Hypoxia-induced stroke tolerance in the mouse is mediated by erythropoietin.
OBJECTIVE
Cellular response to hypoxia is mainly controlled by hypoxia-inducible factor 1 (HIF-1). The HIF-1 target gene erythropoietin (EPO) has been described as neuroprotective. Thus, we hypothesize EPO to be an essential mediator of protection in hypoxic preconditioning.


METHODS
We randomized Sv129 mice into groups for different pretreatments, different hypoxia-ischemia intervals, or different durations of ischemia. For hypoxic preconditioning, the animals were exposed to a hypoxic gas mixture (8% O2 and 92% N2) for 30, 60, 180, 300, or 360 minutes. At 0, 24, 48, 72, or 144 hours later, we performed middle cerebral artery occlusion and allowed reperfusion after 30, 45, 60, or 120 minutes, or occlusion was left to be permanent. We studied EPO gene expression in brain tissue with a real-time reverse transcriptase-polymerase chain reaction and measured HIF-1 DNA-binding activity with an electrophoretic mobility shift assay. To block endogenously produced EPO, we instilled soluble EPO receptor into the cerebral ventricle.


RESULTS
Hypoxic preconditioning for 180 or 300 minutes induced relative tolerance to transient focal cerebral ischemia, as evidenced by a reduction of infarct volumes to 75% or 54% of the control, respectively. Hypoxic pretreatment was effective only when applied 48 or 72 hours before middle cerebral artery occlusion. Sixty minutes after hypoxia, we found a marked activation of HIF-1 DNA-binding activity and a 7-fold induction of EPO transcription. Infusion of soluble EPO receptor significantly reduced the protective effect of hypoxic pretreatment by 40%.


CONCLUSIONS
Endogenously produced EPO is an essential mediator of ischemic preconditioning.
Prass
Konstantin
K
Department of Neurology, Medical Faculty Charité, Humboldt University, Berlin, Germany.
Scharff
Anna
A
Ruscher
Karsten
K
Löwl
Diana
D
Muselmann
Claudia
C
Victorov
Ilya
I
Kapinya
Krisztian
K
Dirnagl
Ulrich
U
Meisel
Andreas
A
D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D002544:Cerebral Infarction / Q000473:pathology / Q000503:physiopathology / Q000517:prevention & control*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004195:Disease Models, Animal; D024202:Electrophoretic Mobility Shift Assay; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006624:Hippocampus / Q000473:pathology; D000860:Hypoxia / Q000378:metabolism / Q000503:physiopathology*; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D020244:Infarction, Middle Cerebral Artery / Q000503:physiopathology; D007276:Injections, Intraventricular; D019194:Ischemic Preconditioning; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D009687:Nuclear Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017467:Receptors, Erythropoietin / Q000008:administration & dosage; D015424:Reperfusion / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020521:Stroke / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D013997:Time Factors; D014157:Transcription Factors
D004268:DNA-Binding Proteins; C497443:Hif1a protein, mouse; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D012333:RNA, Messenger; D017467:Receptors, Erythropoietin; D014157:Transcription Factors; D004921:Erythropoietin; D004247:DNA