Effects of Prenatal Exposure to MRI Magnetic Fields on Protein Expression of Glutamate Receptors in the Hippocampal Formation of Rats?
Mali Jiang, Taizhen Han??, Dongwei Yang, Wen Xie
Abstract: This present study investigated the effects of prenatal exposure to the magnetic resonance imaging (MRI) magnetic fields on protein expression of NMDA receptor subunits NR1, NR2B and AMPA receptor subunit GluR1 in rats' hippocampal formation at different stages of postnatal development. Pregnant rats on gestation of the 12th-18th days were exposed to the magnetic fields used for MRI applications. When the female offspring were 1, 2, and 5-month-old, the SP immunohistochemical staining was made in the paraformaldehyde fixed hippocampal sections. Computer-assisted image was taken for analysis. Compared with the control group, the MRI group showed a significant increase of NR1 immunoreactivity in the hippocampal CA1 region and an increasing tendency in the hippocampal CA3 area at the age of 5 months. No significant effect was detected at the age of 1 and 2 months in the hippocampal formation. No significant difference was observed between the two groups in NR2B and GluR1 subunits at all the ages. The results indicate that prenatal exposure to the MRI magnetic fields induces changes of NMDA receptors in the hippocampus of mature brain.
Key words: prenatal exposure delayed effects; MRI; NMDA receptor; AMPA receptor; hippocampal formation
Scientific researches reported that the central nervous system (CNS) is particularly susceptible to magnetic fields (MFs) [1, 2], which can markedly affect neural electrophysiological and biochemical activities [3, 4]. Exposure to MFs increases the risk of inducing neurodegenerative diseases in humans  and producing deficits in spatial leaning in rats . Therefore, the public concern about the safety of exposure to MFs is increasing. Magnetic resonance imaging (MRI) has been used in studying fetal development [7, 8] and assessing brain injuries , due to its high capacity of visualizing the brain structure. However, whether prenatal exposure to MRI affects the developmental brain is still not clear. Our previous study indicated that prenatal exposure to the MRI magnetic fields not only caused cognitive/behavioral deficits but also induced synaptic ultrastructural changes [10, 11]. The present study investigated the influences of periodic exposure of pregnant rats to MRI magnetic fields on the offspring's immunohistochemical expression of N-Methyl-D-Aspartate (NMDA) receptor subunits NR1, NR2B and ¦Á-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit GluR1 in the hippocampal formation at different postnatal ages.
MATERIALS AND METHODS
1. Subjects and exposure
Fifteen healthy pregnant female Sprague-Dawley rats were used in the present study. The time of exposure to MFs or sham condition started from the 12th to 18th day of gestation. Randomly selected 8 of these pregnant rats were exposed to the MRI field for 7 consecutive days with 40 min a day. The others were exposed to a sham control field with the same time course. The MRI field was 0.35T 1KW power impulse magnetic field that was produced by a Diasonics MRT-35A system (Comp, USA; spin echo T1: TR 500ms/TE 15ms, slice thinness 10mm, Nex 4; T2: TR 2000ms/TE 120ms, Slice thinness 10mm, Nex 2).
After parturition, each of the 15 litters was housed separately in a standard plastic cage (46cm¡Á31.5cm¡Á20cm) with food and water available adlib for the maternal rats. The room temperature was controlled at 21¡À1¡æ. The light-dark cycle was 12/12 h (light on at 7:00AM). On the second postnatal day, the number of pups in each litter was reduced to 8, with a random manner. When the offspring were 21-day old, they were separated from their maternal rats.
Female rats aged 1, 2 and 5-month were examined. The animals of the same age were divided into two groups, control (CON) and MRI groups. There were 3-7 animals in each group.
0.1mol/L paraformaldehyde (pH 7.4), phosphate-buffer (pH 7.4), 0.3% H2O2, 1% Triton X-100, serum albumin, anti-NR1 (1:1,000), NR2B (1:200) and GluR1 (1:100) antibodies (Sigma), phosphate-buffer saline, biotin-goat anti-rabbit IgG (ZhongShan), Streptavidin/Peroxidase complex (ZhongShan), DAB (ZhongShan), ethanol, dimethylbenzene, colophony, glutin.
3. Main instruments
Vibratome (S-1000, TPI Company, USA), light microscope (BH2, Olympus Company, Japan), Image analysis system (Q 550CW, Leica Company, Germany)
Animals were anesthetized by pentobarbital (45mg/kg i.p.) and perfused through the ascending aorta with saline (0.9%) followed by 4% paraformaldehyde in 0.1 mol/L phosphate-buffer (PB), pH 7.4. The brains were removed, postfixed in 4% paraformaldehyde for 12h. Coronal sections (40¦Ìm-thick) were cut with a Vibratome.
For free-floating immunohistochemistry, the brain sections were first treated for 30min in 0.3% H2O2 and then incubated in 1% Triton X-100 and serum albumin for 60 min successively at room temperature, followed by anti-NR1 (1:1,000), NR2B (1:200) and GluR1 (1:100) antibodies (Sigma company) respectively for 72h at 4¡æ. After five washes in phosphate-buffer saline, the sections were then incubated with biotin-goat anti-rabbit IgG (Zhongshan) for 2h then in Streptavidin/Peroxidase complex (Zhongshan) for 2h at room temperature and stained by DAB. The sections were dehydrated in graded ethanol, pellucidumed and coverslipped. In negative control group serum albumin was used instead of primary antibodies.
5. Analysis and statistics
Sections were evaluated and images were taken using the Q550CW system (Leica company). The value of gray was presented as ¡ÀS. Student's test was applied for differences analyzing and p<0.05 was considered to represent significant difference.
1. The expression of NMDA receptor subunit NR1
Expression of NR1 can be detected in the hippocampal pyramidal cells and dentate gyrus granules cells. Positive structures exist mainly in cytoplasm and processes of the neurons. With growing, the positive expression of the control group emerged a decreasing tendency. The mean grey of NR1 in the hippocampal formation was measured. Compared with CON group, the MRI group showed a significant increase of NR1 immunoreactivity at 5-month old in the hippocampal CA1 region. And an increasing tendency was observed in the CA3 area at this age between the two groups (Fig.1). No significant difference was detected on NR1 subunit protein expression at 1- and 2-month old in the three regions of the hippocampal formation between the two groups (Table 1).
Table 1. Immunoreactivity grey value of NR1 subunit in rats' hippocampal formation after
prenatal exposure to MRI magnetic fields (¡ÀS)
One-monthTwo-monthFive-monthCONMRICONMRICONMRICA1110.67¡À35.83129.78¡À31.67128.11¡À19.52121.58¡À25.81158.26¡À11.4133.91¡À24.25*CA3109.53¡À33.81129.48¡À32.36125.17¡À2.29117.96¡À24.47150.03¡À17.86124.57¡À27.14#DG107.54¡À38.85132.88¡À30.92123.42¡À3.75121.12¡À25.11156.38¡À9.17137.02¡À29.84 *p<0.05, # p=0.076, compared with control group
Fig.1 Changes of NR1 subunit protein expression in the hippocampal formation after prenatal exposure to MRI magnetic fields (100¡Á) (A: control group, B: MRI group)
2. Expressions of NMDA receptor subunit NR2B
No difference in all of the ages and hippocampal regions of female offspring was observed between two groups for NR2B subunit (Table 2).
Table 2. Immunoreactivity grey value of NR2B subunit in hippocampal formation of rats (¡ÀS)
One-monthTwo-monthFive-monthCONMRICONMRICONMRICA1139.06¡À27.81157.04¡À30.5141.89¡À28.86120.43¡À7.89137.82¡À16.39121.54¡À24.57CA3132.79¡À22.42151.98¡À31.23139.63¡À32.38120.46¡À6.19137.03¡À18.13112.69¡À23.61DG136.48¡À22.44151.66¡À30.89137.59¡À31.73119.59¡À9.43142.22¡À22.81120.84¡À23.38 p>0.05, compared with control group
3. Expression of AMPA Receptor Subunit GluR1
As in subunit NR2B, no difference in all of the ages and hippocampal regions of the female offspring was observed between two groups for GluR1 subunit (Table 3).
Table 3. Immunoreactivity grey value of AMPA receptor subunit GluR1 in rats' hippocampal formation(¡ÀS)
One-monthTwo-monthFive-monthCONMRICONMRICONMRICA1108.72¡À15.61132.62¡À30.80133.59¡À13.65131.29¡À30.56132.23¡À31.88135.41¡À21.88CA3104.53¡À12.35126.37¡À28.83133.3¡À19.12120.74¡À7.35125.81¡À37.05130.69¡À20.07DG101.01¡À11.76125.02¡À29.53133.57¡À18.97112.86¡À13.66124.02¡À34.41129.89¡À22.93 p>0.05, compared with control group
The experimental results showed that prenatal exposure to MRI magnetic fields induced the changes of NMDA receptors (NMDARs), but not the AMPA receptors (AMPARs). Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system, and it activates both ionotropic and metabotropic glutamate receptors (GluRs). Ionotropic GluRs can be grouped into three categories according to their specific agonists and sequence homology: N-methyl-D-aspartate (NMDA)-type receptors, ¦Á-amino-3-hydroxyl-5- methyl-4-isoxazole propionic acid (AMPA)-type receptors and kainite receptors. Several subunits have been distinguished for each category of ionotropic GluRs, creating great structural and functional heterogeneity [12, 13]. NMDARs are made up of four or five protein subunits which probably contain two or three NR1 subunits (at least one is obligatory for receptor function) and two or three NR2 (A-D) subunits. The NR2B subunit plays more important role in plasticity than other NR2 subunits. In recent years, NMDARs have raised particular interest for two reasons. Firstly, NMDARs are important in variety forms of synaptic plasticity including long-term potentiation, and are known to be critical for normal learning and memory processes. Secondly, NMDARs have been suggested to be involved in excitotoxic cell death and other degenerative processes related to neurological diseases [14-16]. AMPARs are tetrameric or pentameric complexes assembled from any of four different subunits (GluR1-4). Each subunit contains one large extracellular domain and four membrane-associated domains showing considerable homology among different subunits. AMPARs mediate fast synaptic current at most excitatory synapses, and are thought to involve in the bulk of excitatory synaptic transmission during neural activity while NMDARs are particularly important for triggering a number of different forms of synaptic plasticity. GluR1 subunit is a substantial proportion of endogenous AMPARs in hippocampal neurons. Plasticity-inducing stimulation initially caused a net addition of GluR1-containing AMPARs, which may eventually be replaced by GluR2-containing AMPARs, resulting in a long-lasting enhancement in synaptic transmission [17-19]. Combined with the function of these receptors, we suppose that prenatal exposure to MRI magnetic fields is unable to affect the normal synaptic transmission, whereas it influences some forms of synaptic plasticity, such as learning and memory.
It is found that the level of NR1 subunit expression increased in neurodegenarative disease, such as Huntington disease . Therefore, the alterations of NR1 protein in five-month-old female offspring indicate that prenatal exposure to MFs probably interrelate with the occurrence of neurodegenerative diseases. Otherwise, NR1 subunit is involved in multiple formations of learning and memory, such as spatial learning, fear conditioning, inhibitory and active avoidance, olfactory and taste memories, etc. . Alterations of NR1 protein induced by prenatal exposure to MRI could also associate to these formations of learning and memory. The functional significance of NR1 expression changes in prenatal exposure to MFs at 5-month old needs to be investigated.
In summary, the present results suggest that prenatal exposure to the MRI magnetic fields can cause changes of NMDA receptor NR1 subunit in the hippocampus of mature brain. Applying the MRI technology in pregnant women should be chary.
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(Edited by Xiaoman Ling, Xia Gao, Yanhong Wei, Yingqi Zhao and Yanling Xiao)
* Supported by: National Natural Science Foundation of China, No. 30200075; The key laboratory of Environment and Genes related to Diseases (Xi'an Jiaotong University), Ministry of Education Xi'an, Shanxi, China, Postcode: 710061; The key Project of Xi'an Jiaotong University
Mali Jiang, PhD in Department of Physiology, School of Medicine, Xi'an Jiaotong University; Main research fields: molecule mechanism of learning and memory; Tel: 13096938215; Fax: 029-82655274; E-mail: firstname.lastname@example.org
?? Corresponding to Taizhen Han, PhD, professor of Department of Physiology, School of Medicine, Xi'an Jiaotong University; Address: Department of Physiology, Medical School of Xi'an Jiaotong University, 76# Yanta West Street, Xi'an, Shanxi, China, Postcode: 710061; Tel: 029-82655274; Fax: 029-82655274; E-mail: email@example.com