In vivo evidence that erythropoietin protects neurons from ischemic damage

Abstract

Erythropoietin (EPO) produced by the kidney and the liver (in fetuses) stimulates erythropoiesis. In the central nervous system, neurons express EPO receptor (EPOR) and astrocytes produce EPO. EPO has been shown to protect primary cultured neurons from N-methyl-D-aspartate (NMDA) receptor-mediated glutamate toxicity. Here we report in vivo evidence that EPO protects neurons against ischemia-induced cell death. Infusion of EPO into the lateral ventricles of gerbils prevented ischemia-induced learning disability and rescued hippocampal CA1 neurons from lethal ischemic damage. The neuroprotective action of exogenous EPO was also confirmed by counting synapses in the hippocampal CA1 region. Infusion of soluble EPOR (an extracellular domain capable of binding with the ligand) into animals given a mild ischemic treatment that did not produce neuronal damage, caused neuronal degeneration and impaired learning ability, whereas infusion of the heat-denatured soluble EPOR was not detrimental, demonstrating that the endogenous brain EPO is crucial for neuronal survival. The presence of EPO in neuron cultures did not repress a NMDA receptor-mediated increase in intracellular Ca2+, but rescued the neurons from NO-induced death. Taken together EPO may exert its neuroprotective effect by reducing the NO-mediated formation of free radicals or antagonizing their toxicity.

Figures

Figure 1

Effects of intracerebroventricular EPO infusion on the response latency and hippocampal CA1 region of 3-min ischemic gerbils. (A) response latency; (B) CA1 neuronal density. Open columns indicate sham-operated (sham-op) animals and closed columns indicate vehicle- or EPO-infused ischemic animals. Each value represents mean ± SE (n = 8–11). *P < 0.05 and **P < 0.01, significantly different from the corresponding vehicle-infused ischemic group (statistical significance tested by the two-tailed Mann–Whitney U test). (C–H) Photomicrographs of hippocampal sections stained with cresyl violet: (C, E, and G) low magnification; (D, F and H) high magnification corresponding to C, E, and G. (C and D) A sham-operated animal; (E and F) an ischemic animal infused with vehicle; (G and H) an ischemic animal infused with 5 units/day of EPO. [Bar = 1.0 mm (C, E, and G) and 0.1 mm (D, F, and H).]

Figure 2

Effects of intracerebroventricular EPO infusion on the synapses in the hippocampal CA1 region of 3-min ischemic gerbils. (A) the number of synapses within the stratum moleculare (sm), stratum lacunosum/radiatum (sr) and stratum oriens (so). Open columns indicate sham-operated (sham-op) animals and stippled or solid columns indicate vehicle- or EPO-infused ischemic animals. Each value represents mean ± SE (n = 8–11). **P < 0.01, significantly different from the corresponding vehicle-infused ischemic group (statistical significance tested by the two-tailed Mann–Whitney U test). (B–D) Electron micrographs of the stratum radiatum. (B) A sham-operated animal; (C) an ischemic animal treated with vehicle; (D) an ischemic animal treated with EPO (5 units/day). Note that degenerating synapses are extremely electron dense (solid arrowheads) and more numerous in C than in B or D. Intact synapses are indicated by open arrowheads. (Bar = 1 μm.)

Figure 3

Effects of the intracerebroventricular infusion of sEPOR and dsEPOR on the response latency and hippocampal CA1 region of 2.5-min ischemic gerbils. (A) Response latency; (B) CA1 neuronal density. Open columns indicate sham-operated animals and stippled or solid columns indicate vehicle-sEPOR- or dsEPOR-infused ischemic animals. Each value represents mean ± SE (n = 6–8). **P < 0.01, significantly different from the corresponding vehicle-infused ischemic group (statistical significance tested by the two-tailed Mann–Whitney U test). (C and D) Photomicrographs of the hippocampal CA1 field stained with cresyl violet; (C) an ischemic animal infused with sEPOR at a dose of 25 μg/day; (D) an ischemic animal infused with vehicle. Note that the infusion of sEPOR resulted in a significant decrease in hippocampal CA1 pyramidal neurons. (Bar = 0.1 mm.)

Figure 4

TUNEL staining of the hippocampal CA1 field of 2.5-min ischemic gerbils after 7 day infusion of sEPOR. (A) An ischemic animal infused with sEPOR at a dose of 25 μg/day; (B) an ischemic animal infused with vehicle. TUNEL-positive neurons were observed only in the ischemic gerbil infused with sEPOR. (C) TUNEL-positive neurons in the hippocampal CA1 field of 2.5-min ischemic animals. The stippled column indicates vehicle-infused ischemic animals and solid columns indicate sEPOR-infused animals. Each value represents mean ± SE (n = 6–8). **P < 0.01, significantly different from the corresponding vehicle-infused ischemic group (statistical significance tested by the two-tailed Mann–Whitney U test). (Bar = 0.1 mm.)

Figure 5

EPO protects cultured hippocampal neurons from cell toxicity of SNP-derived NO. Neurons were incubated with test materials as described in the Materials and Methods. Glu, glutamate; Me-TC, a NO synthase inhibitor. Total and nonviable cell number was counted. Each value is the mean ± SD of triplicate experiments.