Evaluation of the ECM Structures of Fibrotic Tissues by Using Raman Microspectroscopy

Abstract

Fibrosis is a pathological process of excessive extracellular matrix (ECM) proteins deposition within tissues undergoing chronic inflammation. Fibrosis is highly related to a wide range of disorders across every organ and can cause negative effects on disease prognosis as well as life expectancy. It can be caused by numerous stimuli including implantation, aging, cancer, repetitive tissue damage, or disorders which are highly relevant to the chronic inflammatory reaction. Implantable medical devices can cause fibrotic capsule formation, which is triggered by the foreign body response (FBR). Fibrotic capsules can physically interfere with medical devices, thereby reducing therapeutic outcomes. The diagnostic evaluation of tissue fibrosis relies on the examination of tissue biopsies by gold-standard histochemical analysis. The definition, location and characteristics of the area of the fibrotic tissue can induce bias between individual pathologists with different clinical experiences, expertise and knowledge. Moreover, the limitations of histological staining involve invasive biopsy procedures and staining artifacts, which may cause errors in diagnostic results. The thesis mainly focused on the establishment of a new fibrotic assessment of implantation-driven FBR in streptozotocin (STZ)-induced diabetic animal model by using Raman microspectroscopy (RMS). ECM compositions as well as pro-inflammatory and regenerative macrophage activation states were investigated. We demonstrated the capability of RMS to discriminate collagen type I (Col I) between diabetic and non-diabetic rodent models via advanced glycation end products (AGEs) which were integrated into collagen fibers in diabetic groups. Furthermore, in combination with multivariate analysis (MVA), we revealed that RMS can be used to discern pathological fibrotic tissues and healthy tissues via the secondary structural differences based on the Raman spectrum of Col I, which can provide a non-biased clinical assessment. In addition to ECM characterization, RMS has the potential for immune cell classification. Raman imaging was applied on tissue sections for immunophenotyping in accordance with the fluorescence-guided generation of M1/M2 macrophages. The classification was attributed to the differences in DNA methylation states in the nuclei between M1/M2 phenotypes. In conclusion, Raman imaging and microspectroscopy offer an advanced diagnostic approach to monitor the FBR and fibrosis investigation in a molecular-sensitive and marker-independent manner.