Long-term Effects Of Intravitreal Injection Of Erythropoietin Sustained-release Microspheres On Retinal Ganglion Cells After Optic Nerve Crush In Adult Rats

Chronic ocular disorders such as Optic neuropathy are major causes for blindness. Currently, no effective treatments have been found. Drug therapy, including neurotrophic factors (NTFs), still play an important part. The neuroprotective effects of NTFs on retinal ganglion cells become to be a hopeful therapeutic method in the future. However, it is challenge to achieve a safe, sustained and efficient drug delivery. Intraocular controlled protein delivery system provides an alternative to the traditional administrations (systemic, topical, intraocular, et al).Erythropoietin (EPO) has well-documentd protective neurotrophic activity on many retinal cells such as retinal ganglion cells, photoreceptor, retinal pigment cells and so on. Besides, EPO is rather cheap, easy to obtain, and have a good perspective in the future clinical application. In this study, we microencapsulated EPO in the Poly(L-lactic-co-glycolic acid) (EPO-PLGA) microspheres, injected the microspheres intravitreally to the optic nerve crush rats to evaluate long-term neuroprotective effects of the sustained-release system on the retinal ganglion cells.PartⅠFabrication of EPO-loaded PLGA Microspheres and in vitro release of EPO from PLGA microspheresObjective To microencapsulate EPO into PLGA mocrospheres and explore in vitro release of EPO from PLGA microspheres.Methods EPO-dextran particles were prepared. This formulation process involved an aqueous-aqueous emulsion formed at reduced temperature (Freezing-induced phase aqueous-aqueous emulsion formed at reduced temperature (Freezing-induced phase separation). Then PLGA microspheres containing EPO-dextran particles were prepared in Solid-in-oil-in-water method (S/O/W method), In vitro release of EPO from PLGA microsphere was detected using ELISA.Results The EPO-PLGA microspheres prepared with S/O/W method possess a spherical shape, smooth surfaces and diameters ranging from 40 to 100μm. A sustained-release profile of EPO was achieved with a minimal burst of about 10% below of the total EPO loadings released at day 1 and above 80% of loadings released up to 60 days.Conclusion EPO-PLGA microspheres made from S/O/W method achieved a long-term sustained-release profile of EPO, which provided evidence for further study on the efficacy of EPO from PLGA microspheresPartⅡIn Vitro Activity and Safety of EPO When loaded in PLGA MicrospheresObjective To investigate in vitro activity of EPO microencapsulated in PLGA micropheres and to estimate in vitro safty of PLGA.Methods Establisih three-dimension culture system for retinal explants. Retinal tissues of SD rats aged 1 to 3 days were cut into pieces of 0.5mm×0.5mm and cultured in three-dimension culture system with 1 IU/mL of EPO,1 IU/mL of EPO-PLGA release fluid or the same amount of blank PLGA release fluid. Growth of axions from the retinal tissue edge was detected daily under inverted phase contrast microscope. Data were analyzed at 6 days.Results 6 days after cultured, mean number and density of regrowth axons from the retinal explants were more statistically significant in culture systems containing EPO or EPO-PLGA release fluid (p<0.001). No differences were found between blank PLGA release group and control (p>0.05).Conclusion The activity of EPO was not affected during the process of micropheres preparation and the process of release. PLGA and its degradation product were safe for the retinal tissue in vitro. Part III Long-term Effects of Intravitreal Injection of Erythropoietin Sustained-release Microspheres on Retinal Ganglion Cells after Optic Nerve Crush in Adult RatsObjective To investigate the protective effect of Erythropoietin (EPO) on retinal ganglion cells (RGCs) through single intravitreal injection after optic nerve crush when microencapsulated in PLGA microspheres.Methods Adult SD rats were selected and randomly divided into five groups. Establish optic nerve crush models.①Animals in untreated group:received no treatment.②Animals in EPO group:received intravitreal application of 10IU (5μL) EPO immediately and 4 weeks after optic nerve crush.③Animals in EPO-PLGA group:underwent intravitreal injection of 20mg (5μL, containing 20IU EPO) EPO-PLGA microspheres.④Animals in PBS group:received intravitreal injection of PBS (5μL) immediately and 4 weeks after crush.⑤Animals in PLGA group:received intravitreal injection of 20mg (5μL) blank PLGA microshperes.. At 4 and 8 weeks after the crush, the animals were killed.5 days before sacrifice, RGCs were retrogradely labeled by injecting Dil into the superior colliculi of the brain. Retinal flat mount photographs were assessed for the number of survival RGCs. Apoptotic RGCs were detected with TUNEL labeling in retinal frozen section. Glial fibrilary acid protein (GFAP) expressed in retina was detected with immunohistochemistry.Results Retinal flat mount revealed that the density of the RGCs was 2387.690±164.872(cells/mm2) in normal adult rats,748.30±58.81 (cells/mm2) in untreated group,1418.52±154.91 (cells/mm2) in EPO group,1296.68±157.55 (cells/mm2) in EPO-PLGA group,804.43±86.44 (cells/mm2) in PBS group, 821.72±52.13 (cells/mm2) in PLGA group 4 weeks after operation, and 532.74±35.14 (cells/mm2) in untreated group,1083.66±57.82 (cells/mm2) in EPO group, 913.98±37.72 (cells/mm2) in EPO-PLGA group,548.01±47.00 (cells/mm2) in PBS group,561.96±34.23 (cells/mm2) in PLGA group 8 weeks after crush. Compared to untreated, PBS and PLGA group, EPO and EPO-PLGA group had significantly neuroprotective effect on RGCs(P<0.001), and no significant differences were found between these two groups(P>0.05). TUNEL-positive cells could be identified in all groups 1 day,5 days,2 weeks and 4 weeks after operation. The positive cells in EPO and EPO-PLGA group were significantly fewer than other groups (p<0.001). However, no profound differences were found between groups 2 and 4 weeks after crush (p>0.05). RGCs nucleus DAPI staining revealed that the number of RGCs in EPO and EPO-PLGA group was statistically more than other groups (p<0.01). Immunohistochemistry also suggested that GFAP expressed in retina in EPO and EPO-LGA group were significant difference from other groups (p<0.05).Conclusion Single intravireal injection of EPO-PLGA microspheres appeared to have a long-term neuroprotective effect on retinal ganglion cells after optic nerve crush in adult rats with little ocular damage. It provides a possibility of protein delivery system to retinal disease in the future.