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Timothy A. Sullivan, Justin Templeton, Eldon E. Geisert, Tonia S. Rex; Systemic Gene Delivery of Erythropoietin Protects NeuN Positive Cells in the Retina Following Optic Nerve Crush. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4616.
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Recent evidence points to erythropoietin (EPO) as a putative neuroprotective agent. Our lab has previously tested a modified form EPO containing a single arginine to glutamate mutation (R76E). This form was used to protect photoreceptors in a retinal degeneration slow (rds/rds) mouse model without causing significant increases in hematocrit levels. The goal of this study was to determine if systemic delivery of EPO or EPO-R76E would protect NueN positive cells and retinal ganglion cell axons from optic nerve crush induced cell death.
Two month old Balb/c mice received intramuscular injections of a recombinant adeno-associated virus (rAAV) carrying either eGFP, Epo or EpoR76E under the control of the cytomegalovirus promoter. One month post injection mice were subjected to optic nerve crush. Thirty days post crush isolated retinas were stained with NueN, flat-mounted, and examined at 40X by confocal microscopy. Optic nerve tracts were resin embedded, cut into 1 µm-thick cross sections, stained with 1% parapheynylenediamine, and examined at 60X by light microscopy. Hematocrit levels were determined by capillary centrifugation of blood and serum EPO levels were determined by ELISA.
Retinas from Balb/c mice that received rAAV.Epo had an average of 20% more (n=18: p <0.001) NeuN positive cells present at the RGC layer than control mice (rAAV.eGFP n=18). Treatment with rAAV.EpoR76E resulted in more NeuN positive cells compared to control retinas however the number was not statistically significant (n=20). No significant difference was detected in the number of live axons between the different treatment groups. Hematocrit and EPO levels were 46% and 0mU/ml, 83% and 16.5mU/ml, and 51% and 5.7mU/ml in mice receiving rAAV.eGFP, rAAV.Epo, or rAAV.EpoR76E, respectively.
A single injection of rAAV.Epo protected NeuN positive cells in the RGC layer from death due to optic nerve crush. However, neither treatment had any effect on the axons themselves. Treatment with rAAV.EpoR76E did not significantly increase hematocrit but also did not provide significant levels of protection against optic nerve crush.
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