Purpose-Potent endogenous protection from ischemia can be induced in the retina by ischemic preconditioning (IPC). Protein kinase B/Akt is a cellular survival factor. We hypothesized that Akt was integral to IPC based upon differential effects of Akt subtypes.Methods-Rats were subjected to retinal ischemia after IPC or IPC-mimicking by the opening of mitochondrial KATP (mKATP) channels. The effects of blocking Akt using wortmannin, API-2, or small interfering RNA (siRNA) were examined. Electroretinography assessed functional recovery after ischemia, and TUNEL examined retinal ganglion cell apoptosis. We studied the relationship between Akt activation, and known initiators of IPC, including adenosine receptor stimulation and the opening of mKATP channels.Results-The PI-3 kinase inhibitor wortmannin 1 or 4 mg/kg (i.p.), the specific Akt inhibitor API-2, 5-500 μM in the vitreous, or intravitreal siRNA directed against Akt2 or -3, but not Akt1, significantly attenuated the neuroprotective effect of IPC. Interfering RNA against any of the three Akt subtypes significantly but time-dependently attenuated mKATP channel opening to mimic IPC. Adenosine A1 receptor blockade (DPCPX), A2a blockade (CSC), or the mKATP channel blocker 5-hydroxydecanoic acid significantly attenuated Akt activation after IPC. Interfering RNA directed against Akt subtypes prevented the ameliorative effect of IPC on post-ischemic apoptosis. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (Roth et al. 1998;Zhang et al. 2002;Roth et al. 2006). Such protection offers a model for uncovering pathways and agents to treat retinal ischemia. Ischemic preconditioning (IPC) protects the rat retina against the deleterious effects of a severe ischemic event (Roth et al. 1998;Li et al. 2000;Li et al. 2003). Conclusions-All NIH Public AccessThe cytoplasmic proto-oncogene 57 kD serine/threonine protein kinase Akt (protein kinase B, PKB), is one of the most important kinases, and at the core of numerous and diverse physiological functions. It is activated by receptor tyrosine kinases, integrins, G-protein coupled receptors, and other stimuli, with over 100 reported non-redundant Akt substrates (Manning and Cantley 2007). The Akt pathway has been identified as a cellular survival signal in diverse neuronal cell types and in protection from oxidative stress (Chong et al. 2005). Akt phosphorylates Ser136 on Bad, thus interfering with mitochondrial cytochrome C release (Datta et al. 2000), and it inhibits caspase-9 by phosphorylating Ser196 (Cardone et al. 1998 Because of its involvement in cell survival, Akt is a logi...

Background The purpose of this study was to examine the role of erythropoietin in retinal ischemic preconditioning (IPC). Methods Rats were subjected to retinal ischemia after IPC. Electroretinography assessed functional recovery after ischemia; retinal sections were examined to determine loss of retinal ganglion cells, and Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling was used to assess apoptosis. Levels of downstream mediators were measured in retinal homogenates by Western blotting. To assess the involvement of erythropoietin in IPC, we measured levels of erythropoietin and its receptor (EPO-R) in retinal homogenates following IPC, using Western blotting. To examine erythropoietin’s role in IPC, we studied the impact of blocking erythropoietin via intravitreal injection of soluble EPO-R (sEPO-R) before IPC. Results Erythropoietin levels did not change following IPC, but EPO-R increased. Intravitreal injection of sEPO-R significantly attenuated both the functional and histological neuroprotection produced by IPC in comparison to control injection of denatured sEPO-R. Apoptotic damage after ischemia was enhanced in the sEPO-R treated retinas as indicated by fluorescent Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling. Phosphorylated extracellular-signal-regulated kinase (ERK) and heat shock protein 27 (Hsp27), but not protein kinase B (Akt), upregulated in denatured sEPO-R treated retinae, were attenuated in eyes injected with sEPO-R. Conclusions These results indicate that EPO-R upregulation is a critical component of the functional, histological, and anti-apoptotic protective effect of ischemic preconditioning on ischemia in the retina and that several downstream effectors may be involved in the neuroprotective actions of erythropoietin.