Mechanical stimulation

机械刺激
  • 文章类型: Journal Article
    机械刺激会影响植物生长,发展,和防御。喷水刺激的作用,作为环境中普遍的机械刺激,在作物生长和防御方面不容忽视。在这项研究中,研究了喷水对番茄植物生长和防御咀嚼草食动物棉铃虫和坏死真菌灰霉病的影响。发现超阈值喷水刺激(LS)可增强番茄植物对害虫和病原体的防御能力,同时改变植物结构。植物激素和化学代谢物分析的结果表明,LS通过茉莉酸(JA)信号改善了植物的防御反应。LS显著提高了关键防御代谢物的水平,绿原酸,并减少了番茄植物中挥发性有机化合物(VOCs)的排放,从而防御害虫和病原体的攻击。这项研究中最明显的发现是LS增强了番茄植物对生物胁迫的防御能力,这将为在病虫害管理中应用机械刺激的进一步工作铺平道路。
    Mechanical stimuli can affect plant growth, development, and defenses. The role of water spray stimulation, as a prevalent mechanical stimulus in the environment, in crop growth and defense cannot be overlooked. In this study, the effects of water spray on tomato plant growth and defense against the chewing herbivore Helicoverpa armigera and necrotrophic fungus Botrytis cinerea were investigated. Suprathreshold water spray stimulus (LS) was found to enhance tomato plant defenses against pests and pathogens while concurrently modifying plant architecture. The results of the phytohormone and chemical metabolite analysis revealed that LS improved the plant defense response via jasmonic acid (JA) signaling. LS significantly elevated the level of a pivotal defensive metabolite, chlorogenic acid, and reduced the emissions of volatile organic compounds (VOCs) from tomato plants, thereby defending against pest and pathogen attacks. The most obvious finding to emerge from this study is that LS enhances tomato plant defenses against biotic stresses, which will pave the way for further work on the application of mechanical stimuli for pest management.
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  • 文章类型: Journal Article
    背景:皮质骨适应静力的机制尚不清楚。这是一个重要的过程,因为静力响应软组织的生长以及在正畸和骨科矫正期间施加到皮质骨。这项研究的目的是研究皮质骨对施加于上颌骨的扩张力的反应。
    方法:总的来说,375只成年Sprague-Dawley大鼠分为三组:1)静力组,2)静力加刺激组,和3)假手术组。除了穿过上颌骨的静力,一些动物暴露于抗炎药。在不同的时间点收集样品,并通过显微计算机断层扫描进行评估,荧光显微镜,免疫组织化学,以及基因和蛋白质分析。
    结果:对上颌骨施加扩张力会增加骨膜中的炎症,并激活皮质板表面的破骨细胞。这种激活与牙齿移动的幅度无关,但遵循骨骼位移的模式。皮质板表面的骨形成发生在后期,并导致上颌骨皮质边界的重新定位和皮质漂移。
    结论:这项研究表明,皮质骨对静力的适应源于骨膜,这是一种基于炎症的现象,可以由临床医生操纵。我们的研究结果支持皮质适应静力的新理论和通过骨膜刺激促进皮质漂移的创新临床方法。能够控制皮质漂移可以通过在不需要颌面手术的情况下矫正严重畸形来对临床正畸和牙颌面骨科产生重大影响。
    BACKGROUND: The mechanism of cortical bone adaptation to static forces is not well understood. This is an important process because static forces are applied to the cortical bone in response to the growth of soft tissues and during Orthodontic and Orthopedic corrections. The aim of this study was to investigate the cortical bone response to expanding forces applied to the maxilla.
    METHODS: Overall, 375 adult Sprague-Dawley rats were divided into three groups: 1) static force group, 2) static force plus stimulation group, and 3) sham group. In addition to static force across the maxilla, some animals were exposed to anti-inflammatory medication. Samples were collected at different time points and evaluated by micro-computed tomography, fluorescence microscopy, immunohistochemistry, and gene and protein analyses.
    RESULTS: The application of expansion forces to the maxilla increased inflammation in the periosteum and activated osteoclasts on the surface of the cortical plate. This activation was independent of the magnitude of tooth movement but followed the pattern of skeletal displacement. Bone formation on the surface of the cortical plate occurred at a later stage and resulted in the relocation of the cortical boundary of the maxilla and cortical drifting.
    CONCLUSIONS: This study demonstrates that cortical bone adaptation to static forces originates from the periosteum, and it is an inflammatory-based phenomenon that can be manipulated by the clinician. Our findings support a new theory for cortical adaptation to static forces and an innovative clinical approach to promote cortical drifting through periosteal stimulation. Being able to control cortical drift can have a significant impact on clinical orthodontic and dentofacial orthopedics by allowing corrections of severe deformities without the need for maxillofacial surgery.
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  • 文章类型: Journal Article
    传统的细胞培养不能准确模拟细胞微环境,并证明单个细胞生长的特异性。在这项研究中,我们创建了一个4D细胞培养模型。它是由电磁铁组成的精密仪器,力传感器,和一个悬臂支架。一个培养皿放在磁铁上面,将用磁性纳米颗粒和舌癌细胞封装的凝胶珠置于培养皿中。在这个模型中,当磁体通电时,在培养基中的磁性纳米颗粒上产生磁力以驱动凝胶运动,凝胶对细胞施加外力。当舌头运动时,它可以模拟舌头鳞状细胞癌细胞CAL-27内部的微环境。在水凝胶上进行电子显微镜和流变学分析实验以表征藻酸盐。进行钙黄绿素-AM/PI染色以验证水凝胶培养系统的生物安全性。本研究设立了四个实验组。在此基础上,我们证实,与MTT无机械刺激培养的细胞相比,5天后舌鳞状细胞CAL-27的增殖显著增加.在拍摄的照片上测量不同组的细胞直径,结论是,动态培养环境下的细胞直径大于同期单独的水凝胶培养。此外,舌鳞状细胞癌的细胞形态较好。在流式细胞术实验中,机械刺激后,G0/G1期细胞较少,而G2/M期细胞比例增加。使用RT-PCR检测培养21天后各组之间RNA水平的变化。这个装置,模仿体内舌癌细胞的微环境,可以使体内细胞生长更好的可视化。因此,本研究为后续舌活检提供了可靠依据,舌癌发病机制的研究,和药物治疗。 .
    Traditional cell culture methods often fail to accurately replicate the intricate microenvironments crucial for studying specific cell growth patterns. In our study, we developed a 4D cell culture model-a precision instrument comprising an electromagnet, a force transducer, and a cantilever bracket. The experimental setup involves placing a Petri dish above the electromagnet, where gel beads encapsulating magnetic nanoparticles and tongue cancer cells are positioned. In this model, a magnetic force is generated on the magnetic nanoparticles in the culture medium to drive the gel to move and deform when the magnet is energized, thereby exerting an external force on the cells. This setup can mimic the microenvironment of tongue squamous cell carcinoma CAL-27 cells under mechanical stress induced by tongue movements. Electron microscopy and rheological analysis were performed on the hydrogels to confirm the porosity of alginate and its favorable viscoelastic properties. Additionally, Calcein-AM/PI staining was conducted to verify the biosafety of the hydrogel culture system. It mimics the microenvironment where tongue squamous cell carcinoma CAL-27 cells are stimulated by mechanical stress during tongue movement. Electron microscopy and rheological analysis experiments were conducted on hydrogels to assess the porosity of alginate and its viscoelastic properties. Calcein-AM/PI staining was performed to evaluate the biosafety of the hydrogel culture system. We confirmed that the proliferation of CAL-27 tongue squamous cells significantly increased with increased matrix stiffness after 5 d as assessed by MTT. After 15 d of incubation, the tumor spheroid diameter of the 1%-4D group was larger than that of the hydrogel-only culture. The Transwell assay demonstrated that mechanical stress stimulation and increased matrix stiffness could enhance cell aggressiveness. Flow cytometry experiments revealed a decrease in the number of cells in the resting or growth phase (G0/G1 phase), coupled with an increase in the proportion of cells in the preparation-for-division phase (G2/M phase). RT-PCR confirmed decreased expression levels of P53 and integrinβ3 RNA in the 1%-4D group after 21 d of 4D culture, alongside significant increases in the expression levels of Kindlin-2 and integrinαv. Immunofluorescence assays confirmed that 4D culture enhances tissue oxygenation and diminishes nuclear aggregation of HIF-1α. This device mimics the microenvironment of tongue cancer cells under mechanical force and increased matrix hardness during tongue movement, faithfully reproducing cell growthin vivo, and offering a solid foundation for further research on the pathogenic matrix of tongue cancer and drug treatments.
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  • 文章类型: Journal Article
    当机械刺激应用于自由游泳草履虫时,由于后部机械感觉通道的激活,向前游泳速度短暂增加。行为反应,被称为“逃避反应”,“需要膜超极化和K通道型腺苷酸环化酶的激活。我们的假设是,这种逃逸反应还涉及超极化激活的环核苷酸门控(HCN)通道的激活。HCN通道被超极化激活,并被环核苷酸如cAMP和cGMP调节。它们在高等动物的许多兴奋细胞中起着关键作用。如果HCN通道在草履虫中起作用,这应该有助于增强和延长超极化,从而提高草履虫的游泳速度。这项研究使用RNAi通过产生hcn1基因敲低细胞(hcn1-KD)来检查HCN通道1在逃逸反应中的作用。这些细胞显示出减少的机械刺激逃逸反应和缺乏依赖cGMP的游泳速度增加。电生理实验表明,在hcn1-KD细胞中注入大负电流后,超极化减少。这与HCN1通道活性的降低和逃逸反应的变化是一致的。这些发现表明,HCN1通道是K通道,可通过放大后部机械刺激引起的超极化来调节草履虫的逃逸反应。
    When mechanical stimulation was applied to free swimming Paramecium, forward swimming velocity transiently increased due to activation of the posterior mechanosensory channels. The behavior response, known as \"escape response,\" requires membrane hyperpolarization and the activation of K-channel type adenylate cyclases. Our hypothesis is that this escape response also involves activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. HCN channels are activated by hyperpolarization and are modulated by cyclic nucleotides such as cAMP and cGMP. They play a critical role in many excitable cells in higher animals. If HCN channels act in Paramecium, this should help to enhance and prolong hyperpolarization, thereby increasing the swimming speed of Paramecium. This study used RNAi to examine the role of the HCN channel 1 in the escape responses by generating hcn1-gene knockdown cells (hcn1-KD). These cells showed reduced mechanically-stimulated escape responses and a lack of cGMP-dependent increases in swimming speed. Electrophysiological experiments demonstrated reduced hyperpolarization upon injection of large negative currents in hcn1-KD cells. This is consistent with a decrease in HCN1 channel activity and changes in the escape response. These findings suggest that HCN1 channels are K+ channels that regulate the escape response of Paramecium by amplifying the hyperpolarizations elicited by posterior mechanical stimulation.
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  • 文章类型: Journal Article
    背景:猫骨关节炎(OA)导致慢性疼痛和体感致敏。在人类中,感官暴露可以调节慢性疼痛。最近,脑电图(EEG)揭示了人类OA的特定大脑特征。然而,在OA猫中不存在EEG疼痛表征或其调制,所有的脑电图都是在镇静的猫身上进行的,使用皮内电极,这可以改变感觉(疼痛)知觉。
    方法:使用十个镀金表面电极评估受OA影响的猫(n=11)。感觉刺激以随机顺序呈现:对机械时间总和的反应,葡萄柚气味和单色波长(500nm蓝色,525nm-绿光和627nm-红光)。对记录的EEG进行处理以识别事件相关电位(ERP)并进行频谱分析(z分数)。
    结果:手术耐受性良好。报告了两种机械的ERP(F3、C3、Cz、P3,Pz)和嗅觉刺激(Cz,Pz).主要限制是运动伪影。光谱分析显示EEG频带的功率与光波长之间存在显著的相互作用(p<0.001)。考虑所有波长,α带比例高于δ带和γ带(p<0.044),而后者低于β带(p<0.016)。与绿色和红色相比,暴露于蓝光引起脑电图功率随时间的明显变化(p<0.001)。
    结论:这是有意识的猫的EEG可行性的首次证明,该猫具有表面电极记录大脑活动,同时将其暴露于感官刺激。
    结论:ERPs和光谱模式的鉴定为研究猫慢性疼痛及其通过感觉干预的潜在调节开辟了新的途径。
    BACKGROUND: Feline osteoarthritis (OA) leads to chronic pain and somatosensory sensitisation. In humans, sensory exposure can modulate chronic pain. Recently, electroencephalography (EEG) revealed a specific brain signature to human OA. However, EEG pain characterisation or its modulation does not exist in OA cats, and all EEG were conducted in sedated cats, using intradermal electrodes, which could alter sensory (pain) perception.
    METHODS: Cats (n=11) affected by OA were assessed using ten gold-plated surface electrodes. Sensory stimuli were presented in random orders: response to mechanical temporal summation, grapefruit scent and mono-chromatic wavelengths (500 nm-blue, 525 nm-green and 627 nm-red light). The recorded EEG was processed to identify event-related potentials (ERP) and to perform spectral analysis (z-score).
    RESULTS: The procedure was well-tolerated. The ERPs were reported for both mechanical (F3, C3, Cz, P3, Pz) and olfactory stimuli (Cz, Pz). The main limitation was motion artifacts. Spectral analysis revealed a significant interaction between the power of EEG frequency bands and light wavelengths (p<0.001). All wavelengths considered, alpha band proportion was higher than that of delta and gamma bands (p<0.044), while the latter was lower than the beta band (p<0.016). Compared to green and red, exposure to blue light elicited distinct changes in EEG power over time (p<0.001).
    CONCLUSIONS: This is the first demonstration of EEG feasibility in conscious cats with surface electrodes recording brain activity while exposing them to sensory stimulations.
    CONCLUSIONS: The identification of ERPs and spectral patterns opens new avenues for investigating feline chronic pain and its potential modulation through sensory interventions.
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  • 文章类型: Journal Article
    体感系统被广泛研究以了解其功能机制。多项测试,基于不同的设备和方法,不仅在人类身上进行过,而且在动物和离体模型上也进行过。根据所分析样品的性质和感兴趣的科学目标,已经采用了几种用于实验刺激和用于感觉或疼痛研究的解决方案。在这篇综述论文中,已报告了可用设备和方法的概述,还分析了文献实验中采用的代表性值。在用于研究体感系统的各种物理刺激中,我们只关注机械和热的。在分析其主要特点和文献研究的基础上,我们指出了最适合人类的解决方案,啮齿动物,和离体模型和研究目标(感觉和疼痛)。
    The somatosensory system is widely studied to understand its functioning mechanisms. Multiple tests, based on different devices and methods, have been performed not only on humans but also on animals andex-vivomodels. Depending on the nature of the sample under analysis and on the scientific aims of interest, several solutions for experimental stimulation and for investigations on sensation or pain have been adopted. In this review paper, an overview of the available devices and methods has been reported, also analyzing the representative values adopted during literature experiments. Among the various physical stimulations used to study the somatosensory system, we focused only on mechanical and thermal ones. Based on the analysis of their main features and on literature studies, we pointed out the most suitable solution for humans, rodents, andex-vivomodels and investigation aims (sensation and pain).
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  • 文章类型: Journal Article
    机械应力调节细胞中的各种生物过程,组织,和器官以及有助于各种疾病的发病机理。视网膜受到由眼内压以及视网膜出血和水肿施加的机械应力。已经在视网膜色素上皮细胞和视网膜的Müller细胞中研究了对机械应力的反应,发现前细胞经历应激诱导的血管内皮生长因子(VEGF)表达增加,在视网膜的生理和病理血管生成中起着关键作用。我们在这里研究了拉伸刺激对培养的人Müller细胞中血管生成因子表达的影响。逆转录和定量PCR分析显示,在Müller细胞中,这种刺激增加了VEGF-A基因的表达,而血管生成素1基因的下降。酶联免疫吸附试验表明,拉伸刺激也增加了这些细胞的VEGF分泌。转录因子HIF-1α(缺氧诱导因子-1α)的表达在mRNA和蛋白质水平上通过拉伸刺激增加,HIF-1α抑制剂CAY10585可防止机械应力对VEGF-A基因表达和VEGF分泌的影响。此外,RNA测序分析表明,拉伸刺激可上调血管生成相关途径基因的表达。因此,我们的结果表明,机械应力以依赖于HIF-1α的方式诱导Müller细胞中VEGF的产生,因此,HIF-1α是糖尿病性视网膜病变等疾病的潜在治疗靶点,年龄相关性黄斑变性,和视网膜静脉阻塞.
    Mechanical stress regulates various biological processes in cells, tissues, and organs as well as contributes to the pathogenesis of various diseases. The retina is subjected to mechanical stress imposed by intraocular pressure as well as by retinal hemorrhage and edema. Responses to mechanical stress have been studied in retinal pigment epithelial cells and Müller cells of the retina, with the former cells having been found to undergo a stress-induced increase in the expression of vascular endothelial growth factor (VEGF), which plays a key role in physiological and pathological angiogenesis in the retina. We here examined the effects of stretch stimulation on the expression of angiogenic factors in cultured human Müller cells. Reverse transcription and quantitative PCR analysis revealed that expression of the VEGF-A gene was increased by such stimulation in Müller cells, whereas that of the angiopoietin 1 gene was decreased. An enzyme-linked immunosorbent assay showed that stretch stimulation also increased VEGF secretion from these cells. Expression of the transcription factor HIF-1α (hypoxia-inducible factor-1α) was increased at both mRNA and protein levels by stretch stimulation, and the HIF-1α inhibitor CAY10585 prevented the effects of mechanical stress on VEGF-A gene expression and VEGF secretion. Furthermore, RNA-sequencing analysis showed that the expression of angiogenesis-related pathway genes was upregulated by stretch stimulation. Our results thus suggest that mechanical stress induces VEGF production in Müller cells in a manner dependent on HIF-1α, and that HIF-1α is therefore a potential therapeutic target for conditions such as diabetic retinopathy, age-related macular degeneration, and retinal vein occlusion.
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  • 文章类型: Journal Article
    OBJECTIVE: The aim of this study was to investigate the influence of mechanical strain on clock gene function in periodontal ligament (PDL) cells. Furthermore, we wanted to analyze whether effects induced by mechanical stress vary in relation to the circadian rhythm.
    METHODS: Human PDL fibroblasts were synchronized in their circadian rhythm with dexamethasone and stretched over 24 h. Unstretched cells served as controls. Gene expression of the core clock genes were analyzed at 4 h intervals by quantitative real-time polymerase chain reaction (qRT-PCR). Time points 0 h (group SI1) and 12 h (group SI2) after synchronization served as starting points of a 4 h force application period. Collagen-1α (COL-1α/Col-1α), interleukin-1β (IL1-β), and runt-related transcription factor 2 (RUNX2/Runx2) were assessed by qRT-PCR and enzyme-linked immunosorbent assay (ELISA) after 2 and 4 h. Statistical analysis comprised one-way analysis of variance (ANOVA) and post hoc tests.
    RESULTS: After synchronization, the typical pattern for clock genes was visible in control cells over the 24 h period. This pattern was significantly altered by mechanical strain. Under tensile stress, ARNTL gene expression was reduced, while Per1 and 2 gene expression were upregulated. In addition, mechanical stress had a differential effect on the expression of Col-1α and IL1‑β depending on its initiation within the circadian rhythm (group SI1 vs group SI2). For RUNX2, no significant differences in the two groups were observed.
    CONCLUSIONS: Our results suggest that mechanical stress affects the molecular peripheral oscillator of PDL cells. Vice versa, the circadian rhythm also seems to partially influence the effects that mechanical stress exerts on PDL cells.
    UNASSIGNED: ZIEL: Ziel dieser Arbeit war es, den Einfluss von mechanischer Belastung, wie sie auch im Rahmen einer kieferorthopädischen Zahnbewegung auf das Parodontalligament (PDL) appliziert wird, auf Funktionen von Clock-Genen zu untersuchen. Zusätzlich sollte analysiert werden, ob sich eine mechanische Belastung in Abhängigkeit der zirkadianen Rhythmik unterschiedlich auf wichtige Proteine der PDL-Zellen auswirkt.
    METHODS: Die periphere zirkadiane Rhythmik humaner PDL-Zellen wurde mittels Dexamethason synchronisiert und einer statischen Dehnung von 20% über bis zu 24 h ausgesetzt. Parallel wurde eine Kontrollgruppe ohne Dehnung angesetzt. In Zeitintervallen von 4 h wurde die Genexpression der Clock-Gene mittels qRT-PCR („quantitative real-time polymerase chain reaction“) bestimmt. Weiter wurden die Zellen einem Dehnungsintervall von 4 h zu den Zeitpunkten 0 h (Gruppe SI1) und 12 h (Gruppe SI2) nach Synchronisation ausgesetzt. Die Expression der Gene Collagen-1α (Col-1α), Interleukin-1β (IL1-β) und Runt-verwandter Transkriptionsfaktor 2 (RUNX2) wurde jeweils nach 2 und 4 h mittels qRT-PCR und ELISA („enzyme-linked immunosorbent assay“) quantifiziert. Die statistische Analyse erfolgte über eine einseitige Varianzanalyse (ANOVA) und Post-hoc-Tests.
    UNASSIGNED: In den Kontrollzellen war nach Synchronisation das für die Clock-Gene typische Muster im Verlauf der 24 h erkennbar. Dieses wurde durch die mechanische Belastung signifikant verändert. Unter Zugbelastung wurde eine Verringerung der ARNTL-Genexpression verzeichnet, während die Per1- und Per2-Genexpression hochreguliert wurden. Die mechanische Belastung hatte abhängig von der Initiierung nach Synchronisation (Gruppe SI1 vs. Gruppe SI2) einen unterschiedlichen Einfluss auf die Expression von Col-1α und IL1‑β. Für Runx2 wurde in beiden Gruppen kein Unterschied beobachtet.
    UNASSIGNED: Die Ergebnisse legen nahe, dass mechanische Belastung den molekularen peripheren Oszillator der PDL-Zellen beeinflusst. Umgekehrt scheint auch die zirkadiane Rhythmik die Auswirkungen von mechanischem Stress auf PDL-Zellen teilweise zu beeinflussen.
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  • 文章类型: Journal Article
    气道阻力依次增加的影响:没有,低(5kPa。s/l),高(24千帕。s/l),在16只麻醉(戊巴比妥)自发呼吸猫(3.70±0.15kg,11‰,5)分析了吸气期间的食管压力和diaphragm肌的肌电图活动以及呼气期间的腹部肌肉。咳嗽次数无明显变化。吸气闭塞导致咳嗽吸气期延长,咳嗽吸气膈肌活动,和所有咳嗽相关的活动。吸气闭塞伴随高阻力增加吸气食管压力振幅,总咳嗽周期持续时间和膈肌和腹肌最大活动之间的时间。高呼气阻力和阻塞导致咳嗽呼气食管压力振幅增加,咳嗽呼气的较长的活跃部分,咳嗽腹部活动。呼气闭塞也延长了咳嗽呼气期,所有咳嗽活动,和总咳嗽周期。显著增加的气道阻力和闭塞诱发继发性,除了机械,通过显着调节产生的咳嗽运动模式来改变咳嗽。一定程度的阻力似乎得到了成功的补偿,导致咳嗽特征的最小变化,包括呼气气流和气流的上升时间。来自呼吸道的传入反馈,特别是音量反馈,是调节咳嗽的一个重要因素,主要在呼吸系统的各种病理条件下。
    Effects of sequential increase in airway resistance: no, low (5 kPa.s/l), high (24 kPa.s/l), and complete block in the inspiratory or expiratory phase of mechanically induced cough on the cough motor pattern were studied in 16 anesthetized (pentobarbital) spontaneously breathing cats (3.70±0.15 kg, 11♂, 5♀). Esophageal pressure and electromyographic activities of the diaphragm during inspiration and abdominal muscles during expiration were analyzed. No significant changes in the number of coughs occurred. Inspiratory occlusion caused a prolongation of cough inspiratory phase, cough inspiratory diaphragm activity, and all cough-related activity. Inspiratory occlusion along with high resistance increased inspiratory esophageal pressure amplitude, total cough cycle duration and the time between maximum activity of the diaphragm and abdominal muscles. High expiratory resistance and occlusion resulted in increased cough expiratory esophageal pressure amplitude, a longer active portion of cough expiration, and cough abdominal activity. Expiratory occlusion also prolonged cough expiratory phase, all cough activity, and total cough cycle. Significantly increased airway resistance and occlusion induce secondary, in addition to mechanical, changes in cough by significantly modulating the generated cough motor pattern. A certain level of resistance appears to be successfully compensated, resulting in minimal changes in coughing characteristics, including expiratory airflow and the rising time of the airflow. Afferent feedback from the respiratory tract, particularly volume feedback, represents a significant factor in modulating cough, mainly under various pathological conditions in the respiratory system.
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