The Role of the Fibrotic Scar in Repair Following Neuroinflammation

Abstract

Multiple sclerosis (MS) is a neuroinflammatory disease of the central nervous system (CNS) in which the body's immune system attacks the myelin sheath that surrounds and insulates the axons of neurons. In many cases this myelin is not repaired by myelinating oligodendrocytes, which decreases the efficiency of action potential conduction and leads to neural dysfunction. We hypothesized that a barrier preventing oligodendrocyte lineage cells from interacting with and repairing the damaged myelin is a fibrotic scar. Following CNS injury, a scar consisting of an outer glial scar made up of reactive astrocytes and an inner fibrotic scar made of fibrotic proteins such as collagen I forms around the site of trauma. While these scars prevent toxins and immune cells from exiting areas of blood‐brain barrier breakdown, they remain a barrier for axonal regeneration. In MS the glial scar has also been characterized, but the presence and role of a fibrotic scar have not been investigated. I have shown that following induction of experimental autoimmune encephalomyelitis (EAE) in mice, which leads to the formation of neuroinflammatory lesions experiencing demyelination and is used as a mouse model of MS, an extensive fibrotic scar forms in the lesioned tissue. Scar‐forming cells were visualized in this lesioned tissue using a Col1a1GFP mouse model. The number of these cells increased in the lesion site following symptom onset and peaked at the peak of motor disability of the mice. The objective of this project is to define the molecular mechanisms that cue fibrotic scar formation, with the hopes of identifying potential therapeutics to manipulate the scar in vivo. I have used RNA sequencing to analyze the transcriptional profile of Col1a1GFP+ CNS fibroblasts from healthy mice and mice with EAE. For this experiment, I purified CNS fibroblasts using fluorescence activated cell sorting from single cell suspensions of spinal cords from Col1a1GFP control mice and Col1a1GFP mice either 5 or 10 days after EAE symptom onset. I then performed RNA sequencing and the transcriptome was compared between conditions to identify how gene expression changes during activation of these cells. In addition I analyzed the transcriptome of whole spinal cord homogenate, and thus comparison of the purified Col1GFP+ cells to whole spinal cord allowed for identification of CNS fibroblast enriched genes. These cells are significantly enriched in components of many pathways, including the TGFβ pathway, in comparison to whole spinal cord tissue. There were also many genes and pathways upregulated in these cells in disease. This data will be used to identify potential targets for therapeutics aimed at manipulating scar formation. A primary, in vitro CNS fibroblast model has been optimized to screen various compounds to see what inhibits their growth and collagen production. Future experiments will be guided at investigating the effect of drugs such as TGFβ pathway inhibitors on the proliferation and collagen production of the cells. Overall it has been shown that a fibrotic scar forms following neuroinflammatory lesion formation, the transcriptional profile of these cells changes during disease, and an in vitro model has been developed to study the mechanisms that drive fibrotic cell proliferation and collagen production.Support or Funding InformationThis work is funded by the NIH T32 Pharmacological Sciences Training GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.