Ciliary Beat Frequency Measurements Research Articles

Platelet-activating factor inhibits ciliary beat frequency of human bronchial epithelial cells.

The aim of our study was to investigate the effect of platelet-activating factor on human bronchial ciliary beat frequency (CBF) ex vivo. Brush biopsies were obtained from eight children (mean age 6.6 years) who underwent bronchoscopy for clinical reasons (i.e., foreign body aspiration, chronic cough, stridor). Immediate measurement of CBF by phase-contrast microscopy and photoelectric technique was performed before further treatment, and then on three subsamples: after adding platelet-activating factor (PAF) (10(-5) M); after adding the inhibitor WEB 2086 (10(-4) M); and after adding both PAF and WEB 2086. Addition of 10(-5) M PAF reduced mean CBF by 2.1 Hz to 9.2 Hz (p < 0.05). The inhibitory effect of PAF was completely reversed by the addition of the PAF-inhibitor WEB 2086, whereas WEB 2086 alone did not significantly influence CBF. Optimum PAF concentration had been determined in previous dose-response experiments. Our data indicates that PAF involved in airway inflammation contributes to a decrease in mucociliary clearance in humans.

Characterization of ciliary activity in distal Fallopian tube biopsies of women with obstructive tubal infertility.

Biopsy specimens were obtained from the distal end of the Fallopian tubes of 62 women with tubal infertility and examined by light and electron microscopy. Ciliary beat frequency (CBF) measurements were obtained using laser light spectroscopy. Neither demographic nor behavioural characteristics nor serological evidence of past chlamydial infection were associated with CBF measurements. In contrast, CBF were significantly lower (P < 0.05) in tissues with oedema compared to tissues without oedema (6.7 versus 12.9) and in tissues with erythema compared to tissues without erythema (9.2 versus 13.7). Furthermore, CBF measurements did vary by chlamydial serotype pattern, with lower values observed among the tissues of women with antibodies to serotype C or E (without D) as compared to the tissues of women with other serotypes (P < 0.04). However, these data must be interpreted with caution as the numbers of subjects with chlamydial antibodies to serotype C (n = 3) or E without D (n = 5) were few in number and serotyping of IgG antibodies in blood is not as accurate as it is in bacterial isolates. Confirmation of the suggested association between chlamydial serotype and risk of adverse sequelae could indicate potential new avenues for vaccine research.

Leukotrienes C4 and D4 increase the ciliary beat frequency in human upper airway mucosa in vitro.

It has been suggested that leukotrienes C4 (LTC4) and D4 (LTD4) released from upper respiratory mucosa influence mucociliary transport during allergic reactions. We studied the in vitro effects of leukotrienes C4 and D4 on the ciliary beat frequency (CBF) of human adenoid explants over a 5-hour period. Tissue explants were cultured at 35 degrees C in Minimum Essential Medium Eagle (MEM). The CBF was measured using phase contrast microscopy and microphotometry. Measurements of CBF were recorded in medium alone and in medium containing LTC4 or LTD4 at concentrations of 10(-8) and 10(-6) M. LTC4 and LTD4 increased CBF at concentrations of 10(-8) and 10(-6) M with increases of 20.51% +/- 2.69% and 29.84% +/- 4.06%, respectively. To determine the specificity of the LTC4 and LTD4 effects, the ciliated epithelium was treated with the specific leukotriene receptor antagonist LY-171,883 before administration of LTC4 and LTD4. LY-171,883 (10(-6) M) significantly inhibited the ciliostimulatory effects of both leukotrienes. Our findings indicate that LTC4 and LTD4 increase CBF in vitro by activation of the LTD4 receptor.

Leukotrienes C 4 and D 4 increase the ciliary beat frequency in human upper airway mucosa in vitro

It has been suggested that leukotrienes C 4 (LTC 4 ) and D 4 (LTD 4 ) released from upper respiratory mucosa influence mucociliary transport during allergic reactions. We studied the in vitro effects of leukotrienes C 4 and D 4 on the ciliary beat frequency (CBF) of human adenoid explants over a 5-hour period. Tissue explants were cultured at 35° C in Minimum Essential Medium Eagle (MEM). The CBF was measured using phase contrast microscopy and microphotometry. Measurements of CBF were recorded in medium alone and in medium containing LTC 4 or LTD 4 at concentrations of 10 -8 and 10 -6 M. LTC 4 and LTD 4 increased CBF at concentrations of 10 -8 and 10 -6 M with increases of 20.51% ± 2.69% and 29.84% ± 4.06%, respectively. To determine the specificity of the LTC 4 and LTD 4 effects, the ciliated epithelium was treated with the specific leukotriene receptor antagonist LY-171,883 before administration of LTC 4 and LTD 4 . LY-171,883 (10 -6 M) significantly inhibited the ciliostimulatory effects of both leukotrienes. Our findings indicate that LTC 4 and LTD 4 increase CBF in vitro by activation of the LTD 4 receptor. (Otolaryngol Head Neck Surg 1998;118:472-7.)

Low Levels of Nasal Nitric Oxide (NO) Correlate to Impaired Mucociliary Function in the Upper Airways

Findings in previous studies have suggested nitric oxide (NO) to be a regulator of mucociliary activity in the upper airways. The aim of the present investigation was to study whether a correlation exists between the nasal NO concentration and mucociliary function in patients suffering from respiratory tract diseases such as chronic sinusitis or recurrent pneumonia. Nasal NO was measured with a chemiluminescence analyser, 100 ppb (parts per billion) being adopted as the lower limit of the normal range on the basis of findings in an earlier study of healthy subjects. Mucociliary function was evaluated by measurements of ciliary beat frequency (CBF) in nasal brush samples, and the saccharin transport test. A subnormal level of nasal NO was found in 50% (9/18) of the patients. This correlated with a significantly impaired mucociliary function, regarding both CBF and the saccharin transport time. The median CBF was 10.6 Hz in the group with normal levels of nasal NO, as compared to 8.4 Hz in the subnormal NO group. AH patients with a normal nasal NO concentration had a mean CBF of s9.0 Hz in their nasal brush samples, but in the subnormal group the same measurements yielded a CBF of ≥9.0 Hz in only 22% (2/9) of the cases. As measured with the saccharin test, mucociliary transport was normal in 78% (7/9) in the normal nasal NO group, but the saccharin test was normal only in 11% (1/9) of the subnormal nasal NO group. Nasal NO levels were found to correlate with both CBF measurements (Spearman's rho, 0.80) and the saccharin transport test results (Spearman's rho, -0.61). The results of the present study provide further support for the view that NO is an important regulator of mucociliary function in the upper airways, and that measurements of the nasal NO concentration should be included in investigations of the mucociliary system.

Mucociliary Clearance in Experimental Chronic Sinusitis

Abnormal mucociliary clearance in the paranasal sinuses is a significant factor in the pathogenesis of sinusitis. This study examined effects of chronic sinusitis on ciliary beat frequency (CBF), mucociliary transport (MCT) velocity, mucociliary transport patterns, and histology of sinus mucosa in rabbits. Two groups of rabbits were studied: uninfected (n = 9) and infected (n = 18). Maxillary sinus ostia in all animals were occluded, and sinuses in the infected group were inoculated with 108 cfu of Streptococcus pneumoniae. Chronic sinusitis developed over 6 weeks and was confirmed by histopathology and culture. Evaluation of the MCT mechanism included 1) measurement of CBF using a photometric technique, 2) direct measurement of MCT velocity, and 3) analysis of MCT patterns with inert India ink. In seven of nine (77.8%) uninfected animals, the mean CBF was 11.5 Hz (±.39 Hz) initially and 11.3 Hz (±.32 Hz) 6 weeks after occlusion (P = 0.69). MCT velocity measured 20.6 mm/min (±1.40 mm/min) initially and 16.85 mm/min (±1.96 mm/min) 6 weeks after occlusion (P = 0.16). In the infected group, there were statistically significant decreases in CBF (P &lt; 0.05) and measured MCT velocity (P &lt; 0.05). Examination of MCT in infected animals showed patterns of misdirected flow, pooling, and stagnation. Histopathologic changes included significant losses of ciliated epithelium and mucosal abnormalities consistent with chronic sinusitis. Chronic sinusitis secondary to long-standing bacterial infection leads to alterations in CBF, MCT velocity, and MCT patterns in the paranasal sinuses of rabbits.

The effect of nasal drug formulations on ciliary beating in vitro

In order to estimate the potential of nasal drug formulations to influence ciliary beating, ciliary beat frequency (CBF) measurements were performed in vitro, using chicken embryo trachea and a photo-electric registration device. The effects of nasal prescription and non-prescription drug formulations were studied and compared with a number of investigational nasal drug formulations containing estradiol, dihydroergotamine mesylate and salmon calcitonin. The influence of all formulations on CBF was related to the effects of formulation additives such as preservatives and nasal absorption enhancers on the ciliated tissue. For almost all nasal drug formulations, the preservatives used in the formulations (e.g. benzalkonium chloride, chlorobutanol) play a decisive role in the observed ciliostatic effects. Methylated β-cyclodextrins, used as nasal absorption enhancers/solubilizers in the investigational formulations, appeared to be relatively non-toxic for the ciliated tissue, having similar effects on CBF as physiological saline. After dilution five times, most drug formulations studied showed a moderate ciliostatic effect, but marked differences could still be detected. The present study demonstrates that CBF measurements in vitro are a valuable tool in the design of safe nasal drug formulations.

Chapter 41 Quantification of Ciliary Beat Frequency and Metachrony by High-Speed Digital Video

The quantification of ciliary activity depends on the detection and analysis of the rhythmic variation in light intensity of a microscope image induced by ciliary movement. This approach requires the simplification and optimization of signal contrast and waveform and its success depends on the photo-detection method, the microscope optics, and the type of cell preparation used. For beat frequency measurements, a single detector system consisting of a photomultiplier, phototransistor, or fiberoptic device is sufficient. For the analysis of ciliary metachrony, spatial information is essential and a system requires a multipoint detector such as a digital video camera. This chapter highlights the advantages and limitations of earlier techniques for measuring ciliary activity and describes the principles and methodology of an advanced high-speed digital video technique. Large light signal amplitude can be obtained from coordinated groups of active cilia of isolated or cultured cells with phase-contrast optics. The signal waveform needs to be simple and easily interpretable; however, cilia of isolated cells or epithelial sheets generally beat in a metachronal manner and this can result in a complicated signal waveform because of the orientation of the ciliary beat cycle with respect to the detector and the number of cilia simultaneously contributing to the signal. An improvement in signal waveform can be achieved by monitoring only the effective stroke by recording close to the ciliary tips. The chapter discusses quantification of ciliary metachronism and video techniques for measuring ciliary activity. The high-speed digital video photodetection technique discussed has a high temporal and spatial resolution that can be used for the measurement of ciliary beat frequency and metachrony. The combination of this technique with a fluorescence imaging system that can simultaneously measure intracellular ions or messengers provides a unique opportunity for investigation of the physiological control of ciliary activity.

Chapter 11 Preparation of Explant and Organ Cultures and Single Cells from Airway Epithelium

In this chapter, the methods for preparing explant and organ cultures as well as the enzymatic isolation of cells are described. The inaccessibility of the ciliated epithelium of the mammalian respiratory tract has hampered in vivo studies of its structure and function, including those regarding the regulatory mechanisms of mucociliary transport. Consequently, several in vitro approaches have been developed to maintain and study airway tissues in culture. These include explant cultures, where pieces of tissue are seeded on a substrate to encourage the spreading and outgrowth of the epithelial cells; organ cultures, in which the tissue is maintained to preserve its original structural and functional form; monolayer cell cultures, where enzymatically dissociated cells are plated onto a noncellular substrate; and feeder layer cultures, where isolated epithelial cells are grown on a cellular substrate such as treated fibroblasts. Explant cultures have been utilized for the measurement of ciliary beat frequency, including the timings of the phases of the beat cycle, of single and multiple cells, as well as for the analysis of intercellular Ca 2+ signaling. Isolated epithelial cells have been used in patch–clamp studies and in assays to measure inositol trisphosphate levels. There is description of preparation of rat tail collagen, of collagen-coated coverslips, and tracheal epithelial explant culture—the materials to be used and the procedure. The technique is sterile. There is description of dissection of rabbit trachea, technique is aseptic. Details of preparation of organ culture and preparation of enzymatically isolated cells have been covered. In organ culture, the tracheal mucosa is supported at a liquid-air interface to mimic in vivo conditions of nutrient and gas exchange. Isolated cell preparations are obtained either from freshly dissected tissues or from tissues that had previously been placed in organ culture.

Metachronal activity of cultured mucociliary epithelium under normal and stimulated conditions.

In the present work we measured in real time the metachronism and degree of correlation between beating cilia from cultured mucociliary epithelium. The method is based on simultaneous measurement of ciliary beat frequency, phase shifts, and correlation factors in two directions: parallel and perpendicular to the effective stroke direction (ESD). From the phase shifts the lengths of wave components, and consequently the metachronal wavelength and direction, were evaluated. On active ciliary areas of cultured frog esophagus under normal conditions, a relatively high degree of correlation is observed, but cilia are more correlated in direction parallel to ESD which is also the direction of the mucus propulsion. The length of the wave component parallel to ESD is more than twice as large as that of the perpendicular component. The metachronal wavelength was found to be in the range of 5-9 microns, and the direction of the wave propagation was in the range of 90 degrees-125 degrees clockwise to the ESD. When ciliary beat frequency was rapidly increased by extracellular ATP or acetylcholine, only minor effects were observed on the degree of correlation between beating cilia. The length of the wave component parallel to ESD showed the most dramatic effect increasing up to tenfold. The perpendicular to ESD component was not affected by the stimulation. Consequently, the metachronism became more laeoplectic with the angle between the ESD and the wave directions decreasing by 10 degrees-30 degrees, and the metachronal wavelength remained unaltered.

Nasale mukoziliare Transportzeit und ziliare Schlagfrequenz bei Gesunden und Sinusitispatienten*

The mucociliary system is an important defence mechanism of the respiratory tract. It depends on the interaction between cilia and mucus. To evaluate the value of the saccharin test and a photoelectrical in-vitro method for measurement of ciliary beat frequency for clinical routine diagnosis, we studied twenty patients suffering from acute sinusitis (aged 19-64) and twenty healthy non-smokers (aged 19-50). The nasal mucociliary transport time of saccharin was significantly higher (p < or = 0.01) and the ciliary beat frequency was significantly lower (p < or = 0.001) in patients with acute sinusitis, compared to normals. There was no correlation between nasal mucociliary transport time and ciliary beat frequency in patients with acute sinusitis (r = 0.21) and in healthy non-smokers (r = -0.37). Nasal mucociliary transport time of saccharin and photoelectrical in-vitro method for measurement of ciliary beat frequency measure different aspects of the mucociliary system. They are complementary. Both methods can be carried out together in routine diagnosis.

Absorption enhancers in nasal drug delivery: efficacy and safety

Efficacy and safety of nasal absorption enhancers depend on many different parameters, such as their influence on nasal epithelial membrane barriers, the enzymatic activities in the nasal cavity, and the mucociliary clearance. Large interspecies differences appear to exist in the nasal absorption of drugs. Nasal absorption enhancers, although very effective in some cases, differ substantially in efficacy and safety. For a number of enhancers like surfactants, bile salts and fusidate derivatives a good relationship (similarity in rank order) can be established between the morphological damage and the influence on the ciliary beat frequency (CBF) of nasal epithelial tissue, making CBF measurements a valuable tool in the search for safe nasal absorption enhancers.

Computerized measurement of ciliary beat frequency.

We describe how ciliary beat frequency (CBF) of human respiratory epithelium in vitro can be robustly and quickly measured. The proven photometric technique has been improved by using a microcomputer to calculate and display the results. The software, which runs on an IBM-compatible PC, was developed using Borland Turbo Pascal and employs a fast Fourier transform routine to derive the CBF. Results are displayed almost as soon as a cell is identified under the microscope, and are in accordance with previous work. The technique can now be used diagnostically and for a variety of drug trials on respiratory epithelium.

Mucociliary function, ciliary ultrastructure, and ciliary orientation in Young's syndrome.

Mucociliary clearance is impaired in patients with Young's syndrome (obstructive azoospermia with recurrent sinobronchial disease), cystic fibrosis, and primary ciliary dyskinesia. No defect of cilia or mucus has been detected in Young's syndrome. Ciliary function and ultrastructure, including ciliary orientation, were studied quantitatively in 20 patients with Young's syndrome and 20 normal subjects to determine the incidences of ciliary defects. Nasal ciliated epithelium was obtained from each subject and used for measurement of ciliary beat frequency and ultrastructural analyses. Ciliary orientation was determined by measuring ciliary deviation in electron micrographs; ciliary deviation is a measure of the relative orientation of cilia in relation to each other in which high values indicate ciliary disorientation. Ciliary beat frequency and the incidence of microtubular defects and numbers of dynein arms did not differ between patients with Young's syndrome and control subjects. In patients with Young's syndrome basal ciliary deviation (16.0 degrees) was similar to that in control subjects (14.1 degrees), but at the ciliary tip ciliary deviation (21.9 degrees) was greater than in healthy subjects (14.5 degrees). The relative disorientation of the distal ciliary axoneme in patients with Young's syndrome compared with normal subjects may be due to a structural defect but is more likely to be a consequence of abnormal mucus.

Die pulmonalen Folgen der Langzeitbeatmung von very low birth weight Frühgeborenen*

Fifty six children with a birth weight of less than 1500 g and gestational age less than 34 weeks were studied at a mean age of 7.7 years. All had required mechanical ventilation, 36 suffered from hyaline membrane disease, 17 from atelectasis and three from pneumonia. Of these patients 37 developed broncho-pulmonary dysplasia (BPD). The study included skin prick test, measurement of ciliary beat frequency, chest X-ray, whole-body plethysmography and histamine challenge. Eight patients suffered from recurrent pulmonary infections up to the age of 4 years. Eleven children had a positive skin prick test. Three suffered from allergic asthma and six from hay-fever. The family history of atopy in these premature infants was no different from that of the general population. Ciliary beat frequency was normal in all cases (frequency: 11.8 +/- 1.8 Hz). In 34 patients X-rays showed minimal changes (peribronchial thickening n = 6, mild emphysema n = 28). Pulmonary function was normal except for an increase of thoracic gas volume which was correlated to grade of BPD according to Northway. There was a significant correlation between oxygen-score and radiological stage of BPD (r = 0.7). Histamine challenge showed hyperresponsiveness in 53% of the children. Eight patients showed a threshold similar of mild asthma (less than 1000 micrograms histamine per ml). The degree of airway hyperreactivity was correlated to the grade of BPD (r = 0.7; p less than 0.01) and the oxygen-score (r = -0.63; p less than 0.05). The oxygen-score may be considered a predictor of hyperresponsiveness and pulmonary outcome in later childhood.

Effect of artificial surfactant on ciliary beat frequency in guinea pig trachea

This study defines the effect of artificial surfactant on ciliary beat frequency. We employed guinea pig tracheal rings and a photomultiplier which allows in vitro measurement of ciliary beat frequency. The beat frequency without surfactant decreased continuously while surfactant caused a relative increase in beat frequency. The difference between the relative beat frequency in the presence and absence of surfuctant was significant. The effect of surfuctant was dose dependent. Fifteen minute treatments with 2 mM hydrogen peroxide reduced the beat frequency. The reduction partially recovered in the absence of surfuctant. In contrast, surfuctant markedly accelerated the recovery of the beat frequency. Surfuctant did not influence the effect of terbutaline (a β 2-adrenergic agonist) on beat frequency. These results indicate that artificial surfactant can, in some circumstances, promote ciliary beat frequency. This concept is important to the clearance mechanism of the airways, and its application to some pathophysiological states such as chronic bronchitis and bronchial asthma should be considered.

Dynamic image analysis applied to the study of ciliary beat on cultured ciliated epithelial cells from rabbit trachea

We have developed an automated image analysis method to study the ciliary beat frequency of ciliated cells of the primary culture from rabbit trachea. The ciliated outgrowth image is digitized and the variation in optical density is automatically calculated for each selected area of interest. 32 measurements of ciliary beat frequency are, in this way, calculated simultaneously in 6 min. With this reliable device, some studies on baseline frequency of control culture have been carried out. There was no variation in the mean frequencies of ciliated cells of the primary culture of different tracheas in our culture conditions. Moreover, the values of ciliary beat frequency at the starting point of the outgrowth were similar to those at the periphery of the outgrowth. There is nevertheless a slow decrease in frequencies versus the duration of culture. We have also established that the frequency of ciliary beat of some cells fluctuates in a periodic pattern whereas the majority of the ciliated population beat in a stable way. The image analysis process allows us to perform a cartography of frequencies on the video display. It also allows us to have access to the frequency of one cilium. Our method therefore seems to be reliable and furthermore simple in the evaluation of the potential effect of inhaled toxic compounds on ciliated cells of mammalian respiratory tract.

Stimulation of tracheal ciliary beat frequency by localized tissue incision

Since measurements of basal ciliary beat frequency (CBF) were significantly lower in our intact canine experiments than reports of ciliary activity in rabbits involving surgical intervention, we hypothesized that local tissue trauma stimulates CBF. The effects of minor neck surgery on tracheal CBF in eight barbiturate-anesthetized eucapnically ventilated beagles were investigated. Each dog underwent two studies. Measurements of CBF were made at 1-min intervals on the right lateral midtracheal surface by means of heterodyne mode correlation analysis laser light scattering. In the control study, CBF was measured in each dog for at least 160 min. In the incision study, base-line CBF was measured for at least 40 min. The overlying sternohyoidus muscles were then separated, and a longitudinal 2- to 3-cm incision was made in the trachea caudally from the fourth to the fifth cartilage ring. CBF was measured at least 5 cm distally from the site of tracheal injury for an additional 120 min. Electrocardiogram, rectal temperature, tracheal pressure, exhaled CO2, and arterial blood pressure, PO2, PCO2, and pH remained stable throughout both studies. The mean base-line CBF was 4.7 +/- 0.4 Hz. It increased to 19.5 +/- 2.9 Hz (P less than 0.0001) 100 min after the incision and remained elevated until the end of the study period (P less than 0.0001). The mechanism(s) causing this stimulation may also be responsible for the high "basal" CBF observed in other studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of local anaesthetics (lidocaine) on the structure and function of ciliated respiratory epithelial cells

Sampling for nasal or bronchial ciliated cells requires the use of anaesthetic agents, but such drugs may interfere with the morphological or functional results. Lidocaine is the most frequently used local anaesthetic. In order to study the morphological and functional effects of lidocaine hydrochloride, we designed an experimental study on ciliated cells from guinea pig and bovine trachea. On guinea pig tracheal specimens, different lidocaine concentrations (0.05, 0.25 and 1%) were tested. Tracheal rings were immersed in either culture medium alone (control) or in different lidocaine concentrations. Measurements of ciliary beat frequency (CBF) were performed by the stroboscopic method. Tracheal rings were consecutively incubated in culture medium alone and a second set of measurements was performed. Tracheal rings were studied by light microscopy after incubation in either 1% lidocaine or in culture medium alone. On bovine tracheal specimens, a coton wool swab impregnated with different lidocaine concentrations (0, 0.25, 1, 2.5 and 5%) was placed in contact with the tracheal mucosa. Three different kinds of samples were collected: the first one was used to study CBF, the second one (0.1 and 5%) was studied by scanning electron microscope (SEM) and the third (0.1 and 5%) by transmission electron microscopy (TEM). The results on guinea pig specimens show a significant but reversible CBF diminution for concentrations of 0.25 and 1% lidocaine and cellular lesions for the concentration of 1%. On bovine specimens a diminution in CBF for concentrations of 2.5 and 5% lidocaine was shown and the SEM study demonstrated obvious lesions on the epithelial surface treated with the 5% concentration. The TEM study showed morphological alterations on respiratory epithelium (deciliated areas, cytoplasmic vacuoles and mitochondrial swelling) for 5% lidocaine concentration. However the axonemal structure of cilia was normal for control and 5% concentration. We concluded that in vitro lidocaine can inhibit the CBF and that high concentrations of lidocaine can damage the respiratory epithelium but without modifications of the axonemal ultrastructure.

Effects of local anaesthetics (lidocaine) on the structure and function of ciliated respiratory epithelial cells

Sampling for nasal or bronchial ciliated cells requires the use of anaesthetic agents, but such drugs may interfere with the morphological or functional results. Lidocaine is the most frequently used local anaesthetic. In order to study the morphological and functional effects of lidocaine hydrochloride, we designed an experimental study on ciliated cells from guinea pig and bovine trachea. On guinea pig tracheal specimens, different lidocaine concentrations (0.05, 0.25 and 1%) were tested. Tracheal rings were immersed in either culture medium alone (control) or in different lidocaine concentrations. Measurements of ciliary beat frequency (CBF) were performed by the stroboscopic method. Tracheal rings were consecutively incubated in culture medium alone and a second set of measurements was performed. Tracheal rings were studied by light microscopy after incubation in either 1% lidocaine or in culture medium alone. On bovine tracheal specimens, a cotton wool swab impregnated with different lidocaine concentrations (0, 0.25, 1, 2.5 and 5%) was placed in contact with the tracheal mucosa. Three different kinds of samples were collected: the first one was used to study CBF, the second one (0.1 and 5%) was studied by scanning electron microscope (SEM) and the third (0.1 and 5%) by transmission electron microscopy (TEM). The results on guinea pig specimens show a significant but reversible CBF diminution for concentrations of 0.25 and 1% lidocaine and cellular lesions for the concentration of 1%.(ABSTRACT TRUNCATED AT 250 WORDS)