Nuno R. Grande

Inflammatory response of the lung to tungsten particles: An experimental study in mice submitted to intratracheal instillation of a calcium tungstate powder

Tungsten has been implicated as a cause of a severe form of pneumoconiosis in humans, the so-called “hard metal” lung disease. We have investigated the effect of intratracheal instillation of a powder of calcium tungstate on the pulmonary tissue of CD-1 mice. The tungsten-induced alterations were studied using 3 microanatomical methods: cytologic study of exudates obtained by bronchoalveolar lavage (BAL); histologic examination of paraffin-embedded sections of the lung; and scanning electron microscopic (SEM) examination of lung samples using x-ray microanalysis to detect tungsten in situ. The animals were sacrificed 1, 3, 7, 14, and 21 days after a single intratracheal instillation of 250 µg calcium tungstate particles suspended in 100 µl of saline. We found that the metal particles induced a marked inflammatory response in the bronchoalveolar space characterized by a biphasic attraction of leukocytes with cellular peaks observed at day 1 and 14. More than 50% of the BAL macrophages showed ingested tungsten. In the lung parenchyma, the inflammatory infiltrates were predominantly located at the periphery of the bronchiolar walls. From 7 days on after the tungsten deposition, large inflammatory exudates were seen invading focal areas of the alveolar domain of the lung. SEM views revealed that the tungsten particles could be inside alveolar macrophages, in cells making up the alveolar wall, or inside periacinar lymphatics. Our data document that tungsten particles cause a marked inflammatory response in the lung tissue and that the leukocyte exudates may invade alveolar areas of the lung. This strong inflammatory response may correspond to the early stages of the tungsten-induced “hard-metal” lung disease previously reported in humans.

In Utero and Postnatal Exposure of Wistar Rats to Low Frequency/High Intensity Noise Depletes the Tracheal Epithelium of Ciliated Cells

Chronic exposure of men or rodents to low frequency/high intensity (LFHI) noise causes a number of systemic changes that make up the so-called vibroacoustic disease (VAD), a disorder that includes alterations of the respiratory system, namely, of its epithelial layer. We have investigated here the susceptibility of the tracheal epithelium of Wistar rats to in utero and postnatal exposure to LFHI noise by comparing its ultrastructure with that of the tracheal epithelium of control rats and of animals exposed to LFHI noise only after reaching adulthood (8 weeks of age). Scanning electron microscopy (SEM) of the inner surface of rat trachea was used to determine the relative areas covered by ciliated and non-ciliated cells. In rats that were exposed in utero and postnatally to LFHI noise, we observed that out of 100 microm(2) of tracheal epithelium only 31 +/- 14 microm(2) were covered by cilia, whereas in control rats; ciliated cells occupied an average of 60 +/- 18 microm(2) out of 100 microm(2) of the epithelium; this difference between the two groups was statistically significant (p <0.05). In rats that were exposed to LFHI noise only after reaching adulthood, cilia covered 55 +/- 22 microm(2) out of 100 microm(2) of the luminal surface of the trachea, a value that, although lower than that of controls, was not found to be statistically different. We conclude that (1) the tracheal ciliated cells are damaged by exposure of rats to LFHI noise if the animals are kept under this environmental aggression during in utero and postnatal periods; (2) tracheal ciliated cells from adult rats are more resistant to the deleterious effects of LFHI noise than pleura or lung alveolar cells that were shown before to undergo marked changes upon chronic exposure of rats to LFHI noise. These findings suggest a note of caution regarding pregnant women and young children: they should be prevented from areas where LFHI noise occurs, namely, in aircraft and textile industries where this type of environmental hazard is often present.

Chronic exposure of rats to cotton-mill-room noise changes the cell composition of the tracheal epithelium.

The work environment of cotton mill rooms of modern textile plants is characterized by noise pollution. We have taped and reproduced this noisy environment to study its effects on experimentally exposed rats. Because we have previously documented that chronic noise causes alterations in the respiratory epithelium, we have focused our investigation on the morphology of the tracheal lining. Wistar rats were exposed to the textile-type noise from 1 up to 7 months, with an average 40 hours weekly exposure of the animals. The rats were sacrificed monthly and the tracheas were studied by scanning electron microscopy (SEM) to quantify the areas of the airway lining that were covered by ciliated, serous or other cells of the epithelium. We found that noise exposure of the rats caused a significant loss of tracheal ciliated cells; an increased density of serous cells on the epithelium balanced this change. This modification of the rat trachea was already established after 1 month of noise treatment of the animals; it did not change significantly throughout the 7-month course of the herein investigation. Loss of ciliated cells was more intense in areas of the tracheal epithelium located between the regions of cartilage rings. We conclude that the ciliated cell is an elective target for damage caused on the respiratory epithelium by the workplace noise occurring in cotton mill rooms. This modification of the respiratory epithelium is likely to impair clearance of the airways since this function depends on the activity of ciliated cells.

Tracheal transplantation: cytological changes studied by scanning and transmission electron microscopy in the rabbit.

Objectives Our goal was to offer a comprehensive cytological study of the changes in the trachea after experimental transplantation of the organ.

Zonation of ciliated cells on the epithelium of the rat trachea.

We have used scanning electron microscopy (SEM) to screen the entire epithelial surface of the cervical trachea of the adult rat. This scrutiny revealed that the density of ciliated cells along this epithelium follows a repetitive pattern: circular strips of high density of ciliated cells alternate with areas of low density of the same cells. Cilia-poor strips of the tracheal epithelium were seen on areas of cartilage rings; here, ciliated cells made up 32% of the total surface of the tracheal lining. Cilia-rich areas filled the epithelial surface at the tracheal ligaments (i.e., the regions located in-between the rings); here, ciliated cells occupied 65% of the tracheal lumen. In the cilia-poor zones, the density of ciliated cells decreased from its periphery into its center, where cilia were virtually absent. No differences in this pattern of the tracheal epithelium were seen between young adult and older rats. We conclude that the respiratory epithelium expresses density zonation of ciliated cells on the trachea of adult rats. We propose that the high concentration of ciliated cells on the regions of epithelium located at the tracheal ligaments suggests that these zones are electively committed in the clearance of the respiratory airway.

Cellular Kinetics of Inflammation in the Pleural Space of Mice in Response to the Injection of Exogenous Particles

CD-1 mice were used to study the cellular kinetics of the inflammatory response of the pleural space to the injection of 250 micrograms of silica or of tungsten microparticles. The pleural exudates were collected by lavage of the serous cavity of mice that were sacrificed at 30 min and up to 7 days after the intrapleural instillation of the particles. The samples were studied by light and electron microscopy (transmission and scanning modes); the quantitative cellular kinetics of the inflammation was determined by leukocyte counting in exudates using cytocentrifuge preparations. The normal resident population of cells of CD-1 mice was made up of (2.47 +/- 0.37) x 10(6) cells. It consisted mostly of macrophage-like cells ((2.03 +/- 0.26) x 10(6) cells, 82% of total cells), some lymphocytes ((0.37 +/- 0.07) x 10(6) cells, 15% of total cells), a few mast cells and eosinophilic granulocytes (1-2% of total cells). The initial inflammatory reaction (30-60 min after injection) was characterized by a decrease in the number of cells harvested from the pleural space. This was followed by an intense recruitment of granulocytes and monocytes that resulted in a peak of intrapleural cells at 24 h ((16.8 +/- 4.0) x 10(6) cells induced by silica particles and (18.3 +/- 4.2) x 10(6) cells induced by tungsten particles). In tungsten-injected mice (but not in silica-treated animals) the enhancement in the number of intrapleural macrophages continued up to 72 h after particle injection. The highest percentage of macrophages with ingested tungsten (50% of total macrophages) was found early (6 h) and decreased thereafter; at day 7 it encompassed just 17% of the macrophages. Injection of any of the two particulates led to the disappearance of mast cells from the pleural space of mice. Silica particles attracted a high number of eosinophils to the pleural cavity of mice. Light and electron microscopy documented that pleural macrophages underwent striking morphological changes during the inflammatory response: the phagocytes showed marked increase in size and in number of surface processes, and their cytoplasm often contained large amounts of the injected particles and also of cellular debris. This study establishes the mouse as a reliable animal model to study the dynamics of the pleural space and it offers a precise definition of the cellular kinetics of inflammation in this serous cavity. The data indicate that the kinetics of experimental pleural inflammation induced by particulates may depend on the nature of the injected particles.

REDUCTION OF RAT PLEURAL MICROVILLI CAUSED BY NOISE POLLUTION

Scanning electron microscopy (SEM) was used to investigate whether chronic exposure to noise modifies pleural morphology. Rats were submitted to 8-h/day schedule of noise that is similar to the working hours at cotton-mill rooms. Morphometry of the area occupied by microvilli on the pleural surface showed a decrease in microvilli after 3 months of rat exposure to noise. The reduction of microvilli was 10% after 3 months of noise exposure (reaching 20% after 7 months of noise treatment) and is consistent with pleural effusions found in some of the patients working in noise-polluted environments.

Morphological evidence for migration of particle-laden macrophages through the interalveolar pores of Kohn in the murine lung

We have investigated the topography of particle-laden macrophages in the pulmonary tissue of CD-1 mice after intratracheal instillation of a suspension of 250 micrograms of calcium tungstate. The mice were sacrificed 1, 3, 7 and 14 days after the particle deposition. Lung fragments were studied by scanning electron microscopy (SEM) coupled with X-ray microanalysis that allowed in situ elemental identification of tungsten in the lungs. Tungsten-positive macrophages were distinctly located in the lungs of mice sacrificed at 1-3 days when compared with samples from mice killed 7-14 days after the calcium tungstate instillation. At 1-3 days, the tungsten-carrying macrophages were accumulated near the terminal bronchioles whereas they were seen predominantly in the alveolar ducts and sacs in the 7- to 14-day groups of mice. This suggests that during pulmonary inflammation there is a redistribution of the particle-containing macrophages throughout the deep lung tissue. In high-magnification SEM views, we observed that the tungsten-positive macrophages presented numerous surface microvilli. Tungsten-laden phagocytes were detected in interalveolar fenestrae, at the so-called Kohn pores. This finding documents that the Kohn pores may be used by inflammatory cells as a pathway for the migration of phagocytes in between adjacent alveolar sacs.

Evidence of Drainage of Tungsten Particles Introduced in the Pleural Space through the Visceral Pleura into the Lung Parenchyma

Studies of pleural clearance of calcium tungstate particles were made in the dog. By using scanning electron microscopy and elemental microanalysis, we show that mesothelial cells of the visceral leaflet of the pleura are also involved in the clearance of particles present in the pleural space. The histological study of lung parenchyma shows many macrophages loaded with tungsten particles, and we conclude that this way may be an important pathway for the transmission of pathologic processes from the pleural space to the lung.

Limb immobilization and intimal hyperplasia -an echo-Doppler study in man

The authors studied the circulatory alterations observed in the upper limbs of patients showing muscular atrophy due to flaccid paralysis of traumatic origin. Ten patients who had suffered avulsion of the nerve roots from C4 to D1 that occurred 9 to 216 months previously, presented a significant degree of muscular atrophy of the affected upper limb, despite physiotherapy. We performed echo-doppler examinations of the patients to measure the lumen of the subclavian, axillary, brachial, radial and ulnar arteries and also of the veins of both upper limbs. The measurements from both upper limbs in each patient were statistically compared. The data revealed a significant reduction of the width of the lumen of the arteries and veins and a reduced arterial blood flow in the affected limb in comparison with the normal one. The greater echogenicity and the abnormal Doppler waves of the affected vessels suggest that they presented an increased thickness and a hardened wall. The authors propose that this finding may be related to an intimal hyperplasia brought about by muscular atrophy or by the observed blood flow reduction.