Long-term exposure to 835 MHz RF-EMF induces hyperactivity, autophagy and demyelination in the cortical neurons of mice

Abstract

Radiofrequency electromagnetic field (RF-EMF) is used globally in conjunction with mobile communications. There are public concerns of the perceived deleterious biological consequences of RF-EMF exposure. This study assessed neuronal effects of RF-EMF on the cerebral cortex of the mouse brain as a proxy for cranial exposure during mobile phone use. C57BL/6 mice were exposed to 835 MHz RF-EMF at a specific absorption rate (SAR) of 4.0 W/kg for 5 hours/day during 12 weeks. The aim was to examine activation of autophagy pathway in the cerebral cortex, a brain region that is located relatively externally. Induction of autophagy genes and production of proteins including LC3B-II and Beclin1 were increased and accumulation of autolysosome was observed in neuronal cell bodies. However, proapoptotic factor Bax was down-regulted in the cerebral cortex. Importantly, we found that RF-EMF exposure led to myelin sheath damage and mice displayed hyperactivity-like behaviour. The data suggest that autophagy may act as a protective pathway for the neuronal cell bodies in the cerebral cortex during radiofrequency exposure. The observations that neuronal cell bodies remained structurally stable but demyelination was induced in cortical neurons following prolonged RF-EMF suggests a potential cause of neurological or neurobehavioural disorders.

Figure 1. Behavioural tests of RF-EMF exposed mice.

Basic motor activity (rota-rod, a) and general locomotor activity (rearing frequency, total distance moved, and total duration movement) in the open field (b–d) were measured after RF-EMF exposure. Each bar illuminates the mean ± SEM of value of 6 mice. *P < 0.05.

Figure 2. Expressional levels of autophagy related genes in the cerebral cortex of mice following the chronic RF-EMF exposure.

The cerebral cortical RNA extracted sham-exposed and RF-exposed mice were analysed for the expression level of autophagy genes by quantitative real-time PCR. (a–j) Quantification of AMPK1α, Ulk1, Atg4/B, Beclin1/2, Atg5, Atg9A, and LC3A/B mRNA transcripts by qRT-PCR. (k) Agarose gel electrophoresis showing differential expression of autophagy genes by sqRT-PCR. The expression values of the cerebral cortex of RF-exposed mice were normalized to those of the sham-exposed mice. The relative mRNA levels of each gene were calculated by normalizing to expression of GAPDH by the 2^−ΔΔCt method (n = 6). Each bar represents the mean ± SEM of three independent experiments. Statistical significance was evaluated using a t-test: *P < 0.05, **P < 0.01.

Figure 3. Protein expression of LC3B-II and Beclin1 in the cerebral cortex of mice after chronic RF-EMF exposure.

(a) Total protein extracted from cerebral cortex of mice was subjected to 10–15% SDS–PAGE and immunoblotted with antibody against LC3B-II and Beclin1. α-tubulin was used as the internal loading control. (b) The intensity of bands of western blot was quantified by densitometry. The protein level was normalized relative to α-tubulin. Each bar shows mean of three independent experiments with SEM. Statistical significance was evaluated using two tailed t-test: *P < 0.05.

Figure 4. Expression of apoptosis-related genes in the cerebral cortex of mice following chronic RF-EMF exposure.

(A) The cerebral cortical RNA extracted sham-exposed and RF-exposed mice were analysed for the expression level of autophagy genes by quantitative real-time PCR. (a,b) Quantification of Bcl2 and Bax mRNA transcripts by qRT-PCR. (c) 1.5% Agarose gel electrophoresis showing differential expression of Bcl2 and Bax by sqRT-PCR. The expression values of the cerebral cortex of RF-exposed mice were normalized to those of the sham-exposed mice. The relative mRNA levels of each gene were calculated by normalizing to expression of GAPDH by the 2^−ΔΔCt method (n = 6). (B) (a) Total protein was subjected to 15% SDS–PAGE and immunoblotted with antibodies against Bcl2 and Bax. α-tubulin was used as loading control. (b) The intensity of bands of western blot was quantified by densitometry. The protein level was normalized relative to α-tubulin. Each bar represents the mean ± SEM of three independent experiments. Statistical significance was evaluated using a t-test: *P < 0.05, **P < 0.01.

Figure 5. Representative TEM images showing the autophagy in the neuronal cell body of the cerebral cortex following chronic RF-EMF exposure.

(A) Ultrastructural comparison of autophagy between control and RF-exposed mice. Representative TEM micrographs were acquired from sham control (a) and RF-exposed mice for 12 weeks (b), respectively. In RF-exposed mice, numerous autophagosome (Ap) and autolysosome (Aly) were observed in difference with its sham control. (B) A series of autophagic flux process in cerebral cortical neurons after RF-EMF exposure for 12 weeks. (a) Phagophore containing fragments of cytoplasmic organelles; (b) Early autophagosome; (c) Late autophagosome; (d) Early Autolysosome; and (e) Late autolysosome. Abbreviations are: Ap, autophagosome; Aly, autolysosome; G, Golgi apparatus; M, mitochondria; N, nucleus; Ph, phagophore; RER, rough endoplasmic reticulum. Size bars: 1 μm (A) and 500 nm (B).

Figure 6. Ultrastructural alterations of myelin sheaths in neurons of the cerebral cortex in mice following chronic RF-EMF exposure.

Representative TEM micrographs were acquired from sham control (a,b) and RF-exposed mice for 12 weeks (c,d), respectively. Right side figures showed high power images of each insert (black dotted box) in a–d. Allows indicates damage to myelin sheaths with unusual myelin protrusions into cortical neuron in mice following RF-EMF exposure. Also, allow heads show breakdown sheaths thereby showing blurry myelin. M, mitochondria; My, myelin sheaths. Size bars: 500 nm (Low power images) 100 nm (High power images).