D-valine effect in tracheal stenosis; invitro and invivo experimental study

Objective: In this study, it was aimed to show the place of D-valine, a molecule used to selectively remove fibroblasts in cell culture, in the treatment of tracheal stenosis by inhibiting the proliferation of fibroblasts. Methods: In vitro experimental study; The remaining tissue from the material used by the pathology for diagnosis surgical explants of 10 patients who underwent lobectomy or pneumonectomy for various reasons was used. Both bronchial epithelial cell culture and fibroblast culture were obtained from the same patient. The cultures of the first 4 patients were used for standardization and five D-valine concentrations (10,12.5,15,17.5,20 mM) were determined. The bronchial epithelium and connective tissue samples from each of the next 6 patients were seeded separately, and six groups were formed that were treated with five different D-valine concentrations and saline. Epithelial and connective tissue of each patient were cultured. However, due to technical reasons and different growth rates of the tissues, they could not be cultured and studied simultaneously. Therefore, each tissue was cultured, but cells with 70% dispersal were included in the experiment. When cell cultures became 70-80% surface coverage in 96-well (ml/2000 cells) plates, they were incubated with different concentrations of D-valine (10,12.5,15,17.5,20 mM) for 24 hours. Cell viability was evaluated by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl- tetrazoliumbromide) method. The culture medium was replaced with SF medium containing 1 mg/ml MTT (Sigma) and incubated at 37°C for 30 min. Then, MTT solution was poured and DMSO (Sigma) was placed on the cells. The change in color was read at 550 nm with a colorometric reader (spectrophotometer). The most appropriate inhibitory doses of Dvaline were determined. In vivo Experimental Study: 20 (twenty) adult New Zealand white rabbits (2 to 3.4 kg) were randomly divided into 4 groups. There were 5 rabbits in each group, as the first group was the control group, the second group was administered saline (SF), the third group was administered local steroid (Dexamethasone 2 mg) and the fourth group was the group administered D-Valine. Tracheal stenosis was induced in all animals by performing a standard surgical procedure. On the 15th day, the animals in the control group were sacrificed with an overdose of pentobarbital, and tracheal stenosis was demonstrated histopathologically. In the following days, the same surgical method was applied in the other groups, and saline was administered to the second group, dexamethasone (2 mg/0.5 ml) to the third group, and D-valine (20 mM/0.5 ml) to the fourth group at one time during surgery. At the end of the 15th day, all animals were sacrificed and their tracheas were examined histopathologically. VII Results: Invitro Experimental Study: No significant difference was found between SF and 5 different D-valine concentrations in epithelial and connective tissue cultures. A significant positive correlation was found between epithelial and connective tissue cultures at 20 mM concentration. In vivo Experimental Study: Contrary to the literature, submucosal thickness was observed less in the control group rabbits than in the other groups and it was found to be statistically significant (p=0.004, p=0.006, p=0.022). There was no significant difference between SF, dexamethasone and D-valine groups. When the fibrosis rates were examined, a statistically significant difference was found between the control group and the SF applied group; however, the fibrosis rate was lower in the control group (p=0.001). No significant difference was found between the control group and the other two groups, and when the SF, dexamethasone and D-valine groups were compared among themselves. Conclusions: When the literature was examined, no study was found on the effect of D-valine in tracheal stenosis. Therefore, our study is the first in this regard. It is also important because it is studied both in vitro and in vivo on tracheal stenosis. The suppressive effect of D-valine on fibrosis is known. However, it was observed that it also affected epithelial tissue at concentrations that suppressed fibroblasts under in vitro conditions. In vivo conditions were observed to affect fibrosis relatively, but statistically significant results could not be obtained. Trying more concentrations in in vitro conditions, and repeating the experiment with a larger number of animals and various animals in in vivo conditions will be guiding.