Dhiraj Maskey

Effect of 835 MHz radiofrequency radiation exposure on calcium binding proteins in the hippocampus of the mouse brain

Worldwide expansion of mobile phones and electromagnetic field (EMF) exposure has raised question of their possible biological effects on the brain and nervous system. Radiofrequency (RF) radiation might alter intracellular signaling pathways through changes in calcium (Ca(2+)) permeability across cell membranes. Changes in the expression of calcium binding proteins (CaBP) like calbindin D28-k (CB) and calretinin (CR) could indicate impaired Ca(2+)homeostasis due to EMF exposure. CB and CR expression were measured with immunohistochemistry in the hippocampus of mice after EMF exposure at 835 MHz for different exposure times and absorption rates, 1 h/day for 5 days at a specific absorption rate (SAR)=1.6 W/kg, 1 h/day for 5 days at SAR=4.0 W/kg, 5 h/day for 1 day at SAR=1.6 W/kg, 5 h/day for 1 day at SAR=4.0 W/kg, daily exposure for 1 month at SAR=1.6 W/kg. Body weights did not change significantly. CB immunoreactivity (IR) displayed moderate staining of cells in the cornu ammonis (CA) areas and prominently stained granule cells. CR IR revealed prominently stained pyramidal cells with dendrites running perpendicularly in the CA area. Exposure for 1 month produced almost complete loss of pyramidal cells in the CA1 area. CaBP differences could cause changes in cellular Ca(2+)levels, which could have deleterious effect on normal hippocampal functions concerned with neuronal connectivity and integration.

Chronic 835-MHz radiofrequency exposure to mice hippocampus alters the distribution of calbindin and GFAP immunoreactivity.

Exponential interindividual handling in wireless communication system has raised possible doubts in the biological aspects of radiofrequency (RF) exposure on human brain owing to its close proximity to the mobile phone. In the nervous system, calcium (Ca(2+)) plays a critical role in releasing neurotransmitters, generating action potential and membrane integrity. Alterations in intracellular Ca(2+) concentration trigger aberrant synaptic action or cause neuronal apoptosis, which may exert an influence on the cellular pathology for learning and memory in the hippocampus. Calcium binding proteins like calbindin D28-K (CB) is responsible for the maintaining and controlling Ca(2+) homeostasis. Therefore, in the present study, we investigated the effect of RF exposure on rat hippocampus at 835 MHz with low energy (specific absorption rate: SAR=1.6 W/kg) for 3 months by using both CB and glial fibrillary acidic protein (GFAP) specific antibodies by immunohistochemical method. Decrease in CB immunoreactivity (IR) was noted in exposed (E1.6) group with loss of interneurons and pyramidal cells in CA1 area and loss of granule cells. Also, an overall increase in GFAP IR was observed in the hippocampus of E1.6. By TUNEL assay, apoptotic cells were detected in the CA1, CA3 areas and dentate gyrus of hippocampus, which reflects that chronic RF exposure may affect the cell viability. In addition, the increase of GFAP IR due to RF exposure could be well suited with the feature of reactive astrocytosis, which is an abnormal increase in the number of astrocytes due to the loss of nearby neurons. Chronic RF exposure to the rat brain suggested that the decrease of CB IR accompanying apoptosis and increase of GFAP IR might be morphological parameters in the hippocampus damages.

Genetic Evaluation of ALADIN Gene in Early-Onset Achalasia and Alacrima Patients

Background/Aims ALADIN gene has been known to cause achalasia, alacrima, adrenal abnormalities and a progressive neurological syndrome. A considerable proportion of achalasia patients has been known to show alacrima (decreased secretion of tear). However, the genetic mechanism between achalasia and alacrima has not been defined yet. We postulated that ALADIN gene may be involved in the occurrence of early-onset achalasia; thus, we investigated the correlation of ALADIN gene in early-onset achalasia patients. Methods From 1989 to 2007, patients who were diagnosed as primary achalasia before age 35 were enrolled. All of the enrolled patients were asked for (1) blood sampling for DNA, (2) Shirmer test and (3) dysphagia questionnaires. Results The ALADIN gene in exon 1, 2, 10, 11 and 12 from 19 patients was investigated (M:F = 12:7). The mean age of patients at diagnosis was 27 ± 5 (15-35) years old. Eight out of 19 (42%) showed alacrima by the positive Shirmer test. In spite of thorough exam in the genetic study, there was no definite abnormal genetic finding in this study. Conclusions A considerable number of achalasia patients showed alacrima. Due to the limitation of this study, it is difficult to conclude that early-onset achalasia may have significant correlations with the ALADIN gene.

Calcium-binding proteins and GFAP immunoreactivity alterations in murine hippocampus after 1 month of exposure to 835 MHz radiofrequency at SAR values of 1.6 and 4.0 W/kg.

Widespread use of wireless mobile communication has raised concerns of adverse effect to the brain owing to the proximity during use due to the electromagnetic field emitted by mobile phones. Changes in calcium ion concentrations via binding proteins can disturb calcium homeostasis; however, the correlation between calcium-binding protein (CaBP) immunoreactivity (IR) and glial cells has not been determined with different SAR values. Different SAR values [1.6 (E1.6 group) and 4.0 (E4 group) W/kg] were applied to determine the distribution of calbindin D28-k (CB), calretinin (CR), and glial fibrillary acidic protein (GFAP) IR in murine hippocampus. Compared with sham control group, decreased CB and CR IRs, loss of CB and CR immunoreactive cells and increased GFAP IR exhibiting hypertrophic cytoplasmic processes were noted in both experimental groups. E4 group showed a prominent decrement in CB and CR IR than the E1.6 group due to down-regulation of CaBP proteins and neuronal loss. GFAP IR was more prominent in the E4 group than the E1.6 group. Decrement in the CaBPs can affect the calcium-buffering capacity leading to cell death, while increased GFAP IR and changes in astrocyte morphology, may mediate brain injury due to radiofrequency exposure.

Glutamate co-transmission from developing medial nucleus of the trapezoid body - Lateral superior olive synapses is cochlear dependent in kanamycin-treated rats

Cochlear dependency of glutamate co-transmission at the medial nucleus of the trapezoid body (MNTB)--the lateral superior olive (LSO) synapses was investigated using developing rats treated with high dose kanamycin. Rats were treated with kanamycin from postnatal day (P) 3 to P8. A scanning electron microscopic study on P9 demonstrated partial cochlear hair cell damage. A whole cell voltage clamp experiment demonstrated the increased glutamatergic portion of postsynaptic currents (PSCs) elicited by MNTB stimulation in P9-P11 kanamycin-treated rats. The enhanced VGLUT3 immunoreactivities (IRs) in kanamycin-treated rats and asymmetric VGLUT3 IRs in the LSO of unilaterally cochlear ablated rats supported the electrophysiologic data. Taken together, it is concluded that glutamate co-transmission is cochlear-dependent and enhanced glutamate co-transmission in kanamycin-treated rats is induced by partial cochlear damage.

Immunohistochemical localization of calbindin D28-k, parvalbumin, and calretinin in the cerebellar cortex of the circling mouse

The spontaneous mutant circling mouse has an autosomal recessive pattern of inheritance and is an animal model for deafness, which is characterized by circling, head tossing, and hyperactivity. Since the main pathology in circling mice lies in the organ of Corti, most studies on deaf mice have focused on auditory brain stem nuclei. No studies regarding behavior-related CNS changes in circling mice have been reported. The major center of sensory input for modulation of motor activity is best-studied in the cerebellum. Considering the importance of calcium homeostasis in numerous processes, calcium-binding proteins (CaBPs), such as calbindin D-28k (CB), parvalbumin (PV), and calretinin (CR), may play crucial roles in preserving cerebellar coordinated motor function. Thus, the distribution of CB, PV, and CR was determined in the cerebellum using immunohistochemical methods to compare immunoreactivity (IR) of CaBPs between wild-type (+/+), heterozygous (+/cir), and homozygous (cir/cir) mice. The IR of CB and PV was predominantly observed in the Purkinje cell layer of all three genotypes. Compared with the +/+ genotype, the relative mean density of CB and PV IR in the Purkinje cell layer and CR IR in the granular layer was significantly decreased in the cir/cir genotype. Changes in calcium homeostasis in parallel fiber/Purkinje cell synapses could diminish cerebellar control of motor coordination. A number of deficiencies among the CaBPs lead to distinct alterations in brain physiology, which may affect normal behavior.

Changes in the distribution of calbindin D28-k, parvalbumin, and calretinin in the hippocampus of the circling mouse.

The circling (cir) mouse strain, a murine model of deafness caused by a spontaneous mutation, exhibits characteristic behaviors of circling and hyperactivity. In an induced-noise paradigm, cir mice display a significant loss in their spatial orientation abilities, and this has been suggested to be due at least in part to changes in calcium homeostasis. Auditory information is transferred from the cochlear nucleus to the hippocampus, where it is processed to modulate motor and sensory activity. Such a pathway could be affected at the cellular level by alterations in neurotransmission, including alterations that involve Ca(2+). However, there have been no studies in a hearing deficit model examining the concomitant molecular alterations in the hippocampus. Thus, in the present study we used immunohistochemistry to compare the distribution of the calcium-binding proteins (CaBPs) calbindin D-28k, parvalbumin, and calretinin in the hippocampi of heterozygous (+/cir), homozygous (cir/cir), and wild-type (+/+) mice. The expression of the CaBPs in various hippocampal subfields appeared to be significantly lower in cir mice (+/- and -/-) than in +/+ mice. Such a decrease in CaBP expression in cir/cir mice would alter calcium homeostasis, which in turn could affect the connection of the tri-synaptic circuit of the hippocampus as well as the cortical region. A decrease in CaBPs and the probable resultant glutamate-mediated excitability could contribute to the functional changes that lead to the characteristic behavioral features of cir mice.

Neuroprotective Effect of Ginseng against Alteration of Calcium Binding Proteins Immunoreactivity in the Mice Hippocampus after Radiofrequency Exposure

Calcium binding proteins (CaBPs) such as calbindin D28-k, parvalbumin, and calretinin are able to bind Ca2+ with high affinity. Changes in Ca2+ concentrations via CaBPs can disturb Ca2+ homeostasis. Brain damage can be induced by the prolonged electromagnetic field (EMF) exposure with loss of interacellular Ca2+ balance. The present study investigated the radioprotective effect of ginseng in regard to CaBPs immunoreactivity (IR) in the hippocampus through immunohistochemistry after one-month exposure at 1.6 SAR value by comparing sham control with exposed and ginseng-treated exposed groups separately. Loss of dendritic arborization was noted with the CaBPs in the Cornu Ammonis areas as well as a decrease of staining intensity of the granule cells in the dentate gyrus after exposure while no loss was observed in the ginseng-treated group. A significant difference in the relative mean density was noted between control and exposed groups but was nonsignificant in the ginseng-treated group. Decrease in CaBP IR with changes in the neuronal staining as observed in the exposed group would affect the hippocampal trisynaptic circuit by alteration of the Ca2+ concentration which could be prevented by ginseng. Hence, ginseng could contribute as a radioprotective agent against EMF exposure, contributing to the maintenance of Ca2+ homeostasis by preventing impairment of intracellular Ca2+ levels in the hippocampus.

Immunohistochemical localization of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor in the superior olivary complex of mice after radiofrequency exposure.

Raising health concerns about the biological effects from radiofrequency exposure, even with conflicting results, has prompted calls for formulation of a guideline of the biological safety level. Given the close proximity between a mobile phone and the ear, it has been suggested that the central auditory system may be detrimentally influenced by radiofrequency exposure. In the auditory system, neurotrophins are important in the regulation of neuron survival, especially mammalian cochlear neurons. Neurotrophic factors like brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) present in the auditory system are responsible for the maintenance of auditory neurons. BDNF and GDNF may protect against acoustic trauma and prevent from hearing defect. The present study applied radiofrequency at a specific absorption rate (SAR) of 1.6W/kg (E1.6) or 0W/kg group to determine the distribution of BDNF and GDNF in the nuclei of superior olivary complex (SOC). In the E1.6 group, significant decrements of BDNF immunoreactivity (IR) were noted in the lateral superior olive, medial superior olive, superior paraolivary nucleus and medial nucleus of the trapezoid body. GDNF IR was also significantly decreased (p<0.001) in all SOC nuclei of the E1.6 group. The decrease in the IR of these neurotrophic factors in the SOC of the E1.6 group suggests a detrimental effect of RF exposure in the auditory nuclei.

Decreased Immunoreactivities and Functions of the Chloride Transporters, KCC2 and NKCC1, in the Lateral Superior Olive Neurons of Circling Mice

Objectives We tested the possibility of differential expression and function of the potassium-chloride (KCC2) and sodium-potassium-2 chloride (NKCC1) co-transporters in the lateral superior olive (LSO) of heterozygous (+/cir) or homozygous (cir/cir) mice. Methods Mice pups aged from postnatal (P) day 9 to 16 were used. Tails from mice were cut for DNA typing. For Immunohistochemical analysis, rabbit polyclonal anti-KCC2 or rabbit polyclonal anti-NKCC1 was used and the density of immunolabelings was evaluated using the NIH image program. For functional analysis, whole cell voltage clamp technique was used in brain stem slices and the changes of reversal potentials were evaluated at various membrane potentials. Results Immunohistochemical analysis revealed both KCC2 and NKCC1 immunoreactivities were more prominent in heterozygous (+/cir) than homozygous (cir/cir) mice on P day 16. In P9-P12 heterozygous (+/cir) mice, the reversal potential (Egly) of glycine-induced currents was shifted to a more negative potential by 50 µM bumetanide, a known NKCC1 blocker, and the negatively shifted Egly was restored by additional application of 1 mM furosemide, a KCC2 blocker (-58.9±2.6 mV to -66.0±1.5 mV [bumetanide], -66.0±1.5 mV to -59.8±2.8 mV [furosemide+bumetanide], n=11). However, only bumetanide was weakly, but significantly effective (-60.1±2.9 mV to -62.7±2.6 mV [bumetanide], -62.7±2.6 mV to -62.1±2.5 mV [furosemide+bumetanide], n=7) in P9-P12 homozygous (cir/cir) mice. Conclusion The less prominent immunoreactivities and weak or absent responses to bumetanide or furosemide suggest impaired function or delayed development of both transporters in homozygous (cir/cir) mice.