hon p.1 [100%]YAKUGAKU ZASSHI 130(12) 1751―1754 (2010)  2010 The Pharmaceutical Society of Japan

1751―Note―

Proliferation Changes in Dentate Gyrus of Hippocampus during the First Week
following Kainic Acid-induced Seizures
Peifei GU,,a Yue LI,b Yu SHANG,c Yue HOU,d and Songyan ZHAOd
aCentre of Clinical Pharmacology, Northeast Pharmaceutical Science and Technology Co., Ltd.

Shenyang 110027, P. R. China, bDepartment of Anesthesiology, The First A‹liated Hospitalof Harbin Medical University, Harbin 150001, P. R. China, cDepartment of Anesthesia,
PLA463 Hospital, Shenyang 110042, P. R. China, and dDivision of Pharmacology,
Shenyang Pharmaceutical University, Shenyang 110016, P. R. China(Received July 2, 2010; Accepted September 9, 2010)
Long-term neuroplastic changes in dentate gyrus (DG) have been reported after seizure induction and were shownto contribute to epitogenesis of epilepsy. These changes include increased number of newborn granule cells, sprouted
mossy ˆbers, granule cell layer dispersion, etc. The aim of current study is to determine the acute progression of neuroplastic changes involved newly generated granule cells after kainic acid (KA)-induced seizures. Doublecortion (DCX)
analysis was used to examine the newly generated granule cells morphology 17 days after seizure induction. Quantitative analysis of DCX-labeled cells at diŠerent times shows that there are some rapid changes in the dentate gyrus. At day
7 epileptical mice induced an increase of the number of DCX-labeled cells in DG. At days 3 and 7 after epilepsy induction, the percentage of DCX-labeled cells per DG were signiˆcantly increased. These results show that seizures are capable to increase the number of new granule cell within a short time for function activation in post-seizure period. Therefore, the rapid changes in the DG might be having a potential for hippocampus neuroplastic function.
Key words―kainic acid; epilepsy; dentate gyrus; doublecortion

INTRODUCTION
Epilepsy is a common chronic neurological disorder that is characterized by recurrent unprovokedepileptic seizures. Epilepsy is usually controlled, but
not cured, with medication, even though by surgery.
Epilepsy should not be understood as a single disorder, but rather as a group of syndromes with vastly
divergent symptoms but all involving episodic abnormal electrical activity in the brain.1,2) Among otherpathological ˆndings, an abnormal broadening of thedentate gyrus in epileptic hippocampus is often observable but up to date it remains unclear if this morphological change represents a consequence or a possible cause of epilepsy. Not only mature neurons aresubject to alterations in the epileptic hippocampus
with seizure-activity, also the activity strongly inducescell proliferation and net neurogenesis in rodent
epilepsy models.3,4) Studies of adult rodent models oflimbic epileptogenesis or acute seizures showed that
seizure or seizure-induced injury stimulates neurogenesis in both the SGZ and SVZ of hippocampus.4) In
the dentate gurys, epilepsy increases the proliferation
e-mail: gpeifei9999＠hotmail.com

of progenitors ˆve- to ten-fold after a latent period.5)
Most of these newborn cells diŠerentiate into granuleneurons, some of which mislocate in the hilus region.
Because of proliferation association with neurogenesis processes, there has been recently interested indoublecortin (DCX) as a candidate marker for neurogenesis.35)
Researches suggested that the number of DCX-expressing cells correlates with the level of cellular
proliferation in the dentate gyrus,6) however, itremains unclear that whether this correlation is maintained when levels of neurogenesis are modulated byexternal factors, such as seizures. Recently ˆndings
indicate that seizures induced by various experimental
manipulations (kindling, pilocarpine, kainic acid) increase neurogenesis in the adult rodent DG.7) Previous studies showed that newly generated neurons signiˆcant increase at least 2 weeks after kainic acid(KA) induced seizures, it is unclear whether theseneuroplastic changes happened during the shorter andacuter time-points after KA induced seizures.
Therefore we tried to answer the following questionin this study: whether the newly generated neuronal
proliferation was already appeared during the ˆrst
week post-seizure by DCX staining.

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Vol. 130 (2010)

MATERIALS AND METHODS
Animals
All experimental C57/BL6 mice werepurchased from Shenyang, China. After they arrived,
weighing 2025 g that were housed in individual cages
on 12 h light/dark schedules with water and foodwere available ad libitum. Animal procedures wereapproved by the Veterinary O‹ce of China. AlleŠorts were made to minimize animal suŠering and toreduce the number of animals used.
Chemicals
DCX antibody and Alexa Fluor 594
donkey-anti-goat antibody were purchased from Santa Cruz Biotechology (USA). Hoechst 33258 werepurchased from Molecular Probes (Eugene, OR).
Kainic acid and other chemicals were purchased fromSigma Chemicals (St. Louis, MO).
Kainic Acid Treatment and Seizure Evaluation
KA was administrated to induce epilepsy in mice.
Kainic acid was dissolved in isotonic saline (pH 7.37.4) at a concentration of 24 mg/kg or 6 mg/kg in 10mg/ml volume. Mice were separated into individual
cages and given i.p. injections of KA, the initial dosewas 24 mg/kg, intraperitoneally. After that, whennecessary, mice were given repeat doses of 6 mg/kg
every 30 min until animals had jumping clonus. Seizure intensity was evaluated according to six-point
seizure scale for mice8) which adapted from a ˆvepoint scale for rat.9) Control animals were injectedwith isotonic saline intraperitoneally.
Tissue Preparation
1, 3, 7 Days following seizure induction, mice were deeply anaesthetized withNembutal (100 mg/kg i.p) and then transcardiallyperfused with 0.1 M PBS (pH 7.4) followed by 4％paraformaldehyde in 0.1 M PBS (pH 7.4). The
brains were removed from the skull, and placed in thesame ˆxative for 4 h. After post ˆxation, the brains
were cryoprotected in 30％ sucrose, and serial frozen
coronal sections (20 mm) were cut on cryostat. The
following immuno‰orescent procedures were used:doublecortin (DCX) staining to estimate the newborn cells.
Immuno‰uorescent Staining
For DCX ‰uorescent staining, sections were used then washed threetimes in PBS and serum with 0.3％ TritionX-100 in
PBS was applied for one hour for blocking step andsubsequent DCX (1：500, goat polyclonal) primary
antibody was used. The sections were then rinsed for
20 min in PBS and incubated with corresponding
secondary antibodies: Alexa Fluor 594 donkey-anti-

goat (1：1000) in 0.3％ TX-PBS for 2 h. Then, sections were rinsed according to routine method, washing, Hoechst staining, and coverslips.
In ‰uorescent staining, nuclei were stained with
Hoechst 33258 (Molecular Probes, Eugene, OR).
Sections were mounted out of PBS and coverslippedwith Permount. Control experiments include the incubation of sections in the absence of primary antibodies. Signals were subsequently analyzed under appropriate ˆlter using Leica DM 6000 ‰uorescent
microscope (Germany). All images were captured using a Leica camera or Nikon CCD camera (Japan).
Statistical Analysis
Sections between －1.5 and－2.8 mm to bregma were used in this experiment. To
determine the mean grey values of diŠerent staining in
dentate gyrus, every three areas of one section andthree randomly chosen sections for each mouse wereselected. The reference grey value was determined on
no-staining area. All data in this study are presented
as mean±S.E.M. Cell counted were averaged foreach mouse and were compared among treatmentgroups using one-way ANOVA followed by a LSD
post-hoc test; p＜0.05, p＜0.01. (SPSS 13.0 software).
RESULTS AND DISCUSSION
Seizure Scoring
Forty mice were used in this experiment; 25 to establish KA model, 15 mice with saline injection. All KA-treated animals displayed behavior seizures. According to the method mentioned
above, after 5 times injection (30 mg/kg), 9 (36％)
mice displayed progressive seizures over the next 30min. After 6 times (36 mg/kg) injection, 10 (40％)
treated animals displayed progressive seizures overthe next 30 min. After 7 times injection, 6 (24％)
treated animals of left displayed progressive seizures
over the next 30 min. All animals displayed severe seizure, and 2 mice were dead during the epilepsy duration and one mouse dead following KA injection. The
average dose to induce seizure is 34.64 mg/kg. No seizure activity was observed in animals treated with saline.
The Morphology and Distribution of the DCX-labeled Cells
According to Fig. 1, two parameters
were examined for each time points following KA-induced seizure, which include the number of DCXlabels cells in DG and the percentage of DCX-labeledcells per DG. Only signiˆcant diŠerences in these twoparameters are reported for each time point. The total

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No. 121753Fig. 1.

Histograms of the Numbers of DCX-labeled Cells (a) and the Percentage of DCX-labeled Cells (b) in DG

p ＜0.01 compared with control group (n＝56 animals per group).
All data are showed as mean ±S.E.M. 
p＜0.05, Fig. 2.

Morphology of DCX Expression in the DG of Hippocampus at DiŠerent Time Points

A: control; B: 24 h after KA model established; C: 72 h after KA model established; D: 7 days after KA model established. a: Hoechst staining (100×); b:DCX ‰uorescent staining (100 ×); c: DCX ‰uorescent staining (200×); d: DCX ‰uorescent staining (400×).

number of DCX-positive cells per DG in control
group is 3954±758; and the percentage of DCX-labeled cells per DG in control mice is 11.31±1.62. At

day 1 after KA-induced seizures, the number of DCXlabels cells in DG and the percentage of DCX-labeledcells per DG did not show signiˆcant diŠerence com-hon p.4 [100%]

1754

pared with control group. At day 3 afte seizures, thepercentage of DCX-labeled cells per DG was increased to 20.27±3.18, which has a signiˆcant increase compared with control group (Fig. 1, p＜0.05). At day 7 after seizures, both parameters appeared signiˆcant increased compared with controlgroup; the percentage of DCX-labeled cells per DG
was increased to 23.75±2.30 (Fig. 1, p＜0.01); andthe number of DCX-labels cells in DG was increased
to 5284±500 (Fig. 1, p＜0.05).
The current study demonstrates several rapid neuroplastic changes involving newly generated granulecells during the ˆrst 7 days after KA-induced seizures.
The earliest change observed from the parameters
analyzed indicate that, at day 1 after KA-induced seizures, there was an increase in the percentage ofDCX-labeled cells with basal dendrites and an increase in the progenitor cell population. At day 3 after seizures, the thickness of the DCX-labeled cell layer was increased (Fig. 2). At day 3 after seizures, thenumber of DCX-labeled cells was increased. DCXpositive cells were typically found in the SGZ, at theborder between the SGZ and the granule layer (GL),or in the GL. Consistent with previous studies, epileptic mice have a great number of new-born neurons.
Basal dendrites were observed on DCX-labeled neurons for both control and epileptic mice (Fig. 2). In
the research, morphological analysis of the DCX immunolabeled cells with basal dendrites revealed thatthe new born neurons with basal dendrites have a signiˆcantly increase in the epileptic mice compared withcontrol after day 3 (Fig. 2). These changes using KAinduced seizures are consistent with the results from aprevious study showing that DCX-labeled cells may
become proliferate in response to KA-induced seizures.10) The fact that many of the progenitor cells
were found in dense clusters beyond the hilar borderby 3 days after seizures suggests that they may also
contribute to the thickening of the newly born granulecell layer, because they are generating cells beyond thehilar border region. This neuroplastic change isanalogous to human and primate TLE and rodent
models of TLE, where granule cell layer dispersion

Vol. 130 (2010)and hilar ectopic granule cells are prominent featuresof the epileptic hippocampus.11,12) These new-born
granule cells have been shown to be integrated into
granule cell circuitry functionally and develop protective eŠect in epileptic activity,12,13) but in this research
its speciˆc signiˆcance still needs further elucidation.
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