**2. Materials and methods**

When airplanes took off or landed at Xiaoshan International Airport (Hangzhou, China), aircraft noises on the roof of a residential building standing 100 m from the edge of airport were sampled using an LDS four-channel dynamic signal analyzer (Photon II, Royston, England). Based on the 24-h flight schedule of airport and airplane type, aircraft noises were played through a non-directional dodecahedron sound source (Nor270, Norsonic, Lierskogen, Norway) and the intensity of noise was adjusted by a power amplifier (Nor280, Norsonic, Lierskogen, Norway). The sound absorber and insulation device were optimally assembled so that the *L*WECPN values of the experimental groups I (EG-I) and II (EG-II) were (75 ± 1.0) dB (*L*Aeq = 65.3 dB) and (80 ± 1.0) dB (*L*Aeq =70.3 dB), respectively. In addition, the laboratory was customized to better control acoustics, as the doors were sound-proofed and the vents were installed with mufflers so that background noise was no more than 40 dBA, the highest sound intensity heard by our control group (CG). We measured the intensity of noise exposure with a sound level meter (AWA6291, Hangzhou, China), which was sound-calibrated by a loud‐ speaker before measurement.

**1. Introduction**

164 Recent Progress in Some Aircraft Technologies

of an area near a military aircraft center [3].

it should be further confirmed.

**2. Materials and methods**

Studies have shown that aircraft noise has a great impact on the health status of populations residing in areas near air traffic, particularly citing cardiovascular diseases and the use of sleep and cardiovascular medications [1, 2]. In addition, researchers have concluded that there is a dose-response relationship between aircraft noise levels and blood pressure of the residents

However, most knowledge of the physiological effects of noise is generally obtained through animal experiment. To begin with, the open field test (OFT) is commonly used as a mechanism to assess the neurobehavioral effects of noise. Katz *et al*. [4] showed that white noise of 95 dB increased motor behaviors of rats in OFT and decreased their defecation after 1 h of acute stress. Food intake in OFT is reduced when rats are exposed to white noise of 95 dB, while their defecation increases [5]. In addition, the levels of neurotransmitters or hormones in plasma or brain may reflect the neurobiological effects of noise. Typically, the concentration of norepinephrine (NE) in the brain and cochlea of rats decreases after acute noise stress [6, 7]. Corticosterone levels in mice plasma have been shown to be significantly increased when exposed to acute noise for 10 min [8]. Male rats exposed to broadband white noise of 100 dB have been shown to have a significantly increased level of NE in the brain and corticosterone in plasma [9]. Furthermore, observing the morphological changes in neuronal cells can assess the effects of noise. Outer hair cell apoptosis has been observed in the cochlea of chinchilla after intense impulse noise exposure [10]. In addition, continuous noise stress has been shown to affect both degeneration of epithelial cells and apoptosis of stromal cells in the brain of pig [11]. We have not found any studies indicating that noise exposure induces morphological

damage in the temporal lobe, the lobe related to perception and memory [12].

None of the aforementioned studies studied airport noise. Broadband white noise was diffusely applied in previous experimental studies to examine the physiological effects of noise. However, it is important to note that white noise in normal environment is almost nonexistent. In China, the aircraft-related weighted equivalent continuous perceived noise level (*L*WECPN) in many residential areas around airports overstepped the 75 dB limit stipulated by the "Standard of Aircraft Noise for Environment around Airport" policy. In this study, we thus sampled actual aircraft noise and played it back to laboratory rats. We then systematically

studied their behaviors, plasma NE levels, and cell morphology of the temporal lobe.

Of course, there are some differences in the hearing sensitivity to different sound frequencies and circadian rhythms between rats and humans, which can bring variances in physiological effects under the same noise exposure. Therefore, if this study results are applied into humans,

When airplanes took off or landed at Xiaoshan International Airport (Hangzhou, China), aircraft noises on the roof of a residential building standing 100 m from the edge of airport Fifty male Sprague-Dawley rats (6 weeks old, weighing (150 ± 20) g) were purchased from the Experimental Animal Center of Zhejiang University, and were randomly divided into three groups: CG (*n* = 10), EG-I (*n* = 20), and EG-II (*n* = 20). Rats were housed five per cage and maintained in temperature- controlled ((21 ± 3) °C) rooms with cycles of 12 h of light and 12 h of dark (light on at 8:00 a.m. daily), and allowed free access to water and food. Rats were marked on their fur with picric acid to distinguish individuals. Before experiments were carried out, SD rats were bred for 3 days to adapt to the laboratory environment. After this, SD rats in groups EG-I and EG-II were exposed to aircraft noises, while the CG was not exposed. OFT and blood collection for neurotransmitter determination were carried out at 17:00 on Days 1, 8, 15, 22, 29, and 36 after noise exposure (blood collection excluded on Day 36). All data including OFT and neurotransmitter determination were collected on the same rats in CG (*n* = 5), EG-I (*n* = 10), and EG-II (*n* = 10). For investigating long-term effects of airport noise, after 65 d of continuous (excepting time for OFT/blood collection) noise exposure, four rats each were randomly selected from CG and EG-II, respectively, for additional neuronal morphology studies. Animal breeding and experiments were performed in line with the "Quality Management Approach to Laboratory Animals," and all efforts were made to minimize the number of animals used and their suffering.

The size of open field was 100 × 100 × 50 cm, and its bottom divided into 25 grids (20 × 20 cm) by white lines. We termed the nine grids located in the center of the open field as "center area." The open field was located in a 2.0 × 2.0 m audiometric cabin and lit by a 15-watt red lamp for background lighting. We handled the rats by the base of their tails, carried them to the center of the open field, and allowed them to explore the apparatus for 5 min. The behaviors of the rats were tracked and recorded by the camera fixed above the apparatus. The behaviors measured included line crossing number and center area duration.

In order to test the concentration of neurotransmitters in each group, venous blood (1.0 ml) was sampled from the orbital vein. Blood was transferred into 1.5 ml boil-proof microtubes (Axygen, United States) and was kept quiescence for 10 min, and then centrifuged at 4000 r/min for 10 min at 4°C. Next, 200 μl of supernatant was extracted from each sample and 200 μl of 5% perchloric acid was added to it. Their mixture was shaken, left at room temperature for 20 min to fully precipitate the plasma proteins, and centrifuged at 10,000 r/min for 15 min. Finally, supernatants were filtered with 0.45 μm membrane filters, and high-performance liquid chromatography-fluorimetric detection (HPLC-FLD) was used to measure the concen‐ tration of NE. The instrument parameters used for HPLC-FLD were as follows: column, Agilent Zorbax SB-C18 column (Agilent, US); mobile phase, methanol-buffer (buffer: 0.07 mol NaH2PO4, 10 mmol sodium octanesulfonate, pH 3.5). The gradient procedure of the mobile phase was as follows: at 0 min, 10% methanol and 90% buffer; at 5 min, 10% methanol and 90% buffer; at 30 min, 60% methanol and 40% buffer (1.0 ml/min of flow rate, 20 μl of injection volume, 35.0°C of column temperature, 280 nm of fluorescence excitation wavelength, and 315 nm of emission wavelength). Under these conditions, various substances in plasma were completely separated so that no interference to determination of the targets is experienced.

After being exposed to aircraft noise for 65 d, four rats were randomly selected from the CG and EG-II groups (two per group). We then examined the neuronal and synaptic morphologies of the temporal lobe by transmission electron microscopy (TEM). Rats were anesthetized by administration of an overdose of sodium pentobarbital and then perfused with glutaraldehyde transcardially. The temporal lobe was localized by digital brain stereotaxic instrument (ZH-LanXing B/S, Huaibei, China) with a soft-type cranial drill. After perfusion for about 1 h, we decapitated rats and stripped the whole brain rapidly fixed in glutaraldehyde. After fixed for 24 h, the temporal lobes were removed, cut into thin slices, and further fixed in glutaraldehyde for 3 d. Based on this, slices were washed using PBS, fixed using 1% osmium tetroxide, stained using 2% aqueous solution of uranyl acetate, dehydrated using different concentrations of alcohol and acetone gradient, penetrated and embedded using embedding medium, aggre‐ gated in oven, and finally cut into ultra-thin slices stained using 4% uranyl acetate and citrate. Cell structure in these ultra-thin slices was observed by TEM (Philips Tecnai 10, The Nether‐ lands).
