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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: [email protected] (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
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 (F39,4165
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)
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
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
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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