No evidence of DNA damage by co-exposure to extremely low frequency magnetic fields and aluminum on neuroblastoma cell lines.

Whether exposure to 50-60Hz extremely low frequency magnetic fields (ELF-MF) exerts neurotoxic effects is a debated issue. Analogously, the potential role of Aluminum (Al) in neurodegeneration is a matter of controversial debate. As all living organisms are exposed to ELF-MF and/or Al daily, we found investigating the early effects of co-exposure to ELF-MF and Al in SH-SY5Y and SK-N-BE-2 human neuroblastoma (NB) cells intriguing. SH-SY5Y5 and SK-N-BE-2 cells underwent exposure to 50Hz ELF-MF (0.01, 0.1 or 1mT) or AlCl3 (4 or 40µM) or co-exposure to 50Hz ELF-MF and AlCl3 for 1h continuously or 5h intermittently. The effects of the treatment were evaluated in terms of DNA damage, redox status changes and Hsp70 expression. The DNA damage was assessed by Comet assay; the cellular redox status was investigated by measuring the amount of reduced glutathione (GSH) and glutathione disulfide (GSSG) while the inducible Hsp70 expression was evaluated by western blot analysis and real-time RT-PCR. Neither exposure to ELF-MF or AlCl3 alone induced DNA damage, changes in GSH/GSSG ratio or variations in Hsp70 expression with respect to the controls in both NB cell lines. Similarly, co-exposure to ELF-MF and AlCl3 did not have any synergic toxic effects. The results of this in vitro study, which deals with the effects of co-exposure to 50Hz MF and Aluminum, seem to exclude that short-term exposure to ELF-MF in combination with Al can have harmful effects on human SH-SY5Y and SK-N-BE-2 cells.

Brain hsp70 expression and DNA damage in mice exposed to extremely low frequency magnetic fields: a dose-response study.

PURPOSE: To determine whether a dose-response relationship exists among exposure to extremely low frequency magnetic fields (ELF-MF) at different densities and 70-kDa heat shock protein (hsp70) expression and DNA damage in mouse brain. MATERIALS AND METHODS: Male CD1 mice were exposed to ELF-MF (50 Hz; 0.1, 0.2, 1 or 2 mT) for 7 days (15 h/day) and sacrificed either at the end of exposure or after 24 h. Hsp70 expression was determined in cerebral cortex-striatum, hippocampus and cerebellum by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. Primary DNA damage was evaluated in the same tissues by comet assay. Sham-exposed mice were used as controls. RESULTS: No changes in both hsp70 mRNA and corresponding protein occurred following exposure to ELF-MF, except for a weak increase in the mRNA in hippocampus of exposed mice to 0.1 mT ELF-MF. Only mice exposed to 1 or 2 mT and sacrificed immediately after exposure presented DNA strand breaks higher than controls in all the cerebral areas; such DNA breakage reverted to baseline in the mice sacrificed 24 h after exposure. CONCLUSIONS: These data show that high density ELF-MF only induce reversible brain DNA damage while they do not affect hsp70 expression.

Intravenous administration of pravastatin immediately after middle cerebral artery occlusion reduces cerebral oedema in spontaneously hypertensive rats.

3-hydroxy-3-methyl-glutaryl-coenzyme-A (HMG-CoA) reductase inhibitors (statins) have been shown to protect against ischemic stroke by mechanisms that are independent of lowering serum cholesterol levels. In this study we investigated the potential neuroprotective effect of a single i.v. treatment with four increasing doses of pravastatin on permanent occlusion of middle cerebral artery (MCAo) in spontaneously hypertensive rats. Pravastatin was given 10 min after MCAo and its effect was determined 24 h later. Treatment results were evaluated in terms of infarct volume, homolateral hemisphere oedema, glial fibrillary acid (GFAP), vimentin (Vim) and endothelial NO synthase (eNOS) immunoreactivity and TUNEL positivity. Cerebral levels of eNOS were measured by western blot analysis. Pravastatin did not reduce cerebral infarct while it mitigated homolateral hemisphere oedema in a dose-dependent manner with respect to controls. No differences among groups were found regarding GFAP and Vim immunoreactivity and TUNEL positivity. Instead, pravastatin-treated animals presented a more marked cerebral eNOS immunoreactivity as compared with controls. In agreement with immunohistochemistry, immunoblot revealed dose-dependent increases in cerebral levels of eNOS in pravastatin rats. Our data confirm statin neuroprotection in cerebral ischemia. In particular, it is of great interest that a single i.v. Pravastatin administration reduced cerebral oedema by upregulating eNOS expression/activity. This, by increasing vascular NO bioavailability, could have produced proximal vasodilation and contributed to reducing perfusional deficit. It is worthy stressing how important the anti-oedema action is that pravastatin seems to exert. Indeed, cerebral oedema, when widespread and beyond limits of physiological compensation, causes endocranic hypertension and additional cerebral damage over time.

Brain DNA damage and 70-kDa heat shock protein expression in CD1 mice exposed to extremely low frequency magnetic fields.

PURPOSE: The question of whether exposure to extremely low frequency magnetic fields (ELF-MF), may contribute to cerebral cancer and neurodegeneration is of current interest. In this study we investigated whether exposure to ELF-MF (50 Hz-1 mT) harms cerebral DNA and induces expression of 70-kDa heat shock protein (hsp70). MATERIALS AND METHODS: CD1 mice were exposed to a MF (50 Hz-1 mT) for 1 or 7 days (15 h/day) and sacrificed either at the end of exposure or after 24 h. Unexposed and sham-exposed mice were used as controls. Mouse brains were dissected into cerebral cortex-striatum, hippocampus and cerebellum to evaluate primary DNA damage and hsp70 gene expression. Food intake, weight gain, and motor activity were also evaluated. RESULTS: An increase in primary DNA damage was detected in all cerebral areas of the exposed mice sacrificed at the end of exposure, as compared to controls. DNA damage, as can be evaluated by the comet assay, appeared to be repaired in mice sacrificed 24 h after a 7-day exposure. Neither a short (15 h) nor long (7 days) MF-exposure induced hsp70 expression, metabolic and behavioural changes. CONCLUSIONS: These results indicate that in vivo ELF-MF induce reversible brain DNA damage while they do not elicit the stress response.

Permanent brain ischemia induces marked increments in hsp72 expression and local protein synthesis in synapses of the ischemic hemisphere.

Transient focal ischemia induced in rat brain by occlusion of the middle cerebral artery (MCAo) elicits a generalized induction of the 72 kDa heat-shock protein (hsp72) heralding functional recovery. As this effect implies activation of protein synthesis, and local systems of protein synthesis are present in brain synapses, and may be analyzed in preparations of brain synaptosomes, we evaluated hsp72 expression and protein synthesis in synaptosomal fractions of spontaneously hypertensive rats (SHRs) subjected to permanent MCAo. SHRs were randomly divided in ischemics and sham controls, anaesthesia controls and passive controls. Focal ischemia was induced under chloral hydrate anaesthesia by unilateral permanent MCAo. Protein synthesis was determined by [35S]methionine incorporation into synaptosomal proteins from ischemic and contralateral cortex/striatum, and from cerebellum. Hsp72 expression was measured in the same fractions by immunoblotting. Our data demonstrate that under these conditions synaptic hsp72 markedly increases in the ischemic hemisphere 1 and 2 days after MCAo, progressively declining in the following 2 days, while no significant change occurs in control rats. In addition, in the ischemic hemisphere the rate of synaptic protein synthesis increases more than two-fold between 1 and 4 days after MCAo, without showing signs of an impending decline. The present data provide the first demonstration that synaptic protein synthesis is massively involved in brain plastic events elicited by permanent focal ischemia.

Hippocampal 72-kDa heat shock protein expression varies according to mice learning performance independently from chronic exposure to stress.

The possibility that the inducible 72-kDa heat shock protein (hsp72) is involved in learning-related plasticity mechanisms was investigated in two inbred strains of mice that show spontaneous differences in spatial learning performance as well as an opposite reactivity to stress. Induction of hsp72 after radial maze training was measured by immunoblotting in the hippocampus of C57BL/6 (C57) and DBA/2 (DBA) inbred mice exposed or nonexposed to chronic acoustic stress. In agreement with previous studies, inter-strain differences in radial maze performance were found in nonstressed mice with C57 mice showing the higher scores. Chronic acoustic stress, however, impaired performance in the high-learner C57 strain and improved performance in the low-learner DBA strain. Western blot analysis revealed that post-training expression of hsp72 was low in the condition each strain was showing the higher-performance (nonstressed C57 and stressed DBA) and high in the condition each strain was showing the lower performance (stressed C57 and nonstressed DBA). These findings indicate that expression of hsp72 in the hippocampus varies as a function of the learning performance independently from exposure to chronic acoustic stress.

Gas chromatography-tandem mass spectrometry analysis of gabapentin in serum.

A new highly sensitive analytical method for determining gabapentin [1-(aminomethyl) cyclohexaneacetic acid; Neurontin] in serum using gas chromatography/tandem mass spectrometry (GC-MS/MS) was developed. GC-MS/MS was applied to determine the levels of gabapentin in serum samples of mice at 1 and 6 h after oral or intraperitoneal treatment (300 mg/kg). At 1 h, the concentrations of the drug were 4.02 +/- 0.42 and 4.32 +/- 0.28 microg/mL in mice treated orally and intraperitoneally, respectively. At 6 h, drug levels decreased by about 66% in both groups. The method, coupling two stages of mass analysis, could be very useful in identifying the drug in complex mixtures such as blood and urine. Moreover, it is easy and rapid to perform, and sensitive enough to allow the presence of the drug to be determined at very low detection limits. It is a very reliable method for both clinical and experimental monitoring of gabapentin.

EEG power spectra changes and forebrain ischemia in rats.

BACKGROUND: Several animal models of cerebral ischemia have been developed to investigate both pathophysiology and pharmacological treatment. The aim of this study was to verify the prognostic value of EEG power spectra analysis in a two-vessel plus hypotension rat model of transient global ischemia. METHODS: Spontaneously hypertensive rats (SHRs) and Wistar Kyoto rats (WKYs) were subjected to 20 min bilateral common carotid artery occlusion plus hypotension by sodium nitroprusside followed by reperfusion for seven days. Sham-operated animals served as controls. The changes after ischemia in EEG power spectra, and their relations with neuronal damage and astrocytic response were investigated. RESULTS: The EEG analysis revealed that in SHRs and WKYs, ischemia produced a dramatic increase in delta activity and a decrease in theta, beta and alpha activities derived from both cortical and hippocampal areas. EEG activity reverted to normal values more quickly in WKYs than in SHRs which did not recover cortical and hippocampal alpha and beta activities even at six days of reperfusion. SHRs presented more severe damage and intense astrocytosis than WKYs in almost all the brain regions analyzed. In SHRs, hippocampal delta activity was positively correlated with the degree of neuronal necrosis and astrocytic activation, whereas theta, alpha and beta activities correlated negatively. No correlations were found in WKYs. CONCLUSIONS: These data indicate that the hippocampal bioelectrical activity recorded in SHRs from the beginning of reperfusion could be useful for predicting the ischemic outcome and evaluating the effects of pharmacological interventions.