Glucocorticoid receptor expression in neonatal rat cortex following recurrent seizures The role in developing brain injury**☆
Tao Bo, Lu Yi, Tuanmei Wang, Jian Li, Xingfang Li, Dingan Mao

Department of Pediatrics, Second Xiangya Hospital, Central South University, Changsha  410011, Hunan Province, China

Tao Bo☆, Doctor, Associate professor, Department of Pediatrics, Second Xiangya Hospital, Central South University, Changsha  410011, Hunan Province, China

Corresponding author: Tao Bo, Department of Pediatrics, Second Xiangya Hospital, Central South University, Changsha  410011, Hunan Province, China
bot1114@tom.com

Supported by: the National Natural Science Foundation of China, No. 30400483*; the Natural Science Foundation of Hunan Province, No. 07JJ5020*

Abstract
BACKGROUND: Studies have explored changes in neonatal rat glucocorticoid receptor (GR) ex-pression changes following mature brain injury.
OBJECTIVE: To investigate the temporal and special changes of GR during brain development in rats with recurrent seizures.
DESIGN, TIME AND SETTING: A randomized, controlled animal experiment was performed at the Department of Pediatrics, Second Xiangya Hospital of Central South University, from February 2008 to March 2009.
MATERIALS: Rabbit anti-rat GR monoclonal antibody was purchased from Santa Cruz Biotech-nology, USA; goat anti-rabbit IgG was purchased from Zhongshan Goldenbridge Biotechnology, China.
METHODS: A total of 48 Sprague-Dawley rats, 7 days old, were randomly assigned to control and seizure groups, with 24 animals in each group. Seizures were induced by inhalant flu-rothyl.
MAIN OUTCOME MEASURES: Changes in GR protein expression in the rat cerebral cortex were detected by Western blotting analysis and immunohistochemistry.
RESULTS: GR expression in the cerebral cortex of control rats significantly increased with aging  (P < 0.05), and varied in the frontal lobe, temporal lobe, and parietal lobe. GR was predominantly expressed in the cytoplasm early and rapidly increased in the nuclei. GR protein expression in the cerebral cortex after seizure was lower in the cytoplasm at 15 days and in nuclear protein at 19 days.
CONCLUSION: GR expression displayed temporal and spatial changes in brain development. Recurrent seizures in neonatal rats cause abnormal GR expression and might play an important role in developing brain injury.
Key Words: seizure; neonatal; glucocorticoid receptor; brain; development; rats

INTRODUCTION
  
The role of glucocorticoids in neuronal injury remains controversial[1]. Under physiological conditions, glucocorticoid binds most (60%) mineralocoid receptors to maintain fundamental hypothalamus- pituitary-adrenal gland activity; under acute stress, glucocorticoid secretion is increased, and saturating mineralocorti-coid receptor activates glucocorticoid re-ceptor (GR), producing a series of changes[2-3]. Receptor changes in the brain influence the function of glucocorti-coid, and GR highly correlates with brain development[4].
Here, we measured changes in GR distribu-tion from the nucleus to the cytoplasm in neonatal rats and in recurrent seizures.

MATERIALS AND METHODS

Design
A randomized, controlled animal experi-ment.
Time and setting
The experiment was performed at the De-partment of Pediatrics, Second Xiangya Hospital of Central South University, from February 2008 to March 2009.
Animals
A total of 48 Sprague-Dawley rats, aged 7 days, weighing (10.25 ± 0.83) g, were pro-vided by the Laboratory Animal Center of Second Xiangya Hospital of Central South University (No. SYXK (Xiang) 2004-0013). The experimental procedures were in ac-cordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and
Technology of the People’s Republic of China[5].
Reagents and instruments are listed as follows:

Methods
Establishment of flurothyl-induced seizure model and grouping
Rats were randomly assigned to seizure and control groups, with 24 animals in each group. Seizure group rats were placed in a 40 cm × 20 cm × 20 cm transparent test chamber, and 0.1 mL flurothyl was dropped in the chamber through the hole at the top. The chamber was blocked, and the rats were removed from the chamber 30 minutes fol-lowing seizures and observed for 4 hours. The seizure was induced once a day for 6 consecutive days to establish a model of seizure-induced brain injury[6]. The control rats were not treated with flurothyl.
Eight rats from each group were randomly selected and sacrificed at 13, 15 and 19 days. The brains were harvested; the left cortex was subjected to Western blot test, and the right cortex was used for immunohistochemistry.
Cell protein extraction
The cortex preserved in nuclear protein extraction solu-tion was homogenized with liquid nitrogen, incubated at room temperature for 15 minutes, and centrifuged at 250 × g for 5 minutes. After the supernatant was dis-carded, the sediments were resuspended with a 2-fold volume of cytoplasmic lysate, drawn repetitively with a 27# needle injector, and centrifuged at 8 000 × g for 20 minutes. The supernatant, i.e. cytoplasmic protein, was harvested and stored at -70 °C. The sediments were mixed with 660 μL nuclei extraction solution, drawn repetitively with a 27# needle injector, shaken for 60 minutes, centrifuged at 16 000 × g for 5 minutes, and the supernatant, i.e. nuclear protein, was harvested and stored at -70 °C[7]. Protein concentration was deter-mined using uQuant Microplate Spectrophotometer by ultraviolet absorption.
GR expression detected by Western blot analysis
Protein samples were mixed with the same volume of loading buffer (125 mmol/L pH 6.8 Tris-HCl, 20% glycer-ine, 0.002% bromophenol blue, 10% β-mercaptoethanol and 4% sodium dodecylsulfate) and electrophoresed on a 7% SDS-polyacrylamide gel. The protein sample vol-ume was 50 μg in each lane, and every electrophoresis involved protein samples of all individuals from two groups. The protein was transferred to a nitrocellulose filter by a semi-dry electrophoretic transfer method, blocked by 5% defatted milk powder for 1 hour, and in-cubated with rabbit anti-rat GR monoclonal antibody (1: 500) overnight at 4 °C. The filter was washed with TTBS and incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (1: 2 500) for   1 hour at room tempera-ture. The filter was washed with TTBS, followed by DAB coloration to show the target band[8]. The integral ab-sorbance (IA) was obtained by Image-Pro 5.0 image processing software (Media Cybernetics Inc. USA)[9]. Beta-actin served as cytoplasmic protein internal refer-ence, and Lamin B1 as nuclear protein internal refer-ence.
Immunohistochemical staining
The rat right cerebral hemisphere was prepared as par-affin sections, 10 μm thick, which were deactivated by 3% H2O2, blocked with normal goat serum for 1 hour, incubated with rabbit anti-rat GR monoclonal antibody  (1: 80) overnight at 4 °C. An ABC kit was used for im-munohistochemistry[10]. Briefly, the sections were washed three times with PBS for 5 minutes, incubated with 50 μL biotin-labeled goat anti-rabbit IgG for 1 hour at 37 °C, washed three times with PBS for 5 minutes, incubated with 50 μL horseradish peroxidase-labeled streptavidin for 20 minutes at 37 °C; washed three times with PBS 5 minutes, colored by DAB under the microscope, washed with tap water for 10 minutes, counterstained with he-matoxylin for 2 minutes, mixed with hydrochloric acid alcohol, washed with tap water for 10 minutes, dehy-drated by gradient alcohol, cleared, mounted and ob-served by microscopy. A total of two visual fields (× 400) from each section were harvested. IA was calculated by Image-Pro 5.0 image processing software. A total of two sections of frontal lobe, temporal lobe, and parietal lobe from each rat were harvested, and one visual field (× 400) from each section was selected. Yellow brown staining was regarded as GR-positive. The IA of the positive re-gion was calculated by computer using software.
Main outcome measures
GR expression in rat cerebral cortex.
Statistical analysis
All experimental data were analyzed by SPSS 13.0 software (SPSS Inc. USA. No. GS-35F-5899F). Meas-urement data are expressed as Mean ± SD. Levene’s test was used under homogeneity of variance. Mean comparison between two groups was performed by in-dependent-samples t test, and differences among groups were compared by one-way analysis of variance. With α = 0.05 as test criteria, P < 0.05 was considered statisti-cally significant.

RESULTS

GR expression changes detected by Western blot analysis
Cytoplasmic and nuclear GR expression increased with time in controls (P < 0.05). Seizures delayed GR ex-pression and significantly reduced cytoplasmic proteins at 15 days and nuclear protein at 19 days (P < 0.01 or P < 0.05; Table 1, Figure 1).

GR expression changes detected by immunohisto-chemistry
In the control group, cytoplasmic GR expression was seen at 13 days and nuclear protein at 15 and 19 days in the frontal lobe, parietal lobe, and temporal lobe. In the seizure group, IA was significantly less than the control group in the parietal lobe at 13 days, parietal lobe and temporal lobe at 15 days, as well as frontal, parietal and temporal lobe at 19 days (P < 0.05; Table 2, Figure 2).

Behavior of flurothyl-induced seizure
Seizures happened 1-2 minutes following flurothyl treatment, characterized by dysphoria, head shaking, screaming, moving around, and, tonic clonic seizures. One seizure was maintained for 5-8 minutes at      3-5 minutes intervals. The rats were sleeping and breathless when removed from the test chamber but recovered after 30 minutes. No spontaneous convulsion or overdose-induced death was found in any group.

DISCUSSION

Brain development in rats at 5-7 days is similar to full-term newborn rats[11]. We used 7-day-old rats to es-tablish a model of developing brain injury by flu-rothyl-induced recurrent seizures. Flurothyl can induce seizures, and the brain edema and neuronal necrosis resemble changes in humans following seizures[12].
Here, GR was expressed in the cerebral cortex soon after birth and increased with time. Moreover, GR ex-pression levels varied in the frontal, parietal, and tem-poral lobe, indicating regular changes in brain develop-ment. In addition, GR was predominantly expressed in the cytoplasm early but subsequently increased in nuclei. This indicates that GR regulates target gene expression in neurons and participates in early brain development.
Compared with controls, GR expression levels were sig-nificantly downregulated in the cytoplasm at 15 days and nuclei at 19 days of rat cerebral cortex following seizures. Immunohistochemistry further demonstrated spatial GR expression, indicating that recurrent seizures delayed increased expression in GR in the nuclei and influences target gene expression, ultimately interfering with normal brain development.
The normal hypothalamus-pituitary-adrenal axis is essential to development of the nervous system. Glucocorticoid regulates neuronal survival, growth, and apoptosis by adrenocortical hormone receptor protein[13]. Exogenous glucocorticoid in the neonatal stage disrupts hypothalamus-pituitary-adrenal axis stability and affects brain development. Accordingly, the hypothalamus-pituitary-adrenal axis changes after brain injury. Glucocorticoid in the treatment of seizure-induced brain injury remains controversial[14-17]. Here, recurrent seizures downregulated cortical glucocorticoid expression in the early neonatal stage and overdoses of glucocorticoid cannot inhibit inflammation but leads to many adverse reactions. Therefore, further investigation is required to determine appropriate timelines for exogenous glucocorticoid and its therapeutic effects.

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 (Edited by Niu YJ, He L, Lou Y/Su LL/Song LP)