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Research report

Distinctive amygdala kindled seizures differentially affect

neurobehavioral recovery and lesion-induced

basic fibroblast growth factor (bFGF) expression

1 2

˜

Anthony E. Kline , Sylvia Montanez , Hallie A. Bradley, Courtney J. Millar,

*

Theresa D. Hernandez

Behavioral Neuroscience Program, Department of Psychology, University of Colorado, Campus Box 345, Boulder, CO 80309-0345, USA Accepted 25 July 2000

Abstract

The differing effects of partial seizures on neurobehavioral recovery following anteromedial cortex (AMC) injury in rats have previously been reported. Specifically, convulsive Stage 1 seizures evoked ipsilateral to the lesion during the 6-day post-lesion critical period delayed recovery, while non-convulsive Stage 0 seizures were neutral. The present study was designed to elaborate on that research by examining several potential mechanisms for the seizure-associated difference observed in functional outcome. Anesthetized rats sustained unilateral AMC lesions followed by implantation of a stimulating electrode in the amygdala ipsilateral (Expt. 1) or contralateral (Expt. 2) to the lesion. Beginning 48 h after surgery, animals were kindled to evoke Stage 0 or Stage 1 seizure activity during the critical period. Kindling trials and afterdischarge (AD) were controlled to ascertain their role in functional outcome. Recovery from somatosensory deficits was assessed over a two-month period. The results revealed that (i) Stage 0 seizures did not impact recovery regardless of whether initiated ipsilateral or contralateral to the lesion, (ii) Stage 1 seizures prevented recovery only when initiated in the ipsilateral hemisphere during the post-lesion critical period, and (iii) the detrimental effect of Stage 1 seizures appears to be independent of the number of kindling trials provided and cumulative AD. Thus, to determine why Stage 1 seizures evoked in the hemisphere ipsilateral to the lesion impeded recovery, a separate group of animals (Expt. 3) were kindled accordingly and processed for c-Fos and basic fibroblast growth factor (bFGF) immunohistochemistry. It was hypothesized that Stage 1 seizures evoked in the injured hemisphere prevent recovery by blocking lesion-induced bFGF expression in structures shown to be important for recovery after cortex lesions (e.g., striatum). The results confirmed our hypothesis and suggest that the seizure-associated inhibition of lesion-induced bFGF may alter the growth factor-mediated plasticity necessary for functional recovery.  2000 Elsevier Science B.V. All rights reserved.

Theme: Disorders of the nervous system

Topic: Trauma

Keywords: Anteromedial cortex; c-Fos; Immunohistochemistry; Growth factor; Trauma

1. Introduction of the most common of these trauma-associated secondary

events is the onset of seizures. Posttraumatic seizures

Following brain insult, a diverse and complex series of (PTS) can be classified as either partial or generalized

pathophysiological and behavioral events are initiated. One depending on whether the synchronous neuronal

dis-charges remain localized or spread to both hemispheres, respectively. The latter have been shown in both the

*Corresponding author. Fax:11-303-492-2967. clinical [2,31,33] and experimental [19,32,36,38] settings

E-mail address: terid@psych.colorado.edu (T.D. Hernandez). _{to exert differing effects on the recovering brain. Although} 1

Present address: Brain Trauma Research Center, Department of Neuro- _{generalized seizures are seen in slightly less than half of} surgery, University of Pittsburgh, 3434 Fifth Avenue, Suite 201,

Pitts-PTS patients [76,80], empirical research has focused on

burgh, PA 15260, USA.

2 _{this classification almost exclusively. Thus, to examine the}

Present address: Department of Pharmacology, University of Texas

Health Sciences Center, San Antonio, TX 78284, USA. role of partial seizures on functional outcome, our

labora-0006-8993 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 ( 0 0 ) 0 2 7 6 2 - 1

tory utilized a focal cortical injury model in conjunction Rowntree and Kolb [65] who exquisitely demonstrated that

with electrical kindling of the amygdala, an animal model blocking this trophic factor retarded functional recovery

of epileptogenesis [28]. Unilateral lesions of the anterome- following motor cortex lesions.

dial cortex (AMC) produce an ipsilateral somatosensory The results of the present study provide at least one

deficit. Recovery from this deficit has been found to be plausible mechanism for the distinct recovery patterns

vulnerable to manipulation during the post-lesion ‘critical’ associated with Stage 0 and Stage 1 kindled seizures

period, which has been defined as beginning at 12 h and following AMC lesions in rats.

lasting for 6 days following lesion. That is, drugs or other experimental manipulations that impact the recovery

pro-cess do so only when introduced within the first 6 days 2. Materials and methods (common to all

after AMC lesion [34,35,55,78]. For example, convulsive experiments)

Stage 1 kindled seizures evoked during the 6-day critical

period in the hemisphere ipsilateral to the lesion block 2.1. Subjects

functional recovery. This same degree of seizure activity

on post-lesion day 7 or later has no impact on the recovery Adult male Long–Evans hooded rats (Harlan–Gibco,

process. Moreover, non-convulsive Stage 0 seizure activity Indianapolis, IN) were individually housed in Plexiglas

within the critical period neither disrupts nor facilitates cages and maintained in a temperature (21618C) and light

recovery, and in this respect is similar to non-kindled (on 07:00–19:00 h) controlled environment with ad libitum

controls [38,39]. access to food and water. Handling began 1 day after

Although a marked and unequivocal difference in arrival and consisted of applying slight pressure on the

recovery patterns has been demonstrated between partially head and forepaws simulating attachment of the kindling

kindled Stage 0 and Stage 1 seizures after AMC lesion, a cable and behavioral stimuli, respectively. This taming

viable explanation for the ‘Stage 1 effect’ is lacking. regimen was implemented to habituate the animals to

Therefore, in the present study we implemented a series of experimenters and experimental manipulations such that

experiments designed to expand on previous research by undue stress that could impact kindling [3] or behavior

addressing several potential mechanisms for the observed would be minimized. All experimental procedures were

difference in functional outcome. In Expt. 1, partial conducted during the light phase of the light / dark cycle

seizures were evoked ipsilateral to the lesion during the and conformed to the policies outlined in the National

critical period and methodological variables inherent to Institutes of Health Guide for the Care and Use of

kindling (stimulation trials and epileptiform activity, i.e., Laboratory Animals (NIH Guide) and were approved by

AD) were examined so that we could gain a better the University of Colorado Institutional Animal Care and

understanding of how the number, timing, and severity of Use Committee.

seizure events affect recovery. Experiment 2 was designed

to test the hypothesis that Stage 1 seizures evoked in the 2.2. AMC lesions1kindling electrode implantation

hemisphere contralateral to an AMC lesion would not be

detrimental to recovery, thus showing that the Stage 1 All animals (270–300 g) were anesthetized with

effect is hemisphere dependent, and perhaps associated equithesin (30 mg / kg pentobarbital1140 mg / kg chloral

with localized seizure spread. hydrate, 0.46 cc / 100 g, i.p.) and secured in a stereotaxic

Given that seizures alter growth factors [63,67] and instrument (Stoelting, Wood Dale, IL). A unilateral

elec-trophic factors are an integral part of the recovery process trolytic lesion was produced by passing 1 mA of anodal

following brain injury [51,53,54], we hypothesized that current through the exposed tip (0.5 mm) of an otherwise

Stage 1 seizures evoked ipsilateral to the lesion prevented insulated stainless steel insect pin (gauge 00) for 15 s at

recovery by blocking lesion-induced trophic factor expres- each of three cortical sites comprising the AMC: site 1:

sion in distal (but within the kindled hemisphere) struc- anterior / posterior (AP) to bregma511.5 mm, lateral (L)

tures important for recovery after cortical lesions (e.g., to midline561.0 mm, ventral (V) from dura521.5 mm;

striatum [42]). Hence, in Expt. 3, immunohistochemical site 2: AP513.2 mm, L561.0 mm, V521.6 mm; site 3:

techniques were utilized to map seizure-induced spread AP513.2 mm, L561.0 mm, V523.1 mm [59]. After

using c-Fos activity as a marker, and to determine if completing the lesion a small hole was made (2.4 mm

distinct kindled seizures differentially modulate basic caudal to bregma and64.5 mm lateral to midline) and a

fibroblast growth factor (bFGF) expression. We chose bipolar electrode (0.25 mm diameter; 1.0 mm separation at

seizure-induced c-Fos immunoreactivity to verify seizure the tip; Plastics One, Roanoke, VA) was implanted (8.8 mm

spread in our model based on the numerous studies ventral to the skull surface) in either the left or right

showing that this technique is a useful and reliable tool for amygdala, but in the same hemisphere as the lesion for

mapping seizure propagation in uninjured animals Expts. 1 and 3 and in the hemisphere contralateral to the

[13,15,17,56,66,74]. Using similar logic, the decision to lesion for Expt. 2. The electrode leads were fitted into a

assembly was affixed to the skull with dental cement. After 2.3.2. Magnitude of asymmetry

suturing and applying antibacterial ointment to the wound, Assessment was made by increasing the size of the

the rats were placed in their home cages and monitored stimulus on the contralateral (non-preferred) forelimb

periodically until adequately recovered from anesthesia while simultaneously decreasing the size on the ipsilateral

2

(i.e., moving freely in their cage) before being returned to (preferred) forelimb by 14.1 mm each. Sufficient increase

the colony. in the contralateral / ipsilateral size ratio reverses the

ipsila-teral response bias such that the animal no longer pref-erentially responds to the ipsilateral stimulus, and instead

2.3. Assessment of somatosensory asymmetry: bilateral begins to respond to the contralateral stimulus first. The

tactile stimulation tests contralateral / ipsilateral stimulus size ratio necessary to reverse the response bias denotes the magnitude of

Forty-eight hours after surgery behavioral function was asymmetry. Every incremental change in the contralateral /

assessed with the well-characterized bilateral tactile stimu- ipsilateral stimulus size ratio corresponds to a level or

lation tests that were developed to reliably and quickly score ranging from 1 to 7: level 151.3 / 1, level 251.7 / 1,

detect a somatosensory asymmetry, and the magnitude of level 352.2 / 1, level 453 / 1, level 554.3 / 1, level 657 / 1,

that asymmetry, after unilateral cortical damage in rats and level 7515 / 1. Each level corresponds to the degree of

[69,71]. A modified version of these tests has been shown functional loss or magnitude of asymmetry with lower

to be useful in assessing sensorimotor neglect in mar- levels suggesting minimal impairment and higher levels

mosets [1] and the simultaneous extinction test has been indicating severe impairment or asymmetry. Typically, an

reported to be the best behavioral predictor of functional asymmetry score of ‘4’ is seen in our laboratory following

outcome following unilateral stroke in humans [64]. Test- a unilateral AMC lesion and suggests a deficit of moderate

ing preceded kindling and was conducted in the animal’s severity because the contralateral stimulus (on ignored

home cage in the colony where response to the testing limb) only has to be equal to three times the size of the

stimuli is optimal [7,8,71]. stimulus on the ipsilateral (preferred) side in order to

reverse the ipsilateral response bias. In contrast, level ‘7’ requires that the contralateral stimulus be 15 times the size

2.3.1. Ipsilateral somatosensory asymmetry of the ipsilateral stimulus (see Ref. [68]). Trials were

For each trial the rat was removed from its home cage separated by a minimum of two min and conducted in a

2

for the brief time it took to apply a small (113.1 mm ) semi-random sequence such that no one level was tested

round adhesive-backed label (Avery white multi-purpose consecutively.

labels) to the radial aspect of each forelimb. Because

responsiveness can be influenced by the order of stimulus 2.4. Amygdala kindling

placement [6], the limb each stimulus was applied to first

(right vs. left) was alternated on each trial. Moreover, Approximately 48 h after surgery, and subsequent to

because residual sensation caused by stimulus placement behavioral assessment, kindling was initiated with a 1-s

may also bias a response, both limbs were simultaneously train of 100-Hz biphasic square waves, each 1 ms in

touched prior to returning the rat to its cage. Once in the duration via a Grass S-88 stimulation unit and a constant

cage the rat quickly removed each stimulus one at a time current generator (Grass Instruments, Quincy, MA).

Kindl-with the stimulus on the side ipsilateral to the lesion ing occurred daily, 7 days per week in the home cage until

typically contacted and removed first. The sequence (right a Stage 5 seizure was exhibited. Distinct stimulation

vs. left) and the latency (in seconds) to contact and remove parameters were utilized to reliably elicit the desired

the stimuli were recorded. A contact was recorded when seizure stage within the 6-day post-lesion critical period.

the rat touched and / or removed the patch with its teeth and To this end, animals in the Stage 0 (single) group received

a trial was considered complete when the animal removed a single stimulation each day, with an initial current of 300

both patches or after 2 min had elapsed. Generally five mA (base-to-peak). The Stage 1 group received three

trials were provided during each testing session; however, stimulations (each separated by 2 min) on the first kindling

this number was increased to a maximum of ten if the rat day, two stimulations on the second and third days, and

did not show a response bias during the first five trials. A one stimulation on the fourth and fifth days, with an initial

bias or deficit was said to exist if the animal first contacted current of 150–200 mA. The Stage 0 (multiple) group,

the stimulus on the side ipsilateral to the injury$70% of which was included so that the number of kindling trials

the time. If an asymmetry was detected, the magnitude of could be kept consistent with the Stage 1 group, received

that asymmetry was assessed next. Additionally, if an three stimulations (2 min separation) on the first kindling

asymmetry (bias) was detected prior to surgery the lesion day, two stimulations on the second and third days, and

was produced in the hemisphere contralateral to the one stimulation on the fourth and fifth days, with an initial

preferred side so that post-operative lesion effects would current of 150–250 mA. The stimulation current for

groups was increased by 50mA beyond that of the initial room temperature in blocking solution (0.01 M PB, 0.3%

current of a given day. Behavioral convulsions were rated Triton X-100, 1.5% normal goat serum (PB-G)) followed

according to a modified version of Racine’s stages of by incubation in primary antiserum (rabbit anti-Fos,

behavioral seizures where 05immobility, 15jaw-clonus 1:4000, Santa Cruz, [_{052) at 48C overnight with gentle}

and / or robust chewing, 25head nodding, 35bilateral agitation (Thermolyne Roto-Mix, Dubuque, IA). After two

forelimb clonus, 45rearing onto hind limbs with full 5-min rinses in 0.01 M PB, sections were incubated in

extension of the spine while maintaining balance and / or secondary antiserum (biotinylated goat anti-rabbit Ig —

righting reflex, and 5 corresponds to behaviors described in Vectastain, BA-1000, Vector Laboratories, Burlingame,

the previous stages plus loss of the righting reflex [61]. At CA, 1:400) in PB-G for 60 min at room temperature.

each seizure stage, the epileptiform activity was visualized Sections were rinsed in 0.01 M PB and incubated in

on an oscilloscope and a Macintosh computer interfaced avidin–biotin–HRP (Vectastain Elite) for 30 min then

with a Maclab 4 channel A / D converter (World Precision rinsed again in 0.01 M PB for 5 min followed by rinses in

Instruments, Sarasota, FL). The Non-kindled group was 0.1 M Tris (pH 7.5) for 10 min. Lastly, sections were

treated similarly to the kindled animals, but did not receive incubated in 3,39-diaminobenzidine (DAB; 0.5 mg DAB /

electrical stimulation. ml Tris510 mg DAB, 20 ml Tris, 8 ml H O ) as the2 2

chromagen for 15 min, then rinsed with 0.1 M Tris and

2.5. Histology mounted on gelatinized glass slides. Air-dried sections

were dehydrated in a series of alcohol gradients (70–

2.5.1. Experiments 1 and 2 100%) then placed in Histoclear and coverslipped with

Animals were anesthetized deeply with pentobarbital Permount (Fisher Scientific). Sections incubated without

(50 mg / kg, 0.8 cc, i.p.) and perfused transcardially with primary antibody served as controls.

100–150 ml physiological saline. The electrode assembly

was removed and the brain was carefully extracted and 2.6.2. bFGF immunohistochemistry

immersed in 10% formalin for 1 week, then transferred to The procedure for bFGF immunohistochemistry was

a 10% formalin / 20% sucrose solution for 2 days. After similar to that described for c-Fos after some

modifica-fixation the brain was blocked, frozen at2218C, and every tions: (i) the primary antiserum was rabbit anti-bFGF,

third 40-mm coronal section through the lesion and every 1:400 (Sigma), (ii) the secondary antiserum was diluted

single section through the electrode tract were thaw- 1:200, (iii) the normal goat serum concentration was 3%,

mounted onto gelatinized glass slides and stained with and (iv) sections were incubated in DAB for only 4 min.

Cresyl violet. The location of each electrode tip was

verified based on anatomical parameters in accordance 2.7. Quantitative analyses

with the rat atlas of Paxinos and Watson [59] under light

microscopy (World Precision Instruments, Sarasota, FL) by 2.7.1. c-Fos immunoreactivity

two independent observers who were both blind to ex- Two 50-mm coronal sections (12.20, 22.80 mm

rela-perimental conditions. tive to bregma) with a 4003500 mm rectangular grid

superimposed bilaterally on four cortical regions (piriform,

2.5.2. Experiment 3 perirhinal, infralimbic, and sensorimotor) as well as the

Two hours after the last seizure-evoking stimulation on dorsal striatum and hippocampal CA sector were visual-3

post-injury Day 6 (last day of the critical period), rats were ized with a 43 objective on a VANOX-T Olympus

anesthetized with pentobarbital (50 mg / kg, 0.8 cc, i.p.) microscope (Olympus Optical, Tokyo, Japan). All cells

and perfused transcardially with 0.1 M phosphate-buffered within the grid exhibiting the brown nuclear staining

saline (PBS; pH 7.4, 200 ml) followed by 4% paraformal- characteristic of DAB were considered c-Fos-positive and

dehyde in 0.1 M phosphate buffer (pH 7.4, 450 ml, 48C). subsequently quantified with an NIH imaging system

Brains were carefully extracted and post-fixed in the same (version 1.6) by setting the threshold for an intermediately

solution for 2 days. Two sets of serial coronal sections (50 stained cell and automatically counting all cells above that

mm) through the brain and electrode tract were cut on a value. Although this automated technique underestimates

Vibratome and immersed in wells containing 0.1 M PBS the total number of cells, one of its major advantages is

(pH 7.4) in preparation for c-Fos and bFGF immuno- that adjusting threshold density is highly consistent, which

histochemistry. translates to a reduction in counting error. It is possible

that the underestimation may present a problem in reaching

2.6. Immunohistochemical procedures statistical significance in cases where c-Fos is minimally

induced. However, as the data will show this was not an

2.6.1. c-Fos immunohistochemistry issue in the present study. The sensorimotor and

infralim-To minimize variability in staining intensity, tissue from bic cortices were quantified at 12.20 mm relative to

each experimental group was simultaneously processed. bregma, the piriform and perirhinal cortices as well as the

quantified at 11.6 mm. The rationale for choosing these 2.9. Statistical analyses structures was based on previous research on non-injured

brains showing c-Fos expression in these regions after All statistical analyses were performed using Statview

partial and / or generalized seizure activity [16]. software (Abacus Concepts, 4.51) on a Macintosh

com-puter. One- and two-factor analyses of variance (ANOVAs) ´

2.7.2. bFGF immunoreactivity were used followed by Scheffe post hoc tests when

Basic FGF positive astrocytes were viewed with a 203 appropriate. The behavioral data are expressed as the group

objective on the same imaging equipment used for observ- mean6standard error (S.E.) and the immunohistochemical

ing c-Fos. Two 50-mm coronal sections (11.60, 11.20 data are reported as difference scores derived by

subtract-mm relative to bregma) with a 4003300 mm rectangular ing the mean of the control groups (e.g., Non-kindled,

¨

grid superimposed on the frontal cortex (areas 1 and 2, Non-kindled, and Naıve) from the main groups (Stage 0,

lateral to the lesion), the corpus callosum (directly under Stage 1, and Lesion-only, respectively). The data were

the cingulum, including the forceps of the minor corpus considered significant when probability values were less

callosum), and the dorsal striatum (directly below the than 0.05.

corpus callosum) were quantified in the lesioned-hemi-¨

sphere. In the case of the Naıve group, which did not have

a lesion or electrode, the hemisphere for quantification was 3. Results

randomly chosen. Only cells displaying the morphology of

reactive astrocytes and exhibiting distinct processes and 3.1. Histology (common to all experiments)

soma [65] were physically counted by an observer blind to

group conditions. An expert neuroanatomist in our depart- Regions of the prefrontal cortex sustaining damage due

ment (Dr Eva Fifkova) observed several random sections to the AMC lesion included the anterior cingulate and

and agreed with our findings. medial agranular cortices as well as portions of the

prelimbic and infralimbic areas. These regions are

con-2.8. Experimental design sistently damaged to the same degree following our AMC

injury model and have been depicted elsewhere [38].

2.8.1. Experiment 1. AMC lesion1ipsilateral hemisphere Kindling electrode tips were located in the following

kindling nuclei of the amygdala with equal representation among

Thirty-six rats (Non-kindled510, Stage 0 (single)57, groups: central, basolateral, lateral and medial.

Stage 0 (multiple)59, Stage 1510) underwent AMC lesion

and kindling electrode placement in the same hemisphere 3.2. Experiment 1. AMC lesion1ipsilateral hemisphere

as described in Section 2.2. kindling

2.8.2. Experiment 2. AMC lesion1contralateral 3.2.1. Magnitude of asymmetry

hemisphere kindling A repeated measures ANOVA revealed a significant

Thirty-eight rats (Non-kindled510, Stage 0 (single)510, difference among groups (F 3,3257.841, P50.0005), a

Stage 0 (multiple)59, Stage 159) were used. With the significant difference over days (F _13,416538.909, P,

exception of placing the kindling electrode in the hemi- 0.0001) and a significant group3day interaction (F_39,4165

sphere contralateral to the AMC lesion all procedures were 2.337, P,0.0001). Fig. 1 illustrates that Stage 1 seizures

identical to those described for Expt. 1. elicited during the critical period following a unilateral

AMC lesion significantly impeded functional recovery. ´

2.8.3. Experiment 3. AMC lesion1ipsilateral hemisphere Scheffe comparisons demonstrated that the Stage 1 group

kindling: c-Fos and bFGF immunohistochemistry differed significantly from the Stage 0 (single), Stage 0

Eighteen (Lesion only54, Non-kindled54, Stage 055, (multiple), and Non-kindled groups (all Ps,0.0001), but

Stage 155) of 22 rats underwent surgery as described for the latter groups were not significantly different from one

Expt. 1. The remaining four were not subjected to any of another (all Ps.0.05, not significant (n.s.)). Further

inspec-the surgical or behavioral manipulations and thus served as tion of Fig. 1 reveals that despite all groups displaying

¨

Naıve controls so that a distinction between basal and similar magnitudes of asymmetry initially (Non-kindled5

lesion-induced c-Fos and bFGF could be made. Addition- 4.5560.28, Stage 0 (single)54.0060.45, Stage 0

ally, because the results of Expts. 1 and 2 showed that (multiple)54.2760.32, Stage 154.7560.36; P50.5006,

functional recovery was not significantly different between one-factor ANOVA, n.s.), Stage 1 animals did not recover

the Stage 0 (single) and Stage 0 (multiple) groups, we even after 2 months of behavioral testing. A one-factor

evaluated in this experiment only the Stage 0 group ANOVA on the last day of behavioral testing (Day 63)

receiving multiple stimulations so that the number of revealed an asymmetry score of 2.4560.49 for the Stage 1

kindling trials could be kept consistent with the Stage 1 group that was significantly different from the 0.4060.40

Fig. 1. Mean (6S.E.) magnitude of asymmetry following a unilateral Fig. 2. Mean (6S.E.) magnitude of asymmetry following a unilateral anteromedial cortex lesion in amygdala-kindled rats. Animals were AMC lesion in amygdala-kindled rats. Animals were stimulated contrala-stimulated ipsilateral to the lesion either once per day (Stage 0 (single)), teral to the lesion either once per day (Stage 0 (single)), or 1–3 times per or 1–3 times per day (Stage 0 (multiple) and Stage 1), during the critical day (Stage 0 (multiple) and Stage 1), during the critical period, and once period, and once a day thereafter until a Stage 5 seizure was attained. per day thereafter until a Stage 5 seizure was attained. There were no Despite all groups displaying similar asymmetries initially, the group differences in recovery patterns among the groups throughout the exhibiting a Stage 1 seizure during the critical period remained markedly evaluation period (all Ps.0.05). This finding is unlike that seen in Fig. 1 impaired throughout the 63-day neurobehavioral evaluation period. where the initiation of Stage 1 seizures ipsilateral to the lesion sig-*Significantly different from Non-kindled, Stage 0 (single), and Stage 0 nificantly impaired recovery. Hash-marks denote the defined critical

´

(multiple) (all Ps,0.0001, Scheffe). Hash-marks denote the defined period of 12 h to 6 days following injury. critical period of 12 h to 6 days post-injury.

the critical period in the hemisphere contralateral to a

and the 0.3860.38 asymmetry score of the Stage 0 unilateral AMC lesion did not impact functional recovery.

(multiple) groups (P50.0005). Analysis of the latency (in An ANOVA did not yield a significant difference among

seconds) to respond to equal-sized adhesive stimuli did not groups (F 3,3451.873, P50.8648), nor did it reveal a

reveal a significant difference among groups (P50.3047), group3day interaction (F39,44250.382, P50.9998). There

indicating that the difference in recovery rates was not was, however, a significant difference over days as the

attributed to a deficit in general behavioral responsiveness. animals recovered (F 13,442567.102, P,0.0001). All

groups displayed equivalent magnitudes of asymmetry on

3.2.2. Amygdala kindling the first testing day (Non-kindled53.8260.43, Stage 0

As designed, the distinct stimulation parameters utilized (single)53.7560.27, Stage 0 (multiple),53.9460.44,

for each group within the critical period resulted in the Stage 154.0560.28) (F3,3450.134, P50.9392, one-factor

desired kindled seizure stage reliably occurring within the ANOVA; n.s.). Similarly, all groups recovered from

som-first 6 days after lesion. During this same time, the amount atosensory asymmetries by Day 42 post-surgery

(Non-of accumulated AD differed only between the Stage 0 kindled50.0060.00, Stage 0 (single)50.0060.00, Stage 0

(single) (35.52463.532 s) group and the groups receiving (multiple)50.0060.00, Stage 150.3860.38). This finding

multiple stimulations: Stage 0 (multiple) (73.7765.2 s*) is in marked contrast to that seen in Expt. 1 where Stage 1

and Stage 1 (89.566.80 s*), *Ps,0.0001, each vs. Stage 0 seizures elicited in the ipsilateral hemisphere prevented

´

(single), Scheffe. These data suggest that neither the functional recovery for more than 2 months.

number of kindling trials nor cumulative AD during the critical period following an AMC lesion were factors

influencing the Stage 1 seizure-mediated neurobehavioral 3.3.2. Amygdala kindling

impairment. Instead, the functional consequences of kin- The distinct stimulation parameters utilized for each

dled seizures appear to be related to the degree of group, again resulted in the desired kindled seizure stage

convulsive behavior (Stage 0 vs. Stage 1) experienced occurring within the post-lesion critical period. During this

during the critical period. same time, the accumulated AD was significantly lower in

the Stage 0 (single) group (40.0063.48 s) when compared

3.3. Experiment 2. AMC lesion1contralateral to the multiple-stimulation groups: Stage 0 (multiple)

hemisphere kindling (101.9568.25 s*) and Stage 1 (101.7568.69 s*), *Ps,

´

0.0001, each vs. Stage 0 (single), Scheffe. This finding is

3.3.1. Magnitude of asymmetry not surprising given that the groups receiving multiple

between the Stage 0 (multiple) or Stage 1 groups (as is in agreement with Expts. 1 and 2 above).

3.4.3. Quantification of c-Fos immunoreactive cells Analysis of c-Fos positive cells expressed in the piriform cortex revealed a significant difference among

groups (F 9,3259.928, P,0.0001), which was attributed to

Stage 1 seizures significantly increasing c-Fos induction in the kindled hemisphere compared to all other groups (all

´

Ps,0.05, Scheffe) (Fig. 3). There were no significant

differences revealed by the other comparisons (all Ps.

´

0.05, Scheffe). The mean (6S.E.) difference score for each

of the main groups in the ipsilateral piriform cortex was

7.7567.84 for the Lesion-only group, 6.25614.69 for the

Stage 0, and 142.65643.47 for the Stage 1 group.

Seizure-Fig. 3. Mean (6S.E.) difference scores in c-Fos expression in the

induced c-Fos did not change significantly in the

contrala-piriform cortex 2 h after a Stage 0 or Stage 1 amygdala-kindled seizure in

teral piriform cortex from that observed ipsilaterally for rats with a unilateral AMC lesion. Animals experiencing Stage 1 seizures

exhibited marked c-Fos expression ipsilateral, but not contralateral, to the the Lesion-only and Stage 0 groups (6.5069.52 and

lesion. In contrast, Stage 0 seizures did not induce c-Fos above that of the _{5.2567.62, respectively), but was dramatically reduced in} Lesion-only (denoted in legend as ‘Lesion’) or Non-kindled groups (data

the Stage 1 group (16.7565.93).

not shown). These data demonstrate that Stage 1 seizures propagate from

As shown in Table 1, Stage 1 seizures also significantly

the site of stimulation, but remain confined to the kindled hemisphere.

enhanced c-Fos induction in the ipsilateral perirhinal and

´ *Significantly different from all other groups (all Ps,0.05, Scheffe).

Horizontal dashed line separates ipsilateral from contralateral. infralimbic cortices (all Ps,0.05). With the exception of the Stage 0 seizure group markedly inducing c-Fos in the

ipsilateral infralimbic cortex (P,0.05), there were no

3.4. Experiment 3. AMC lesion1Ipsilateral hemisphere significant differences among the remaining groups in

´

kindling: c-Fos and bFGF immunohistochemistry either structure (all Ps.0.05, Scheffe). The fact that Stage 1 seizures did not significantly elevate c-Fos induction in the contralateral structures suggests that this seizure type 3.4.1. Magnitude of asymmetry

remained relatively localized in the ipsilateral hemisphere. Animals were assessed on post-operative days 2 and 5 to

Although both the ipsilateral and contralateral sen-determine if all groups sustained a similar degree of injury

sorimotor cortices expressed more c-Fos following Stage 1 prior to immunohistochemical analyses. Analysis of the

seizures in comparison to the Stage 0 and the Lesion-only data revealed that all groups displayed equivalent

mag-groups, the differences were not statistically significant (all

nitudes of asymmetry on each testing day (Day 2 range5

´

Ps.0.05, Scheffe). Additionally, while the Stage 1 group

3.50–4.60 and Day 5 range52.50–3.75).

also induced more c-Fos in the ipsilateral striatum when compared to the other groups, the variability was

suffi-3.4.2. Amygdala kindling ciently large to yield no statistical differences (Ps.0.05).

The distinct kindling parameters utilized resulted in the Similarly, c-Fos expression was increased in the ipsilateral

desired kindled seizure stages being evoked within the 6 CA of the Stage 0 and Stage 1 groups, but presumably3

´

day critical period, prior to sacrifice. There were no because of excessive variability, the Scheffe post hoc tests

differences, however, in the amount of cumulative AD did not detect significant differences (Ps.0.05, n.s.).

Table 1

a Summary of seizure and lesion-induced c-Fos expression

Seizure Stage 0 (I) Stage 0 (C) Stage 1 (I) Stage 1 (C) Lesion (I) Lesion (C) b

Piriform 6.25614.69 5.256 7.62 142.60643.47 16.756 5.93 7.7567.84 6.5069.52 b

Perirhinal 4.006 4.25 3.256 2.35 76.20628.95 14.956 5.19 20.5065.40 20.5063.95 c

Infralimbic 56.50634.22 9.00612.09 96.50644.17 17.25613.28 17.7567.61 7.5065.06 Sensorimotor 5.506 5.05 6.006 5.47 10.106 4.95 13.756 3.90 0.0562.43 3.5062.93 Striatum 0.756 6.38 25.506 3.82 10.256 5.71 20.056 4.92 0.0563.42 0.2563.07 CA3 48.92630.70 6.176 5.29 26.67619.39 6.276 1.96 2.7562.45 2.2561.99 a

Quantification of c-Fos immunoreactive cells in the kindled hemisphere (ipsilateral to the lesion). The Lesion group was not kindled and did not have an indwelling amygdala electrode. (I) hemisphere ipsilateral to the lesion; (C) hemisphere contralateral to the lesion. Values are expressed as difference scores

¨

derived by subtracting the mean of the control groups (Non-kindled, Non-kindled, and Naıve) from the main groups (Stage 0, Stage 1, and Lesion-only,

b c

respectively). Five animals were used in each group. Superscript letters reflect significant differences from all conditions (P,0.05), from all conditions except Stage 0 (I) (P,0.05).

Taken together, the results show that Stage 1 seizures for the Stage 0, and22.2063.54 for the Stage 1 group. A ´

markedly increased the expression of c-Fos positive cells Scheffe analysis revealed that both the Stage 0 and

Lesion-in the piriform, perirhLesion-inal, and Lesion-infralimbic cortices of the only groups were significantly different from the Stage 1

kindled hemisphere beyond that of Stage 0 seizures. Stage group (P,0.0001 and P50.0062, respectively), but were

1 seizures also increased c-Fos in the sensorimotor cortex, not significantly different from each other (P50.0691,

dorsal striatum, and CA sector of the hippocampus, but to3 n.s.). The results show both a lesion-induced expression of

a lesser extent than in the aforementioned regions. Thus, bFGF-positive astrocytes that is consistent with other

despite spreading beyond the site of stimulation, Stage 1 studies [22,24,41] and an inhibition of endogenous (or

seizures remain relatively confined to the kindled hemi- lesion-induced) bFGF-positive astrocytes following Stage

sphere (Figs. 3 and 4). 1 seizures (Fig. 5). The latter finding is exclusive to our

study and may provide an explanation as to why Stage 1

3.4.4. Quantification of bFGF immunoreactive astrocytes seizures evoked in the hemisphere ipsilateral to the AMC

The expression of bFGF-positive astrocytes in the dorsal lesion impeded somatosensory recovery.

striatum following a unilateral AMC lesion coupled with As seen in Table 2, lesion-induced expression of

bFGF-amygdala-kindled seizures was significantly different positive astrocytes was decreased in the sensorimotor

among groups (F 4,39522.522, P,0.0001). The mean cortex following Stage 0 and Stage 1 seizures. Indeed,

(6S.E.) difference scores of immunoreactive astrocytes bFGF immunoreactivity was significantly different among

´

were 19.0063.94 for the Lesion-only group, 17.3064.38 groups (P,0.0001). A Scheffe analysis revealed that none

Fig. 4. Photomicrograph providing a visual corroboration of the data in Fig. 3 showing c-Fos immunoreactivity in the piriform cortex 2 h after amygdala-kindled seizures in AMC lesioned rats. c-Fos expression in animals experiencing Stage 0 seizures (C,D) was not significantly different from that of Non-kindled (A,B) or Lesion-only (not depicted) groups. Conversely, animals experiencing Stage 1 seizures exhibited marked c-Fos staining ipsilateral (E), but not contralateral (F), to the lesion. Scale bar5250mm.

exhibited fewer bFGF-positive astrocytes than the Non-kindled control group as reflected by the negative scores of the kindled groups. The Lesion-only group displayed significantly more bFGF immunoreactive astrocytes than its control group, but not significantly more than either the

´

Stage 0 or Stage 1 groups (Ps.0.05, Scheffe, n.s.).

4. Discussion

The present study was undertaken to elucidate the potential mechanistic underpinnings of a behavioral phe-nomenon discovered in our laboratory in which convulsive (Stage 1) seizures, but not non-convulsive (Stage 0)

Fig. 5. Mean (6S.E.) kindling induced expression of bFGF

immuno-seizures, initiated ipsilateral to an AMC lesion during the

reactive astrocytes in the ipsilateral dorsal striatum six days after a

6-day critical period severely disrupted functional recovery

unilateral AMC lesion. A significant increase in bFGF immunoreactivity

is observed in both the Lesion-only (denoted in legend as ‘Lesion’) and _{[38]. To this end, we evaluated the effects of kindling trials} Stage 0 groups. In marked contrast, Stage 1 seizures evoked during the _{and cumulative AD, as well as seizure focus and bFGF} 6-day critical period significantly inhibited the expression of

lesion-expression as potential influencing factors. When initiated

induced bFGF-positive astrocytes. *Significantly different from

Lesion-in the amygdala ipsilateral to the lesion (Expt. 1), Stage 0

only and Stage 0.

seizures had a neutral impact on recovery regardless of whether evoked by single or multiple stimulations. Stage 1 of the main groups were significantly different from one

seizures within the critical period, were associated with

another (Lesion-only vs. Stage 0, P50.6599; Lesion-only

sustained impairment as evidenced by that group still

vs. Stage 1, P50.7428; Stage 0 vs. Stage 1, P50.9999),

exhibiting a functional deficit (asymmetry score .2, Fig.

indicating that the significant difference detected by

1) two months after injury. Additionally, animals in the ANOVA was due to differences between the main groups

Stage 0 (multiple) group recovered at a rate similar to the and their respective controls. That is, both of the kindled

Non-kindled and Stage 0 (single) groups despite receiving groups were significantly different from the Non-kindled

as many kindling trials and accruing as much AD as the control group (data not shown) and the Lesion-only group

Stage 1 group within the 6-day critical period. In contrast, ¨

was significantly different from the Naıve group (data not

when initiated contralateral to the lesion (Expt. 2), neither shown). The negative difference scores reported for the

Stage 0 nor Stage 1 seizures were detrimental to the Stage 0 and Stage 1 groups are a direct result of the

recovery process (Fig. 2). Hence, in addition to replicating Non-kindled control group exhibiting more bFGF

immuno-the main outcome from immuno-the earlier study in our laboratory reactive astrocytes in the sensorimotor cortex. This finding

[38], the results presented here reveal that neither the indicates that endogenous or basal levels of bFGF-positive

number of kindling trials nor cumulative AD were con-astrocytes, at least in this region, were decreased by

founding variables regarding why Stage 1 seizures impede seizures.

recovery. Factors that were influential in determining how ¨

When compared to the Naıve group, bFGF

immuno-functional recovery progressed were seizure severity reactive astrocytes were increased in the corpus callosum

(Stage 0 vs. Stage 1), the temporal presentation of the in all groups. However, calculation of mean difference

seizure activity (i.e., whether the distinct seizure type scores revealed that both the Stage 0 and Stage 1 groups

occurred during or after the critical period) and whether the seizure focus was ipsilateral or contralateral to the

Table 2 _{lesion. This latter finding corroborates other work}

demon-a

Summary of seizure and lesion-induced bFGF expression _{strating that the hemisphere ipsilateral to the injury is} Seizure Stage 0 Stage 1 Lesion important for the ensuing functional recovery [12,46].

bc The neutral impact of Stage 0 or Stage 1 seizures

Corpus callosum 24.13610.14 26.12610.12 63.0069.04

evoked in the contralateral hemisphere after the critical

Sensorimotor 23.486 2.03 23.286 3.45 6.2561.44

c bd c

Striatum 17.306 4.38 22.206 3.54 19.0063.94 _{period substantiates other findings in PTS / epilepsy}

re-a _{search suggesting that seizures per se are not always}

Quantification of bFGF-positive astrocytes in the kindled hemisphere

(ipsilateral to the lesion). The Lesion group was not kindled and did not detrimental to functional outcome. Haltiner and colleagues

have an indwelling amygdala electrode. Values are expressed as difference _{have shown that when injury severity is controlled, late} scores derived by subtracting the mean of the control groups (Non- _{PTS reportedly have no influence on neurobehavioral}

¨

kindled, Non-kindled, and Naıve) from the main groups (Stage 0, Stage 1,

outcome [31]. In fact, several studies have reported

and Lesion-only, respectively). Five animals were used in each group.

b c _{improved functional recovery being associated with}

post-Superscript letters reflect significant differences from Stage 0, Stage 1, d

[69] suggested that the enhanced recovery may be due to importance of bFGF following brain insult. Basic FGF, an

seizure-associated attenuation of post-traumatic neural 18-kDa, 154-amino-acid protein with potent trophic

ac-depression [18,77]. This postulation is supported by re- tions, is expressed endogenously and in response to injury

search showing that central nervous system (CNS) stimul- [22,24,41]. It has been shown to support the survival and

ant drugs that enhance functional recovery outgrowth of a variety of cells both in vitro and in vivo

([20,29,30,44,45]; see Ref. [21] for review) increase [5,62]. Additionally, bFGF is reported to promote recovery

cerebral metabolism and / or glucose utilization following focal cerebral infarction [43,49], contusive

spi-[27,40,60,72], thereby reversing post-traumatic neural de- nal cord injury [4] or traumatic brain injury [52,75].

pression. Furthermore, bFGF confers neuroprotection against

Despite the demonstrated importance of general CNS seizure-associated hippocampal damage [50], protects

stimulation promoting a beneficial outcome, a facilitation striatal neurons from NMDA-receptor mediated

excitotox-of recovery was not observed in the present study. Instead, icity [25], and attenuates histopathological damage

follow-animals experiencing Stage 0 seizures in either the ipsila- ing fluid percussion brain injury [14] or spinal cord

teral or contralateral hemisphere, or Stage 1 seizures contusion [48,73]. In many instances, bFGF appears to

contralateral to the lesion during the critical period re- produce its effects whether administered before or after

covered at the same rate as the Non-kindled controls. injury. The endogenous expression of bFGF following

Improved recovery in the Stage 0 groups may not have brain injury may reflect the brain’s attempt to prevent

been observed in the present study for at least two reasons. further degeneration and / or provide regeneration. This

First, Stage 0 kindled animals experienced subconvulsive, latter point is of particular interest given that

bFGF-partial seizures within the critical period, whereas the prior positive astrocytes were significantly decreased in the

research showing improved recovery employed generalized ipsilateral striatum and the sensorimotor cortex following

seizure activity with convulsions via electroconvulsive Stage 1 seizures (Expt. 3). The failure of this group to

shock [19] or the chemoconvulsant, pentylenetetrazol recover may have resulted from seizure-associated

inhibi-[32,36]. Second, Stage 0 seizures did not increase bFGF- tion of lesion-induced bFGF, thereby diminishing the

positive astrocytes in the striatum beyond that of the growth factor-mediated plasticity important for functional

Lesion-only group. Thus, unlike more generalized seizure recovery. That bFGF is susceptible to such modulation is

activity [23,26,63], partially kindled seizures may not supported by the findings that diazepam, a CNS

depres-increase bFGF expression to sufficient levels to improve sant, blocks astrocyte mitosis [58], suppresses the

induc-functional outcome. In particular, facilitated recovery may tion of bFGF [10] and blocks functional recovery when

rely on elevated bFGF levels in structures like the striatum administered during the critical period after AMC lesion

that have been shown to be important to the recovery [34,35,69].

process [42]. Still somewhat puzzling, however, was the observed

Post-injury CNS stimulation, therefore, can be influen- decrease in bFGF-positive astrocytes in the sensorimotor

tial following brain insult and the consequences of that cortex following Stage 0 seizures, given that behavioral

influence depend upon the nature of the activation. Spe- recovery in this group occurred unimpeded. These data

cifically, in addition to CNS activation, partially kindled suggest that, at least for the time point examined, the

(convulsive) seizures (Stages 1–2) are followed by a striatum may play a greater role than the sensorimotor

period of post-ictal depression as evidenced by depressed cortex in mediating functional recovery following AMC

neocortical metabolism in the kindled hemisphere [57]. damage. This possibility is supported by studies showing

Inducing such regional neural depression pharmacological- that AMC lesion-induced bFGF and c-Fos expression are,

ly has been shown to disrupt functional outcome [9,37,79]. at peak time points, consistently greater in the striatum

For example, administration of the GABA agonist, mus- than in the cortex, and that this pattern is associated with

cimol, into the adjacent cortex produces long term impair- functional recovery [11]. At the same time, because

ment of behavioral recovery following AMC damage [37]. surviving striatal tissue experiences increased vulnerability

Thus, if the Stage 1 seizures in our paradigm produced a to excitotoxicity and behavioral events following cortical

similar post-ictal depressive state during the critical period, insult [47,70], the significant decrease in bFGF-positive

this may explain why functional outcome was so severely astrocytes in the ipsilateral striatum of Stage 1 kindled

impaired. The results of our c-Fos data are suggestive of a animals may have prevented growth factor-induced

at-Stage 1-induced depressive state in that the striatum and tenuation of subcortical excitotoxicity [25]. Alternatively,

sensorimotor cortex exhibited significantly fewer c-Fos secondary plasticity necessary for recovery may have been

positive cells compared to other brain regions in this prevented or hampered. Such plasticity could include

group. Because CNS depression has been associated with synaptogenesis involved in reestablishing cortico-striatal

decreased growth factor expression [10], this could have connectivity disrupted by injury-induced damage. Indeed

contributed to the diminished bFGF expression observed in synaptogenesis has been reported following the exogenous

the Stage 1 group. administration of bFGF [43].

re-search investigating the functional effects of seizure activi- ses. The assistance of Kimberley Buytaert, M.A. and two

ty on brain injury and subsequent recovery. Understanding exceptional undergraduate students, Stephanie Butler and

the impact of distinct seizure types secondary to brain Jim Suozzi, is also greatly appreciated.

insult has been hindered by the ability to control important variables such as injury and seizure severity, as well as

seizure number and timing. The present study overcomes _References

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Taken together, the results show that Stage 1 seizures

for the Stage 0, and

2

2.20

6

3.54 for the Stage 1 group. A

´

markedly increased the expression of c-Fos positive cells

Scheffe analysis revealed that both the Stage 0 and

Lesion-in the piriform, perirhLesion-inal, and Lesion-infralimbic cortices of the

only groups were significantly different from the Stage 1

kindled hemisphere beyond that of Stage 0 seizures. Stage

group (P

,

0.0001 and P

5

0.0062, respectively), but were

1 seizures also increased c-Fos in the sensorimotor cortex,

not significantly different from each other (P

5

0.0691,

dorsal striatum, and CA sector of the hippocampus, but to

3

n.s.). The results show both a lesion-induced expression of

a lesser extent than in the aforementioned regions. Thus,

bFGF-positive astrocytes that is consistent with other

despite spreading beyond the site of stimulation, Stage 1

studies [22,24,41] and an inhibition of endogenous (or

seizures remain relatively confined to the kindled hemi-

lesion-induced) bFGF-positive astrocytes following Stage

sphere (Figs. 3 and 4).

1 seizures (Fig. 5). The latter finding is exclusive to our

study and may provide an explanation as to why Stage 1

3.4.4. Quantification of bFGF immunoreactive astrocytes

seizures evoked in the hemisphere ipsilateral to the AMC

The expression of bFGF-positive astrocytes in the dorsal

lesion impeded somatosensory recovery.

striatum following a unilateral AMC lesion coupled with

As seen in Table 2, lesion-induced expression of

bFGF-amygdala-kindled

seizures

was

significantly

different

positive astrocytes was decreased in the sensorimotor

among groups (F

4,39

5

22.522, P

,

0.0001). The mean

cortex following Stage 0 and Stage 1 seizures. Indeed,

(

6

S.E.) difference scores of immunoreactive astrocytes

bFGF immunoreactivity was significantly different among

´

were 19.00

6

3.94 for the Lesion-only group, 17.30

6

4.38

groups (P

,

0.0001). A Scheffe analysis revealed that none

Fig. 4. Photomicrograph providing a visual corroboration of the data in Fig. 3 showing c-Fos immunoreactivity in the piriform cortex 2 h after amygdala-kindled seizures in AMC lesioned rats. c-Fos expression in animals experiencing Stage 0 seizures (C,D) was not significantly different from that of Non-kindled (A,B) or Lesion-only (not depicted) groups. Conversely, animals experiencing Stage 1 seizures exhibited marked c-Fos staining ipsilateral (E), but not contralateral (F), to the lesion. Scale bar5250mm.

exhibited fewer bFGF-positive astrocytes than the

Non-kindled control group as reflected by the negative scores of

the kindled groups. The Lesion-only group displayed

significantly more bFGF immunoreactive astrocytes than

its control group, but not significantly more than either the

´

Stage 0 or Stage 1 groups (Ps

.

0.05, Scheffe, n.s.).

4. Discussion

The present study was undertaken to elucidate the

potential mechanistic underpinnings of a behavioral

phe-nomenon discovered in our laboratory in which convulsive

(Stage 1) seizures, but not non-convulsive (Stage 0)

Fig. 5. Mean (6S.E.) kindling induced expression of bFGF

immuno-seizures, initiated ipsilateral to an AMC lesion during the

reactive astrocytes in the ipsilateral dorsal striatum six days after a

6-day critical period severely disrupted functional recovery

unilateral AMC lesion. A significant increase in bFGF immunoreactivity

is observed in both the Lesion-only (denoted in legend as ‘Lesion’) and

_{[38]. To this end, we evaluated the effects of kindling trials}

Stage 0 groups. In marked contrast, Stage 1 seizures evoked during the

_{and cumulative AD, as well as seizure focus and bFGF}

6-day critical period significantly inhibited the expression of

lesion-expression as potential influencing factors. When initiated

induced bFGF-positive astrocytes. *Significantly different from

Lesion-in the amygdala ipsilateral to the lesion (Expt. 1), Stage 0

only and Stage 0.

seizures had a neutral impact on recovery regardless of

whether evoked by single or multiple stimulations. Stage 1

of the main groups were significantly different from one

seizures within the critical period, were associated with

another (Lesion-only vs. Stage 0, P

5

0.6599; Lesion-only

sustained impairment as evidenced by that group still

vs. Stage 1, P

5

0.7428; Stage 0 vs. Stage 1, P

5

0.9999),

exhibiting a functional deficit (asymmetry score

.

2, Fig.

indicating that the significant difference detected by

1) two months after injury. Additionally, animals in the

ANOVA was due to differences between the main groups

Stage 0 (multiple) group recovered at a rate similar to the

and their respective controls. That is, both of the kindled

Non-kindled and Stage 0 (single) groups despite receiving

groups were significantly different from the Non-kindled

as many kindling trials and accruing as much AD as the

control group (data not shown) and the Lesion-only group

Stage 1 group within the 6-day critical period. In contrast,

¨

was significantly different from the Naıve group (data not

when initiated contralateral to the lesion (Expt. 2), neither

shown). The negative difference scores reported for the

Stage 0 nor Stage 1 seizures were detrimental to the

Stage 0 and Stage 1 groups are a direct result of the

recovery process (Fig. 2). Hence, in addition to replicating

Non-kindled control group exhibiting more bFGF

immuno-the main outcome from immuno-the earlier study in our laboratory

reactive astrocytes in the sensorimotor cortex. This finding

[38], the results presented here reveal that neither the

indicates that endogenous or basal levels of bFGF-positive

number of kindling trials nor cumulative AD were

con-astrocytes, at least in this region, were decreased by

founding variables regarding why Stage 1 seizures impede

seizures.

recovery. Factors that were influential in determining how

¨

When compared to the Naıve group, bFGF

immuno-functional recovery progressed were seizure severity

reactive astrocytes were increased in the corpus callosum

(Stage 0 vs. Stage 1), the temporal presentation of the

in all groups. However, calculation of mean difference

seizure activity (i.e., whether the distinct seizure type

scores revealed that both the Stage 0 and Stage 1 groups

occurred during or after the critical period) and whether

the seizure focus was ipsilateral or contralateral to the

Table 2

_{lesion. This latter finding corroborates other work}

demon-a

Summary of seizure and lesion-induced bFGF expression

_{strating that the hemisphere ipsilateral to the injury is}

Seizure Stage 0 Stage 1 Lesion

important for the ensuing functional recovery [12,46].

bc

The neutral impact of Stage 0 or Stage 1 seizures

Corpus callosum 24.13610.14 26.12610.12 63.0069.04

evoked in the contralateral hemisphere after the critical

Sensorimotor 23.486 2.03 23.286 3.45 6.2561.44

c bd c

Striatum 17.306 4.38 22.206 3.54 19.0063.94

_{period substantiates other findings in PTS / epilepsy}

re-a

_{search suggesting that seizures per se are not always}

Quantification of bFGF-positive astrocytes in the kindled hemisphere

(ipsilateral to the lesion). The Lesion group was not kindled and did not

detrimental to functional outcome. Haltiner and colleagues

have an indwelling amygdala electrode. Values are expressed as difference

_{have shown that when injury severity is controlled, late}

scores derived by subtracting the mean of the control groups (Non-

_{PTS reportedly have no influence on neurobehavioral}

¨

kindled, Non-kindled, and Naıve) from the main groups (Stage 0, Stage 1,

outcome [31]. In fact, several studies have reported

and Lesion-only, respectively). Five animals were used in each group.

b c

_{improved functional recovery being associated with}

post-Superscript letters reflect significant differences from Stage 0, Stage 1,

d

[69] suggested that the enhanced recovery may be due to

importance of bFGF following brain insult. Basic FGF, an

seizure-associated attenuation of post-traumatic neural

18-kDa, 154-amino-acid protein with potent trophic

ac-depression [18,77]. This postulation is supported by re-

tions, is expressed endogenously and in response to injury

search showing that central nervous system (CNS) stimul-

[22,24,41]. It has been shown to support the survival and

ant

drugs

that

enhance

functional

recovery

outgrowth of a variety of cells both in vitro and in vivo

([20,29,30,44,45]; see Ref. [21] for review) increase

[5,62]. Additionally, bFGF is reported to promote recovery

cerebral

metabolism

and / or

glucose

utilization

following focal cerebral infarction [43,49], contusive

spi-[27,40,60,72], thereby reversing post-traumatic neural de-

nal cord injury [4] or traumatic brain injury [52,75].

pression.

Furthermore,

bFGF

confers

neuroprotection

against

Despite the demonstrated importance of general CNS

seizure-associated hippocampal damage [50], protects

stimulation promoting a beneficial outcome, a facilitation

striatal neurons from NMDA-receptor mediated

excitotox-of recovery was not observed in the present study. Instead,

icity [25], and attenuates histopathological damage

follow-animals experiencing Stage 0 seizures in either the ipsila-

ing fluid percussion brain injury [14] or spinal cord

teral or contralateral hemisphere, or Stage 1 seizures

contusion [48,73]. In many instances, bFGF appears to

contralateral to the lesion during the critical period re-

produce its effects whether administered before or after

covered at the same rate as the Non-kindled controls.

injury. The endogenous expression of bFGF following

Improved recovery in the Stage 0 groups may not have

brain injury may reflect the brain’s attempt to prevent

been observed in the present study for at least two reasons.

further degeneration and / or provide regeneration. This

First, Stage 0 kindled animals experienced subconvulsive,

latter point is of particular interest given that

bFGF-partial seizures within the critical period, whereas the prior

positive astrocytes were significantly decreased in the

research showing improved recovery employed generalized

ipsilateral striatum and the sensorimotor cortex following

seizure activity with convulsions via electroconvulsive

Stage 1 seizures (Expt. 3). The failure of this group to

shock [19] or the chemoconvulsant, pentylenetetrazol

recover may have resulted from seizure-associated

inhibi-[32,36]. Second, Stage 0 seizures did not increase bFGF-

tion of lesion-induced bFGF, thereby diminishing the

positive astrocytes in the striatum beyond that of the

growth factor-mediated plasticity important for functional

Lesion-only group. Thus, unlike more generalized seizure

recovery. That bFGF is susceptible to such modulation is

activity [23,26,63], partially kindled seizures may not

supported by the findings that diazepam, a CNS

depres-increase bFGF expression to sufficient levels to improve

sant, blocks astrocyte mitosis [58], suppresses the

induc-functional outcome. In particular, facilitated recovery may

tion of bFGF [10] and blocks functional recovery when

rely on elevated bFGF levels in structures like the striatum

administered during the critical period after AMC lesion

that have been shown to be important to the recovery

[34,35,69].

process [42].

Still somewhat puzzling, however, was the observed

Post-injury CNS stimulation, therefore, can be influen-

decrease in bFGF-positive astrocytes in the sensorimotor

tial following brain insult and the consequences of that

cortex following Stage 0 seizures, given that behavioral

influence depend upon the nature of the activation. Spe-

recovery in this group occurred unimpeded. These data

cifically, in addition to CNS activation, partially kindled

suggest that, at least for the time point examined, the

(convulsive) seizures (Stages 1–2) are followed by a

striatum may play a greater role than the sensorimotor

period of post-ictal depression as evidenced by depressed

cortex in mediating functional recovery following AMC

neocortical metabolism in the kindled hemisphere [57].

damage. This possibility is supported by studies showing

Inducing such regional neural depression pharmacological-

that AMC lesion-induced bFGF and c-Fos expression are,

ly has been shown to disrupt functional outcome [9,37,79].

at peak time points, consistently greater in the striatum

For example, administration of the GABA agonist, mus-

than in the cortex, and that this pattern is associated with

cimol, into the adjacent cortex produces long term impair-

functional recovery [11]. At the same time, because

ment of behavioral recovery following AMC damage [37].

surviving striatal tissue experiences increased vulnerability

Thus, if the Stage 1 seizures in our paradigm produced a

to excitotoxicity and behavioral events following cortical

similar post-ictal depressive state during the critical period,

insult [47,70], the significant decrease in bFGF-positive

this may explain why functional outcome was so severely

astrocytes in the ipsilateral striatum of Stage 1 kindled

impaired. The results of our c-Fos data are suggestive of a

animals may have prevented growth factor-induced

at-Stage 1-induced depressive state in that the striatum and

tenuation of subcortical excitotoxicity [25]. Alternatively,

sensorimotor cortex exhibited significantly fewer c-Fos

secondary plasticity necessary for recovery may have been

positive cells compared to other brain regions in this

prevented or hampered. Such plasticity could include

group. Because CNS depression has been associated with

synaptogenesis involved in reestablishing cortico-striatal

decreased growth factor expression [10], this could have

connectivity disrupted by injury-induced damage. Indeed

contributed to the diminished bFGF expression observed in

synaptogenesis has been reported following the exogenous

the Stage 1 group.

administration of bFGF [43].

re-search investigating the functional effects of seizure activi-

ses. The assistance of Kimberley Buytaert, M.A. and two

ty on brain injury and subsequent recovery. Understanding

exceptional undergraduate students, Stephanie Butler and

the impact of distinct seizure types secondary to brain

Jim Suozzi, is also greatly appreciated.

insult has been hindered by the ability to control important

variables such as injury and seizure severity, as well as

seizure number and timing. The present study overcomes

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