Influence Of Infrasound Exposure On Cultured Neuronal Axons And Relative Mechanism

?Infrasound is a kind of public noise, and its oscillation frequency is below 20Hz. Due to its characteristics of strong penetration, little propagation attenuation, ubiquitous existence, and difficulty in protection, it has been recognized as an important element of environmental and industrial noise.Infrasound is capable of eliciting certain biological resonance responsible on human. The inherent frequencies of human's organs, such as viscera and brain, are within the range of infrasound. Hence, when under a circumstance of infrasound, it will induce intensive resonance of organs, then functional or even structural injury. Among all the organs that are fragile to infrasound, the most vulnerable one is brain. Long time exposure to infrasound can cause decreased cognitive competence and neurological/mental symptoms such as debility, distraction, vertigo, nausea, and so on. Therefore, it is of great significance to explore on infrasound triggered biological effect, especially focus on the brain injury. To understand the underlie mechanism of infrasound injury is the prerequisite of successful shielding and treatment against it. The previous studies on infrasound induced brain injury were mainly on the cell soma of neuron, while the change of the axons has long been ignored. In fact, axons often extend for great distances from their cell bodies of origin, and are therefore susceptible to a diverse range of insults, including injury, toxins and ischemia, without the death of the parent neuron. In the current study, we employed the cultured hippocampal neurons as the model to investigate the influence of infrasound on axons. We also sought to disclose the relationship between the infrasound elicited alteration on neurons and the axons. In addition, we studied the potential mechanism involved in infrasound injury by using inhibitors on Ca2+, UPS, and RhoA/ROCK signaling pathway, respectively.Then we investigated whether pretreatment with Gs-Rd, a Ca2+ channel inhibitor/neuroprotectant would be effective on saving the axons after IE. The major results are as below: first, we successfully cultured low-density primary hippocampal neurons for an optimized observation on the morphological alterations of single axon and its parent neuron after infrasound exposure. Second, after 1h exposure of 16Hz/130dB infrasound, we observed the axons on time point 0, 4, 8, 12, and 24 h via immunocytochemistry. We found that anti-?III-tubulin stained axons began to develop focal swelling and beading approximately at 4~8 hr, and completely degenerated and disconnected at 12~24hr after infrasonic exposure (IE). Immunofluorensce of anti-APP, a marker for impaired axonal transport, showed that the target protein strongly accumulated along the axons as early as 4h after IE. Moreover, the signals of Glu-tubulin which is a marker of stable microtubules began to attenuate significantly and disappear multi-focally in the axons from 4 h to 24 h post-exposure on. Third, we utilized TUNEL, Hoechst staining and western blot of cleaved caspase-3 to assess the apoptosis induced by IE. We found that although the percent of apoptotic cells on each time point were significantly increased compared to control group, they were much fewer than that of bead-bearing neurons from 4 h post-exposure on. Our immunoblot assay further confirmed our observation. Compared to the controls, the expression of stable Glu-tubulin gradually decreased with time after IE, and that of apoptosis-associated protein cleaved caspase-3 was intact by 4 h post-exposure and increased from 8 to 24 h post-exposure on. Then, to investigate the mechanism(s) underlying infrasound-induced axon degeneration, we analyzed Ca2+, UPS and RhoA pathways involved in axon degeneration as reported previously. Our results manifested that axon degeneration induced by infrasound is profoundly hampered by EGTA, ALLN and Fasudil, while MG132 did not show a significant prevention as expected. Finally, we found that the potent neuroprotectant Gs-Rd can profoundly protect the axons after IE and rescue the cell soma at the mean time.Conclusion: Taken together, our results indicate that infrasound can result in axon degeneration of the cultured neurons; this axonal event seems to be independent of infrasound-induced neuronal apoptosis, and can be significantly blocked by calcium chelator EGTA and calpain inhibitor ALLN, suggesting that Ca2+ signaling is likely to account for infrasound-induced axon degeneration. Treatment of Gs-Rd may save as a novel avenue to protect the IE induced neuronal injury.