Neuroprotective effects of sevoflurane against electromagnetic pulse-induced brain injury through inhibition of neuronal oxidative stress and apoptosis nanoxia ncore retro

Electromagnetic pulse (EMP) causes central nervous system damage and neurobehavioral disorders, and sevoflurane protects the brain from ischemic injury. We investigated the effects of sevoflurane on EMP-induced brain injury. Rats were exposed to EMP and immediately treated with sevoflurane. The protective effects of sevoflurane were assessed by nissl staining, fluoro-jade C staining and electron microscopy. The neurobehavioral effects were assessed using the open-field test and the morris water maze. Finally, primary cerebral cortical neurons were exposed to EMP and incubated with different concentration of sevoflurane.


The cellular viability, lactate dehydrogenase (LDH) release, superoxide dismutase (SOD) activity and malondialdehyde (MDA) level were assayed.Nanoxia ncore retro TUNEL staining was performed, and the expression of apoptotic markers was determined. The cerebral cortexes of EMP-exposed rats presented neuronal abnormalities. Sevoflurane alleviated these effects, as well as the learning and memory deficits caused by EMP exposure. In vitro, cell viability was reduced and LDH release was increased after EMP exposure; treatment with sevoflurane ameliorated these effects. Additionally, sevoflurane increased SOD activity, decreased MDA levels and alleviated neuronal apoptosis by regulating the expression of cleaved caspase-3, bax and bcl-2. These findings demonstrate that sevoflurane conferred neuroprotective effects against EMP radiation-induced brain damage by inhibiting neuronal oxidative stress and apoptosis.Nanoxia ncore retro

Introduction

Most electrical equipment and wireless communication devices produce electromagnetic radiation. There is widespread concern regarding the adverse effects on human health caused by exposure to many types of electromagnetic fields (emfs) [1], [2]. The potential for EMF exposure to damage the central nervous system (CNS) has been discussed in-depth. Previous studies indicate that the non-thermal effects of EMF exposure can lead to cellular changes [3], [4], [5]. Additionally, EMF can increase reactive oxygen species (ROS) and reactive nitrogen species (RNS) in organs and cause histopathological damage and oxidative stress [6], [7], [8], [9]; for example, under particular circumstances, exposure to a GSM-modulated, 900-mhz signal acts as a co-stressor for oxidative damage of neural cells [10].Nanoxia ncore retro several studies also suggest that occupational exposure to electromagnetic fields may be associated with increased risk of neurodegenerative diseases [11], [12].

Electromagnetic pulse (EMP), a specific type of EMF, is a short high-voltage pulse with an extremely fast rising time and a broad bandwidth from extremely low frequencies up to 1.5 ghz [13], [14]. EMP is widely applied in medical therapies, such as those targeting osteoporosis, and is also used in military campaigns. However, the biological effects and potential harm to humans in an environment of electromagnetic radiation have not been well studied. Brain tissue is sensitive to EMP, which increases cerebral microvascular permeability in rats [15] and can disrupt the blood-brain barrier (BBB) [5].Nanoxia ncore retro additionally, EMP exposure can cause long-term impairments in rat learning and memory [12]. However, the non-thermal effects of EMP remain controversial [2]. It is unknown whether the non-thermal effects of EMP can induce short-term histopathological damage and ultrastructural changes in cerebral cortex neurons.

Recently, the neuroprotective effect of inhaled anesthetics has attracted increased attention. Sevoflurane is an inhaled anesthetic that is broadly used in clinical applications [16], [17], . The neuroprotective effect of sevoflurane may be related to its inhibitory effects on oxidative stress, apoptosis and excitatory amino acid release and its stabilizing effects on cell metabolism [19], [20], [21], [22].Nanoxia ncore retro however, it is unclear whether sevoflurane exerts neuroprotective effects against EMP radiation-induced brain injury.

The present study was designed to investigate whether exposure to EMP (400 kv/m, 200 pulses) has detrimental effects on cerebral cortex neurons and cognitive ability and to further elucidate whether sevoflurane exerts neuroprotective effects against EMP radiation-induced cerebral damage in vivo and in vitro.

3. EMP exposure

An all-solid-state nanosecond generator was developed and tested as described in previous studies [5], [13]. Briefly, EMP (peak intensity 400 KV/m, rise time 10 ns, pulse width 350 ns, 0.5 pps, 1 hz, 200 pulses total) was generated by a spark gap generator and transmitted into a gigahertz transverse electromagnetic (GTEM) cell.Nanoxia ncore retro the electric field in the exposure area was uniform within 30×30×30 cm. The animals were whole-body exposed to EMP at 400 kv/m for 200 pulses ( fig. 1G). During exposure, the rats were awake and not restrained in the exposure chamber. The temperature measurements were made immediately before and after EMP exposure. The exposure caused a 0.2°C increase in the rectal temperature of control and exposed rats. The cells were also exposed to EMP at 400 kv/m for 200 pulses. After exposure, no significant change in the temperature of the medium was noted. A tektronix 7000B oscilloscope (tektronix, beaverton, OR) was used to observe and record the pulse waveform.

6. Water maze task

At 24 h after EMP injury, the rats were submitted to behavioral testing to analyze spatial learning and memory in the morris water maze.Nanoxia ncore retro the water maze was a circular pool (painted black, 1.8 m in diameter, 0.4 m high) constructed of fiberglass. The water was maintained at 22±2°C. The pool was geographically divided into four equal quadrants, with release points in each quadrant designated as southwest, northwest, northeast and southeast. During testing in the water maze, a transparent platform (8 cm in diameter) was placed 1 cm beneath the water surface. This escape platform location was kept constant in the middle of the southwest quadrant. Rats were placed into the water facing the wall at each release point in a random order. A video camera was mounted in the ceiling above the pool and was connected to a video recorder and tracking device (S-MART; pan-lab, barcelona, spain), which permitted online and offline automated tracking of the paths taken by the rats.Nanoxia ncore retro the animals were subjected to four trials per session. The rats were trained to locate the hidden escape platform, which remained in a fixed location throughout the testing. Trials lasted a maximum of 120 s and the latency and the experimenter placed rats in platform for 15 sec. The intertrial interval was 60 sec. Each rat performed 4 trials daily for 4 days. During the training trials, the length of the path by which each animal found the platform was measured. On the fifth day, the rats were subjected to a 2-min probe trial in which the platform was removed. The start point for the probe trial was randomly selected for each subject. The swimming time and trajectory of the rats were recorded.Nanoxia ncore retro

3. Sevoflurane protected neuronal ultrastructure in rats exposed to EMP radiation

Electron micrographs of normal cerebral cortex neurons are shown in fig. 3A. At 24 h after EMP exposure, the ultrastructure of cortical neurons was characterized by nuclear membrane folds, collapse, blurred boundaries, nuclear condensation and increased heterochromatin. A large amount of nuclear chromatin was accumulated under the nuclear membrane. Additionally, obvious swelling of mitochondria in the cytoplasm was noted, and many mitochondria were spherical in shape. Some mitochondrial cristae were fractured. The rough endoplasmic reticulum was cystic and degranulated. Moreover, the structure of the cerebral cortex was disrupted.Nanoxia ncore retro the cytoplasm became more highly concentrated as the electron density increased, the nucleus shrank significantly, the chromatin was concentrated and aggregated at the edge of the nucleus and apoptotic bodies were observed that were similar to those observed in apoptotic cells ( figs. 3B and 3C). The damage to the neuronal ultrastructure in the 2% sevoflurane treatment group was significantly alleviated, especially in the mitochondria and the rough endoplasmic reticulum ( fig. 3D).

Discussion

The CNS is sensitive to electromagnetic radiation, and pathological damage and neurobehavioral disorders have been observed after EMP exposure [30]. Exposure to electromagnetic fields is a potential health hazard to humans, especially military personnel and/or researchers who work with or can be exposed to this type of electromagnetic field.Nanoxia ncore retro therefore, it is of great importance to investigate the biological effects of electromagnetic fields on the CNS and to develop potential preventive strategies.

There is no consensus regarding whether EMP exposure could cause potential detrimental effects in whole animals or isolated cells. Chavdoula et al. Reported that global system for mobile telecommunications (GSM)-900 mhz mobile phone electromagnetic radiation caused a large decrease in insect reproductive capacity. This effect was found to be non-thermal and correlated with an increased percentage of induced fragmented DNA and induced cell death in egg chamber cells at early and mid-oogenesis [10]. Although no histopathological changes occurred in rat brains following long-term EMP exposure from GSM-900 mobile phone radiation [31], several studies demonstrated that EMP exposure increased the permeability of the BBB [13] and altered the localization and decreased the levels of tight junction proteins [5].Nanoxia ncore retro the negative effect of EMP exposure may depend on the frequency of the electromagnetic field, the type of electrical field applied, the intensity of the power and the thermal or non-thermal effects [32], [33], [34].

In the present study, cerebral cortex neuron injury and neurocognitive impairment were evaluated within 24 h after exposure to EMP radiation. Morphological changes were first observable in cortex neurons at 24 h after EMP exposure. We found many abnormal neurons, and we noted cell bodies that exhibited pyknosis. Furthermore, nissl staining, FJC staining [35], and neuronal ultrastructures revealed that EMP caused cortical neuron morphological damage. Importantly, learning and memory deficits were also observed after EMP exposure.Nanoxia ncore retro therefore, our present study showed that EMP exposure caused acute damage to neurons and short-term neurocognitive impairment. These results differ from our previous study, which demonstrated that EMP exposure could cause long-term neurocognitive impairment in rats at 12 and 18 months after EMP exposure [12].

Sevoflurane is one of the most frequently used volatile general anesthetic agents used during surgical procedures. Sevoflurane is especially useful for pediatric anesthesia because it allows rapid induction and recovery and is less irritating to the airway than other inhaled anesthetics. Although the effects of sevoflurane on neuronal oxidative stress and apoptosis are still controversial, several lines of evidence have suggested that sevoflurane possesses potent neuroprotective effects against oxidative stress injury and apoptosis in the central nervous system.Nanoxia ncore retro the neuroprotective effects of sevoflurane are related to its ability to decrease the cerebral utilization of oxygen and glucose, inhibit oxidative metabolism in neutrophils [36] and scavenge ROS [37]. Furthermore, sevoflurane shows acute neuroprotective effects by inhibiting lipid peroxidation, lowering MDA levels, and increasing normal pyramidal neuron density in cerebral ischemia reperfusion rats [22]. Another study revealed that exposure with 3.7% sevoflurane for 20 min upregulated the activities of CAT and SOD and induced acute neuroprotection against spinal cord ischemic injury [38]. Meanwhile, sevoflurane alleviated cell apoptosis after brain injury by upregulating bcl-2 [39]. Wang, J K et al reported that postconditioning with sevoflurane markedly improved spatial learning and memory and reduced apoptotic cell numbers by upregulating bcl-2 and downregulating bax, suggesting that the underlying acute protective mechanism of sevoflurane might be linked to reduced apoptosis [40].Nanoxia ncore retro thus, sevoflurane may be an attractive candidate for preventing EMP-induced brain damage.

In this study, rats or cultured cortex neurons were treated with sevoflurane for 20 min after EMP exposure. Our results showed that 2% or 4% sevoflurane reduced neuronal degeneration and alleviated neuronal ultrastructural damage to the mitochondria, rough endoplasmic reticulum and cell nucleus. Interestingly, treatment with sevoflurane improved the cognitive ability of rats exposed to EMP radiation. Several studies have verified that sevoflurane preconditioning protects mitochondria or other organelles from cerebral ischemia/reperfusion injury. Thus, we speculated that the beneficial effects of sevoflurane against EMP exposure may result from the neuroprotective effect of sevoflurane.Nanoxia ncore retro

We also explored the underlying mechanism of the protective effect of sevoflurane against EMP damage. We demonstrated that at 24 h after EMP exposure, the ultrastructure of cortical neurons changed markedly and was characterized by nuclear membrane folds, collapse and blurred boundaries. All changes in neuronal ultrastructure reflected imbalances in neuronal metabolic functions. Therefore, we speculated that this type of EMP-induced neuronal damage may be related to impaired cell function and metabolic disorders. In vitro, we found that EMP exposure reduced SOD activity and increased the MDA level, which was consistent with the previous finding that EMP enhanced oxidative stress [7]. However, sevoflurane increased SOD activity and reduced the MDA level, which implied that sevoflurane exerted a neuroprotective effect against EMP exposure by alleviating oxidative stress.Nanoxia ncore retro according to a previous study, general anesthetics may abrogate oxidative injury to neurons by preventing the initiation of free radical chain reactions or terminating the propagation of highly reactive radicals [37].

Apoptosis is characterized by specific cell structural changes, including cell shrinkage, nuclear enrichment and DNA breakage [41]. TUNEL staining is a sensitive method for marking early apoptotic cells. In the present study, the number of TUNEL-positive cells increased at 24 h after EMP exposure, indicating that electromagnetic radiation induced DNA damage or apoptotic cell death as reported previously [42], [43]. Caspase-3 can enzymatically digest specific substrates and inhibit DNA repair enzymes, thus destroying cytoskeletal proteins and ribonucleic protein, leading to breakage of the chromosome into small fragments and eventual apoptosis.Nanoxia ncore retro moreover, it is known that the pro-apoptotic protein bax and the anti-apoptotic protein bcl-2 can migrate from the cytoplasm to mitochondria, which are distributed in a manner that is consistent with mitochondrial release of cytochrome C and caspase [44]. The mitochondrial apoptotic pathway plays an important role in neuronal injury [45], and sevoflurane inhibits cellular apoptosis after brain cerebral ischemic injury by upregulating bcl-2 expression and reducing bax expression [39], [40]. Our results showed that sevoflurane reduced caspase-3 and bax expression and enhanced the expression of the anti-apoptosis protein bcl-2 after EMP exposure, suggesting that the mitochondrial apoptotic pathway is involved in the protective effects against EMP-induced brain injury.Nanoxia ncore retro however, the underlying mechanism of the neuroprotective effects of sevoflurane requires further investigation.

In summary, the current study demonstrated that exposure to 400 kv/m EMP induced cerebral cortical neuronal damage and degeneration, apoptosis and cognitive impairment. Sevoflurane conferred protective effects against EMP-induced brain injury by inhibiting neuronal oxidative stress and apoptosis.