Effects of posttraumatic carbamylated erythropoietin therapy on reducing lesion volume and hippocampal cell loss, enhancing angiogenesis and neurogenesis, and improving functional outcome in rats following traumatic brain injury

Abstract

Object: Carbamylated erythropoietin (CEPO) is a modified erythropoietin molecule that does not affect hematocrit. In this study, the authors compared the efficacy of a single dose with a triple dose of CEPO treatment for traumatic brain injury (TBI) in rats.

Methods: Traumatic brain injury was induced by controlled cortical impact over the left parietal cortex. Carbamylated erythropoietin (50 μg/kg) was administered intraperitoneally in rats with TBI at 6 hours (CEPO × 1) or at 6, 24, and 48 hours (CEPO × 3) postinjury. Neurological function was assessed using a modified neurological severity score and foot fault and Morris water maze tests. Animals were killed 35 days after injury, and brain sections were stained for immunohistochemical analysis to assess lesion volume, cell loss, cell proliferation, angiogenesis, and neurogenesis after CEPO treatment.

Results: Compared with the vehicle treatment, single treatment of CEPO (6 hours) significantly reduced lesion volume and hippocampal cell loss, enhanced angiogenesis and neurogenesis in the injured cortex and hippocampus, and significantly improved sensorimotor functional recovery and spatial learning in rats after TBI. Importantly, triple dosing of CEPO (6, 24, and 48 hours) further reduced lesion volume and improved functional recovery and neurogenesis compared with the CEPO × 1 group.

Conclusions: The authors' results indicate that CEPO has considerable therapeutic potential in TBI and related pathologies and furthermore that repeated dosing in the subacute phase might have important pharmacological relevance.

Fig. 1

Effect of CEPO on hematocrit and lesion volume. a: CEPO treatment does not affect hematocrit in all groups. b: CEPO treatment significantly reduces lesion volume after TBI. Lesion volume was examined after H&E staining of brain sections on Day 35 after TBI. Data represent mean ± SD. ^#p < 0.05 vs. the vehicle group. ^$p < 0.05 vs CEPO x 1 group. N (rats/group) = 8.

Fig. 2

Effect of CEPO on functional recovery after TBI. a: Effect of CEPO on spatial learning function 31–35 days after TBI. TBI significantly impairs spatial learning on Days 32–35 compared to sham controls (p < 0.05). Delayed treatment with CEPO improves spatial learning performance measured by a recent version of the water maze test on Days 32–35 (CEPO x 3) or Days 33–35 (CEPO x 1) compared with the vehicle group (p < 0.05). CEPO x 3 treatment shows better spatial learning compared to CEPO x 1 treatment on Days 32–35 after TBI (p < 0.05). b: The line graph shows the functional improvement detected on the modified neurological severity scores (mNSS). CEPO treatment significantly lowers mNSS scores on Days 4–35 (CEPO x 3) or Days 7–35 (CEPO x 1) compared to the vehicle group (p < 0.05). CEPO x 3 treatment shows significantly lowered mNSS scores compared to CEPO x 1 treatment on Days 14–35 after TBI (p < 0.05). c: Effect of CEPO on sensorimotor function (forelimb footfault) after TBI. “Pre” represents pre-injury level. TBI significantly impairs sensorimotor function on Days 1–35 compared to sham controls (p < 0.05). Delayed CEPO treatment significantly reduces forelimb foot faults on Days 4–35 (CEPO x 3) or Days 7–35 (CEPO x 1) compared with the vehicle treated group (p < 0.05). However, CEPO x 3 treatment has a much lower incidence of forelimb footfaults than CEPO x 1 treatment on Days 4–14 after TBI (p < 0.05). d: Effect of CEPO on sensorimotor function (hindlimb footfault) after TBI. “Pre” represents pre-injury level. TBI significantly impairs sensorimotor function on Days 1–35 compared to sham controls (p < 0.05). Delayed CEPO treatment significantly reduces hindlimb foot faults on Days 7–35 (CEPO x 3 and CEPO x 1) compared with the vehicle-treated group (p < 0.05). However, CEPO x 3 treatment has much lower incidence of hindlimb footfaults than CEPO x 1 treatment on Days 7–35 after TBI (p < 0.05). Data represent mean ± SD. *p < 0.05 vs Sham group. ^#p < 0.05 vs Vehicle group. ^$p < 0.05 vs CEPO x 1 group. N (rats/group) = 8.

Fig. 3

Effect of CEPO on cell number in the ipsilateral DG and CA3 region 35 days after TBI. H&E staining: a–h. Delayed treatment with CEPO (c, d, g, and h) significantly reduces cell loss as compared with the vehicle-treated group (b and f) (p < 0.05). The cell number in the DG and CA3 region is shown in (i). Scale bar = 50 μm (a, applicable to a–h). Data represent mean ± SD. *p < 0.05 vs Sham group. ^#p < 0.05 vs Vehicle group. N (rats/group) = 8.

Fig. 4

Effect of CEPO on cell proliferation in the injured cortex and ipsilateral DG 35 days after TBI. TBI alone (b and f) significantly increases the number of BrdU⁺ cells (brown-stained) in the ipsilateral cortex and DG compared to sham controls (a and e) (p < 0.05). CEPO treatment significantly increases the number of BrdU⁺ cells in these regions compared to the vehicle-treated groups (p < 0.05). The number of BrdU⁺ cells is shown in (i). Scale bar = 50μm (a, applicable to a–h). Data represent mean ± SD. *p < 0.05 vs Sham group. ^#p < 0.05 vs Vehicle group. N (rats/group) = 8.

Fig. 5

Effect of CEPO on vWF-staining vascular structure in the injured cortex and ipsilateral DG 35 days after TBI. TBI alone (b and f) significantly increases the vascular density (brown-stained) in these regions compared to sham controls (p < 0.05). CEPO treatment (c, d, g, and h) further enhances angiogenesis after TBI compared to the vehicle-treated groups (p < 0.05). The density of vWF-stained vasculature is shown in (i). Scale bar = 50 μm (a, applicable to a–h). Data represent mean ± SD. *p < 0.05 vs Sham group. ^#p < 0.05 vs Vehicle group. N (rats/group) = 8.

Fig. 6

Double fluorescent staining for BrdU (red) and NeuN (green) to identify newborn neurons (yellow after merge) in the ipsilateral DG 35 days after TBI (f) and CEPO treatment (i and l). The total number of NeuN/BrdU-colabeled cells is shown in (m). Scale bars = 50μm. Data represent mean ± SD. ^#p < 0.05 vs. the vehicle group. ^$p < 0.05 vs CEPO x 1 group. N (rats/group) = 8.

Fig. 7

Line graphs showing the correlation of lesion volume, angiogenesis and neurogenesis with functional recovery. The lesion volume was significantly and positively correlated to functional deficits assessed by the mNSS score and the incidence of forelimb and hindlimb footfaults examined on day 35 after TBI (a). The cortical angiogenesis was highly and inversely correlated to functional deficits evaluated by the mNSS score and the incidence of forelimb and hindlimb footfaults examined on day 35 after TBI (b). Spatial learning was significantly and positively correlated to neurogenesis, cell number and vessels in the DG examined 35 days after TBI (c).