Recombinant human erythropoietin induces neuroprotection, activates MAPK/CREB pathway, and rescues fear memory after traumatic brain injury with delayed hypoxemia in mice

Abstract

Therapeutic interventions targeting secondary insults, such as delayed hypoxemia, provide a unique opportunity for treatment in severe traumatic brain injury (TBI). Erythropoietin (EPO) is a hypoxia-responsive cytokine with important roles in neurodevelopment, neuroprotection and neuromodulation. We hypothesized that recombinant human erythropoietin (rhEPO) administration would mitigate injury in a combined injury model of TBI and delayed hypoxemia. Utilizing a clinically relevant murine model of TBI and delayed hypoxemia, we characterized how ongoing rhEPO administration influenced neurogenesis, neuroprotection, synaptic density and, behavioral outcomes early after TBI, and the impact on long-lasting outcomes 6 months after injury. We employed novel object recognition (NOR) and fear conditioning to assess long-term memory. At 1-month post-injury, we observed a significant increase in cued-fear memory response in the rhEPO-injured mice compared with vehicle-injured mice. This was associated with neuroprotection and neurogenesis in the hippocampus and mitogen-activated protein kinase (MAPK)/cAMP response element-binding protein (CREB) signaling activation and increased of excitatory synaptic density in the amygdala. Early rhEPO treatment after injury reduced neurodegeneration and increased excitatory synaptic density in the hippocampus and amygdala at 6 months post-injury. However at 6 months post-injury (4 months after discontinuation of rhEPO), we did not observe changes in behavioral assessments nor MAPK/CREB pathway activation. In summary, these data demonstrate that ongoing rhEPO treatment initiated at a clinically feasible time point improves neurological, cognitive, and histological outcomes after TBI in the setting of secondary hypoxemic insults.

Keywords: Erythropoietin; Fear conditioning; Hypoxemia; Neurogenesis; Neuroprotection; Traumatic brain injury.

Fig. 1.

Delayed hypoxemia increased EPO and EPOr synthesis in the blood and brain, respectively, following TBI. A, Experimental design: brain and blood collection 22h, 24h, 26h, 32h and 48h after injury inducing 1h of hypoxemia at 24h post-injury. B, Endogenous mouse EPO in plasma over time analyzed by ELISA after TBI with delayed hypoxemia. C, Endogenous mouse EPO in the brain over time analyzed by ELISA after TBI with delayed hypoxemia. D, Representative western blot of the hippocampus membrane fraction probed for EPOr and (E) densitometric analysis. F, Representative image of EPOr-positive cells in CA3 region of injured hippocampi. G, Representative western blot of the hippocampus cytoplasm fraction probed for pJAK2 and (H) densitometric analysis. β-actin was used as a loading control. For western blot images, 2 samples per condition always from the same gel, using the same two samples throughout the figure. Two-way (Time and hypoxemia) ANOVA were used to determine statistical differences for hypoxemia effect followed by Tukey multiple comparison post-hoc test were used to determine statistical differences, n=6–7 mice per group. Time F_(5,57) = 3.424 p = 0.009. Hypoxemia F_(1,57) = 10.53 p < 0.0020. Scale bar: 20 μm. Abbreviations: CCI, controlled cortical impact; EPO, erythropoietin, EPOr, erythropoietin receptor.

Fig. 2.

Ongoing rhEPO administration improved fear memory response but not novel memory. A, Experimental design performing NOR 15 days and fear conditioning 1 month post-injury. B, NOR paradigm. C, time in the center during the habituation day. Unpaired t-test *p< 0.05 (D) total distance during the second habituation day. (E) discrimination index during the test day. F, Fear conditioning scheme. Quantification of percentage of freezing time on G, conditioning, H, contextual memory I, cued memory. Two-way (Treatment and Tone) repeated measures ANOVA for tone freezing (n = 15/group). Tone F_(4,112) = 31.11 p < 0.0001. Treatment*Tone interaction F_(4,112) = 5.21 p = 0.0007 *p = 0.015. Abbreviations: CCI, controlled cortical impact; rhEPO, recombinant human erythropoietin; EPOr, erythropoietin receptor; Veh, vehicle.

Fig. 3

Ongoing rhEPO administration induced neuroprotection, increased neurogenesis in hippocampus and, increased synaptic density in amygdala. A, Experimental design. B, Representative image of NeuN+ cells in CA3 region of injured hippocampi (indicated by the dotted line) and (C) stereological quantification. D, Hippocampal volume quantification. E, Representative immunofluorescence image of the SGZ in the DG of the hippocampus labeled with NeuN (red), DCX (green) and BrdU (magenta) with a zoomed in insert. White arrows indicate BrdU-positives cells. F Summation of total neuronal lineage cells per area of hippocampi. G, Puncta detection of PSD95 (red) synapsin (green) images in DG (left) and CeA (right). H, Quantification of synaptic loci (% vehicle) in DG and (I) CeA. Unpaired t-test *p< 0.05, **p< 0.01, n=4–8 mice per group. Scale bar: 50 μm. Abbreviations: CCI, controlled cortical impact; rhEPO, recombinant human erythropoietin; Veh, vehicle; SGZ, subgranular zone; DG, dentate gyrus; DCX, doublecortin; BrdU, 5-bromo-2’-deoxyuridine. CeA, central amygdala.

Fig. 4.

MAPK/CREB signaling pathway activation after rhEPO treatment in amygdala but not in hippocampus. A, Experimental design. B, Representative western blot of the amygdalar cytoplasm fraction probed for total and phosphorylated MAPK1 and 2. C, Densitometric analysis of pMAPK1 expression normalized by total MAPK1. D, Densitometric analysis of pMAPK2 expression normalized by total MAPK2. E, Representative western blot of the amygdalar nuclear fraction probed for total and phospho-CREB. F, Densitometric analysis of pCREB expression normalized by total CREB. β-actin was used as a loading control. G, Representative immunofluorescence image of the BLA labeled with pCREB (green) and NeuN (red) with a zoomed in insert. H, Quantification of pCREB density expression in neurons in the BLA. I, Representative western blot of the hippocampal cytoplasm fraction probed for total and phosphorylated MAPK1 and 2. J, Densitometric analysis of pMAPK1 expression normalized by total MAPK1. K, Densitometric analysis of pMAPK2 expression normalized by total MAPK2. L, Representative western blot of the hippocampal nuclear fraction probed for total and phospho-CREB. M, Densitometric analysis of pCREB expression normalized by total CREB. β-actin was used as a loading control. For western blot images, 2 samples per condition always from the same gel, using the same two samples throughout the figure. N, Representative immunofluorescence image of the BLA labeled with pCREB (green) and NeuN (red) with a zoomed in insert. O, Quantification of pCREB density expression in neurons in the BLA. Mean values are plotted ± SEM, One-way ANOVA followed by Tukey multiple comparison post hoc test were used to determine statistical differences; *p<0.05, n=5–7 mice per group. Mean values are plotted ± SEM, unpaired t-test *p<0.05, n=5–6 mice per group. Scale bar: 50 μm. Abbreviations: CCI, controlled cortical impact; rhEPO, recombinant human erythropoietin; Veh, vehicle; pCREB, phosphorylated cAMP-response element binding protein; pMAPK, phosphorylated mitogen-activated protein kinase; BLA, basolateral amygdala; CeA, central amygdala.

Fig 5.

rhEPO treatment did not enhance behavioral improvement 6 months post-injury. A, Experimental design. NOR on day 1 quantification of (B) time in the center. One-way ANOVA followed by Tukey multiple comparison post hoc test F_{(2, 40)} = 9.68, p = 0.0004. **p<0.01 (C) total distance. F_{(2, 40)} = 8.91, p = 0.0006. **p<0.01. On day 3 (familiarization) quantification of (D) discrimination index. On day 4 (novel recognition test) quantification of (E) discrimination index. F, Open field arenas with exploration traces. Fear conditioning 3-day paradigm quantification of percentage freezing time of (G) conditioning, (H) contextual and (I) cued memory. n=13–16 mice per group. Abbreviations: CCI, controlled cortical impact; rhEPO, recombinant human erythropoietin; Veh, vehicle.

Fig 6.

rhEPO reduces neurodegeneration and improves excitatory synaptic plasticity 6 months post-injury. A, Experimental design. B, Representative image of NeuN+ cells in CA3 region of injured hippocampi (indicated by the dotted line) and (C) stereological quantification. Scale bar: 20 μm. D, Representative image of cresyl violet in the injured side of the brain. E, Hippocampal volume quantification. F, Top images, puncta detection of PSD95 (red) synapsin (green) in DG and, bottom images, in CeA. Scale bar: 5 μm. G, Quantification of synaptic loci (% sham-EPO) in DG. One-way ANOVA followed by Tukey multiple comparison post hoc test. F_(2,17) = 27.1, p < 0.0001. ****p < 0.0001, ***p < 0.001, **p<0.01. H, Quantification of synaptic loci (% sham-EPO) in CeA. F_(2,17) = 5.16, p = 0.018, *p<0.05, n=5–8 mice per group. Abbreviations: CCI, controlled cortical impact; rhEPO, recombinant human erythropoietin; Veh, vehicle; DG, dentate gyrus. CeA, central amygdala.