Skin nitric oxide and its metabolites are increased in nonburned skin after thermal injuries.

Local and systemic inflammation can lead to progression of burn wounds, converting second- to third-degree wounds or extending the burn to adjacent areas. Previous studies have suggested that the skin is an important site of production of nitric oxide (NO), synthesized by inducible nitric oxide synthase (iNOS) activation after injury. NO increases in burned wounds, but its formation in noninjured skin has not been investigated. We hypothesized that after severe burns, NO and cytotoxic peroxynitrite would increase in noninjured skin. We also tested the hypothesis that BBS-2, a specific inhibitor of iNOS, would impair NO formation after burn. Thirteen female sheep were randomized into burn injury and smoke inhalation (n = 5, group 1), burn and smoke treated with BBS-2 (n = 3, group 2), and sham (saline treatment, no injury) (n = 5, group 3). All the animals, including the sham-injury group, were mechanically ventilated for 48 h. Samples of nonburned skin and plasma were collected from each animal, and levels of NO and its metabolites were evaluated using a NO chemiluminescent detector. Nitrotyrosine and iNOS expression were determined in the skin by Immunoperoxidase staining, and scoring of masked slides (epidermis, hair follicles, vessels, glands, and stroma) was performed. Skin NO and metabolites significantly increased in the burn and smoke injury group, and this was inhibited by BBS-2. Nitrotyrosine expression also increased significantly in the skin of burned animals. BBS-2 prevented the increase of NOx but not the increase of nitrotyrosine expression in skin. Plasma levels of NO increased in burned animals when compared with sham, but this increase was not significant. The increase of NO and its metabolites after burn in noninjured skin is followed by a significant increase in peroxynitrite, a potent cytotoxic mediator.

Enhanced pulmonary expression of endothelin-1 in an ovine model of smoke inhalation injury.

Recent studies suggest a role of endothelin-1 (ET-1) in mediating airway inflammation and lung injury. The aim of this study was to assess the immunohistochemical expression of ET-1 in the lung following smoke inhalation injury. ET-1 immunoreactivity was assessed in normal sheep (N = 4) and in sheep at 1 (N = 2), 6 (N = 3), 12 (N = 3), and 24 (N = 3) hours after inhalation injury. In normal animals, ET-1 expression was limited to the basal cell layer of the tracheal epithelium, main bronchi, and associated mucous glands. One hour after injury, ET-1 immunoreactivity was enhanced in upper airway epithelium and mucus glands with new expression in bronchioles. Airway smooth muscle, vascular tissue, and alveolar duct smooth muscle cells expressed moderate levels of ET-1 at 12 and 24 hours. ET-1 immunoreactivity was absent in areas of parenchymal edema and inflammation. The pattern of ET-1 expression following inhalation injury suggests that this peptide may contribute to the airway inflammation, mucus secretion, pulmonary hypertension, increased airway resistance, and decreased lung compliance, which are evident in our ovine model of inhalation injury.

In situ preparation of rat tracheal basal cells.

The primary function of basal cells is to attach columnar epithelium to the basal lamina. They may also have limited stem cell potential, but very little is known of other biological functions. Basal cells lie on the basal lamina beneath the ciliated and secretory cells and do not reach the surface of the epithelium. The position of the cell beneath the ciliated and secretory cell epithelium makes their in situ study difficult. In order to further aid in the study of basal cells, we have developed an in situ preparation technique in which ciliated and secretory cells are removed. Treatment of rat tracheas with a 20 mM Na2 EDTA solution, pH 7.4, results in partial removal of columnar epithelium from the basal lamina. The percent of denuded columnar epithelial cells per mm of basal lamina is 43.9 +/- 7.8% at 60 min, 47.6 +/- 8.4% at 90 min, and 52.6 +/- 2.7% at 120 min. The viability of the exposed basal cells was the same at both 60 and 90 min of treatment (79.4 +/- 7.8 and 78.0 +/- 8.5, respectively). Morphologically, the exposed basal cells are attached to the basal lamina by hemidesmosomes and are similar to those in the intact animal.

Organization of cytokeratin intermediate filaments in basal cells of growing rat trachea.

The basal cell in airway epithelium plays a major role in attachment of ciliated and nonciliated columnar cells to the basal lamina. As the airway grows in diameter and the columnar epithelium in height, the number of basal cells and the amount of tonofilaments (cytokeratin filaments) and anchoring junctions increase. In this way they maintain a constant attachment strength between the increased volume of the epithelium and the basal lamina. The purpose of this study was to determine which cytokeratins (CKs) are expressed in growing basal cells of the rat and demonstrate where they are localized in the cytoskeleton. Sprague Dawley rats 10, 30 and 90 days of age were used in this study. For light microscopy, tracheal samples were fixed in 95% alcohol or 4% formalin for 2 hr and then embedded in paraffin. For electron microscopy, the tracheal samples were placed in 20 mM EDTA in HBSS media minus Ca++ and Mg++ at pH 7.4 for 60 min to permeabilize the cells and expose the intracellular structures. Antibodies to cytokeratins 7, 8, 10, 13 and 18 did not react to basal cells at any age studied. Antibodies to CKs 5 + 8, 14, 16 + 13, and 19 gave a positive reaction with basal cells at each age studied. Immunogold particles representing antibodies to CK 14 were heavily distributed over intermediate filaments making up the cytoskeleton. Both CK 16 + 13 and 19 were also over intermediate filaments but at a much lower density.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphologic changes in basal cells during repair of tracheal epithelium.

Basal cells are differentiated with respect to junctional adhesion mechanisms and play a role in attachment of columnar epithelium to the basal lamina. Although much is known about nonciliated and ciliated cell differentiation during the repair process after injury, little is known about the basal cell. We studied the morphology of basal cells and quantitated junctional adhesion structures during repair of tracheal epithelium exposed to toxic cotton smoke. Ten adult ewes were given a smoke injury to a portion of the upper cervical trachea and were killed at 4, 6, 8, 10, and 18 days after injury for morphometric studies. At 4 days, there was a stratified reparative epithelium over the basal lamina, which was two to four cells in depth. The basal cells were identified by their hemidesmosome (HD) attachment to the basal lamina. Basal cells were about 69% larger than controls and flattened rather than columnar. The amount of HD attachment was 192% greater than controls. In contrast, volume density of cytokeratin filaments had decreased about 47%. Basal cells had returned to normal numbers and size and a columnar shape by day 18. The amount of desmosome (D) and HD attachment and volume density of cytokeratins had also reached control levels by day 18. These data indicate that morphology of basal cells changes during the initial stages of reparative regeneration but returns to normal by 18 days. Morphologic changes appear to reflect changes in size of the cell associated with cell division rather than differentiation of recently divided basal cells.

Differentiation of anchoring junctions in tracheal basal cells in the growing rat.

A function of airway basal cells is to attach ciliated and nonciliated columnar cells to the basal lamina. The significance of the basal cell in attachment is related to the height of the columnar epithelium. In taller epithelia, basal cells are more numerous and differentiated with respect to anchoring junctional adhesion mechanisms (desmosomes, hemidesmosomes, and the cytoskeleton) than in shorter epithelia. In this study, we determined if basal cell anchoring junctional adhesion mechanisms differentiated during growth of the airway. Tracheas from five 3-day-old, five 30-day-old, and five 90-day-old rats were prepared for electron microscopy and morphometrically studied by standard techniques. The circumference of the trachea increased from 2.5 +/- 0.2 to 7.5 +/- 0.4 mm during growth. The height of the columnar cell increased from 13.4 +/- 1.5 to 24.6 +/- 3.9 microns, and the number of basal cells per millimeter increased from 3.2 +/- 0.7 to 9.6 +/- 1.8 during growth. The number of desmosomes per basal cell profile increased significantly from 1.5 +/- 0.1 to 2.1 +/- 0.1, as did keratin filament volume density from 0.046 +/- 0.05 to 0.098 +/- 0.032. The amount of hemidesmosome attachment per basal cell did not increase significantly during growth of the airway. These data demonstrate that as tracheas grow in circumference, the columnar cells increase in height, basal cells increase in number, and anchoring junctional adhesion mechanisms differentiate in the basal cells. These changes are closely related to the height of the epithelium and result in maintaining a constant amount of attachment between the columnar epithelium and the basal lamina as the epithelium increases in height.