Fig 1.
Time flow of the different experimental settings.
First row: Fluorogold (FG) labeling seven days prior to IRI, subsequent treatment with or without p38 inhibitor (SB203580) and/or iALF-186/ALF-186 and enucleation another seven days later, giving FG the chance to promote into RGC via axonal transport. Second row: IRI with or without p38 inhibitor (SB203580) and/or iALF-186/ALF-186 treatment and enucleation 24 or 48 hours after IRI for molecular analysis. Third row: IRI with or without p38 inhibitor (SB203580) and/or iALF-186/ALF-186 treatment and enucleation 24 or 48 hours after IRI for immunohistochemical analysis.
Fig 2.
Effect of ALF-186 on vital retinal ganglion cells after ischemia reperfusion injury (IRI) in vivo.
(A) Representative flat mount images (n = 8) of fluorogold-labeled retinal ganglion cells 7 days after IRI, with immediate (0 h) and delayed (3 h) ALF-186 treatment, inactivated ALF-186 treatment and p38 inhibition using SB203580. Scale bar is 100 μm. (B) Quantification of retinal ganglion cell density (cells/mm2; data are mean±SD; n = 8; IRI vs. IRI+ALF-186 0h and 3h, *** = p<0.001, IRI+ALF-186 0h vs. IRI+ALF-186+SB203580, * = p<0.05).
Fig 3.
Effect of ALF-186 treatment on the phosphorylation of the mitogen-activated protein (MAP) kinases ERK1/2 and JNK after IRI.
(A) Representative western blot image (n = 8) showing the suppression of the phosphorylated MAPK ERK1/2 compared to total ERK1/2 after immediate ALF-186 treatment. Enucleation was either performed 24 hours or 48 hours after IRI. (B) Densitometric analysis (n = 8) of western blots for phosphorylated ERK1/2 compared to its total protein formation 24 and 48 hours after IRI+ALF-186 (data are mean±SD; IRI vs. IRI+ALF-186 24h, ** = p<0.01 and IRI vs. IRI+ALF-186 48h, * = p<0.05). (C) Representative western blot image (n = 8) showing no effect of the phosphorylated MAPK JNK compared to total JNK after immediate ALF-186 treatment. Enucleation was either performed 24 hours or 48 hours after IRI. (D) Densitometric analysis (n = 8) of western blots for phosphorylated JNK compared to its total protein formation 24 and 48 hours after IRI+ALF-186.
Fig 4.
Effect of ALF-186 treatment on the phosphorylation of the MAP kinase p38 after IRI.
(A) Representative western blot image (n = 8) showing the increase of phosphorylated p38 compared to total p38 after immediate ALF-186 treatment. Enucleation was either performed 24 hours or 48 hours post IRI. (B) Densitometric analysis of n = 8 western blots for phosphorylated p38 compared to its total protein formation after IRI+ALF-186 (data are mean±SD; IRI vs. IRI+ALF-186 24h, *** = p<0.001 and IRI vs. IRI+ALF-186 48h, * = p<0.05). (C) Representative western blot image (n = 8) showing the abolishment of p38 phosphorylation due to either iALF-186 treatment or inhibition of p38 using SB203580 compared to total p38 after immediate ALF-186 treatment. Enucleation was performed 24 hours post IRI. (D) Densitometric analysis of n = 8 western blots for phosphorylated p38 compared to its total protein formation after IRI and respective treatment.
Fig 5.
Effect of ALF-186 treatment on retinal mRNA expression and protein cleavage of Caspase-3 and protein and mRNA expression of Bax.
(A) Fold induction of Caspase-3 mRNA after IRI+ALF-186 and p38 inhibition in ischemic retinal tissue compared to GAPDH in relation to the corresponding non-ischemic retinae analyzed by RT-PCR (n = 8; data are mean±SD; IRI vs. IRI+ALF-186, ** = p<0.01 and IRI+ALF-186 vs. SB203580+IRI+ALF-186, ** = p<0.01). (B) Representative western blot image showing the induction of caspase-3 cleavage due to IRI (lane 2) and its suppression (lane 4) compared to uncleaved caspase-3 after ALF-186 treatment. This effect was abrogated by p38 inhibition using SB203580 (lane 6). (C) Densitometric analysis of n = 8 western blots for Caspase-3 cleavage (data are mean±SD; IRI vs. IRI+ALF-186, * = p<0.05 and IRI+ALF-186 vs. IRI+SB203580+ALF-186, * = p<0.05). (D) Fold induction of Bax mRNA after IRI+ALF-186 and p38 inhibition in ischemic retinal tissue compared to GAPDH in relation to the corresponding non-ischemic retinae analyzed by RT-PCR (n = 8; data are mean±SD; IRI vs. IRI+ALF-186, * = p<0.05 and IRI+ALF-186 vs. SB203580+IRI+ALF-186, * = p<0.05). (E) Representative western blot image showing the induction of Bax due to IRI (lane 2) and its suppression (lane 4) compared to β-Actin after ALF-186 treatment. This effect was abrogated by p38 inhibition using SB203580 (lane 6). (F) Densitometric analysis of n = 8 western blots for Bax expression (data are mean±SD; IRI vs. IRI+ALF-186, * = p<0.05 and IRI+ALF-186 vs. SB203580+IRI+ ALF-186, * = p<0.05).
Fig 6.
Effect of ALF-186 treatment on retinal protein and mRNA expression of Bcl-2.
(A) Fold induction of Bcl-2 mRNA after IRI+ALF-186 and p38 inhibition in ischemic retinal tissue compared to GAPDH in relation to the corresponding non-ischemic retinae analyzed by RT-PCR (n = 8; data are mean±SD; IRI vs. IRI+ALF-186, *** = p<0.001 and IRI+ALF-186 vs. SB203580+IRI+ALF-186, *** = p<0.001). (E) Representative western blot image showing no change of Bcl-2 due to IRI (lane 2) but its strong induction (lane 4) compared to β-Actin after ALF-186 treatment. This effect was abrogated by p38 inhibition using SB203580 (lane 6). (F) Densitometric analysis of n = 8 western blots for Bax expression (data are mean±SD; IRI vs. IRI+ALF-186, ** = p<0.01 and IRI+ALF-186 vs. SB203580+IRI+ ALF-186, * = p<0.05).
Fig 7.
ALF-186 induced p38 MAPK in retinal ganglion cell layer.
Retinal cross sections were evaluated for p38 immunoreactivity to answer the question, which cells were responsible for p38 upregulation after IRI+ALF. In controls and after IRI+PBS, there was only a weak p38 expression, pronounced in the area of the ganglion cell layer (GCL) (Fig 5, right side, A, D, G). Co-staining with Brn3a (B, E, H) revealed a low degree of double-immunoreactivity, the p38 signal was seen in a low level in the area of the nerve fibers on top of the GCL (C, F, I). ALF treatment directly after IRI induced upregulation of p38 (J) in the GCL and inner nuclear layer (INL), as expected from the results of the p38 mRNA expression. Double-immunoreactivity with Brn3a (K+L) was more frequent and more intense but not exclusively expressed in RGCs. Scale bar in L is 100 μm.
Fig 8.
Double staining of p38 and Brn3a.
Magnification of the GCL of Fig 7, picture L; demonstrating the double staining of p38 and Brn3a. The arrow points to a double stained retinal ganglion cell.
Fig 9.
Diagram showing the proposed mechanism of ALF-186 mediated anti-apoptotic effect on retinal ganglion cells.
ALF treatment affects mitogen-activated protein kinases ERK1/2 and p38. Due to increased p38 phosphorylation ALF-186 reduced pro-apoptotic Bax protein expression and caspase-3 cleavage while increasing anti-apoptotic Bcl-2, thus promoting neuroprotection.