Beryl Ojwang

ERYTHROPOIETIN PROTEIN AND RECEPTOR EXPRESSION IN MOUSE RETINA AFTER PRECONDITIONING WITH HYPOXIA AND DEFERROXAMINE. Beryl Ojwang¹, Yanli Zhu², Jeffrey M. Gidday², Department of Biology¹and Department of Neurosurgery², Washington University, St. Louis, MO.

Brief exposure of mammals to moderate hypoxia, or low oxygen levels, causes an adaptive response characterized by an upregulation of certain type of proteins in their tissues, so that they are better prepared to cope with hypoxia again. It was discovered that the adaptive response induced by hypoxic “preconditioning” actually rendered tissues in the central nervous system resistant to injury caused by complete ischemia. This phenomenon is called “ischemic tolerance”.

The mechanism for this hypoxia-induced tolerance to ischemic injury has been attributed, in part, to the induction of the transcription factor called hypoxia-inducible factor-1α (HIF-1α) that is responsible for activating the expression of a family of genes that promote cell survival under stress. The current investigation is designed to study the role of erythropoietin (EPO), one of the downstream gene targets of HIF-1α that exhibits trophic and other survival-promoting, neuroprotective effects. I hypothesized that changes in EPO and/or its receptor contribute to the protection against retinal ischemic injury following hypoxic preconditioning (HPC).

To test this hypothesis, I utilized a mouse model of retinal ischemic tolerance already established in our laboratory wherein HPC or deferroxamine (DFX) pretreatment lead to ischemic tolerance from 24 h to 1 week later. Adult mice were preconditioned with moderate hypoxia (2 h of 11% oxygen) and then retinae were obtained from mice at 4 hours, 24 hours, and 7 days after this exposure and prepared either for Western blot to determine and quantify EPO expression, or paraffin embedded for EPO immunocytochemistry to determine the cellular localization of EPO expression, for both EPO itself and the EPO receptor. Control animals were run for all endpoint variables. Other adult mice were administered DFX, a drug that mimics hypoxia’s effects by stabilizing HIF-1α. Mouse retinae from these animals were also prepared for Western blot and immunohistochemical analysis. Temporal changes in retinal EPO and EPO receptor expression levels in response to hypoxic and DFX preconditioning will be compared with one another, relative to untreated controls.

An additional study involved elucidating changes in the expression of EPO and EPO receptors after retinal ischemia, in normal, untreated mice and in mice previously preconditioned with hypoxia or DFX, to examine whether EPO and/or EPO receptor expression must remain elevated after ischemia to account for retinal ischemic tolerance.

Results showed that both EPO and its receptor are upregulated after hypoxic and DFX preconditioning, with expression peaking at 24 hrs after treatment and remaining elevated even after 7 days, when ischemic tolerance is evident. However, in mice without prior HPC or DFX preconditioning, ischemia caused a decrease of EPO and its receptor after 24 hrs. Surprisingly, when ischemia was preceded by hypoxic or DFX preconditioning, the EPO and EPO receptor expression showed greater reduction at this time point. My results test that hypoxic and chemical preconditioning-induced upregulation of EPO and its receptor prior to ischemia may contribute to retinal ischemic tolerance.