Dose-dependent neurorestorative effects of delayed treatment of traumatic brain injury with recombinant human erythropoietin in rats

Abstract

Object: Delayed (24 hours postinjury) treatment with erythropoietin (EPO) improves functional recovery following experimental traumatic brain injury (TBI). In this study, the authors tested whether therapeutic effects of delayed EPO treatment for TBI are dose dependent in an attempt to establish an optimal dose paradigm for the delayed EPO treatment.

Methods: Experimental TBI was performed in anesthetized young adult male Wistar rats using a controlled cortical impact device. Sham animals underwent the same surgical procedure without injury. The animals (8 rats/group) received 3 intraperitoneal injections of EPO (0, 1000, 3000, 5000, or 7000 U/kg body weight, at 24, 48, and 72 hours) after TBI. Sensorimotor and cognitive functions were assessed using a modified neurological severity score and foot fault test, and Morris water maze tests, respectively. Animals were killed 35 days after injury, and the brain sections were stained for immunohistochemical analyses.

Results: Compared with the saline treatment, EPO treatment at doses from 1000 to 7000 U/kg did not alter lesion volume but significantly reduced hippocampal neuron loss, enhanced angiogenesis and neurogenesis in the injured cortex and hippocampus, and significantly improved sensorimotor function and spatial learning. The animals receiving the medium dose of 5000 U/kg exhibited a significant improvement in histological and functional outcomes compared with the lower or higher EPO dose groups.

Conclusions: These data demonstrate that delayed (24 hours postinjury) treatment with EPO provides dose-dependent neurorestoration, which may contribute to improved functional recovery after TBI, implying that application of an optimal dose of EPO is likely to increase successful preclinical and clinical trials for treatment of TBI.

Fig. 1

Body weight and hematocrit. “Pre” represents preinjury level. Data represent mean ± SD. *p < 0.05 vs. corresponding Pre. N (rats/group) = 8.

Fig. 2

Effect of EPO treatment on functional outcomes. (A) Spatial learning measured by a recent version of the Morris water maze test at Days 31–35 after TBI. TBI significantly impaired spatial learning at Days 32–35 compared to sham controls (p < 0.05). Delayed treatment with EPO improves spatial learning performance at Days 33–35 compared with the saline group (p < 0.05). However, the spatial learning performance at Days 34 and 35 in the EPO5K group is better than that in other EPO groups (p < 0.05). (B) Effect of EPO on sensorimotor function (forelimb footfault) before and after TBI. Delayed EPO treatment significantly reduces forelimb foot faults at Days 7–35 while EPO3K and 5K treatment significantly reduces them at Day 4 compared with the saline group (p < 0.05). EPO5K shows better effects on reducing forelimb footfaults compared to other EPO groups at Days 4–35 (p < 0.05). (C) Effect of EPO on sensorimotor function (hindlimb footfault) before and after TBI. Delayed EPO treatment significantly reduces hindlimb foot faults at days 4–35 while EPO5K treatment significantly reduces them at Days 7 –35 compared with the other EPO groups (p < 0.05). (D) The plot shows the functional improvement detected on the modified neurological severity scores (mNSS). EPO treatment significantly lowers mNSS scores at Days 7–35 compared to saline group (p < 0.05). EPO3K and 5K significantly reduces mNSS scores at Day 4 (p < 0.05). However, the functional recovery (lowered mNSS score) at Days 4–35 in the EPO5K group is better than that in the EPO3K group (p < 0.05). Data represent mean ± SD. *p < 0.05 vs. Saline group. ^#p < 0.05 vs. other EPO groups. N (rats/group) =8.

Fig. 3

Effect of EPO on cell loss in the ipsilateral hippocampus at 35 days after TBI. H&E staining: A–I. TBI caused significant cell loss in the CA3, DG and CA1 regions (B, E, and H, p < 0.05) of the ipsilateral hippocampus compared to sham controls (A, D, and G). Delayed treatment with EPO (C, F, and I) significantly reduced cell loss as compared with the saline group (p < 0.05). The cell number in the DG, CA3 and CA1 region is shown in (J). As compared to other EPO groups, the cell number in the EPO5K group was significantly higher (J, p < 0.05). Data represent mean ± SD. Scale bar = 25μm (a–h). *p < 0.05 vs. Saline. ^#p < 0.05 vs. other EPO groups. N (rats/group) = 8.

Fig. 4

Effect of EPO on vWF-staining vascular structure in the injured cortex, ipsilateral DG and CA3 region 35 days after TBI. TBI alone (B, E and H, p < 0.05) significantly increased the vascular density in these regions compared to sham controls (A, D and G, p < 0.05). EPO treatment further enhanced angiogenesis after TBI compared to saline groups (C, F and I, p < 0.05). The density of vWF-stained vasculature is shown in (J). As compared to other EPO groups, the EPO5K group had a significantly higher vascular density in these regions (J, p < 0.05). Data represent mean ± SD. Scale bar = 50μm (a); 25 μm (e). *p < 0.05 vs. Saline. ^#p < 0.05 vs. other EPO groups. N (rats/group) = 8.

Fig.5

Effect of EPO on BrdU-positive cells and NeuN/BrdU-positive cells in the injured cortex and ipsilateral DG 35 days after TBI. The cells with BrdU (brown stained) that clearly localized to the nucleus (hematoxylin stained) were counted as BrdU-positive cells (arrows in C and F). TBI alone (B and E) significantly increased the number of BrdU-positive cells in the ipsilateral cortex and DG compared to sham controls (A and D, p < 0.05). The number of BrdU-positive cells is shown in M). EPO treatment significantly increased the number of BrdU-positive cells in these regions (p < 0.05) compared to saline groups. As compared to other EPO groups, the EPO5K group had a significantly larger number of BrdU-positive cells in these regions (M, p < 0.05). Double fluorescent staining for BrdU (red, H and K) and NeuN (green, G and J) to identify newborn neurons (yellow after merge, I and L) in the injured cortex (G–I) and the ipsilateral DG (J–L) at 35 days after TBI and EPO treatment. The newborn neuron number in the injured cortex and DG is shown in (N). EPO treatment significantly increased the number of NeuN/BrdU-positive cells in these regions (p < 0.05) compared to saline groups. As compared to other EPO groups, the EPO5K group had a significantly larger number of NeuN/BrdU-positive cells in these regions (N, p < 0.05). Data represent mean ± SD. Scale bar = 25μm. *p < 0.05 vs. Saline. ^#p < 0.05 vs. other EPO groups. N (rats/group) = 8.

Fig. 6

Correlation of functional outcomes with cell loss, angiogenesis, cell proliferation and neurogenesis. The top panel line graphs show that the functional outcomes (hindlimb, forelimb footfault and mNSS scores) are significantly and inversely correlated with the number of vessels (A), BrdU-positive cells (B) and NeuN/BrdU-positive cells (C) in the injured cortex measured at Day 35 after TBI and EPO treatment (p < 0.05). The bottom panel line graphs show that spatial learning performance is significantly and positively correlated with the number of neuron cells (D), vessels (E), BrdU-positive and NeuN/BrdU-positive cells (F) in the ipsilateral hippocampus measured at Day 35 in rats after TBI and EPO treatment (p < 0.05). Data represent mean ± SD. N (rats/group) = 8.