A new deformation model of hard alpha-keratin fibers at the nanometer scale: implications for hard alpha-keratin intermediate filament mechanical properties.

The mechanical behavior of human hair fibers is determined by the interactions between keratin proteins structured into microfibrils (hard alpha-keratin intermediate filaments), a protein sulfur-rich matrix (intermediate filaments associated proteins), and water molecules. The structure of the microfibril-matrix assembly has already been fully characterized using electron microscopy and small-angle x-ray scattering on unstressed fibers. However, these results give only a static image of this assembly. To observe and characterize the deformation of the microfibrils and of the matrix, we have carried out time-resolved small-angle x-ray microdiffraction experiments on human hair fibers stretched at 45% relative humidity and in water. Three structural parameters were monitored and quantified: the 6.7-nm meridian arc, which is related to an axial separation between groups of molecules along the microfibrils, the microfibril's radius, and the packing distance between microfibrils. Using a surface lattice model of the microfibril, we have described its deformation as a combination of a sliding process and a molecular stretching process. The radial contraction of the matrix is also emphasized, reinforcing the hydrophilic gel nature hypothesis.

Prolonged active glottic closure after barbiturate-induced respiratory arrest in lambs.

We recently showed that the glottis is actively closed throughout post-hyperventilation, hypocapnic central apnea in lambs. The present study was designed to test whether the glottis is also closed in non-hypocapnic central apnea. Twenty-seven lambs aged 2 to 30 days were intravenously injected with 325 mg of sodium pentobarbital, so as to obtain breathing arrest. Airflow was recorded via a facial mask and pneumotachograph, along with the electromyographic activity (EMG) of the thyroarytenoid muscle (TA, a glottic adductor). With the onset of apnea, continuous TA EMG appeared in a few seconds and rose rapidly. Brief inspiratory gasps were observed in eight lambs, and TA EMG was abruptly inhibited for the exact duration of the gasps. The continuous TA EMG then disappeared after 115 to 230 sec. We conclude that the glottis is actively closed during fatal non-hypocapnic central apnea in lambs. Our data suggest that active glottic closure occurs with major depression of central inspiratory drive.

Active expiratory glottic closure during permeability pulmonary edema in nonsedated lambs.

It has long been claimed that the active expiratory glottic closure observed in newborns, especially during hyaline membrane disease, is related to hypoxia. However, we recently showed that hypoxia does not lead to active expiratory glottic closure in nonsedated lambs. In this study, we test the hypothesis that glottic closure is related to an excess of lung water present at birth. We studied 17 nonsedated lambs after inducing a permeability pulmonary edema via intravenous of either oleic acid (8 lambs) or halothane (9 lambs). We recorded airflow via a facial mask and pneumotachograph, as well as the electromyographic activity (EMG) of the thyroarytenoid muscle (TA), a glottic adductor. Blood gases were measured in 8 lambs via a brachial artery catheter. We identified laryngeal expiratory airflow braking on the breath-by-breath computed flow-volume loop and TA expiratory EMG as evidence of active expiratory glottic adduction. After the injection of oleic acid or halothane, an active expiratory glottic closure was recorded in all lambs but 1, usually throughout the recording period (60 to 300 min). The active expiratory glottic closure was not inhibited after correction of the hypoxia. We conclude that, in nonsedated lambs, a permeability pulmonary edema induces an active expiratory glottic closure. We hypothesize that the expiratory glottic closure commonly observed in newborns could help to ameliorate the alveolocapillary gas exchange by reopening the flooded alveoli.

Laryngeal response to hypoxia in awake lambs during the first postnatal days.

We recently showed that hypoxia does not induce active expiratory glottic adduction in awake lambs more than 10 d old. To reconcile our results with previous data from other researchers, we hypothesized that an active expiratory glottic closure might still be part of the response to hypoxia in the very first postnatal days. The present study was undertaken to test this hypothesis. We studied 22 awake, nonsedated lambs during hypocapnic hypoxia (fraction of inspired O2 = 0.08 during 15 min) induced during the first 72 h of postnatal life. We recorded airflow via a facial mask and pneumotachograph, along with the electromyographic activity (EMG) of the thyroarytenoid muscle (a glottic adductor) in 10 lambs. We also recorded the EMG of both the posterior cricoarytenoid (n = 4) and cricothyroid (n = 5) muscles (glottic abductors), as well as the abdominal muscles (n = 4). We identified typical expiratory airflow braking on the breath-by-breath computed flow-volume loop and thyroarytenoid muscle expiratory EMG as evidence of active expiratory glottic adduction. We found that hypoxia induced a biphasic ventilatory response, with an early peak and a subsequent decrease, and that active expiratory glottic adduction was absent during baseline room-air breathing and hypoxia. We also found that the glottic abductor phasic inspiratory and tonic expiratory EMG as well as the abdominal muscle phasic expiratory EMG, all of which were present during baseline recording, increased during hypoxia. We conclude that hypoxia does not induce expiratory glottic closure in the very first days of life in awake lambs.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroarytenoid muscle activity during hypoxia in awake lambs.

It is generally accepted that hypoxia in early life results in active laryngeal braking of expiratory airflow via the recruitment of glottic adductor muscles. We examined the electromyogram expiratory activity of the thyroarytenoid muscle in seven 11- to 18-day-old awake nonsedated lambs exposed to an inspired O2 fraction of 0.08 for 18 min. The lambs breathed through a face mask and a pneumotachograph. During baseline prehypoxic breathing, the thyroarytenoid muscle was largely inactive in each awake lamb. Unexpectedly, no recruitment of the thyroarytenoid muscle was recorded during hypoxia in any of the seven lambs; simultaneous examination of the flow-volume curves revealed an absence of expiratory airflow braking. Also unexpectedly, marked expiratory activity of the thyroarytenoid muscle was recorded, with each expiration occurring within less than 10 s after the return to room air. The resulting delay of expiration was apparent in the flow-volume loops. Thus, in awake 11- to 18-day-old lambs, 1) active expiratory glottic adduction is absent during hypoxia and 2) a return from hypoxia to room air results in prolonged expiration as well as active glottic adduction that controls end-expiratory lung volume.

Measurement of pulmonary mechanics in the newborn lamb: a comparison of three techniques.

Although recent interest in neonatal respiratory mechanics has led to the development of a plethora of techniques for measuring lung compliance and resistance, a critical appraisal of the limitations of these techniques in the newborn has not been performed to date. We evaluated three techniques of measuring respiratory mechanics in the newborn lamb, with the reference method (method 1) being the Mead-Whittenberger technique using flow, volume, and esophageal pressure (Pes) by water-filled catheter, and the other two methods entailing the measurement of mouth pressure (Pm) during airway occlusion (method 2 using end-expiratory occlusion; method 3 using end-inspiratory occlusion). Each technique was evaluated during eupnea and tachypnea in intubated and nonintubated newborn lambs. We found that the use of Pes for the measurement of resistance and compliance gave the most reliable results during both eupnea and tachypnea in both the intubated and nonintubated subjects. The airway occlusion techniques that use Pm to derive resistance and compliance (methods 2 and 3) gave more variable results under all conditions of testing. Method 2 was the least precise method of measurement with a variability of greater than 30% compared with a variation of less than 20% for method 1. For all three methods, it was found that the number of breaths needed for reproducible measurements of mechanics was four to six during eupnea and seven to nine during tachypnea.

Asbestos exposure and retention as determinants of airway disease and asbestos alveolitis.

To evaluate the relationships of asbestos exposure, retention, airway response, and the asbestos alveolitis, we exposed 2 groups of sheep every 2 wk for 3 yr to either 100 ml phosphate-buffered saline (PBS) or 100 mg UICC chrysotile fibers in 100 ml PBS. The sheep were evaluated periodically by pulmonary function tests (PFT), chest radiograph (CR), bronchoalveolar lavage (BAL), and transbronchial lung biopsy (TLB). At Month 24 of the study, all asbestos-exposed sheep had significant increases in lung resistance and upstream resistance. However, only 9 of the 16 asbestos-exposed sheep had significant changes in TLB, CR, Cst, and VC, which clearly separated them from the other 6 sheep in these parameters. The 2 groups, however, had similar air-flow limitation. At lung biopsy, all asbestos-exposed sheep had significant peribronchiolar fibrosis, with significant alveolitis only in the group of 9 sheep with radiographic and functional changes of early asbestosis. The 9 sheep also had significant changes in BAL cellularity and biochemical profile, which differentiated them from the other 6 asbestos-exposed sheep. Analysis of BAL fiber content at that point revealed that despite identical exposure, the group with interstitial lung disease had significantly more fiber retention (p less than 0.01). The data demonstrate that whereas asbestos airway disease appears to be primarily an exposure-dose-related response, the lung response appears to be more closely related to alveolar retention of the dust.

Exponential and diphasic ventilatory response to hypoxia in conscious lambs.

This study was undertaken to test the hypothesis that in the neonate the hypoxic chemoreflex drive adapts to steady-state hypoxia but not to progressive hypoxia. First we have compared the ventilatory (VE) response of 2-day-old conscious lambs to steady-state hypoxia with their response to progressive hypoxia. Second, we have quantified the chemoreceptor excitatory function operating at the end of each period of hypoxia by studying the immediate VE response to the withdrawal of the hypoxic stimulus. Lambs responded to steady-state hypoxia [fractional concentration of inspired O2 (FIO2) = 0.08] by a diphasic VE response but responded to progressive hypoxia (FIO2 0.21-0.08) by an exponential VE increase. Hyperventilation in steady-state hypoxia was transient; VE increased immediately from 532 to a mean peak response of 712 ml X kg-1 X min-1 and decreased to 595 ml X kg-1. min-1 within 10 min. With progressive hypoxia, VE increased within 13 min from 514 to 705 ml X kg-1 X min-1. At the end of steady-state and progressive hypoxia the abrupt withdrawal of the hypoxic drive caused an instantaneous VE decrease to 390 and 399 ml X kg-1 X min-1, respectively; the VE decrease was respectively 306 and 205 ml X kg-1 X min-1 (P less than 0.05). This demonstrates that during steady-state hypoxia the lambs had suffered a loss of one third of the chemoreceptor excitatory function.(ABSTRACT TRUNCATED AT 250 WORDS)

Gallium-67 uptake in the lung of asbestos exposed sheep: early association with enhanced macrophage-derived fibronectin accumulation.

To evaluate the time course and mechanisms of enhanced 67Ga lung uptake in asbestosis, we exposed two groups of sheep every 2 wk to either 100 ml saline (controls) or 100 mg UICC chrysotile fibers in 100 ml saline. The sheep were evaluated periodically by pulmonary function tests (PFT), thoracic radiograph (TR), 67Ga lung scan bronchoalveolar lavage (BAL), and transbronchial lung biopsy (TLB). By month 24 of the study, 9/15 exposed sheep had developed the initial alveolitis and had significant changes in PFT, TR, and TLB. The other six exposed sheep differed from controls only by a 75% increase in BAL fibronectin until month 30, where significant changes in albumin occurred and 67Ga scan score increased. The nine sheep with alveolitis had significant sustained increases in 67Ga scan and BAL levels from month 6, associated with a 150% increase in BAL fibronectin and other parameters of disease activity changed from month 18 to 30. We concluded that in the sheep model of asbestosis, significant changes in 67Ga scan, 67Ga BAL counts, and excessive elevation of BAL fibronectin preceded other parameters of disease activity. The data suggest that excessively activated macrophages are primarily responsible for the early 67Ga lung uptake.

Postnatal maturation of respiration in intact and carotid body-chemodenervated lambs.

The contribution of the carotid body chemoreceptor to postnatal maturation of breathing was evaluated in lambs from 7 to 70 days of age. The study was conducted by comparing the eupneic ventilation and resting pneumograms in intact conscious lambs with those of lambs that were carotid body chemodenervated (CBD) at birth. In comparison to the 1-wk-old intact lambs, the CBD lambs had significant decreases in minute ventilation (VE, 313 vs. 517 ml/kg), tidal volume (VT, 7.2 vs. 10.5 ml/kg), respiratory rate (f, 44 vs. 51 breaths/min), and occlusion pressure (P0.1, 2.8 vs. 7.2 cmH2O). Arterial PO2's were 59 vs. 75 Torr (P less than 0.05) and arterial PCO2's 47 vs. 36 Torr (P less than 0.05), respectively, in CBD and intact lambs. In intact lambs from 7 to 70 days, resting VE decreased progressively from 517 to 274 ml/kg (P less than 0.01) due to a fall in VT, mean inspiratory flow (VT/TI), and f, whereas the ratio of inspiratory time to total breath duration remained constant. P0.1 decreased from 7.2 to 3.9 cmH2O from 7 to 42 days. In contrast the CBD lambs experienced only minimal changes in VE, VT, VT/TI, and f during the same period. VE only decreased from 313 to 218 and P0.1 from 2.8 to 2.4 cmH2O. In contrast to that of intact lambs the resting pneumogram of CBD lambs remained relatively fixed from 7 to 70 days. Three CBD lambs died unexpectedly, without apparent cause, in the 4th and 5th wk of life.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective exposure and analysis of the sheep tracheal lobe as a model for toxicological studies of respirable particles.

A conscious sheep model, recently developed to study sequentially the bronchoalveolar milieu, was further refined to use in the rapid in vivo assessment of the biological effects of respirable particles. In this model, the anatomically isolated tracheal lobe was selectively exposed to either 100 ml phosphate buffered saline (PBS) (control group of 12 sheep), 100 mg of 0.1-micron latex beads in 100 ml PBS (latex group of 12 sheep), or 100 mg of UICC Canadian chrysotile fibers in 100 ml PBS (asbestos group of 12 sheep). Bronchoalveolar lavages (BAL) of the tracheal lobe were obtained prior to exposure and at Days 1, 8, 15, 21, 29, 45, and 60 after exposure. Whole-lung detailed pulmonary function tests (PFT) were performed at the same times and the histopathology of the lobe was examined in six sheep in each group at Days 29 and 60. In the control sheep, there were no significant changes over time in any of the measures, and lung morphology remained normal. In the latex group, there was no significant change in PFT, the BAL analyses documented early transient increase in cellularity (macrophages and neutrophils at Day 1) and only macrophages after; lung histology documented an early macrophagic alveolitis which decreased to less than 10% of the initial inflammatory reaction at Day 60, without other distortion of the lung and airway architecture. In the asbestos sheep, the only change in whole-lung PFT was a 10-torr fall in arterial O2 pressure. BAL analyses documented persistent increases in macrophages, neutrophils, and lactate dehydrogenase as well as increasing gamma-globulins. Lung histology revealed a macrophagic and neutrophilic peribronchiolar alveolitis at Day 30, which regressed substantially by Day 60, but persistent peribronchiolar alveolitis, early fibrosis, and severe distortion of the small airways, lesions comparable to those of early asbestosis in sheep or humans. Thus selective exposure and sequential analyses of the sheep tracheal lobe in terms of BAL histomorphology should be valuable for rapid in vivo assessment of toxicity of respirable particles.

The ventilatory response to hypoxia in the newborn lamb after carotid body denervation.

Neonates of various species including lambs respond to hypoxia by a transient hyperventilation followed by a VE depression (diphasic response). To better delineate the role of the carotid chemoreceptors and that of the central depressive/inhibitive effect of hypoxia on minute ventilation, we have studied the VE response of 4-day-old carotid body-deprived lambs (CBD) during successive exposure to moderate and severe (0.12 and 0.07 FIO2) hypoxia. The carotid body denervation was done to abolish most of the chemoreceptor stimulating effect on VE during hypoxia and to allow for central depression/inhibition of VE during hypoxia. In the CBD lambs, baseline VE was 461 +/- 81 (SE) ml X (kg X min)-1. It increased to 532 +/- 79 ml X (kg X min)-1 and to 541 +/- 75 ml X (kg X min)-1, to 0.12 FIO2 and 0.07 FIO2. These VE increases did not reach level of significance (P greater than 0.05). After 2-5 min of both levels of hypoxia VE dropped respectively to 460 +/- 60 ml X (kg X min)-1 and to 459 +/- 38 ml X (kg X min)-1. No marked ventilatory depressions were noted but VE had only returned to baseline. It is concluded that, in the denervated newborn lamb, the centrally mediated depressive effect of hypoxia is small and not sufficient to explain the diphasic VE response of the intact lamb to steady state hypoxia. Analysis of the magnitude of the hyperventilation and the VE damping pre-hypoxic levels occurring with sustained hypoxia in newborns of various species suggests that the immaturity of the O2-sensitive chemoreceptor rather than the central effect of hypoxia is the determinant factor of the diphasic response of newborn mammals to hypoxic hypoxia.