In vitro modelling of glial and fibrotic scar following spinal cord injury

Spinal cord injury (SCI) is a condition that has caused disability worldwide. Following the injury, a scar composed of inner glial scar and outer fibrotic scar is formed. The glial scar is made of inhibitory extracellular matrix (ECM) molecules while the fibrotic one contains the ECM proteins, which both becomes upregulated in response to injury. The structure and composition of the ECM vary after the injury, inhibiting the regeneration. Hence, it is of pivotal importance to propose new model systems and protocols that allow the cells to produce ECM in vitro to assess therapeutic strategies for targeting inhibitory molecules. Exposing cells to an environment crowded with macromolecular crowders (MMC) is an effective strategy to mimic the physiological cellular milieu and develop an in vitro model of glial and fibrotic scar. A three-dimensional (3D) environment for scarring was generated using cells within a collagen hydrogel followed by adding a small molecule to both 2D and 3D in vitro models with the focus on suppressing the expression of inhibitory factors. Chapter 3 aimed to evaluate the impact of different concentrations of MMCs, including dextran sulphate (DxS) and carrageenan (Ca), and Ficoll (FC) cocktail 70 kDa/FC 400 kDa, on Neu7 astrocyte and primary astrocyte cell morphology, metabolic activity, viability, and ECM molecules deposition. Furthermore, the expression of various ECM proteins of the glial and fibrotic scar as a function of MMCs concentration was explored. FC was adaptable to either Neu7 astrocytes or primary astrocytes, leading to enhanced expression of glial and fibrotic scar proteins. Chapter 4 examined the effect of FC cocktail 70 kDa/FC 400 kDa on morphology, migration pattern, and ECM molecule deposition in Neu7 astrocytes, primary astrocytes, leptomeningeal (LPG) cells, and mixed glial cultures (MGC). FC had no negative impact on the morphology and migration pattern of Neu7 astrocytes, primary astrocytes, LPG cells, and MGC. In addition, FC increased the expression of glial scar markers in Neu7 astrocytes, primary astrocytes, and MGC, as well as the deposition of fibrotic scar markers in Neu7 astrocytes, primary astrocytes, LPG cells. Treating cells with FC was proposed as an effective strategy to create an in vitro model of glial and fibrotic scar formation without applying physical (scratch) or chemical injury (cytokine cocktail). Primary astrocytes were found to be more suitable for use in this in vitro model of SCI, as they not only accurately reflect the protein composition observed in actual SCI scars but also offer more stable growth over extended periods. Importantly, upregulation of MMPs and reduction in DRG neurite length suggests a dynamic interplay between ECM deposition and degradation. Chapter 5 details the developed 3D in vitro model of glial and fibrotic scar using collagen hydrogel. Cell viability results revealed that primary astrocytes were alive in the collagen hydrogel. By adding FC to both collagen solution and culture media, expression of glial and fibrotic scar markers were enhanced. Additionally, TGFb1, known to induce astrocyte reactivity, was introduced into the culture media to see its effect on ECM molecules. TGFb1 and FC synergistically increased the expression of scar markers. These findings highlight the importance of both FC and TGFβ1 in modulating astrocyte behaviour and ECM production, offering a more accurate model to study glial scar formation and potential therapeutic strategies for SCI. The small molecule Sp-8-BnT-cAMPS (S220) was loaded into microparticles (MPs) with uniform morphology and was shown to release over 48 hours. Notably, the S220 released from the MPs reduced the expression of glial scar markers in primary astrocytes in both 2D and 3D in vitro models. S220 offers a targeted approach to modulate the inhibitory microenvironment of glial scars. This novel MMC in vitro model of glial and fibrotic scar formation mimics the cellular and molecular changes that occur following SCI and provides a new direction for screening of therapeutic compounds to modulate the environment of the glial and fibrotic scar in spinal cord repair.

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University of Galway

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Attribution-NonCommercial-NoDerivatives 4.0 International

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University of Galway Theses (PhD Theses)