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Craig Pearson, Herbert M. Geller, Keith R Martin; Modifying chondroitin sulfate proteoglycans enhances retinal ganglion cell axon regeneration in the mouse optic nerve. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1739.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal ganglion cell (RGC) axon regeneration can be induced by inflammation, genetic manipulation, or other stimuli, but regrowth to central targets is limited in part due to the inhibitory extracellular environment of the optic pathway. We aimed to characterize the spatial and temporal expression of inhibitory chondroitin sulfate proteoglycans (CSPGs) in the mouse optic nerve after optic nerve crush (ONC) and to test if modifying CSPG enhances RGC axon regeneration in vivo.
CSPG expression was assessed by immunohistochemistry 1, 3, 7 and 21 days after ONC using antibodies against CSPGs (including a specific sulfated epitope (4S) known to be inhibitory to axon growth in vitro), and markers for reactive astrocytes (GFAP) and activated microglia (Iba1) (n=3 per group). To assess the effects of CSPGs on RGC axon regeneration, mice with a cre-inducible mutation in the phosphoinositide 3-kinase (PI3K) protein, which stimulates axon regeneration, also underwent ONC. Animals received either chondroitinase ABC (ChABC) to inactivate CSPGs (n=4) or PBS control injection (n=4) at the lesion site. Intravitreally injected Choleratoxin-B (CTB) was used to visualize regenerating RGC axons.
Elevation of CSPGs, including the 4S epitope, was observed at the lesion site from between 3 and 7d until 21d post crush. Reactive astrocytes withdrew from the lesion site, creating a GFAP-negative zone filled with Iba1-positive microglia and macrophages. CSPGs occupied spaces between Iba1-positive cells, alongside RGC axons, suggesting that axons must travel through CSPG-rich regions while growing past the lesion. PI3K mutant mice showed significant regeneration of RGC axons. Inactivating CSPGs with ChABC enhanced this effect, with more CTB-positive axons observed at 0.5 mm distal to the lesion site in the enzyme-treated group (252.32 +/- 96.35 axons per nerve) versus controls (119.23 +/- 112.50, p=0.049).
CSPG expression increased after mouse optic nerve crush, and levels remained high within the lesion site for at least 3 weeks. Inactivating CSPGs with ChABC enhances intrinsically-stimulated RGC axon regeneration, suggesting that CSPGs at the lesion site inhibit growing axons and that modifying the extracellular environment may improve intrinsic regenerative therapies.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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