Cerebellar norepinephrine infusions facilitate recovery after sensorimotor cortex injury

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Brain Research Bulletin,Vol. 29, pp. 435-439, 1992 Copyright

Printed in the USA. All rights reserved.

0 1992 Pergamon

Press Ltd.

Cerebellar Norepinephrine Infusions Facilitate Recovery After Sensorimotor Cortex Injury MICHAEL G. BOYESON*’ AND KURT A. KROBERTt *Department

qf Rehabilitution Medicine. University qf Wisconsin Medical School, Madison, WI 53706, and TDepartment of Physiology, University of New Mexico, Albuquerque, NM 87131 Received 18 September 199 1; Accepted 27 February 1992

BOYESON. M. G. AND K. A. KROBERT. Cerebellar norepinephrine i&ions ,facilitate recovery after sensorimotor cortex injury. BRAIN RES BULL 29(3/4) 435-439, 1992.-This study reports the effects of norepinephrine infusions into cerebellum after unilateral sensorimotor cortex injury. The results demonstrate an immediate and permanent acceleration in motor recovery in awake rats infused with I50 rg norepinephrine into the cerebellum contralateral to a right sensorimotor cortex ablation. A vehicle infusion or infusion of norepinephrine into the ipsilateral cerebellum produced no beneficial effects on functional recovery. Norepinephrine

Brain injury

Cerebellum

Sensorimotor cortex

RECOVERY of function from brain injury represents a tremendous financial, physical, and emotional burden on the patient and family. Seldom, if at all, are pharmacological agents employed to facilitate recovery of function. even though several promising scientific bases exist for such treatment in animal models of brain injury (1,5.7,19,20) and in the clinic (I 1,25). Instead, efforts have been directed toward maintenance of homeostatic functions, e.g.. controlling edema, agitation, hemorrhaging, rather than toward intervening and speeding the recovery process. For example, Feeney and his collaborators have published numerous results showing that d-amphetamine, when given under appropriate conditions, rapidly facilitates recovery from hemiplegia induced by sensorimotor cortex (SMCX) injuries in animals [see (l,l3,14) for review]. and these results have been favorably extended to stroke patients (I I). A recent study has indicated that amphetamine may be stimulating recovery of function through actions on the noradrenergic (NE) system, since intraventricular NE infusions facilitate recovery, but infusions of dopamine or dopamine plus a dopaminebeta-hydroxylase inhibitor have no beneficial effect on recovery outcome (5). Disturbances in NE functioning in the cerebellum contralateral to the SMCX injury have been reported in animals (4) presumably due to bifurcating locus ceruleus (LC) axons to both areas from the same neuron (12). Such disturbances may be related to observations of crossed-cerebellar diaschisis in humans after parietal cortex damage (13). Unilateral cerebellar ablation injury, depending on the extent, results in severe motor deficits as measured by a beam-walking task, and both NE agonists and antagonists interfere with the recovery process (6). If it is the case that subtle disturbances in NE functioning in the cerebellum contralateral to an SMCX injury contribute

largely to the manifestation of motor deficits, then efforts directed towards increasing NE transmission should facilitate recovery from the cortical injury. In order to assess this possibility, localized microinfusions of NE were given by cannula into the cerebellar cortex contralateral or ipsilateral to the SMCX injury in awake animals to assess the possible therapeutic benefits on recovery from hemiplegia. METHOD

Prior to surgery, all animals were trained on the beam-walking task. The apparatus used to assess beam-walking ability was a long (22 cm), narrow (2.5 cm), elevated (36 cm) beam. The beam-walking task has provided a reproducible motor deficit that may be termed hemiplegic if induced by a unilateral (SMCX) injury ( 18). Twenty-two male Sprague-Dawley rats weighing 300-324 g were handled on a daily basis (for approximately 30 seconds) for I week prior to the beam-training period. Eight additional animals were used to assess different doses of intracerebellar infusions of NE. All animals were individually housed and on a 12-h light/dark cycle (lights on 0600) and fed ad lib. Daily behavioral assessments of beam walking took place between 1000-1400 h. A large, dark goal box (24.8 X 20.3 X 17.8 cm) was positioned at the end of the beam. Training on the beam-walk task began after a I -week habituation period. On the first training day, the animal was given three trials. On trial one the animal was placed just outside the goal box; on trial two, at the midpoint of the beam; and on trial three, at the start position. Motivation for the animal to move consisted of a tail flick combined with a hiss. Escape from this stimulation to the darkened box was easily learned by the animal. Single trials were then

’ Requests for reprints should be addressed to Michael G. Boyeson, Ph.D., Department of Rehabilitation Medicine, University of Wisconsin Medical School, Room 3490 Medical Science Center, 1300 University Avenue, Madison, WI 53706.

435

436

BOYESON

n

1

hrs

RT SMCX INJ

AND

KROBERT

I.1 CER “E”

days POST

INFUSION

FIG. 1.The eflects on beam-walking recovery of a single infusion of NE ( 150 pg/5 ~1) into the right cerebellum at 24 h after a right SMCX ablation. Only the infusion of NE into the cerebellum contralateral to the SMCX injury facilitated recovery compared to a control infusion. Abbreviations: NE = I-norepinephrine: SMCX = sensorimotor cortex: VEH = vehicle: vertical bars z SEM. or left

conducted every other day until criterion on the beam-walking task was reached on two consecutive trials. The criterion consisted of acheiving two consecutive 7s on a 7-point rating scale using two assessors, one blind to all treatment conditions. The rating scale has been described previously ( 14). In brief, however, ifthe animal traversed the beam with no more than two hindlimb footslips, he was rated the maximum score of 7 (usually within two to four trials). Once criterion was reached, no further training was conducted so that slower animals were able to attain the same presurgery performance level. This was done to avoid overtraining some animals. A score of 6 indicated that the animal could traverse the beam using the effected hindlimb for greater than 50% of the distance across the beam. A 5 consisted of the animal using the effected limb for less than 50% of the distance. A 4 indicated that the animal could get the limb up on the vertical surface of the beam but could not push off the surface without slipping. A 3 describes an animal that can pull itself down the beam without getting the effected hindlimb on the vertical surface. A 2 represented an animal that could not traverse the beam but if placed sideways on the beam could bring the limb up to the vertical surface. A I indicated an animal that could not maintain its balance on the beam. On the day of surgery all animals were given a single test trial to insure continued performance at a 7 level. For surgery the animals were anesthetized with pentobarbital sodium (Nembutal: 21 mg/kg, IP) preceded by ketamine hy-

drochloride (Ketacet: 60 mg/kg. IM) and placed in small animal stereotaxic apparatus (David Kopf Instruments). A craniotomy was performed over the right sensorimotor cortex. the dura was excised, and the animal received a right unilateral sensorimotor cortex ablation extending from 2 mm anterior to 4 mm posterior to bregma. and 5 mm lateral from the sagittal sinus to the depth of the white matter (see Fig. 2). The cavity was filled with sterile gelfoam. At the same time as the SMCX injury. a small burr hole was drilled through the cranium of the left or right cerebellar hemisphere for the later infusion of NE or vehicle, and a cannula inserted and cemented with dental acrylic. The top ofthe cannula was capped. The lengths of all cannulae inserted into all animals were the same, with the length of the inserting syringe measured to project to 0.3 mm below the tip of the cannula. The cannula was inserted at the following coordinates according the the rat atlas of Paxinos and Watson (1983): lateral 2.8 mm from the midline: AP - I .3 mm from the interaural line: vertical. 0.5 mm from dorsal surface of the cerebellum (see Fig. 3). Based on a previous study indicating that I50 fig of NE was effective in facilitating recovery compared to 50. 100, and 200 pg administred intraventricularly (5) 75 pg (N = 3) 100 fig 02: = 3) and I50 pg (N = 2) was slowly infused into the cerebellum at 24 h post-SMCX injury to assess the relative effectiveness of the infusions. The dose of 75 fig/5 ~1 was found to be less effective than 100 Fug/5 ~1, and both these doses were less effective than

CEREBELLAR

NE AND

CORTICAL

INJURY

431

tralateral cerebellum facilitated recovery compared to vehicle controls as measured by the beam-walking task, F( I, 14) = 8.9, p < 0.01. To assess diffusion effects of NE, NE was infused into the cerebellum ipsilateral to the SMCX injury. No significant difference in recovery was noted for this group compared to vehicle controls. A schematic drawing of the extent of the SMCX injury is presented in Fig. 2. No significant differences in the volume of tissue (*SEM) removed from SMCX (compiled from serial sections) were noted between groups (66 + 4.2 cubic mm NE ipsalat. cerebellum; 67 + 3.3 cubic mm VEH contralat. cerebellum; 7 I + 3.9 cubic mm NE contralat. cerebellum). Histological examination of the brain revealed that the cannula implant into cerebellum penetrated to a depth of 0.5 to 0.7 mm below the surface of the cerebellum. In the Fig. 3A, B photomicrograph, the extent and location ofthe cannula implant are depicted for each cerebellar hemisphere. Comparable implant areas were observed for both cerebellar hemispheres. Across all groups, some pathology to cerebellar tissue was noted with the infusion procedure at points below the cannula insert. DISCUSSION

FIG. 2. The above photograph represents the extent of the right sensorimotor cortex ablation (see text for additional details). Abbreviations: CC = corpus callosum: CP = caudate-putamen; SN = substantia nigra; V = ventricle: IP = interpeduncular nucleus: HP = hippocampus.

I50 &5 ~1. [In a previous study (5) a 200 p(g dose resulted in a temporary flaccid paralysis of the hindlimbs that lasted approximately 30 min and interfered with testing on the beam in some animals.] Following these initial studies for the determination of the effective NE dose level, a dose of 150 pg/5 (.d was chosen and was slowly infused (over 2 min) into the left or right cerebellum (2.8 mm lateral from midline suture; -I .3 mm posterior to interaural line; 0.5 mm vertical from surface of cerebellum: see Fig. 3). Vehicle consisted of sterile 0.9% NaCl with ascorbic acid 0.1% added to retard oxidation of NE. Following a single predrug measurement at 24 h postinjury, the cannula was uncapped and NE was slowly infused into the cannula while the animal was passively restrained. Multiple postdrug measurements of beam-walking performance were assessed at 0.5, I, 3, 6. and 24 h postinfusion, and every other day through I5 days. Animals were sacrificed for verification of infusion point and comparability of SMCX injury at the conclusion of the experiment. The behavioral results were analyzed with a repeated measures ANOVA. RESULTS

The results for the infusions of NE or vehicle into cerebellum contralateral of ipsalateral to a unilateral SMCX injury are presented in Fig. I. The overall main effect of all groups was significant as was the interaction, F(2, 19) = 10.6, p < 0.01; interaction, F = 3.6, p < 0.05. Only the NE infusion into con-

The results obtained from the present study complement a previous study showing that intraventricular infusions of NE facilitate recovery of motor function (5) and indicate that the NE effect is modulated, in part, by NE projections to cerebellum. The NE system appears to modulate the changes in motor behavior rather than directly expressing the hemiparesis, since animals with prior removal of the LC (with either 6-OHDA or DSP-4) eventually recover (although with considerable delay) on the beam following a SMCX injury (3,7,8, IO). It is not clear whether the NE-induced facilitation of recovery is due to activation of a particular receptor subtype. For example, blockade of alpha-2 receptors by idazoxan facilitates recovery. whereas stimulation appears deleterious (I 7,19). Presumably these actions are via presynaptic effects of inhibition or excitation on locus ceruleus neurons, respectively. Additionally, blockade of alpha-l postsynaptic receptors by phenoxybenzamine administered IP or into the contralateral cerebellum has deleterious effects on recovery of function after SMCX injury (4,9). The facilitated recovery effect appears to be specific to the contralateral cerebellum since microinfusions of NE into the cerebellum ipsilateral to the SMCX injury failed to accelerate recovery. One possible mechanism through which changes in NE functioning might affect recovery outcome may relate to observations that the same LC neuron bifurcates and projects simultaneously to SMCX and contralateral cerebellum (12). Injury to one axonal projection would be expected to produce changes in functioning to the intact axonal projection (24), perhaps eventually leading to compensatory increases in NE functioning of the intact branches (I ,23). These changes in NE functioning may also parallel changes in functional recovery. For example. evidence from this laboratory has indicated that, if the cerebellum contralateral to the SMCX injury is made supersensitive to NE prior to a SMCX injury, the NE infusions at 24 h postinjury result in total recovery on the beam as early as I h postinfusion (IO). These results clearly suggest that pharmacological intervention after brain injury using NE agonists might have beneficial effects on recovery [e.g., as in enhancement of recovery by am-

438

BOYESON

AND

KR0BER-l

FIG. 3. The above photomicrograph depicts a representative cannula insertion pomt (arrows) for the infusion of NE or vehicle into the cerebellum contralateral (A) or ipsalateral (B) to the SMCX injury of two different animals.

( I I) and animals (I 4)] and that phetan tine in strol te patients when administered on NE ant agonists ma mybe contraindicated a contil nuous basis. The results also indicate that the functional motor I-ecovery obs ,erved after SMCX injury is mediated in large part by changes in cerebellar output. Although it is presently unclear which out1xtt areas are involved, it is likely that com-

pensatory

changes

and rubroolivary

between deep cerebellar nucle i. Iubrc rspi nal. are involved in the motor recoj :er Y (6 22) ACKNOWLEDCitMtN

This work was supported

by NIH Grant

I

#NS 23003

to M.G .B.

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NE AND

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INJURY

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