Ocular Erythropoietin Penetration after Subconjunctival Administration in Glaucomatous Rats

Abstract

Purpose: The present study aimed to determine whether the subconjunctival administration of recombinant human erythropoietin (rHuEPO) reached the retina in glaucoma conditions. After subconjunctival rHuEPO administration, in a rat glaucoma model, erythropoietin (EPO) distribution in the rat's retina was studied by immunohistochemistry. Methods: Female Wistar Hannover albino rats (n = 15) were divided into 2 groups, control (n = 3) and treated (n = 12). The animals' unilateral glaucoma was induced by coagulation of episcleral veins, under general anaesthesia. After vein coagulation, 1,000 IU of rHuEPO were administered by the subconjunctival route to the treated group (n = 12). The control group (n = 3) received only a subconjunctival saline injection. The contralateral eye of each animal remained untouched. Treated group animals were euthanized at different time points, i.e. days 1, 3, 7 and 14. Bilateral enucleation was performed, and EPO distribution in the rat's retina was assessed by immunohistochemistry. Results: Glaucoma was confirmed by results of repeated intraocular pressure measurements over the experimental period. In the test group, EPO was identified in different neuroretinal cells, showing a stronger immunostaining signal during the first 2 time points in the retinal ganglion cell (RGC) layer. EPO protein was still present on day 14 after the subconjunctival injection. EPO was not detected in any of the control eyes or in any contralateral eye of the treated group. Conclusion: When administered subconjunctivally to glaucomatous eyes, rHuEPO reached the RGC layer and was still present at least 14 days after administration. The subconjunctival route was shown to be a promising alternative for ocular EPO delivery in glaucomatous conditions in a rat animal model.

Functional and Structural Effects of Erythropoietin Subconjunctival Administration in Glaucomatous Animals

“…Since glaucoma is a chronic disease, multiple treatments to protect RGCs are required, and using the subconjunctival route seems to be more feasible. Concerning subconjunctival EPO administration, we also demonstrated that rHuEPO reached the RGC layers when administered by this route both in physiological and glaucomatous conditions without significant unwanted side effects [20,21].…”

“…It has been previously demonstrated that EPO reached the RGC layer both in physiological and glaucoma conditions in a rat animal model when administered through the subconjunctival route [20,21]. The subconjunctival route for EPO ocular administration has been demonstrated to be a safe and easy procedure with few associated risks [19,22].…”

Chitosan and Hyaluronic Acid Nanoparticles as Vehicles of Epoetin Beta for Subconjunctival Ocular Delivery

“…A recent review article describes the antiapoptotic, angiogenic, antiinflammatory, antioxidant, and neuroprotective effects of EPO in the ocular tissues, including the retina [25]. Subconjunctival administration of EPOβ in solution (NeoRecormon ® ) in rats was already performed by our team, which proved that EPOβ reached the inner layers of the retina with one single dose [8][9][10]. Due to the minor-invasive nature of this route of administration, it was selected to administer CS/HA-EPOβ nanoparticles to rats to assess its safety and ocular penetration in healthy animals.…”

“…The nanoparticles allowed EPOβ to trespass the outer layers of the ocular globe to reach the inner layers after only 12 h. This fast delivery to the retina might be attributed to the hydrophilic nature of EPOβ, due to its high degree of glycosylation [26], which means that the preferential route of EPOβ absorption would be the conjunctival-scleral pathway, as it is more successful to the posterior segment delivery [35]. In previous studies, EPOβ was also detected in the retina after subconjunctival administration of a commercial solution (NeoRecormon ® ) [8,9]. Moreover, in this study, EPOβ was likewise detected in the corneal endothelium and ciliary body, and the conjunctival-scleral absorption would be unlikely to contribute to this finding due to the presence of the blood-aqueous barrier [36].…”

“…However, novel EPO secreting sites were discovered, like the brain [3] and the retina [4], which have also demonstrated the expression of EPO receptors [5,6]. Recent studies demonstrated that EPO has tissue-protective properties in the brain, heart, inner ear, and retina [7], and the subconjunctival administration of EPO, as an alternative delivery method aiming at the posterior ocular segment, has been explored with promising results [8,9]. Recent investigation with glaucomatous rats demonstrated that epoetin beta (EPOβ), a recombinant version of EPO, administered by subconjunctival route, reached the retina and improved its condition when compared to non-treated animals [10].…”