Developing a canine vocal fold scar model by CO₂ laser and studying the LOX, HSP70 and HA expression in its extra celluar matrix

Abstract

Objective:Building a canine vocal fold scar model and analyzing the changes in morphology, histopathology and related factors of extra cellular matrix (ECM) of vocal cord healing at different time periods.Method:Five experimental dogs were randomly divided into the control group(one dog) and the experimental group (four dogs). No special treatment was done on the control group, and the experimental group was given CO₂ laser through laryngoscope with minimally invasive surgery on bilateral vocal cords. Observation of the morphological changes of injured vocal cords was made at five different time, pre-operation and 6 h, 3 w, 8 w and 12 w post-operation. HE staining, immunofluorescence, were used respectively to observe the histopathological and ultrastructural changes of each layer of vocal cord. Observation were made on the changing pattern of lysyl oxidase (LOX), heat shock proteins70 (HSP70), and the integrated optical density index (IOD) of Hyaluronic acid (HA) in vocal cord tissues. Result:①Laryngoscope shows there were mild congestion and edema and inflammatory exudation on the wound surface of vocal cord 3w post-operation. On the wound surface of vocal cord 8 w post-operation, the congestion and edema disappear, the local contraction sink, and no adhesion and granulation form. 12 w post-operation, the surface of the vocal cord was smooth, there were local contractures, vocal fold scar form, and hoarseness in the bilateral vocal cords was obvious. ②HE staining shows 6 h post-operation there were a large number of inflammatory cell infiltration, red blood cell leakage, and cell congestion and edema on the wound surface of vocal cord. 3 w post-operation, there were fibroblast proliferation, angiogenesis, and a large number of fibrous tissues disorderly arranged on the wound surface of vocal cord. 8w post-operation, each layer of fibrous tissues were in hyperplasia and the blood vessels was thicken after on the wound surface of vocal cord, 12 w post-operation, a large number of collagens increases and were in group or fasciculation disorder. There were many irregular gaps in fibers, and blood vessels and glands become rare or disappear. ③The immunofluorescence showed LOX, HSP70 and HA were all localized in the cellular cytoplasm and nucleus. The expression levels were different at different postoperative time, and they were mainly relatively strong expressed in the inflammatory cells, vascular endothelial cells and the glands. ④The comparison of IOD values: The IOD values of LOX were different at different time periods (P< 0.05). There were no significant differences in IOD of HSP70 between pre-operation and 12 w post-operation, but there are significant differences among other groups (P< 0.05). There were no significant differences in IOD of HA between pre-operation and 12 w post-operation, but there were significant differences among other groups (P< 0.01). ⑤The expression of LOX decreased 6h post-operation and increased 3-12 w post-operation. The expression of HSP70 post-operation reached the peak 6 h post-operation and decreased 3-12 w post-operation. The expression of HA decreased 6 h post-operation, increased to the peak 8 w post-operation, and decreased 8-12 w post-operation.⑥The transmission electron microscope showed 3 w to 8 w post-operation, in the intrinsic layer of the vocal cord, the fibroblasts were extremely active, the cells swelled, and the organelles were abundant.12 w post-operation, there were a small number of fibroblasts in the active state, and larger gaps between the fibers and fewer and thinner elastic fibers. Conclusion:CO₂ laser ablation of vocal cords under the Micro Post-Laryngoscope could establish reliable animal model of vocal fold scar, and 12 w reticular the vocal fold scars basically form. LOX, HSP70, HA play different roles at early, middle, and late stages in the vocal fold scar formation, and they can be used as a more sensitive index for vocal fold scar formation.