Status Epilepticus-Related Hippocampal Injury in the Immature Rat Brain
Status epilepticus (SE)-related neuronal degeneration and glial activation in different regions of the developing rat hippocampus were investigated in an age- and time-dependent manner. Wistar rat pups of postnatal day (P) 7, 15, and 21 were injected i.p. with lithium+pilocarpine to induce SE or...
Gespeichert in:
| Datum: | 2015 |
|---|---|
| Hauptverfasser: | , , , |
| Format: | Artikel |
| Sprache: | English |
| Veröffentlicht: |
Інститут фізіології ім. О.О. Богомольця НАН України
2015
|
| Schriftenreihe: | Нейрофизиология |
| Online Zugang: | http://dspace.nbuv.gov.ua/handle/123456789/148155 |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Zitieren: | Status Epilepticus-Related Hippocampal Injury in the Immature Rat Brain / B. Li, L. Yang, H. Zhou, R. Sun // Нейрофизиология. — 2015. — Т. 47, № 1. — С. 38-44. — Бібліогр.: 14 назв. — англ. |
Institution
Digital Library of Periodicals of National Academy of Sciences of Ukraine| id |
irk-123456789-148155 |
|---|---|
| record_format |
dspace |
| spelling |
irk-123456789-1481552019-02-18T01:24:30Z Status Epilepticus-Related Hippocampal Injury in the Immature Rat Brain Li, B. Yang, L. Zhou, H. Sun, R. Status epilepticus (SE)-related neuronal degeneration and glial activation in different regions of the developing rat hippocampus were investigated in an age- and time-dependent manner. Wistar rat pups of postnatal day (P) 7, 15, and 21 were injected i.p. with lithium+pilocarpine to induce SE or saline to make controls. Rats were sacrificed at 2, 12, 24 h, 3 days (d) and 7 d after SE induction. Neurodegeneration in the hippocampus was assessed by Fluoro-Jade B staining. The expressions of the astrocyte marker (GFAP) and microglia marker (Iba-1) were evaluated by immunohistochemistry. In P7 rats, there was no neuronal damage at any time points in SE. Two hours after SE induction, the number of degenerating neurons in the hippocampus significantly increased in the CA1 region of P15 rats and in both CA1 and CA3 regions of P21 rats. Degenerating neurons in the dentate gyrus appeared at 24 h after SE in P15 and P21 rats. In P7 rats, there was no up-regulation of GFAP- or Iba-1-positive cells in SE. The expression of GFAP was dramatically elevated at 12 h in the CA1 and CA3 regions of P15 rats. The number of GFAP-positive cells did not increase in the dentate gyrus until 24 h after SE induction in P15 rats. In P21 SE rats, the mentioned index increased in the CA1, CA3, and dentate gyrus at 2 h. The number of Iba-1-positive cells increased significantly in the CA1, CA3, and dentate gyrus at 12 h in P15 rats and as early as at 2 h in P21 rats. These findings suggest that SE-related neuronal damage and glial activation in the immature brain are, in general, less intense than in the adult one, and the development of these processes in different structures of the hippocampus demonstrates significant temporal and spatial specificity Досліджено залежність дегенерації нейронів та активації глії, індукованих епілептичним статусом (ЕС) у різних ділянках гіпокампа щурів від віку тварини і часу після розвитку ЕС, що розвивається. Щурятам лінії Вістар (постнатальні дні П7, П15 та П21) внутрішньоочеревинно ін'єкували сіль літію + пілокарпін для індукції ЕС або фізіологічний розчин для створення контролю. Щурят піддавали евтаназії через 2, 12, 24 год та через 3 або 7 діб після індукції ЕС. Нейродегенерацію в гіпокампі виявляли за допомогою забарвлення Флуоро-Джейд Б. Рівні експресії маркера астроцитів GFAP та маркера мікроглії Iba-1 оцінювали з використанням імуногістохімічних методів. У П7-щурят в ЕС будь-якого ушкодження нейронів на усіх відрізках часу не виявлялося. Через 2 год після індукції ЕС кількість дегенеруючих нейронів у гіпокампі щурят П15 суттєво підвищувалася в зоні CA1, а у П12-щурят це відбувалося в зонах CA1 і CA3. У щурят П15 та П21 дегенеруючі нейрони в зубчастій звивині з'являлися через 24 год. У щурят П7 не спостерігалося збільшення кількості GFAP- або Iba-1-позитивних клітин, зумовленого ЕС. Експресія GFAP у щурят П15 дуже сильно зростала в зонах CA1 та CA3 через 12 год. У той же час у таких щурят не відмічалося збільшення імунореактивності цього білка в зубчастій звивині. У щурят П21 даний індекс збільшувався в зонах CA1, CA3 та зубчастій звивині вже через 2 год після індукції ЕС. Кількість Iba-1-позитивних клітин у 3-х згаданих регіонах суттєво збільшувалась у щурят П15 через 12, а у тварин П21 - вже через 2 год. Ці результати вказують на те, що пов'язані з ЕС ушкодження нейронів та активація глії в незрілому мозку в цілому є менш інтенсивними, ніж у дорослому, а розвиток даних процесів у гіпокампі є специфічним у просторовому та часовому аспектах. 2015 Article Status Epilepticus-Related Hippocampal Injury in the Immature Rat Brain / B. Li, L. Yang, H. Zhou, R. Sun // Нейрофизиология. — 2015. — Т. 47, № 1. — С. 38-44. — Бібліогр.: 14 назв. — англ. 0028-2561 http://dspace.nbuv.gov.ua/handle/123456789/148155 616.853:612.82 en Нейрофизиология Інститут фізіології ім. О.О. Богомольця НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
English |
| description |
Status epilepticus (SE)-related neuronal degeneration and glial activation in different regions
of the developing rat hippocampus were investigated in an age- and time-dependent manner.
Wistar rat pups of postnatal day (P) 7, 15, and 21 were injected i.p. with lithium+pilocarpine
to induce SE or saline to make controls. Rats were sacrificed at 2, 12, 24 h, 3 days (d) and
7 d after SE induction. Neurodegeneration in the hippocampus was assessed by Fluoro-Jade B
staining. The expressions of the astrocyte marker (GFAP) and microglia marker (Iba-1)
were evaluated by immunohistochemistry. In P7 rats, there was no neuronal damage at any
time points in SE. Two hours after SE induction, the number of degenerating neurons in the
hippocampus significantly increased in the CA1 region of P15 rats and in both CA1 and CA3
regions of P21 rats. Degenerating neurons in the dentate gyrus appeared at 24 h after SE in
P15 and P21 rats. In P7 rats, there was no up-regulation of GFAP- or Iba-1-positive cells in
SE. The expression of GFAP was dramatically elevated at 12 h in the CA1 and CA3 regions
of P15 rats. The number of GFAP-positive cells did not increase in the dentate gyrus until 24
h after SE induction in P15 rats. In P21 SE rats, the mentioned index increased in the CA1,
CA3, and dentate gyrus at 2 h. The number of Iba-1-positive cells increased significantly in
the CA1, CA3, and dentate gyrus at 12 h in P15 rats and as early as at 2 h in P21 rats. These
findings suggest that SE-related neuronal damage and glial activation in the immature brain
are, in general, less intense than in the adult one, and the development of these processes
in different structures of the hippocampus demonstrates significant temporal and spatial
specificity |
| format |
Article |
| author |
Li, B. Yang, L. Zhou, H. Sun, R. |
| spellingShingle |
Li, B. Yang, L. Zhou, H. Sun, R. Status Epilepticus-Related Hippocampal Injury in the Immature Rat Brain Нейрофизиология |
| author_facet |
Li, B. Yang, L. Zhou, H. Sun, R. |
| author_sort |
Li, B. |
| title |
Status Epilepticus-Related Hippocampal Injury in the Immature Rat Brain |
| title_short |
Status Epilepticus-Related Hippocampal Injury in the Immature Rat Brain |
| title_full |
Status Epilepticus-Related Hippocampal Injury in the Immature Rat Brain |
| title_fullStr |
Status Epilepticus-Related Hippocampal Injury in the Immature Rat Brain |
| title_full_unstemmed |
Status Epilepticus-Related Hippocampal Injury in the Immature Rat Brain |
| title_sort |
status epilepticus-related hippocampal injury in the immature rat brain |
| publisher |
Інститут фізіології ім. О.О. Богомольця НАН України |
| publishDate |
2015 |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/148155 |
| citation_txt |
Status Epilepticus-Related Hippocampal Injury in the Immature Rat Brain / B. Li, L. Yang, H. Zhou, R. Sun // Нейрофизиология. — 2015. — Т. 47, № 1. — С. 38-44. — Бібліогр.: 14 назв. — англ. |
| series |
Нейрофизиология |
| work_keys_str_mv |
AT lib statusepilepticusrelatedhippocampalinjuryintheimmatureratbrain AT yangl statusepilepticusrelatedhippocampalinjuryintheimmatureratbrain AT zhouh statusepilepticusrelatedhippocampalinjuryintheimmatureratbrain AT sunr statusepilepticusrelatedhippocampalinjuryintheimmatureratbrain |
| first_indexed |
2025-07-12T18:28:29Z |
| last_indexed |
2025-07-12T18:28:29Z |
| _version_ |
1837466822233817088 |
| fulltext |
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 138
UDC 616.853:612.82
B. LI,1 L. YANG,1 H. ZHOU,1 and R. SUN1
STATUS EPILEPTICUS-RELATED HIPPOCAMPAL INJURY
IN THE IMMATURE RAT BRAIN
Received November 17, 2013
Status epilepticus (SE)-related neuronal degeneration and glial activation in different regions
of the developing rat hippocampus were investigated in an age- and time-dependent manner.
Wistar rat pups of postnatal day (P) 7, 15, and 21 were injected i.p. with lithium+pilocarpine
to induce SE or saline to make controls. Rats were sacrificed at 2, 12, 24 h, 3 days (d) and
7 d after SE induction. Neurodegeneration in the hippocampus was assessed by Fluoro-Jade B
staining. The expressions of the astrocyte marker (GFAP) and microglia marker (Iba-1)
were evaluated by immunohistochemistry. In P7 rats, there was no neuronal damage at any
time points in SE. Two hours after SE induction, the number of degenerating neurons in the
hippocampus significantly increased in the CA1 region of P15 rats and in both CA1 and CA3
regions of P21 rats. Degenerating neurons in the dentate gyrus appeared at 24 h after SE in
P15 and P21 rats. In P7 rats, there was no up-regulation of GFAP- or Iba-1-positive cells in
SE. The expression of GFAP was dramatically elevated at 12 h in the CA1 and CA3 regions
of P15 rats. The number of GFAP-positive cells did not increase in the dentate gyrus until 24
h after SE induction in P15 rats. In P21 SE rats, the mentioned index increased in the CA1,
CA3, and dentate gyrus at 2 h. The number of Iba-1-positive cells increased significantly in
the CA1, CA3, and dentate gyrus at 12 h in P15 rats and as early as at 2 h in P21 rats. These
findings suggest that SE-related neuronal damage and glial activation in the immature brain
are, in general, less intense than in the adult one, and the development of these processes
in different structures of the hippocampus demonstrates significant temporal and spatial
specificity.
Keywords: pilocarpine, epilepsy, hippocampus, gilal fibrillary acidic protein (GFAP),
Iba-1.
1 Department of Pediatry, Qilu Hospital of the Shandong University, Jinan,
China.
Correspondence should be addressed to B. Li
(e-mail: lbmsqf@163.com),
L. Yang (e-mail: srpyl2007@sina.com.cn),
H. Zhou (e-mail: zhouhaiyan0822@163.com),
R. Sun (e-mail: srpyl2008@sina.com.cn).
INTRODUCTION
Status epilepticus (SE) is a life-threatening emergency
inducing high mortality. After successful rescue, per-
manent brain damage and the risk for the epilepsy de-
velopment are two main concerns. Neuronal death and
glial activation are characteristic pathological changes
after SE in the adult brain. Neuronal excitation during
SE triggers glial activation via various extracellular
signals such as glutamate, ATP, and IL-1в [1]. Acti-
vated glial cells might contribute to neuronal dam-
age by releasing inflammatory factors. An immature
brain is rather prone to seizure activity. It is, however,
less vulnerable to irreversible seizure-induced dama-
ge, which indicates that the respective pathological
changes are age-dependent.
In our study, we observed pathological changes at
different time points in the immature rat hippocampus
after induction of the SE, to find the manner in which
neuronal degeneration and glial activation occur in the
developing brain.
METHODS
Animals. Male Wistar rats of postnatal day (P) 7,
P15, and P21 were purchased from the Shandong
University. P7 and P15 rat pups were housed with
their dams until weaning or sacrificed. P21 rats were
maintained in quiet uncrowded facilities and given
unlimited access to food and water. All rats were kept
in a room at constant temperature (23°C) and humidity
(60%) on a 12-h light schedule. Rats in each age group
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 1 39
STATUS EPILEPTICUS-RELATED HIPPOCAMPAL INJURY IN THE IMMATURE BRAIN
were divided into the SE and control groups.
Lithium+Pilocapine-Induced SE Model. All Wistar
rats of the SE group were injected intraperitoneally
(i .p.) with 127 mg/kg lithium chloride. After
18 to 24 h, P7, P15, and P21 rats were administered
i.p. with pilocarpine at dosages of 120, 80, and
60 mg/kg, respectively. Atropine (1 mg/kg) was i.p.
injected 30 min before pilocarpine. Animals of the
control group were given with the same volumes of
saline. Seizure activity was graded, as previously
described, according to the following manifestations
[2]: Mouth and facial movements corresponded to
level I, head nodding, to level II, forelimb clonus,
to level III, rearing with forelimb clonus, to level
IV, and rearing and falling with forelimb clonus, to
level V. Rats that exhibited grade-IV and V seizures
were included in the experiment. Seizures could be
terminated with 400 mg/kg i.p chloral hydrate. The
pilocarpine dosages used led to approximately 20%
mortality in rats of all groups. Finally, 50 rats were
included in the SE group, and 25 rats were included
in the control group. Both groups were further divided
into subgroups and sacrificed at 2, 6, and 24 hours and
3 or 7 days after the SE induction.
Tissue Preparation. Rats of both groups were per-
fused transcardially with chilled PBS followed by 4%
paraformaldehyde. The brains were taken off, manu-
ally dissected from the calvarium, and immersed in
4% paraformaldehyde for 24 h at 4°C before paraffin
embedding. Embedded brains were sectioned coronal-
ly with a microtome into 5 mm-thick sections and col-
lected on gelatin-coated microscope slides.
Fluoro Jade B Stain. The slides were first im-
mersed in a solution containing 1% sodium hydr-
oxide in 80% alcohol (20 ml of 5% NaOH added to
80 ml absolute alcohol) for 5 min. This was followed
by a 2 min-long-stay in 70% alcohol and 2 min in dis-
tilled water. The slides were then transferred to 0.06%
potassium permanganate for 10 min and rinsed in dis-
tilled water for 2 min. After 20 min in 0.0004% Fluo-
ro Jade B staining solution, the slides were rinsed for
1 min in each of three distilled-water washes and then
placed on a slide warmer set at approximately 50°C,
until they were fully dry. Finally, the tissue was exa-
mined using an epifluorescent microscope with blue
(450-490 nm) excitation light.
Immunohistochemistry. Hippocampal sections
were incubated with diluted mouse anti-GFAP antibody
(1:100; Millipore Corporation, USA) or mouse anti-
Iba1 antibody (1:100; Epitomics Inc., USA) overnight
at 4°C. Subsequently, they were exposed to HRP-
conjugated anti-mouse antibody (1:200; ZSGB-
BIO, China). After treatment with DAB (ZSGB-
BIO, China), the slides were counterstained with
hematoxylin and mounted with permanent mounting
medium. Immunohistochemical staining was observed
under a light microscope. An image-Pro Plus 6.0 set
was used for quantitative analysis of GFAP- and Iba-1-
positive cells.
Statistical Analysis. Numerical values were ex-
pressed as means ± s.d. for each group; SPSS 11.0 was
used for all statistical analyses. The t-test was per-
formed to compare the SE and control groups for Flu-
oro Jade B and immunohistochemistry; P values less
than 0.05 were considered statistically significant.
RESULTS
Fluoro Jade B. In the SE P7 age group, there were no
positive cells at all time points. In the SE P15 group,
there were some positive cells in the CA1 zone at 2 h,
12 h, 24 h, 3d, and 7 d. No positive cells were seen in
the CA3 region in the P15 group at any time interval.
There were positive cells in the dentate gyrus at 24 h,
3 d, and 7 d in P15 rats in the above status. In the SE
P21 group, there were positive cells in the CA1 and
CA3 zones at 2 h, 12 h, 24 h, 3 d, and 7 d. In SE P21
rats, positive cells were observed in the dentate gyrus
only at 24 h, 3 d, and 7 d (Fig. 1; Table 1).
Immunohistochemistry. There was no significant
difference in the number of GFAP-positive cells
between the control and SE groups at all five time
intervals in P7 rats. In SE P15 rats, the numbers of such
cells in the CA1 and CA3 regions were significantly
greater at 12 h, 24 h, 3 d, and 7 d after SE, while this
index was higher in the dentate gyrus at 24 h, 3 d and
7 d. In the SE P21 group, GFAP-positive cells were
more numerous at all time points in the CA1, CA3, and
dentate gyrus (Fig. 2; Table 2).
In the P7 group, the number of Iba-1-positive cells
did not differ significantly between control and SE
groups at any time. Iba-1-positive cells were more
numerous at 12 h, 24 h, 3 d, and 7 d in the CA1, CA3
and dentate gyrus in the SE P15 group. The respective
values were greater in P21 SE than in the control at 2
h, 12 h, 24 h, 3 d, and 7 d in the CA1, CA3, and dentate
gyrus (Fig. 3; Table 3).
DISCUSSION
Available reports concerning the effects of status epi-
lepticus on the immature brain were focused on cer-
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 140
B. LI, L. YANG, H. ZHOU, and R. SUN
F i g. 2. GFAP staining in the hippocampus at 3 d after SE in P15
rats. A) CA3 region, control, B) CA3 region, group SE, C) CA1
region, control, D) CA1 region, group SE, E) dentate gyrus, control,
F) dentate gyrus, group SE.
Р и с. 2. Результати імуногістохімічного забарвлення гіпокампа
щодо GFAP через три доби після індукції епілептичного статусу
у П15-щурят.
F i g. 1. Fluoro Jade B staining at 24 h after
SE. A) CA3 region in P7 rats, B) CA3 region
in P15 rats, C) CA3 region in P21 rats, D)
CA1 region in P7 rats, E) CA1 region in P15
rats, F) CA1 region in P21 rats.
Р и с. 1. Результати забарвлення гіпокампа
Fluoro Jade B через 24 год після індукції
епілептичного стану.
tain specific aspects such as recurrent seizures, EEG,
neuronal death, and mossy fiber sprouting. It should
be mentioned that studies of neuronal death and glial
activation in this state are still patchy. In our experi-
ments, we found that there was no SE-related neuro-
nal damage or glial activation in the hippocampus of
P7 rats. Degenerating neurons in the hippocampus ap-
peared at 2 h after SE induction in P15 and P21 rats.
Astrocytes and microglia were activated at 12 h after
SE in P15 animals and at 2 h in P21 rats.
Results of Fluoro Jade B staining suggest that
the neonatal brain is more resistant to SE-related
neurodegeneration; significant changes could only be
detected after postnatal day 15. There are several possible
mechanisms that could explain different vulnerability
to SE within different developmental stages. Status
epilepticus produces neuronal damage mostly by
excessive activation of glutamate receptors. Functional
studies in the rat indicated that the glutamate receptor
density appears to peak around P10 [3]. Thus, increased
expression of functional glutamate receptors is likely
to play a major role in increased vulnerability of the
brain only after two weeks of life. Immediate early gene
А
А B
C D
E F
B C
FED
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 1 41
STATUS EPILEPTICUS-RELATED HIPPOCAMPAL INJURY IN THE IMMATURE BRAIN
TABLE 1. Density of Fluoro-Jade B-positive cells in the hippocampus in P15 and P21 rats
Т а б л и ц я 1. Щільність клітин, позитивних щодо Fluoro-Jade B, у гіпокампі щурят віком P15 та P21
P15 P21
Time after
induction of
the SE
2 h 12 h 24 h 3 d 7 d 2 h 12 h 24 h 3 d 7 d
CA1 7±2 14±3 17±4 18±3 19±4 12±2 19±4 20±6 22±5 23±6
CA3 Neg Neg Neg Neg Neg 11±1 14±3 18±4 19±5 21±3
DG Neg Neg 8±1 7±2 11±2 Neg Neg 10±2 12±4 15±3
Footnotes. Numbers of positive cells (means ± s.d.) per 0.25 mm2 area are shown. Neg corresponds to the absence of positive cells
TABLE 2. Density of GFAP-positive cells in rats of different ages
Т а б л и ц я 2. Щільність клітин, позитивних щодо GFAP, у щурят різного віку
Age Region Group 2 h 12 h 24 h 3 d 7 d
P7
CA1
Contr. 24.4±2.9 23.8±5.1 26.4±4.2 24.8±2.6 27.3±4.0
SE 25.3±3.5 24.25±3.0 25.6±3.9 25.0±5.2 26.2±3.5
CA3
Contr. 22.0±2.7 22.7±3.6 23.4±3.1 25.7±5.1 24.9±4.3
SE 20.8±3.1 23.4±5.6 22.5±5.0 26.2±3.5 25.9±3.0
DG
Contr. 27.8±2.3 29.5±4.6 30.6±5.0 34.7±3.7 35.0±3.3
SE 26.8±2.2 31.1±3.7 32.3±4.5 35.7±4.1 32.3±3.0
P15
CA1
Contr. 27.4±4.1 28.6±3.4 28.1±4.3 29.6±4.4 30.1±4.2
SE 29.7±4.3 37.6±2.6* 39.1±3.9* 40.3±3.0* 45.8±3.5*
CA3 Contr. 25.2±4.5 26.8±3.7 27.6±3.8 29.6±3.3 30.5±4.0
SE 24.5±3.0 34.5±3.5* 36.4±3.6* 44.3±4.2* 42.3±4.1*
DG
Contr. 34.4±3.0 32.3±3.8 35.9±4.7 33.1±3.5 33.6±3.8
SE 32.4±2.9 35.3±2.8 44.2±4.5* 49.8±3.1* 55.3±4.7*
P21
CA1
Contr. 29.1±4.0 30.0±3.6 31.2±3.5 32.8±4.0 35.6±4.2
SE 36.5±4.2* 36.0±3.2* 39.2±4.5* 49.7±5.6* 40.3±4.0*
CA3 Contr. 31.4±4.9 33.6±4.1 32.3±3.9 35.7±3.6 34.7±4.3
SE 37.6±5.3* 39.5±3.5* 42.9±3.6* 47.6±5.1* 49.6±3.6*
DG
Contr. 34.8±2.7 36.8±4.0 33.6±2.9 35.1±3.2 34.9±4.6
SE 45.6±5.0* 44.2±4.3* 47.1±4.2* 46.4±5.5* 49.9±4.7*
Footnotes. Contr. and SE are controls and status epilepticus groups, respectively. Difference is significant compared with the control group
(P < 0.05) in a specific region in each age group. Other designations are similar to those in Table 1.
responses could be determinants of neuronal survival;
such responses in the immature and mature brains are
dissimilar. For example, there is no c-fos transcription
in the neonatal hippocampal and cortical structures after
kainic acid (KA)-induced SE. At the same time, c-fos
induction could be detected after postnatal day 13 [4].
Greater GABA synthesis [5] and less mitochondrial
oxidative stress [6] under SE conditions in the immature
brain are also possible mechanisms providing higher
resistance of the immature brain to SE.
May be, a more important finding is that neurons
in different regions of the hippocampus respond in
a dissimilar manner to excitation signals. Neurons
in the CA1 and dentate gyrus degenerated after SE
in both P15 and P21 rats, whereas the CA3 region
underwent neurodegeneration only in P21 rats. Intrinsic
characteristics of hippocampal neurons could explain
this age-dependent and region-specific mode of changes.
Vulnerable neurons have higher expression of the genes
related to stress. The CA1 neurons in organotypic cultures
maintained in vitro have a higher level of oxidative
activity under baseline conditions when compared with
the CA3 neurons [7]. As was reported, extracellular
potassium regulation was less effective in the CA1
compared to the CA3 region in immature rabbits after
repetitive stimulation [8]; this is why the CA1 region
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 142
B. LI, L. YANG, H. ZHOU, and R. SUN
F i g. 3. Iba-1 staining in the hippocampus at 3 d after SE in P15
rats. A) CA3 region, control, B) CA3 region, group SE, C) CA1
region, control, D) CA1 region, group SE, E) dentate gyrus, control,
F) dentate gyrus, group SE.
Р и с. 3. Результати імуногістохімічного забарвлення гіпокампа
щодо Iba-1 через три доби після індукції епілептичного статусу
у П15-щурят.
TABLE 3. Density of Iba-1-positive cells rats of different ages
Т а б л и ц я 3. Щільність клітин, позитивних щодо Iba-1, у щурят різного віку
Age Region Group 2 h 12 h 24 h 3 d 7 d
P7
CA1 Contr. 7.8 ± 1.2 8.1 ± 1.3 9.2 ± 1.7 13.4 ± 2.1 16.9 ± 3.2
SE 8.4 ± 1.8 8.2 ± 1.6 10.0 ± 2.7 15.2 ± 2.5 14.5 ± 1.9
CA3 Contr. 8.7 ± 1.7 8.4 ± 1.4 10.0 ± 2.1 13.1 ± 2.3 13.6 ± 2.0
SE 6.8 ± 2.0 7.9 ± 1.6 11.2 ± 2.8 13.8 ± 2.8 14.8 ± 2.1
DG
Contr. 4.4 ± 0.9 6.9 ± 0.8 7.3 ± 1.1 9.7 ± 1.6 11.3 ± 1.5
SE 5.1 ± 1.3 5.2 ± 1.0 7.7 ± 2.0 11.4 ± 2.0 10.0 ± 1.4
P15
CA1 Contr. 18.5 ± 2.6 23.8 ± 2.9 24.5 ± 3.0 29.4 ± 3.2 32.8 ± 3.9
SE 19.4 ± 3.2 28.2 ± 3.3* 36.7 ± 3.7* 39.7 ± 4.2* 44.2 ± 4.6*
CA3 Contr. 15.3 ± 3.1 18.7 ± 3.4 19.6 ± 4.0 22.6 ± 3.4 27.4 ± 2.7
SE 16.7 ± 3.6 24.2 ± 2.9* 26.4 ± 3.0* 33.4 ± 2.4* 38.8 ± 4.0*
DG
Contr. 11.7 ± 1.8 12.6 ± 1.4 18.7 ± 2.1 22.9 ± 2.4 27.3 ± 1.8*
SE 13.1 ± 2.6 19.2 ± 3.3* 25.2 ± 3.4* 32.6 ± 2.6* 37.0 ± 4.8*
P21
CA1 Contr. 31.5 ± 2.4 35.6 ± 3.2 36.8 ± 3.5 37.4 ± 4.1 40.9 ± 4.6
SE 39.0 ± 3.0* 46.3 ± 2.9* 49.1 ± 3.1* 53.9 ± 3.9* 58.1 ± 5.0*
CA3 Contr. 25.5 ± 1.8 24.3 ± 2.1 26.7 ± 2.4 33.7 ± 3.5 34.3 ± 3.7
SE 37.2 ± 3.7* 39.7 ± 3.8* 38.1 ± 2.3* 51.3 ± 3.0* 55.7 ± 3.6*
DG Contr. 28.9 ± 2.1 29.3 ± 2.6 35.5 ± 3.7 36.1 ± 4.2 39.4 ± 3.6
SE 37.8 ± 2.4* 37.3 ± 2.6* 40.2 ± 2.1* 55.0±2.5* 59.6 ± 4.2*
Footnote. Designations are similar to those in Table 2.
is more vulnerable to such excitation. The neuronal
excitability reaches the level typical of mature animals
in the CA1 region after postnatal day 14, while inhibitory
processes did not reach such level even several weeks
later [9]. The development of synaptic inhibition lag in
the CA1 compared to the CA3 can explain preferential
CA1 damage in 2-week-old animals [10].
Not only neuronal degeneration but also glial
activation are age-dependent. Microglia and astrocytes
in the rat hippocampus were not activated until
two weeks after birth. These gliocytes types were
simultaneously activated at 12 h in P15 rats and at 2 h in
P21 rats after SE induction, which means that microglia
and astrocytes are involved in the pathological
process in SE practically simultaneously. The precise
mechanism of seizures-related glial activation remains
unknown. After neuronal excitation, an increased
extracellular K+ concentration and various signals (such
as glutamate, ATP, and calcitonin gene-related peptide)
are possible factors triggering microglial activation [1,
11-13]. IL-1в, produced mainly by activated microglia,
А B
C D
E F
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 1 43
STATUS EPILEPTICUS-RELATED HIPPOCAMPAL INJURY IN THE IMMATURE BRAIN
is one of the most powerful inducers of reactive
astrogliosis [14]. Swann et al. [10] reported that, after
penicillin-induced epileptogenesis, the extracellular K+
concentration in the CA3 region of hippocampal slices
taken from 9- to 16-day-old rats is higher than that in
more mature rats (30-35 days old). This finding seems
to be contradictory to our observation of quiescent glial
cells in P7 rats. Oxidative substances from degenerating
neurons are, however, also strong stimulators of glial
activation, and this explains well the inactivated status
of glial cells in the immature brain.
Furthermore, glial activation showed a region-
specific pattern at different time points after SE.
In our experiments, astrocytes were activated at
12 h in the CA1 and CA3 regions and later (at 24 h)
in the dentate gyrus of P15 rats. Astrocytes in all
three regions in P21 rats were activated 2 h after SE
induction. Microglial cells were activated in the CA1,
CA3, and dentate gyrus at 12 h after SE in P15 rats
and at 2 h after such influence in P21 rats. Whether
glia activation leads to neuronal death, or neuronal
death results in long-term glial activation after SE,
has not been clarified in previous studies. In our
research, neuronal degeneration was detected earlier
than glial activation in the CA1 region in P15 rats,
while glial cells were activated earlier than neuronal
injury developed in the dentate gyrus in P21 rats.
Microglia and astrocytes were all activated in the CA3
region at 12 h after SE in P15 rats, but no degenerating
neurons were observed at any time point in this region.
According to this time-sequence analysis, we suppose
that the cause/effect relationship between neuronal
death and glial activation depends on the region
specificity, time point, and developmental stage. In
addition, glial activation without neuronal death in the
CA3 region in P15 rats suggests that there must be
some factor other than glial activation precipitating
neuronal degeneration.
Thus, pathological changes after SE in the immature
brain differ from those in the adult brain. Neuronal
degeneration and glial activation in the hippocampus
demonstrate age, time, and regional specificity. De-
tailed observations in our research probably can pro-
vide some help for further studies of the pathological
processes in the immature brain under SE conditions.
Acknowledgment. This work was supported by the
Shandong Province Natural Science Foundation (Grant
No. ZR2009CM072) and Shandong Province Science and
Technology Development Project (Grant No.2010GSF10201).
The study was carried out in accordance with the statements
of the Council Directive regarding the protection of animals
used for experimental and other scientific purposes (86/609/
EEC, 1986, Strasbourg) and respective regulations of the local
Ethics Committee.
The authors, B. Li, L. Yang, H. Zhou, and R. Sun, confirm
that the research and publication of the results were not
associated with any conflicts regarding commercial or financial
relations, relations with organizations and/or individuals who
may have been related to the study, and interrelations of co-
authors of the article.
Б. Лі1, Л. Янг1, Х. Жоу1, Р. Сун1
ІНДУКОВАНІ ЕПІЛЕПТИЧНИМ СТАТУСОМ УШКОД-
ЖЕННЯ ГІПОКАМПА В НЕЗРІЛОМУ МОЗКУ ЩУРІВ
1 Лікарня Квілу при Шандонському університеті, Чжінань
(Китай).
Р е з ю м е
Досліджували залежність дегенерації нейронів та актива-
ції глії, індукованих епілептичним статусом (ЕС) у різних ді-
лянках гіпокампа щурів від віку тварини і часу після розви-
тку ЕС, що розвивається. Щурятам лінії Вістар (постнатальні
дні П7, П15 та П21) внутрішньоочеревинно ін’єкували сіль
літію+пілокарпін для індукції ЕС або фізіологічний розчин
для створення контролю. Щурят піддавали евтаназії через
2, 12, 24 год та через три або сім діб після індукції ЕС. Не-
йродегенерацію в гіпокампі виявляли за допомогою забарв-
лення Флуоро-Джейд Б. Рівні експресії маркера астроцитів
GFAP та маркера мікроглії Iba-1 оцінювали з використанням
імуногістохімічних методів. У П7-щурят в ЕС будь-якого
ушкодження нейронів на усіх відрізках часу не виявляло-
ся. Через 2 год після індукції ЕС кількість дегенеруючих
нейронів у гіпокампі щурят П15 істотно підвищувалася в
зоні CA1, а у П12-щурят це відбувалося в зонах CA1 і CA3.
У щурят П15 та П21 дегенеруючі нейрони в зубчастій зви-
вині з’являлися через 24 год. У щурят П7 не спостерігалося
збільшення кількості GFAP- або Iba-1-позитивних клітин,
зумовленого ЕС. Експресія GFAP у щурят П15 дуже сильно
зростала в зонах CA1 та CA3 через 12 год. У той же час у
таких щурят не відмічалося збільшення імунореактивності
цього білка в зубчастій звивині. У щурят П21 даний індекс
збільшувався в зонах CA1, CA3 та зубчастій звивині вже
через 2 год після індукції ЕС. Кількість Iba-1-позитивних
клітин у трьох згаданих регіонах істотно збільшувалась у
щурят П15 через 12, а у тварин П21 – вже через 2 год. Ці ре-
зультати вказують на те, що пов’язані з ЕС ушкодження не-
йронів та активація глії в незрілому мозку в цілому є менш
інтенсивними, ніж у дорослому, а розвиток даних процесів
у гіпокампі є специфічним у просторовому та часовому ас-
пектах.
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 144
B. LI, L. YANG, H. ZHOU, and R. SUN
REFERENCES
1. K. Biber, H. Neumann, K. Inoue, and H. W. Boddeke,
“Neuronal ‘on’ and ‘off ’ signals control microglia,” Trends
Neurosci., 30, No. 11, 596-602 (2007).
2. R. J. Racine, “Modification of seizure activity by electrical
stimulation. II. Motor seizure,” Electroencephalogr. Clin.
Neurophysiol., 32, No. 3, 281-294 (1972).
3. F. E. Jensen, H. Blume, S. Alvarado, et al., “NBQX blocks
acute and late epileptogenic effects of perinatal hypoxia,”
Epilepsia, 36, No. 10, 966-972 (1995).
4. S. S. Schreiber, G. Tocco, I. Najm, et al., “Absence of c-fos
induction in neonatal rat brain after seizures,” Neurosci. Lett.,
136, No. 1, 31-35 (1992).
5. R. Sankar, D. H. Shin, and C. G. Wasterlain, “GABA
metabolism during status epilepticus in the developing rat
brain,” Dev. Brain Res., 98, No. 1, 60-64 (1997).
6. M. Patel and Q. Y. Li, “Age dependence of seizure-induced
oxidative stress,” Neuroscience, 118, No. 2, 431-437 (2003).
7. X. Wang, A. Zaidi, R. Pal, et al., “Genomic and biochemical
approaches in the discovery of mechanisms for selective
neuronal vulnerability to oxidative stress,” BMC Neurosci.,
19, doi: 10.1186/1471-2202-10-12 (2009).
8. M. M. Haglund and P. A. Schwartzkroin, “Role of Na-K pump
potassium regulation and IPSPs in seizures and spreading
depression in immature rabbit hippocampal slices,” J.
Neurophysiol., 63, No. 2, 225-239 (1990).
9. H. B. Michelson and E. W. Lothman, “An in vivo
electrophysiological study of the ontogeny of excitatory and
inhibitory processes in the rat hippocampus,” Dev. Brain Res.,
47, No. 1, 113-122 (1989).
10. J. W. Swann, R. J. Brady, and D. L. Martin, “Postnatal
development of GABA-mediated synaptic inhibition in rat
hippocampus,” Neuroscience, 28, No. 3, 551-561 (1989).
11. J. Priller, C. A. Haas, M. Reddington, and G. W. Kreutzberg,
“Calcitonin gene-related peptide and ATP induce immediate
early gene expression in cultured rat microglial cells,” Glia,
15, No. 4, 447-457 (1995).
12. Y. Taniwaki, M. Kato, T. Araki, and T. Kobayashi, “Microglial
activation by epileptic activities through the propagation
pathway of kainic acid-induced hippocampal seizures in the
rat,” Neurosci. Lett., 217, No. 1, 29-32 (1996).
13. W. J. Streit, S. A. Walter, and N. A. Pennell, “Reactive
microgliosis,” Prog. Neurobiol., 57, No. 6, 563-581 (1999).
14. M. Oprica, C. Eriksson, and M. Schultzberg, “Inflammatory
mechanisms associated with brain damage induced by kainic
acid with special reference to the interleukin-1 system,” J. Cell
Mol. Med., 7, No. 2, 127-140 (2003).
|