The development of a rat in vitro model of spinal cord injury demonstrating the additive effects of rho and ROCK inhibitors on neurite outgrowth and myelination

Abstract

It is currently thought that treatment for spinal cord injury (SCI) will involve a combined pharmacological and biological approach; however, testing their efficacy in animal models of SCI is time-consuming and requires large animal cohorts. For this reason we have modified our myelinating cultures as an in vitro model of SCI and studied its potential as a prescreen for combined therapeutics. This culture comprises dissociated rat embryonic spinal cord cells plated onto a monolayer of astrocytes, which form myelinated axons interspaced with nodes of Ranvier. After cutting the culture, an initial cell-free area appears persistently devoid of neurites, accompanied over time by many features of SCI, including demyelination and reduced neurite density adjacent to the lesion, and infiltration of microglia and reactive astrocytes into the lesioned area. We tested a range of concentrations of the Rho inhibitor C3 transferase (C3) and ROCK inhibitor Y27632 that have been shown to promote SCI repair in vivo. C3 promoted neurite extension into the lesion and enhanced neurite density in surrounding areas but failed to induce remyelination. In contrast, while Y27632 did not induce significant neurite outgrowth, myelination adjacent to the lesion was dramatically enhanced. The effects of the inhibitors were concentration-dependent. Combined treatment with C3 and Y27632 had additive affects with an enhancement of neurite outgrowth and increased myelination adjacent to the lesion, demonstrating neither conflicting nor synergistic effects when coadministered. Overall, these results demonstrate that this culture serves as a useful tool to study combined strategies that promote CNS repair.

An in vitro spinal cord injury model to screen neuroregenerative materials

“…Such synergistic approaches have the potential to regenerate the injured spinal cord with varying degrees of efficacy, but none have been successfully translated into the clinic [41]. The full potential of combinatorial strategies utilising aligned nanofibers with combinations of cells and biomolecules has yet to be elucidated, due in part to a heavy reliance on in vivo SCI models, in the absence of highthroughput, biologically-relevant in vitro screening models of SCI [5,42]. Two-dimensional reductionist tools in current widespread use e.g.…”

The Development of a ɛ-Polycaprolactone Scaffold for Central Nervous System Repair

“…35 Importantly, these cultures allow the study of myelin sheath formation, rather than relying entirely on the expression of late myelin markers. Myelin ensheathment observed in these cultures is similar to myelination and sheath formation in vivo.…”

Using a 3-D multicellular simulation of spinal cord injury with live cell imaging to study the neural immune barrier to nanoparticle uptake

“…A few different approaches have been taken to mimic SCI pathology in vitro including: monoculture systems (primary and cell lines), co-culture systems, microfluidic devices and organotypic slice cultures [31][32][33][34][35][36][37]. A number of factors must be taken into consideration when evaluating the utility of each model system including the ability to mimic intercellular uptake dynamics, definition of cellular ratios to model specific CNS regions/disease states, standardisation of protein coronas around introduced nanoparticles, ease of nanoparticle delivery, and amenability to various (including real-time) imaging techniques [9].…”

“…However, cardinal neuropathological responses were only characterised in the neurons, neglecting neuroglial responses. An elegant model has been established that contains neurons and the major neuroglial cells, by seeding dissociated rat embryonic spinal cord on top of an astrocyte monolayer [37]. The mechanical induction of injury resulted in a lesion site with many of the cardinal pathological responses displayed by multiple cell types (demyelination, reactive astrocytosis, neurite degeneration and, importantly, microglial infiltration).…”