlation coefficient. Unexpectedly, the percent of retrieved pellets in the left forelimb on day 14 in the Montoya staircase test did not correlate with the volume of infarcted subcortex, including striatum, ventral pallidum, lateral globus pallidus, and internal capsule (Fig. 3a), but correlated well with the infarction volume limited to the striatum (Fig. 3b). The number of methamphetamine-induced rotations also correlated well with the volume of both infarcted subcortex (Fig. 3c) and infarcted striatum (Fig. 3d). Finally, the correlation between the results of the Montoya staircase test and methamphetamine-induced rotation was analyzed, but there was no significant correlation (Fig. 3e). These data suggest that the Montoya staircase test and methamphetamine-rotation test each reflect different neurological deficits in this model.
3.
Discussion
3
16
abnormal findings. In training sessions of the Montoya staircase test, the number of retrieved pellets increased day by day, and the total number of eaten pellets was the same in tMCAo and sham-operated rats (Fig. 2a). In testing sessions, sham-operated rats temporarily showed a reduction in skillfulness of the left forelimb at 8 days after operation when the number of pellets retrieved by each forelimb was compared to that in training sessions (day −3 to −1, Fig. 2b), while the number of retrieved pellets gradually increased from day 9 to 14 without a significant difference between the left and right forelimbs. In contrast, the number of retrieved pellets in tMCAo rats increased more slowly than in sham-operated rats, especially in the left forelimb (Fig. 2c). There was a significant difference in the ability to retrieve pellets between the left and right forelimbs on day 10 and days 12–14. Since appetite would have an influence on the Montoya staircase test (Montoya et al., 1991), we evaluated the ratio of body weight change through the testing sessions (Fig. 2d), and found no significant difference between sham-operated and tMCAo rats. After methamphetamine (2.5 mg/kg) was injected intraperitoneally at 21 days after tMCAo, rats subjected to right tMCAo showed turning toward the right side. The number of rotations was significantly increased in tMCAo rats (59.4 ± 26.7/ 20 min) as compared with sham rats (4.5 ± 0.9/20 min, p < 0.01 vs. tMCAo rats).
2.2. Correlation between infarction volume and behavioral test results As both the Montoya staircase test and methamphetaminerotation test were useful for the evaluation of neurological
To assess various behavioral dysfunctions caused by cerebral infarction, rodent models have been used to evaluate the effectiveness of drugs, gene therapy, and cell therapy. Most previous studies evaluated neurological deficit reflecting the function of the neocortex, which could be induced in Tamura's model (Tamura et al., 1981) or a photochemical embolic model (Watson et al., 1985), or both the neocortex and subcortex, which could be induced by 60–120-min intraluminal thread occlusion (Memezawa et al., 1992). However, infarction limited to even the subcortex also causes various neurological deficits such as parkinsonism, sensori-motor deficit, or dementia clinically. Thus, it is noteworthy to establish useful methods to assess the neurological deficit after subcortical infarction in rodent models. As expected, rats subjected to 30-min tMCAo clearly showed a decrease in their ability to retrieve pellets in the Montoya staircase test in the chronic stage of subcortical infarction, when the commonly used neurological severity score and adhesive-removal test could not detect any neurological deficit (Zhang et al., 2000). Our data indicated that the disability correlated with the extent of striatal injury, which is compatible with previous reports that the striatal lesion induced by intrastriatal injection of 6-OHDA causes disability of the forelimb (Cousins and Salamone, 1996a,b; Sabol et al., 1985). In contrast, subcortical infarction, involving globus pallidus, did not correlate with the disability although bilateral globus pallidus lesions induced by electrocauterization were reported to cause obvious disruption in forelimb reaching task (Schneider and Olazabal, 1984). It is unclear why globus pallidus lesion did not correlate with the results of Montoya staircase tests in the present study, but we speculate that the damage both in the caudate putamen and globus pallidus might cause different result from that in the lesion limited in globus pallidus. Unexpectedly, the ipsilateral forelimb was also affected to some degree. One possible explanation could be that the bilateral input pathway to the striatum and cross connection of the output pathway from the basal ganglia cause ipsilateral disability (Fricker et al., 1996).
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Fig. 2 – (a) Total retrieved pellets during training sessions in Montoya staircase test. Both sham-operated and 30-min tMCAo rats could not take pellets at the start of training (day −8), but gradually became able to retrieve pellets (day −1). There was no statistically significant difference in their ability to retrieve pellets. (b) Percent of retrieved pellets in sham-operated rats. If the number of retrieved pellets is the same as the average number of retrieved pellets from day −3 to −1, the percent of retrieved pellets is 100%. If the number is less than the average number of retrieved pellets from day −3 to − 1, the percent of retrieved pellets is less than 100%. The number of retrieved pellets increased gradually from day 9 to 14 without a significant difference between the left and right forelimbs. (c) Percent of retrieved pellets in testing sessions in tMCAo rats. The number of eaten pellets in rats was not increased compared to that in sham-operated rats, especially in the left forelimb. There were significant differences in the ability to retrieve pellets between the left and right forelimbs on day 10 and days 12–14. *p < 0.05, **p < 0.01 vs. right forelimb. (d) Ratio of body weight change. There was no significant difference.
Another possible explanation is that rats with a unilateral lesion in the striatum might find it difficult to adjust their posture to maintain equilibrium on the platform of the staircase (Barneoud et al., 1995; Montoya et al., 1990), causing ipsilateral difficulty in retrieving pellets. Since basal ganglia, particularly dorsal striatum, has been considered to participate in formation of learning and memory (Packard and Knowlton, 2002), subcortical infarction, in this study, might also affect the clumsiness not only in the contralateral side but also in the ipsilateral side. Further behavioral study about memory is necessary to clarify the relationship between the memory and Montoya staircase test. Although appetite loss would also cause an ipsilateral decrease in retrieving pellets, it might have had no influence in the present study, since there was no difference in the ratio of body weight change in the testing sessions. Similarly, methamphetamine caused ipsilateral rotation in 30-min tMCAo rats. The number of rotations was well correlated with striatal infarction volume, which is similar to the previous report showing the close correlation between striatal infarction size and ipsilateral rotations in focal ischemia in mongolian gerbils (Ishibashi et al., 2004). Additionally, the data in the present study also implies that infarction in a subcortical region other than the striatum also has an influence on ipsilateral rotation, which is compatible
with previous studies showing that a globus pallidus lesion induced by a dopaminergic agonist or dopamine release stimulant resulted in ipsilateral rotation (Konitsiotis et al., 1998; Miwa et al., 1998). In the present study, these behavioral tests were performed from 8 days to 21 days after tMCAo. It might be also center of interest that these two tests could be used in the late stage of cerebral infarction, such as 2 or 3 months after tMCAo. We speculate that these tests might be useful in the late stage of subcortical infarction, since they were used 4–5 months after striatal lesion induced by quinolinic acid (Dobrossy and Dunnett, 2003). Further studies are necessary to determine whether these tests are useful in the late stage of subcortical infarction. Recently, another model of subcortical infarction has been produced by injections of endothelin-1 into two sites in the striatum using a 23 G-needle (Whitehead et al., 2005a,b). However, hemorrhage or inflammation is usually evoked by a needle in the sensory or motor cortex, which might have some influence on behavioral tests. Also, it is clear that intracerebral injection of endothelin-1 might not cause typical cerebral ischemia as compared to an arterial occlusion model, since endothelin-1 induces efflux of glutamate from astrocytes, which could cause neuronal death (Sasaki et al., 1997) as well as vasoconstriction. In comparison, the 30-min tMCAo model
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Fig. 3 – Correlation between infarction volume and behavioral test results (a–d). Although there was no significant correlation between subcortical infarction volume and staircase test result (a), there was a significant correlation between striatal infarction volume and staircase test result (b). The number of methamphetamine-induced rotations was correlated with the volume of both infarcted subcortex (c) and infarcted striatum (d). No significant correlation was seen between the results of methamphetamine-induced rotation and staircase test (e). used in the present study caused subcortical infarction without widespread neocortical lesions, as described previously (Goto et al., 1993, 1994; Memezawa et al., 1992). Although the present study demonstrated that the Montotya staircase test and methamphetamine-induced rotation are useful to evaluate neurological deficit after 30-min tMCAo, there was no correlation between the results of the staircase test and methamphetamine-induced rotation. Which is more suitable to assess the neurological deficit after 30-min tMCAo? In methamphetamine-induced rotation, the therapeutic effects of target substances should be carefully evaluated, since methamphetamine itself has some influence on cerebral ischemic injury (Wang et al., 2001). In contrast, the staircase test can be performed without any drugs, but requires training of rats and special apparatus. Since several previous studies reported dissociation between rotational behavior and skilled motor tasks in 6-OHDA-treated striatal or nigrostriatal bundle-injured rats (Castaneda et al., 2005; Dunnett et al., 1987; Metz and Whishaw, 2002; Montoya et al., 1990), the combination of these two tests might be most appropriate to assess the therapeutic effects of various drugs on the functional recovery after subcortical infarction.
4.
Experimental procedures
4.1.
Surgical procedure
All procedures used in this study were carried out in accordance with protocols complying with local institutional
guidelines for animal care of the University of Tokyo. Adult male Wistar rats (200–250 g; Charles River Laboratories Japan, Kanagawa, Japan) were housed at three per cage under standardized humidity, temperature, and lighting conditions (light on at 8 AM and off at 8 PM). The rats were given free access to food pellets and water except during both the training and testing sessions. In these sessions, rats had restricted access to food to keep their body weight at 85–90% of the free feeding level so as to maintain their motivation to eat. To generate a transient MCAo model, the right middle cerebral artery was transiently occluded by placement of poly-L-lysinecoated 4-0 nylon around the origin of the MCA, as described previously (Belayev et al., 1996). Thirty minutes after the start of occlusion, rats were re-anesthetized and the nylon was removed.
4.2.
Modified neurological severity score (mNSS)
Neurological function was graded as 0 to 16 (normal score, 0; maximal deficit score, 16) of the modified NSS, by which motor and sensory function, balance impairment, and reflex abnormality were evaluated (Table 1).
4.3. Montoya staircase test and methamphetamine-induced rotation Montoya staircase test was performed as described before (Whitehead et al., 2005b). Briefly, the apparatus consists of the removal double staircase and box (Montoya et al., 1991). Each stair has 3-mm-depth well, where 45-mg pellets (Bioserv,
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Frenchtown, NJ, USA) are placed. With this apparatus, we trained rats before surgery (training sessions: day −9 to −1) and checked the ability of forelimb task after tMCAo (testing sessions: day 8 to 14). During the training sessions, rats were deprived of food to stimulate their appetite on day −9 and −8, followed by food restriction by feeding 15 g normal food pellets per rat (day −7 to −1). From day −7 to −1, rats were placed on the apparatus once a day and trained for 20 min. The number of retrieved pellets was counted in each trial. In the end of training sessions, the rats that could not habit in the apparatus and retrieve pellets were excluded. In the testing sessions (days 8 to 14), the rats were also examined in the same way that performed in training sessions. To calculate the ability of forelimb task, the number of retrieved pellets in testing sessions on each day was compared to that in training sessions using the following equation: (% of retrieved pellets) = [(Eaten pellets on each day in testing session)/(Average number of eaten pellets on day − 3 to −1)] × 100 (%). The ratio of body weight change was calculated as follows: (Ratio of body weight change) = (Body weight) / (Body weight on day 8). In the methamphetamine-induced rotational test on day 21, methamphetamine (1 mg/ml in saline) was injected intraperitoneally at 2.5 mg/kg as described before (Kirik et al., 1998). The rats were returned to the holding cage. Five minutes later, they were moved into a round opaque holder (300 mm diameter, 240 mm high), and the frequency of turning 360° in a direction toward the right was counted for 20 min. Since methamphetamine was reported to be more sensitive than apomorphine to detect behavioral abnormality (Kirik et al., 1998), we used methamphetamine in the present study.
4.4.
Histological examination
On day 21, all rats were sacrificed and perfused transcardially first with normal saline followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). The fixed brain was removed from the skull and immersed overnight in fixative solution. It was cryoprotected and serial coronal sections (30 μm) were cut on a freezing microtome at nine levels; +1.7, +1.2,+0.7,+0.2, −0.3, −0.8, −1.3, −1.8, −2.3 and −2.8 mm from the bregma. To evaluate infarction, immunostaining for GFAP was performed as described previously (Hoehn et al., 2005). Sections were treated with 0.3% H2O2 to block endogenous peroxidase. They were incubated with a primary antibody against GFAP (1:1000; mouse monoclonal; Sigma-Aldrich, USA) at 25 °C for 1 h, followed by incubation with biotinylated antimouse antibody (Vectastain Elite ABC; Vector Laboratories, USA). Then, they were incubated with streptavidin–horseradish peroxidase (Vectastain Elite ABC; Vector Laboratories), and the biotin–streptavidin–peroxidase complex was detected with diaminobenzidine solution (Vector Peroxidase Substrate Kit; Vector Laboratories). Images were digitized with a Fujix Digital Camera (HC-2500, Fuji Film Co., Japan) on a BX51 microscope (Olympus, Japan). To quantify the cerebral ischemic area delineated by the immunoreactivity for GFAP, the acquired images were imported into Adobe Photoshop (Version 7.0, Adobe System, USA). To measure the area of brain ischemia in each slice, the number of pixels was recorded and converted into an area presented in square millimeters. The
infarcted areas were evaluated in serial sections in 500-μm steps. The infarction volume was calculated from the infarction areas in nine sections as follows: Infarction volume = Σ(Infarcted area) × 500 (mm3).
4.5.
Statistical analysis
Statistical analysis was performed using Stat View Version 5.0 (SAS Institute Inc., USA). All values are expressed as mean ± S.E.M. Statistical analysis of body weight ratio, staircase test (training sessions) and methamphetamine rotation behavior was performed using Mann–Whitney U-test, a nonparametric test. The staircase test (testing sessions) was analyzed with paired t-test. Correlations between infarction volume and results of behavioral tests were assessed by Pearson's correlations coefficient. The level of statistical significance was set at p < 0.05.
Acknowledgments This work was partially supported by a Grant-in-Aid from the Organization for Pharmaceutical Safety and Research, a Grantin-Aid from The Ministry of Public Health and Welfare, a Grantin-Aid from Japan Promotion of Science, and a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government.
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Research Report
Quantitative measurement of neurological deficit after mild (30 min) transient middle cerebral artery occlusion in rats Kouji Wakayama a,b , Munehisa Shimamura a , Masataka Sata a , Naoyuki Sato c , Koji Kawakami a , Hirotsugu Fukuda d , Takuji Tomimatsu d , Toshio Ogihara b , Ryuichi Morishita c,⁎ a
Department of Advanced Clinical Science and Therapeutics, Graduate School of Medicine, The University of Tokyo, Japan Department of Geriatric Medicine, Graduate School of Medicine, Osaka University, Japan c Department of Clinical Gene Therapy, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita 565-0871, Japan d Department of Obstetrics and Gynecology, Graduate School of Medicine, Osaka University, Japan b
A R T I C LE I N FO
AB S T R A C T
Article history:
Although 30-min transient middle cerebral artery occlusion (30-min tMCAo) causes
Accepted 4 October 2006
reproducible subcortical infarction in rats, it is difficult to evaluate the resulting
Available online 14 December 2006
neurological deficit using common behavioral tests such as the rota-rod test, adhesiveremoval test, or narrow beam test. Establishment of a method of quantitative evaluation
Keywords:
would help to develop a novel therapeutic approach to treat cerebral infarction. To solve this
Transient middle cerebral
problem, we examined whether the neurological deficit could be detected by the Montoya
artery occlusion
staircase test or methamphetamine-induced rotation, which are commonly used in a
Subcortical infarction
Parkinson disease model induced by intrastriatal injection of 6-hydroxydopamine (6-
Behavior test
OHDA). From 10 to 14 days after tMCAo, the Montoya staircase test showed significant
Methamphetamine
clumsiness in forelimb tasks contralateral to the lesion side, whereas sham-operated rats
Montoya staircase test
showed no significant clumsiness in both forelimbs. The number of ipsilateral rotations induced by methamphetamine was also increased in tMCAo-rats at 21 days after tMCAo. Although Pearson's correlations coefficient showed that the results of these tests were correlated with the infarction volume, there was no significant correlation between the results of these two tests. These findings imply that the neurological deficit detected by both tests might reflect the severity of ischemic injury, but each test might evaluate different aspects of neurological deficit. Thus, the Montoya staircase test and methamphetamineinduced rotation are useful to evaluate neurological deficit in the chronic stage of subcortical infarction induced by 30-min tMCAo. © 2006 Published by Elsevier B.V.
1.
Introduction
Although 30-min transient middle cerebral artery occlusion (30-min tMCAo) in rats, which causes subcortical infarction without widespread neocortical ischemic damage (Goto et al., ⁎ Corresponding author. Fax: +81 6 6879 3409. E-mail address: [email protected] (R. Morishita). 0006-8993/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.brainres.2006.10.088
1994; Zhang et al., 2000), is a reliable model for research, it is difficult to evaluate neurological deficit in this model, since the sensori-motor deficit assessed by the commonly used neurological severity score, rota-rod test, grid walking test, cylinder test, ledged beam walking, or adhesive-removal test
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quickly recovers to the level in sham-operated rats (Rogers et al., 1997; Susanne et al., 2005; Zhang et al., 2000). Therefore, the development of a novel therapeutic approach to treat cerebral infarction is limited by methodological issues in evaluation. From this perspective, the development of other behavioral tests is necessary to evaluate neurological deficit quantitatively. In the present study, we focused on the Montoya staircase test and methamphetamine-induced rotation, which are commonly used in a Parkinson disease model in rats induced by intrastriatal injection of 6-hydroxydopamine (6-OHDA) (Barneoud et al., 2000; Jeyasingham et al., 2001; Kirik et al., 1998), for the evaluation of neurological deficit in a cerebral infarction model. The Montoya staircase test evaluates independent forelimb reaching tasks and grasping ability in rats (Montoya et al., 1990, 1991), and reflects the extent of striatal damage (Montoya et al., 1990). It is applied not only in Parkinson disease models, but also in focal cerebral ischemic rats such as 60, 90, and 120-min tMCAo models and neonatal hypoxic– ischemic brain injury model (Colbourne et al., 2000; Hudzik et al., 2000; Palmer et al., 2001; Tomimatsu et al., 2002; Virley et al., 2000), which exhibit widespread infarction of the neocortex and basal ganglia. However, this test has not been evaluated in subcortical infarction induced by 30-min tMCAo. Alternatively, methamphetamine-induced rotation examines postural instability and reflects subcortical damage (Pycock, 1980). Theoretically, methamphetamine stimulates the release of dopamine from neurons in the intact side of the brain and induces rotation ipsilateral to the lesion (Deumens et al., 2002). Similarly to the Montoya staircase test, this test has been applied in permanent MCAo (Grabowski et al., 1993) and 60–120 min tMCAo models (Hudzik et al., 2000; Veizovic et al., 2001), but it has not been examined in the 30-min tMCAo
model. From these viewpoints, the present study examined whether these two tests could detect the neurological deficit in the chronic stage of subcortical infarction in rats induced by 30-min tMCAo. Here, we demonstrated that the Montoya staircase test and methamphetamine-induced rotation were useful tests for the quantitative assessment of neurological deficit after 30-min tMCAo, and that these two tests reflected the severity of ischemic damage.
2.
Results
2.1.
Behavioral tests
After training sessions (day − 8 to −1, Fig. 1a), rats were exposed to 30-min tMCAo (day 0, sham: n = 4, tMCAo: n = 7). The staircase test was performed from day 8 to 14 (testing sessions). The commonly used neurological severity score (Table 1) was also determined on day 14. Then, rats were subjected to methamphetamine-induced rotation test on day 21. To confirm the severity of cerebral infarction, infarction volume was assessed by examining immunoreactivity for glial fibrillary acid protein (GFAP) as described previously (Hoehn et al., 2005). Rats exposed to 30-min tMCAo showed strong immunoreactivity for GFAP in subcortical regions, including the striatum, ventral pallidum, lateral globus pallidus, and internal capsule (Fig. 1b). Although small areas of cortical infarction could also be detected, most of them were restricted to a part of the granular insular cortex, dysgranular cortex, or agranular insular cortex. The sensory or motor cortex was not damaged. The modified neurological severity score (Table 1) was 0.0± 0.0 on day 14 in rats subjected to tMCAo, which corresponded to a previous report (Zhang et al., 2000). Sham rats also exhibited no
Fig. 1 – (a) Timing of behavioral tests and surgery. MCAo: middle cerebral artery occlusion, MP: methamphetamine, mNSS: modified neurological severity score. (b) Immunohistochemical staining for glial fibrillary acid protein (GFAP). Ischemic lesions showed strong GFAP immunoreactivity. In the 30-min tMCAo rat, the striatum was widely injured throughout the rostrocaudal axis accompanied by damage in the ventral pallidum, lateral globus pallidus, and internal capsule. Although a small cortical lesion was observed, no obvious GFAP immunoreactivity could be detected in the motor and somatosensory cortex. Bar= 2 mm.
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Table 1 – Modified neurological severity score Points Motor tests 1 Flexion of forelimb 1 Flexion of hindlimb Placing rat on the floor (normal = 0; maximum = 3) 0 Normal walk 1 Inability to walk straight 2 Circling toward the paretic side 3 Fall down to the paretic side Sensory test 1 Placing test (visual tactile test) 2 Proprioceptive test (deep sensation, pushing the paw against the table edge) Beam balance test (normal = 0; maximum = 6) 0 Balance with steady posture 1 Grasps side of beam 2 Hugs the beam and one limb falls down from the beam 3 Hugs the beam and two limbs fall down from the beam, or spins on beam (> 60 s) 4 Attempts to balance on the beam but falls off (> 40 s) 5 Attempts to balance on the beam but falls off (> 20 s) 6 Falls off: No attempt to balance or hang on the beam (< 20 s) Reflexes absent and abnormal movements 1 Corneal reflex (eye blink when lightly touching the cornea with cotton) 1 Startle reflex (motor response to a brief noise) 1 Seizures, myoclonus, myodystony Maximum points
2
3
2
6
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deficit in the tMCAo model, we further focused on the relation between the two tests. Thus, we examined the correlations between infarction volume and the results of behavioral tests as assessed by Pearson's correlation coefficient. Unexpectedly, the percent of retrieved pellets in the left forelimb on day 14 in the Montoya staircase test did not correlate with the volume of infarcted subcortex, including striatum, ventral pallidum, lateral globus pallidus, and internal capsule (Fig. 3a), but correlated well with the infarction volume limited to the striatum (Fig. 3b). The number of methamphetamine-induced rotations also correlated well with the volume of both infarcted subcortex (Fig. 3c) and infarcted striatum (Fig. 3d). Finally, the correlation between the results of the Montoya staircase test and methamphetamine-induced rotation was analyzed, but there was no significant correlation (Fig. 3e). These data suggest that the Montoya staircase test and methamphetamine-rotation test each reflect different neurological deficits in this model.
3.
Discussion
3
16
abnormal findings. In training sessions of the Montoya staircase test, the number of retrieved pellets increased day by day, and the total number of eaten pellets was the same in tMCAo and sham-operated rats (Fig. 2a). In testing sessions, sham-operated rats temporarily showed a reduction in skillfulness of the left forelimb at 8 days after operation when the number of pellets retrieved by each forelimb was compared to that in training sessions (day −3 to −1, Fig. 2b), while the number of retrieved pellets gradually increased from day 9 to 14 without a significant difference between the left and right forelimbs. In contrast, the number of retrieved pellets in tMCAo rats increased more slowly than in sham-operated rats, especially in the left forelimb (Fig. 2c). There was a significant difference in the ability to retrieve pellets between the left and right forelimbs on day 10 and days 12–14. Since appetite would have an influence on the Montoya staircase test (Montoya et al., 1991), we evaluated the ratio of body weight change through the testing sessions (Fig. 2d), and found no significant difference between sham-operated and tMCAo rats. After methamphetamine (2.5 mg/kg) was injected intraperitoneally at 21 days after tMCAo, rats subjected to right tMCAo showed turning toward the right side. The number of rotations was significantly increased in tMCAo rats (59.4 ± 26.7/ 20 min) as compared with sham rats (4.5 ± 0.9/20 min, p < 0.01 vs. tMCAo rats).
2.2. Correlation between infarction volume and behavioral test results As both the Montoya staircase test and methamphetaminerotation test were useful for the evaluation of neurological
To assess various behavioral dysfunctions caused by cerebral infarction, rodent models have been used to evaluate the effectiveness of drugs, gene therapy, and cell therapy. Most previous studies evaluated neurological deficit reflecting the function of the neocortex, which could be induced in Tamura's model (Tamura et al., 1981) or a photochemical embolic model (Watson et al., 1985), or both the neocortex and subcortex, which could be induced by 60–120-min intraluminal thread occlusion (Memezawa et al., 1992). However, infarction limited to even the subcortex also causes various neurological deficits such as parkinsonism, sensori-motor deficit, or dementia clinically. Thus, it is noteworthy to establish useful methods to assess the neurological deficit after subcortical infarction in rodent models. As expected, rats subjected to 30-min tMCAo clearly showed a decrease in their ability to retrieve pellets in the Montoya staircase test in the chronic stage of subcortical infarction, when the commonly used neurological severity score and adhesive-removal test could not detect any neurological deficit (Zhang et al., 2000). Our data indicated that the disability correlated with the extent of striatal injury, which is compatible with previous reports that the striatal lesion induced by intrastriatal injection of 6-OHDA causes disability of the forelimb (Cousins and Salamone, 1996a,b; Sabol et al., 1985). In contrast, subcortical infarction, involving globus pallidus, did not correlate with the disability although bilateral globus pallidus lesions induced by electrocauterization were reported to cause obvious disruption in forelimb reaching task (Schneider and Olazabal, 1984). It is unclear why globus pallidus lesion did not correlate with the results of Montoya staircase tests in the present study, but we speculate that the damage both in the caudate putamen and globus pallidus might cause different result from that in the lesion limited in globus pallidus. Unexpectedly, the ipsilateral forelimb was also affected to some degree. One possible explanation could be that the bilateral input pathway to the striatum and cross connection of the output pathway from the basal ganglia cause ipsilateral disability (Fricker et al., 1996).
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Fig. 2 – (a) Total retrieved pellets during training sessions in Montoya staircase test. Both sham-operated and 30-min tMCAo rats could not take pellets at the start of training (day −8), but gradually became able to retrieve pellets (day −1). There was no statistically significant difference in their ability to retrieve pellets. (b) Percent of retrieved pellets in sham-operated rats. If the number of retrieved pellets is the same as the average number of retrieved pellets from day −3 to −1, the percent of retrieved pellets is 100%. If the number is less than the average number of retrieved pellets from day −3 to − 1, the percent of retrieved pellets is less than 100%. The number of retrieved pellets increased gradually from day 9 to 14 without a significant difference between the left and right forelimbs. (c) Percent of retrieved pellets in testing sessions in tMCAo rats. The number of eaten pellets in rats was not increased compared to that in sham-operated rats, especially in the left forelimb. There were significant differences in the ability to retrieve pellets between the left and right forelimbs on day 10 and days 12–14. *p < 0.05, **p < 0.01 vs. right forelimb. (d) Ratio of body weight change. There was no significant difference.
Another possible explanation is that rats with a unilateral lesion in the striatum might find it difficult to adjust their posture to maintain equilibrium on the platform of the staircase (Barneoud et al., 1995; Montoya et al., 1990), causing ipsilateral difficulty in retrieving pellets. Since basal ganglia, particularly dorsal striatum, has been considered to participate in formation of learning and memory (Packard and Knowlton, 2002), subcortical infarction, in this study, might also affect the clumsiness not only in the contralateral side but also in the ipsilateral side. Further behavioral study about memory is necessary to clarify the relationship between the memory and Montoya staircase test. Although appetite loss would also cause an ipsilateral decrease in retrieving pellets, it might have had no influence in the present study, since there was no difference in the ratio of body weight change in the testing sessions. Similarly, methamphetamine caused ipsilateral rotation in 30-min tMCAo rats. The number of rotations was well correlated with striatal infarction volume, which is similar to the previous report showing the close correlation between striatal infarction size and ipsilateral rotations in focal ischemia in mongolian gerbils (Ishibashi et al., 2004). Additionally, the data in the present study also implies that infarction in a subcortical region other than the striatum also has an influence on ipsilateral rotation, which is compatible
with previous studies showing that a globus pallidus lesion induced by a dopaminergic agonist or dopamine release stimulant resulted in ipsilateral rotation (Konitsiotis et al., 1998; Miwa et al., 1998). In the present study, these behavioral tests were performed from 8 days to 21 days after tMCAo. It might be also center of interest that these two tests could be used in the late stage of cerebral infarction, such as 2 or 3 months after tMCAo. We speculate that these tests might be useful in the late stage of subcortical infarction, since they were used 4–5 months after striatal lesion induced by quinolinic acid (Dobrossy and Dunnett, 2003). Further studies are necessary to determine whether these tests are useful in the late stage of subcortical infarction. Recently, another model of subcortical infarction has been produced by injections of endothelin-1 into two sites in the striatum using a 23 G-needle (Whitehead et al., 2005a,b). However, hemorrhage or inflammation is usually evoked by a needle in the sensory or motor cortex, which might have some influence on behavioral tests. Also, it is clear that intracerebral injection of endothelin-1 might not cause typical cerebral ischemia as compared to an arterial occlusion model, since endothelin-1 induces efflux of glutamate from astrocytes, which could cause neuronal death (Sasaki et al., 1997) as well as vasoconstriction. In comparison, the 30-min tMCAo model
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Fig. 3 – Correlation between infarction volume and behavioral test results (a–d). Although there was no significant correlation between subcortical infarction volume and staircase test result (a), there was a significant correlation between striatal infarction volume and staircase test result (b). The number of methamphetamine-induced rotations was correlated with the volume of both infarcted subcortex (c) and infarcted striatum (d). No significant correlation was seen between the results of methamphetamine-induced rotation and staircase test (e). used in the present study caused subcortical infarction without widespread neocortical lesions, as described previously (Goto et al., 1993, 1994; Memezawa et al., 1992). Although the present study demonstrated that the Montotya staircase test and methamphetamine-induced rotation are useful to evaluate neurological deficit after 30-min tMCAo, there was no correlation between the results of the staircase test and methamphetamine-induced rotation. Which is more suitable to assess the neurological deficit after 30-min tMCAo? In methamphetamine-induced rotation, the therapeutic effects of target substances should be carefully evaluated, since methamphetamine itself has some influence on cerebral ischemic injury (Wang et al., 2001). In contrast, the staircase test can be performed without any drugs, but requires training of rats and special apparatus. Since several previous studies reported dissociation between rotational behavior and skilled motor tasks in 6-OHDA-treated striatal or nigrostriatal bundle-injured rats (Castaneda et al., 2005; Dunnett et al., 1987; Metz and Whishaw, 2002; Montoya et al., 1990), the combination of these two tests might be most appropriate to assess the therapeutic effects of various drugs on the functional recovery after subcortical infarction.
4.
Experimental procedures
4.1.
Surgical procedure
All procedures used in this study were carried out in accordance with protocols complying with local institutional
guidelines for animal care of the University of Tokyo. Adult male Wistar rats (200–250 g; Charles River Laboratories Japan, Kanagawa, Japan) were housed at three per cage under standardized humidity, temperature, and lighting conditions (light on at 8 AM and off at 8 PM). The rats were given free access to food pellets and water except during both the training and testing sessions. In these sessions, rats had restricted access to food to keep their body weight at 85–90% of the free feeding level so as to maintain their motivation to eat. To generate a transient MCAo model, the right middle cerebral artery was transiently occluded by placement of poly-L-lysinecoated 4-0 nylon around the origin of the MCA, as described previously (Belayev et al., 1996). Thirty minutes after the start of occlusion, rats were re-anesthetized and the nylon was removed.
4.2.
Modified neurological severity score (mNSS)
Neurological function was graded as 0 to 16 (normal score, 0; maximal deficit score, 16) of the modified NSS, by which motor and sensory function, balance impairment, and reflex abnormality were evaluated (Table 1).
4.3. Montoya staircase test and methamphetamine-induced rotation Montoya staircase test was performed as described before (Whitehead et al., 2005b). Briefly, the apparatus consists of the removal double staircase and box (Montoya et al., 1991). Each stair has 3-mm-depth well, where 45-mg pellets (Bioserv,
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Frenchtown, NJ, USA) are placed. With this apparatus, we trained rats before surgery (training sessions: day −9 to −1) and checked the ability of forelimb task after tMCAo (testing sessions: day 8 to 14). During the training sessions, rats were deprived of food to stimulate their appetite on day −9 and −8, followed by food restriction by feeding 15 g normal food pellets per rat (day −7 to −1). From day −7 to −1, rats were placed on the apparatus once a day and trained for 20 min. The number of retrieved pellets was counted in each trial. In the end of training sessions, the rats that could not habit in the apparatus and retrieve pellets were excluded. In the testing sessions (days 8 to 14), the rats were also examined in the same way that performed in training sessions. To calculate the ability of forelimb task, the number of retrieved pellets in testing sessions on each day was compared to that in training sessions using the following equation: (% of retrieved pellets) = [(Eaten pellets on each day in testing session)/(Average number of eaten pellets on day − 3 to −1)] × 100 (%). The ratio of body weight change was calculated as follows: (Ratio of body weight change) = (Body weight) / (Body weight on day 8). In the methamphetamine-induced rotational test on day 21, methamphetamine (1 mg/ml in saline) was injected intraperitoneally at 2.5 mg/kg as described before (Kirik et al., 1998). The rats were returned to the holding cage. Five minutes later, they were moved into a round opaque holder (300 mm diameter, 240 mm high), and the frequency of turning 360° in a direction toward the right was counted for 20 min. Since methamphetamine was reported to be more sensitive than apomorphine to detect behavioral abnormality (Kirik et al., 1998), we used methamphetamine in the present study.
4.4.
Histological examination
On day 21, all rats were sacrificed and perfused transcardially first with normal saline followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). The fixed brain was removed from the skull and immersed overnight in fixative solution. It was cryoprotected and serial coronal sections (30 μm) were cut on a freezing microtome at nine levels; +1.7, +1.2,+0.7,+0.2, −0.3, −0.8, −1.3, −1.8, −2.3 and −2.8 mm from the bregma. To evaluate infarction, immunostaining for GFAP was performed as described previously (Hoehn et al., 2005). Sections were treated with 0.3% H2O2 to block endogenous peroxidase. They were incubated with a primary antibody against GFAP (1:1000; mouse monoclonal; Sigma-Aldrich, USA) at 25 °C for 1 h, followed by incubation with biotinylated antimouse antibody (Vectastain Elite ABC; Vector Laboratories, USA). Then, they were incubated with streptavidin–horseradish peroxidase (Vectastain Elite ABC; Vector Laboratories), and the biotin–streptavidin–peroxidase complex was detected with diaminobenzidine solution (Vector Peroxidase Substrate Kit; Vector Laboratories). Images were digitized with a Fujix Digital Camera (HC-2500, Fuji Film Co., Japan) on a BX51 microscope (Olympus, Japan). To quantify the cerebral ischemic area delineated by the immunoreactivity for GFAP, the acquired images were imported into Adobe Photoshop (Version 7.0, Adobe System, USA). To measure the area of brain ischemia in each slice, the number of pixels was recorded and converted into an area presented in square millimeters. The
infarcted areas were evaluated in serial sections in 500-μm steps. The infarction volume was calculated from the infarction areas in nine sections as follows: Infarction volume = Σ(Infarcted area) × 500 (mm3).
4.5.
Statistical analysis
Statistical analysis was performed using Stat View Version 5.0 (SAS Institute Inc., USA). All values are expressed as mean ± S.E.M. Statistical analysis of body weight ratio, staircase test (training sessions) and methamphetamine rotation behavior was performed using Mann–Whitney U-test, a nonparametric test. The staircase test (testing sessions) was analyzed with paired t-test. Correlations between infarction volume and results of behavioral tests were assessed by Pearson's correlations coefficient. The level of statistical significance was set at p < 0.05.
Acknowledgments This work was partially supported by a Grant-in-Aid from the Organization for Pharmaceutical Safety and Research, a Grantin-Aid from The Ministry of Public Health and Welfare, a Grantin-Aid from Japan Promotion of Science, and a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government.
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