Erythropoietin regulates immune/inflammatory reaction and improves neurological function outcomes in traumatic brain injury

Abstract

Introduction: Traumatic brain injury (TBI) remains a leading cause of disability and death among young people in China. Unfortunately, no specific pharmacological agents to block the progression of secondary brain injury have been approved for clinical treatment. Recently, neuroprotective effects of erythropoietin (EPO) have been demonstrated in addition to its principal function in erythropoiesis, and hence it is viewed as a potential drug for TBI. In this study, we have investigated the neuroprotective effects of EPO associated with immune/inflammatory modulation in a mouse experimental TBI model.

Methods: EPO (5000 U/kg body weight, i.p.) was injected at 1 hr, 1, 2, and 3 days after TBI, and its effect on cognitive function, brain edema, immune/inflammatory cells including regulatory T cells (Tregs), neutrophils, CD3⁺ T cells, and microglia, cytokines including interleukin-10 (IL-10), transforming growth factor-β (TGF-β), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) were evaluated at different time points after treatment.

Results: EPO treatment significantly decreased brain edema and improved cognitive function when compared to Saline-treated mice (p < .05). EPO treatment also significantly increased Tregs level in spleen and injured brain tissue as well as significantly reduced the infiltration and activation of immune/inflammatory cells (neutrophils, CD3⁺T cells, and microglia) in the injured hemisphere compared to Saline-treated control animals (p < .05). In addition, ELISA analysis demonstrated that EPO treatment increased the expression of anti-inflammatory cytokine IL-10, but decreased the expression of proinflammatory cytokine IL-1β and TNF-α in the injured brain tissue (p < .05).

Conclusions: These findings suggest that EPO could improve neurological and cognitive functional outcomes as well as regulate immune/inflammatory reaction in TBI.

Keywords: erythropoietin; neuroimmune; neuroinflammation; regulatory T cell (Treg); traumatic brain injury.

Figure 1

Brain edema measured through water content measurement. TBI significantly increased brain edema at 1 and 3 days after TBI; EPO treatment significantly decreased brain edema at 1 and 3 days after TBI compared to TBI + Saline control. n = 6/group, *p < .05 TBI + EPO group versus TBI + Saline group, #p < .05 versus Sham group

Figure 2

Detect the changes in Treg in spleen by FACS. Tregs were stained by CD4 and Foxp3 antibodies. After mononuclear cells being gated, the double positive cells in outer and upper quadrant were defined as Tregs. TBI significantly decreased the level of Tregs at 3 and 7 days after injury, but EPO treatment significantly increased Tregs level at 3 and 7 days after injury. n = 6/group, *p < .05 TBI + EPO group versus TBI + Saline group, #p < .05 versus Sham group

Figure 3

Detect the changes in immuno/inflammatory cells in injured brain tissue by immunohistochemistry staining. The positive cells are brown and directed by black arrowheads. (a) Foxp3+ Tregs mainly appeared around the lesions at 3 and 7 days after TBI. EPO treatment increased Foxp3+ Tregs expression in the injured brain. (b) MPO+ neutrophils promptly appeared in and around lesions at 1 day after TBI, and then gradually decreased. Compared with TBI + Saline group, EPO treatment significantly decreased numbers of MPO +  neutrophils at 1 and 3 days after TBI. (c) TBI increased the infiltration of CD3+ T cells in the injured brain tissue at 3 and 7 days after TBI. EPO treatment significantly decreased CD3+ T cells levels compared with TBI + Saline group. n = 3/group, *p < .05 TBI + EPO group versus TBI + Saline group, #p < .05 TBI + EPO group or TBI + Saline group versus Sham group

Figure 4

Detect the activation of microglia in injured hemisphere by immunofluorescence staining. The microglia was stained by IBA1 antibody (red) and cell nucleus was stained by DAPI (blue). The cells directed by white arrowheads are positive cells. (a) Sham group, (b) TBI + Saline group, (c) TBI + EPO group, (d) TBI increased IBA1 expression in the injured brain compared to sham control; EPO treatment significantly decreased IBA1 expression at 3 and 7 days after TBI. n = 3/group, *p < .05 TBI + EPO group versus TBI + Saline group, #p < .05 TBI + EPO group or TBI + Saline group versus Sham group

Figure 5

Detect immuno/inflammatory cytokines in the brain tissue of injured hemisphere by ELISA at 1, 3, and 7 days after TBI. (a and b), respectively, show that TBI increased the expression of proinflammatory cytokines IL‐1β and TNF‐α at postinjury 1 and 3 days compared to sham control group; EPO treatment of TBI significantly decreased IL‐1β and TNF‐α levels at 1 and 3 days after TBI compared to TBI control group. (c) EPO treatment increased the expression of anti‐inflammatory cytokine IL‐10 after TBI compared to TBI + Saline group. (d) There was no significant difference between EPO treatment and TBI + Saline on the regulation of TGF‐β expression. n = 6/group, *p < .05 TBI + EPO group vs. TBI + Saline group, #p < .05. TBI + EPO group or TBI + Saline group versus Sham group

Figure 6

Detect cognitive function by MWM. (a) The spatial learning test at postinjury days 7–11. It shows that latency (time to reach the platform) was significantly shortened during the 5 days test, which suggests that spatial memory was developed in all mice (repeated measures ANOVA, n = 10, F = 69.014, p < .001). (b) The probe test on postinjury day 12 shows that TBI + EPO group spent significantly higher percentage of time in goal quadrant than the TBI + Saline group. n = 10/group, *p < .05 TBI + EPO group versus TBI + Saline group