Enhanced oligodendrogenesis and recovery of neurological function by erythropoietin after neonatal hypoxic/ischemic brain injury

Abstract

Background and purpose: Neuronal replacement has recently gained attention as a potential therapeutic target under ischemic conditions. However, the oligodendrogenic infrastructure is equally critical for restoration of brain function and is also sensitive to ischemic injury. Erythropoietin (EPO) is a neuroprotective molecule that stimulates neuronal replacement after neonatal hypoxia/ischemia (H/I) when delivered soon after the onset of reperfusion. Because EPO can improve recovery of neurological function in the absence of tissue protection, we hypothesize that EPO may improve neurological function via enhancement of white matter recovery after H/I. Thus, we sought to determine the effects of delayed administration of EPO on white matter injury and recovery of neurological function after neonatal H/I.

Methods: EPO (1000 U/kg) was injected intraperitoneally at multiple time points beginning 48 hours after H/I in postnatal day 7 rats. The effects of EPO on oligodendrogenesis, white matter injury, and neurogenesis were evaluated using bromodeoxyuridine incorporation and cell-specific immunohistochemistry. Neurological function was assessed by sensorimotor behavioral tests.

Results: Delayed administration of EPO was incapable of reducing brain volume loss but significantly increased oligodendrogenesis and maturation of oligodendrocytes and attenuated white matter injury after H/I. These effects occurred concurrently with enhanced neurogenesis. Delayed EPO treatment improved behavioral neurological outcomes 14 days after H/I injury.

Conclusions: Our study demonstrates that delayed administration of EPO promotes oligodendrogenesis and attenuates white matter injury concurrently with increased neurogenesis. These effects likely contribute to the observed improvement in neurological functional outcomes.

Figure 1

Delayed EPO injection increases proliferation and differentiation of OPCs after H/I. A, Diagram of EPO and BrdU injection. B, Representative pictures and quantification of brain volume loss after H/I. Scale bar, 1 mm. C–E, Assessment of NG2⁺ cells 5d post-H/I. C, Immunostaining of NG2 (red) and BrdU (green) in the CC, CTX and ST of ipsilateral hemisphere. Scale bar, 100 μm. D, Confocal and 3D images of BrdU⁺/NG2⁺cells. Scale bar, 10 μm. E, Quantification of NG2⁺ (Top) and BrdU⁺/NG2⁺ (bottom) cells. F–H, Assessment of APC⁺ cells 14 d post-H/I. Immunostaining (F, scale bar 100 μm) and confocal images (G, scale bar 10 μm) of APC (red) and BrdU (green) in the CC, CTX and ST of ipsilateral hemisphere. H, Quantification of APC⁺ (Top) and BrdU⁺/APC⁺ (bottom) cells. *p<0.05, **p<0.01 versus control; #p<0.05, ##p<0.01 versus H/I vehicle.

Figure 2

Delayed EPO administration attenuates white matter injury after H/I. A–C, MBP (green) and NF-200 (red) immunostaining 5 d and 14 d after H/I. Boxes in A indicate the selected area of CC, CTX and ST. Scale bar, 1 mm. B, Immunostaining of MBP and NF-200 in the CC, CTX and ST of ipsilateral hemisphere. Scale bar, 100 μm. C, Confocal image of MBP and NF-200 staining. Scale bar, 10 μm. D and E, Quantification of the relative ratio of MBP versus NF-200 immunostaining intensity in ipsilateral (D) and contralateral (E) hemispheres. *p<0.05, **p<0.01 versus control; ##p<0.01 versus H/I vehicle.

Figure 3

Delayed EPO administration induces neurogenesis. Representative images and quantification of DCX and BrdU double-labeling in the CTX (A, B) and ST (C, D) following H/I. Scale bar, 500 μm. E, Confocal (left) and 3D (middle) image of BrdU⁺/DCX⁺ cells in the CTX and confocal image of BrdU⁺/DCX⁺ cell in the ST (right). Scale bar, 10 μm. *P<0.05, **p<0.01 versus control; ##p<0.01 versus H/I vehicle.

Figure 4

Delayed EPO administration improves neurological outcomes after H/I. A, Quantification of total steps among groups. Foot fault testing from contralateral forelimb (B) and hindlimb (C). D, Assessment of negative geotaxis. Control (n=15), vehicle- (n=25) and EPO-treated animals (n=21). *P<0.05, **P<0.01 versus control. ##p<0.01 versus H/I vehicle.