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Mohor Biplab Sengupta, Christo Kole, Ben Mead, Naoki Nakaya, Stanislav I Tomarev; Olfactomedin1 provides retinal ganglion cell neuroprotection and stimulates axon regeneration after optic nerve crush in rodents. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5516.
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© ARVO (1962-2015); The Authors (2016-present)
Olfactomedin1 (Olfm1) is a secreted glycoprotein preferentially expressed in the neuronal tissues including retina. Studies in zebrafish larvae indicate that Olfm1 facilitates axon growth. Here, we analyzed effects of Olfm1 on axon growth and regeneration, and neuronal cell survival using in vitro mouse neuronal cell cultures and in vivo rat and mouse optic nerve crush (ONC) models.
Primary neuronal cells were prepared from the P7 cortex of C57BL/6J mice and cultured in the presence or absence of 20-40 nM Olfm1 and 300 nM neuritin for 3 days. Neurite length was measured after staining with antibodies against βIII-tubulin. Adult Sprague-Dawley rats and four to six weeks-old C57BL/6J mice were used for ONC experiments. A recombinant virus was constructed by cloning the AMZ domain of Olfm1 into the pAAV2/2 vector. To test in vivo effects of Olfm1, AAV2-Olfm1 virus was injected intravitreally into rat or mouse eyes 7 days prior to ONC. Forskolin (0.5 µg) was injected simultaneously with ONC. For the retinal ganglion cell (RGC) count, wholemounted retinae were stained with an antibody against RBPMS and imaged using a Zeiss 700 confocal microscope. Regenerating RGC axons were counted after staining optic nerve cryosections with antibodies against GAP43.
Addition of Olfm1 or neuritin to neuronal cultures led to 60% and 100% increase (p<0.0001), respectively, in the average neurite length after 3 days in culture. Simultaneous addition of both proteins did not produce a synergistic effect. Overexpression of Olfm1 in rat eyes enhanced survival of RGCs and led to a modest increase in the number of regenerating axons measured 21 days after ONC at 100 µm from the crush site. To check whether the enhanced RGC survival and/or regeneration of their axons by Olfm1 could be further increased by the elevation of cAMP, forskolin was injected at the time of ONC in mice. Forskolin and Olfm1 injection led to a statistically significant increase in the amount of RBPMS+ RGCs as compared with Olfm1 injection alone, while the increase in the number of regenerating axons did not reach statistically significant values 10 days after ONC.
Overexpression of Olfm1 provides modest RGC survival and stimulates axon regeneration after ONC. Combination of Olfm1 with neuritin or forskolin did not show synergistic stimulation of axon regeneration.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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