Physiology & Behavior, Vol. 50, pp. 263-269. © Pergamon Press pic, 1991. Printed in the U.S.A.
0031-9384491 $3.00 + .00
Neurological Correlates of Unilateral and Bilateral "Strokes" of the Middle Cerebral Artery in the Rat C H E R Y L S. A N D E R S E N , A N D E R S B. A N D E R S E N A N D S T A N L E Y F I N G E R 1
Department o f Psychology, Washington University, St. Louis, M O 63130 R e c e i v e d 18 January 1991 ANDERSEN, C. S., A. B. ANDERSEN AND S. FINGER. Neurological correlates of unilateral and bilateral "strokes" of the middle cerebral artery in the rat. PHYSIOL BEHAV 50(2) 263-269, 1991.--Rats with unilateral lesions of the middle cerebral artery (MCA) were tested for the ability to detect (touch) and remove a square of adhesive tape from each forepaw, and for performance on a number of neurological tests (e.g., placing and hopping reflexes, activity). Rats with MCA damage showed deficits in both touching and removing the tape from the paw conWalateral to the damage, but not ipsilateral to the damage, while performing within normal limits on the other tests. After scores of the MCA rats dropped into the control group range on the adhesive tape test, they sustained damage to the opposite MCA. This did not reinstate the original deficit, suggesting that the recovery seen after the unilateral lesion was not mediated by the opposite cortex. The second lesion, however, caused a deficit in removing the adhesive tape from the limb opposite the new stroke. Some of the rats that originally had sham operations received bilateral MCA lesions at this time. These animals showed much more severe deficits on the adhesive tape test than the rats with sequential strokes. Rats with bilateral MCA damage (simultaneous or sequential) also slipped on a long narrow plank more often than control animals. Nimodipine did not enhance recovery on any of the behavioral measures. Nimodipine Stroke Middle cerebral artery (MCA) Calcium channel blocker (antagonist) Motor function Brain damage Sensory neglect Motor cortex Somatosensory cortex Placing Hopping Open field
Somesthesis Activity
least transient deficits in detecting and removing the sticky paper from the paw contralateral to the damage (4, 18, 30, 31). This basic finding, which seems analogous to observations made on simultaneous extinction and obscuration with brain-damaged human patients (6), was recently replicated and extended in our laboratory in an experiment in which adhesive tape was put on the forepaws of rats with sensorimotor cortical lesions (3). The present experiment was primarily designed to determine whether performance on this test, which demands high levels of attention and sensorimotor processing and integration, but which can be conducted in a short amount of time, would be disrupted by MCA stroke. A second purpose of the current experiment was to see whether the central calcium channel blocker, nimodipine (Bay e 9736), might decrease the effects of MCA stroke on this test and on a number of more traditional behavioral measures (e.g., activity, paw reflexes). Nimodipine was expected to be effective in reducing the deficit because it has the potential to reduce secondary cell death by blocking excessive influx of calcium into neurons and preventing vasospasm after stroke (3, 14, 19, 22, 32, 34). For example, Sauter and Rudin (28) cauterized the MCA and found that infarct size was smaller in rats treated with nimodipine than in those given placebo. This effect was confinned by Paschen et al. (24) and by G-ermano et al. (15), who
FOR the most part, experimenters studying stroke models in rodents have ligated, cauterized, coagulated or cut middle cerebral arteries (MCA) or common carotid arteries [e.g., (5, 24-27, 36, 37)]. Most studies have used anatomical or physiological measures as dependent measures. Among those studies where behavioral measures have been obtained, experimenters have concentrated upon the general appearance of the animals, activity scores, and simple reflexive responses. There has been a notable paucity of behavioral studies utilizing measures that demand higher levels of sensory-motor integration which show improvement with practice. The relative absence of tests of more complex sensorimotor functions is surprising because damaging the MCA near the rat's rhinal fissure would be expected to affect the homologue of the Rolandic cortex, especially around the facial and forepaw motor and sensory areas, and to yield sensorimotor deficits (20). The need for such behavioral measures is clear if rats are to serve as a model for human stroke effects. Schallert et al. (29) developed a procedure in which sticky paper was put simultaneously on the forepaws of rats with unilateral nigrostriatal damage, and measurements were made of how long it took the animals to touch and remove the paper from each limb. They found deficits contralateral to the damage and, in subsequent studies, it was noted that rats with hemispberectomies or unilateral sensorimotor cortex ablations also showed at IRequests for reprints should be addressed to Dr. Stanley Finger.
263
264
ANDERSEN, ANDERSEN AND FINGER
also reported better neurological outcomes among the nimodipine-treated animals. It should be noted, however, that not all experimenters employing nimodipine in stroke studies have obtained positive findings [e.g., (7, 16, 37)]. A third purpose of the present study was to learn more about the recovery that was expected on the adhesive tape test after unilateral MCA damage [see (3, 30, 31)]. Two questions were of particular interest. One was whether or not the expected diminution of the unilateral deficit would be reversed by a subsequent stroke of the opposite MCA. The other was whether or not simultaneous, bilateral MCA strokes would result in more severe deficits than sequential unilateral strokes [i.e., the "serial lesion effect"; (8, 12, 13)]. METHOD
Subjects The experiments were conducted on 64 female albino rats (Charles River, CD) that were maintained in individual cages on a cycle of 12 h light/12 h dark with free access to food and water. The animals were 80 days of age when they began genfling for three weeks prior to surgery. Prior to the first surgery, 42 rats were randomly assigned to the following four conditions: l) Right MCA/Nimodipine, 2) Right MCA/Vehicle, 3) Sham Operation/Nimodipine, and 4) Sham Operation/Vehicle. At the time of testing, the groups contained 9, 10, 9, and 9 rats, respectively. Twenty-two additional rats were given sham operations followed by behavioral testing (in preparation for bilateral lesions in the second part of the investigation). Eleven of these additional rats received nimodipine and the other 11 received vehicle alone. These animals were not used for the analyses of unilateral lesion effects.
First Surgeries All animals were anesthetized with sodium pentobarbital (50 mg/kg). For animals in the lesion groups, the cranium was opened with a drill and rongeurs to expose the right MCA ventral to its division with anterior and posterior branches. The main stem of the artery was then broken with a pair of free forceps approximately 1 mm above the rhinal fissure. Gelfoam was put on the wound, and the skin was sutured with nylon thread. The sham operation consisted of opening the scalp, scraping the fascia, and suturing the skin.
Drug Administration The rats in the drug groups were intubated with 15 mg/kg nimodipine (in 5% methyl-cellulose) early each day for two weeks, beginning the morning after surgery. Fresh solutions were made just prior to administration. This oral dosage, administered through infant feeding tubes, was found to enhance behavioral recovery in rats with hippocampal lesions (10) and somatosensory cortex ablations (3), and to promote the growth of neural grafts (9). The rats in the vehicle groups were intubated with an equivalent amount of methyl-cellulose without the drug.
Adhesive Tape Task The adhesive tape test, which is a variant of Schallert's sticky paper test, has been described in a recent publication (3). The procedure required one experimenter to lift a rat from its cage
and to hold it steady while a second experimenter attached a piece of 1/2 cm2 Johnson & Johnson's waterproof medical tape to the ventral surface of each forepaw. Both forepaws were rubbed simultaneously, and the animal was put back in its cage. The lid was immediately closed and latencies to touch each square of tape, and to remove it from each paw, were recorded. Testing took place soon after the beginning of the dark cycle, in dim light, on postoperative Days 2, 4, 6, 8, 10, 12, 14, 16 and 18.
Other Behavioral Measures The animals were examined for: (a) hopping and placing reflexes on Day 7; (b) the ability to hang from an 11-mm diameter wood dowel on Days 5, 9, 13, 17; and (c) squares crossed in an open field (5-min sessions) on Days 3, 7, 11, 15 and 19 [see (2,3) for descriptions of the open field apparatus, the tests, and their scoring]. In addition, the rats were tested for the ability to run on a long narrow wooden plank (3 x 100 cm) from a brightly lit starting point to a dimly lit safety platform. Each rat received 3 trials on this "bridge" test on Day 6. Latencies and number of slips were recorded.
Second Surgeries and Drug Administration The rats were subjected to a second surgery 28 days after the first surgery. The 19 right MCA animals (9 nimodipine, 10 vehicle) received a lesion of the left MCA using procedures identical to those described above. The 18 rats in the original sham operation (9 nimodipine, 9 vehicle) groups received another sham operation. The other 22 animals (11 nimodipine, 11 vehicle) that had been given sham operations and that were tested strictly in preparation for this part of the experiment now had their MCAs cut bilaterally in a single operation. No animals changed between drug and vehicle conditions after the second surgery. Those rats that received nimodipine after the first surgery also received the drug (15 mg/kg, 2 weeks) after the second surgery, while the rats that had received only the vehicle continued to be intubated with just methyl-cellulose. Data were obtained on the following animals that survived the second surgery: 2-Stage/Nimodipine (n = 5), 2-Stage/Vehicle (n = 8), 1-Stage/Nimodipine (n = 10), 1-Stage/Vehicle (n = 8), Sharn/Nimodipine (n = 9), and Sham/Vehicle (n = 8).
Behavioral Testing After the Second Surgery The adhesive tape test was conducted on Days 2, 4, 6, 8, 10, 14, 18 and 27 after the second surgery. In addition, hopping and placing reflexes were measured on Day 9; open field activity on Days 3, 7, 11, 15, and 19; hanging onto the dowel on Days 5, 9, 13 and 17; and traversing the narrow bridge on Day 10.
Histology The rats were deeply anesthetized with pentobarbital and perfused through the aorta with 0.9% NaCI followed by a 2% solutino of 2,3,5-triphenyltetrazolium chloride (TTC) upon completion of behavioral testing (15). Photographs were taken of the brains, and drawings were made on graph paper. Squares were counted to estimate the surface area of the lesions. After the brains were allowed to harden in buffered formalin solution, frozen sections were cut at 40 p~m. Every fifth section was saved and stained with cresyl-violet for assessment with a light microscope.
MIDDLE CEREBRAL ARTERY STROKE AND BEHAVIOR
CONTRALATERAL (Left)PAW
--25 ~-~ 20[
,,-, MCA(..19)
. . . . . . . . .
30
25
20
IPSILATERAL fRight) PAW
[
265
~
110[
~ ~
100~ 50
~ linS:l~ Bao~rolMCA o-o
-N
o. . . . . . . . . .
30
...,~" 25
25
~.
0031-9384491 $3.00 + .00
Neurological Correlates of Unilateral and Bilateral "Strokes" of the Middle Cerebral Artery in the Rat C H E R Y L S. A N D E R S E N , A N D E R S B. A N D E R S E N A N D S T A N L E Y F I N G E R 1
Department o f Psychology, Washington University, St. Louis, M O 63130 R e c e i v e d 18 January 1991 ANDERSEN, C. S., A. B. ANDERSEN AND S. FINGER. Neurological correlates of unilateral and bilateral "strokes" of the middle cerebral artery in the rat. PHYSIOL BEHAV 50(2) 263-269, 1991.--Rats with unilateral lesions of the middle cerebral artery (MCA) were tested for the ability to detect (touch) and remove a square of adhesive tape from each forepaw, and for performance on a number of neurological tests (e.g., placing and hopping reflexes, activity). Rats with MCA damage showed deficits in both touching and removing the tape from the paw conWalateral to the damage, but not ipsilateral to the damage, while performing within normal limits on the other tests. After scores of the MCA rats dropped into the control group range on the adhesive tape test, they sustained damage to the opposite MCA. This did not reinstate the original deficit, suggesting that the recovery seen after the unilateral lesion was not mediated by the opposite cortex. The second lesion, however, caused a deficit in removing the adhesive tape from the limb opposite the new stroke. Some of the rats that originally had sham operations received bilateral MCA lesions at this time. These animals showed much more severe deficits on the adhesive tape test than the rats with sequential strokes. Rats with bilateral MCA damage (simultaneous or sequential) also slipped on a long narrow plank more often than control animals. Nimodipine did not enhance recovery on any of the behavioral measures. Nimodipine Stroke Middle cerebral artery (MCA) Calcium channel blocker (antagonist) Motor function Brain damage Sensory neglect Motor cortex Somatosensory cortex Placing Hopping Open field
Somesthesis Activity
least transient deficits in detecting and removing the sticky paper from the paw contralateral to the damage (4, 18, 30, 31). This basic finding, which seems analogous to observations made on simultaneous extinction and obscuration with brain-damaged human patients (6), was recently replicated and extended in our laboratory in an experiment in which adhesive tape was put on the forepaws of rats with sensorimotor cortical lesions (3). The present experiment was primarily designed to determine whether performance on this test, which demands high levels of attention and sensorimotor processing and integration, but which can be conducted in a short amount of time, would be disrupted by MCA stroke. A second purpose of the current experiment was to see whether the central calcium channel blocker, nimodipine (Bay e 9736), might decrease the effects of MCA stroke on this test and on a number of more traditional behavioral measures (e.g., activity, paw reflexes). Nimodipine was expected to be effective in reducing the deficit because it has the potential to reduce secondary cell death by blocking excessive influx of calcium into neurons and preventing vasospasm after stroke (3, 14, 19, 22, 32, 34). For example, Sauter and Rudin (28) cauterized the MCA and found that infarct size was smaller in rats treated with nimodipine than in those given placebo. This effect was confinned by Paschen et al. (24) and by G-ermano et al. (15), who
FOR the most part, experimenters studying stroke models in rodents have ligated, cauterized, coagulated or cut middle cerebral arteries (MCA) or common carotid arteries [e.g., (5, 24-27, 36, 37)]. Most studies have used anatomical or physiological measures as dependent measures. Among those studies where behavioral measures have been obtained, experimenters have concentrated upon the general appearance of the animals, activity scores, and simple reflexive responses. There has been a notable paucity of behavioral studies utilizing measures that demand higher levels of sensory-motor integration which show improvement with practice. The relative absence of tests of more complex sensorimotor functions is surprising because damaging the MCA near the rat's rhinal fissure would be expected to affect the homologue of the Rolandic cortex, especially around the facial and forepaw motor and sensory areas, and to yield sensorimotor deficits (20). The need for such behavioral measures is clear if rats are to serve as a model for human stroke effects. Schallert et al. (29) developed a procedure in which sticky paper was put simultaneously on the forepaws of rats with unilateral nigrostriatal damage, and measurements were made of how long it took the animals to touch and remove the paper from each limb. They found deficits contralateral to the damage and, in subsequent studies, it was noted that rats with hemispberectomies or unilateral sensorimotor cortex ablations also showed at IRequests for reprints should be addressed to Dr. Stanley Finger.
263
264
ANDERSEN, ANDERSEN AND FINGER
also reported better neurological outcomes among the nimodipine-treated animals. It should be noted, however, that not all experimenters employing nimodipine in stroke studies have obtained positive findings [e.g., (7, 16, 37)]. A third purpose of the present study was to learn more about the recovery that was expected on the adhesive tape test after unilateral MCA damage [see (3, 30, 31)]. Two questions were of particular interest. One was whether or not the expected diminution of the unilateral deficit would be reversed by a subsequent stroke of the opposite MCA. The other was whether or not simultaneous, bilateral MCA strokes would result in more severe deficits than sequential unilateral strokes [i.e., the "serial lesion effect"; (8, 12, 13)]. METHOD
Subjects The experiments were conducted on 64 female albino rats (Charles River, CD) that were maintained in individual cages on a cycle of 12 h light/12 h dark with free access to food and water. The animals were 80 days of age when they began genfling for three weeks prior to surgery. Prior to the first surgery, 42 rats were randomly assigned to the following four conditions: l) Right MCA/Nimodipine, 2) Right MCA/Vehicle, 3) Sham Operation/Nimodipine, and 4) Sham Operation/Vehicle. At the time of testing, the groups contained 9, 10, 9, and 9 rats, respectively. Twenty-two additional rats were given sham operations followed by behavioral testing (in preparation for bilateral lesions in the second part of the investigation). Eleven of these additional rats received nimodipine and the other 11 received vehicle alone. These animals were not used for the analyses of unilateral lesion effects.
First Surgeries All animals were anesthetized with sodium pentobarbital (50 mg/kg). For animals in the lesion groups, the cranium was opened with a drill and rongeurs to expose the right MCA ventral to its division with anterior and posterior branches. The main stem of the artery was then broken with a pair of free forceps approximately 1 mm above the rhinal fissure. Gelfoam was put on the wound, and the skin was sutured with nylon thread. The sham operation consisted of opening the scalp, scraping the fascia, and suturing the skin.
Drug Administration The rats in the drug groups were intubated with 15 mg/kg nimodipine (in 5% methyl-cellulose) early each day for two weeks, beginning the morning after surgery. Fresh solutions were made just prior to administration. This oral dosage, administered through infant feeding tubes, was found to enhance behavioral recovery in rats with hippocampal lesions (10) and somatosensory cortex ablations (3), and to promote the growth of neural grafts (9). The rats in the vehicle groups were intubated with an equivalent amount of methyl-cellulose without the drug.
Adhesive Tape Task The adhesive tape test, which is a variant of Schallert's sticky paper test, has been described in a recent publication (3). The procedure required one experimenter to lift a rat from its cage
and to hold it steady while a second experimenter attached a piece of 1/2 cm2 Johnson & Johnson's waterproof medical tape to the ventral surface of each forepaw. Both forepaws were rubbed simultaneously, and the animal was put back in its cage. The lid was immediately closed and latencies to touch each square of tape, and to remove it from each paw, were recorded. Testing took place soon after the beginning of the dark cycle, in dim light, on postoperative Days 2, 4, 6, 8, 10, 12, 14, 16 and 18.
Other Behavioral Measures The animals were examined for: (a) hopping and placing reflexes on Day 7; (b) the ability to hang from an 11-mm diameter wood dowel on Days 5, 9, 13, 17; and (c) squares crossed in an open field (5-min sessions) on Days 3, 7, 11, 15 and 19 [see (2,3) for descriptions of the open field apparatus, the tests, and their scoring]. In addition, the rats were tested for the ability to run on a long narrow wooden plank (3 x 100 cm) from a brightly lit starting point to a dimly lit safety platform. Each rat received 3 trials on this "bridge" test on Day 6. Latencies and number of slips were recorded.
Second Surgeries and Drug Administration The rats were subjected to a second surgery 28 days after the first surgery. The 19 right MCA animals (9 nimodipine, 10 vehicle) received a lesion of the left MCA using procedures identical to those described above. The 18 rats in the original sham operation (9 nimodipine, 9 vehicle) groups received another sham operation. The other 22 animals (11 nimodipine, 11 vehicle) that had been given sham operations and that were tested strictly in preparation for this part of the experiment now had their MCAs cut bilaterally in a single operation. No animals changed between drug and vehicle conditions after the second surgery. Those rats that received nimodipine after the first surgery also received the drug (15 mg/kg, 2 weeks) after the second surgery, while the rats that had received only the vehicle continued to be intubated with just methyl-cellulose. Data were obtained on the following animals that survived the second surgery: 2-Stage/Nimodipine (n = 5), 2-Stage/Vehicle (n = 8), 1-Stage/Nimodipine (n = 10), 1-Stage/Vehicle (n = 8), Sharn/Nimodipine (n = 9), and Sham/Vehicle (n = 8).
Behavioral Testing After the Second Surgery The adhesive tape test was conducted on Days 2, 4, 6, 8, 10, 14, 18 and 27 after the second surgery. In addition, hopping and placing reflexes were measured on Day 9; open field activity on Days 3, 7, 11, 15, and 19; hanging onto the dowel on Days 5, 9, 13 and 17; and traversing the narrow bridge on Day 10.
Histology The rats were deeply anesthetized with pentobarbital and perfused through the aorta with 0.9% NaCI followed by a 2% solutino of 2,3,5-triphenyltetrazolium chloride (TTC) upon completion of behavioral testing (15). Photographs were taken of the brains, and drawings were made on graph paper. Squares were counted to estimate the surface area of the lesions. After the brains were allowed to harden in buffered formalin solution, frozen sections were cut at 40 p~m. Every fifth section was saved and stained with cresyl-violet for assessment with a light microscope.
MIDDLE CEREBRAL ARTERY STROKE AND BEHAVIOR
CONTRALATERAL (Left)PAW
--25 ~-~ 20[
,,-, MCA(..19)
. . . . . . . . .
30
25
20
IPSILATERAL fRight) PAW
[
265
~
110[
~ ~
100~ 50
~ linS:l~ Bao~rolMCA o-o
-N
o. . . . . . . . . .
30
...,~" 25
25
~.
