Erythropoietin (EPO) is a cytokine known to have effective cytoprotective action in the brain, particularly in ischemic, traumatic, inflammatory, and neurodegenerative conditions. We previously reported the neuroprotective effect of a low sialic form of EPO, Neuro-EPO, applied intranasally in rodent models of stroke or cerebellar ataxia and in a non-transgenic mouse model of Alzheimer's disease (AD). Here we analyzed the protective effect of Neuro-EPO in APPSwe mice, a reference transgenic mouse model of AD. Mice were administered 3 times a day, 3 days in the week with Neuro-EPO (125, 250 μg/kg) intranasally, between 12 and 14 months of age. Motor responses, general activity, and memory responses were analyzed during and after treatment. The deficits in spontaneous alternation, place learning in the water-maze, and novel object recognition observed in APPSwe mice were alleviated by the low dose of Neuro-EPO. Oxidative stress, neuroinflammation, trophic factor levels, and a synaptic marker were analyzed in the hippocampus or cortex of the animals. The increases in lipid peroxidation or in GFAP and Iba-1 contents in APPSwe mice were significantly reduced after Neuro-EPO. Activation of intrinsic and extrinsic apoptotic pathways was analyzed. The increases in Bax/Bcl-2 ratio, TNFα, or Fas ligand levels observed in APPSwe mice were reduced by Neuro-EPO. Finally, immunohistochemical and ELISA analyses of Aβ1-42 levels in the APPSwe mouse cortex and hippocampus showed a marked reduction in Aβ deposits and in soluble and insoluble Aβ1-42 forms. This study therefore confirmed the neuroprotective activity of EPO, particularly for an intranasally deliverable formulation, devoid of erythropoietic side effects, in a transgenic mouse model of AD. Neuro-EPO alleviated memory alterations, oxidative stress, neuroinflammation, apoptosis induction, and amyloid load in 14-month-old APPSwe mice.

Erythropoietin (EPO) promotes neurogenesis and neuroprotection. We here compared the protection induced by two EPO formulations in a rodent model of Alzheimer's disease (AD): rHu-EPO and a low sialic form, Neuro-EPO. We used the intracerebroventricular administration of aggregated Aβ₂₅₋₃₅ peptide, a non-transgenic AD model. rHu-EPO was tested at 125-500 µg/kg intraperitoneally and Neuro-EPO at 62-250 µg/kg intranasally (IN). Behavioural procedures included spontaneous alternation, passive avoidance, water-maze and object recognition, to address spatial and non-spatial, short- and long-term memories. Biochemical markers of Aβ₂₅₋₃₅ toxicity in the mouse hippocampus were examined and cell loss in the CA1 layer was determined. rHu-EPO and Neuro-EPO led to a significant prevention of Aβ₂₅₋₃₅-induced learning deficits. Both EPO formulations prevented the induction of lipid peroxidation in the hippocampus, showing an antioxidant activity. rHu-EPO (250 µg/kg) or Neuro-EPO (125 µg/kg) prevented the Aβ₂₅₋₃₅-induced increase in Bax level, TNFα and IL-1β production and decrease in Akt activation. A significant prevention of the Aβ₂₅₋₃₅-induced cell loss in CA1 was also observed. EPO is neuroprotective in the Aβ₂₅₋₃₅ AD model, confirming its potential as an endogenous neuroprotection system that could be boosted for therapeutic efficacy. We here identified a new IN formulation of EPO showing high neuroprotective activity. Considering its efficacy, ease and safety, IN Neuro-EPO is a new promising therapeutic agent in AD.

Vascular illness of the brain constitutes the third cause of death and the first cause of disability in Cuba and many other countries. Presently, no medication has been registered as a neuroprotector. Neuroprotection with intranasal Neuro-EPO (EPO, erythropoietin) has emerged as a multifunctional therapy that plays a significant role in neural survival and functional recovery in an animal model of stroke. On the other hand, there is limited access to the brain through the blood brain barrier (BBB) for intravenously applied EPO, and the high EPO dosages needed to obtain a protective effect increase the danger of elevated hematocrit levels and practically exclude chronic or subchronic treatment with EPO. A promising approach has been recently developed with a nonerythropoietic variant of EPO, Neuro-EPO, with low sialic acid content, a very short plasma half-life, and without erythropoietic activity, probably similar to endogenous brain EPO. The objective of this work was to determine the neuroprotective effect of intranasal Neuro-EPO in comparison with the human recombinant EPO injected intraperitoneally in the acute phase of cerebral ischemia, employing the common carotid artery occlusion model in gerbils. Neuro-EPO has demonstrated a better neuroprotective effect, evidenced through increased viability, improvements of the neurological state and cognitive functions, as well as protection of the CA3 region of the hippocampus, temporal cortex, and the thalamus. In conclusion, the intranasal application of Neuro-EPO has a better neuroprotective effect than intraperitoneal EPO, evidenced by the significant improvement of neurological, cognitive, and histological status in the animal model of stroke employed.

The purpose of this study was to evaluate the neuroprotective effects of intranasally delivered recombinant human neuronal erythropoietin (Neuro-EPO) on brain injury induced by unilateral permanent ischemia in the Mongolian gerbil. Expression of EPO receptor (EPOR) and neuroglobin (Ngb) over 5 weeks after intranasal treatment with Neuro-EPO was determined using immunohistochemistry. Mortality of Neuro-EPO-treated gerbils decreased after surgery, and the sensory and motor function was significantly improved. Histopathological mapping showed that Neuro-EPO significantly reduced delayed neuronal death in the brain. Expression of Ngb was upregulated in the cerebral cortex at most time points (expect for 10 min and 48 hr) and in the hippocampus at 10 min and from 48 hr to 5 weeks, whereas EPOR was almost downregulated or unchanged in the brain (expect for 48 hr). The 10 min and 48 hr seemed to be two time points for the brain to switch the expression of both Ngb and EPOR to early and late recovery phase, respectively. In addition, there were two phases, 10 min to 1 hr and 24 hr to 72 hr, respectively, closing to the "golden hour" of about 60 min and the "silver day" of 1 to 3 days, for the brain to recover from stroke onset with intranasal Neuro-EPO treatment. Therefore, the results suggest that the intranasal administration of Neuro-EPO is effective in the treatment of acute brain ischemia. The different expression patterns of Ngb and EPOR is probably due to ischemic tolerance in the cerebral cortex and ischemic sensitivity in the hippocampus.

Cerebrovascular disease is the third leading cause of death and the leading cause of disability in Cuba and in several developed countries. A possible neuroprotective agent is the rHu-EPO, whose effects have been demonstrated in models of brain ischemia. The Neuro-EPO is a derivative of the rHu-EPO that avoids the stimulation of erythropoiesis. The aim of this study was to determine the Neuro-EPO delivery into the central nervous system (CNS) to exert a neuroprotective effect in cerebral ischemia model of the Mongolian gerbil. The Neuro-EPO in a rate of 249.4 UI every 8 hours for 4 days showed 25% higher viability efficacy (P > 0.01), improving neurological score and behavior of the spontaneous exploratory activity, the preservation of CA3 areas of the hippocampus, the cortex, and thalamic nuclei in the focal ischemia model of the Mongolian gerbil. In summary, this study, the average dose-used Neuro-EPO (249.4 UI/10 μL/every 8 hours for 4 days), proved to be valid indicators of viability, neurological status, and spontaneous exploratory activity, being significantly lower than that reported for the systemically use of the rHu-EPO as a neuroprotectant. Indeed, up to 12 h after brain ischemia is very positive Neuro-EPO administration by the nasal route as a candidate for neuroprotection.