Abstract
Purpose :
Fibroblast Growth Factor (FGF) secretion has been shown to be upregulated in response to retinal injury, nevertheless, its role during regeneration remains controversial. In this study we aim to examine the efficiency of FGF to promote Müller cell proliferation in mouse retinas as well as understand the downstream signalling cascade that is essential during this process.
Methods :
To assess the efficiency of FGF signaling to induce Müller cell proliferation following N-methyl-D-aspartate (NMDA) injury, exogenous bFGF was injected intravitreally in P60 wild type mice. To validate the nessecity of FGF signalling during this process conditional FGFR1/2 knock out mice (RaxCreERT2FGFR1flox/floxFGFR2flox/flox) were injected intravitreally with established regeneration inducers (EGF, Activin A and Chir99021). Regeneration efficiency was assessed though the colocalisation of EdU and Sox2 on both flat mount retinas and cryosectioning. Cell signalling propagation following the injections was analysed using both immunohistochemistry and western blot. Retinal physiology was tested using optical coherence tomography and electroretinography
Results :
Exogenous supply of bFGF through intravitreal injection was not sufficient to induce Müller cell proliferation following NMDA injury (n=8). Conditional deletion of FGFR1/2 in Müller cells, dramatically decreased their proliferation when induced with the regeneration inducers (Chir99021 n=18, p=0.015, SE=0.001; EGF n=9, p=0.009, SE=0.002; Activin A n=8, p=0.04, SE=0.003). This result was reproduced through intravitreal injections of MEK inhibitor (Chir99021 n=4, p= 0.017, SE=0.001; EGF n=4, p=0.002, SE=0.001; Activin A n=4, p=0.045, SE=0.006) but not with PI3K inhibitor. Elevated activation of STAT3 was detected in the conditional knockout mice when compared to wild type. Inhibition of STAT3 increased the efficiency of Müller cell proliferation (n=6, p=0.037, SE=0.013) but failed to rescue the lack of regeneration in the conditional knockout mice.
Conclusions :
We show that FGF signaling is upregulated in response to injury. FGF alone is not adequate to promote regeneration but it is an obligatory mitogen during this process. The capacity of FGF to maintain Müller cell proliferation is achieved through MEK/ERK transduction and the lack of FGF signaling increases STAT activity. Finally, inhibition of cell proliferation in the absence of FGF signaling is likely independent of STAT3
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.