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Aging and disease    2017, Vol. 8 Issue (4) : 410-419     DOI: 10.14336/AD.2016.1209
Original Article |
Effects of Erythropoietin on Gliogenesis during Cerebral Ischemic/Reperfusion Recovery in Adult Mice
Wang Rongliang1,2,3, Li Jincheng4, Duan Yunxia2,3, Tao Zhen1,2,3, Zhao Haiping1,2,3,*, Luo Yumin1,2,3,*
1Cerebrovascular Diseases Research Institute and Department of Neurology, Xuanwu Hospital of Capital Medical University, Beijing 100053, China
2Beijing Institute for Brain Disorders, Beijing 100053, China
3Beijing Key Laboratory of Translational Medicine for Cerebrovascular Diseases, Beijing 100053, China
4Department of Neurology, Zibo Central Hospital, Zibo 255036, China
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Abstract  

Erythropoietin (EPO) promotes oligodendrogenesis and attenuates white matter injury in neonatal rats. However, it is unknown whether this effect extends to adult mice and whether EPO regulate microglia polarization after ischemic stroke. Male adult C57BL/6 mice (25–30g) were subjected to 45 min of middle cerebral artery occlusion (MCAO). EPO (5000 IU/kg) or saline was injected intraperitoneally every other day after reperfusion. Neurological function was evaluated using the rotarod test at 1, 3, 7 and 14 days after MCAO. Brain tissue loss volume was determined by hematoxylin-eosin staining. Immunofluorescence staining and Western blot were also used to assess the severity of white matter injury and phenotypic changes in microglia/macrophages. Bromodeoxyuridine (BrdU) was injected intraperitoneally daily for 1 week to analyze the number of newly proliferating glia cells (oligodendrocytes, microglia, and astrocytes). We found that EPO significantly reduced Brain tissue loss volume, ameliorated white matter injury, and improved neurobehavioral outcomes at 14 days after MCAO (P<0.05). In addition, EPO also increased the number of newly generated oligodendrocytes and attenuated the rapid hypertrophy and hyperplasia of microglia and astrocytes after ischemic stroke (P<0.05). Furthermore, EPO reduced M1 microglia and increased M2 microglia (P<0.05). Taken together, our results suggest that EPO treatment improves white matter integrity after cerebral ischemia, which could be attributed to EPO attenuating gliosis and facilitating the microglial polarization toward the beneficial M2 phenotype to promote oligodendrogenesis.

Keywords Brain ischemia      Erythropoietin      Microglial polarization      Gliogenesis      White matter injury     
Corresponding Authors: Zhao Haiping,Luo Yumin   
About author:

these authors equally contributed to this work

Issue Date: 01 August 2017
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Wang Rongliang
Li Jincheng
Duan Yunxia
Tao Zhen
Zhao Haiping
Luo Yumin
Cite this article:   
Wang Rongliang,Li Jincheng,Duan Yunxia, et al. Effects of Erythropoietin on Gliogenesis during Cerebral Ischemic/Reperfusion Recovery in Adult Mice[J]. Aging and disease, 2017, 8(4): 410-419.
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http://www.aginganddisease.org/EN/10.14336/AD.2016.1209     OR     http://www.aginganddisease.org/EN/Y2017/V8/I4/410
Figure 1.  EPO reduces brain tissue loss volume and improves recovery of neurological function after MCAO

Experimental design. Mice received EPO or saline intraperitoneally (i.p.) every other day until day 11 (A). Representative pictures and quantification of brain tissue loss volume at 14 days after I/R. (B, C). The brain tissue loss for each section was measured using this equation: mean brain tissue loss volume (%) = (Area a - Area b)/Area a × 100%. Scale bar, 1 mm. n=4 per group. Neurological function was assessed by the Rotarod test (D). *P≤0.05, **P≤0.01 vs. sham; #P≤0.05 vs. I/R vehicle. n=9 per group.

Figure 2.  EPO attenuates white matter injury after MCAO

MBP (red) and NF-200 (green) immunostaining 14 days after I/R (A-C). Boxes in (A) indicate the selected areas of the cortex (CTX), corpus callosum (CC), and striatum (ST). Scale bar, 1 mm. Immunostaining of MBP and NF-200 in the CTX, CC, and ST in the ipsilateral hemisphere (B). Scale bar, 50 μm. Quantification of the relative ratio of NF-200 vs. MBP immunostaining intensity in ipsilateral hemispheres (C). Relative expression levels of myelin proteins (CNPase and MBP) in the ipsilateral brain tissue of different groups (D). β-actin served as a loading control. n=4 per group. Representative western blots for results in D (E-F). *P≤0.05, **P≤0.01 vs. sham; #P≤0.05 vs. I/R vehicle. n=4 per group.

Figure 3.  EPO promotes the M2 microglial phenotype after MCAO

Double immunofluorescent staining for M1 marker (CD16) or M2 marker (CD206) (red) with Iba1 marker (green) for activated microglia in the peri-infarct region at 14 days following I/R injury (A). Scale bar, 100 μm. Quantification of microglia cells immunopositive for CD16 and CD206 (B). Protein expression levels of M1 (CD16 and CD11b) and M2 (CD206) markers at 14 days following I/R injury (C, D). *P≤0.05, **P≤0.01 vs. sham; #P≤0.05 vs. I/R vehicle. n=4 per group.

Figure 4.  EPO enhances the generation of new oligodendrocytes and decreases gliogenesis after MCAO

Colocalization of 5′-bromo-deoxyuridine (BrdU; green) and glia cells (CNPase, Iba1 and GFAP; red) in the peri-infarct region at 14 d following I/R injury (A). Scale bar, 100 μm. Representative image showing colocalization of BrdU+/CNPase+, BrdU+/Iba1+, BrdU+/GFAP+, and DAPI (blue) staining at high magnification (B). Scale bar, 20 μm. Numbers of BrdU and glia double-positive cells expressed as cells/mm2 (C). *P≤0.05, **P≤0.01 vs. sham; #P≤0.05 vs. I/R vehicle. n=4 per group.

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