Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury

Abstract

Erythropoietin (EPO), recognized for its central role in erythropoiesis, also mediates neuroprotection when the recombinant form (r-Hu-EPO) is directly injected into ischemic rodent brain. We observed abundant expression of the EPO receptor at brain capillaries, which could provide a route for circulating EPO to enter the brain. In confirmation of this hypothesis, systemic administration of r-Hu-EPO before or up to 6 h after focal brain ischemia reduced injury by approximately 50-75%. R-Hu-EPO also ameliorates the extent of concussive brain injury, the immune damage in experimental autoimmune encephalomyelitis, and the toxicity of kainate. Given r-Hu-EPO's excellent safety profile, clinical trials evaluating systemically administered r-Hu-EPO as a general neuroprotective treatment are warranted.

Figure 1

EPO-R is found within and around human brain capillaries. (A) Anti-EPO-R staining in white matter (human hippocampal fimbria) is primarily localized to capillaries (arrows). (B) High-power view of capillaries illustrates the distinctly fibrous quality of EPO-R immunoreactivity around the capillary wall. (C) This immunoreactivity at the capillary (c) often could be identified as an astrocytic process (a). The entire cytoplasmic volume of such stellate astrocytes contain EPO-R immunoreactivity. (D) Transmission electron microscopy confirms that the predominant EPO-R immunoreactivity is within astrocytic foot processes (*), but it is also present within endothelial cells (arrows).

Figure 2

Systemically administered biotinylated r-Hu-EPO labels capillaries within the mouse brain. (A) Localization of biotinylated r-Hu-EPO (b-EPO) is around capillaries 5 h after i.p. injection into mice but is not observed (C) if given with 100 times excess of unlabeled r-Hu-EPO (bEPO + EPO). Tissue sections are from the striatum. (B) Biotinylated r-Hu-EPO surrounds the lumen of capillaries (arrow) 5 h after administration.

Figure 3

Systemic administration of r-Hu-EPO reduces infarct volume after cerebral artery occlusion. Animals given r-Hu-EPO (5,000 units/kg BW i.p.) before, during, or 3 h after carotid artery occlusion showed significant (*, P < 0.01) and equivalent reduction of necrosis volume compared with controls. In contrast, animals receiving r-Hu-EPO 6 h after the onset of reversible ischemia exhibited a significant, but substantially smaller, decrease in injury volume compared with animals receiving r-Hu-EPO sooner (†, P < 0.05). High-dose r-Hu-EPO given 9 h after the onset of occlusion was ineffective in reducing the cortical volume of injury. Numbers in parentheses indicate number of animals studied under each condition.

Figure 4

Systemic administration of r-Hu-EPO attenuates injury after blunt trauma. (A) Mice receiving a nonpenetrating blow to the frontal cortex exhibited extensive cavitary necrosis when examined 10 days after injury if treated with saline (Upper) in contrast to the minimal injury observed if they had received r-Hu-EPO (Lower). Cresyl violet stain of representative brain sections through site of injury. (B) Results of a representative experiment for r-Hu-EPO (5,000 units/kg BW) given 24 h before delivery of the impact. n = 6 animals each group; P < 0.05. The experiment was repeated four times with similar results.

Figure 5

Systemic administration of r-Hu-EPO ameliorates EAE. Lewis rats receiving r-Hu-EPO (5,000 units/kg BW) beginning at day 3 after immunization with myelin basic protein demonstrate both a delay in onset and a marked reduction of clinical symptoms (n = 18 animals in each group, three separate experiments; **, P < 0.01; *, P < 0.05).

Figure 6

Systemic administration of r-Hu-EPO delays and lessens kainate-induced seizures. (A) Mortality subsequent to a convulsant dosage of kainate (20 mg/kg BW i.p.) is significantly reduced (P < 0.01) by a 24-h pretreatment of mice with r-Hu-EPO (5,000 units/kg BW i.p.). (B) A single dose of r-Hu-EPO (5,000 units/kg BW) administered on day 0, 1, 2, 3, or 7 before testing continues to provide protection from kainate (n = 18, 36, 17, 8, or 8 respectively; *, P < 0.05 compared with control).