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The Responses of Microglia to Infrasound and Its Effects on Primary Cultured Neuron Cell

Author: ZhangGuoFeng
Tutor: ZhaoGang；DuFang
School: Fourth Military Medical University
Course: Neurology
Keywords: Infrasound Microglia Astrocytes Neurons Cytokines Apoptosis
CLC: R741
Type: Master's thesis
Year: 2009
Downloads: 83
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Abstract

Infrasound (infrasound) is generated by the mechanical vibration of objects (substances), 0.0001 ~ 20Hz frequency sound waves, has penetrating, propagation distance and slow decay. Widely found in nature and artificial environments, including tidal waves, earthquakes, tsunamis, large machinery, and rocket launchers. Although the human ear can not hear infrasound, but experimental studies found that high-intensity infrasound can produce significant damage the central nervous system of the human body, and a variety of animal, including the hypothalamus, cortical and subcortical wide area. With the development of modern industrial and transport sectors, infrasound play an important role in the noise pollution. Studies have shown that: infrasound damage CNS mechanisms is one cause inflammation, but inflammation of the central nervous system caused by infrasound specific mechanisms are still poorly understood. MI as immune cells within the central nervous system, play an important role in the inflammatory process, its research is particularly important. MI injury of the central nervous system will soon be moving to the detriment of regional, on the one hand, the protection of the central nervous system, restriction and removal of exotic microorganisms, phagocytosis, and elimination of hazardous substances and the fragments of necrotic brain; on the other hand can produce a variety of cytokines MI and participation in the process of antigen-presenting constitute the immune system of the brain, involved in a variety of diseases, development and outcome of the process. They play a protective role, the disease outcome healed; or disease-causing persistent aggravate the injury makes the disease progression of the disease. The preliminary work of our laboratory has found that the infrasound can significantly activated brain MI. Some studies have shown that activation of the MI neurons is a double-edged sword, both caused neuronal damage, also can damage neurons play a protective role. Infrasound role of microglia specific to what happens as well as the exact role of infrasound injury remain unclear. Through the use of of 16Hz130dB high intensity infrasound in vitro cultured rat MI, and design experiments on the above two issues a preliminary study. Experimental one outside cultured microglia infrasound reaction. The experimental secondary sound activation keratinocytes supernatant neurons. The role of the experimental three-vitro simulation infrasound activated microglia cells secrete TNF-α, IL-1β, the neuronal damage test, and TNF-α, IL-1β cytokines in neutralizing antibodies protect neurons . Experiment four sound role of microglia and astrocytes secrete several cytokines changes. The experimental results are as follows: Experiment integrally cultured microglia infrasound reaction. (1) changing the cell: the normal MI extracellular body smaller, less cytoplasm, round and protruding less. The infrasound immediately see the the MI cell body larger. Infrasound 4h amoeba-like cells increased after the active cell division. Infrasound 12h after MI swelling, increased cytoplasmic vacuoles. Infrasound 24h after MI cytoplasmic richer, more dual-core cells. (2) Cytokines: immediately after 30min infrasound MI in the supernatant of the IL-10 starting to rise to 12h reaches the peak. Expression of TNF-α and IL-1β in the role of infrasound 4h after began to rise to the 12h rise to the peak, and then decreased. Infrasound 1h, MI inflammatory cytokine TNF-α and IL-1β content within 24h in culture medium was persistent elevated secretion of both is not synchronized. IL-1β increased immediately after the active significantly increased; 12h after TNF-α has been to the role only see significantly increased and has lagged far behind in the secretion of IL-1β. Protective cytokine IL-10 double peak in 4h and 24h, and the value of each observation point, compared with the control group were significantly increased. The experimental secondary sound activation keratinocytes supernatant neurons. Collected at different time points after the infrasound 30min and 1h MI supernatant of neurons: (a) MTT results: of infrasound exposure 30min after MI supernatant collected at each time point of primary cultured rat neurons does not produce injury; the supernatant the subsonic role 1h after MI collected at each time point in primary cultured rat neurons will produce significant injury. (B) TUNEL detection of apoptosis results: the infrasound exposure 30min collected at each time point after MI supernatant of primary cultured rat nerve neuronal cell does not produce pro-apoptotic role; the infrasound exposure 1h each time point MI supernatant collected obvious pro-apoptotic role of primary cultured rat neurons. 24h time point collected after 1h infrasound MI supernatant caused neuronal apoptosis is the most obvious. The experimental three-vitro simulation infrasound activated microglia cells secrete TNF-α, IL-1β, the neuronal damage test, and TNF-α, IL-1β cytokines protective effect of neutralizing antibodies. (1) infrasound 1h after MI secretion of TNF-α, IL-1β and other simulation on neurons damage test as well as the role of TNF-α, IL-1β and other cytokines and antibodies protect neurons. Flow cytometry results: subsonic 1h after MI secretion of TNF-α, IL-1β simulation damage neuron cell apoptosis rate average were 443.2% and 57 ± 4.8%, while both induced withered the death rate is an average of 67.5 ± 6.4%; 5μg/ml TNF-α, cytokines such as IL-1β neutralizing antibodies can completely neutralize out after infrasound microglia secretion of TNF-α, IL-1β The biological activity of 5μg/ml of TNF-α, IL-1β and other cytokines and antibody concentration, and the application does not affect the state of the neuron cells. (2) were added to in infrasound 24h time point collected after 1h MI supernatant of TNF-αIL-1β and TNF-α/IL-1β the neutralizing antibodies after a certain amount of replacement rat neurons in cell culture liquid neurons after 48h of: (a) flow cytometry detection of apoptosis results: added IL-1β and TNF-α/IL-1β and antibodies of the two groups and the control group (24 h time point group) and simply added to the TNF-α neutralizing antibodies between the groups were statistically significant differences; simply adding TNF-α neutralizing antibodies and control groups was no significant difference. Experiment four sound role of microglia and astrocytes secrete several cytokines changes. (1) astrocytes change: visible normal control group astrocytes shape irregular, abundant cytoplasm and nuclear pulp is relatively small, the cell body many branches nucleus often biased towards the side of the cell body is oval, star shaped glial cells. The astrocytes infrasound 1h under a microscope to see the cells did not change significantly change within 24h. MI change such as the experimental one, obviously from the resting state into the activated state. (2) astrocyte supernatant cytokine changes: In the infrasound 1h astrocytes supernatant cytokines TNF-α, IL-1β, and IL-10 were seen at 24h after a slight rise. MI supernatant multiple cytokines within 4h after infrasound visible rise significantly. In this study, the following conclusions: (1) combined with the basis of previous work, further confirmed exposure to 16Hz 130dB infrasound rat microglia significantly from resting into activated state, and this reaction earlier in astrocytes cells. Therefore, microglia may be the reaction of the earliest cells within the central nervous system of infrasound. (2) 16Hz 130dB infrasound 30min activated microglia, the secretion of cytokines does not produce damage to neurons; 16Hz 130dB sound 1h activated microglia in 4h, 12h and 24h time point The supernatant were interested neurons produce damage, mainly caused neuronal apoptosis 24h time point of apoptosis is the most obvious. (3) exposure to 16Hz 130dB infrasound 1h activated microglia by proinflammatory cytokines cause neuronal apoptosis, which may mainly within the first 24h through the secretion of IL-1β to cause neurons apoptosis; TNF-α may not play a major role within the first 24h.

The Responses of Microglia to Infrasound and Its Effects on Primary Cultured Neuron Cell

Abstract

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