Erythropoietin mediates neurobehavioral recovery and neurovascular remodeling following traumatic brain injury in rats by increasing expression of vascular endothelial growth factor

Abstract

Erythropoietin (EPO) improves functional recovery after traumatic brain injury (TBI). Here, we investigated the role of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) on EPO-induced therapeutic efficacy in rats after TBI. Young male Wistar rats were subjected to unilateral controlled cortical impact injury and then infused intracerebroventricularly with either a potent selective VEGFR2 inhibitor SU5416 or vehicle dimethyl sulfoxide. Animals from both groups received delayed EPO treatment (5,000 U/kg in saline) administered intraperitoneally daily at 1, 2, and 3 days post injury. TBI rats treated with saline administered intraperitoneally daily at 1, 2, and 3 days post injury served as EPO treatment controls. 5-bromo-2-deoxyuridine was administered to label dividing cells. Spatial learning and sensorimotor function were assessed using a modified Morris water maze test and modified neurological severity score, respectively. Animals were sacrificed at 4 days post injury for measurement of VEGF and VEGFR2 or 35 days post injury for evaluation of cell proliferation, angiogenesis and neurogenesis. EPO treatment promoted sensorimotor and cognitive functional recovery after TBI. EPO treatment increased brain VEGF expression and phosphorylation of VEGFR2. EPO significantly increased cell proliferation, angiogenesis and neurogenesis in the dentate gyrus after TBI. Compared to the vehicle, SU5416 infusion significantly inhibited phosphorylation of VEGFR2, cell proliferation, angiogenesis, and neurogenesis as well as abolished functional recovery in EPO-treated TBI rats. These findings indicate the VEGF/VEGFR2 activation plays an important role in EPO-mediated neurobehavioral recovery and neurovascular remodeling after TBI.

Fig. 1

The effect of EPO and SU5416 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 32–35 compared with the saline group (P<0.05). However, the spatial learning performance at days 32–35 in the EPO+SU5416 group is worse than that in the EPO+DMSO group (P<0.05). (b) The plot shows the functional improvement detected on the modified neurological severity scores (mNSS). EPO treatment significantly lowers mNSS at days 4–35 compared to saline group (P<0.05). However, the functional recovery (mNSS) at days 4–35 in the EPO+SU5416 group is worse than that in the EPO+DMSO group (P<0.05). Data represent mean ± SD. *P<0.05 vs. Sham group. ^#P<0.05 vs. Saline group. $P<0.05 vs. EPO+DMSO. n (rats/group) =8.

Fig. 2

Effect of EPO and SU5416 on lesion volume examined at 35 days after TBI. TBI caused a significant cortical lesion compared to sham controls (H&E staining). Delayed treatment with EPO combined with DMSO had a tendency to reduce lesion volume without a significance compared to the saline group (P<0.05). EPO+SU5416 treatment had a tendency to increase the lesion volume but did not reach a significant level compared to the EPO+DMSO group. Data represent mean ± SD. Scale bar = 3 mm. *P<0.05 vs. Sham group. n (rats/group) = 8.

Fig. 3

Effect of EPO and SU5416 on cell loss in the ipsilateral hippocampus at 35 days after TBI. H&E staining: a-h. TBI caused significant cell loss in the dentate gyrus (DG), CA3 and CA1 regions (b, f, and i; P<0.05) of the ipsilateral hippocampus compared to sham controls (a and e). Arrows indicate cell loss. Delayed treatment with EPO (c, g, 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 (i). As compared to the EPO+DMSO group, the cell number in the EPO+SU5416 group was significantly decreased (d, h, and i; P<0.05). Data represent mean ± SD. Scale bar = 50 µm (a–h). *P < 0.05 vs. Sham group. ^#P<0.05 vs. Saline group. $P<0.05 vs. EPO+DMSO. n (rats/group) = 8.

Fig. 4

Effect of EPO and SU5416 on EBA-staining vascular structure in the injured cortex, ipsilateral DG and CA3 region 35 days after TBI. Arrows in a (as an example) indicate EBA-positive vascular structure. TBI alone (b, f, and g; P<0.05) significantly increased the vascular density in these regions compared to sham controls (a, e, and i; P < 0.05). EPO treatment further enhanced angiogenesis after TBI compared to saline groups (c, g and k; P<0.05). As compared to the EPO+DMSO group, the EPO+SU5416 group had a significantly decreased vascular density in these regions (d, h, and i; P<0.05). The density of EBA-stained vasculature is shown in (m). Data represent mean ± SD. Scale bar = 25 µm (i). *P<0.05 vs. Sham group. ^#P<0.05 vs. Saline group. $P<0.05 vs. EPO+DMSO. n (rats/group) = 8.

Fig. 5

Effect of EPO and SU5416 on cell proliferation 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 (arrow in a as an example). TBI alone (b and f) significantly increased the number of BrdU-positive cells in the ipsilateral cortex and DG compared to sham controls (a and e; P<0.05). The number of BrdU-positive cells is shown in i. EPO treatment significantly increased the number of BrdU-positive cells in these regions (c and g; P<0.05) compared to the saline group. As compared to the EPO+DMSO group, the EPO+SU5416 group had a significantly smaller number of BrdU-positive cells in these regions (d and h; P<0.05). Data represent mean ± SD. Scale bar = 25µm. *P<0.05 vs. Sham group. ^#P<0.05 vs. Saline group. $P<0.05 vs. EPO+DMSO. n (rats/group) = 8.

Fig.6

Effect of EPO and SU5416 on NeuN/BrdU-positive cells in the ipsilateral DG 35 days after TBI. Double fluorescent staining for BrdU (red) and NeuN (green) to identify newborn neurons (yellow after merge, as arrows indicate) in the ipsilateral DG. TBI significantly increased the newborn neuron number in the injured DG (b) compared to sham (a; P<0.05). EPO treatment significantly increased the number of NeuN/BrdU-positive cells (c; P<0.05) compared to the saline group. As compared to the EPO+DMSO group, the EPO+SU5416 group had a significantly smaller number of NeuN/BrdU-positive cells (d; P<0.05). The bar graphs show the number (e) and the percentage (f) of newborn neurons in the DG. The number of newborn neurons (NeuN/BrdU-colocalized cells) was counted in the DG and expressed per mm². The percentage of newborn neurons was the ratio of the number of NeuN/BrdU-positive cells to the total number of BrdU-positive cells in the DG. Data represent mean ± SD. *P<0.05 vs. Sham group. ^#P<0.05 vs. Saline group. n (rats/group) = 8.

Fig. 7

Correlation of functional outcomes with lesion volume, cell loss, angiogenesis, and neurogenesis. Data from all four groups were included to generate the correlations between functional and histological outcomes. The top panel line graphs show that the functional outcomes (mNSS scores) are significantly and positively correlated with the lesion volume (a; P<0.05) but inversely correlated with the vascular density (b; P<0.05). The other panel line graphs show that spatial learning performance is significantly and positively correlated with the number of neuron cells (c), vascular density (d), and NeuN/BrdU-positive cells (e) 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.

Fig. 8

Effect of EPO and SU5416 on VEGF and VEGFR2 level in the brain after TBI. Western blot analyses show the effects of EPO and SU5416 on VEGF and phosphorylation of VEGFR2 in brain tissue after TBI (a and c). Bar graphs show the quantitative data of protein band density (b and d). TBI significantly increased VEGF level and p-VEGFR2 in the injury boundary zone (a) and hippocampus (c) compared to sham (a and e; P<0.05). EPO treatment significantly increased the VEGF expression and p-VEGFR2 (P<0.05) compared to the saline group. As compared to the EPO+DMSO group, the EPO+SU5416 group did not significantly affect the VEGF level in the brain (c and d; P>0.05); SU5416 significantly decreased the p-VEGFR2 level in the brain (P<0.05). The bar graphs (i) show the density of VEGF-positive astrocytes. Data represent mean ± SD. *P<0.05 vs. Sham group. ^#P<0.05 vs. Saline group. $P<0.05 vs. EPO+DMSO. n (rats/group) = 4.

Fig. 9

Effect of EPO and SU5416 on VEGF expression in astrocytes after TBI. Triple fluorescent staining for VEGF (red), GFAP (green) and DAPI (blue) to identify VEGF-positive astrocytes (yellow after merge, as arrows indicate). TBI significantly increased the number of VEGF-positive astrocytes in the hippocampal CA3 region (b) and injury boundary zone (f) compared to sham (a and e; P<0.05). EPO treatment significantly increased the number of VEGF-positive astrocytes (c and g; P<0.05) compared to the saline group. As compared to the EPO+DMSO group, the EPO+SU5416 group did not significantly affect the number of VEGF-positive astrocytes (d and h; P>0.05). The bar graphs (i) show the density of VEGF-positive astrocytes. Data represent mean ± SD. Scale bar = 25µm (d). *P<0.05 vs. Sham group. ^#P<0.05 vs. Saline group. n (rats/group) = 4.