Mitogen-activated protein kinase p38alpha and retinal ischemic preconditioning

Abstract

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.

Figure 1

IPC changed retinal levels of phosphorylated p38 and increased p38 downstream kinase activity/signaling. (A) Quantitation of Western blots for phosphorylated p38. IPC significantly changed phosphorylated p38 levels at 6 and 24 h after IPC as compared to 1 h after IPC in the IPC-treated retinae. (B) Representative blots of phosphorylated and total p38 used to quantify data for part A, indicate p38 phosphorylation, equal protein loading and stable total p38 protein levels following IPC. (C) Representative Western blot non-radioactive kinase activity assay indicating that IPC induced ATF2 phosphorylation. IPC significantly increased p38 kinase activity and potential for downstream signaling 24 h after IPC.

Figure 2

SB203580, an inhibitor of phosphorylated p38 phosphorylation of downstream targets, significantly decreased IPC protection. (A) Intravitreal injection of SB203580 prior to IPC resulted in a decreased electroretinography (ERG) b-wave recovery 7 d after ischemia compared to PBS (phosphate-buffered saline) vehicle. PBS response was similar to the protection observed with IPC previously and SB203580 response was similar to non-IPC responses seen previously (see text). ERG a-wave recovery was not significantly affected. (B) Representative ERG b-wave responses obtained from PBS- and SB203580-treated rats used to quantitate data shown in (A).

Figure 3

p38α siRNA specificity. Representative images of (A) in vitro immunocytochemical analysis of siRNA-treated PC12-T cultured cells. (B) In vivo immunohistochemical analysis of siRNA treated frozen retinal sections. The retinal layers are labeled RGC (retinal ganglion cell layer), INL (inner nuclear layer) and PRL (photoreceptor layer). (C) Western blot analysis of the p38α kinase assay on paired p38α siRNA and non-silencing siRNA injected retinal homogenates.

Figure 4

p38α siRNA significantly decreased IPC protection of the retina. (A) Intravitreal injection of p38α siRNA prior to IPC decreased ERG b-wave recovery 7 d after ischemia compared to non-silencing siRNA. ERG a-wave recovery was not significantly affected. Thus, p38α siRNA exhibited an effect profile similar to the p38 inhibitor SB203580 (see Fig. 2). (B) Representative ERG traces obtained from non-silencing siRNA- and p38α siRNA -treated rats used to quantitate data shown in (A).

Figure 5

Retinal histopathology (5 μm paraffin stained sections) following ischemia. Representative images of the ischemic eyes harvested 7 d after ischemia comparing non-silencing siRNA-treated retina (left) to p38α siRNA-treated retina (right) with IPC 6 h after injection followed by ischemia 24 h later.

Figure 6

Anisomycin produced a rapid increase in retinal p38 downstream signaling, mimicked IPC when injected systemically 24 h prior to ischemia, and the neuroprotection was attenuated by SB203580. (A) Western blot analysis of phosphorylated ATF2 expression showed that anisomycin significantly increased p38 kinase activity as early as 1 h after its administration. Thus, anisomycin exhibited an increase in p38 signaling similar to IPC (see Fig. 1). Representative Western blot from vehicle or anisomycin-treated rats used to quantitate data (on right). (B) Intravitreal injection of SB203580 6 h before anisomycin, followed 24 h later by ischemia, significantly decreased the ERG b wave recovery 7 d after ischemia compared to intravitreal PBS (phosphate-buffered saline) + anisomycin. ERG a-wave recovery was not significantly affected. Thus, anisomycin exhibited a p38 dependent profile similar to IPC (see Figs. 2 & 3). Representative ERG traces obtained from anisomycin and intravitreal PBS- or anisomycin and intravitreal SB203580-treated rats used to quantitate data (on right).

Figure 7

Retinal p38 phosphorylation increases following administration of the mitochondrial KATP channel opener diazoxide; also shown is siRNA block of IPC mimicking by diazoxide. (A) Western blot demonstrated increased phosphorylation of p38 at 1 and 6 h following diazoxide injection. Thus, diazoxide injection (mimicking IPC) exhibited an early increase in p38 phosphorylation/activation relatively similar to IPC (see Fig. 1). Representative Western blots from 1% DMSO (Vehicle) - or diazoxide-treated rats used to quantitate data (on right). (B) Intravitreal p38α siRNA injected 24 h prior to diazoxide, followed by ischemia 24 h later, attenuated the IPC-mimicking of post-ischemic recovery at 7 d after ischemia by diazoxide compared to non-silencing siRNA. Results are similar to SB203580 and p38α siRNA effects on IPC (see Figs. 2 & 3) and p38α siRNA on anisomycin (see Fig. 5). Representative baseline and 7 d post-ischemia ERG responses for non-silencing siRNA or p38α siRNA treated rats used in the quantitation of data (on right).