| Background High-intensity acoustics direct injure the auditory system,and affect central nervous system by the auditory organs,and then caused the nervous system to produce varying degrees of non-specific damage.The nervous system disorders and injuries caused by High-intensity acoustics exposure,were mostly established in the clinical High-intensity noise exposure experience and on the research work with rodent animal model.However,nervous system disorders and injuries induced by pure tone high-intensity acoustics,especially by medium/low-frequency pure tone high-intensity acoustics have not been fully studied.In view of the mechanism of nervous system injury from the High-intensity acoustics exposure,the current study focused on the neurotransmitters and related receptor synthesis of brain area,released and transported etc.For the impact mechanism of brain neurotransmitters and related receptors were less.The signaling mechanism of the relevant impact mechanism is not explicitly stated.Recently studies have shown that neuronal synapses in hippocampus and cortical neurons are induced by long-term potentiation and decreased synaptic susceptibility to hippocampal neurotransmitters.Its most prominent performance is a declination in spatial learning and memory ability.However,the mechanism of the transmitter and the receptor were changed has not been elucidated.Therefore we focuses on the effect of high intensity sound on the important second messenger Ca2+ and the mechanism on nerve receptors and neurotransmitters is described.,The Bama miniature pigs were used as the research model,and the medium/low-frequency pure tone high-intensity acoustics were used as the exposure condition.The characteristics of the nervous system injury were analyzed,and the molecular mechanism was also discussed,The results of this study will provide the basis for the study of the mechanism of high-intensity acoustics damage.Materials and Methods30 bama miniature pigs were divided into three groups randomly: 130 d B high-Intensity acoustics group(n=10),142 d B high-Intensity acoustics group(n=10)and blank control group(n=10).In the animal compartment of academy of military medical sciences,the same feeding conditions were given to all miniature pigs.The miniature pigs of 130 d B group was stimulated with 900 Hz-130 d B high-intensity acoustics for 15 min,and the miniature pigs of142 d B group were treated with 900 Hz-142 d B high-intensity acoustics for 15 min.Miniature pigs in control group were put outside the high-intensity acoustics exposition circumstance.After high-intensity acoustics exposition,half miniature pigs of each groups were sacrificed and the 10 ml venous blood was collected.The hippocampus and cortex of the animals were taken in liquid nitrogen and 4% formalin solution respectively.One week later,all miniature pigs were sacrificed in the same way.1.The peripheral blood of the miniature pigs was collected after high-intensity acoustics stimulation,and the biochemical indexes were analyzed with the AW5800 automatic hematology analyzer(specific mode for miniature pigs).The serum biochemical indexes including ammonia acid aminotransferase,creatine phosphate,lactate dehydrogenase,a-hydroxybutyrate dehydrogenase,total bile acid,cholinesterase,alkaline phosphatase,leucine aminopeptidase and other biochemical indicators.2.The blood samples were collected and serum ACTH,CORT,CRP,norepinephrine,NGB,NSE and other molecules concentration were measured according to their ELISA kit manual on the microplate reader.3.The hippocampus and cortical tissues were cut into sections,and the tissues sections were incubated with Fluo-4 at 37° C for 30 min,incubated at room temperature for 10 min thereafter and washed twice with PBS,sections were observed with laser confocal microscopy.Fluorescence signal was analyzed by ZEN2011 software.4.Morphology analysis of miniature pig brain: post-fixed brain were dehydrated gradiently and embedded by paraffin,then brain were sectioned at the 4 μm and stained.Sections were observed and photographed.5.The frozen hippocampus and cortex tissues were weighted about 150 mg,and then adding RIPA lysate buffer and protease inhibitor,extracted animal tissue protein,adding loading buffer,carried out protein electrophoresis and protein transfer,PKC and Ca2+ receptor antibody 4°C incubation overnight,then secondary antibody incubate for 30 min.The films were developed under darkroom.6.The 150 mg animal frozen tissue were add the tissue lysate buffer,according to the Qiagen kit operating instructions to extract the total m RNA,and then the total RNA quantitative,according to the reverse transcription kit operation instructions to reverse the RNA into c DNA,the primers of PKC,PI3 K and CACNA2D1 were synthesized.The expression of PKC,PI3 K and CACNA2D1 genes analyzed by real time PCR.Results1.130 d B and 142 d B medium/low-frequency pure tone high-intensity acoustics for 15 minutes exposure resulted into miniature pig neurological damage and significant behavioral changes.2.The levels of SOD and NSE in peripheral blood were increased,while the level of CK was decreased,which indicated that SOD,NSE and CK could be used as bio-markers of injury under these conditions,and high-intensity acoustics exposure also caused the liver injury and elevated immune response.3.Morphology analysis of miniature pig brain by HE staining showed that 900 Hz-130 d B high-intensity acoustics had no significantly effect on the hippocampus,900 Hz-142 d B high-intensity acoustics injured hippocampus significantly,with shrunk cell nucleus,cell vacuolation and pyramidal cells edema,which indicates apoptosis of theses cells.However,no change was observed in cortex.4.The effect of high-intensity acoustics on the changes of intracellular Ca2+ concentration,in the hippocampus,the intracellular Ca2+ concentration was significantly increased in the high-intensity acoustics groups compared with the control group.In the cortical tissue,the intracellular Ca2+ concentration of high-intensity acoustics groups was significantly lower in the high-intensity acoustics groups compared with the control group.5.Effects of high-intensity acoustics treatment on PKC,PI3 K and CACNA2D1 in tissue cells:The expression of PKC,PI3 K and CACNA2D1 in the hippocampus was significantly higher than that in the control group at the m RNA level in the hippocampus.In the cortical tissue,The expression of PKC,PI3 K and CACNA2D1 in the high-intensity acoustics group was significantly lower than that in the control group.6.The effect of high-intensity acoustics on PKC and calcium receptor in the tissue: At the protein level,the expression of PKC and calcium receptor in the high-intensity acoustics group was significantly higher than that in the control group,however,in the cortical tissue,the expression of PKC and calcium receptor in the high-intensity acoustics group was significantly lower.Conclusion Our research showed that both 900 Hz-130 d B and 900 Hz-142 d B high-intensity acoustics exposure for 15 minutes results in abnormal behavior in miniature pigs and changes of biochemical analysis and hormone in serum.Of which,NSE and SOD got up-regulated,while CK got down-regulated significantly,which indicate that these indexes may be used as a bio-marker of damage under this condition.900 Hz-142 d B high-intensity acoustics exposure injured hippocampus but not cortex in the brain.900 Hz-142 d B high-intensity acoustics activated PI3 K and PKC signaling pathway thus to up-regulate CACNA2D1 gene to increase the intracellular calcium concentration in hippocampus.However,inverse phenomenon was observed in cortex.900 Hz-142 d B high-intensity acoustics depressed PI3 K and PKC signaling pathway thus to down-regulate CACNA2D1 gene to decrease the intracellular calcium concentration in cortex.These results not only reveal the possible mechanism of nerve damage caused by high-intensity acoustics,but also provide new insights of prevention and treatment of neurological damage induced by high-intensity acoustics. |
