Recombinant Erythropoietin Induces Oligodendrocyte Progenitor Cell Proliferation After Traumatic Brain Injury and Delayed Hypoxemia

Abstract

Traumatic brain injury (TBI) can result in axonal loss and demyelination, leading to persistent damage in the white matter. Demyelinated axons are vulnerable to pathologies related to an abnormal myelin structure that expose neurons to further damage. Oligodendrocyte progenitor cells (OPCs) mediate remyelination after recruitment to the injury site. Often this process is inefficient due to inadequate OPC proliferation. To date, no effective treatments are currently available to stimulate OPC proliferation in TBI. Recombinant human erythropoietin (rhEPO) is a pleiotropic neuroprotective cytokine, and its receptor is present in all stages of oligodendroglial lineage cell differentiation. Therefore, we hypothesized that rhEPO administration would enhance remyelination after TBI through the modulation of OPC response. Utilizing a murine model of controlled cortical impact and a primary OPC culture in vitro model, we characterized the impact of rhEPO on remyelination and proliferation of oligodendrocyte lineage cells. Myelin black gold II staining of the peri-contusional corpus callosum revealed an increase in myelinated area in association with an increase in BrdU-positive oligodendrocytes in injured mice treated with rhEPO. Furthermore, morphological analysis of OPCs showed a decrease in process length in rhEPO-treated animals. RhEPO treatment increased OPC proliferation after in vitro CSPG exposure. Erythropoietin receptor (EPOr) gene knockdown using siRNA prevented rhEPO-induced OPC proliferation, demonstrating that the rhEPO effect on OPC response is EPOr activation dependent. Together, our findings demonstrate that rhEPO administration may promote myelination by increasing oligodendrocyte lineage cell proliferation after TBI.

Keywords: Erythropoietin; Myelination; Oligodendrocyte progenitor cells; Traumatic brain injury; White matter injury.

Fig. 1

RhEPO administration reduces myelin loss after TBI and delayed hypoxemia. a Experimental design: rhEPO injections were performed at days 1, 2, and 3 followed by a weekly injection for a month after TBI; hypoxia was induced 8 h after the first rhEPO administration. b Representative images of the CC (white dashed line) and hippocampus (black dashed line) areas stained with BGII. c Quantification of percentage of the myelinated area of CC F_(2,15) = 19.92 ***p < 0.005. d Quantification of percentage of the myelinated area of the hippocampus F_(2,15) = 9.294 **p = 0.024. e Representative images of the CC and hippocampus areas stained with dMBP. f Quantification of percentage of the dMBP-stained area of the CC F_(2,10) = 4.265 *p = 0.0458. Mean values are plotted ± SEM. One-way ANOVA followed by Tukey multiple comparison post hoc test was used to determine statistical differences; n = 4–7 mice per group. Scale bar = 200 µm and 50 µm in the inserts. Abbreviations: CCI: controlled cortical impact; BGII: myelin black gold II; rhEPO: recombinant human erythropoietin, Veh: vehicle. dMBP: degraded myelin basic protein

Fig. 2

RhEPO stimulates oligodendrocyte lineage cell proliferation after TBI in the CC. a Representative fluorescent images of Olig1⁺/BrdU⁺ cells (white arrows) in the pericontusional CC. b Quantification of Olig1⁺ and BrdU⁺ cell density F_(2,15) = 34.65 p < 0.005. c Representative fluorescent images of PDGFr-α and Olig1 in the CC. d Quantification of PDGFr-α⁺ cell density. e Representative three-dimensional reconstruction images of OPCs. f Quantification of PDGFr-α⁺ Olig1⁻ cell density. g Representative high-magnification images of Olig1 and BrdU images. h Quantification of the Olig1 percent area of staining. i Representative high-magnification images of Olig1 and PDGFr-α. Quantification of OPC morphology j process length, k number of segments, l number of terminal points, and m volume. One-way ANOVA followed by Tukey multiple comparison post hoc test were used to determine statistical significance. ***p < 0.0005, n = 3–7 mice per group. Scale bar = 200 µm in a and c: scale bar = 10 µm in e. Abbreviations: CCI: controlled cortical impact; BrdU: 5-bromo-2′-deoxyuridine, EPO: erythropoietin, Veh: vehicle

Fig. 3

RhEPO increases early OPC populations after TBI in the DG. a Representative fluorescent images of Olig1⁺/BrdU⁺ cells (white arrows) in DG. b Quantification of Olig1⁺ and BrdU⁺ cell density F_{(2, 9)} = 12.53, p = 0.0025. c Representative fluorescent images of PDGFr-α and Olig1 in DG. d Quantification of PDGFr-α⁺ cell density. e Representative three-dimensional reconstruction images of OPCs. f Quantification of PDGFr-α⁺ Olig1⁻ cell density F_{(2, 26)} = 13.08, p < 0.0001. g Representative high-magnification images of Olig1 and BrdU images. h Quantification of the Olig1 percent area of staining F_{(2, 9)} = 13.40, p = 0.002. i Representative high-magnification images of Olig1 and PDGFr-α. Quantification of OPC morphology j process length, F_{(2, 24)} = 77.90, p < 0.0001, k number of segments, F_{(2, 24)} = 33.05, p < 0.0001, l number of terminal points, F_{(2, 24)} = 35.32, p < 0.0001, m volume, F_{(2, 24)} = 12.15, p = 0.0002. Mean values are plotted ± SD; one-way ANOVA followed by Tukey multiple comparison post hoc test was used to determine statistical significance, ****p < 0.0001; *p < 0.05, n = 4–12 mice per group. Scale bar = 200 µm in a and c: scale bar = 10 µm in e. Abbreviations: CCI: controlled cortical impact, EPO: erythropoietin, Veh: vehicle

Fig. 4

CSPG-induced OPC injury is rescued by rhEPO treatment in vitro. a Experimental design. b Representative fluorescent image of PDGFr-α and EPOr cells. c Representative fluorescent images of PDGFr-α- and Olig1-positive cells. d Quantification of Olig1⁺ cell density, F_{(1, 34)} = 14.88, p < 0.0005. e Quantification of PDGFr-α⁺ cell density F_{(1, 36)} = 12.89, p < 0.001. f Quantification of BrdU⁺ cell density, F_{(1, 34)} = 7.205, p < 0.011. g Representative fluorescent images of BrdU-positive cells. h Mander’s coefficient of BrdU and DAPI, F_{(1, 34)} = 7.173, p < 0.0112. i Quantification of DAPI density F_{(1, 34)} = 10.96, p < 0.0022. Mean values are plotted ± SEM; two-way ANOVA followed by Tukey multiple comparison post hoc test was used to determine statistical significance. *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001, n = 10 wells per group. Abbreviations: CSPG: chondroitin sulfate proteoglycan, EPO: erythropoietin, FOV: field of view, PFA: paraformaldehyde

Fig. 5

RhEPO rescues oligodendrocyte precursor cells after CSPG exposure. a Gating strategy. b Schematic representation of oligodendrocyte differentiation. c Representation of the % from CD45⁻CD11b⁻ mature oligodendrocytes, premyelinating oligodendrocytes, pre-oligodendrocytes, late OPCs, and early OPCs. d Quantification of the % from CD45⁻CD11b⁻ late OPCs (PDGFr-α⁺O4⁺), F_{(1, 15)} = 36.97, p < 0.0001. e Quantification of the % from CD45⁻CD11b⁻ pre-oligodendrocytes (PDGFr-α⁻O4⁺), F_{(1, 15)} = 19.54, p < 0.0005. f Quantification of the % from PDGFr-α⁻ mature oligodendrocytes (O4⁻O1⁺), F_{(1, 15)} = 5.656, p < 0.0311. g Quantification of the ZombieNir cells F_{(1, 15)} = 68.22, p < 0.0001. Mean values are plotted ± SEM; two-way ANOVA followed by Tukey multiple comparison post hoc test was used to determine statistical differences, ****p < 0.0001; ***p < 0.0005, n = 5 wells per group. Abbreviations: CSPG: chondroitin sulfate proteoglycan, EPO: erythropoietin, OPCs: oligodendrocyte progenitor cells

Fig. 6

EPOr knockdown reverses rhEPO rescue after CSPG exposure. a Representative fluorescent images of BrdU⁺, Olig1⁺, and EPOr⁺ cells. b Quantification of Olig1⁺ cell density. c Quantification of BrdU⁺ cell density. d Quantification of EPOr percent of the stained area p < 0.0001. e Experimental design. f Representative images of BrdU⁺ and Olig1⁺ cells. g Quantification of Olig1 + cell density, F_{(1, 16)} = 5.603, p < 0.0309. h Quantification of BrdU⁺ cell density, F_{(1, 16)} = 12.23, p < 0.003. i Mander’s coefficient of BrdU and DAPI, F_{(1, 16)} = 30.30, p < 0.0001. Mean values are plotted ± SEM; two-way ANOVA followed by Tukey multiple comparison post hoc test was used to determine statistical significance. *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001, n = 4–5 wells per group. Scale bar = 5 µm. Abbreviations: CSPG: chondroitin sulfate proteoglycan, EPO: erythropoietin, EPOr: erythropoietin receptor, FOV: field of view, PFA: paraformaldehyde