The present study describes, for the first time, a temporal and spatial cellular expression of erythropoietin (Epo) and Epo receptor (Epo-R) with the evolution of a cerebral infarct after focal permanent ischemia in mice. In addition to a basal expression of Epo in neurons and astrocytes, a postischemic Epo expression has been localized specifically to endothelial cells (1 day), microglia/macrophage-like cells (3 days), and reactive astrocytes (7 days after occlusion). Under these conditions, the Epo-R expression always precedes that of Epo for each cell type. These results support the hypothesis that there is a continuous formation of Epo, with its corresponding receptor, during the active evolution of a focal cerebral infarct and that the Epo/Epo-R system might be implicated in the processes of neuroprotection and restructuring (such as angiogenesis and gliosis) after ischemia. To support this hypothesis, a significant reduction in infarct volume (47%; P < 0.0002) was found in mice treated with recombinant Epo 24 hours before induction of cerebral ischemia. Based on the above, we propose that the Epo/Epo-R system is an endogenous mechanism that protects the brain against damages consequent to a reduction in blood flow, a mechanism that can be amplified by the intracerebroventricular application of exogenous recombinant Epo.

Tolerance to cerebral ischemia is achieved by preconditioning sublethal stresses, such as ischemia or hypoxia, paradigms in which the decrease of O₂ availability may constitute an early signal inducing tolerance. In accordance with this concept, this study shows that hypoxia induces tolerance against focal permanent ischemia in adult mice. Normobaric hypoxia (8% O₂ of 1-hour, 3-hour, or 6-hour duration), performed 24 hours before ischemia, reduces infarct volume by approximately 30% when compared with controls. To elucidate the mechanisms underlying this neuroprotection, the authors investigated the effects of preconditioning on cerebral expression of hypoxia-inducible factor-1α (HIF-1α) and its target genes, erythropoietin and vascular endothelial growth factor (VEGF). Hypoxia, whatever its duration (1 hour, 3 hours, 6 hours), rapidly increases the nuclear content of HIF-1α as well as the mRNA levels of erythropoietin and VEGF. Furthermore, erythropoietin and VEGF are upregulated at the protein level 24 hours after 6 hours of hypoxia. The authors' findings show that (1) hypoxia elicits a delayed, short-lasting (<72 hours) tolerance to focal permanent ischemia in the adult mouse brain; (2) HIF-1 target genes could contribute to the establishment of tolerance; and (3) this model might be a useful paradigm to further study the mechanisms of ischemic tolerance, to identify new therapeutic targets for stroke.

Hypoxic preconditioning (8% O₂, 3 h) produces tolerance 24 h after hypoxic-ischemic brain injury in neonatal rats. To better understand the ischemic tolerance mechanisms induced by hypoxia, we used oligonucleotide microarrays to examine genomic responses in neonatal rat brain following 3 h of hypoxia (8% O₂) and either 0, 6, 18, or 24 h of re-oxygenation. The results showed that hypoxia-inducible factor (HIF)-1- but not HIF-2-mediated gene expression may be involved in brain hypoxia-induced tolerance. Among the genes regulated by hypoxia, 12 genes were confirmed by real time reverse transcriptase-PCR as follows: VEGF,EPO, GLUT-1, adrenomedullin,propyl 4-hydroxylase α, MT-1,MKP-1, CELF, 12-lipoxygenase,t-PA, CAR-1, and an expressed sequence tag. Some genes, for example GLUT-1, MT-1,CELF, MKP-1, and t-PA did not show any hypoxic regulation in either astrocytes or neurons, suggesting that other cells are responsible for the up-regulation of these genes in the hypoxic brain. These genes were expressed in normal and hypoxic brain, heart, kidney, liver, and lung, with adrenomedullin,MT-1, and VEGF being prominently induced in brain by hypoxia. These results suggest that a number of endogenous molecular mechanisms may explain how hypoxic preconditioning protects against subsequent ischemia, and may provide novel therapeutic targets for treatment of cerebral ischemia.