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Seizures and recovery from experimental brain damage
EXPERIMENTALNEUROLOGY
102,318-324
(1988)
Seizures and Recovery from Experimental Brain Damage’ THERESA D. HERNANDEZ’
AND
TIMOTHY
SCHALLERT~
Department of Psychology and Institute for Neurological Science, The University of Texas at Austin, Austin, Texas 78712
26). By enhancing the inhibitory GABAergic transmission after brain damage, diazepam may be augmenting neural depression (diaschisis; (66)) that occurs secondary to the lesion. Many experimental findings confirm that disruption of neuronal activity frequently occurs in regions remote from the site of brain injury (31, 47, 48, 51) and that reinstatement of normal neuronal activity accompanies recovery of certain behavioral functions (11, 13, 14, 23, 38, 68). Post-traumatic events may be responsible in part for some of the observed behavioral deficits. Diazepam may have affected recovery by preventing the termination of these events. Investigators have long considered the possibility that pharmacological manipulations might have an impact upon behavioral recovery (9, 10, 17, 28, 29, 34, 52, 69, 70). In 1945, Watson and Kennard (70) found that daily exposure to phenobarbital following unilateral motor cortex ablation profoundly retarded recovery from behavioral deficits in monkeys. Several decades later, Brailowsky and colleagues (9) reported that intracortical infusions of GABA potentiated the hemiplegia produced by motor cortex damage in rats. It was suggested that the effects were due to enhancement of depression in brain areas related to, but undamaged by, the lesion. Assuming that diazepam-induced potentiation of GABAergic activity can interfere with recovery of function after some brain lesions (52), it is possible that attenuating GABAergic activity might have the opposite effect of relative facilitation of recovery of function. We have hypothesized that post-traumatic seizures, if mild and infrequent, might serve as an endogenous source of stimulation that potentially could counteract neural depression (52). Based on this hypothesis and studies showing that amphetamine promotes recovery of motor function in rats and people (12, 19), Feeney and colleagues (18) experimentally tested the effects of electroconvulsive shock (ECS) on recovery from cortical contusions and they reported improvements in beam walking behavior. They concluded that the seizures, which may augment catecholamine activity (36, 71) and cerebral metabolism (44, 62), promoted recovery by altering “remote functional depression” of catecholaminergic and metabolic processes occurring in parallel with behavioral deficits (6, 7,21).
The effects of the y-aminobutyric acid antagonist, pentylenetetrazol (PTZ), on recovery from somatosensory and motor asymmetries after unilateral sensorimotor cortex lesions were investigated. Behavior was assessed using a bilateral tactile stimulation test and a measure of forelimb motor coordination. Immediately after surgery, the PTZ-treated and saline (SAL) control groups both exhibited severe ipsilateral behavioral asymmetries. Rats receiving PTZ recovered significantly faster from somatosensory asymmetry than those receiving SAL. Recovery was complete in the PTZ group within 3 postoperative weeks, while the SAL group failed to reach a comparable level until 2 months after surgery. There was no difference between PTZ and SAL groups on recovery of forelimb motor coordination. No difference in lesion size between the SAL and the PTZ groups could be found. These data are consistent with the hypothesis that post-traumatic neuronal depression may contribute to the severity of sensorimotor deficits observed after brain damage. o less Academic
Press,
Inc.
INTRODUCTION Chronic exposure to the anticonvulsant, diazepam, after unilateral anterior medial cortex damage has been shown to retard recovery from somatosensory asymmetry (28,29,52). We hypothesized that diazepam’s effect on recovery of function might be due in part to its enhancement of the inhibitory neurotransmitter y-aminobutyric acid (GABA) at the GABA/benzodiazepine receptor complex. This complex houses GABAA, benzodiazepine (BZ), and picrotoxin recognition sites, each of which are coupled to a chloride (Cl-) channel (25, 42, 58, 63). Diazepam, which has a high affinity for the BZ recognition site, enhances GABAergic activity by altering the sensitivity of the GABAA recognition site (25, i This work was supported by funds from National Institute of Health Grant NS-23964. We thank Dr. Tim Barth, Theresa A. Jones, and Dayne Mayfield for their help. ’ Present address: Department of Psychiatry, Yale University School of Medicine, New Haven, CT. 3 To whom reprint requests should be addressed.
0014s4&%36/38 $3.00 Copyright 0 1988 by Academic Press, All rights of reproduction in any form
318 Inc.
reserved.
EXPERIMENTAL
The present study, which was initiated prior to the publication of Feeney et al. (18), was conducted to determine the effects of a chronic regimen of the chemoconvulsant, pentylenetetrazol (PTZ), on recovery from somatosensory and motor asymmetry after unilateral sensorimotor cortex lesions in rats. PTZ is widely used in the assessment of the efficacy of anticonvulsant drugs and in animal models of epilepsy (1, 32, 39-41). When administered chronically at low doses (as in the present study), PTZ yields mild seizures, presumably by blocking GABA-induced inhibition via the picrotoxin recognition site in selected brain regions (35,42,59,63,64). METHODS
Animals In this experiment, 16 male Long-Evans hooded rats (300-450 g) were used. Animals were housed individually in wire mesh cages and maintained on a 12:12 h 1ight:dark cycle. Behavioral testing was conducted during the light portion of the cycle. Food and water were available ad Zibitum. Surgery After being anesthetized with equithesin (0.33 ml/100 g), all rats sustained unilateral sensorimotor cortex (SMC) lesions. The SMC includes the sensorimotor forepaw area described by Hall and Lindholm (27) and the more anterior forelimb motor area described by Neafsey and Sievert (37). It has been established that rats with lesions in this area show a somatosensory asymmetry for at least 6 weeks on the bilateral stimulation test (2, 3, 5). This relatively long recovery period allowed for measurement of possible facilitatory effects of the drug. Lesions were done according to the procedure of Barth and Schallert (5): A rectangular area of skull and dura overlying the forepaw SMC was removed (between 0 to +2.2 AP and 2.0 to 4.0 L) and a bare platinum electrode lowered 1.7 mm below the surface of the cortex. The electrode was lowered into one corner of the exposed cortex and 1 mA of anodal current delivered during 16 equally spaced horizontal traverses throughout the SMC for 120 s. This method has been found to be more consistent than the suction technique in yielding hemorrhage-free cortical lesions of uniform dimension (4,5). Drugs PTZ crystals, purchased from Sigma (St. Louis, MO) were dissolved in a vehicle of isotonic saline at concentrations of 45 and 30 mg/ml. The injectable solutions were made fresh weekly. Drug Regimen Postoperatively, eight of the animals received a daily regimen of PTZ and eight were given matching vehicle
BRAIN
319
DAMAGE
(SAL) injections. The initial injection of PTZ or SAL was administered approximately 16-18 h after surgery. This delay was imposed to make sure that PTZ did not differentially affect the eventual size of the brain lesion, which can change rapidly during the first 8 postoperative hours (56,57,65, 72). The rats were injected intraperitoneally with PTZ or SAL three times a day for the first 3 days and twice per day over the next 19 days. The first injection of PTZ was 45 mg/kg and the second and third injections were 30 mg/kg on the first day. Thereafter, all injections were 15 mg/kg each. A 45 mg/kg dose is 75% of the EDs0 to induce a full seizure (40) and was chosen to decrease the likelihood of brain damage often associated with severe seizures (16, 60). In addition, the dose was decreased over days in an effort to avoid “pharmacological kindling” (i.e., intensified responses leading to seizures following repeated administration of certain drugs) which may lead to brain damage (33,45,46,50). So that intoxication would not be a factor, all behavioral tests were conducted before daily drug treatment (at 0900 h, 16 h after the last injection of the previous day). Drug administration was discontinued after 22 postoperative days; however, behavioral testing continued for 60 to 90 days to determine whether the effects were enduring. Assessment
of Post-injection
Convulsive
Behavior
Using a seizure rating scale similar to that of Ito et al. (30), animals were observed for 20-30 min following each injection and given a rating from 0 to 4. The scale used was as follows: 0 = no convulsive behavior; 1 = head or body twitching; 2 = clonic forelimb convulsions; 3 = rearing (“violent convulsion”); 4 = falling back. Somatosensory
Tests
Somatosensory asymmetry was assessed both preand postoperatively using the bilateral tactile stimulation tests originally developed to assess sensory function following damage to the dopaminergic input to the striaturn (53, 54). The experimenters were blind as to the treatment of the animals being examined. The first test indicates the presence or absence of an asymmetry. The second test measures the magnitude of that asymmetry. The procedures have been described in detail elsewhere (52, 54, 55). Briefly, animals were removed from their home cage so that adhesive stimuli (Avery adhesivebacked labels) could be attached to the distal-radial region of both forelimbs. Upon return to the home cage, rats typically contact and remove each stimulus one at a time using their teeth. The latency and order of stimulus contact/removal were recorded on at least four trials. Using the first test (in which adhesive stimuli are of equal size: 113 mm2), it has been reported that after unilateral cortical lesions, rats will consistently respond
320
HERNANDEZ
AND
first to an adhesive stimulus located on the forelimb ipsilateral to the lesion, despite the simultaneous presence of contralateral stimulation (11, 29, 49, 52, 55). As an animal recovers, there is a gradual reduction in the ipsilaterally biased asymmetry. Preoperatively, if an animal preferentially responded to either the right or the left stimulus, lesions were made in the opposite hemisphere so that any endogenous asymmetry did not mask postoperative lesion effects. All rats with an ipsilateral bias (i.e., on greater than 70% of the trials the ipsilateral stimulus was contacted first) were given an additional test in which the magnitude of their asymmetry was measured. In this more sensitive test, the size of the contralateral (C) stimulus was progressively increased and the size of the ipsilateral (I) stimulus was simultaneously decreased by an equal amount (14.1 mm2; see vertical axis, Fig. 1). With sufficient increase in the C/I ratio, an ipsilateral bias can be neutralized; i.e., the rat will no longer respond preferentially to the stimulus ipsilateral to the lesion. This latter test provides a more sensitive measure of somatosensory asymmetry for two reasons. First, the C/1 ratio necessary to neutralize an ipsilateral bias is proportional to the degree of brain damage, and second, as an animal recovers, this ratio progressively decreases (3,54,55). Foot-Fault
Test
Animals were placed on an elevated, (8.5 mm) grid floor (34 X 29 mm) for 2 min (11). The openings in the grid were 3 mm2. While moving around on the grid, animals typically placed their paws on the wire frame for foot holds. Occasionally, intact animals inaccurately placed a limb (fore or hind), and it fell through one of the openings in the grid (i.e., “foot fault”). Limb misplacements in intact animals were typically symmetrical. The number of “foot faults” for both fore- and hindlimbs and the total number of forelimb steps were recorded for each animal pre- and postoperatively. A primary measure of interest was the percentage of contralateral forelimb foot faults per forelimb step minus the percentage ipsilateral forelimb foot faults per forelimb step (i.e., the contralateralfoot-fault index, adjusted for ipsilateral forelimb foot faults and calculated as a percentage of total forelimb steps). This test may be similar to the elevated narrow beam discussed in the literature (8-10, 19) in that it is designed to assess limb use and placing deficits during locomotion. RESULTS
Statistical
Analyses
A two-way analysis of variance (ANOVA) between the drug and the vehicle groups was performed to determine whether PTZ affected recovery from sensorimotor asymmetry. Post hoc univariate F tests were used to
SCHALLERT
TABLE
1
Percentage of Trials in Which the Ipsilateral Adhesive Stimulus Was Contacted First” Days Groups
2
SAL
100 (+o)
PTZ
95.0 (k3.5)
after
SMC
lesion
6
16
22
97.2 (22.7)
96.0 (k4.0)
77.5 (f12.5)
70.0 (+13.8)
’ Data are means f SEM. * Significantly different from ate F test, P < 0.05.
SAL-treated
45
60
92.5 (k3.9)
83.5 (k7.0)
67.0 (k10.2)
5&o* (e13.5)
56.2* (k9.0)
67.5 (f7.8)
group,
post hoc univari-
compare group differences on several postoperative days. Convulsive Behavioral Rating The initial injection of 45 mg/kg (75% of the ED5,-,for a full seizure) produced clonic forelimb convulsions and/ or rearing in all animals (rating score = 2.5). It is possible that 18 h after electrolytic lesion, the seizure threshold was lower than it would have been in intact rats. However, as the dose was decreased over days, there was a concomitant decrease in convulsive behavior. The ratings following the second and third injections were 1.5 and 1.0, respectively. Between Days 2 and 22 the majority of the animals exhibited head and/or body twitching (score of 1) and the remainder received scores of 0 (rating = 0.75). Thus, there was no behavioral evidence to suggest that pharmacological kindling occurred. The behavioral effects of this regimen could be evidence of low-level EEG seizure activity. Repeated administration of a 40 mg/kg dose of PTZ produces characteristic behavioral changes (head twitching and clonic movement) that correlate with a ~-HZ spike and wave EEG pattern (30). In addition, an even lower dose (10 or 20 mg/kg) of PTZ produces EEG patterns similar to those of spontaneous seizures (32). This evidence suggests, but does not confirm, that the PTZ regimen used in the present study produced neuronal seizure activity. Somatosensory Tests Behavioral data from representative postoperative days are shown in Table 1. Two-way ANOVA revealed a significant difference between the groups (Fl,14 = 8.04, P < 0.05) and a significant difference over postoperative days (F7,9s= 5.96, P < 0.05). However, there was not a significant group X day interaction (F7,98 = 1.99, P > 0.05). When tested 2 days after lesion, both groups exhibited an equivalent, severe ipsilateral asymmetry (post hoc univariate F tests; Fl,14 = 3.09, P > 0.05). Both PTZ- and
EXPERIMENTAL
BRAIN
OIllI
246
I
I
I
I
I
L
IO
16
22
30
45
60
Days
AfterSMC
Lesion
FIG. 1. The mean magnitude (HEM) of an ipsilateral somatosensory asymmetry is plotted across days after unilateral lesions of the sensorimotor cortex (SMC) in rats receiving a regimen of saline (SAL) or pentylenetetrazol (PTZ). Adjacent to each magnitude level (l-7) are representative stimulus pairs.
SAL-treated rats contacted the stimulus ipsilateral to the lesion first on over 95% of the trials. Over a 3-week period, only the PTZ group displayed a significant reduction in ipsilateral asymmetry (Day 22, PTZ = 58.0 + 13.5% vs SAL = 92.5 + 3.9%). By Day 60 postlesion, the SAL group (67.0 + 10.2%) was not significantly different from the PTZ group (67.5 t 7.8%). Figure 1 shows the magnitude of asymmetry over days following SMC lesions. Two-way ANOVA revealed a significant main effect for group (F1,14= 25.2, P < 0.05), days (F8,112= 24.1, P < 0.05), and interaction (Fs,lln = 2.8, P < 0.05). Two days after lesion, the magnitude of ipsilateral asymmetry for the PTZ group (5.3 + 0.33) was not significantly different from that of the SAL-treated rats (5.5 + 0.34) (post hoc univariate Ftests; Fl,14 = 0.28, P > 0.05). This level of ipsilateral asymmetry meant that the contralateral stimulus needed to be at least seven times larger than the simultaneously applied ipsilateral stimulus in order to neutralize the ipsilateral bias. The magnitude decreased within the first week after surgery in the PTZ-treated rats (from 5.3 + 0.33 to 2.1 & 0.76). During this time there was little change in the SAL group (from 5.5 -t 0.34 to 5.3 ? 0.40). Two weeks later, the PTZ group (0.25 f 0.53) recovered to 0.25 f 0.53, which was near preoperative level. This was significantly different from SAL rats (3.4 f 0.64) (post hoc univariate F tests; Fl,14 = 14.06, P < 0.05). Although the SAL-treated rats continued to recover (1.61 f 0.65 at Day 60), they had not achieved the same level of recovery as those in the PTZ group (0.44 f 0.24) 2 months after lesion (post hoc univariate F tests; FI,14 = 2.83, P < 0.05). Foot-Fault Test It is important to note that PTZ administration was discontinued on Day 22, yet recovery from ipsilateral
321
DAMAGE
asymmetry endured through 60 postoperative days. This suggests that the neutralizing effects of PTZ on sensorimotor asymmetry were not dependent upon PTZ recently being in the system. In Table 2, the percentage of contralateral forelimb foot faults per forelimb step minus the percentage of ipsilateral forelimb foot faults per forelimb step is displayed for selected days after SMC lesion in PTZ- and SAL-treated rats. Two days after lesion, the lesion-induced contralateral forelimb placing deficit did not differ between groups (group effect, F,,,,, = 2.38, P > 0.05). Both groups recovered significantly (day effect, F5,50= 3.26, P < 0.05) and at an equivalent rate (group X day effect F5,50= 0.46, P > 0.05) during the next 3 weeks and this level of recovery endured through 45 days after surgery. Histological Analysis All animals with lesions were given a lethal dose of anesthesia and perfused intracardially with saline followed by a 10% formalin solution. Their brains were extracted and frozen sections (40 pm) beginning anterior to the lesion and continuing through the hippocampus were mounted and stained with cresyl violet. Analysis of the hippocampus was necessary because of studies reporting “epileptic” brain damage in the hippocampus due to central injections of excitatory amino acids (60, 61). Sections were compared with rat brain atlases (43, 74). Using a Bausch and Lomb microprojector, an investigator who was blind to the treatment condition traced the total amount of damage (as measured by remaining lesioned and unlesioned cortex between +1.7 and -0.3 AP) onto a bit pad connected to an IBM AT computer. There was no significant group difference in the extent of damage (as assessedby the area of remaining dorsal cortex on the lesioned side, two-way ANOVA, F,,,, = 0.73, P > 0.05). Figure 2 shows the location of the sensorimotor cortex lesions. The common areas damaged included the posterior aspect of the forelimb sensorimotor area (27) and the more rostra1 forelimb motor area (37).
TABLE
2
Contralateral Forelimb Foot-Fault IndexaJ’ Daysafter Groups
SMC
lesion
2
4
7
14
21
SAL
22.8 (k7.0)
17.6 (s3.0)
17.3 (T9.2)
14.1 (k9.0)
2.8 (k1.2)
PTZ
14.4 (f5.0)
15.0 (k3.0)
11.0
14.9 (210.0)
5.2
’ Percentage of contralateral minus the percentage ipsilateral * Data are means + SEM.
(k2.0)
forelimb forelimb
foot faults foot faults
(21.0) per forelimb per forelimb
45
(20, 3.0 C-r-2.0)
step step.
322 HERNANDEZ
FIG. 2. lesions.
Schematic
representation damage;
n = area of common
of sensorimotor •4 = largest extent
AND
cortex @MC) of damage.
DISCUSSION
A chronic regimen of PTZ appeared to enhance the rate of recovery from somatosensory asymmetry following unilateral lesions of the sensorimotor cortex. That is, relative to saline control treatment, daily administration of PTZ diminished the severity of somatosensory deficit, as measured by the bilateral tactile stimulation test. Without PTZ, recovery proceeded at a rate comparable to that observed in untreated SMC-damaged rats in a previous investigation (5). PTZ did not significantly affect recovery from forelimb foot-fault deficits. This could be related to the different nature of each task. The rate of forelimb motor coordination recovery observed in brain-damaged animals may be influenced greatly by practice, or motor learning (28, 29). Conversely, the rate of recovery from somatosensory asymmetry measured by the bilateral tactile stimulation tests does not seem to be susceptible to practice (49, 55). Recovery from unilateral SMC lesions may involve two distinct behaviorally relevant processes,one of which is sensitive to PTZ and one which is resistant. These data mirror the effects of diazepam on behavioral recovery (29). That is, diazepam disrupted recovery from somatosensory asymmetry measured by
SCHALLERT
the bilateral stimulation test, but had no effect on recovery measured by a forelimb placing test. The apparent resistance of forelimb motor coordination deficits to PTZ might seem surprising when compared to the study of Feeney et al. (19), who reported amphetamine-induced facilitation of recovery from a beam walking deficit. However, it is noteworthy that this facilitation occurred only if the animal had experience with the task while it was under the influence of the drug. Recall that our animals were not tested for at least 16 h after the last injection of the previous day and were probably not intoxicated at this time. The half-life of PTZ in the rodent brain appears to be approximately 16-21 min after intraperitoneal injection and brain PTZ levels drop 90% within 60 min after injection (73). Perhaps if we had allowed experience with our test of motor ability soon after PTZ injection, recovery of motor function might have been affected significantly. Chemoconvulsants can augment cerebral metabolism (44), perhaps by attenuating GABAergic activity at the GABA-BZ receptor complex (35,42,59,63,64). Attenuation of GABA-induced inhibition may alleviate posttraumatic neuronal disruption associated with behavioral deficits (11, 13, 14,20,68), thereby aiding in recovery from those deficits. Previous experimental evidence showing that the potentiation of GABA-induced inhibition is detrimental to recovery of function is not inconsistent with this hypothesis (9, 10,28,29, 70). Alternatively, it is possible that PTZ facilitated recovery from somatosensory asymmetry by interfering with neuronal activity in the contralateral hemisphere. Bilateral cortical damage would produce somatosensory symmetry with no reduction (relative to controls) in the latency to respond to the adhesive stimuli (2). However, there was no histological evidence to suggest nonspecific damage in the contralateral cortex, striatum, or hippocampus of either hemisphere. Nevertheless, it cannot be ruled out that PTZ caused undetectable dysfunction in the SMC or related tissue in the contralateral hemisphere. In people, seizures frequently accompany brain injury (22, 24,67). The use of anticonvulsants to prevent posttraumatic seizures, though not universally accepted, is rather common despite a lack of systematic studies of the effects on subsequent behavioral recovery (15). If seizures, as sequelae of brain injury, can attenuate neuronal depression (l&52), then a partial, rather than complete, prevention of seizures (by limiting the dose or frequency of anticonvulsant therapy) might favorably affect behavioral recovery in certain cases.A clinical characterization of post-traumatic behavioral deficits in relation to the occurrence of seizures and/or the use of anticonvulsants is necessary for the resolution of this issue. REFERENCES 1. ALBERTSON,T.E.,S.L.PETERSON,ANDL.G.STARK.~~C~~.T~~ anticonvulsant
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102,318-324
(1988)
Seizures and Recovery from Experimental Brain Damage’ THERESA D. HERNANDEZ’
AND
TIMOTHY
SCHALLERT~
Department of Psychology and Institute for Neurological Science, The University of Texas at Austin, Austin, Texas 78712
26). By enhancing the inhibitory GABAergic transmission after brain damage, diazepam may be augmenting neural depression (diaschisis; (66)) that occurs secondary to the lesion. Many experimental findings confirm that disruption of neuronal activity frequently occurs in regions remote from the site of brain injury (31, 47, 48, 51) and that reinstatement of normal neuronal activity accompanies recovery of certain behavioral functions (11, 13, 14, 23, 38, 68). Post-traumatic events may be responsible in part for some of the observed behavioral deficits. Diazepam may have affected recovery by preventing the termination of these events. Investigators have long considered the possibility that pharmacological manipulations might have an impact upon behavioral recovery (9, 10, 17, 28, 29, 34, 52, 69, 70). In 1945, Watson and Kennard (70) found that daily exposure to phenobarbital following unilateral motor cortex ablation profoundly retarded recovery from behavioral deficits in monkeys. Several decades later, Brailowsky and colleagues (9) reported that intracortical infusions of GABA potentiated the hemiplegia produced by motor cortex damage in rats. It was suggested that the effects were due to enhancement of depression in brain areas related to, but undamaged by, the lesion. Assuming that diazepam-induced potentiation of GABAergic activity can interfere with recovery of function after some brain lesions (52), it is possible that attenuating GABAergic activity might have the opposite effect of relative facilitation of recovery of function. We have hypothesized that post-traumatic seizures, if mild and infrequent, might serve as an endogenous source of stimulation that potentially could counteract neural depression (52). Based on this hypothesis and studies showing that amphetamine promotes recovery of motor function in rats and people (12, 19), Feeney and colleagues (18) experimentally tested the effects of electroconvulsive shock (ECS) on recovery from cortical contusions and they reported improvements in beam walking behavior. They concluded that the seizures, which may augment catecholamine activity (36, 71) and cerebral metabolism (44, 62), promoted recovery by altering “remote functional depression” of catecholaminergic and metabolic processes occurring in parallel with behavioral deficits (6, 7,21).
The effects of the y-aminobutyric acid antagonist, pentylenetetrazol (PTZ), on recovery from somatosensory and motor asymmetries after unilateral sensorimotor cortex lesions were investigated. Behavior was assessed using a bilateral tactile stimulation test and a measure of forelimb motor coordination. Immediately after surgery, the PTZ-treated and saline (SAL) control groups both exhibited severe ipsilateral behavioral asymmetries. Rats receiving PTZ recovered significantly faster from somatosensory asymmetry than those receiving SAL. Recovery was complete in the PTZ group within 3 postoperative weeks, while the SAL group failed to reach a comparable level until 2 months after surgery. There was no difference between PTZ and SAL groups on recovery of forelimb motor coordination. No difference in lesion size between the SAL and the PTZ groups could be found. These data are consistent with the hypothesis that post-traumatic neuronal depression may contribute to the severity of sensorimotor deficits observed after brain damage. o less Academic
Press,
Inc.
INTRODUCTION Chronic exposure to the anticonvulsant, diazepam, after unilateral anterior medial cortex damage has been shown to retard recovery from somatosensory asymmetry (28,29,52). We hypothesized that diazepam’s effect on recovery of function might be due in part to its enhancement of the inhibitory neurotransmitter y-aminobutyric acid (GABA) at the GABA/benzodiazepine receptor complex. This complex houses GABAA, benzodiazepine (BZ), and picrotoxin recognition sites, each of which are coupled to a chloride (Cl-) channel (25, 42, 58, 63). Diazepam, which has a high affinity for the BZ recognition site, enhances GABAergic activity by altering the sensitivity of the GABAA recognition site (25, i This work was supported by funds from National Institute of Health Grant NS-23964. We thank Dr. Tim Barth, Theresa A. Jones, and Dayne Mayfield for their help. ’ Present address: Department of Psychiatry, Yale University School of Medicine, New Haven, CT. 3 To whom reprint requests should be addressed.
0014s4&%36/38 $3.00 Copyright 0 1988 by Academic Press, All rights of reproduction in any form
318 Inc.
reserved.
EXPERIMENTAL
The present study, which was initiated prior to the publication of Feeney et al. (18), was conducted to determine the effects of a chronic regimen of the chemoconvulsant, pentylenetetrazol (PTZ), on recovery from somatosensory and motor asymmetry after unilateral sensorimotor cortex lesions in rats. PTZ is widely used in the assessment of the efficacy of anticonvulsant drugs and in animal models of epilepsy (1, 32, 39-41). When administered chronically at low doses (as in the present study), PTZ yields mild seizures, presumably by blocking GABA-induced inhibition via the picrotoxin recognition site in selected brain regions (35,42,59,63,64). METHODS
Animals In this experiment, 16 male Long-Evans hooded rats (300-450 g) were used. Animals were housed individually in wire mesh cages and maintained on a 12:12 h 1ight:dark cycle. Behavioral testing was conducted during the light portion of the cycle. Food and water were available ad Zibitum. Surgery After being anesthetized with equithesin (0.33 ml/100 g), all rats sustained unilateral sensorimotor cortex (SMC) lesions. The SMC includes the sensorimotor forepaw area described by Hall and Lindholm (27) and the more anterior forelimb motor area described by Neafsey and Sievert (37). It has been established that rats with lesions in this area show a somatosensory asymmetry for at least 6 weeks on the bilateral stimulation test (2, 3, 5). This relatively long recovery period allowed for measurement of possible facilitatory effects of the drug. Lesions were done according to the procedure of Barth and Schallert (5): A rectangular area of skull and dura overlying the forepaw SMC was removed (between 0 to +2.2 AP and 2.0 to 4.0 L) and a bare platinum electrode lowered 1.7 mm below the surface of the cortex. The electrode was lowered into one corner of the exposed cortex and 1 mA of anodal current delivered during 16 equally spaced horizontal traverses throughout the SMC for 120 s. This method has been found to be more consistent than the suction technique in yielding hemorrhage-free cortical lesions of uniform dimension (4,5). Drugs PTZ crystals, purchased from Sigma (St. Louis, MO) were dissolved in a vehicle of isotonic saline at concentrations of 45 and 30 mg/ml. The injectable solutions were made fresh weekly. Drug Regimen Postoperatively, eight of the animals received a daily regimen of PTZ and eight were given matching vehicle
BRAIN
319
DAMAGE
(SAL) injections. The initial injection of PTZ or SAL was administered approximately 16-18 h after surgery. This delay was imposed to make sure that PTZ did not differentially affect the eventual size of the brain lesion, which can change rapidly during the first 8 postoperative hours (56,57,65, 72). The rats were injected intraperitoneally with PTZ or SAL three times a day for the first 3 days and twice per day over the next 19 days. The first injection of PTZ was 45 mg/kg and the second and third injections were 30 mg/kg on the first day. Thereafter, all injections were 15 mg/kg each. A 45 mg/kg dose is 75% of the EDs0 to induce a full seizure (40) and was chosen to decrease the likelihood of brain damage often associated with severe seizures (16, 60). In addition, the dose was decreased over days in an effort to avoid “pharmacological kindling” (i.e., intensified responses leading to seizures following repeated administration of certain drugs) which may lead to brain damage (33,45,46,50). So that intoxication would not be a factor, all behavioral tests were conducted before daily drug treatment (at 0900 h, 16 h after the last injection of the previous day). Drug administration was discontinued after 22 postoperative days; however, behavioral testing continued for 60 to 90 days to determine whether the effects were enduring. Assessment
of Post-injection
Convulsive
Behavior
Using a seizure rating scale similar to that of Ito et al. (30), animals were observed for 20-30 min following each injection and given a rating from 0 to 4. The scale used was as follows: 0 = no convulsive behavior; 1 = head or body twitching; 2 = clonic forelimb convulsions; 3 = rearing (“violent convulsion”); 4 = falling back. Somatosensory
Tests
Somatosensory asymmetry was assessed both preand postoperatively using the bilateral tactile stimulation tests originally developed to assess sensory function following damage to the dopaminergic input to the striaturn (53, 54). The experimenters were blind as to the treatment of the animals being examined. The first test indicates the presence or absence of an asymmetry. The second test measures the magnitude of that asymmetry. The procedures have been described in detail elsewhere (52, 54, 55). Briefly, animals were removed from their home cage so that adhesive stimuli (Avery adhesivebacked labels) could be attached to the distal-radial region of both forelimbs. Upon return to the home cage, rats typically contact and remove each stimulus one at a time using their teeth. The latency and order of stimulus contact/removal were recorded on at least four trials. Using the first test (in which adhesive stimuli are of equal size: 113 mm2), it has been reported that after unilateral cortical lesions, rats will consistently respond
320
HERNANDEZ
AND
first to an adhesive stimulus located on the forelimb ipsilateral to the lesion, despite the simultaneous presence of contralateral stimulation (11, 29, 49, 52, 55). As an animal recovers, there is a gradual reduction in the ipsilaterally biased asymmetry. Preoperatively, if an animal preferentially responded to either the right or the left stimulus, lesions were made in the opposite hemisphere so that any endogenous asymmetry did not mask postoperative lesion effects. All rats with an ipsilateral bias (i.e., on greater than 70% of the trials the ipsilateral stimulus was contacted first) were given an additional test in which the magnitude of their asymmetry was measured. In this more sensitive test, the size of the contralateral (C) stimulus was progressively increased and the size of the ipsilateral (I) stimulus was simultaneously decreased by an equal amount (14.1 mm2; see vertical axis, Fig. 1). With sufficient increase in the C/I ratio, an ipsilateral bias can be neutralized; i.e., the rat will no longer respond preferentially to the stimulus ipsilateral to the lesion. This latter test provides a more sensitive measure of somatosensory asymmetry for two reasons. First, the C/1 ratio necessary to neutralize an ipsilateral bias is proportional to the degree of brain damage, and second, as an animal recovers, this ratio progressively decreases (3,54,55). Foot-Fault
Test
Animals were placed on an elevated, (8.5 mm) grid floor (34 X 29 mm) for 2 min (11). The openings in the grid were 3 mm2. While moving around on the grid, animals typically placed their paws on the wire frame for foot holds. Occasionally, intact animals inaccurately placed a limb (fore or hind), and it fell through one of the openings in the grid (i.e., “foot fault”). Limb misplacements in intact animals were typically symmetrical. The number of “foot faults” for both fore- and hindlimbs and the total number of forelimb steps were recorded for each animal pre- and postoperatively. A primary measure of interest was the percentage of contralateral forelimb foot faults per forelimb step minus the percentage ipsilateral forelimb foot faults per forelimb step (i.e., the contralateralfoot-fault index, adjusted for ipsilateral forelimb foot faults and calculated as a percentage of total forelimb steps). This test may be similar to the elevated narrow beam discussed in the literature (8-10, 19) in that it is designed to assess limb use and placing deficits during locomotion. RESULTS
Statistical
Analyses
A two-way analysis of variance (ANOVA) between the drug and the vehicle groups was performed to determine whether PTZ affected recovery from sensorimotor asymmetry. Post hoc univariate F tests were used to
SCHALLERT
TABLE
1
Percentage of Trials in Which the Ipsilateral Adhesive Stimulus Was Contacted First” Days Groups
2
SAL
100 (+o)
PTZ
95.0 (k3.5)
after
SMC
lesion
6
16
22
97.2 (22.7)
96.0 (k4.0)
77.5 (f12.5)
70.0 (+13.8)
’ Data are means f SEM. * Significantly different from ate F test, P < 0.05.
SAL-treated
45
60
92.5 (k3.9)
83.5 (k7.0)
67.0 (k10.2)
5&o* (e13.5)
56.2* (k9.0)
67.5 (f7.8)
group,
post hoc univari-
compare group differences on several postoperative days. Convulsive Behavioral Rating The initial injection of 45 mg/kg (75% of the ED5,-,for a full seizure) produced clonic forelimb convulsions and/ or rearing in all animals (rating score = 2.5). It is possible that 18 h after electrolytic lesion, the seizure threshold was lower than it would have been in intact rats. However, as the dose was decreased over days, there was a concomitant decrease in convulsive behavior. The ratings following the second and third injections were 1.5 and 1.0, respectively. Between Days 2 and 22 the majority of the animals exhibited head and/or body twitching (score of 1) and the remainder received scores of 0 (rating = 0.75). Thus, there was no behavioral evidence to suggest that pharmacological kindling occurred. The behavioral effects of this regimen could be evidence of low-level EEG seizure activity. Repeated administration of a 40 mg/kg dose of PTZ produces characteristic behavioral changes (head twitching and clonic movement) that correlate with a ~-HZ spike and wave EEG pattern (30). In addition, an even lower dose (10 or 20 mg/kg) of PTZ produces EEG patterns similar to those of spontaneous seizures (32). This evidence suggests, but does not confirm, that the PTZ regimen used in the present study produced neuronal seizure activity. Somatosensory Tests Behavioral data from representative postoperative days are shown in Table 1. Two-way ANOVA revealed a significant difference between the groups (Fl,14 = 8.04, P < 0.05) and a significant difference over postoperative days (F7,9s= 5.96, P < 0.05). However, there was not a significant group X day interaction (F7,98 = 1.99, P > 0.05). When tested 2 days after lesion, both groups exhibited an equivalent, severe ipsilateral asymmetry (post hoc univariate F tests; Fl,14 = 3.09, P > 0.05). Both PTZ- and
EXPERIMENTAL
BRAIN
OIllI
246
I
I
I
I
I
L
IO
16
22
30
45
60
Days
AfterSMC
Lesion
FIG. 1. The mean magnitude (HEM) of an ipsilateral somatosensory asymmetry is plotted across days after unilateral lesions of the sensorimotor cortex (SMC) in rats receiving a regimen of saline (SAL) or pentylenetetrazol (PTZ). Adjacent to each magnitude level (l-7) are representative stimulus pairs.
SAL-treated rats contacted the stimulus ipsilateral to the lesion first on over 95% of the trials. Over a 3-week period, only the PTZ group displayed a significant reduction in ipsilateral asymmetry (Day 22, PTZ = 58.0 + 13.5% vs SAL = 92.5 + 3.9%). By Day 60 postlesion, the SAL group (67.0 + 10.2%) was not significantly different from the PTZ group (67.5 t 7.8%). Figure 1 shows the magnitude of asymmetry over days following SMC lesions. Two-way ANOVA revealed a significant main effect for group (F1,14= 25.2, P < 0.05), days (F8,112= 24.1, P < 0.05), and interaction (Fs,lln = 2.8, P < 0.05). Two days after lesion, the magnitude of ipsilateral asymmetry for the PTZ group (5.3 + 0.33) was not significantly different from that of the SAL-treated rats (5.5 + 0.34) (post hoc univariate Ftests; Fl,14 = 0.28, P > 0.05). This level of ipsilateral asymmetry meant that the contralateral stimulus needed to be at least seven times larger than the simultaneously applied ipsilateral stimulus in order to neutralize the ipsilateral bias. The magnitude decreased within the first week after surgery in the PTZ-treated rats (from 5.3 + 0.33 to 2.1 & 0.76). During this time there was little change in the SAL group (from 5.5 -t 0.34 to 5.3 ? 0.40). Two weeks later, the PTZ group (0.25 f 0.53) recovered to 0.25 f 0.53, which was near preoperative level. This was significantly different from SAL rats (3.4 f 0.64) (post hoc univariate F tests; Fl,14 = 14.06, P < 0.05). Although the SAL-treated rats continued to recover (1.61 f 0.65 at Day 60), they had not achieved the same level of recovery as those in the PTZ group (0.44 f 0.24) 2 months after lesion (post hoc univariate F tests; FI,14 = 2.83, P < 0.05). Foot-Fault Test It is important to note that PTZ administration was discontinued on Day 22, yet recovery from ipsilateral
321
DAMAGE
asymmetry endured through 60 postoperative days. This suggests that the neutralizing effects of PTZ on sensorimotor asymmetry were not dependent upon PTZ recently being in the system. In Table 2, the percentage of contralateral forelimb foot faults per forelimb step minus the percentage of ipsilateral forelimb foot faults per forelimb step is displayed for selected days after SMC lesion in PTZ- and SAL-treated rats. Two days after lesion, the lesion-induced contralateral forelimb placing deficit did not differ between groups (group effect, F,,,,, = 2.38, P > 0.05). Both groups recovered significantly (day effect, F5,50= 3.26, P < 0.05) and at an equivalent rate (group X day effect F5,50= 0.46, P > 0.05) during the next 3 weeks and this level of recovery endured through 45 days after surgery. Histological Analysis All animals with lesions were given a lethal dose of anesthesia and perfused intracardially with saline followed by a 10% formalin solution. Their brains were extracted and frozen sections (40 pm) beginning anterior to the lesion and continuing through the hippocampus were mounted and stained with cresyl violet. Analysis of the hippocampus was necessary because of studies reporting “epileptic” brain damage in the hippocampus due to central injections of excitatory amino acids (60, 61). Sections were compared with rat brain atlases (43, 74). Using a Bausch and Lomb microprojector, an investigator who was blind to the treatment condition traced the total amount of damage (as measured by remaining lesioned and unlesioned cortex between +1.7 and -0.3 AP) onto a bit pad connected to an IBM AT computer. There was no significant group difference in the extent of damage (as assessedby the area of remaining dorsal cortex on the lesioned side, two-way ANOVA, F,,,, = 0.73, P > 0.05). Figure 2 shows the location of the sensorimotor cortex lesions. The common areas damaged included the posterior aspect of the forelimb sensorimotor area (27) and the more rostra1 forelimb motor area (37).
TABLE
2
Contralateral Forelimb Foot-Fault IndexaJ’ Daysafter Groups
SMC
lesion
2
4
7
14
21
SAL
22.8 (k7.0)
17.6 (s3.0)
17.3 (T9.2)
14.1 (k9.0)
2.8 (k1.2)
PTZ
14.4 (f5.0)
15.0 (k3.0)
11.0
14.9 (210.0)
5.2
’ Percentage of contralateral minus the percentage ipsilateral * Data are means + SEM.
(k2.0)
forelimb forelimb
foot faults foot faults
(21.0) per forelimb per forelimb
45
(20, 3.0 C-r-2.0)
step step.
322 HERNANDEZ
FIG. 2. lesions.
Schematic
representation damage;
n = area of common
of sensorimotor •4 = largest extent
AND
cortex @MC) of damage.
DISCUSSION
A chronic regimen of PTZ appeared to enhance the rate of recovery from somatosensory asymmetry following unilateral lesions of the sensorimotor cortex. That is, relative to saline control treatment, daily administration of PTZ diminished the severity of somatosensory deficit, as measured by the bilateral tactile stimulation test. Without PTZ, recovery proceeded at a rate comparable to that observed in untreated SMC-damaged rats in a previous investigation (5). PTZ did not significantly affect recovery from forelimb foot-fault deficits. This could be related to the different nature of each task. The rate of forelimb motor coordination recovery observed in brain-damaged animals may be influenced greatly by practice, or motor learning (28, 29). Conversely, the rate of recovery from somatosensory asymmetry measured by the bilateral tactile stimulation tests does not seem to be susceptible to practice (49, 55). Recovery from unilateral SMC lesions may involve two distinct behaviorally relevant processes,one of which is sensitive to PTZ and one which is resistant. These data mirror the effects of diazepam on behavioral recovery (29). That is, diazepam disrupted recovery from somatosensory asymmetry measured by
SCHALLERT
the bilateral stimulation test, but had no effect on recovery measured by a forelimb placing test. The apparent resistance of forelimb motor coordination deficits to PTZ might seem surprising when compared to the study of Feeney et al. (19), who reported amphetamine-induced facilitation of recovery from a beam walking deficit. However, it is noteworthy that this facilitation occurred only if the animal had experience with the task while it was under the influence of the drug. Recall that our animals were not tested for at least 16 h after the last injection of the previous day and were probably not intoxicated at this time. The half-life of PTZ in the rodent brain appears to be approximately 16-21 min after intraperitoneal injection and brain PTZ levels drop 90% within 60 min after injection (73). Perhaps if we had allowed experience with our test of motor ability soon after PTZ injection, recovery of motor function might have been affected significantly. Chemoconvulsants can augment cerebral metabolism (44), perhaps by attenuating GABAergic activity at the GABA-BZ receptor complex (35,42,59,63,64). Attenuation of GABA-induced inhibition may alleviate posttraumatic neuronal disruption associated with behavioral deficits (11, 13, 14,20,68), thereby aiding in recovery from those deficits. Previous experimental evidence showing that the potentiation of GABA-induced inhibition is detrimental to recovery of function is not inconsistent with this hypothesis (9, 10,28,29, 70). Alternatively, it is possible that PTZ facilitated recovery from somatosensory asymmetry by interfering with neuronal activity in the contralateral hemisphere. Bilateral cortical damage would produce somatosensory symmetry with no reduction (relative to controls) in the latency to respond to the adhesive stimuli (2). However, there was no histological evidence to suggest nonspecific damage in the contralateral cortex, striatum, or hippocampus of either hemisphere. Nevertheless, it cannot be ruled out that PTZ caused undetectable dysfunction in the SMC or related tissue in the contralateral hemisphere. In people, seizures frequently accompany brain injury (22, 24,67). The use of anticonvulsants to prevent posttraumatic seizures, though not universally accepted, is rather common despite a lack of systematic studies of the effects on subsequent behavioral recovery (15). If seizures, as sequelae of brain injury, can attenuate neuronal depression (l&52), then a partial, rather than complete, prevention of seizures (by limiting the dose or frequency of anticonvulsant therapy) might favorably affect behavioral recovery in certain cases.A clinical characterization of post-traumatic behavioral deficits in relation to the occurrence of seizures and/or the use of anticonvulsants is necessary for the resolution of this issue. REFERENCES 1. ALBERTSON,T.E.,S.L.PETERSON,ANDL.G.STARK.~~C~~.T~~ anticonvulsant
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of diazepam
and phenobarbital
in prekin-
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