| Infrasound is a kind of sound wave whose frequency of mechanical vibration ranges from 0.0001 to 20 Hz, and is the main source of public noise. A group of evidence has proven that infrasound can act as a kind of stressor, leading to a variety of functional abnormalities in neuroendocrine system, such as hypothalamic-pituitary-adrenal (HPA) axis. It is reported that after exposure of rats to infrasound, the mRNA of corticotrophin releasing hormone (CRH), which is one of the most important hormones involving in the stress and secreted from the paraventricular nucleus in hypothalamus (PVN), was significantly increased. The CRH exerts its function through binding to its receptor CRH-R1. Apart from its effects on neurons, infrasound also affects glial cells. For instance, 8Hz, >90dB infrasound promoted the activition of microglia and infiltration to the lesion sites. Our previous studies also revealed that the exposure to infrasound was able to activate the microglia in paraventricular nucleus of hypothalamus. Moreover, these responses of microglia to infrasound seemed to be prior to that of astrocytes.Microglia cells are the resident immune cells in the central nervous system (CNS) in physiological condition. Microglia activation and migration play an important role in neuroinflammation propagation. It is actively involved in pathogenesis of a number of neurodegenerative diseases including multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD) et al. Microglia responded to brain injury by migrating to sites of tissue damage, undergoing marked changes in morphology, proliferating and engulfing tissue debris. In addition, microglia can release potent neurotoxins, which may cause neuronal damage. Microglia is also the potential promoter of the migration, axonal growth, and terminal differentiation of different neuronal subsets, through the release of extracellular matrix components, soluble factors, and direction of cell–cell contact.It was used to think that neurons are the ultimate target cells responsed to stress, and thus stress-associated CRH-R1 was thought to be only expressed in neurons. However, recent studies have revealed that CRH-R1 was also highly expressed in the microglia exposed to endotoxin and hypoxia in vitro. Addition of exogenous CRH to cultured microglia promoted their proliferation and secretion of TNF-α, and these effects were reversed by CRH-R1antagonist antalarmin, suggesting that microglia also plays an important role in the stress. However, so far we lack the knowledge that whether infrasound as a kind of stressor can promote the expression of CRH-R1 in microglia and in turn caused stressed injury to the central nerve system. Thus, in present study we exposed microglia to the infrasound with high intensity (16Hz 130dB) in vivo and in vitro, and explored the changes of CRH-R1 expression in microglia and analyzed underlying mechamism of microglia in the stress.Experiment 1Objective To observe the changes of CRH-R1expression in microglial cells in the PVN of rats in vivo induced by infrasound of 16Hz 130dB for 2h. Methods Rats were exposed to infrasound of 16Hz 130dB for 2h. The changes of CRH-R1expression in microglia were investigated by immunofluorescence with OX42. Results Only a few of microglial cells were rare in normal and control groups . The amplitude of the microglia (OX42-positive) cells significantly activated and the expression of CRH-R1 in them significantly increased after exposure to infrasound of 16Hz 130dB for 2h, and reached a peak level at 6h after exposure, while there was no significant changed in astrocytes (GFAP-positive) and the expression of CRH-R1 not significant. Conclusion These results imply that the exposure to infrasound induced activation and upregulation of CRH-R1 expression in rat microglia cells of the PVN.Experiment 2Objective To observe activation and CRH-R1 expression of cultured microglial cells induced by infrasound of 16Hz 130 dB for 0.5h, 1h or 2h. Methods The rat microglia cells were cultured and purified. Immunofluorescence of OX42 and CRH-R1 was used to observe the morphological changes and the changes of CRH-R1 expression of the microglia at different time points (0.5, 1, 2, 4, 8, 12 and 24h) after exposure to infrasound. Results The percentages of activated microglia cells were increased gradually along with the prolonged exposure period of infrasound. In activated microglia cells, the expression of CRH-R1 were upregraduated significantly at 0.5h and reached a peak level at 4h, then showed a descending tendency at 8,12 or 24h. In contrast, there are no significant changes in control cells. The expression of CRH-R1 was upregraduated gradually along with prolonged exposure time of infrasound. Infrasound stress exerted a time-dependent effect on activation and CRH-R1 expression in cultured microglia. Conclusion Infrasound induces activation of microglia and upregulation of CRH-R1 expression in vitro.Experiment 3Objective To observe the effect of antalarmin on the changes of CRH-R1expression in microglial cells in the PVN of rats in vivo induced by infrasound of 16Hz 130dB for 2h. Methods Antalarmin was intraperitoneally (i.p.) at the dose of 20 mg/kg 20 min before the exposure to infrasound. The changes of CRH/CRH-R1 expression in microglia were investigated by immunostaining. Results Increased intracellular expressions of CRH and CRH-R1 in the PVN were observed after infrasound exposure for 2h. Antalarmin significantly blocked the expressions of CRH and CRH-R1. However, the activated states of microglia cells were remined after antalarmin administration, indicating that antalarmin has no effects on activation of microglia induced by infrasound. Conclusion Infrasound-induced increased expression of CRH-R1 and CRH can be blocked by antalarmin, suggesting that CRH-R1 antagonist is able to protect CNS against the stress injury induced by infrasound.
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