A .E. Kline et al. Brain Research 880 2000 38 –50
39
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 and 64.5 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 evaluate bFGF expression was based on a report by
plastic pedestal MS363, Plastics One and the entire
40 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 injury70 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
not be masked by the endogenous bias. subsequent kindling trials in the multiple stimulation
A .E. Kline et al. Brain Research 880 2000 38 –50
41
groups was increased by 50 mA 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 the
2 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 at 2218C, 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
Briefly, free-floating sections were incubated for 30 min at CA were quantified at 22.80, and the dorsal striatum was
3
42 A
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
