Brain Research, 508 (1990) 305-309 Elsevier
305
BRES 23940
Clonidine impairs recovery of beam-walking after a sensorimotor cortex lesion in the rat L a r r y B. G o l d s t e i n I a n d J a m e s N. D a v i s 2 1'2Neurology Research Laboratory, Durham Department of Veterans' Affairs Medical Center and Departments of 1'2Medicine (Neurology), 2pharmacology, and 2Neurobiology, Duke University, Durham, N, C. (U.S.A.) (Accepted 17 October 1989)
Key words: Motor recovery; Clonidine; Beam-walking; Brain injury; Stroke; Cortical lesion; Rats
Beam-walking in the rat is a useful model for studying the effects of drugs on motor recovery following brain injury. In the present experiment, the effect of clonidine HCI on beam-walking recovery was investigated. Groups of rats were first trained to traverse a narrow elevated beam and then subjected to a right sensorimotor cortex suction-ablation injury. After 24 h, each rat received a single dose of clonidine HC1 (20, 60, or 200/~g/kg, i.p., salt weight) or saline. Recovery of beam-walking ability was scored over the next 12 days. Treatment with clonidine significantly slowed the rate of recovery (Kruskal-Wallis H = 8.755, df = 3; 0,02 < P < 0.05). Furthermore, the impairment persisted for at least 5 days after the rats were treated (Kruskal-Wallis H = 8.47, df = 3; 0.02 < P < 0.05). These data are consistent with the hypothesis that norepinephrine, working through central az-adrenergic receptors, influences motor recovery after a unilateral sensorimotor cortex lesion in the rat. Since many stroke patients are treated with centrally acting antihypertensive drugs, the potential effects of specific classes of these drugs during the recovery period, should be carefully considered. R e c e n t investigations suggest that a variety of drugs m a y influence functional recovery following brain injury 13. While some drugs may interfere with the r e c o v e r y process, o t h e r drugs may improve functional outcome. F o r e x a m p l e , d i a z e p a m impedes recovery from the sensory a s y m m e t r y caused by unilateral anteriorm e d i a l n e o c o r t e x d a m a g e in the rat 29. Systemic administration of the anticonvuisant drug, phenytoin, increases the h e m i p l e g i a resulting from intracortical infusion of ~-aminobutyric acid 4, A m p h e t a m i n e is perhaps the most extensively studied drug with the capacity to enhance recovery following brain injury. T r e a t m e n t with Da m p h e t a m i n e transiently restores placing responses in n e o d e c o r t i c a t e cats H'25"27, facilitates the reestablishment of visual discrimination p e r f o r m a n c e in visually decorticate rats 5, reinstates binocular depth perception following bilateral visual cortex lesions in cats 12'21'22, reduces spatial learning deficits resulting from bilateral striatal lesions in rats s, and enhances recovery from the sensory deficit after infarction of the primary s o m a t o s e n s o r y cortex in rats 23. Beam-walking has been used to investigate the effects of a m p h e t a m i n e on m o t o r recovery following brain injury. A unilateral lesion of the sensor i m o t o r cortex in the rat results in an i m p a i r m e n t in the animal's ability to traverse a narrow elevated b e a m 6,26. A single dose of ~ - a m p h e t a m i n e a d m i n i s t e r e d 24 h after
cortex injury increases recovery9,1o,16,18,19.
the
rate
of
beam-walking
A m p h e t a m i n e may exert its influence on m o t o r recovery through its effects on central noradrenergic, dopaminergic, or serotonergic neurons as. We hypothesize that a m p h e t a m i n e ' s facilitatory effect on m o t o r recovery is m e d i a t e d , at least in part, through central n o r a d r e n e r g i c neurons since p r e l i m i n a r y data suggest that centrallyacting ct2-adrenergic r e c e p t o r antagonists also enhance b e a m - w a l k i n g recovery t6'2°'31'33 and a centrally-acting az-adrenergic r e c e p t o r agonist reinstates the b e a m walking deficit in recovered rats 3°'31'33. In o r d e r to further test this hypothesis, the effect on m o t o r recovery of the a2-adrenergic r e c e p t o r agonist, clonidine HCI, was investigated. Specifically, it was h y p o t h e s i z e d that clonidine would impair b e a m - w a l k i n g recovery in rat following a unilateral s e n s o r i m o t o r cortex injury. Male S p r a g u e - D a w l e y rats (n = 120) weighing 250300 g o b t a i n e d from Charles River B r e e d i n g L a b o r a t o ries, Inc. (Raleigh, N.C.) were used in these experiments. A n i m a l s were housed in a vivarium with a 12 h l i g h t - d a r k cycle and controlled t e m p e r a t u r e and humidity. F o o d (Purina Rat Chow) and water were p r o v i d e d ad libitum. The behavioral testing a p p a r a t u s , behavioral protocols, and surgical p r o c e d u r e s were previously described 1~'19. Briefly, the b e h a v i o r a l testing apparatus
Correspondence: L.B. Goldstein, Building 16, Room 28, Durham Department of Veteran's Affairs Medical Center, Durham, N.C. 27705, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
306 consisted of a goal box located at one end of a 2.5 × 122 cm elevated wooden beam. A switch-activated source of bright light and white noise were located at the start-end of the beam and served as avoidance/activating stimuli. M o t o r performance was rated on a 7 point scale as previously described a°. For each training or testing trial,
as necessary)• The rats underwent craniotomy extending from 2 mm rostral to 2 mm caudal to the coronal suture and from 1 m m lateral of the sagittal suture to the temporal ridge. The cortex underlying the craniotomy site was removed by gentle suction through a fine glass
the rat was placed at the start-end of the beam opposite
Pasteur pipet until the underlying white matter was visualized. The skin incision was closed with surgical
to the goal box. If the animal did not begin to traverse the beam after 10 s, the light and noise stimuli were
staples and the animal returned to a holding cage to recover from the procedure. Following recovery from
activated and continued until the rat's nose entered the goal box or for a total of 80 s at which time the trial was
anesthesia, the rats were returned to their home cages. The experiment was replicated 5 times (n = 120).
terminated. In each replication of the experiment, groups of 24 rats were first trained on the beam-walking task. O n the first
A baseline measure of each rat's motor ability following cortex injury was provided by a single trial on the beam performed before the drug or saline-vehicle ad-
day of training, each rat was given a series of 3
ministration the day following surgery. Only rats which were unable to traverse the beam (score o f ' l ' or '2') were
approximate trials. Subsequent training consisted of one trial o n the beam each day until the rat achieved a score of '7' on two successive days. Following training, surgical procedures were carried out on groups of rats u n d e r complete pentobarbital anesthesia (50 mg/kg; additional doses were administered
7
used in the experiment. The rats were then randomly assigned to receive a single dose of either clonidine HCI (20, 60, or 200 ~g/kg) or saline• Clonidine HC1 (Sigma) was dissolved in saline (0.9% w/v NaC1) and administered by i.p. injection at the indicated doses (salt weight).
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Fig. 1. Beam-walking recovery in rats given a single dose of: A, Saline (n = 26); B, Clonidine, 20/~g/kg (n = 22); C, Clonidine, 60/~g/kg (n = 24); or D, Clonid;ne 200 ~g/kg (n = 18). A single dose of clonidine or saline vehicle was administered 24 h after a right sensorimotor cortex suction-ablation injury (arrows). The mean (+ S.E.M.) beam-walking scores are given for each trial. Motor performance was rated on a 7 point scale as previously described1°: 1, the rat is unable to place the affected hindpaw on the horizontal surface of the beam; 2, the rat places the affected hindpaw on the horizontal surface of the beam and maintains balance for at least 5 s; 3, the rat traverses the beam while dragging the affected hindpaw; 4, the rat traverses the beam and at least once places the affected hindpaw on the horizontal surface of the beam; 5, the rat crosses the beam and places the affected hindlimb on the horizontal surface of the beam to aid less than half its steps; 6, the rat uses the affected hindpaw to aid more than half its steps, and 7, the rat traverses the beam with no more than two footslips.
307 C o n t r o l rats received the saline vehicle. Each rat was then tested at 1-h intervals beginning 1 h after drug administration for 6 h ( ' H o u r s 2 - 6 ' ) . The rats were subsequently tested over a p e r i o d of 12 days ( ' D a y s 1-12'). In addition to b e a m - w a l k i n g ratings, each rat was also r a t e d on a qualitative 4 point scale for evidence of sedation: 0, none; 1, mild - - arouses with minimal tactile stimuli; 2, m o d e r a t e - - difficult to arouse; 3, unarousable. A l l behavioral m e a s u r e m e n t s were r e c o r d e d by an o b s e r v e r blind to the rats' t r e a t m e n t status. Following the behavioral m e a s u r e m e n t s , rats were sacrificed by an o v e r d o s e of p e n t o b a r b i t a l and the brains were dissected and fixed by immersion in 4% formalin. A f t e r fixation, a v i d e o c a m e r a - g e n e r a t e d image of the dorsal aspect of each brain was digitized and lesion sizes (areas) and m a x i m u m medial extents (closest approxim a t i o n of the lesion to the inter-hemispheric fissure) were m e a s u r e d with an image analysis system (Jandel V i d e o Analysis Software). The brains of rats from one replication of the e x p e r i m e n t (19 of 90 brains) were not analyzed because of a tissue handling accident. In o r d e r to facilitate comparison with previous work, m e a n ( + S . E . M . ) b e a m - w a l k i n g scores are given in the figures. H o w e v e r , since an ordinal scale was e m p l o y e d to rate b e a m - w a l k i n g p e r f o r m a n c e , the data were analyzed with n o n - p a r a m e t r i c methods. Performances on the ' p r e - d r u g ' trial (to d e t e r m i n e whether c o m p a r a b l e postsurgical deficits occurred across groups) was analyzed with the K r u s k a l - W a l l i s test. In o r d e r to c o m p a r e the rates of i m p r o v e m e n t of beam-walking p e r f o r m a n c e
among d r u g - t r e a t m e n t groups, the Trapzoidal rule l was used to calculate the areas u n d e r the curves f o r m e d when each rat's scores were p l o t t e d against trial n u m b e r (i.e. area u n d e r the t i m e - e f f e c t curve). T h e resulting data was analyzed with the K r u s k a l - W a l l i s test. T h e Dunn Procedure was subsequently applied to the ranks of the data in o r d e r to d e t e r m i n e the significance of differences b e t w e e n groups 2s. Lesion size and medial extent were analyzed with A N O V A . All tests of significance are two-tailed. Fifteen of 120 rats did not reach training criteria, 2 rats died i m m e d i a t e l y following surgery, and 13 rats had baseline postsurgical b e a m - w a l k i n g scores > 2 indicating a less severe m o t o r deficit. The remaining rats (n = 90) served as subjects for the e x p e r i m e n t . The b e a m - w a l k i n g recoveries in rats given a single dose of saline (n = 26) or clonidine (20 ~tg/kg, n = 22; 60 Bg/kg, n = 24; 200 /~g/kg, n = 18) are given in Fig. 1. T h e r e were no differences a m o n g the groups prior to the administration of drug/placebo ( ' P r e d r u g ' trial; K r u s k a l - W a l l i s H = 0.87, df = 3; P > 0.20). Fig. 2 gives the overall rates of recovery of saline- and c l o n i d i n e - t r e a t e d rats. Rats which had received clonidine had slower recoveries as comp a r e d to the saline-treated controls ( K r u s k a l - W a l l i s H -= 8.755, df = 3; 0.02 < P < 0.05; Dunn P r o c e d u r e P < 0.05 for each clonidine dose c o m p a r e d with saline). There were no differences in recovery rates among the 3 groups of clonidine-treated rats (Dunn P r o c e d u r e , P > 0.05 for each comparison). I m p a i r m e n t by clonidine was present for all doses 5 days after the administration of the single dose of the drug (Fig. 1 ; K r u s k a l - W a l l i s H = 8.47, df = 3; 0.02 < P < 0,05; D u n n P r o c e d u r e P < 0.05 for each
65
30
A,
20
g,) (0
OE
B.
45
I Saline
i 20pg/kg
i i 60pg/kg 200tJg/kg
Clonidine Dose
Fig. 2. The relationship between a single dose of saline or clonidine administered 24 h after sensorimotor cortex suction-ablation injury and the rate of recovery of beam-walking over a 12-day period. The mean (+ S.E.M.) recovery rates are given.
Saline
20pg/kg.
60lJg/kg
200JJg/k 9
Clonidine Dose
Fig. 3. The mean (+_ S.E.M.) lesion sizes (A) and maximum medial lesion extents (B) for the brains of rats in each of the 4 treatment groups.
308 clonidine dose c o m p a r e d with saline). By day 6, the difference in beam-walking scores among saline- and c l o n i d i n e - t r e a t e d rats was no longer significant ( K r u s k a l Wallis H = 7.339, df = 3; 0.05 < P < 0.10). N o n e of the rats showed any signs of sedation following the administration of clonidine/saline. There were no differences a m o n g the replications of the experiment with respect to ' P r e d r u g ' b e a m - w a l k i n g scores or rates of beam-walking recovery. T h e r e were no significant differences in mean lesion size (Fig. 3; A N O V A , F3,67 = 0 . 7 8 5 ; P = 0 . 5 5 ) o r in m e a n m e d i a l lesion extent ( A N O V A , F3,~7 = 1.01; P = 0.39) a m o n g the 4 t r e a t m e n t groups, The central finding of the present investigation is that a single dose of the a2-adrenergic r e c e p t o r agonist clonidine HCI given 24 h after a unilateral s e n s o r i m o t o r cortex lesion results in a significant and p r o l o n g e d i m p a i r m e n t of m o t o r recovery in rats. This effect of clonidine occurred with doses as low as 20/~g/kg. These studies are part of a series of experiments designed to further elucidate the p h a r m a c o l o g y of amp h e t a m i n e - f a c i l i t a t e d recovery of beam-walking. Preliminary evidence suggests that the a m p h e t a m i n e effect is m e d i a t e d t h r o u g h central n o r a d r e n e r g i c neurons. A n intraventricular infusion of n o r e p i n e p h r i n e but not dopamine mimics a m p h e t a m i n e - f a c i l i t a t e d recovery of beam-
walking in rats 2. Infusion of n o r e p i n e p h r i n e into the cerebellum contralateral to a s e n s o r i m o t o r cortex lesion also accelerates m o t o r recovery in rats 3. Lesions of the locus coeruleus m a y influence b e a m - w a l k i n g recovery 3' 14.24,32 Treatment with centrally-acting a~-adrenergic r e c e p t o r antagonists interfere with m o t o r recovery 3~'33. The present results are also consistent with o t h e r preliminary reports which suggest that ae-adrenergic receptor antagonists may enhance b e a m - w a l k i n g recovery 16' 20,31.33 while t r e a t m e n t with clonidine may reinstate beam-walking deficits in recovered rats 3°'3t'33
1 Beyer, W.H,, CRC Standard Mathematical Tables, 26th edn., CRC Press, Boca Raton, FL, 1981, p. 125. 2 Boyeson, M.G. and Feeney, D.M., The role of norepinephrine in recovery from brain injury, Soc. Neurosci. Abstr., 10 (1984) 68. 3 Boyeson, M.G., Krobert, K.A. and Hughes, J.M., Norepinephfine infusions into cerebellum facilitate recovery from sensorimotor cortex injury in the rat, Soc. Neurosci. Abstr., 12 (1986) 1120. 4 Brailowsky, S., Knight, R.T. and Efron, R., Phenytoin increases the severity of cortical hemiplegia in rats, Brain Research, 376 (1986) 71-77. 5 Braun, J.J., Meyer, P.M. Meyer, D.R., Sparing of a brightness habit in rats following visual decortication, J. Cornp. Physiol. Psychol., 61 (1986) 79-82. 6 Buytendijk, F.J.J., An experimental investigation into the influence of cortical lesions on the behaviour of rats, Arch, Neerl. Physiol. L'Homme Anita., 17 (1932)370-434. 7 Crisostomo, E.A., Duncan, P.W., Propst, M.A., Dawson, D.B. and Davis, J.N., Evidence that amphetamine with physical therapy promotes recovery of motor function in stroke patients, Ann~ NeuroL, 23 (1988) 94-97. 8 Dunbar, G.L., Hecht, S.A., Merbaum, S.L., DeAngelis, M.M. and Stein, D.G., Use of gangliosides and amphetamines to promote behavioral recovery following bilateral caudate nucleus lesions. In R.L. Masland, A. Portera-Sanchez and G. Toffano (Eds.), Neuroplasticity: A New Therapeutic Tool in the CNS, Liviana Press, Padova, 1987, pp. 117-124. 9 Feeney, D.M., Gonzalez, A. and Law, W.A., Amphetamine restores locomotor function after motor cortex injury in the rat, Proc. West. Pharmacol. Soc., 24 (1981) 15-17. 10 Feeney, D.M., Gonzalez, A. and Law, W.A., Amphetamine haloperidol and experience interact to affect the rate of recovery after motor cortex injury, Science. 217 (1982) 855-857.
11 Feeney, D.M. and Hovda, D.A., Amphetamine restores tactile placing after motor cortex lesions, Fed. Proc. 39 (t980) 1095. 12 Feeney, D.M. and Hovda, D.A., Reinstatement of binocular depth perception by amphetamine and visual experience after visual cortex ablation, Brain Research, 342 (t985) 352-356. 13 Feeney, D.M. and Sutton, R.L., Pharmacotherapy for recovery of function after brain injury, CRC Crit. Rev. Neurobiol., 3 (1987) 135-197. 14 Feeney, D.M., Sutton, R.L., Boyeson, M.G., Hovda, D.A. and Dail, W.G., The locus-coeruleus and cerebral metabolism: recovery of function after cortical injury, Physiol. PsychoL, 13 (1985) 197-203. 15 Fuxe, K. and Ungerstedt, U., Histochemical, biochemical and functional studies on central monoamine neurons after acute and chronic amphetamine administration. In E. Costa and S. Garattini (Eds.), Amphetamines and Related Compounds, Raven, New York, 1970, pp. 257-288. 16 Goldstein, L.B., Amphetamine-facilitated functional recovery after stroke. In M.D. Ginsberg and W.D. Dietrich (Eds.), The
Beam-walking in the rat has b e e n used as an animal model which m a y he predictive o f the effects of drugs on m o t o r recovery in humans following stroke 7. Based on these studies, a clinical trial was p e r f o r m e d which found that t r e a t m e n t with a m p h e t a m i n e facilitated short-term m o t o r recovery in a small group of highly-selected stroke patients 17. Clonidine is frequently prescribed as an antihypertensive in this group of patients. If the present results are also relevant to humans, then clonidine should be avoided during the recovery p e r i o d after stroke.
The authors are indebted to Ms. Holli Poe for her excellent technical assistance. This work was supported in part by the NIH (NS 06233 and NS 01162) and by the Department of Veterans Affairs.
16th Princeton Conference on Cerebral Vascular Diseases,
17 18 19 20
21
Raven, New York, 1988, pp. 303-308. Goldstein, L.B. and Davis, J.N., Physician prescribing patterns after ischemic stroke, Neurology, 38 (1988) 1806-1809. Goldstein, L.B. and Davis, J.N., The influence of lesion size and location on amphetamine-facilitated recovery of beam-walking in rats, Behav. Neurosci., in press. Goldstein, L.B. and Davis, J.N., Beam-walking in rats: studies towards developing an animal model of functional recovery after brain injury, J. Neurosci. Methods, in press. Goldstein, L.B., Poe, H.V. and Davis, J.N., An animal model of recovery of function after stroke: facilitation of recovery by an alpha2-adrenergic receptor antagonist. Ann. NeuroL, 26 (1989) 157. Hovda, D.A. and Feeney, D.M., Haloperidol blocks amphet-
309
22
23
24
25
26 27
amine induced recovery of binocular depth perception after bilateral visual cortex ablation in the cat, Proc. West. Pharmacol. Soc., 28 (1985)209-211. Hovda, D.A., Sutton, R.L. and Feeney, D.M., Amphetamineinduced recovery of visual cliff performance after bilateral visual cortex ablation in cats: measurements of depth perception thresholds, Behav. Neurosci., 103 (1989) 574-584. Hurwitz, B.E., Dietrich, W.D., McCabe, P.M., Watson, B.D., Ginsberg, M.D. and Schneiderman, N., Amphetamine enhances behavioral recovery from sensory-motor deficit resulting from infarction of primary somatosensory cortex, Soc. Neurosci. Abstr., 14 (1988) 1132. Krobert, K.A., Boyeson, M.G. and Scherer, P.J., The role of the locus ceruleus in recovery of function from sensorimotor cortex injury, Soc. Neurosci. Abstr., 13 (1987) 1664. Macht, M.B., Effects of D-amphetamine on hemi-decorticate, decorticate, and decerebrate cats, Am. J. Physiol., 163 (1950) 731-732. Maier, N.R.F., The cortical area concerned with coordinated walking in the rat., J. Comp. Neurol., 61 (1935) 395-405. Meyer, P.M., Horel, J.A. and Meyer, D.R., Effects of DLamphetamine upon placing responses in neocorticate cats, J.
Comp. Physiol. Psychol., 56 (1963) 402-404. 28 Rosner, B., Fundamentals of Biostatistics, 512th edn., Duxburry Press, Boston, 1986, pp. 470-471. 29 Schallert, T., Hernandez, T.D. and Barth, T.M., Recovery of function after brain damage: severe and chronic disruption by diazepam, Brain Research, 379 (1986) 104-1ll. 30 Stephens, J., Goldberg, G. and Demopoulos, J.T., Clonidine reinstates deficits following recovery from sensorimotor cortex lesion in rats, Arch. Phys. Med. Rehabil., 67 (1986) 666-667. 31 Sutton, R.L. and Feeney, D.M., Yohimbine accelerates recovery and clonidine and prazosin reinstate deficits after recovery in rats with sensorimotor cortex ablation, Soc. Neurosci. Abstr., 13 (1987) 913. 32 Weaver, M.S,, Chen, M.J., Westerberg, V.S. and Feeney, D.M., Locus coeruleus lesions facilitate recovery of locomotor function after sensorimotor cortex contusion in the rat, Soc. Neurosci. Abstr., 14 (1988) 405. 33 Weaver, M.S., Farmer, L.J. and Feeney, D.M., Norepinephrine agonists and antagonists influence rate and maintenance of recovery of function after sensorimotor cortex contusion in the rat, Soc. Neurosci. Abstr., 13 (1987) 477.
305
BRES 23940
Clonidine impairs recovery of beam-walking after a sensorimotor cortex lesion in the rat L a r r y B. G o l d s t e i n I a n d J a m e s N. D a v i s 2 1'2Neurology Research Laboratory, Durham Department of Veterans' Affairs Medical Center and Departments of 1'2Medicine (Neurology), 2pharmacology, and 2Neurobiology, Duke University, Durham, N, C. (U.S.A.) (Accepted 17 October 1989)
Key words: Motor recovery; Clonidine; Beam-walking; Brain injury; Stroke; Cortical lesion; Rats
Beam-walking in the rat is a useful model for studying the effects of drugs on motor recovery following brain injury. In the present experiment, the effect of clonidine HCI on beam-walking recovery was investigated. Groups of rats were first trained to traverse a narrow elevated beam and then subjected to a right sensorimotor cortex suction-ablation injury. After 24 h, each rat received a single dose of clonidine HC1 (20, 60, or 200/~g/kg, i.p., salt weight) or saline. Recovery of beam-walking ability was scored over the next 12 days. Treatment with clonidine significantly slowed the rate of recovery (Kruskal-Wallis H = 8.755, df = 3; 0,02 < P < 0.05). Furthermore, the impairment persisted for at least 5 days after the rats were treated (Kruskal-Wallis H = 8.47, df = 3; 0.02 < P < 0.05). These data are consistent with the hypothesis that norepinephrine, working through central az-adrenergic receptors, influences motor recovery after a unilateral sensorimotor cortex lesion in the rat. Since many stroke patients are treated with centrally acting antihypertensive drugs, the potential effects of specific classes of these drugs during the recovery period, should be carefully considered. R e c e n t investigations suggest that a variety of drugs m a y influence functional recovery following brain injury 13. While some drugs may interfere with the r e c o v e r y process, o t h e r drugs may improve functional outcome. F o r e x a m p l e , d i a z e p a m impedes recovery from the sensory a s y m m e t r y caused by unilateral anteriorm e d i a l n e o c o r t e x d a m a g e in the rat 29. Systemic administration of the anticonvuisant drug, phenytoin, increases the h e m i p l e g i a resulting from intracortical infusion of ~-aminobutyric acid 4, A m p h e t a m i n e is perhaps the most extensively studied drug with the capacity to enhance recovery following brain injury. T r e a t m e n t with Da m p h e t a m i n e transiently restores placing responses in n e o d e c o r t i c a t e cats H'25"27, facilitates the reestablishment of visual discrimination p e r f o r m a n c e in visually decorticate rats 5, reinstates binocular depth perception following bilateral visual cortex lesions in cats 12'21'22, reduces spatial learning deficits resulting from bilateral striatal lesions in rats s, and enhances recovery from the sensory deficit after infarction of the primary s o m a t o s e n s o r y cortex in rats 23. Beam-walking has been used to investigate the effects of a m p h e t a m i n e on m o t o r recovery following brain injury. A unilateral lesion of the sensor i m o t o r cortex in the rat results in an i m p a i r m e n t in the animal's ability to traverse a narrow elevated b e a m 6,26. A single dose of ~ - a m p h e t a m i n e a d m i n i s t e r e d 24 h after
cortex injury increases recovery9,1o,16,18,19.
the
rate
of
beam-walking
A m p h e t a m i n e may exert its influence on m o t o r recovery through its effects on central noradrenergic, dopaminergic, or serotonergic neurons as. We hypothesize that a m p h e t a m i n e ' s facilitatory effect on m o t o r recovery is m e d i a t e d , at least in part, through central n o r a d r e n e r g i c neurons since p r e l i m i n a r y data suggest that centrallyacting ct2-adrenergic r e c e p t o r antagonists also enhance b e a m - w a l k i n g recovery t6'2°'31'33 and a centrally-acting az-adrenergic r e c e p t o r agonist reinstates the b e a m walking deficit in recovered rats 3°'31'33. In o r d e r to further test this hypothesis, the effect on m o t o r recovery of the a2-adrenergic r e c e p t o r agonist, clonidine HCI, was investigated. Specifically, it was h y p o t h e s i z e d that clonidine would impair b e a m - w a l k i n g recovery in rat following a unilateral s e n s o r i m o t o r cortex injury. Male S p r a g u e - D a w l e y rats (n = 120) weighing 250300 g o b t a i n e d from Charles River B r e e d i n g L a b o r a t o ries, Inc. (Raleigh, N.C.) were used in these experiments. A n i m a l s were housed in a vivarium with a 12 h l i g h t - d a r k cycle and controlled t e m p e r a t u r e and humidity. F o o d (Purina Rat Chow) and water were p r o v i d e d ad libitum. The behavioral testing a p p a r a t u s , behavioral protocols, and surgical p r o c e d u r e s were previously described 1~'19. Briefly, the b e h a v i o r a l testing apparatus
Correspondence: L.B. Goldstein, Building 16, Room 28, Durham Department of Veteran's Affairs Medical Center, Durham, N.C. 27705, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
306 consisted of a goal box located at one end of a 2.5 × 122 cm elevated wooden beam. A switch-activated source of bright light and white noise were located at the start-end of the beam and served as avoidance/activating stimuli. M o t o r performance was rated on a 7 point scale as previously described a°. For each training or testing trial,
as necessary)• The rats underwent craniotomy extending from 2 mm rostral to 2 mm caudal to the coronal suture and from 1 m m lateral of the sagittal suture to the temporal ridge. The cortex underlying the craniotomy site was removed by gentle suction through a fine glass
the rat was placed at the start-end of the beam opposite
Pasteur pipet until the underlying white matter was visualized. The skin incision was closed with surgical
to the goal box. If the animal did not begin to traverse the beam after 10 s, the light and noise stimuli were
staples and the animal returned to a holding cage to recover from the procedure. Following recovery from
activated and continued until the rat's nose entered the goal box or for a total of 80 s at which time the trial was
anesthesia, the rats were returned to their home cages. The experiment was replicated 5 times (n = 120).
terminated. In each replication of the experiment, groups of 24 rats were first trained on the beam-walking task. O n the first
A baseline measure of each rat's motor ability following cortex injury was provided by a single trial on the beam performed before the drug or saline-vehicle ad-
day of training, each rat was given a series of 3
ministration the day following surgery. Only rats which were unable to traverse the beam (score o f ' l ' or '2') were
approximate trials. Subsequent training consisted of one trial o n the beam each day until the rat achieved a score of '7' on two successive days. Following training, surgical procedures were carried out on groups of rats u n d e r complete pentobarbital anesthesia (50 mg/kg; additional doses were administered
7
used in the experiment. The rats were then randomly assigned to receive a single dose of either clonidine HCI (20, 60, or 200 ~g/kg) or saline• Clonidine HC1 (Sigma) was dissolved in saline (0.9% w/v NaC1) and administered by i.p. injection at the indicated doses (salt weight).
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Fig. 1. Beam-walking recovery in rats given a single dose of: A, Saline (n = 26); B, Clonidine, 20/~g/kg (n = 22); C, Clonidine, 60/~g/kg (n = 24); or D, Clonid;ne 200 ~g/kg (n = 18). A single dose of clonidine or saline vehicle was administered 24 h after a right sensorimotor cortex suction-ablation injury (arrows). The mean (+ S.E.M.) beam-walking scores are given for each trial. Motor performance was rated on a 7 point scale as previously described1°: 1, the rat is unable to place the affected hindpaw on the horizontal surface of the beam; 2, the rat places the affected hindpaw on the horizontal surface of the beam and maintains balance for at least 5 s; 3, the rat traverses the beam while dragging the affected hindpaw; 4, the rat traverses the beam and at least once places the affected hindpaw on the horizontal surface of the beam; 5, the rat crosses the beam and places the affected hindlimb on the horizontal surface of the beam to aid less than half its steps; 6, the rat uses the affected hindpaw to aid more than half its steps, and 7, the rat traverses the beam with no more than two footslips.
307 C o n t r o l rats received the saline vehicle. Each rat was then tested at 1-h intervals beginning 1 h after drug administration for 6 h ( ' H o u r s 2 - 6 ' ) . The rats were subsequently tested over a p e r i o d of 12 days ( ' D a y s 1-12'). In addition to b e a m - w a l k i n g ratings, each rat was also r a t e d on a qualitative 4 point scale for evidence of sedation: 0, none; 1, mild - - arouses with minimal tactile stimuli; 2, m o d e r a t e - - difficult to arouse; 3, unarousable. A l l behavioral m e a s u r e m e n t s were r e c o r d e d by an o b s e r v e r blind to the rats' t r e a t m e n t status. Following the behavioral m e a s u r e m e n t s , rats were sacrificed by an o v e r d o s e of p e n t o b a r b i t a l and the brains were dissected and fixed by immersion in 4% formalin. A f t e r fixation, a v i d e o c a m e r a - g e n e r a t e d image of the dorsal aspect of each brain was digitized and lesion sizes (areas) and m a x i m u m medial extents (closest approxim a t i o n of the lesion to the inter-hemispheric fissure) were m e a s u r e d with an image analysis system (Jandel V i d e o Analysis Software). The brains of rats from one replication of the e x p e r i m e n t (19 of 90 brains) were not analyzed because of a tissue handling accident. In o r d e r to facilitate comparison with previous work, m e a n ( + S . E . M . ) b e a m - w a l k i n g scores are given in the figures. H o w e v e r , since an ordinal scale was e m p l o y e d to rate b e a m - w a l k i n g p e r f o r m a n c e , the data were analyzed with n o n - p a r a m e t r i c methods. Performances on the ' p r e - d r u g ' trial (to d e t e r m i n e whether c o m p a r a b l e postsurgical deficits occurred across groups) was analyzed with the K r u s k a l - W a l l i s test. In o r d e r to c o m p a r e the rates of i m p r o v e m e n t of beam-walking p e r f o r m a n c e
among d r u g - t r e a t m e n t groups, the Trapzoidal rule l was used to calculate the areas u n d e r the curves f o r m e d when each rat's scores were p l o t t e d against trial n u m b e r (i.e. area u n d e r the t i m e - e f f e c t curve). T h e resulting data was analyzed with the K r u s k a l - W a l l i s test. T h e Dunn Procedure was subsequently applied to the ranks of the data in o r d e r to d e t e r m i n e the significance of differences b e t w e e n groups 2s. Lesion size and medial extent were analyzed with A N O V A . All tests of significance are two-tailed. Fifteen of 120 rats did not reach training criteria, 2 rats died i m m e d i a t e l y following surgery, and 13 rats had baseline postsurgical b e a m - w a l k i n g scores > 2 indicating a less severe m o t o r deficit. The remaining rats (n = 90) served as subjects for the e x p e r i m e n t . The b e a m - w a l k i n g recoveries in rats given a single dose of saline (n = 26) or clonidine (20 ~tg/kg, n = 22; 60 Bg/kg, n = 24; 200 /~g/kg, n = 18) are given in Fig. 1. T h e r e were no differences a m o n g the groups prior to the administration of drug/placebo ( ' P r e d r u g ' trial; K r u s k a l - W a l l i s H = 0.87, df = 3; P > 0.20). Fig. 2 gives the overall rates of recovery of saline- and c l o n i d i n e - t r e a t e d rats. Rats which had received clonidine had slower recoveries as comp a r e d to the saline-treated controls ( K r u s k a l - W a l l i s H -= 8.755, df = 3; 0.02 < P < 0.05; Dunn P r o c e d u r e P < 0.05 for each clonidine dose c o m p a r e d with saline). There were no differences in recovery rates among the 3 groups of clonidine-treated rats (Dunn P r o c e d u r e , P > 0.05 for each comparison). I m p a i r m e n t by clonidine was present for all doses 5 days after the administration of the single dose of the drug (Fig. 1 ; K r u s k a l - W a l l i s H = 8.47, df = 3; 0.02 < P < 0,05; D u n n P r o c e d u r e P < 0.05 for each
65
30
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20
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45
I Saline
i 20pg/kg
i i 60pg/kg 200tJg/kg
Clonidine Dose
Fig. 2. The relationship between a single dose of saline or clonidine administered 24 h after sensorimotor cortex suction-ablation injury and the rate of recovery of beam-walking over a 12-day period. The mean (+ S.E.M.) recovery rates are given.
Saline
20pg/kg.
60lJg/kg
200JJg/k 9
Clonidine Dose
Fig. 3. The mean (+_ S.E.M.) lesion sizes (A) and maximum medial lesion extents (B) for the brains of rats in each of the 4 treatment groups.
308 clonidine dose c o m p a r e d with saline). By day 6, the difference in beam-walking scores among saline- and c l o n i d i n e - t r e a t e d rats was no longer significant ( K r u s k a l Wallis H = 7.339, df = 3; 0.05 < P < 0.10). N o n e of the rats showed any signs of sedation following the administration of clonidine/saline. There were no differences a m o n g the replications of the experiment with respect to ' P r e d r u g ' b e a m - w a l k i n g scores or rates of beam-walking recovery. T h e r e were no significant differences in mean lesion size (Fig. 3; A N O V A , F3,67 = 0 . 7 8 5 ; P = 0 . 5 5 ) o r in m e a n m e d i a l lesion extent ( A N O V A , F3,~7 = 1.01; P = 0.39) a m o n g the 4 t r e a t m e n t groups, The central finding of the present investigation is that a single dose of the a2-adrenergic r e c e p t o r agonist clonidine HCI given 24 h after a unilateral s e n s o r i m o t o r cortex lesion results in a significant and p r o l o n g e d i m p a i r m e n t of m o t o r recovery in rats. This effect of clonidine occurred with doses as low as 20/~g/kg. These studies are part of a series of experiments designed to further elucidate the p h a r m a c o l o g y of amp h e t a m i n e - f a c i l i t a t e d recovery of beam-walking. Preliminary evidence suggests that the a m p h e t a m i n e effect is m e d i a t e d t h r o u g h central n o r a d r e n e r g i c neurons. A n intraventricular infusion of n o r e p i n e p h r i n e but not dopamine mimics a m p h e t a m i n e - f a c i l i t a t e d recovery of beam-
walking in rats 2. Infusion of n o r e p i n e p h r i n e into the cerebellum contralateral to a s e n s o r i m o t o r cortex lesion also accelerates m o t o r recovery in rats 3. Lesions of the locus coeruleus m a y influence b e a m - w a l k i n g recovery 3' 14.24,32 Treatment with centrally-acting a~-adrenergic r e c e p t o r antagonists interfere with m o t o r recovery 3~'33. The present results are also consistent with o t h e r preliminary reports which suggest that ae-adrenergic receptor antagonists may enhance b e a m - w a l k i n g recovery 16' 20,31.33 while t r e a t m e n t with clonidine may reinstate beam-walking deficits in recovered rats 3°'3t'33
1 Beyer, W.H,, CRC Standard Mathematical Tables, 26th edn., CRC Press, Boca Raton, FL, 1981, p. 125. 2 Boyeson, M.G. and Feeney, D.M., The role of norepinephrine in recovery from brain injury, Soc. Neurosci. Abstr., 10 (1984) 68. 3 Boyeson, M.G., Krobert, K.A. and Hughes, J.M., Norepinephfine infusions into cerebellum facilitate recovery from sensorimotor cortex injury in the rat, Soc. Neurosci. Abstr., 12 (1986) 1120. 4 Brailowsky, S., Knight, R.T. and Efron, R., Phenytoin increases the severity of cortical hemiplegia in rats, Brain Research, 376 (1986) 71-77. 5 Braun, J.J., Meyer, P.M. Meyer, D.R., Sparing of a brightness habit in rats following visual decortication, J. Cornp. Physiol. Psychol., 61 (1986) 79-82. 6 Buytendijk, F.J.J., An experimental investigation into the influence of cortical lesions on the behaviour of rats, Arch, Neerl. Physiol. L'Homme Anita., 17 (1932)370-434. 7 Crisostomo, E.A., Duncan, P.W., Propst, M.A., Dawson, D.B. and Davis, J.N., Evidence that amphetamine with physical therapy promotes recovery of motor function in stroke patients, Ann~ NeuroL, 23 (1988) 94-97. 8 Dunbar, G.L., Hecht, S.A., Merbaum, S.L., DeAngelis, M.M. and Stein, D.G., Use of gangliosides and amphetamines to promote behavioral recovery following bilateral caudate nucleus lesions. In R.L. Masland, A. Portera-Sanchez and G. Toffano (Eds.), Neuroplasticity: A New Therapeutic Tool in the CNS, Liviana Press, Padova, 1987, pp. 117-124. 9 Feeney, D.M., Gonzalez, A. and Law, W.A., Amphetamine restores locomotor function after motor cortex injury in the rat, Proc. West. Pharmacol. Soc., 24 (1981) 15-17. 10 Feeney, D.M., Gonzalez, A. and Law, W.A., Amphetamine haloperidol and experience interact to affect the rate of recovery after motor cortex injury, Science. 217 (1982) 855-857.
11 Feeney, D.M. and Hovda, D.A., Amphetamine restores tactile placing after motor cortex lesions, Fed. Proc. 39 (t980) 1095. 12 Feeney, D.M. and Hovda, D.A., Reinstatement of binocular depth perception by amphetamine and visual experience after visual cortex ablation, Brain Research, 342 (t985) 352-356. 13 Feeney, D.M. and Sutton, R.L., Pharmacotherapy for recovery of function after brain injury, CRC Crit. Rev. Neurobiol., 3 (1987) 135-197. 14 Feeney, D.M., Sutton, R.L., Boyeson, M.G., Hovda, D.A. and Dail, W.G., The locus-coeruleus and cerebral metabolism: recovery of function after cortical injury, Physiol. PsychoL, 13 (1985) 197-203. 15 Fuxe, K. and Ungerstedt, U., Histochemical, biochemical and functional studies on central monoamine neurons after acute and chronic amphetamine administration. In E. Costa and S. Garattini (Eds.), Amphetamines and Related Compounds, Raven, New York, 1970, pp. 257-288. 16 Goldstein, L.B., Amphetamine-facilitated functional recovery after stroke. In M.D. Ginsberg and W.D. Dietrich (Eds.), The
Beam-walking in the rat has b e e n used as an animal model which m a y he predictive o f the effects of drugs on m o t o r recovery in humans following stroke 7. Based on these studies, a clinical trial was p e r f o r m e d which found that t r e a t m e n t with a m p h e t a m i n e facilitated short-term m o t o r recovery in a small group of highly-selected stroke patients 17. Clonidine is frequently prescribed as an antihypertensive in this group of patients. If the present results are also relevant to humans, then clonidine should be avoided during the recovery p e r i o d after stroke.
The authors are indebted to Ms. Holli Poe for her excellent technical assistance. This work was supported in part by the NIH (NS 06233 and NS 01162) and by the Department of Veterans Affairs.
16th Princeton Conference on Cerebral Vascular Diseases,
17 18 19 20
21
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