An ultrastructural comparison between the nasal cavities of healthy rabbits and those suffering from two forms of spontaneous infection with
The nasal cavity is considered the natural habitat of
Al-Haddawi et al. [
On the other hand, histological or ultrastructural studies documenting lesions in the respiratory epithelium of the nasal cavities of rabbits affected with a natural respiratory disease are not frequent. Histologically, we reported various changes in this region in rabbits infected with
The main purpose of this study was to describe and compare the transmission electron microscopy (TEM) findings of the respiratory epithelium and the propria of the nasal septum from healthy rabbits of different ages under commercial production conditions with those of rabbits affected with the rhinitic and septicemic forms of
After the animals were euthanized, nasal and tracheal swabs as well as lung samples were collected for microbiology. The isolates were cultured in brain-heart agar (BHI) at 37°C for 24 h. Isolates were identified as
Twelve New Zealand White rabbits of different ages from a commercial rabbitry located at the Savannah of Bogotá at 2600 m.a.s., Colombia, were divided into 4 groups: 2 healthy rabbits between 0 and 21 days of age (group 1), 2 healthy rabbits between 23 and 49 days (group 2), 2 healthy rabbits between 51 and 69 days of age (group 3), and 6 acute diseased rabbits, 3 with septicemia (clinically showing cyanosis of the ears and the ocular mucosa, polypnea, dyspnea and orthopnea, and a dilated abdomen) and 3 with rhinitis (group 4). The animals were anesthetized i.m. with 35 mg/kg of ketamine and 5 mg/kg of xylazine then instilled with 2.5% glutaraldehyde (SPI Supplies, West Chester, PA, USA) in Millonig’s buffer (4°C) via the nasal cavity. The animals were euthanized with an overdose of anesthesia. The nasal cavity was cross-sectioned at the level of the first molar, and the nasal septum was taken. The samples were divided into two parts: one was immersed in buffered formaldehyde (3.7%) for paraffin embedding and the other in glutaraldehyde (2.5%, 4°C) for TEM. The experimental procedures were approved by the Veterinary School Bioethics Committee of the National University of Colombia.
Tissues were processed by routine paraffin wax embedding and sectioned at 5
The tissues for TEM were postfixed in osmium tetroxide (1%) and embedded in epoxy resins. Five semithin sections per animal were obtained and stained with toluidine blue. The normal and the pathological morphologies were described, and a 2 mm2 area of tissue that included the epithelium, propria, and glandular epithelium was selected for ultrastructural evaluation. Ultrafine sections were made, contrasted with uranyl acetate and lead citrate and evaluated in a Phillips 110 TEM.
Bacteria could not be isolated from the rabbits in groups 1 and 2, but
TEM findings. Severity of changes in cells of respiratory epithelium of nasal cavity.
Changes/groups | Intercellular |
Vacuoles in |
Detritus | Heterophils | Bacteria | Activity |
No. of goblet cells |
---|---|---|---|---|---|---|---|
Group 1 | + | ++ | − | + | − | − | + |
Group 2 | + | − | − | + | − | − | + |
Group 3 | ++ +++ | ++ +++ | − | ++ | − | + | |
Group 4 S | +++ | +++ | ++1 +++2 | ++2 +++1 | + | ++2 | +++1 |
Group 4 R | + ++ | + ++ | ++ | +++ | ++1 +++2 |
− No change; + light; ++ moderate; +++ severe; 1number of animals; S: Septicemic; R: Rhinitic.
Animals from these groups showed similar findings and will therefore be described together. In the H&E and semithin sections, the rabbits (
Respiratory nasal epithelium from a rabbit of group 2. (a) Intercellular spaces (arrows) that extent from the basement membrane to approximately 2/3 of the height of the epithelial cells, semi-thin section. 1000x. (b) Electron micrograph of (a): The intercellular spaces (arrows) are more abundant around basal cells. 2500x.
Despite being clinically healthy animals (rabbits at most risk of developing the disease), they showed different types of lesions in varying degrees. Interepithelial spaces were more obvious in these animals and were not limited to the basal region of the cells, with some cells completely separated, detached, and desquamated. Moderate to severe vacuolation of the epithelial cells was also present in both animals. Vacuoles of different sizes were observed, some of which filled the whole cytoplasm. Ultrastructurally, in one rabbit, the epithelium was lower, with apparent loss of cilia and excessive cytoplasmic vacuolation. The vacuoles were of different sizes and contained a membranous or granular material. In the second animal, the ciliated cells showed large vacuoles with granular content, which was heavily electrodense and resembled a typical phagosome in a phagocytic cell (Figure
Electron micrograph of respiratory epithelium from a rabbit of group 3. Ciliated cell with electrodense material (D) within a cytoplasmic vacuole, also visible is a severe vacuolation of the cytoplasm. N: nucleus of a ciliated cell. Arrows indicate the interepithelial space. 15500x.
Animals in this group had the most intense and numerous lesions. In two of the rabbits, copious amounts of detritus, inflammatory cells, and mucus accumulated in the lumen, but this accumulation was moderate in the remaining rabbit. A small number of bacteria were also observed free in the lumen. The intercellular spaces were severely dilated (Figure
Electron micrograph of nasal epithelium from a rabbit of group 4. Apical opening between two ciliated cells (arrow). The cytoplasm is moderately vacuolated. 10000x.
Electron micrograph of nasal epithelium from a rabbit of group 4. A PMN migrating through an intercellular space and separating the apical border of two ciliated cells (arrows). The PMN cytoplasm is less electrodense and with lesser number of granules (large arrow). 5200x.
Electron micrograph of nasal epithelium from a rabbit of group 4 (septicemia). (a) Severe mitochondrial vacuolation (arrows) of several ciliated cells. 10000x. (b) Severe vacuolation of the apical cytoplasm of a ciliated cell. Some vacuole (v) contain myelin-like material. 11500x.
The lesions differed in quantity but not in quality from those of septicemic rabbits, though some of them were milder in the rhinitic rabbits. Migrating inflammatory cells were associated with widened interepithelial spaces and cytoplasmic vacuolation, with both changes ranging from mild to moderate. More significant in the epithelium was the increased number of goblet cells, which in some cases were the only cells in a large area. Two rabbits were severely affected, while the remaining one was moderately so. Ultrastructurally, the goblet cells displayed high activity. They were larger than and out-numbered the ciliated cells over large areas of tissue. In between, lymphocytes and heterophils migrated through the intercellular spaces.
The presence of interepithelial spaces in both healthy and diseased rabbits was one of the most important findings of group three. In healthy animals, however, such spaces were always restricted to the basal region of the epithelium (Figures
At least two potential mechanisms would explain the changes in the permeability and morphology of the cell monolayers: changes in the actin cytoskeleton or modifications to the tight junction proteins. It is likely that actin-myosin contraction creates intercellular gaps in both epithelial and endothelial cell monolayers [
For the mucociliary apparatus to function properly as the first defense mechanism against different invaders, it is important that the fluid surrounding the cilia as well as the viscoelasticity of the mucus blanket over the cilia maintains a correct hydration and concentration of electrolytes. However, both of these defense responses are altered during infectious and inflammatory reactions. Three mechanisms for water secretion onto the surface of the airway epithelium have been elucidated: by serous cells in the surface epithelium (Rogers et al., 1993 in [
Despite that the animals of group 3 were taken during the age of most frequent development of the respiratory entity, none of them showed clinical signs of the disease; although it is not possible to state that they would not develop the disease later, there was a significant change in rabbits of group 4 that was negligible or absent in the healthy groups, namely, the number and the activity of goblet cells, making this event unlikely. Recent findings from our group [
Hypertrophy, hyperplasia, and metaplasia of the mucous cells in the epithelium and of the mucous and serous glands in the submucosa are associated with oversecretion of mucus and are some of the most prevalent lesions in rhinitis, sinusitis, and tracheobronchitis [
The cytoplasmic vacuolation of epithelial cells in the nasal cavity was more severe in diseased animals, particularly, those with septicemia (Figure
In our study, three patterns of vacuolation were observed in the epithelial cells: one corresponded to swollen or exploded mitochondria, the most common one had the appearance of digested cellular components, and the final one contained material resembling myelin figures. These changes might be induced by the
In addition, there appeared to be a direct relationship between the number and size of vacuoles, the extension of intercellular spaces, and the number of migrating heterophils. The heterophils of the diseased rabbits seemed to be degranulating, which was reflected by the decreased quantity and smaller size of their granules, leaving clear spaces in the cytoplasm. Klut et al. [
In asthma, bronchial hyperreactivity, infectious pneumonia, endotoxemia, and so forth, authors have demonstrated that heterophils and eosinophils are responsible for tissue damage [
Ultrastructural changes similar to those observed here in the upper airways of rabbits inoculated experimentally with
Our findings do not guarantee that
Endotoxins are commonly found in different environments [
In summary, to our knowledge, this is the first description of the nasal cavity of healthy rabbits and of rabbits afflicted with the spontaneous form of the disease caused by