Biologicals and biotherapeutics
MEPO promotes neurogenesis and angiogenesis but suppresses gliogenesis in mice with acute ischemic stroke

https://doi.org/10.1016/j.ejphar.2019.01.066Get rights and content

Abstract

Previously study has proved the non-erythropoietic mutant erythropoietin (MEPO) exerted neuroprotective effects against ischemic cerebral injury, with an efficacy similar to that of wild-type EPO. This study investigates its effects on neurogenesis, angiogenesis, and gliogenesis in cerebral ischemic mice. Male C57BL/6 mice were subjected to middle cerebral artery occlusion (MCAO) and reperfusion. EPO (5000 U/kg), MEPO (5000 U/kg) or equal volume of normal saline was injected intraperitoneally. Neurological function was evaluated by Rota-rod test, Neurological severity scores (NSS) and Adhesive removal test. After ischemia and reperfusion (I/R), the survival rate, brain tissue loss, neurogenesis, angiogenesis and gliogenesis were detected by Nissl staining, Immunofluorescence and Western blot, respectively. The results shown that MEPO significantly increased survival rate, reduced brain tissue loss, and improved neurological function after MCAO (P < 0.05). Furthermore, MEPO obviously enhanced the proliferation of neuronal precursors (DCX) and promoted its differentiation into mature neurons (NeuN) (P < 0.05). In addition, compared to normal saline treatment mice, MEPO increased the number of BrdU-positive cells in the cerebral vasculature (P < 0.05). Whereas, MEPO treatment also reduced the numbers of newly generated astrocytes (GFAP) and microglia (Iba1) (P < 0.05). Among all the tests in this study, there was no significant difference between EPO group and MEPO group. Taken together, MEPO promoted the regeneration of neurons and blood vessels in peripheral area of infarction, and suppressed the gliogenesis, thus promoting neurogenesis, improving neurological function and survival rate. Our findings suggest that the MEPO may be a therapeutic drug for ischemic stroke intervention.

Introduction

Erythropoietin (EPO) is a hematopoiesis factor, which is mainly secreted by the kidney and liver that regulates erythropoiesis. As a neuroprotective agent in cerebral ischemia, its protective effects have been proved by large number of preclinical and some clinical studies in the last two decades (Ehrenreich et al., 2002, Wu et al., 2009, Nguyen et al., 2014, Tsai et al., 2015). Although in the German Multicenter Erythropoietin Stroke Trial (NCT00604630) and the following experimental studies indicated that the combination of rtPA and EPO treatment maybe not suitable for patients with cerebral ischemic stroke (Ehrenreich et al., 2009, Jia et al., 2010, Zechariah et al., 2010). However, only less than 5% of stroke patients fit the criteria for tPA therapy due to its seriously side effects of intracranial hemorrhage and its narrow therapeutic time-window (less than 6 h) (National Institute of Neurological and Stroke rt, 1995, Soeteman et al., 2017). Therefore, EPO is still an alternative neuroprotectant for most patients with acute cerebral ischemia (Ehrenreich et al., 2002).

Less than 1% efficient EPO can across the blood-brain barrier into the brain parenchyma through systemic administration, consequently, high-doses EPO are required to achieve effective therapeutically concentrations (Brines et al., 2000). However, high-dose and multiple administration of EPO was associated with unexpected side effects in clinical research, such as increases risk of thrombosis and secondary infarction, which restricts its application in clinical treatment. In light of recently studies have shown that EPO's hematological and tissue-protective activities were distinct and separate through interaction with different receptors (Brines et al., 2004, Leist et al., 2004). Accordingly, alternate strategies to abolish its erythropoietic activity and reserve the tissue-protective through chemical and genetic modification will greatly improve its clinical applicability for ischemic stroke treatment. Recent years, several types of EPO derivatives that lack hematopoietic but retain tissue-protection activity have been generated, which include asialo-EPO (Erbayraktar et al., 2003), carbamylated EPO (Leist et al., 2004), neuro-EPO (Parra and Rodriguez, 2012) and ARA290, a peptide derived from erythropoietin (Swartjes et al., 2014). However, the very short plasma half-life, complex chemical modifications and high production costs restricted its application in brain injuries. We constructed the MEPO (S104I-EPO) containing a single amino acid mutation within the erythropoietic motif, which completely lacks erythropoietic activity but retains neuroprotective effects against ischemic brain injury in mice, with an efficacy similar to that of wild-type EPO (Gan et al., 2012). In this study, we used a well-established mouse model of transient focal cerebral ischemia to further evaluate the effects of MEPO on neurogenesis, angiogenesis, and gliogenesis after cerebral ischemia. The results demonstrated that MEPO treatment enhanced the proliferation of neuronal precursors and mature neurons, increased the newly generated cells in blood vessels, and suppressed newly proliferating cells of astrocytes and microglia in peri-infarct area, thus improved neurological function and survival rate after cerebral ischemic injury. Our investigation will provide a reliable basis for clinical trials of MEPO.

Section snippets

Experiment animals and middle cerebral artery occlusion (MCAO) model

Male C57BL/6 mice (25–30 g, 2-month-old) were purchased from Vital River Laboratory Animal Technology Co. Ltd and approved by the Institutional Animal Care and Use Committee of Capital Medical University. Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO), with a nylon filament silicon tip (0.19 mm diameter) inserted to the right middle cerebral artery (MCA) to obstruct the blood flow. Forty-five min after occlusion, the filament was removed to allow for reperfusion.

MEPO provides neuroprotective effects against cerebral ischemic injury

We first verified whether MEPO treatment could exert similar neuroprotective effects as EPO in the early recovery periods of cerebral ischemic injury. The result shown that MEPO treatment remarkedly improved the survival rate compared to I/R+Veh group until 28 days after MCAO surgery (Fig. 1A).

Neurobehavioral tests were performed at 1, 3, 7, 14, 21, and 28 days after MCAO by two investigators who were blinded to the experimental groups. The Rota-rod test indicated MEPO treatment obviously

Discussion

Exogenously administered EPO has reported to provide multiple neuroprotective effects in the animal models of cerebral ischemia and in ischemic stroke patients for many years, through the anti-inflammatory, anti-oxidant and anti-apoptotic effects, as well as through the stimulation of neurogenesis and angiogenesis (Villa et al., 2003, Iwai et al., 2007, Patel et al., 2012). Unfortunately, the serious adverse effects of wide type EPO or recombinant human EPO severely limits its clinical

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81471340, 81801149, 81641054), China, and National Institutes of Health/NINDS grants NS079345, United States.

Declarations of interest

None.

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    These authors contributed equally to the paper.

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