4.2.1 Granulation tissue in glottis observed under an endoscope
Steven M. et al. [16] reported that intubation is the most commonly observed etiology of acquired laryngeal stenosis at present, and for inhalational thermal injury, Allison Reid et al. [17] suggested that it is difficult to distinguish laryngeal stenosis that arise from intubation or inhalational injury. The study conducted by Bruce Benjamin et al. [18] revealed that granuloma on the vocal process of the posterior glottis, which proceeds from granulation tissues, is one of the main sequelae of intubation. In the present study, it was observed that the neoformation was located in the posterior glottis in group 2, and that its histologic demonstration was compatible with the manifestation of granulation tissues that comprised of fibroblasts, new born capillaries and inflammatory cells. This finding indicates that inhalational thermal injury could separately induce the formation of laryngeal granulation tissues from intubation. However, few cases of inhalational thermal injury have been found with the existence of granulation tissues in the glottis in ordinary clinical work, and reports on these are rarely found. In order to explain this, three reasons were considered: (1) In most cases of inhalational injury, the epithelium of laryngeal structures completely regenerated, while the epithelium would only heal in the manner of forming granulation tissues when the injury was severe. On the other hand, when the injury was excessively severe, the symptoms of laryngeal obstruction appeared, and immediate intubation would conducted so that it would be hard to detect the granulation tissues in the posterior glottis due to the inhalational injury. For sufferers who rapidly died during burn accidents of airway obstruction or gas exchange disability, lesions such as granulation tissues could also not be detected. (2) In the present study, the granulation tissues were first detected at week two after the injury, while merely oedema, exudation, ulceration and necrosis could be observed on the surface of the mucosa when immediately examined after injury. However, recurrent laryngoscopy examinations have been rarely performed after the acute stage of inhalational injury, unless symptoms of voice change or difficult breathing emerges. Thus, in some cases without obvious symptoms, the formation of granulation tissues would escape the examination. (3) Sometimes, the formation of granulation tissues in the posterior glottis would be contributed to intubation or reflux diseases in clinic. Rintaro et at. [19] found through an experiment that more rats in the gastroesophageal reflux disease (GERD) group developed granuloma in the vocal process, when compared to the control group, while Petros D et al. [20] suggested that antireflux treatment was the main treatment strategy for vocal process granuloma. In the present study, it was found that inhalational thermal injury is one factor that could induce the formation of granulation tissues in the larynx, which might have been previously underestimated. Hence, the molecular mechanisms behind this might need further studies.
It was also found in the present study that the granulation tissue shrank at week four and six after injury in groups 3 and 4 to a size of 2/3 and 1/3, respectively, and this was considered a consequence of the decrease in oedema and the possible granulation tissue organization. Nevertheless, the disappearance or size change of the granulation tissues in group 5 were not observed. Thus, it was inferred that the granulation tissues in the posterior glottis would not gradually resolve. Conversely, granuloma would occur in this place, or in some cases, this would undergo further organization, fibrosis and scarring. Phillip B et al. [15] once reported 11 cases of laryngeal stenosis caused by inhalational thermal injury, and most of the cases had posterior glottic webs developing from the granulation tissue. This was somehow in accordance with what was found in the present experiment. In addition, for the granulation tissue or granuloma obstructive effect, interarytenoid adhesion from the granulation tissue proceeded when this formed, and the subsequent scarring contracture, different extents of laryngeal stenosis would also occur [18].
The behavior of experimental animals in the present study was in accordance with endoscopic morphologies, since the diet and body movements were observed to return to normal at week two after injury, while the voice change persisted up to the end of the experiment.
4.2.2 Histologic demonstrations related to laryngeal stenosis
For the histologic examinations, the glottis epithelium exhibited obvious repair in groups 3 and 4. However, the epithelium did not appear to completely regenerate, even at the end of the present experiment. Instead, the appearance of numerous fibroblasts, new born capillaries, and inflammatory cells indicated the formation of granulation tissues. This healing process involved granulation tissue forming, as observed under a microscope, and this was consistent with what was observed under an endoscope. Another impressive histologic examination in the glottis was the severe damage in muscle fibers and cartilage cells, and the high severitymight be attributable to the special structures - the thin mucosa and poor blood supply in vocal process of the posterior glottis [21], while the protective effects might be attributable to the narrow space and reflex closure that holds the saturated steam for a longer period of time in the glottis. More impressively, muscle fibers and cartilage cells were not observed to be repaired during the experiment at eight weeks, and the reason might be the complex structures and reflex closure of the glottis. However, since the regeneration ability of muscle fiber and cartilage cells was weaker than that of the epithelium, in such a severe condition, fibrosis and scarring in glottis might occur with lack of repair, which might result in abnormity of vocal cords mobility, or collapse of cartilage or muscle framework, thereby leading to glottic stenosis.
Henning A et al. [4] reported more subglottic than glottic stenosis cases in their research. Furthermore, they found that subglottic stenosis was more common than supraglottic or glottic stenosis in children [22]. Nevertheless, the histologic demonstrations of cricoid cartilage damage in the present study was much slighter, with local epithelium exfoliation, few epithelial cell necrosis, slight oedema, congestion in the lamina propria and submucosa, when observed immediately after injury in group 1, while in group 2, all these pathological changes returned to normal. In addition, cartilage cells and muscle fibers appeared totally normal, even when observed immediately after injury. This may suggest that the model animals in the present experiment would not develop subglottic stenosis in the cricoid cartilage where this usually observed, because the totally normal tissues appeared at week two after the injury. Previously, in a rabbit model established to study inhalational injury in the larynx [7], the researcher found that elevated experimental temperature was followed by increased severity of injury or depth in injured tissues. In the present study, 100℃ saturated steam was used to induce injury in the larynx, and it could be inferred that when given a larger quantity of heat by extending the time of inducing injury or using other methods to induce injury, the cricoid cartilage should be observed to have severer damage, which is possibly deep to the cartilage or muscle lamina. Thus, stenosis in this portion might occur due to the framework collapse. In some cases, the granulation tissue would form from the injured subglottic mucosa, and led to stenosis from the obstruction or scar contracture. On the other hand, it was considered that burn accidents that fail to conduct the reflex closure of the glottis might lead to severe injury in the subglottis, or possibly in the lower airway.
The epiglottis of animals in the present study appeared to have severe damages in the mucosa, submucosa and cartilage cells, while complete epithelium repair and some new born cartilage cells could be observed in group 2. It was considered that the reason for such severe damages was the first and unceasing exposure to saturated steam [13]. As this was injured deeply into cartilage, the healing process might result in epiglottis abnormality, which is one manifestation of laryngeal stenosis.