Sıçan Modelinde Polikromatik Işık ve Adipoz Doku Kökenli Mezenkimal Kök Hücre Uygulamasının Kornea Yara İyileşmesi Üzerine Etkisi

Abstract

The aim of the presented thesis was to evaluate the therapeutic efficacy and safety of subconjunctival adipose tissue-derived mesenchymal stem cell (AdMSC) injection and photobiomodulation (PBM) treatment via polychromatic light source in the wavelength range of 600-1200 nm alone and in combination, on corneal opacity, inflammation, and extracellular matrix organization in corneal wound healing using a rat corneal opacity model. In the first part of the study, the isolation, culture, and characterization of rat AdMSCs were performed and optimization studies were carried out to establish the parameters of the duration and energy dose of the FBM treatment to be applied. In the second part, a corneal opacity model was created by mechanical debridement of rat corneas with a diamond burr. Rats with corneal scar were divided into 4 treatment groups: negative control (NC), AdMSC treatment, PBM treatment and combined (AdMSC + PBM) treatment (n=12 eyes in each group). Finally, on the 10th and 30th days following the injury, corneal opacity score (COS) and degree of neovascularization (NVD) were evaluated by slit lamp examination, and central corneal thickness (CCT) was measured by anterior segment optical coherence tomography. On the 30th day, corneas were harvested and analyzed by transmission electron microscopy (TEM), hematoxylin and eosin (H&E), immunohistochemical (IHC) staining, and real-time polymerase chain reaction (RT-PCR). On day 30, COS and CCT values decreased significantly in all treatment groups compared to the NC group. While the NVD decreased significantly in the AdMSC and combined treatment groups, the change in the PBM group was similar to the NC group. In TEM and H&E staining, the best results in corneal wound healing were observed in the combined treatment compared to the application of PBM and AdMSCs alone. The increased synthesis of collagen type 1 (COL1) and 3 (COL3) after injury was significantly reduced in the combined treatment group compared to the NC group. Compared to the NC group, a significant decrease in transforming growth factor beta 1 (TGF-β1) value was observed only in the PBM + AdMSC group. With combined treatment, interleukin 1 beta (IL-1β) level was found to be significantly lower compared to the NC, PBM and AdMSC groups. As a result, the best results in suppressing inflammation were obtained in the combined treatment group. In all treatment groups, α-smooth muscle actin (α-SMA) level was found to be significantly lower compared to the corneas in the NC group. Keratocan levels in the combined treatment and AdMSC groups were found to be similar to those in healthy corneas. In the RT-PCR analysis, COL1, COL3, IL-1β and α-SMA were significantly decreased in all treatment groups compared to the NC group. Polychromatic light did not cause any damage to the rat lens, retina and endothelium according to TEM and H&E staining analyses. In this study, in vivo imaging and in vitro tissue analysis demonstrated that subconjunctival AdMSC injection and PBM treatment with polychromatic light (3 sessions with total energy density of 4.6 J/cm2 in each session) were effective and safe in corneal tissue regeneration and PBM could be considered as a potential treatment for corneal wound healing. Moreover, PBM therapy can enhance the therapeutic efficacy of stem cells when combined with ADMSCs.