| Infrasound (IS) is the sound below 20 Hz . It is acoustic energy with physical characters of strong penetration and less attenuation in long distance propagating. A large variety of natural and man-made phenomena produce infrasound, including avalanches, meteors, ocean waves, tornadoes, auroras, earthquakes, atmospheric nuclear tests, rockets, supersonic aircraft and so on.It is usually inaudible,but humans can perceive infrasound if the level is sufficiently high. Experimental studies reported that humans or various species of animals such as rats and mice exposed to infrasound at 90 dB or higher for short or long terms up to several months exhibited significant toxicological effects.Involvement of the limbicoreticular complex, hypothalamus, and other subcortical structures into responses to higher factor levels on human beings stipulates a sharply prominent pathological discomfort as a manifestation of the infrasonic diencephalic hypothalamic syndrome with sensorisomatic and autonomic visceral symptoms. Microglia are the resident immune cells of the central nervous system (CNS) .Microglial 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'sdisease (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 are potentially also promotors of the migration, axonal growth, and terminal differentiation of different neuronal subsets, through the release of extracellular matrix components, soluble factors and direct cell–cell contact.With the development of modern industry and transportation, infrasound plays a more and more important role in noise pollution. Although there have been some studies on infrasonic toxicology, relative little is known about the adverse effects of infrasound on inflammation of CNS.Since microglia as an important cell in the inflammation of CNS is becoming topical in society, the need for further research in this field is clearly indicated.In the present study, we sought to determine whether infrasound could induce microglial activation in the brain.Experiments were carried out in rats both in vitro and in vivo.Experiment 1:Rats were exposed to infrasound for 2 hours daily during 1days, 7days and 14days. Exposure to infrasound with a main frequency of 16 Hz and a sound pressure level of 130 dB. The changes of microglia were investigated utilizing the immunohistochemical and electron microscope methods.The results were showed as the following:(1) Light microscopic observation:the OX42-positive cells and GFAP-positive cells were rare in normal group.The amplitude of the OX42-positive cells significantly increased at once when rats were exposed to infrasound after exposure to 16 Hz, 130 dB. and then decreased after 7 days,while GFAP-positive cells were increased on the 7st day,and peaked on the 14st day , the double labeling between two cells significant increased on the 14st day.(2) Electron microscopic observation: Microglia was rare in normal group. On the 1st day of infrasound exposing, the number of Microglia significantly increased and the cytoplasm of microglia cell formed a thin rim around nucleus, but extended out in quite broad processes. When cells was in perineuronal positions these process seem partially to encompass the neuron.The activated microglia had nuclei with an irregular outline in which the chromatin was clumped beneath the nuclear envelope.The cytoplasm was rather pale and the process extending from the lower pole of the cell shows the long cisternae of the granular endoplasmic reticulum , which were the characteristic of these cells.The upper pole of the cell was largely occupied by inclusion bodies,some of which had granular contents,and others of which had membranous inclusion.These were swollen processes and contain a variety of vesicles and tubules.Experiment 2:Microglia were exposed to infrasound for 30min or 1h ,and then observe changes of microglia and cytokine through cytochemistry and Luminex liquid phase chip.The results were showed as the following:(1) The changes of cell morphology: Twenty-four hours after isolation, microglia showed a vacuolated cytoplasm and circular shape. Microglia enlarged at once after IS exposed. 4h after IS exposed, ameboid cells thrived and binucleated ameboid cells were often observed with occasional telophase pairs and mitotic figures present general. We can see microglia were densely packed with vesicles 12 hr after IS exposed and meoid microglia had multiple short spinous processes which remained on cell surfaces well. In contrast, there is no significant changes in control cells. 24h after IS expose, we can see some cells began to develop thin cytoplasmic projections. These"process-bearing"cells showed a complex multipolar pattern or, more commonly, a uni- or bipolar morphology.(2) The changes of cytokine: 30min exposure of microglia to IS has increased the concentrations of TNF-α, IL-1β, IL-10 and IL-18 in the supernatant. We observed the expected strong induction of TNF-α, IL-1β, IL-10 and IL-18 in response. IL-10 was released as early as 0h after exposing, and the level of its concentration reached the higlhest at 12 hours after exposing. While the significant upregulation in release of TNF-αor IL-1βwas detected at 12 hours and then degraded. The concentration of IL-18 was detected at a lower level at 0h but showed a gradually increasing process until 24h after exposing. We also found the inhibiting effect of INF-γon the 4 Cytokines introduced above by adding it in supernatant 2h before esposing and detecting at 12h after exposing, but in the supernatant of each groups INF-γhad not been found. After 1h exposing to IS, the level of TNF-αand IL1-βelevated gradually until the end point of observation. IL-18 reached its peak level right after exposing and showed a descending tendency in the following 24h. IL-10, previously known as a protective factor, showed two peak levels at 4h and 24h and the level was higher in all observing points compared to control. In addition, by immunocytochemistry,we found that the expression of TGF-βupragulate continuously after 30min exposing and downragulated after 1h exposing.The following is the conclusion of experiments:(1) In vivo, microglia could be activated by exposing to infrasound of 16 Hz, 130 dB. The microglial reactions were ahead of the activation of astrocyte.(2) The activated microglia distributed around neuron and vessels and formed synapse with astrocyte. (3) In vitro, Infrasound could also induce activation of microglia. After 30min exposing, microglia secreted protective cytokines mainly but the level of inflammatory cytokines increased after 1h exposing.
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