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Rock inhibition for the promotion of spinal cord regeneration Chan, Carmen

Abstract

Axonal regeneration within the injured spinal cord is hampered by multiple inhibitory molecules in the glial scar and in the surrounding disrupted myelin. Of these the chondroitin sulfate proteoglycans [i.e chondroitin sulphate proteoglycans] (CSPGs) have an important role in the regeneration failure but its signaling [i.e. signalling] is not fully understood. Therefore, the effects of CSPG on neurite growth were analyzed by growing embryonic chick dorsal root ganglion (DRG) explants on aggrecan (a CSPG of the hyalectan family). Aggrecan aggregate, aggrecan monomer, and hyaluronic acid inhibited neurite growth from nerve growth factor- and neurotrophin-3-responsive DRG neurons. Chondroitinase ABC digestion reversed aggrecan inhibition. ROCK inhibition with Y27632 increased neurite growth on some, although not all of the aggrecan components tested, suggesting that some of them activated the Rho/ROCK pathway. Many of the inhibitory molecules found in the injured spinal cord activate the Rho pathway, providing a strong rationale to target it following spinal cord injuries (SCI). A dorsal column transection model in rats was used to analyze the efficacy of the ROCK inhibitor, Y27632, in stimulating axonal regeneration. Acute treatment with Y27632 stimulated sprouting of corticospinal tract and dorsal column tract axons, and accelerated functional recovery/compensation. However, lower doses of Y27632 appeared to be detrimental, as low dose-treated animals had decreased axonal regrowth and impaired functional recovery. Y27632 treatment caused negative side effects potentially by acting on the non-neuronal cells in the injured spinal cord. In vivo spinal cord tissue and in vitro astrocyte cultures were used to test if ROCK inhibition increased axonal growth inhibition by astrocytes. In vivo, Y27632 treatment enhanced the upregulation of GFAP and neurocan after SCI. In vitro, the ECM derived from Y27632-treated astrocytes contained higher levels of CSPGs, on which neurite growth from cortical neurons was inhibited. The increased expression of inhibitory CSPGs with Y27632 treatment may contribute to the negative side effects observed in the rat model of SCI. Results here demonstrate that more basic science research must be done to determine the range of dosage of Y27632 that is safe for use in treating SCI, and to design methods for cell specific targeting of ROCK inhibition.

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