Afzhal R. Shaikh

The role of Akt/protein kinase B subtypes in retinal ischemic preconditioning.

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. 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. The PI-3 kinase inhibitor wortmannin 1 or 4 mg/kg (i.p.), the specific Akt inhibitor API-2, 5-500 microM 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. All three Akt subtypes are involved in functional retinal neuroprotection by IPC or IPC-mimicking. Akt is downstream of adenosine A1 and A2a receptors and mKATP channel opening. The results indicate the presence in the retina of robust and redundant endogenous neuroprotection based upon subtypes of Akt.

Mitogen-activated Protein Kinase p38α and Retinal Ischemic Preconditioning

In previous studies, inhibition of mitogen-activated protein kinase (MAP) p38 significantly improved recovery and attenuated apoptosis after retinal ischemia in rats. Yet, ischemic preconditioning (IPC) attenuated the ischemia-induced increase in p38 expression. We hypothesized that p38 was required for induction of ischemic tolerance by IPC. We examined the mechanisms of involvement of p38 in IPC neuroprotection. IPC or ischemia was induced in rat retina in vivo. Recovery after ischemia performed 24h after IPC was assessed functionally (electroretinography) and histologically at 7d after ischemia in the presence or absence of inhibition of p38. We examined the role of p38alpha in the mimicking of IPC produced by opening mitochondrial KATP channels using diazoxide, or stimulation of p38 activation by anisomycin. The importance of adenosine receptors in p38 activation after IPC was assessed using specific blockers of adenosine A1 and A2a receptors. Interfering RNA (siRNA) or SB203580 was used to block p38alpha. Phosphorylated p38 levels were measured. Phosphorylated p38 protein increased with IPC. Interfering RNA (siRNA) to p38alpha prior to IPC, or inhibiting p38 activation with SB203580, with ischemia following 24h later, significantly attenuated the neuroprotective effect of IPC. Anisomycin administered to increase p38 mimicked IPC, an effect blocked by SB203580. IPC-mimicking with diazoxide, an opener of mitochondrial KATP channels, was diminished with p38alpha siRNA. Adenosine receptor blockade did not decrease the elevated levels of phosphorylated p38 after IPC. Specific inhibition of p38alpha suggests that this MAPK is involved in the protective effects of IPC, and that p38 is downstream of mitochondrial KATP channels, but not adenosine receptors, in this neuroprotection.

Involvement of Erythropoietin in Retinal Ischemic Preconditioning

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, Western blotting was used to measure levels of erythropoietin and its receptor (EPO-R) in retinal homogenates after IPC. To examine erythropoietin’s role in IPC, the impact of blocking erythropoietin via intravitreal injection of soluble EPO-R (sEPO-R) before IPC was studied. Results:Erythropoietin levels did not change after IPC, but EPO-R increased. Intravitreal injection of sEPO-R significantly attenuated both the functional and histologic 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 and heat shock protein 27, but not protein kinase B, 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, histologic, and antiapoptotic protective effect of IPC on ischemia in the retina and that several downstream effectors may be involved in the neuroprotective actions of erythropoietin.

Protein kinase C subtypes and retinal ischemic preconditioning.

The purpose of our study was to determine the specific subtypes of protein kinase C involved in the neuroprotection afforded by retinal ischemic preconditioning (IPC), their relationship to the opening of mitochondrial KATP (mKATP) channels, and their role in apoptosis after preconditioning and ischemia. Rats were subjected to retinal ischemia after IPC, or retinas were rendered ischemic after pharmacological opening of mKATP channels. Using immunohistochemistry and image analysis, we determined cellular localization of PKC subtypes. We blocked PKC-delta and -epsilon to study the effect on protection with IPC or with IPC-mimicking by the opening of mKATP channels. PKC subtypes were inhibited pharmacologically or with interfering RNA. Electroretinography assessed functional recovery after ischemia. IPC was effectively mimicked by injection of diazoxide to open the mKATP channel. IPC and/or its mimicking were attenuated by the PKC-delta inhibitor rottlerin and by interfering RNA targeting PKC-delta or -epsilon. Using TUNEL staining and Western blotting for caspase-3 and fodrin breakdown we assessed apoptosis. The injection of interfering RNA to PKC-delta and -epsilon before preconditioning significantly enhanced TUNEL staining as well as the cleavage of caspase-3 and fodrin after ischemia. In summary, our experiments have shown that both PKC-delta and -epsilon subtypes are involved in the cellular signaling that results in neuroprotection from IPC and that both are downstream of the opening of mKATP channels.

Delayed administration of bone marrow mesenchymal stem cell conditioned medium significantly improves outcome after retinal ischemia in rats.

PURPOSE Delayed treatment after ischemia is often unsatisfactory. We hypothesized that injection of bone marrow stem cell (BMSC) conditioned medium after ischemia could rescue ischemic retina, and in this study we characterized the functional and histological outcomes and mechanisms of this neuroprotection. METHODS Retinal ischemia was produced in adult Wistar rats by increasing intraocular pressure for 55 minutes. Conditioned medium (CM) from rat BMSCs or unconditioned medium (uCM) was injected into the vitreous 24 hours after the end of ischemia. Recovery was assessed 7 days after ischemia using electroretinography, at which time we euthanized the animals and then prepared 4-μm-thick paraffin-embedded retinal sections. TUNEL and Western blot were used to identify apoptotic cells and apoptosis-related gene expression 24 hours after injections; that is, 48 hours after ischemia. Protein content in CM versus uCM was studied using tandem mass spectrometry, and bioinformatics methods were used to model protein interactions. RESULTS Intravitreal injection of CM 24 hours after ischemia significantly improved retinal function and attenuated cell loss in the retinal ganglion cell layer. CM attenuated postischemic apoptosis and apoptosis-related gene expression. By spectral counting, 19 proteins that met stringent identification criteria were increased in the CM compared to uCM; the majority were extracellular matrix proteins that mapped into an interactional network together with other proteins involved in cell growth and adhesion. CONCLUSIONS By restoring retinal function, attenuating apoptosis, and preventing retinal cell loss after ischemia, CM is a robust means of delayed postischemic intervention. We identified some potential candidate proteins for this effect.

Post-ischemic conditioning in the rat retina is dependent upon ischemia duration and is not additive with ischemic pre-conditioning.

Ischemic pre-conditioning (IPC) provides neuroprotection in the rat retina from the damaging effects of severe ischemia. Recently, neuroprotection by retinal ischemic post-conditioning (Post-C), i.e., transient ischemia after more lengthy, damaging ischemia, was described, but its mechanisms are not yet known. One possible explanation of the effectiveness of Post-C is that it augments intrinsic neuroprotective mechanisms initiated during ischemia. Increasing duration of the damaging ischemic insult may therefore impact the effectiveness of Post-C. IPC, in contrast, sets in motion a series of neuroprotective events prior to the onset of ischemia. Thus, IPC and Post-C may operate by differing mechanisms. Accordingly, we examined the effect of retinal ischemic duration on post-ischemic outcome in vivo in rats after adding Post-C, and the impact of combining pre- and post-conditioning. Recovery after ischemia performed 24 h after IPC, or after Post-C performed 5 min after ischemia ended, was assessed functionally (electroretinography) and histologically at 7 days after ischemia. Durations of ischemia of 45 and 55 min were studied. Since recovery with IPC or Post-C alone, with 55 min of ischemia, did not achieve the same degree of effect (i.e., not complete recovery) exhibited in our previous studies of IPC using a different ischemia model, we also combined IPC and Post-C to test the hypothesis of the possible additive effects of the IPC and Post-C. We found that the recovery after Post-C was enhanced to a greater degree when ischemia was of longer duration. Post-C led to greater post-ischemic recovery compared to IPC. Both IPC and Post-C also attenuated structural damage to the retina. Contrary to our hypothesis, IPC and Post-C did not combine to enhance recovery after ischemia. In earlier studies, IPC attenuated post-ischemic apoptosis. To begin to examine the mechanism of Post-C, we studied its impact on apoptosis following ischemia. We examined apoptosis by determining the percentage of TUNEL-positive cells at 24 h after ischemia. Post-C attenuated apoptosis, but when combined with IPC, TUNEL was similar in the combined group to that of ischemia alone. We also examined the role of the recruitment of an inflammatory response in ischemia and Post-C. We found that inflammatory markers increased by ischemia were not altered by Post-C. We conclude that Post-C effectiveness depends upon the duration of ischemia; Post-C is not additive with IPC, and Post-C functions, in part, by preventing apoptotic damage to the inner retina. Post-C has considerable promise for clinical translation to eye diseases that cause blindness by ischemia.