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Comparative Study
. 2010 Sep;113(3):598-608.
doi: 10.3171/2009.9.JNS09844.

Delayed administration of erythropoietin reducing hippocampal cell loss, enhancing angiogenesis and neurogenesis, and improving functional outcome following traumatic brain injury in rats: comparison of treatment with single and triple dose

Ye Xiong  1 , Asim Mahmood, Yuling Meng, Yanlu Zhang, Changsheng Qu, Timothy Schallert, Michael Chopp
Affiliations
Comparative Study

Delayed administration of erythropoietin reducing hippocampal cell loss, enhancing angiogenesis and neurogenesis, and improving functional outcome following traumatic brain injury in rats: comparison of treatment with single and triple dose

Ye Xiong et al. J Neurosurg. 2010 Sep.

Abstract

Object: This efficacy study was designed to investigate traumatic brain injury (TBI) in rats treated with delayed erythropoietin (EPO) administered in a single dose compared with a triple dose.

Methods: Young adult male Wistar rats were randomly divided into the following groups: 1) sham group (6 animals); 2) TBI/saline group (6 animals); 3) TBI/EPOx1 group (6 animals); and 4) TBI/EPOx3 group (7 animals). Traumatic brain injury was induced by controlled cortical impact over the left parietal cortex. Erythropoietin (5000 U/kg) or saline was administered intraperitoneally on Day 1 (EPOx1 group) or on Days 1, 2, and 3 (EPOx3 group) postinjury. Neurological function was assessed using a modified neurological severity score, foot-fault, and Morris water maze tests. Animals were killed 35 days after injury and brain sections were stained for immunohistochemistry.

Results: Compared with the saline treatment, EPO treatment in both the EPOx1 and EPOx3 groups significantly reduced hippocampal cell loss, enhanced angiogenesis and neurogenesis in the injured cortex and hippocampus, and significantly improved neurological functional outcome. The EPOx3 group exhibited significantly improved functional and histological outcomes compared with the EPOx1 group.

Conclusions: These data demonstrate that delayed posttraumatic administration of EPO significantly improved histological and long-term functional outcomes in rats after TBI. The triple doses of delayed EPO treatment produced better histological and functional outcomes in rats, although a single dose provided substantial benefits compared with saline treatment.

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Figures

Fig. 1
Fig. 1
Changes in hematocrit before and after TBI. “Pre” represents preinjury level. *p < 0.05 vs. corresponding Pre. Data represent mean ± SD. #p < 0.05 vs. the EPOx1 group at Day 14 after injury. N (rats/group) = 6 (Sham); 6 (Saline); 6 (EPOx1); 7 (EPOx3).
Fig. 2
Fig. 2
Effect of EPO on spatial learning function 31–35 days after TBI. TBI significantly impaired spatial learning at Days 32–35 compared to sham controls (p < 0.05). Delayed treatment with EPO (EPOx1 and EPOx3) improves spatial learning performance measured by a recent version of the water maze test at Days 33–35 compared with the saline group (p < 0.05). However, the spatial learning performance at Days 34 and 35 in the EPOx3 group is better than that in the EPOx1 group (p < 0.05). Data represent mean ± SD. *p < 0.05 vs. Saline group. #p < 0.05 vs. sham controls or EPOx3 group. N (rats/group) = 6 (Sham); 6 (Saline); 6 (EPOx1); 7 (EPOx3).
Fig. 3
Fig. 3
Effect of EPO on sensorimotor function (forelimb footfault) before and after TBI. “Pre” represents pre-injury level. Delayed EPOx3 treatment significantly reduces forelimb foot faults at Days 7–28 while EPOx1 treatment significantly reduces them at Days 7 and 14 compared with the saline group (p < 0.05). Data represent mean ± SD. * p < 0.05 vs. EPOx1 or EPOx3. N (rats/group) = 6 (Saline); 6 (EPOx1); 7 (EPOx3).
Fig. 4
Fig. 4
Effect of EPO on sensorimotor function (hindlimb footfault) before and after TBI. “Pre” represents pre-injury level. Delayed EPOx3 treatment significantly reduces hindlimb foot faults at days 7–28 while EPOx1 treatment significantly reduces them at Days 7 and 21 compared with the saline group (p < 0.05). Data represent mean ± SD. * p < 0.05 vs. EPOx1 or EPOx3. N (rats/group) = 6 (Saline); 6 (EPOx1); 7 (EPOx3).
Fig. 5
Fig. 5
The plot shows the functional improvement detected on the modified neurological severity scores (mNSS). EPOx3 treatment significantly lowers mNSS scores at Days 7–35 compared to saline group (*p < 0.05). However, the functional recovery (lowered mNSS score) at Days 7 and 14 in the EPOx3 group is better than that in the EPOx1 group (#p < 0.05). N (rats/group) = 6 (Saline); 6 (EPOx1); 7 (EPOx3).
Fig. 6
Fig. 6
Effect of EPO on cell loss in the ipsilateral DG and CA3 region at 35 days after TBI. H&E staining: a–h. Delayed treatment with EPOx1 (c, g) and EPOx3 (d, h) significantly reduced cell loss as compared with the saline group (b, f) (p < 0.05). As compared to EPOx1 group, the cell number in the EPOx3 group was significantly higher (p < 0.05). The cell number in the DG and CA3 region is shown in (i). Data represent mean ± SD. Scale bar = 50μm (a–h). *p < 0.05 vs. corresponding sham. #p < 0.05 vs. the saline group. @ p < 0.05 vs EPOx1 group. N (rats/group) = 6 (Sham); 6 (Saline); 6 (EPOx1); 7 (EPOx3).
Fig. 7
Fig. 7
Effect of EPO on vWF-staining vascular structure in the injured cortex, ipsilateral DG and CA3 region 35 days after TBI. TBI alone (d, g, and i) significantly increased the vascular density in these regions compared to sham controls (p < 0.05). EPO treatment (EPOx1 and EPOx3) further enhanced angiogenesis after TBI compared to saline groups (p < 0.05). The density of vWF-stained vasculature is shown in (p). Data represent mean ± SD. Scale bar = 50 μm (a); 25 μm (e). *p < 0.05 vs. Sham. #p < 0.05 vs. the saline group. N (rats/group) = 6 (Sham); 6 (Saline); 6 (EPOx1); 7 (EPOx3).
Fig. 8
Fig. 8
Effect of EPO on BrdU-positive cells in the injured cortex and ipsilateral DG 35 days after TBI. The cells with BrdU (brown stained) that clearlylocalized to the nucleus (hematoxylin stained) were counted as BrdU-positive cells (arrows in e and j). TBI alone (b, g) significantly increased the number of BrdU-positive cells in the ipsilateral cortex and DG compared to sham controls (p < 0.05). EPO treatment (EPOx1 and EPOx3) significantly increased the number of BrdU-positive cells in these regions (c, d, h, and i) compared to saline groups (p < 0.05). The number of BrdU-positive cells is shown in (k). Data represent mean ± SD. Scale bar = 50μm (a); 25μm (e). *p < 0.05 vs. Sham. #p < 0.05 vs. the saline group. N (rats/group) = 6 (Sham); 6 (Saline); 6 (EPOx1); 7 (EPOx3).
Fig. 9
Fig. 9
Double fluorescent staining for BrdU (red) and NeuN (green) to identify newborn neurons (yellow after merge, k and n) in the injured cortex (a-d) and the ipsilateral DG (e-h) at 35 days after TBI (b, f) and EPO treatment (c, d, g, and h). Newborn BrdU-positive cells (red, j and m) differentiate into neurons expressing NeuN (yellow, k and n). The total number and percentage of NeuN/BrdU-colabeled cells are shown in (o) and (p), respectively. Data represent mean ± SD. Scale bars = 50 μm (a); 25 μm (i). *p < 0.05 vs. corresponding sham. #p < 0.05 vs. the saline group. @ p < 0.05 vs EPOx1 group. N (rats/group) = 6 (Sham); 6 (Saline); 6 (EPOx1); 7 (EPOx3).
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