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Establishment of an In vitro Central Nervous System Fibrotic Scar Model to Screen Drugs That Can Modulate Fibrosis

초록/요약

The spinal cord functions as an important link between the brain and the rest of the body. Spinal cord injury (SCI) affects the anatomical and functional connections between the brain and the spinal cord, resulting in lifelong neurological challenges such as sensory and motor dysfunction, aberrant reflexes, and autonomic abnormalities. Axon regeneration and functional recovery after SCI continue to be among the most difficult medical concerns in neuroscience. An inhibitory, non-permissive extracellular environment and a weak intrinsic axon growth capacity are widely accepted as primary reasons for axon regeneration failure in adult central nervous system (CNS). During the acute phase of SCI, excessive cell death and degeneration result in a reduction in tissue volume and formation of cysts. Previous research has shown that cystic cavities create a physical barrier that inhibits signal transduction and limits neurotrophic factor infiltration, making them an unfavorable environment for cell migration and coordinated axonal regeneration. Using a clinically relevant SCI model, I previously showed that a Polyethyleneimine (PEI) based PEI-Mannose injectable hydrogel completely eliminated cystic cavities by bridging the tissue with a fibrotic extracellular matrix (ECM). The assessment of the hydrogel-created matrix illustrated that, hydrogel is replaced by an ECM that is similar to fibrotic scar seen in mouse contusion SCI and was surrounded by scar-forming astrocytes. The corticospinal tract descending axon tracing from our laboratory and examination of 5-HT axon innervation to hydrogel matrix showed that host axons were not able to regenerate through hydrogel-created fibrotic ECM. Therefore, although the hydrogel-created fibrotic matrix was successfully eliminating cystic cavities in was a barrier for the growth of axonal connections from the brain into it. As a result, I hypothesized that modifying hydrogel-created ECM would facilitate axonal regeneration into the matrix, greatly improving the hydrogel's performance as a conduit for restoring axonal connectivity. To modulate the fibrotic environment, I decided to search for potential anti-fibrotic drugs that can be complemented with PEI-Mannose hydrogel to enhance axonal connections between host tissue and hydrogel created matrix. Trying all potential drug candidates in vivo can be time consuming, costly, and raise ethical and technical concerns when routine analyses are conducted to elucidate molecular mechanisms of potential therapeutics. On the other hand, in vitro assays allow time dose-response studies, synergistic activity studies, can provide the necessary information in a matter of days or weeks. As a result, I constructed an in vitro fibrotic scar model using primary meningeal fibroblasts and cerebral astrocytes in the first part of my dissertation. Initially, I decided to replicate previous research that had been successful in producing in vitro fibrotic scar model. However, when I attempted to recreate those findings, I discovered that the clusters formed after TGF-β1 treatment did not entirely resemble the in vivo hydrogel-created fibrotic scar. The hydrogel-created FN-rich matrix was easily differentiated from GFAP-positive astrocytes, yet when I treated TGF-β1, I experienced aggregates of fibroblasts and astrocytes. Therefore, I sought to establish an in vitro fibrotic scar model that can create fibrotic clusters separated by surrounding astrocytes and resembles the in vivo hydrogel created fibrosis. I concluded that the novel in vitro fibrotic scar model I established, resembles the hallmarks of hydrogel induced fibrotic matrix. Moreover, I found that this in vitro model also represents the glial scar formed around hydrogel-created ECM. I confirmed that in vitro co-cultured astrocytes transformed into scar-forming phenotype upon TGF-β1 treatment and was positive with various scar-forming astrocyte markers that represented in vivo. The second portion of my thesis research focused on modulating the fibrotic milieu in order to facilitate axonal growth into the hydrogel-created matrix. I searched for anti-fibrotic drugs that can be complemented with biomaterials. Previous studies showed that microtubule stabilization decreased scarring and chondroitin sulfate proteoglycan levels, and impacted sensory and serotonergic axon regeneration eventually led functional improvement after SCI. In consideration of these findings, I evaluated the effects of microtubule stabilizer Taxol and Epothilone B on in vitro fibrotic scar model and confirmed that Taxol and Epothilone B can target FN production of co-cultured meningeal fibroblasts while altering palisading scar-forming astrocyte morphology. Additionally, I tested tyrosine kinase inhibitors frequently used for the treatment of idiopathic pulmonary fibrosis, however not utilized for the biomaterial induced fibrosis. Tyrosine kinase inhibitors were successfully reduced the immunoreactivity of fibrotic scar markers fibronectin, collagen 1 and PDGFRβ. Moreover, tyrosine kinase inhibitors also altered GFAP positive scar-forming astrocytes’ morphology, therefore, making them a promising drug candidate that can be complemented with biomaterials modulate glial-fibrotic scar environment in vivo.

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목차

I. INTRODUCTION 1
A. Spinal cord injury 1
B. The mechanism of primary and secondary damage following SCI 2
1. Primary Injury 2
2. Secondary Injury 2
C. Challenges in SCI Regeneration 4
1. Extrinsic Influences on Neural Repair 4
a. Cystic cavity formation after SCI 4
b. Glial Scar 4
c. Fibrotic Scar 5
2. Intrinsic Influences on Neural Repair 7
D. Therapeutic Strategies for Spinal Cord Injury 8
1. Surgical Techniques for SCI 8
2. Cellular therapeutic interventions after SCI 9
3. Pharmacological therapy for SCI 10
4. Biomaterials for SCI repair 11
E. Aims of this study 12
II. MATERIALS AND METHODS 14
1. Animal and surgical procedures 14
2. Injection of PEI-Mannose hydrogel 14
3. Tissue processing and histological assessments 14
4. Primary meningeal fibroblast, cerebral astrocyte cultures 15
5. Astrocyte-fibroblast co-culture system 16
6. In vitro drug testing 16
7. Quantitative assessment of co-culture size and numbers 17
8. Quantitative assessment of astrocytic process lengths 17
9. Quantitative analysis of immunoreactivity 17
10. Immunocytochemistry 17
11. Statistical analysis 18
III. RESULTS 19
Part A. Creating biologically meaningful in vitro fibrotic scar model that resembles the hydrogel-created matrix 19
1. PEI-Mannose hydrogel eliminates the cystic cavities that are formed after SCI with a growth incompetent fibronectin-rich matrix 19
2. Hydrogel mediated fibrotic scar can be reconstructed in vitro 22
3. Meningeal fibroblasts form clusters when they are cultured on top of astrocytes 24
4. TGF-β1 treatment increased FN and GFAP immunoreactivity of co-cultured cells 27
5. TGF-β1 treatment to co-cultures enhances fibrotic scar markers vastly present in in vivo hydrogel induced fibrotic scar 29
6. TGF-β1 promotes scar fibroblasts proliferation and enhances the fibrotic scar marker expression 31
7. TGF-β1 is a modulator of astrocytic GFAP expression, and phenotypic alterations when co-cultured with primary fibroblasts 33
8. Addition of TGF-β1 to co-cultures induces the phenotype of the scar-forming astrocytes 35
Part B. Screening anti-fibrotic drugs that can modulate hydrogel-induced fibrotic matrix 37
1. Inhibiting fibrotic scar formation with microtubule stabilizers 37
2. Targeting tyrosine kinases and PDGFRβ in the pathogenesis of fibrosis 40
3. Second screening with promising anti-fibrotic drugs using common fibrotic scar markers 45
IV. DISCUSSION 48
V. CONCLUSION 54
REFERENCES 55

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