- Email: [email protected]
Phenytoin increases the severity of cortical hemiplegia in rats
BrainResearch, 376(1986)71-77 Elsevier
71
BRE 11780
Phenytoin Increases the Severity of Cortical Hemiplegia in Rats SIMON BRAILOWSKY*, ROBERT T. KNIGHT and ROBERT EFRON
Department of Neurology, University of California, Davis and Veterans Administration Medical Center, Martinez, CA 94553 (U.S.A.) (Accepted October 29th, 1985)
Key words: hemiplegia - - ~,-aminobutyric acid (GABA) - - phenytoin - - motor cortex
The effects of systemic phenytoin administration on the motor deficit resulting from a cortical lesion were studied in rats trained to walk coordinately on a narrow beam. The somatomotor cortex lesion was produced by an indwelling cannula through which saline or GABA were infused chronically via an osmotic minipump. Phenytoin (50 mg/kg i.p.) administered between days 3 and 5 after the intracortical catheter implantation produced a significant increase in the severity of the resulting hemiplegic syndrome. This DPH effect was more noticeable in those animals also receiving intracortical GABA infusions. The anticonvulsant at the dose used had no effect on motor performance when administered preoperatively or when given to the animals 14 days after surgical intervention when their hemiplegic syndrome had cleared. These findings suggest that phenytoin administration to brain-damaged individuals in the initial postlesion stage may be deleterious. INTRODUCTION D i p h e n y l h y d a n t o i n ( D P H ) is among the first 50 drugs most frequently prescribed in the U n i t e d States 27 and is most c o m m o n l y used to reduce seizure frequency including seizures associated with acute brain lesions 31. T h e mechanism by which D P H acts, however, is still uncertain. A m o n g the p r o p o s e d mechanisms of action of D P H , the e n h a n c e m e n t of G A B A - m e d i a t e d inhibition has been r e p e a t e d l y suggested (for review, see ref. 19). The interactions b e t w e e n D P H and the G A B A system have p r o d u c e d , however, conflicting results. Phenytoin administration increases the concentration of G A B A in rat cerebral cortex 28 and in mice brain 3,22, and increases the duration of G A B A m e d i a t e d IPSPs in the crayfish stretch r e c e p t o r neuron t'2'1°, decreases p y r a m i d a l responses to m o t o r cortex stimulation 5'24, prolongs recurrent inhibition in cat p y r a m i d a l tract neurons zl, enhances postsynaptic inhibition in rat cuneate nucleus zS, in frog spinal cord 9 and in isolated mouse spinal cord neurons 19. Phenytoin also has protective effects against picrotoxin-induced b l o c k a d e of the G A B A r e c e p t o r 1°,
and against seizures induced by allyglycine, an inhibitor of G A B A synthesis 26. Conversely, D P H has been r e p o r t e d to have no effect on G A B A responses of frog spinal m o t o n e u r o n s z°, of rat 8 and cat dorsal root ganglion neurons 13, or on rat h i p p o c a m p a l pyramidal cells 15. F u r t h e r m o r e , D P H has been r e p o r t e d to diminish the K+-induced release of G A B A from rat cerebral cortex slices, without effect on the resting release of G A B A 4, and has been r e p o r t e d to augment picrotoxin-induced seizure activity 16. Thus, the role of G A B A in the effects of D P H is still a m a t t e r of controversy. In previous reports we have shown that surface application of G A B A to the s o m a t o m o t o r cortex of anesthetized cats p r o d u c e d a reversible inhibition of the negative c o m p o n e n t (N20) of the primary somatosensory e v o k e d potential 6. F u r t h e r , we have shown 7 that intracortical G A B A administration (via osmotic minipump) to the s o m a t o m o t o r area of unanesthetized young and aged rats p r o d u c e d a hemiplegic synd r o m e significantly m o r e m a r k e d than the one produced by the same cannula-induced lesion but without intracortical G A B A infusion. Thus, intracortical G A B A infusion increases the functional deficit with-
* On leave of absence from the National Institute of Sciences and Technology (INCYTAS, DIF), Mexico. Correspondence: R.T. Knight, Department of Neurology, Veterans Administration Medical Center, 150 Muir Road, Martinez, CA 94553, U.S.A. 0006-8993/86/$03.50 t~) 1986 Elsevier Science Publishers B.V. (Biomedical Division)
72 out changing the lesion size. This technique of intracortical G A B A infusion permits the evaluation of the effect of phenytoin at different levels of hemiplegic severity without introducing the complication associated with varying lesion size. MATERIALS AND METHODS
Subjects Adult male F-344 rats weighing 240-270 g were used. They were housed in groups of 6 at constant temperature (21 °C) and maintained on a standard (12:12 h) light-dark cycle, so that the light cycle occurred during working hours. The animals had free access to food and water.
Training One week after arrival, the animals were trained to walk on an elevated (45 cm above table level), narrow (2.5 cm) beam (2 m long) until they reached stable performance criteria (no slips or falls from the beam, no asymmetric positioning of the paws while walking, and no defects in paw placing on the surface of the wooden bar). Motor function on the beam was evaluated daily as described previously 7. Typically, after a motor cortex aspiration or chronic G A B A infusion into the somatomotor region, the progression of the resultant motor deficit evolves from an animal totally unable to perform the task (score = 24), to animals who show falls, slips or defects in paw placing (score range = 8-20), to animals who walk without touching the lateral aspects of the bar (score = 0). Sensory function testing was performed after completion of the motor task, and consisted of stimulation of vibrissae, trunk and dorsal and lateral surfaces of both extremities. Orientation to or withdrawal from the stimulus were considered to indicate normal sensory function.
Surgical procedures After pretreatment with atropine methylbromide (6 mg/kg s.c.), the animals were anesthetized with ketamine (40 mg/kg i.p.) followed by pentobarbital (20 mg/kg i.p.). The scalp was shaved, cleaned and infiltrated with lidocaine hydrochloride (2%). The animals were then positioned in a stereotaxic apparatus. After opening the scalp, the bone sutures were visualized. A small (2 mm) trephine was then made
over the hindlimb representation of the motor cortex 11A4'23,2 mm posterior to bregma and 2 mm lateral to the midline. After opening the dura with a needle, a beveled cannula (PE60) to which an osmotic minipump (Alza 2001) was attached was inserted through the trephine for 2 mm tangentially to the skull at an angle of approximately -30 ° to the horizontal plane. The cannula was then attached to the skull with dental acrylic, using a bone screw to assure fixation. All implants were performed on the left side. The minipumps had been previously filled with either saline or G A B A and inserted subcutaneously on the interscapular space. These devices are rated to deliver 1 pl/h for 7 days. The GABA-filled pumps were prepared to deliver 1 ktl/h of a 100 #g//d solution for 1 week. The wound was sutured and antibiotics were administered both locally and systemically.
Drug administration Once the animals had achieved stable scores on the bar running task, they were divided in 3 groups of 12 animals each. After motor and sensory testing, they were given 0.1 ml/100 g of body weight, i.p., of one of 3 solutions (prepared beforehand by a colleague and randomly marked A, B, and C): phenytoin solvent (40% propylene glycol and 10% alcohol in distilled water, adjusted to p H 12), or phenytoin at 25 and 50 mg/ml concentrations. Animals were retested on the beam 30, 60 and 120 min after injection. The purpose of this presurgical control was two-fold. Firstly, it was intended to determine the dosage level of D P H which would not result in any ataxia or other alteration of motor performance in normal rats. Secondly, it was intended to determine the effects, if any, of the D P H solvent itself. The solvent did produce a relative 'improvement' in the scores of these normal rats which was significant at the P < 0.025 level. Since no significant changes in motor skills were observed with D P H we used the larger (50 mg/kg) concentration in the main experiment. One week after this preliminary drug testing, the animals were subdivided in two groups: in one group saline-filled minipumps were implanted, and in another GABA-filled minipumps were used. These two groups were further subdivided; half of them received phenytoin solvent, and the other half received the anticonvulsant at a 50 mg/kg concentration. The response to drug administration was studied again on
73 20
[~ Before inlection I-
[]
Ll. Ill (3 rr 0 I-0
After injection
tographed and measured. The brain was then removed for further histological analysis (Cresyl violet staining). Minipumps were opened to verify for complete emptying of the reservoir.
A = GABA DPH B= S A L - S O L V
10
C= LESION-DPH D= L E S I O N - S O L V
Data evaluation The data were analyzed using an A N O V A test for repeated measures 29.
5'
RESULTS
SOLV
DPH 25
A
B
C
D
A
B
C
D
5o
PBE-SURGERY
bAYS 3-5
DAY 14
Fig. 1. Effects of phenytoin (DPH) or its solvent (SOLV) on motor performance. Scores obtained before (Pre-surgery) and after surgical implantation of an intracortical cannula (Days 3-5 and Day 14). * P < 0.05, ** P < 0.005, *** P < 0.001. Presurgery condition: no significant effects were found in animals treated with either DPH (25 or 50 mg/kg) or its solvent (n = 12/group). Days 3-5: Group A, animals implanted (on day 0) with an osmotic minipump delivering GABA (100 /~g/pl/h for 7 days) and injected with DPH (50 mg/kg) on days 3-5. Scores obtained before and 30 min after drug administration; Group B, rats infused with saline-filled minipumps implanted at day 0 and injected with DPH solvent on days 3-5; Group C, rats implanted with intracortical cannulae identical to those of groups A and B but without drug infusion. DPH (50 mg/kg) administered on days 3-5; Group D, animals treated as group C (intracortical cannulae without infusion) and injected on days 3-5 with DPH solvent. Day 14: animals treated with DPH (50 mg/kg) 14 days after cannulae implantation. Group distribution as in Days 3-5 condition. No statistically significant effects were observed in these animals.
days 3, 4 and 5 postsurgery. Motor and sensory functions were evaluated before and 30 min after injection. The experimenter was unaware of either the minipumps' content or the identity of the drugs used. Fourteen days after minipump implantation, all animals were given phenytoin (50 mg/kg i.p.) and tested as previously described.
Histology The animals were sacrificed with a barbiturate overdose and perfused intracardially with a cold buffered formaldehyde solution (10%). After perfusion, the rats were positioned on a stereotaxic apparatus, and after removal of the cannula fixture, two insect pins were inserted in symmetrical positions (2 m m from the midline) over bregma. After bone and dura were removed, the implantation site lesion was pho-
All animals reached performance criteria (a stable score on at least 3 consecutive days) after 2 weeks of training. The training was given every other day during the first week and daily thereafter.
Presurgery testing As shown in Fig. 1, neither dose of phenytoin (25 or 50 mg/kg) produced any significant changes in motor performance. In contrast, D P H solvent decreased the motor deficit score.
Minipump implantation In approximately half of the animals the extracranial end of the cannula was found to be detached from the minipump. In our previous work 7 the severity and course of recovery of animals with saline injection into motor cortex did not differ from those who had simple aspiration of motor cortex. Since the animals found to have detached cannulas had the same severity and time course of recovery as we have found in those with saline injections, it can be assumed that the cannula disconnection occurred shortly after surgery and that no intracortical solution was delivered. Thus we can consider these animals to have only a cannula induced motor cortex lesion. Accordingly, 4 groups of animals were designated as: Group A, GABA-filled minipump with D P H treatment; Group B, saline-filled minipump with solvent treatment; Group C, motor cortex lesion with D P H treatment; and Group D, motor cortex lesion with solvent treatment. The motor deficits observed in Group A ( G A B A filled minipump with phenytoin treatment) in the first 3 days after implantation were similar to the ones reported previously 7. Administration of D P H between days 3 and 5 after surgery in this group produced a transient but significant (FI, 5 = 73.42, P < 0.001) en-
74
GABA-DPH
SALINE-SOLVENT
LESION-SOLVENT
LESION-DPH
2":
Fig. 2. Dorsal view of the superficial extent of the lesions in animals from all 4 experimental groups. Two insect pins positioned at 2 mm from the midline, bilaterally, mark the bregma suture. Scale in mm.
75 hancement of the deficit (see Fig. 1). These deficits consisted in an increased incidence of slips and falls, limping and defective paw placing. All these deficits were contralateral to the implanted site for all 3 days of systemic drug administration. Animals with saline-filled minipumps (Group B) showed motor deficits in the first 3 days after implantation similar to those previously reportedT: administration of phenytoin solvent to these animals did not produce significant changes in motor performance. In the animals treated with phenytoin but without GABA infusion (Group C), a significant (F1,5 = 24.04, P < 0.005) increase in the severity of the lateralized motor deficits was observed (see Fig. 1). These abnormalities were less severe than the ones observed in group A (who had received G A B A infusions) and consisted of slips and falls, limping and abnormal positioning of the contralateral hindlimb. In Group D (motor cortex lesion and phenytoin solvent treatment), the systemic administration of the DPH solvent actually resulted in a decreased motor deficit score. It will be noted that the DPH solvent also 'improved' performance even presurgery. Although the cause of this effect is uncertain, it does suggest that the presence of the solvent in the DPH groups (A and C) actually may have attenuated the hemiplegia enhancing effects of diphenylhydantoin itself. In all groups, the observed deficits were primarily motor deficits, and no persistent sensory abnormalities were detected.
Postrecovery testing Fourteen days after surgery, and when the animals had recovered from their motor deficits, phenytoin (50 mg/kg i.p.) was administered. As seen in Fig. 1, no significant effects were produced by the drug. However, some of the animals had a transient generalized ataxia after drug administration without any sign of lateralization.
Histology All lesions were localized in a comparable area - in reference to the bregma m a r k s - - corresponding to the hindlimb representation of the somatomotor cortex 11'14'23. The structural lesions were of similar extent in all groups. In length, width and depth they measured 2.7 x 2.2 x 1.8 mm in group A, 3.2 x 2.3
x 1.9 mm in group B, 2.4 x 1.9 x 1.7 mm in group C and 2.5 x 2.1 x 1.9 mm in group D. None of these lesion dimensions were significantly different between groups (see Fig. 2). DISCUSSION All groups of animals in this study had structural lesions of the somatomotor cortex induced by the cannula which were comparable in size. However, the group of animals which received G A B A through the cannula had a significantly more dense hemiplegia than the other 3 groups that did not have G A B A infusions. The 3 non-GABA infused animal groups had a less severe hemiplegia which was not statistically different in Groups B, C and D. This finding replicates our previous observation 7 on the potentiation of hemiplegia by cortical G A B A infusions. The purpose of the present experiments was to determine whether DPH administration would increase the functional deficit produced by an acute cortical lesion as might be anticipated if DPH potentiated the inhibitory effects of GABA. In the two groups receiving DPH (A and C) a statistically significant worsening of the functional deficit was observed. This DPH effect was reversible, however, since the animals returned to their prephenytoin motor performance score by the next day. When G A B A was infused (Group A) with increase in the severity of the hemiplegia (but not changing the size of the lesion), the deleterious DPH effect on motor performance was more noticeable (see Fig. 1). It should be emphasized, however, that the larger increment in the DPHenhanced motor deficit in the G A B A treated group (A) compared to the non-GABA treated group (C) does not necessarily reflect a synergistic interaction between G A B A and DPH. The larger increment in the motor deficit score in Group A could be due in part to the non-linear characteristics of the scoring system used. For this reason, the present experiment does not provide unequivocal evidence about G A B A involvement in the reversible exacerbation of the hemiplegic syndrome produced by DPH. Nevertheless, the results suggest that DPH reversibly increases the functional deficit produced by cortical damage in the early stages of the lesion. The less severe the deficit, the less noticeable are the drug effects, and in a very mildly impaired animal, the DPH
76 effect may not be detected. The advantage of the present technique of G A B A infusion is that it can generate a m o r e m a r k e d functional deficit without the necessity of creating larger lesions 7. It is of particular interest to note that on day 14, when the m o t o r deficit score of our animals had returned to presurgical levels, no effect of D P H was detected, even in the G A B A - t r e a t e d animals. In contrast, we have r e p o r t e d 17 that systemic administration of haloperidol, a dopaminergic blocker, on day 14 after surgery caused a r e - e m e r g e n c e of the hemiplegic s y n d r o m e in animals who had G A B A infused in a p r o c e d u r e identical to the G r o u p A animals of the present study. Thus, those mechanisms involved in the expression of the effects of injury in the first week after cortical d a m a g e were refractory to D P H 2 weeks later but still c a p a b l e of being activated by haloperidol, indicating some specificity of the D P H effect. It has been r e p o r t e d that cerebral ischemia in various brain regions 12'18'3° results in increased intracere-
bral G A B A levels. W e can reasonably assume that the cerebral lesions m a d e by the cannula in all our animal groups also caused an elevation of e n d o g e n o u s G A B A . W h e t h e r or not the p o t e n t i a t i o n of the h e m p plegia by D P H is related to intracerebral G A B A levels cannot be ascertained in the present experiment, H o w e v e r , the fact that G A B A infusions increase the severity of the hemiplegia raises the interesting possibility that the deleterious D P H effects on acutely brain-injured animals might be m e d i a t e d by G A B A or G A B A - l i k e inhibitory neurotransmitters. W h e t h er or not the effects of D P H are G A B A - m e d i a t e d , the present results m a y be of clinical i m p o r t a n c e as they suggest that some p r u d e n c e be exercised in the administration of D P H in the initial days following an acute brain injury.
REFERENCES
(1981) 357-369. 9 Davidoff, R., Diphenylhydantoin increases spinal presynaptic inhibition, Trans. Amer. Neurol. Ass., 97 (1972) 193-196. 10 Deisz, R. and Lux, H., Diphenylhydantoin prolongs postsynaptic inhibition and iontophoretic GABA action in the crayfish stretch receptor, Neurosci. Lett., 5 (1977) 199-203. 11 Donoghue, J. and Wise, S., The motor cortex of the rat: cytoarchitecture and microstimulation mapping, J. Comp. Neurol., 212 (1982) 76-88. 12 Erecinska, M., Nelson, D., Wilson, D. and Silver, I., Neurotransmitter amino acids in the CNS. I. Regional changes in amino acid levels in rat brain during ischemia and reperfusion, Brain Research, 304 (1984) 9-22. 13 Gallagher, D., Inokuchi, H., Nakamura, J. and ShinnickGallagher, P., Effects of anticonvulsants on excitability and GABA sensitivity of cat dorsal root ganglion cells, Neuropharmacology, 20 (1981) 427-433. 14 Hall, R. and Lindholm, E., Organization of motor and somatosensory neocortex in the albino rat, Brain Research, 66 (1974) 23-38. 15 Hershkowitz, N. and Ayala, G., Effects of phenytoin in pyramidal neurons of the rat hippocampus, Brain Research, 208 (1981) 487-492. 16 Jones, G., Phenytoin: basic and clinical pharmacology, Med. Res. Rev., 3 (1983) 383-434. 17 Knight, R.T. and Brailowsky, S., Possible role of dopamine in the functional recovery from hemiplegia in aged rats, Soc. Neurosci. Abstr., 10 (1984) 448.
1 Aiken, C., Deisz, R. and Lux, H., On the action of the anticonvulsant 5,5-diphenylhydantoin and the convulsant picrotoxin in crayfish stretch receptor, J. Physiol. (London), 315 (1981) 157-173. 2 Ayala, G., Johnston, D., Lin, S. and Dichter, H., The mechanism of action of diphenylhydantoin on invertebrate neurons. II. Effects on synaptic mechanisms, Brain Research, 121 (1977) 259-270. 3 Battistin, L., Varotto, M., Berlese, G. and Roman, G., Effects of some anticonvulsant drugs on brain GABA level and GAD and GABA-T activities, Neurochem. Res., 9 (1984) 225-231. 4 De Belleroche, J., Dick, A. and Wyrley-Birch, A., Anticonvulsants and trifluoperazine inhibit the evoked release of GABA from cerebral cortex of rat at different sites, Life Sci., 31 (1982) 2875-2882. 5 Blum, B., A differential action of diphenylhydantoin on the motor cortex of the cat, Arch. Int. Pharmacodyn. Ther., 149 (1964) 45-55. 6 Brailowsky, S. and Knight, R., Inhibitory modulation of cat somatosensory cortex: a pharmacological study, Brain Research, 322 (1984) 310-315. 7 Brailowsky, S., Knight, R., Blood, K. and Scabini, D., GABA-induced potentiation of cortical hemiplegia, Brain Research, 362 (1986) 322-330. 8 Connors, B., A comparison of the effects of pentobarbital and diphenylhydantoin on the G A B A sensitivity and excitability of adult sensory ganglion cells, Brain Research, 207
ACKNOWLEDGEMENT This research was s u p p o r t e d by the R e s e a r c h Service of the V e t e r a n s A d m i n i s t r a t i o n .
77 18 Lust, W., Mrsulja, B., Mrsulja, B., Passonneau, J. and Klatzo, I., Putative neurotransmitters and cyclic nucleotides in prolonged ischemia of the cerebral cortex, Brain Research, 98 (1975) 394-399. 19 Macdonald, R., Barbiturate and hydantoin anticonvulsant mechanisms of action. In H.H. Jasper and N.M. van Gelder (Eds.), Basic Mechanisms of Neuronal Hyperexcitability, A.R. Liss, New York, 1983, pp. 361-387. 20 Nicoll, R. and Wojtowicz, J., The effects of pentobarbital and related compounds on frog motoneurons, Brain Research, 191 (1980) 225-237. 21 Raabe, W. and Ayala, G., Diphenylhydantoin increases cortical postsynaptic inhibition, Brain Research, 105 (1976) 597-601. 22 Saad, S., Masry, A.E. and Scott, P., Influence of certain anticonvulsants on the concentration of 7-aminobutyric acid in the cerebral hemispheres of mice, Eur. J. Pharmacol., 17 (1972) 386-392. 23 Sanderson, K., Welker, W. and Shambes, G., Reevaluation of motor cortex and of sensorimotor overlap in cerebral cortex of albino rats, Brain Research, 292 (1984) 251-260. 24 Sherwin, I., Suppressant effects of diphenylhydantoin on the cortical epileptogenic focus, Neurology, 23 (1973)
274-281. 25 Simmonds, M., Distinction between the effects of barbiturates, benzodiazepines and phenytoin on responses to 7-aminobutyric acid receptor activation and antagonism by bicuculline and picrotoxin, Br. J. Pharmacol., 73 (1981) 739-747. 26 Snead, O.C., On the sacred disease: the neurochemistry of epilepsy, Int. Rev. Neurobiol., 24 (1983) 93-180. 27 Tallarida, R., Top 200, Springer Verlag, New York, 1981, 246 pp. 28 Vernadakis, A. and Woodbury, D., Effects of diphenylhydantoin and adrenocortical steroids on free glutamic acid, glutamate and gamma-aminobutyric acid concentrations of the rat cerebral cortex. In E. Roberts (Ed.), Inhibition in the Nervous System and Gamma-Aminobutyric Acid, Pergamon, Oxford, 1960, pp. 242-248. 29 Winer, B., Statistical Principles in Experimental Design, McGraw Hill, New York, 1971. 30 Wood, J., Watson, W. and Ducker, A., The effect of hypoxia on brain gamma-aminobutyric acid levels, J. Neurochem., 15 (1968) 603-608. 31 Woodbury, D., Penry, J. and Pippenger, C. (Eds.), Antiepileptic Drugs, Raven Press, New York, 1982.
71
BRE 11780
Phenytoin Increases the Severity of Cortical Hemiplegia in Rats SIMON BRAILOWSKY*, ROBERT T. KNIGHT and ROBERT EFRON
Department of Neurology, University of California, Davis and Veterans Administration Medical Center, Martinez, CA 94553 (U.S.A.) (Accepted October 29th, 1985)
Key words: hemiplegia - - ~,-aminobutyric acid (GABA) - - phenytoin - - motor cortex
The effects of systemic phenytoin administration on the motor deficit resulting from a cortical lesion were studied in rats trained to walk coordinately on a narrow beam. The somatomotor cortex lesion was produced by an indwelling cannula through which saline or GABA were infused chronically via an osmotic minipump. Phenytoin (50 mg/kg i.p.) administered between days 3 and 5 after the intracortical catheter implantation produced a significant increase in the severity of the resulting hemiplegic syndrome. This DPH effect was more noticeable in those animals also receiving intracortical GABA infusions. The anticonvulsant at the dose used had no effect on motor performance when administered preoperatively or when given to the animals 14 days after surgical intervention when their hemiplegic syndrome had cleared. These findings suggest that phenytoin administration to brain-damaged individuals in the initial postlesion stage may be deleterious. INTRODUCTION D i p h e n y l h y d a n t o i n ( D P H ) is among the first 50 drugs most frequently prescribed in the U n i t e d States 27 and is most c o m m o n l y used to reduce seizure frequency including seizures associated with acute brain lesions 31. T h e mechanism by which D P H acts, however, is still uncertain. A m o n g the p r o p o s e d mechanisms of action of D P H , the e n h a n c e m e n t of G A B A - m e d i a t e d inhibition has been r e p e a t e d l y suggested (for review, see ref. 19). The interactions b e t w e e n D P H and the G A B A system have p r o d u c e d , however, conflicting results. Phenytoin administration increases the concentration of G A B A in rat cerebral cortex 28 and in mice brain 3,22, and increases the duration of G A B A m e d i a t e d IPSPs in the crayfish stretch r e c e p t o r neuron t'2'1°, decreases p y r a m i d a l responses to m o t o r cortex stimulation 5'24, prolongs recurrent inhibition in cat p y r a m i d a l tract neurons zl, enhances postsynaptic inhibition in rat cuneate nucleus zS, in frog spinal cord 9 and in isolated mouse spinal cord neurons 19. Phenytoin also has protective effects against picrotoxin-induced b l o c k a d e of the G A B A r e c e p t o r 1°,
and against seizures induced by allyglycine, an inhibitor of G A B A synthesis 26. Conversely, D P H has been r e p o r t e d to have no effect on G A B A responses of frog spinal m o t o n e u r o n s z°, of rat 8 and cat dorsal root ganglion neurons 13, or on rat h i p p o c a m p a l pyramidal cells 15. F u r t h e r m o r e , D P H has been r e p o r t e d to diminish the K+-induced release of G A B A from rat cerebral cortex slices, without effect on the resting release of G A B A 4, and has been r e p o r t e d to augment picrotoxin-induced seizure activity 16. Thus, the role of G A B A in the effects of D P H is still a m a t t e r of controversy. In previous reports we have shown that surface application of G A B A to the s o m a t o m o t o r cortex of anesthetized cats p r o d u c e d a reversible inhibition of the negative c o m p o n e n t (N20) of the primary somatosensory e v o k e d potential 6. F u r t h e r , we have shown 7 that intracortical G A B A administration (via osmotic minipump) to the s o m a t o m o t o r area of unanesthetized young and aged rats p r o d u c e d a hemiplegic synd r o m e significantly m o r e m a r k e d than the one produced by the same cannula-induced lesion but without intracortical G A B A infusion. Thus, intracortical G A B A infusion increases the functional deficit with-
* On leave of absence from the National Institute of Sciences and Technology (INCYTAS, DIF), Mexico. Correspondence: R.T. Knight, Department of Neurology, Veterans Administration Medical Center, 150 Muir Road, Martinez, CA 94553, U.S.A. 0006-8993/86/$03.50 t~) 1986 Elsevier Science Publishers B.V. (Biomedical Division)
72 out changing the lesion size. This technique of intracortical G A B A infusion permits the evaluation of the effect of phenytoin at different levels of hemiplegic severity without introducing the complication associated with varying lesion size. MATERIALS AND METHODS
Subjects Adult male F-344 rats weighing 240-270 g were used. They were housed in groups of 6 at constant temperature (21 °C) and maintained on a standard (12:12 h) light-dark cycle, so that the light cycle occurred during working hours. The animals had free access to food and water.
Training One week after arrival, the animals were trained to walk on an elevated (45 cm above table level), narrow (2.5 cm) beam (2 m long) until they reached stable performance criteria (no slips or falls from the beam, no asymmetric positioning of the paws while walking, and no defects in paw placing on the surface of the wooden bar). Motor function on the beam was evaluated daily as described previously 7. Typically, after a motor cortex aspiration or chronic G A B A infusion into the somatomotor region, the progression of the resultant motor deficit evolves from an animal totally unable to perform the task (score = 24), to animals who show falls, slips or defects in paw placing (score range = 8-20), to animals who walk without touching the lateral aspects of the bar (score = 0). Sensory function testing was performed after completion of the motor task, and consisted of stimulation of vibrissae, trunk and dorsal and lateral surfaces of both extremities. Orientation to or withdrawal from the stimulus were considered to indicate normal sensory function.
Surgical procedures After pretreatment with atropine methylbromide (6 mg/kg s.c.), the animals were anesthetized with ketamine (40 mg/kg i.p.) followed by pentobarbital (20 mg/kg i.p.). The scalp was shaved, cleaned and infiltrated with lidocaine hydrochloride (2%). The animals were then positioned in a stereotaxic apparatus. After opening the scalp, the bone sutures were visualized. A small (2 mm) trephine was then made
over the hindlimb representation of the motor cortex 11A4'23,2 mm posterior to bregma and 2 mm lateral to the midline. After opening the dura with a needle, a beveled cannula (PE60) to which an osmotic minipump (Alza 2001) was attached was inserted through the trephine for 2 mm tangentially to the skull at an angle of approximately -30 ° to the horizontal plane. The cannula was then attached to the skull with dental acrylic, using a bone screw to assure fixation. All implants were performed on the left side. The minipumps had been previously filled with either saline or G A B A and inserted subcutaneously on the interscapular space. These devices are rated to deliver 1 pl/h for 7 days. The GABA-filled pumps were prepared to deliver 1 ktl/h of a 100 #g//d solution for 1 week. The wound was sutured and antibiotics were administered both locally and systemically.
Drug administration Once the animals had achieved stable scores on the bar running task, they were divided in 3 groups of 12 animals each. After motor and sensory testing, they were given 0.1 ml/100 g of body weight, i.p., of one of 3 solutions (prepared beforehand by a colleague and randomly marked A, B, and C): phenytoin solvent (40% propylene glycol and 10% alcohol in distilled water, adjusted to p H 12), or phenytoin at 25 and 50 mg/ml concentrations. Animals were retested on the beam 30, 60 and 120 min after injection. The purpose of this presurgical control was two-fold. Firstly, it was intended to determine the dosage level of D P H which would not result in any ataxia or other alteration of motor performance in normal rats. Secondly, it was intended to determine the effects, if any, of the D P H solvent itself. The solvent did produce a relative 'improvement' in the scores of these normal rats which was significant at the P < 0.025 level. Since no significant changes in motor skills were observed with D P H we used the larger (50 mg/kg) concentration in the main experiment. One week after this preliminary drug testing, the animals were subdivided in two groups: in one group saline-filled minipumps were implanted, and in another GABA-filled minipumps were used. These two groups were further subdivided; half of them received phenytoin solvent, and the other half received the anticonvulsant at a 50 mg/kg concentration. The response to drug administration was studied again on
73 20
[~ Before inlection I-
[]
Ll. Ill (3 rr 0 I-0
After injection
tographed and measured. The brain was then removed for further histological analysis (Cresyl violet staining). Minipumps were opened to verify for complete emptying of the reservoir.
A = GABA DPH B= S A L - S O L V
10
C= LESION-DPH D= L E S I O N - S O L V
Data evaluation The data were analyzed using an A N O V A test for repeated measures 29.
5'
RESULTS
SOLV
DPH 25
A
B
C
D
A
B
C
D
5o
PBE-SURGERY
bAYS 3-5
DAY 14
Fig. 1. Effects of phenytoin (DPH) or its solvent (SOLV) on motor performance. Scores obtained before (Pre-surgery) and after surgical implantation of an intracortical cannula (Days 3-5 and Day 14). * P < 0.05, ** P < 0.005, *** P < 0.001. Presurgery condition: no significant effects were found in animals treated with either DPH (25 or 50 mg/kg) or its solvent (n = 12/group). Days 3-5: Group A, animals implanted (on day 0) with an osmotic minipump delivering GABA (100 /~g/pl/h for 7 days) and injected with DPH (50 mg/kg) on days 3-5. Scores obtained before and 30 min after drug administration; Group B, rats infused with saline-filled minipumps implanted at day 0 and injected with DPH solvent on days 3-5; Group C, rats implanted with intracortical cannulae identical to those of groups A and B but without drug infusion. DPH (50 mg/kg) administered on days 3-5; Group D, animals treated as group C (intracortical cannulae without infusion) and injected on days 3-5 with DPH solvent. Day 14: animals treated with DPH (50 mg/kg) 14 days after cannulae implantation. Group distribution as in Days 3-5 condition. No statistically significant effects were observed in these animals.
days 3, 4 and 5 postsurgery. Motor and sensory functions were evaluated before and 30 min after injection. The experimenter was unaware of either the minipumps' content or the identity of the drugs used. Fourteen days after minipump implantation, all animals were given phenytoin (50 mg/kg i.p.) and tested as previously described.
Histology The animals were sacrificed with a barbiturate overdose and perfused intracardially with a cold buffered formaldehyde solution (10%). After perfusion, the rats were positioned on a stereotaxic apparatus, and after removal of the cannula fixture, two insect pins were inserted in symmetrical positions (2 m m from the midline) over bregma. After bone and dura were removed, the implantation site lesion was pho-
All animals reached performance criteria (a stable score on at least 3 consecutive days) after 2 weeks of training. The training was given every other day during the first week and daily thereafter.
Presurgery testing As shown in Fig. 1, neither dose of phenytoin (25 or 50 mg/kg) produced any significant changes in motor performance. In contrast, D P H solvent decreased the motor deficit score.
Minipump implantation In approximately half of the animals the extracranial end of the cannula was found to be detached from the minipump. In our previous work 7 the severity and course of recovery of animals with saline injection into motor cortex did not differ from those who had simple aspiration of motor cortex. Since the animals found to have detached cannulas had the same severity and time course of recovery as we have found in those with saline injections, it can be assumed that the cannula disconnection occurred shortly after surgery and that no intracortical solution was delivered. Thus we can consider these animals to have only a cannula induced motor cortex lesion. Accordingly, 4 groups of animals were designated as: Group A, GABA-filled minipump with D P H treatment; Group B, saline-filled minipump with solvent treatment; Group C, motor cortex lesion with D P H treatment; and Group D, motor cortex lesion with solvent treatment. The motor deficits observed in Group A ( G A B A filled minipump with phenytoin treatment) in the first 3 days after implantation were similar to the ones reported previously 7. Administration of D P H between days 3 and 5 after surgery in this group produced a transient but significant (FI, 5 = 73.42, P < 0.001) en-
74
GABA-DPH
SALINE-SOLVENT
LESION-SOLVENT
LESION-DPH
2":
Fig. 2. Dorsal view of the superficial extent of the lesions in animals from all 4 experimental groups. Two insect pins positioned at 2 mm from the midline, bilaterally, mark the bregma suture. Scale in mm.
75 hancement of the deficit (see Fig. 1). These deficits consisted in an increased incidence of slips and falls, limping and defective paw placing. All these deficits were contralateral to the implanted site for all 3 days of systemic drug administration. Animals with saline-filled minipumps (Group B) showed motor deficits in the first 3 days after implantation similar to those previously reportedT: administration of phenytoin solvent to these animals did not produce significant changes in motor performance. In the animals treated with phenytoin but without GABA infusion (Group C), a significant (F1,5 = 24.04, P < 0.005) increase in the severity of the lateralized motor deficits was observed (see Fig. 1). These abnormalities were less severe than the ones observed in group A (who had received G A B A infusions) and consisted of slips and falls, limping and abnormal positioning of the contralateral hindlimb. In Group D (motor cortex lesion and phenytoin solvent treatment), the systemic administration of the DPH solvent actually resulted in a decreased motor deficit score. It will be noted that the DPH solvent also 'improved' performance even presurgery. Although the cause of this effect is uncertain, it does suggest that the presence of the solvent in the DPH groups (A and C) actually may have attenuated the hemiplegia enhancing effects of diphenylhydantoin itself. In all groups, the observed deficits were primarily motor deficits, and no persistent sensory abnormalities were detected.
Postrecovery testing Fourteen days after surgery, and when the animals had recovered from their motor deficits, phenytoin (50 mg/kg i.p.) was administered. As seen in Fig. 1, no significant effects were produced by the drug. However, some of the animals had a transient generalized ataxia after drug administration without any sign of lateralization.
Histology All lesions were localized in a comparable area - in reference to the bregma m a r k s - - corresponding to the hindlimb representation of the somatomotor cortex 11'14'23. The structural lesions were of similar extent in all groups. In length, width and depth they measured 2.7 x 2.2 x 1.8 mm in group A, 3.2 x 2.3
x 1.9 mm in group B, 2.4 x 1.9 x 1.7 mm in group C and 2.5 x 2.1 x 1.9 mm in group D. None of these lesion dimensions were significantly different between groups (see Fig. 2). DISCUSSION All groups of animals in this study had structural lesions of the somatomotor cortex induced by the cannula which were comparable in size. However, the group of animals which received G A B A through the cannula had a significantly more dense hemiplegia than the other 3 groups that did not have G A B A infusions. The 3 non-GABA infused animal groups had a less severe hemiplegia which was not statistically different in Groups B, C and D. This finding replicates our previous observation 7 on the potentiation of hemiplegia by cortical G A B A infusions. The purpose of the present experiments was to determine whether DPH administration would increase the functional deficit produced by an acute cortical lesion as might be anticipated if DPH potentiated the inhibitory effects of GABA. In the two groups receiving DPH (A and C) a statistically significant worsening of the functional deficit was observed. This DPH effect was reversible, however, since the animals returned to their prephenytoin motor performance score by the next day. When G A B A was infused (Group A) with increase in the severity of the hemiplegia (but not changing the size of the lesion), the deleterious DPH effect on motor performance was more noticeable (see Fig. 1). It should be emphasized, however, that the larger increment in the DPHenhanced motor deficit in the G A B A treated group (A) compared to the non-GABA treated group (C) does not necessarily reflect a synergistic interaction between G A B A and DPH. The larger increment in the motor deficit score in Group A could be due in part to the non-linear characteristics of the scoring system used. For this reason, the present experiment does not provide unequivocal evidence about G A B A involvement in the reversible exacerbation of the hemiplegic syndrome produced by DPH. Nevertheless, the results suggest that DPH reversibly increases the functional deficit produced by cortical damage in the early stages of the lesion. The less severe the deficit, the less noticeable are the drug effects, and in a very mildly impaired animal, the DPH
76 effect may not be detected. The advantage of the present technique of G A B A infusion is that it can generate a m o r e m a r k e d functional deficit without the necessity of creating larger lesions 7. It is of particular interest to note that on day 14, when the m o t o r deficit score of our animals had returned to presurgical levels, no effect of D P H was detected, even in the G A B A - t r e a t e d animals. In contrast, we have r e p o r t e d 17 that systemic administration of haloperidol, a dopaminergic blocker, on day 14 after surgery caused a r e - e m e r g e n c e of the hemiplegic s y n d r o m e in animals who had G A B A infused in a p r o c e d u r e identical to the G r o u p A animals of the present study. Thus, those mechanisms involved in the expression of the effects of injury in the first week after cortical d a m a g e were refractory to D P H 2 weeks later but still c a p a b l e of being activated by haloperidol, indicating some specificity of the D P H effect. It has been r e p o r t e d that cerebral ischemia in various brain regions 12'18'3° results in increased intracere-
bral G A B A levels. W e can reasonably assume that the cerebral lesions m a d e by the cannula in all our animal groups also caused an elevation of e n d o g e n o u s G A B A . W h e t h e r or not the p o t e n t i a t i o n of the h e m p plegia by D P H is related to intracerebral G A B A levels cannot be ascertained in the present experiment, H o w e v e r , the fact that G A B A infusions increase the severity of the hemiplegia raises the interesting possibility that the deleterious D P H effects on acutely brain-injured animals might be m e d i a t e d by G A B A or G A B A - l i k e inhibitory neurotransmitters. W h e t h er or not the effects of D P H are G A B A - m e d i a t e d , the present results m a y be of clinical i m p o r t a n c e as they suggest that some p r u d e n c e be exercised in the administration of D P H in the initial days following an acute brain injury.
REFERENCES
(1981) 357-369. 9 Davidoff, R., Diphenylhydantoin increases spinal presynaptic inhibition, Trans. Amer. Neurol. Ass., 97 (1972) 193-196. 10 Deisz, R. and Lux, H., Diphenylhydantoin prolongs postsynaptic inhibition and iontophoretic GABA action in the crayfish stretch receptor, Neurosci. Lett., 5 (1977) 199-203. 11 Donoghue, J. and Wise, S., The motor cortex of the rat: cytoarchitecture and microstimulation mapping, J. Comp. Neurol., 212 (1982) 76-88. 12 Erecinska, M., Nelson, D., Wilson, D. and Silver, I., Neurotransmitter amino acids in the CNS. I. Regional changes in amino acid levels in rat brain during ischemia and reperfusion, Brain Research, 304 (1984) 9-22. 13 Gallagher, D., Inokuchi, H., Nakamura, J. and ShinnickGallagher, P., Effects of anticonvulsants on excitability and GABA sensitivity of cat dorsal root ganglion cells, Neuropharmacology, 20 (1981) 427-433. 14 Hall, R. and Lindholm, E., Organization of motor and somatosensory neocortex in the albino rat, Brain Research, 66 (1974) 23-38. 15 Hershkowitz, N. and Ayala, G., Effects of phenytoin in pyramidal neurons of the rat hippocampus, Brain Research, 208 (1981) 487-492. 16 Jones, G., Phenytoin: basic and clinical pharmacology, Med. Res. Rev., 3 (1983) 383-434. 17 Knight, R.T. and Brailowsky, S., Possible role of dopamine in the functional recovery from hemiplegia in aged rats, Soc. Neurosci. Abstr., 10 (1984) 448.
1 Aiken, C., Deisz, R. and Lux, H., On the action of the anticonvulsant 5,5-diphenylhydantoin and the convulsant picrotoxin in crayfish stretch receptor, J. Physiol. (London), 315 (1981) 157-173. 2 Ayala, G., Johnston, D., Lin, S. and Dichter, H., The mechanism of action of diphenylhydantoin on invertebrate neurons. II. Effects on synaptic mechanisms, Brain Research, 121 (1977) 259-270. 3 Battistin, L., Varotto, M., Berlese, G. and Roman, G., Effects of some anticonvulsant drugs on brain GABA level and GAD and GABA-T activities, Neurochem. Res., 9 (1984) 225-231. 4 De Belleroche, J., Dick, A. and Wyrley-Birch, A., Anticonvulsants and trifluoperazine inhibit the evoked release of GABA from cerebral cortex of rat at different sites, Life Sci., 31 (1982) 2875-2882. 5 Blum, B., A differential action of diphenylhydantoin on the motor cortex of the cat, Arch. Int. Pharmacodyn. Ther., 149 (1964) 45-55. 6 Brailowsky, S. and Knight, R., Inhibitory modulation of cat somatosensory cortex: a pharmacological study, Brain Research, 322 (1984) 310-315. 7 Brailowsky, S., Knight, R., Blood, K. and Scabini, D., GABA-induced potentiation of cortical hemiplegia, Brain Research, 362 (1986) 322-330. 8 Connors, B., A comparison of the effects of pentobarbital and diphenylhydantoin on the G A B A sensitivity and excitability of adult sensory ganglion cells, Brain Research, 207
ACKNOWLEDGEMENT This research was s u p p o r t e d by the R e s e a r c h Service of the V e t e r a n s A d m i n i s t r a t i o n .
77 18 Lust, W., Mrsulja, B., Mrsulja, B., Passonneau, J. and Klatzo, I., Putative neurotransmitters and cyclic nucleotides in prolonged ischemia of the cerebral cortex, Brain Research, 98 (1975) 394-399. 19 Macdonald, R., Barbiturate and hydantoin anticonvulsant mechanisms of action. In H.H. Jasper and N.M. van Gelder (Eds.), Basic Mechanisms of Neuronal Hyperexcitability, A.R. Liss, New York, 1983, pp. 361-387. 20 Nicoll, R. and Wojtowicz, J., The effects of pentobarbital and related compounds on frog motoneurons, Brain Research, 191 (1980) 225-237. 21 Raabe, W. and Ayala, G., Diphenylhydantoin increases cortical postsynaptic inhibition, Brain Research, 105 (1976) 597-601. 22 Saad, S., Masry, A.E. and Scott, P., Influence of certain anticonvulsants on the concentration of 7-aminobutyric acid in the cerebral hemispheres of mice, Eur. J. Pharmacol., 17 (1972) 386-392. 23 Sanderson, K., Welker, W. and Shambes, G., Reevaluation of motor cortex and of sensorimotor overlap in cerebral cortex of albino rats, Brain Research, 292 (1984) 251-260. 24 Sherwin, I., Suppressant effects of diphenylhydantoin on the cortical epileptogenic focus, Neurology, 23 (1973)
274-281. 25 Simmonds, M., Distinction between the effects of barbiturates, benzodiazepines and phenytoin on responses to 7-aminobutyric acid receptor activation and antagonism by bicuculline and picrotoxin, Br. J. Pharmacol., 73 (1981) 739-747. 26 Snead, O.C., On the sacred disease: the neurochemistry of epilepsy, Int. Rev. Neurobiol., 24 (1983) 93-180. 27 Tallarida, R., Top 200, Springer Verlag, New York, 1981, 246 pp. 28 Vernadakis, A. and Woodbury, D., Effects of diphenylhydantoin and adrenocortical steroids on free glutamic acid, glutamate and gamma-aminobutyric acid concentrations of the rat cerebral cortex. In E. Roberts (Ed.), Inhibition in the Nervous System and Gamma-Aminobutyric Acid, Pergamon, Oxford, 1960, pp. 242-248. 29 Winer, B., Statistical Principles in Experimental Design, McGraw Hill, New York, 1971. 30 Wood, J., Watson, W. and Ducker, A., The effect of hypoxia on brain gamma-aminobutyric acid levels, J. Neurochem., 15 (1968) 603-608. 31 Woodbury, D., Penry, J. and Pippenger, C. (Eds.), Antiepileptic Drugs, Raven Press, New York, 1982.