Abstract
Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disease of the central nervous system. Neurological deficits are attributed to inflammatory demyelination, which compromises axonal function and survival. These are mitigated in experimental models by rapid and often complete remyelination of affected axons, but in MS this endogenous repair mechanism frequently fails, leaving axons increasingly vulnerable to the detrimental effects of inflammatory and metabolic stress. Understanding the molecular basis of remyelination and remyelination failure is essential to develop improved therapies for this devastating disease. However, recent studies suggest that this is not due to a single dominant mechanism, but rather represents the biological outcome of multiple changes in the lesion microenvironment that combine to disrupt oligodendrocyte differentiation. This identifies a pressing need to develop technical platforms to investigate combinatory and/or synergistic effects of factors differentially expressed in MS lesions on oligodendrocyte proliferation and differentiation. Here we describe protocols using primary oligodendrocyte cultures from Bl6 mice on 384-well nanofiber plates to model changes affecting oligodendrogenesis and differentiation in the complex signaling environment associated with multiple sclerosis lesions. Using platelet-derived growth factor (PDGF–AA), fibroblast growth factor 2 (FGF2), bone morphogenetic protein 2 (BMP2) and bone morphogenetic protein 4 (BMP4) as representative targets, we demonstrate that we can assess their combinatory effects across a wide range of concentrations in a single experiment. This in vitro model is ideal for assessing the combinatory effects of changes in availability of multiple factors, thus more closely modelling the situation in vivo and furthering high-throughput screening possibilities.
Highlights
Multiple Sclerosis (MS) is a chronic inflammatory, demyelinating disease of the central nervous system (CNS) with diverse clinical presentations and a heterogeneous histopathology
To demonstrate the effectiveness of this platform we focused on four factors differentially regulated in MS lesions that influence oligodendrocyte progenitor cells (OPCs) proliferation, differentiation, and myelination: platelet-derived growth factor subunit A dimer (PDGF–AA [20]), fibroblast growth factor 2 (FGF2 [15,21]), bone morphogenetic protein 2 (BMP2 [22]), and bone morphogenetic protein 4 (BMP4 [22,23])
Primary mouse oligodendrocytes were cultivated as spheres and plated on three 384-well nanofiber plates for 14 days with Platelet-derived growth factor (PDGF)–AA and FGF2 in combination or independently concentrated at 0, 0.625, 1.25, 2.5, 5, 10, or 20 ng/mL and BMP2 or BMP4 concentrated at 0, 10, 50, or 100 ng/mL (Figure 1a,b)
Summary
Multiple Sclerosis (MS) is a chronic inflammatory, demyelinating disease of the central nervous system (CNS) with diverse clinical presentations and a heterogeneous histopathology. It is suggested that demyelination and failure of remyelination lead to axonal degeneration and functional impairment This axonal pathology is the underlying cause of disability in patients with MS and is attributed to the detrimental effects of inflammatory demyelination on the functional and structural integrity of affected axons [1,2]. In this context, demyelination disrupts axonal conduction per se and disrupts metabolic support provided via the myelin sheath whilst simultaneously enhancing axonal susceptibility to damage by inflammatory mediators, a combination of effects predicted to exacerbate. In MS, this repair mechanism frequently fails, leaving affected axons increasingly vulnerable to inflammatory and metabolic stress [3]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
