The effect of ATP on the ciliary activity of normal and pathological human respiratory mucosa in vitro

The effect of adenosine triphosphate (ATP) on the airway ciliary beating frequency (CBF) of mucosal explants excised from human maxillary sinuses was studied by measuring CBF photoelectrically before and after immersion of the explants in a solution containing ATP. In samples from 64 patients with chronic sinusitis the CBF was not significantly different from the CBF in mucosal specimens from healthy tissue of 22 patients without infection. During 15 min immersion, ATP (1 mg/ml) slightly (by 5%, p less than 0.05 in healthy tissue; by 2.7%, p less than 0.01, in tissue from sinusitis patients) increased the CBF. The effect of 10 mg/ml concentration was more pronounced (19.6%). It is concluded that the impairment in ciliary function caused by chronic sinusitis is reversible when the mucosal endothelium is cleansed of the infected mucus, and that the ciliostimulatory action of ATP seen in animals is also present in human respiratory mucosa.     

Validation of animal experiments on ciliary function in vitro. II. The influence of absorption enhancers, preservatives and physiologic saline

Ciliary beat frequency (CBF) is one of the most important parameters of mucociliary clearance. Previously, we demonstrated that mucosa from chicken embryo trachea is a good substitute for human ciliated epithelium to study the effects on CBF of substances that are used clinically. In this study, we examined the effect on CBF of four excipients for nasal drug formulations: the absorption enhancers methylated beta-cyclodextrin 2% and sodium taurodihydrofusidate 1%, the preservative benzalkonium chloride 0.01%, and physiologic saline. We also examined the effect on CBF of the cryopreservative dimethyl sulfoxide, which is used to protect ciliated epithelium prior to storage in liquid nitrogen. Results obtained with chicken embryo trachea were compared with those of cryopreserved human mucosa taken from the sphenoidal sinus. For all of the substances tested, the effects on CBF of chicken material were comparable to those measured on human material. Benzalkonium chloride had a stronger ciliostatic effect on human tissue. After 60 min, however, the effect of that substance on CBF was similar in both tissues. We conclude that chicken embryo trachea can be used as a substitute for human ciliated mucosa when studying ciliary activity in vitro.     

In vivo and in vitro effect of ozone and formaldehyde on human nasal mucociliary transport system.

The effect of ozone and formaldehyde on the nasal mucociliary transport system after short-term exposure was comparatively evaluated in human by using in vivo and in vitro test systems. Concentrations of ozone used were 10, 100, 500 and 1000 micrograms/m3 of ozone and of formaldehyde 100, 500 and 5000 micrograms/m3. The in vivo effect of ozone was monitored by measuring the saccharin transport time before and two hours after exposure to ozone. The in vitro effect of ozone and formaldehyde was evaluated by quantifying the ciliary beat frequency (CBF) of isolated respiratory epithelial cells before and after one, two, and three hours of exposure. Control experiments were performed using synthetic air. Ozone had no effect on the human nasal mucociliary transport system under the conditions tested here. Neither in vivo nor in vitro any significant changes of saccharin transport time nor CBF were measured. In contrast, formaldehyde significantly reduced (67.1%) CBF at the highest dosis (2 hours, 5000 micrograms/m3). These results will be discussed according to the environmental impact of ozone and formaldehyde in air pollutants and compared to sulphur dioxide and nitric oxide, which were tested under similar conditions, and to results revealed from animal experiments.     

游离空肠重建气管对气道黏液清除功能影响的实验研究

目的　探讨游离空肠重建长段环形缺损气管后对气管的黏液 纤毛清除功能的影响 ,为该方法应用于临床提供依据。方法　通过显微外科游离空肠联合镍钛合金网重建 12条犬气管的动物实验模型 ,纤维支气管镜下于重建气道的下切缘用注射器滴入示踪剂 ,计算示踪剂从滴入到到达声门的时间 (mucociliarytransittime ,MTT)除该段长度 (mucociliarytransportlength ,MTL) ,为黏液清除率(mucociliarytransportrate,MTR) ,对游离空肠重建 6 5cm长段环形缺损气管后新气道的黏液 纤毛清除功能进行术前 ,术后 7d、1个月、3个月、6个月多时间点研究。结果　除了术前的MTR与术后 1个月的MTR相比差异有统计学意义外 (P <0 0 5 ) ,其余术前MTR与术后 7d、3个月、6个月及术后 7d与术后 1个月、3个月、6个月相比均无统计学意义 (P >0 0 5 )。结论　游离空肠重建气管中 ,新气道的黏液 纤毛清除功能随着呼吸道炎症的消退术后 3个月恢复正常水平     

Bioelectric properties of excised rabbit nasal mucosa

The water flow across the respiratory epithelia is an important determinant of the efficiency of mucociliary clearance. Bulk water flow has been shown to be coupled to net ion flux. We studied ion transport across rabbit nasal mucosa by measuring bioelectric properties using Ussing chambers. Results were summarized as follows. (1) Compared with tracheal mucosa, nasal mucosa exhibited lower potential difference (p less than 0.01), lower short-circuit current (p less than 0.05), and higher conductance (p less than 0.01). (2) Ouabain 10(-4)M inhibited short-circuit current when added to the submucosal bath of Ussing chambers, and amiloride decreased short-circuit current to about 40% when added to the mucosal bath. (3) When the bubbling of the solution was changed from 95%O2, 5%CO2 to 100%N2, short-circuit current remarkably decreased. (4) A significant positive correlation existed between temperature ranging from 33 degrees C to 41 degrees C and short-circuit current (r = 0.46, p less than 0.02).     

Effect of oxymetazoline on healthy human nasal ciliary beat frequency measured with high-speed digital microscopy and mucociliary transport time

OBJECTIVES: We investigated the effects of oxymetazoline hydrochloride on the regulation of healthy human nasal ciliary beat frequency (CBF) and its influence on nasal mucociliary transport time (MTT). METHODS: Changes in (cultured) human nasal CBF in response to increasing concentrations of oxymetazoline within 20 minutes were quantified by use of high-speed digital microscopy. Moreover, the MTT before and after application of 0.05% oxymetazoline was determined by use of the saccharin test. RESULTS: Whereas no statistically significant difference was identified when compared to basal CBF at the concentration of 0.025% or 0.05%, both 0.10% and 0.20% oxymetazoline induced a significantly lower CBF at the end of the observation period. The decrement induced by 0.20% oxymetazoline appeared earlier. At concentrations ranging from 0.025% to 0.20%, the inhibitory effect was dependent on the concentration of oxymetazoline. In addition, the use of 0.05% oxymetazoline increased the mean (+/- SD) human nasal MTT from 474 +/- 21 seconds to 572 +/- 41 seconds (n = 29). CONCLUSIONS: The clinical concentration of oxymetazoline, 0.05%, has no obvious inhibitory effect on human nasal CBF in vitro. The increased MTT caused by 0.05% oxymetazoline in vivo is within the normal range.     

A Vasoactive Agent Enhances the Effect of ATP on Cochlear Blood Flow

Adenosine 5-triphosphate (ATP) has a dual effect on cochlear blood flow (CBF). It induces on initial, transient decrease, which is followed by a long-lasting increase in CBF. Dilazep dihydrochloride (DZ) is a unique vasoactive agent, which increases CBF as well as decreases systemic blood pressure (BP). This agent is thought to keep the concentration of extracellular adenosine higher than normal by preventing the absorption of adenosine into the intracellular space. Thus, it may potentiate the effect of ATP on CBF. In this study, the effects are reported of ATP on CBF in the presence of DZ as measured with a laser Doppler flowmeter in the guinea pig cochlea. In the presence of DZ, the effect of ATP on CBF was amplified and prolonged. Specifically, the mean value of CBF initially decreased to 30.0% of the baseline, determined prior to administration of any agent, and increased gradually up to 287.8% of the baseline. This pharmaceutical effect of DZ might involve a longer activation of both P_2x- and P_2y-purinoceptors.     

Classification of Cilio‐Inhibiting Effects of Nasal Drugs

Objective/Hypothesis Nasal drug formulations are widely used for a local therapeutic effect, but are also used for systemic drug delivery. In the development of new nasal drugs, the toxic effects on the mucociliary clearance and therefore on the ciliated tissue is of importance. In this study, the effect of nasal drugs and their excipients on the ciliary beat frequency (CBF) is investigated. Study Design Experimental, in vitro. Methods CBF is measured by a photograph–electric registration method. Excised ciliated chicken trachea tissue is incubated for 15 minutes in the formulation, followed by a reversibility test. To estimate the ciliostatic potential, a classification is given of all tested formulations. According to the CBF, after 60 minutes every drug or excipient could be classified as follows:cilio‐friendly: after 60 minutes the CBF has regained 75% or more of its initial frequency;cilio‐inhibiting: after 60 minutes the CBF has regained between 25% and 75% of its initial frequency; or ciliostatic: after 60 minutes the CBF has regained 25% or less of its initial frequency. Results Most formulations used are cilio‐friendly or cilio‐inhibiting. Only some are ciliostatic. Preservatives have a major role in the cilio‐inhibiting effect of the drug. Also, other additives can contribute to the toxicity profile of nasal drug formulations. Conclusion This classification of the cilio‐inhibiting potential of nasal drug formulations is a valuable tool in the design of safe nasal drugs. The number of animal studies in vivo can be reduced substantially by using this in vitro screening technique. This study demonstrates that the effect on ciliary movement of most drug formulations is due to the preservatives and/or additives and mostly not to the drug itself.     

A vasoactive agent enhances the effect of ATP on cochlear blood flow

Adenosine 5'-triphosphate (ATP) has a dual effect on cochlear blood flow (CBF). It induces on initial, transient decrease, which is followed by a long-lasting increase in CBF. Dilazep dihydrochloride (DZ) is a unique vasoactive agent, which increases CBF as well as decreases systemic blood pressure (BP). This agent is thought to keep the concentration of extracellular adenosine higher than normal by preventing the absorption of adenosine into the intracellular space. Thus, it may potentiate the effect of ATP on CBF. In this study, the effects are reported of ATP on CBF in the presence of DZ as measured with a laser Doppler flowmeter in the guinea pig cochlea. In the presence of DZ, the effect of ATP on CBF was amplified and prolonged. Specifically, the mean value of CBF initially decreased to 30.0% of the baseline, determined prior to administration of any agent, and increased gradually up to 287.8% of the baseline. This pharmaceutical effect of DZ might involve a longer activation of both P2x- and P2y-purinoceptors.     

Mucociliary Clearance Following Segmental Tracheal Reversal

Ciliated tracheal epithelium is arranged in a polarized pattern oriented according to the inferior-superior axis of the trachea and is responsible for the transport of mucus toward the larynx. In this study, ciliary beat orientation and the influence of external factors on mucociliary clearance direction were studied in rabbit inverted cervical tracheas. The animals displayed normal respiration postoperatively. After 16 weeks, airway clearance was studied by observation of the movement of silicone particles placed in the inverted segment and in normal parts of the ciliated epithelium. Cilia exhibited unidirectional and coordinated movement within inverted tracheal segments. As shown by the direction of effective flow produced by beating cilia and by scanning electron microscopy, the cilia in the inverted segment beat in the opposite direction from the cilia in the remainder of the trachea. This study demonstrated that ciliary orientation is irreversibly determined, but the reversal of ciliary beating within the cervical trachea had no adverse effects on the survival of the animals.     

缓释载药聚乳酸凝胶预防气管纤维组织增生

目的探讨丝裂霉素C（mitomycin C,MMC）与地塞米松磷酸钠（dexamethasone sodium phosphate,DSP）的聚乳酸凝胶对气管瘢痕组织形成的抑制作用。方法在体外缓释实验中,将MMC与DSP聚乳酸凝胶后置于恒温水浴摇床中,药物的累积浓度通过紫外分光光度法测定。在体内缓释试验中,通过喉气管外壁损伤建立瘢痕形成的动物模型。将42只新西兰大白兔进行气管外壁刮伤后分成7组：实验组包括载MMC组3组（0.1、0.2、0.4 mg）、载DSP组3组（1、2、3 mg）和空白对照组1组。0.4 mg MMC组动物于术后不同时间抽血,通过质谱测量体内药物浓度及血常规。术后4周处死动物,取气管外壁标本,HE染色观察纤维组织增生厚度。结果体外实验1 mg MMC、2 mgDSP凝胶缓释分别可达35 d及28 d以上。动物体内两种药物凝胶在术后4周未完全降解。两种药物剂量组（除0.1 mgMMC组外）与对照组比较,对气管壁纤维组织增生有明显抑制作用;在MMC各剂量组中,0.4 mg MMC组比其他组对纤维组织增生的抑制程度更明显;在DSP不同剂量组中,抑制作用无统计学意义;0.4 mg MMC与3种剂量的DSP组纤维组织增生厚度差异均无统计学意义。术后1、7、14 d白细胞计数无统计学差异。结论 MMC及DSP聚乳酸凝胶在动物体内外释放时间均长达4周,可作为预防瘢痕形成的缓释用药。本研究为未来研制聚乳酸聚乙醇酸支架涂层药物缓释载体对喉气管狭窄的治疗提供了实验基础。     

Validation of animal experiments on ciliary function in vitro. I. The influence of substances used clinically

In vitro studies of ciliary activity require specimens of healthy epithelium in relatively large quantities. Since human material is difficult to obtain, fresh chicken trachea samples have frequently been used in function experiments. The aim of the present study was to investigate whether several substances had comparable effects on the ciliary beat frequency (CBF) of chicken trachea and cryopreserved human respiratory epithelium obtained from the sphenoidal sinus. For this study, we used two topical anaesthetics: cocaine (3% and 7%) and lidocaine (2%). These anaesthetic substances were adjusted to pH 6 and pH 7. We also used two decongestants, namely xylometazoline 0.1% and oxymetazoline 0.1%, and the beta-blocking agent propranolol. Topical anaesthetics appeared to be more ciliostatic in solutions with pH 7 compared to pH 6. Complete ciliostatic effects were reversible, with the exception of the ciliostasis induced by propranolol. The effects of these substances on the CBF of fresh chicken trachea and cryopreserved human tissue did not differ significantly. These experiments show that chicken trachea constitutes a valid substitute for human material in studying ciliary activity in vitro. Moreover, the experiments provide evidence in support of the assumption that cryopreservation has no effect on ciliary reactivity as expressed by the CBF.     

Altered sinonasal ciliary dynamics in chronic rhinosinusitis

BACKGROUND: Although multiple etiologies contribute to the development of rhinosinusitis, a common pathophysiological sequelae is ineffective sinonasal mucociliary clearance, resulting in stasis of sinonasal secretions, with subsequent infection, and persistent inflammation. The respiratory cilia beat continually at a basal rate, while during times of stress, such as exercise or infection, ciliary beat frequency (CBF) increases, accelerating mucus clearance. Previous investigations have led to conflicting results with some authors reporting decreased CBF while others have found normal values of CBF in patients with chronic rhinosinusitis (CRS). Additionally, these studies have only analyzed basal CBF. The goal of this study was to compare the basal as well as the stimulated sinonasal CBF in patients with CRS versus controls. METHODS: A dual temperature controlled perfusion chamber, differential interference contrast microscopy, and high-speed digital video were used to analyze both basal and adenosine triphosphate (100 microM)-stimulated CBF in human sinonasal mucosal explants. RESULTS: Although no difference in basal CBF was detected between control and CRS patients, a marked difference in stimulated CBF was noted. Exogenously applied adenosine triphosphate resulted in a 50-70% increase of CBF in control tissue with a minimally observed CBF increase in explants from CRS patients. CONCLUSION: Dynamic regulation of respiratory ciliary activity is critical for the respiratory epithelium to adapt to varying environmental situations. Thus, diminished or absent adaptation could predispose the sinonasal cavity to accumulation of inhaled infectious and noxious particulate matter resulting in infection/inflammation. Our findings suggest that CRS patients have decreased sinonasal ciliary adaptation to environmental stimuli.     

Larynx and cervical trachea in humidification and heating of inhaled gases

To evaluate the participation of the larynx and cervical trachea in conditioning inspired gases, we randomly allocated 16 mixed-breed dogs to two groups: group TT (tracheal tube; n = 8) and group LMA (laryngeal mask airway; n = 8). The dogs were anesthetized with pentobarbital sodium and mechanically ventilated for 3 hours. The parameters studied were temperature and absolute humidities of ambient, inhaled, and tracheal air. There was a small increase in tracheal air temperature compared to inhaled air temperature, but no significant difference between groups. The absolute humidity of tracheal air was greater in group LMA than in group TT (23 mg H2O * L(-1) and 14 mg H2O * L(-1), respectively; p < .0001). The difference in absolute humidity between the tracheal air and the inhaled air was higher in group LMA at all times (p < .0001). We conclude that the larynx and cervical trachea of the dog participate in humidification and heating of inhaled air by means of air contact with mucosa in this airway segment.     

Mucociliary transport pathway on laryngotracheal tract and stented glottis in guinea pigs

We investigated the laryngotracheal mucociliary transport pathway of guinea pigs in vivo and immediately postmortem. Only intraperitoneal anesthesia was used during the procedure to avoid the disturbance of mucociliary function. Resin particles were used as the marking substance. A microcolpohysteroscope was placed at different levels in the laryngotracheal region for observing the marking particles and recording the transport pattern. The tracheal mucociliary transport flow primarily moved along the posterior wall and both lateral walls in a zigzag trace. Upon reaching the subglottis, the resin particles stayed underneath the vocal cords, and a whirlpool phenomenon developed. The majority of the particles were shifted and directed onto the posterior glottic area. With a short delay, some resin particles crossed over the free edge of the vocal cords and turned posteriorly along the medial upper cordal margin. No mucociliary transport could be observed on the entire upper surface of the true vocal cords, which is covered by squamous epithelium. Occasionally, a few resin particles in the vicinity of the epiglottic root traveled along the aryepiglottic folds toward the posterior commissure. All streams of mucociliary transport finally joined together in the interarytenoid area. After leaving the glottis, the resin particles traveled to the hypopharynx and entered the esophagus through the motion of deglutition. The pattern of mucociliary clearance in the laryngotracheal region was not delayed by stenting.     

部分去黏膜游离空肠重建气管对气道黏液清除功能影响的实验研究

目的 通过建立部分去除黏膜层的游离空肠段重建6.5cm袖状气管缺损动物模型,从而研究部分去黏膜游离空肠修复犬气管缺损前后气管黏液清除率的变化。方法 比格犬8只,从腹部取长约7cm的带蒂游离空肠段,部分去除空肠的黏膜层。将部分去黏膜游离空肠联合肠腔内放置硅胶管内支架,空肠外面放置形状记忆镍钛合金(SMA)外支架,应用显微吻合技术,将游离空肠的系膜动静脉分别与右侧颈总动脉及颈内静脉相吻合,建立长段气管重建动物模型。分别于术前1周、术后1周、术后1个月、术后3个月,静脉全麻下犬仰卧位,纤维支气管镜下于重建气道(术前大约为环状软骨下约7.0cm处)的下切缘用注射器滴入示踪剂,计算示踪剂从滴入到到达声门的时间(MTT)除该段长度(MTL)为黏液清除率(MTR),并对结果进行统计学分析。结果 8只犬黏液清除功能观测术前与术后1个月、3个月,术后1个月与术后3个月的MTR相比差异无统计学意义(P＞0.05),术前MTR与术后1周、术后1个月与术后1周、术后3个月与术后1周相比差异均有统计学意义(P＜0.01)。结论 部分去黏膜游离空肠重建气管后气管黏液清除率术后1个月可恢复到术前的水平,术后3个月保持与术前、术后1个月相当的黏液清除功能。     

体外培养的人鼻腔黏膜纤毛的摆动频率

目的通过测量体外培养的人鼻腔纤毛上皮细胞的纤毛摆动频率（ciliray beat frequency,CBF）,为人鼻腔黏液纤毛运输系统功能的研究提供理论依据。方法应用组织块培养法和高速数字化摄像系统,测量体外培养的人鼻腔钩突、下鼻甲黏膜的CBF。结果体外培养的正常鼻腔黏膜上皮细胞和鼻窦炎鼻腔黏膜上皮细胞的CBF,两者之间差异无统计学意义（t=0.419,P=0.678）;测量来源相同体外培养的钩突和下鼻甲纤毛上皮细胞的CBF,两者的CBF是一致的,之间的差异无统计学意义（t=-0.087,P=0.931）。结论体外培养的鼻窦炎患者的鼻腔黏膜可代表正常人的鼻腔黏膜进行黏液纤毛运动的研究;钩突黏膜更容易获得,可代替下鼻甲黏膜进行体外培养。     

Effect of Ringer-Lactate and isotonic saline solutions on mucociliary clearance of tracheal epithelium: an experimental study in rats

Isotonic saline solution is frequently used in nasal and tracheal lavage. In a previous clinical study, it was found that Ringer-Lactate solution, as a nasal lavage, was better for mucociliary clearance function than isotonic saline solution after nasal septal surgery. In this experimental study, the effects of Ringer-Lactate and isotonic saline solutions on mucociliary clearance of healthy rat tracheal epithelium were investigated by measuring the transport of carbon particles. We found that tracheal segments that were irrigated with Ringer-Lactate had better mucociliary transport than those irrigated with isotonic saline (p = 0.035).     

Effects of oxymetazoline on isolated rat’s tracheal smooth muscle

Oxymetazoline is often used as a decongestant in rhinitis patients who are suffering from nasal obstruction. It is used as a nasal drop or spray solution. The effect on nasal mucosa in vitro or in vivo is well known. However, the effect of the drug on tracheal smooth muscle has rarely been explored. During administration of the drug to the nose, it might affect the trachea via inhalation. We used our preparation to test the effectiveness of oxymetazoline on isolated rat’s tracheal smooth muscle. A 5 mm long portion of rat trachea was submersed in 30 ml Kreb’s solution in a muscle bath at 37°C. Changes in tracheal contractility in response to the application of parasympathetic mimetic agents were measured using a transducer connected to a Pentium III computer equipped with polygraphy software. The following assessments were performed: (1) effect on tracheal smooth muscle resting tension; (2) effect on contraction caused by 10⁻⁶ M methacholine as a parasympathetic mimetic; (3) effect of oxymetazoline on electrically induced tracheal smooth muscle contractions. Addition of parasympathetic mimetics to the incubation medium caused the trachea to contract in a dose-dependent manner. Addition of oxymetazoline induced a significant relaxation response when the preparation was up to 10⁻⁴ M. At the same concentration, the drug also could inhibit EFS induced spike contraction. Oxymetazoline had negligible effect on the basal tension of trachea as the concentration increased. The degree of drug-induced tracheal contraction or relaxation was dose-dependent. The study indicated that high concentrations of oxymetazoline might actually antagonize cholinergic receptors of the trachea.     

Quantification of ciliary beat frequency in sinonasal epithelial cells using differential interference contrast microscopy and high-speed digital video imaging

BACKGROUND: Mucociliary clearance is a critical upper airway host defense mechanism. Ciliated epithelium in the mammalian airway continually beat at a baseline frequency. Importantly, during times of stress such as exercise or infection, the cilia beat faster to increase clearance. Nasal epithelial ciliary beat frequency (CBF) has been analyzed previously in ex vivo specimens using a variety of methods including photodiode detectors and conventional video recording. Recent studies performed using lower airway ciliated mucosa have shown poor correlation between CBF quantified by photodiode/conventional video and those using high-speed digital video capture at temperatures close to physiological temperatures. Thus, to more rigorously interrogate sinonasal CBF at physiological conditions, we have incorporated a high-speed digital video camera to our CBF analysis system. This is the first report of sinonasal epithelial CBF analysis performed using high-speed video digital analysis. METHODS: Ex vivo samples of sinonasal epithelium were placed in lactated Ringer's in a temperature-controlled microscope stage chamber. An edge of tissue containing beating cilia was observed at a magnification of 630x using differential interference contrast microscopy. The images were captured using a high-speed digital camera with a sampling rate of 250 frames per second. CBF was determined using computerized data analysis. RESULTS: The mean nasal CBF was calculated from a minimum of five regions for each sample. Temperature curves were generated from tissue obtained from chronically infected subjects. CONCLUSION: Analysis of high-speed digital video capture of sinonasal CBF observed under differential interference contrast microscopy is a powerful method to investigate environmental as well as host influences on mucociliary clearance within the upper airways.