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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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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   
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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
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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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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.

[1] Chen Y, Yi Q, Liu G, Shen X, Xuan L, Tian Y (2013). Cerebral white matter injury and damage to myelin sheath following whole-brain ischemia. Brain Res, 1495: 11-17.
[2] Ho PW, Reutens DC, Phan TG, Wright PM, Markus R, Indra I,et al. (2005). Is white matter involved in patients entered into typical trials of neuroprotection? Stroke, 36: 2742-2744.
[3] Matute C, Domercq M, Pérezsamartín A, Ransom BR (2012). Protecting white matter from stroke injury. Stroke, 44: 1204-1211.
[4] Mciver SR, Muccigrosso M, Gonzales ER, Lee JM, Roberts MS, Sands MS,et al. (2010). Oligodendrocyte degeneration and recovery after focal cerebral ischemia. Neuroscience, 169: 1364-1375.
[5] Goldman SA, Osorio J (2014). So many progenitors, so little myelin. Nat Neurosci, 17: 483-485.
[6] Han L, Cai W, Mao L, Liu J, Li P, Leak RK,et al. (2015). Rosiglitazone Promotes white matter integrity and long-term functional recovery after focal cerebral ischemia. Stroke, 46: 2628-2636.
[7] Perry VH, Nicoll JA, Holmes C (2010). Microglia in neurodegenerative disease. Nat Rev Neurol, 6: 193-201.
[8] Hu X, Li P, Guo Y, Wang H, Leak RK, Chen S,et al. (2012). Microglia/macrophage polarization dynamics reveal novel mechanism of injury expansion after focal cerebral ischemia. Stroke, 43: 3063-3070.
[9] Wang G, Zhang J, Hu X, Zhang L, Mao L, Jiang X,et al. (2013). Microglia/macrophage polarization dynamics in white matter after traumatic brain injury. J Cereb Blood Flow Metab, 33: 1864-1874.
[10] Hao Z, Garton T, Keep RF, Hua Y, Xi G, (2015). Microglia/Macrophage polarization after experimental intracerebral hemorrhage. Transl Stroke Res, 6: 407-409.
[11] Miron VE, Boyd A, Zhao JW, Yuen TJ, Ruckh JM, Shadrach JL,et al. (2013). M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination. Nat Neurosci, 16: 1211-1218.
[12] Hu X, Leak RK, Shi Y, Suenaga J, Gao Y, Zheng P,et al. (2015). Microglial and macrophage polarization—new prospects for brain repair. Nat Rev Neurol, 11: 56-64.
[13] Iwai M, Stetler RA, Xing J, Hu X, Gao Y, Zhang W,et al. (2010). Enhanced oligodendrogenesis and recovery of neurological function by erythropoietin after neonatal hypoxic/ischemic brain injury. Stroke, 41: 1032-1037.
[14] Bond WS, Rex TS (2014). Evidence that erythropoietin modulates neuroinflammation through differential action on neurons, astrocytes, and microglia. Front Immunol, 5: 523.
[15] Ma Q, Zhao H, Tao Z, Wang R, Liu Pet, Han Z,et al. (2016). MicroRNA-181c exacerbates brain injury in acute ischemic stroke. Aging Dis, 7:705-714.
[16] Matute C, Domercq M, Pérezsamartín A, Ransom BR (2012). Protecting white matter from stroke injury. Stroke, 44: 1204-1211.
[17] Shi H, Hu X, Leak RK, Shi Y, An C, Suenaga J,et al. (2015). Demyelination as a rational therapeutic target for ischemic or traumatic brain injury. Exp Neurol, 272: 17-25.
[18] Back SA, Tuohy TM, Chen H, Wallingford N, Craig A, Struve J,et al. (2005). Hyaluronan accumulates in demyelinated lesions and inhibits oligodendrocyte progenitor maturation. Nat Med, 11: 966-972.
[19] Zanier ER, Pischiutta F, Riganti L, Marchesi F, Turola E, Fumagalli S, et al. (2014). Bone marrow mesenchymal stromal cells drive protective M2 microglia polarization after brain trauma. Neurotherapeutics. 11: 679-695.
[20] Aungst SL, Kabadi SV, Thompson SM, Stoica BA, Faden AI (2014). Repeated mild traumatic brain injury causes chronic neuroinflammation, changes in hippocampal synaptic plasticity, and associated cognitive deficits. J Cereb Blood Flow Metab, 34: 1223-1232.
[21] David S, Kroner A (2011). Repertoire of microglial and macrophage responses after spinal cord injury. Nat Rev Neurosci, 12: 388-399.
[22] Suenaga J, Hu X, Pu H, Shi Y, Hassan SH, Xu M,et al. (2015). White matter injury and microglia/macrophage polarization are strongly linked with age-related long-term deficits in neurological function after stroke. Exp Neurol, 272: 109-119.
[23] Wang G, Shi Y, Jiang X, Leak RK, Hu X, Wu Y,et al. (2015). HDAC inhibition prevents white matter injury by modulating microglia/macrophage polarization through the GSK3β/PTEN/Akt axis. Proc Natl Acad Sci U S A, 112: 2853-2858.
[24] Iwai M, Cao G, Yin W, Stetler RA, Liu J, Chen J (2007). Erythropoietin promotes neuronal replacement through revascularization and neurogenesis after neonatal hypoxia/ischemia in rats. Stroke, 38: 2795-2803.
[25] Kuhn HG (2015). Control of cell survival in adult mammalian neurogenesis. Cold Spring Harb Perspect Biol.7. pii: a018895.
[26] Frisén J (2016). Neurogenesis and gliogenesis in nervous system plasticity and repair. Annu Rev Cell Dev Biol, 32: 127-141.
[27] Lynch AM, Murphy KJ, Deighan BF, O’Reilly JA, Gun’Ko YK, Cowley TR,et al. (2010). The impact of glial activation in the aging brain. Aging Dis, 1: 262-278.
[28] McKeon RJ, Schreiber RC, Rudge JS, Silver J (1991). Reduction of neurite outgrowth in a model of glial scarring following CNS injury is correlated with the expression of inhibitory molecules on reactive astrocytes. J Neurosci, 11: 3398-3411.
[29] Vitellaro-Zuccarello L, Mazzetti S, Madaschi L, Bosisio P, Fontana E, Gorio A,et al. (2008). Chronic erythropoietin-mediated effects on the expression of astrocyte markers in a rat model of contusive spinal cord injury. Neuroscience, 151: 452-466.
[30] Susarla BT, Villapol S, Yi JH, Geller HM1, Symes AJ. (2014). Temporal patterns of cortical proliferation of glial cell populations after traumatic brain injury in mice. ASN Neuro, 6: 94-107.
[31] Xu W, Mu X, Wang H, Song C, Ma W, Jolkkonen J. (2016). Chloride co-transporter NKCC1 inhibitor bumetanide enhances neurogenesis and behavioral recovery in rats after experimental stroke. Mol Neurobiol, 1-9.
[32] Ma M, Ma Y, Yi X, Guo R, Zhu W, Fan X,et al. (2008). Intranasal delivery of transforming growth factor-beta1 in mice after stroke reduces infarct volume and increases neurogenesis in the subventricular zone. BMC Neurosci, 9: 11
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