Periventricular leukomalacia (PVL) is a common white matter lesion affecting the neonatal brain. PVL is closely associated with cerebral palsy (CP) and characterized by increase in the number of astrocytes, which can be detected by positivity for glial fibrillary acidic protein (GFAP). Change in myelin basic protein (MBP) is an early sign of white matter abnormality. Maternal or placental infection can damage the neonatal brain. In the present study, we investigated the effects of treadmill walking exercise on GFAP and MBP expressions in rats with maternal lipopolysaccharide (LPS)-induced PVL. Immunohistochemistry was performed for the detection of GFAP and MBP. The present results showed that intracervical maternal LPS injection during pregnancy increased GFAP expression in the striatum and decreased MBP expression in the corpus callosum of rats. The results also showed that treadmill walking exercise suppressed GFAP expression and enhanced MBP expression in the brains of rats with maternal LPS-induced PVL. The present study revealed that treadmill walking exercise is effective for the suppressing astrogliosis and hypomyelination associated with PVL. Here in this study, we showed that treadmill walking exercise may be effective therapeutic strategy for alleviating the detrimental effects of CP.
Approximately 10% of newborns are born prematurely, and severe disturbances in mental development and perinatal brain injuries have been observed in premature infants (
Astroglia and myelin are important components of white matter. Reactive astrogliosis, typified by astrocyte proliferation and/or astrocytic hypertrophy, is a common phenomenon in the central nerve system (CNS) following brain trauma or inflammation (
Maternal LPS administration increases the number of GFAP-positive astrocytes, decreases MBP expressions and alters microglia immunoreactivity in the brain of offspring (
It is well known that exercise induces several physiological and biochemical changes in the brain (
In the present study, we investigated whether treadmill walking would have a protective effect on astrogliosis and hypomyelination in rats with PVL. The animal model of PVL was induced by maternal intracervical injection of LPS. In the present study, immunohistochemistry was performed for the detection of GFAP in the striatum and MBP in the corpus callosum.
This study was performed in accordance with the guidelines of the National Institutes of Health (NIH) and the Korean Academy of Medical Sciences. Female Sprague-Dawley rats (180±10 g, 8 weeks old, n=20) were allowed to mate with male rats for a period of 24 h. The female rats were then individually housed in plastic home cages under controlled temperature (20±2°C) and a light –dark cycle consisting of 12 h of light and 12 h of darkness (lights on from 07:00 to 19:00 h). Food and water were made available
The pregnant rats were divided into two groups: the sham group and the LPS-treated group (n=4 in each group). On the 9th, 13th, and 17th day of pregnancy, the pregnant rats in the LPS-treated group received intracervical injections of 1.0 mg/kg LPS (from
Eight weeks after birth, the rats in the exercise group were forced to walk on a motorized treadmill for 30 min three times a week for 4 weeks. The exercise load consisted of walking at the speed of 8 meters/min at an inclination of 0°.
The animals were sacrificed immediately after the completion of the last exercise. The rats were weighed and given an overdose of Zoletil 50® (10 mg/kg i.p.; Vibac Laboratories, Carros, France). After a complete lack of response was observed, the rats were transcardially perfused with 50 mM phosphate-buffered saline (PBS) and subsequently fixed with freshly prepared 100 mM phosphate buffer (PB, pH 7.4) containing 4% paraformaldehyde. The brains were removed and fixed in the same fixative overnight and then transferred to a 30% sucrose solution for cryoprotection. Serial coronal sections of 40-μm thickness were obtained using a freezing microtome (Leica, Nussloch, Germany).
An average of six sections within the striatum spanning from Bregma 1.20 mm to 0.96 mm and six sections within the corpus callosum spanning from Bregma 0.84 mm to 0.60 mm were obtained from each brain. Tissue sections were incubated overnight with rabbit anti-GFAP antibody or anti-MBP antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:500.
The sections were then incubated with anti-rabbit secondary antibody (1:200, Vector Laboratories, Burlingame, CA, USA). The sections were subsequently incubated with avidin- biotin-peroxidase complex (1:100, Vector Laboratories) for 1 h at room temperature. Immunoreactivity was visualized by incubating the sections in a solution containing 0.02% 3, 3′-diaminobenzidine tetrahydrochloride (DAB) and 0.03% H2O2 in 50 mM Tris-HCl (pH 7.6) for approximately 5 min. The sections were then washed three times with PBS and mounted onto gelatin-coated slides. The slides were air-dried overnight at room temperature, and coverslips were mounted using Permount® (Fisher Scientific, New Jersey, NJ, USA).
The intensities of GFAP and MBP staining were quantitatively assessed using a microdensitometrical method based on optical density (the mean gray scale) with the use of an image analyzer (Multiscan, Fullerton, CA, USA). Before starting the image analysis, the light source was adjusted to the brightness that generated the best possible contrast between immunopositive and immunonegative cells.
Statistically significant differences were determined by one-way analysis of variance (ANOVA) followed by Duncan’s
Eight weeks after birth, the mean body weight of the rats was 297±3.85 g in the sham group, 246±24.33 g in the LPS-treated group, and 299±2.00 g in the LPS-treated and exercise group. After 4 weeks of exercise, the mean body weight of the rats was 394±4.60 g in the sham group, 347±7.54 g in the LPS-treated group, 342±7.50 g in the LPS-treated and exercise group. There was no significant difference in body weight between the groups.
Photomicrographs of GFAP expression in the striatum are presented in
Photomicrographs of MBP expression in the corpus callosum are presented in
Maternal infection as well as increased levels of pro-inflammatory cytokines in amniotic fluid and fetal blood is associated with preterm birth and neonatal neurological disorders (
In the present results, intracervical injection of LPS into the maternal rats induced astrogliosis in the striatum of rats that was represented by enhanced expression of GFAP in the striatum. Many studies have shown that PVL is closely associated with the increased number of GFAP-positive astrocytes in the white matter lesion of infant brains (
In the present results, intracervical injection of LPS into the maternal rats induced hypomyelination in the corpus callosum of rats, that was represented by suppressed expression of MBP in the corpus callosum. MBP is the most abundant protein in the myelin sheath. One possible reason for the reduced MBP staining is a reduction MBP production by the oligodendrocyte or a reduction in the number of oligodendrocytes after maternal LPS treatment. Reduced myelination and oligodendrocytes are concomitantly observed in the infant brains with PVL (
In the present results, we investigated the effects of treadmill walking exercise on astrogliosis and hypomyelination in rats with PVL. Treadmill walking exercise suppressed LPS-induced astrogliosis in the striatum and alleviated LPS-induced hypomyelination in the corpus callosum of the rats with PVL. The present study revealed that treadmill walking exercise is effective for the suppressing astrogliosis and hypomyelination associated with PVL. In the present study, we showed that treadmill walking exercise may be effective therapeutic strategy for alleviating the detrimental effects of CP.
This work was supported by the research grant of the Jeju National University in 2011.
No potential conflict of interest relevant to this article was reported.
Photomicrographs of glial fibrillary acidic protein (GFAP)-positive cells in the striatum. The scale bar represents 250 μm.
Effect of treadmill walking exercise on the expression of glial fibrillary acidic protein (GFAP) in the striatum of rats with maternal endotoxin-induced periventricular leukomalacia (PVL). (A) Sham group, (B) lipopolysaccharide (LPS)-treated group, (C) LPS-treated and exercise group. The data are presented as the mean± SEM. *Represents
Photomicrographs of myelin basic protein (MBP)-positive cells in the corpus callosum. The scale bar represents 250 μm.
Effect of treadmill walking exercise on the expression of myelin basic protein (MBP) in the corpus callosum of rats with maternal endotoxin-induced periventricular leukomalacia (PVL). (A) Sham group, (B) lipopolysaccharide (LPS)-treated group, (C) LPS-treated and exercise group. The data are presented as the mean± SEM. *Represents