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Excerpt
Methods: Rats were subjected to IPC or to retinal ischemia after IPC. Levels of putative downstream signal transduction mediators Akt (protein kinase B), extracellular-regulated kinase (p44/42 mitogen-activated kinase, ERK), heat shock protein 27 (HSP27), and endothelial nitric oxide synthase (eNOS) were measured and semi-quantitated by Western blotting. Levels of EPO and of EPO-R were measured and quantitated after IPC using immunohistochemistry. Proteins were localized to specific retinal cells using double-labeling immunohistochemistry with flourescent secondary antibodies. Retinal function after ischemia was evaluated using electroretinography (ERG). To test the functional role of Akt, interfering RNA (RNAi) to Akt subtypes was injected into the vitreous prior to IPC and ischemia and compared to results using a non-silencing RNAi sequence.
Results: EPO increased 1.5 fold in retinal ganglion cells (RGCs) at 1 h after IPC (P<0.05), with a trend for significance at 24 h.
EPO-R showed a trend for increase at 6 h after IPC. EPO and EPO-R localized to RGCs, amacrine cells, and photoreceptor cells.
Intravitreal injection of sEPO-R completely attenuated IPC. Injection of RNAi to Akt2 partially attenuated IPC.
Conclusions: EPO produces neuroprotective effects by activating Akt. Widespread distribution of EPO and EPO-R, taken together with increases after IPC and the blunting of neuroprotection by EPO blockade, indicate an essential role for EPO in ischemic tolerance.
Acknowledgment: The research was supported by National Institutes of Health Grants EY10343, EY11253, by our state Society for the Prevention of Blindness, and by a grant from the Office of Education of the University. JCH was the recipient of a medical student fellowship from the American Academy of Neurology.
Summary: EPO plays an essential role in the induction of neuronal tolerance to ischemia in the retinal ischemic preconditioning model.
