The question of whether microwave radiation that is emitted by mobile phones (radiofrequency-modulated electromagnetic fields) might have any detrimental health effects remains unanswered [1,2].
As mobile phones are usually held close to the head when in use, part of the microwave they emit is absorbed by the brain .
Therefore, it is conceivable that microwaves from mobile phones could affect brain functions. In fact, several studies have already reported that mobile phones affect brain functioning and behaviors [4,5].
Moreover, the microwaves emitted have been shown to affect brain EEG activity  and cause gene expression alterations in the cortex and hippocampus .
Moreover, epidemiological studies have indicated that long-term use (longer than 10 years) may increase the risk for acoustic neuroma or meningioma  and malignant glioblastoma multiforme .
Tight junctions are important structural components of the blood–brain barrier; they are present in the apical region of interendothelial clefts and restrict paracellular permeability . Among other tight junction proteins, the transmembrane protein occludin is critically involved in sealing the tight junctions. It was thought that disruption of occludin alone was enough to cause functional changes in the tight junctions .
Data on the effect of exposure to an electromagnetic field on the permeability of the blood–brain barrier are still controversial, with both positive  and negative  findings.
Ascorbic acid (AA) is an important enzyme cofactor, antioxidant, and neuromodulator in the brain . It plays a crucial role in the physiological and pathological processes, that is, learning and memory and cerebral ischemia .
Thus, the objectives of this study were to evaluate, using light and electron microscopic examination, the possible toxic effects of long-term mobile phone radiation (42 days) on rat thalamic neurons. This study also aimed to assess blood–thalamic barrier integrity using immunostaining by the vascular endothelial cell tight junction protein occludin. The study also investigated the hypothesis that AA as an antioxidant could ameliorate mobile wave-induced changes.
Materials and methods
This study was carried out on 40 adult male albino rats, weighing 200–250 g. The animals were kept in plastic cages with mesh wire covers with adequate ventilation and at an optimal temperature and were fed standard laboratory food and water ad libitum. They were housed in the animal house at the Medical Research Center, Faculty of Medicine, Ain Shams University.
Mobile wave exposure protocol 
Animals were exposed to microwave radiation for 42 days (4 h/day in the light) over their entire bodies. The microwave radiation was produced by a mobile test phone (model NOKIA 3110; Nokia mobile phones Ltd, Finland). The mobile phone was placed over the cage of the animals to prevent damage. The whole-body specific energy absorption rate was estimated at 0.043–0.135 W/kg. These data were previously reported in a similar protocol, in which rats were exposed to the same duration of microwave radiation emitted from a similar mobile phone . During the exposure, the rats were awake and not restrained in their cages. The nonexposed groups were held separate in a different room.
Duration of experiment
The experiment was carried out for 42 days.
The animals were divided into four equal groups of 10 animals each.
The control group was kept under standard laboratory conditions without any intervention.
The mobile wave-exposed group was exposed to mobile waves according to the protocol mentioned  (for 42 days) (4 h/day in the light).
The AA intake with mobile wave-exposed group was exposed to mobile waves according to the protocol mentioned . They also received AA orally at a dose of 250 mg/kg/day . AA was supplied in the form of tablets from Ameyria Company, Cairo, Egypt. Tablets were dissolved in tap water.
The AA intake group received AA orally at a dose of 250 mg/kg/day  for 42 days daily.
At the end of the experiment, the animals were anesthetized and the brains were coronally sectioned at −2 mm from the Bregma and +7 mm from the interaural line  to visualize and obtain the thalamus.
The specimens were then prepared for the following:
For the light microscopic study, samples were fixed in 10% neutral-buffered formalin; paraffin blocks were generated; and 5-μm-thick sections were stained with hematoxylin and eosin (H&E) .
For the immunohistochemical study, staining was performed using the vascular endothelial cell tight junction protein occludin as an indicator of blood–brain barrier integrity.
Occludin is a protein that is encoded by the OCLN gene in humans. It is a 65 kDa (522-amino acid polypeptide) integral plasma-membrane protein located on the tight junctions .
Avidin–biotin peroxidase immunohistochemical staining technique 
Formalin-fixed paraffin-embedded sections were used for immunoperoxidase staining of the thalamus using the avidin–biotin peroxidase complex method . This technique was used to detect the tight junction protein occludin. Antibodies were purchased from Sigma (St. Louis, Missouri, USA). Sections were placed on positively charged glass slides. The paraffin sections were deparaffinized, hydrated, and placed in 10% H2O2 to block endogenous peroxidase activity. Unmasking of antigenic sites was carried out by transferring sections into a jar containing 0.001 mol/l citrate buffer and boiling in a microwave for 4 min. Blocking was carried out for 10 min with a serum blocking solution. Slides were incubated with the primary antibody (1 : 500 monoclonal goat anti-occludin protein) at room temperature for 2 h and, after washing, they were incubated with biotinylated secondary antibodies [ABC kit, 1 : 200 (St. Louis, Missouri, USA)]. Freshly prepared diaminobenzidine was used as a chromogen. Sections were incubated with diaminobenzidine for 10 min, washed with tap water, counterstained with hematoxylin, dehydrated, and mounted. For the negative control, the primary antibody was replaced by PBS and it was found that the cell border of blood vessels appeared brown.
For electron microscopy, small specimens (1×1 mm) were taken from all groups, fixed in a 3% cold phosphate-buffered glutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated in graded alcohol and embedded in epoxy resin. Semithin sections were cut and stained with toludine blue. Ultrathin sections were cut and stained with uranyl acetate, followed by lead citrate . Examination of the grids and photography were carried out using a 201 Philips transmission electron microscope (Netherlands) at the Anatomy Department, Faculty of Medicine, Ain Shams University.
For the morphometric study, five different nonoverlapping fields from five different sections of different rats were examined in each group (five high powers ×40 fields hpf per section). The measurements were performed by the image analyzer using Leica Q500 MC program installed on a PC. The PC was connected to a camera attached to a Zeiss universal microscope (Germany).
- (1) Mean area percentage for vascular tight junction occludin protein expression.
- (2) Number of histologically damaged neurons in H&E-stained sections according to the histological necrosis index (HNI) .
The HNI was defined and calculated as follows:
Neurons were classified morphologically into four categories, in which N1 through N4 are the number of neurons in the corresponding categories:
- (1) N1=morphologically intact neurons with vesicular nuclei and basophilic cytoplasm (Fig. 2).
- (2) N2=neurons with microvacuolization or eosinophilic cytoplasm (Fig. 8).
- (3) N3=neurons destroyed, with marked vacuolization of the cytoplasm or homogenization of the cytoplasm and an occasional faint outline of the nuclei (Fig. 5).
- (4) N4=shrunken neurons with pyknotic or darkly stained nuclei (Fig. 5).
For statistical analysis
All data were collected, revised, and then subjected to statistical analysis using student's t-test. The significance of data was determined by P value, where P<0.05 is considered significant (S).
Group I (control group)
H&E-stained sections of the thalamus showed that it is located just inferior to the hippocampus and the superior horn of lateral ventricles (Fig. 1). The thalamic neurons appeared in different sizes and shapes. Some were round and others were oval. They showed a basophilic cytoplasm and vesicular nuclei, whereas the smaller neuroglia cells were scattered in between neurons (Fig. 2).
Immunocytochemistry of the vascular endothelial cell tight junction occludin protein demonstrated that the cell borders of the thalamic microvessels were intensely immunoreactive (Fig. 3).
The ultrastructural examination of the thalamic neurons demonstrated large euchromatic nuclei surrounded by a well-defined regular nuclear envelope. The cytoplasm appeared covered with numerous ribosomes, mitochondria, and rough endoplasmic reticulum (Fig. 4).
Group II (mobile wave-exposed group)
H&E-stained sections of the thalamus showed that some neurons had marked cytoplasmic vacuolization. Others were irregular in shape and shrunken, with darkly stained nuclei. They appeared to be enveloped by halos (Fig. 5).
Immunohistochemical staining showed that the expression of the tight junction protein occludin in thalamic microvessels was reduced and nearly depleted compared with the control group (Fig. 6).
Ultrastructurally, the nuclei of the affected neurons appeared to have an irregular nuclear envelope. Their cytoplasm showed prominent vacuolization (Fig. 7).
Group III (ascorbic acid intake with mobile wave-exposed group)
H&E-stained sections of the thalamus showed many neurons with cytoplasmic microvacuolization (Fig. 8).
Immunohistochemical-stained sections revealed discontinuous immune-expression on the cell borders of thalamic microvessels (Fig. 9).
The ultrastructural examination of the thalamic neurons revealed slight cytoplasmic vacuolation. However, numerous ribosomes and mitochondria were evident in these neurons (Fig. 10).
The histological profile of group IV animals was almost identical to that of the control group I.
- (1) Mean area percentage for tight junction occludin protein expression: Microvessels in the microwave exposure group (group II) showed statistically significantly less mean area percentage for occludin protein than the control group P<0.001 (Table 1 and Histogram 1), whereas the mean area percentage for protein occludin in microvessels in group III (microwave exposure plus AA intake) was significantly elevated P<0.001 compared with group II (Table 1), although significantly reduced compared with control group P<0.001 (Table 1 and Histogram 1).
- (2) Number of histologically damaged neurons in H&E-stained sections according to the HNI/hpf: The thalamic neurons of microwave-exposed rats showed statistically significant higher HNI than the control rats P<0.001 (Table 2 and Histogram 1). In contrast, rats subjected to wave exposure with intake of AA showed significantly decreased HNI P<0.001 compared with group II (Table 2), but still significantly higher than the control group P<0.001 (Table 2 and Histogram 1).
In recent years, the widespread use of mobile phones has given rise to a growing concern over the possible adverse effects of the emitted electromagnetic waves on human health . Thus, the specific aim of this study was to determine whether long-term exposure to mobile waves (for 42 days) would lead to cytopathological effects on rat thalamus. Moreover, the study investigated the potential role of AA administration in ameliorating these changes.
In this study, examination of H&E-stained sections of wave-exposed rats revealed the presence of many degenerated neurons that appeared markedly vacuolated, irregular in shape, and shrunken, with darkly stained nuclei. Moreover, there was a significant elevation in HNI as compared with the control group. Ultrastructurally, the thalamic neurons showed an irregular nuclear envelope and a vacuolated cytoplasm.
Similarly, other authors reported that, 7 days after exposure to radiation from GSM-900 mobile phones, the damaged neurons were often shrunken and homogenized, with loss of discernable internal cell structures. They added that some damaged neurons showed microvacuoles in the cytoplasm. They believe that these vacuoles are signs of severe neuropathy, indicating an active pathological process .
Previous investigators also reported that microwaves from mobile phones induced neuronal death both in in-vivo rat brain and in-vitro cultured cells. Trypan blue staining and terminal deoxynucleotidy transferase-mediated nick-end labeling staining and immunohistochemistry for Bcl-2 and Bax expression were used in their study .
A few studies have revealed in detail the cellular signaling pathways involved in the induction of neuronal damage. They have shown that radiofrequency radiation could activate calcium channels by means of a specialized family of receptors, which in turn allowed calcium-ion efflux into the extracellular space. These changes in calcium homeostasis might be responsible for the increase in membrane conductivity and prolongation of the refractory period following depolarization .
Immunohistochemistry performed in this study using vascular tight junction occludin protein enabled the assessment of blood–thalamic barrier permeability. Occludin protein expression in thalamic microvessels was significantly reduced following microwave exposure.
Similar findings were obtained by many researchers. An increase in blood–brain barrier permeability was detected after 7 , 14, and 28 days  of exposure to microwaves from GSM mobile phones. Histologically, albumin extravasation, neuronal albumin uptake and neuronal damage were assessed by these researchers.
Interestingly, the molecular mechanism involved in the destruction of blood–brain barrier integrity was studied by many investigators. Some authors presented evidence suggesting that a pinocytotic-like mechanism was responsible for the microwave-induced increase in barrier permeability . Others hypothesized that mobile phone radiation could affect cytoplasmic distribution and the stability of F-actin and stress fibers of vascular endothelial cells. They added that relocalization of F-actin to endothelial cell ruffles caused these cells to round up. Consequently, these cells could only establish contact through thin pseudopods, thus increasing vascular permeability .
AA is one of the most important enzyme cofactors, antioxidants, and neuromodulators. It was found that it could prevent and treat a number of neurological disorders including vertigo and Meniere's disease . This study revealed that chronic administration of AA was able to confer a degree of protection against mobile wave-induced neuronal damage, which led to a significant reduction in HNI in comparison with group II. In addition, immunohistochemical examination revealed that AA partially preserved the integrity of the blood–thalamic barrier, which was detected by a significant increase in the mean area percentage of tight junction protein occludin expression in comparison with group II.
However, little is known about the mechanisms by which AA exerts its effects. An explanation for the protective effect of AA was that, as it is an oxygen-radical scavenger, it could thus prevent neuronal damage by scavenging free radicals in the central nervous system [32,33].
Accumulated evidence suggested that AA is highly concentrated in extracellular and intracellular brain spaces, and appears to undergo dynamic changes in response to a variety of physiological and pathophysiological conditions. Observations have implicated that AA can regulate brain dopaminergic and glutamatergic transmission [14,32].
Investigations of the effects of electromagnetic waves on humans, and biology as a whole, are increasingly important. This study showed the potential importance of AA in the prevention, at least partially, of microwave-induced injury.
2. Khurana UG, Teo C, Kundi M, Hardell L, CarLberg M. Cellphones and brain tumors: a review including the long term epidemiologic data. Surg Neurol 2009; 72:205–214.
3. Lang S. Recent advances in bioelectromagnetics research on mobile telephony and health – an introduction PIERS. 2006;2:192–196
4. Hamblin DL, Wood AW. Effects of mobile phone emissions on human brain activity and sleep variables Int J Radiat Biol. 2002;78:659–669
5. Ferreri F, Curcio G, Pasqualetti P, De Gennaro L, Fini R, Rossini PM. Mobile phone emissions and human brain excitability Ann Neurol. 2006;60:188–196
6. Vecchio F, Babiloni C, Ferreri F, Curcio G, Fini R, Del Percio C, Rossini PM. Mobile phone emission modulates interhemispheric functional coupling of EEG alpha rhythms Eur J Neurosci. 2007;25:1908–1913
7. Nittby H, Widegren B, Krogh M, Grafström G, Berlin H, Rehn G, et al. Exposure to radiation from global system for mobile communications at 1800 MHz significantly changes gene expression in rat hippocampus and cortex Environmentalist. 2008;28:458–465
8. Hardell L, Carlberg M, Hansson Mild K. Case–control study on cellular and cordless telephones and the risk for acoustic neuroma or meningioma in patients diagnosed 2000–2003 Neuroepidemiology. 2005;25:120–128
9. Hardell L, Carlberg M, Hansson Mild K. Pooled analysis of two case–control studies on the use of cellular and cordless telephones and the risk of benign brain tumours diagnosed during 1997–2003 Int J Oncol. 2006;28:509–518
10. Wolburg H, Lippoldt A. Tight junctions of the blood–brain barrier
: development, composition and regulation Vascul Pharmacol. 2002;38:323–337
11. Persidsky Y, Ramirez SH, Haorah J, Kanmogne GD. Blood–brain barrier
: structural components and function under physiologic and pathologic conditions J Neuroimmune Pharmacol. 2006;1:223–236
12. Hossmann KA, Hermann DM. Effects of electromagnetic radiation of mobile phones on the central nervous system Bioelectromagnetics. 2003;24:49–62
13. Gruenau SP, Oscar KJ, Folker MT, Rapoport SI. Absence of microwave effect on blood–brain barrier
permeability to [14C] sucrose in the conscious rat Exp Neurol. 1982;75:299–307
14. Rebec GV, Pierce RC. A vitamin as neuromodulator: ascorbate release into the extracellular fluid of the brain regulates dopaminergic and glutamatergic transmission Prog Neurobiol. 1994;43:537–565
15. Liu K, Lin Y, Xiang L, Yu P, Su L, Mao L. Comparative study of change in extracellular ascorbic acid
in different brain ischemia/reperfusion models with in vivo microdialysis combined with on-line electrochemical detection Neurochem Int. 2008;52:1247–1255
16. Sokolovic D, Djindjic B, Nikolic J, Bjelakovic G, Pavlovic D, Kocic G, et al. Melatonin reduces oxidative stress induced by chronic exposure of microwave radiation from mobile phones in rat brain J Radiat Res. 2008;49:579–586
17. Yanardag R, Ozsoy Sacan O, Ozdil S, Bolkent S. Combined effects of vitamin C, vitamin E and sodium selenate supplementation on absolute ethanol-induced injury in various organs of rats Int J Toxicol. 2007;26:513–523
18. Paxinos G, Watson C The rat brain in stereotaxic coordinates. 20055th ed San Diego Elsevier Academic Press
19. Bancroft JD, Stevens A Theory and practice of histological techniques. 19964th ed New York Churchill Livingstone
20. Ando Akatsuka Y, Saitou M, Hirase T, Kishi M, Sakakibara A, Itoh M, et al. Interspecies diversity of the occludin sequence: cDNA cloning of human, mouse, dog and rat-kangaroo homologues J Cell Biol. 1996;133:43–47
21. Kiernan JA Histological and histochemical methods: theory and practice. 20003rd ed Oxford Butterworth-Heinemann
22. Glauret AM, Lewis PR Biophysical, specimen preparation for transmission electron microscopy. 199817th ed London Parhand Press
23. Nogami M, Takatsu A, Ishiyama I. Immunohistochemical study of neuron-specific enolase in human brains from forensic autopsies Forensic Sci Int. 1998;94:97–109
24. Barnett J, Timotijevic L, Shepherd R, Senior V. Public responses to precautionary information from the Department of Health (UK) about possible health risks from mobile phones Health Policy. 2007;82:240–250
25. Nittby H, Brun A, Eberhardt J, Malmgren L, Persson BRR, Salford LG. Increased blood–brain barrier
permeability in mammalian brain 7 days after exposure to the radiation from a GSM-900 mobile phone Pathophysiology. 2009;16:103–112
26. Zhu Y, Gao F, Yang X, Shen H, Liu W, Chen H, Jiang X. The effect of microwave emission from mobile phones on neuron survival in rat central nervous system Prog Electromagnet Res. 2008;82:287–298
27. Blackman CF, Benane SG, Elder JA. Induction of calcium-ion efflux from brain tissue by radiofrequency radiation: effect of sample number and modulation frequency on the power-density window Bioelectromagnetics. 1980;1:35–43
28. Eberhardt JL, Persson BRR, Brun AE, Salford LG, Malmgren LOG. Blood–brain barrier
permeability and nerve cell damage in rat brain 14 and 28 days after exposure to microwaves from GSM mobile phones Electromagnet Biol Med. 2008;27:215–229
29. Neubauer C, Phelan AM, Kues H, Lange DG. Microwave irradiation of rats at 2.45 GHz activates pinocytotic-like uptake of tracer by capillary endothelial cells of cerebral cortex Bioelectromagnetics. 1990;11:261–268
30. Leszczynski D, Joenvaara S, Reivinen J, Kuokka R. Non-thermal activation of the hsp27/p38MAPK stress pathway by mobile phone radiation in human endothelial cells: molecular mechanism for cancer- and blood–brain barrier
-related effects Differentiation. 2002;70:120–129
31. Zhang N, Liu JX, Ma FR, Yu LS, Lin YQ, Liu K, Mao LQ. Change of extracellular ascorbic acid
in the brain cortex following ice water vestibular stimulation: an on-line electrochemical detection coupled with in vivo microdialysis sampling for guinea pigs Chin Med J. 2008;121:1120–1125
32. Ciani E, Groneng L, Voltattorni M, Rolseth V, Contestabile A, Paulsen RE. Inhibition of free radical production or free radical scavenging protects from the excitotoxic cell death mediated by glutamate in cultures of cerebellar granule neurons Brain Res. 1996;728:1–6
33. Rice ME. Ascorbate regulation and its neuroprotective role in the brain Trends Neurosci. 2000;23:209–216