Axotomy-induced STAT3 activation is dependent on RA.

<p><b>A</b>. Representative Western blots of phosphorylated-STAT3 and total STAT3 from extracts of retinas from uncut animals, and animals at 1 week after axotomy. Approximate molecular weight in kDa is indicated. The order of some of the bands was rearranged to correspond to the bar chart below. <b>B.</b> Quantification of Western blots, standardized to control values. pSTAT3 levels are increased at 1 week after axotomy. Application of ATRA increases the levels of pSTAT3 further, but other RAR agonists do not. Inhibition of RA synthesis, or application of RAR antagonists, prevents the axotomy-induced increase in pSTAT3. One-way ANOVA followed by <i>post-hoc</i> Tukey-Kramer tests: * p<0.05, ** p<0.01, *** p<0.001, N = 4, 4, 3, 3, 3, 3, 3, 4, 4, and 3 pools, each of 2 animals and with duplicate measurements. Error bars represent the S.E.M.</p

Retinoic signaling agonists promote long-term survival of axotomized RGCs.

<p><b>A-F.</b> Representative fields of whole-mount retinas in which RGCs have been retrogradely labled with Texas Red dextran amine (TDA). <b>A.</b> Uncut normal retina. <b>B.</b> Retina from animal at 6 weeks after axotomy, eye injected with PBS and DMSO (vehicle). <b>C.</b> Retina from animal at 6 weeks after axotomy, eye injected with PBS and DMSO with the addition of all-trans retinoic acid (ATRA). <b>D.</b> Retina from animal at 6 weeks after axotomy, eye injected with PBS and DMSO with the addition of the RARα agonist AM80 (RARα agon). <b>E.</b> Retina from animal at 6 weeks after axotomy, eye injected with PBS and DMSO with the addition of the RARβ agonist CD2314 (RARβ agon). <b>F.</b> Retina from animal at 6 weeks after axotomy, eye injected with PBS and DMSO with the addition of the RARγ agonist CD1530 (RARγ agon). <b>G.</b> Quantification of RGC percentage survival in the temporal region of the retina at 6 weeks after axotomy. Vehicle-treated retinas show a significant 40% decrease in RGC numbers. Treatment with ATRA or RAR agonists rescues many of these RGCs. One-way ANOVA followed by <i>post-hoc</i> Tukey tests: * p < 0.05, ** p < 0.01, *** p < 0.001, N = 4 animals in all cases. Error bars represent the S.E.M. Scale bar in A = 50 μm.</p

Increased activity of signaling pathways is localized to RGCs.

<p>Immunostaining was carried out against pMAPK, pAKT and pSTAT3 in frozen sections of retinas from uncut animals (A, E, I), animals at 1 week after the optic nerve was cut and the eyeball injected with vehicle (B, F, J), animals at 1 week after the optic nerve was cut and the eyeball injected with ATRA (C, G, K), and animals at 1 week after the optic nerve was cut and the eyeball injected with RALDH inhibitor (D, H, L). In A, the inner plexiform layer (IPL) and ganglion cell layer (GCL) are indicated. <b>A, E, I.</b> In control frog retinal sections, low levels of pMAPK, pAKT and pSTAT3 immunoreactivity are present in the GCL and the INL. <b>B, F, J.</b> One week after axotomy, immunostaining of all three proteins has increased in intensity, particularly in the cell bodies of RGCs of the GCL and their axons, and in the cells in the INL. <b>C, G, K.</b> One week after axotomy and ATRA treatment, immunostaining of all three proteins is intense in the cell bodies of RGCs of the GCL and their axons, and in the cells in the INL. <b>D, H, L.</b> One week after axotomy and RALDH inhibitor treatment, faint immunostaining of all three proteins is present in the GCL and the INL. Scale bar in A: 50 μm.</p

Retinoic signaling antagonists reduce long-term survival of axotomized RGCs.

<p><b>A-D.</b> Representative fields of whole-mount retinas in which RGCs have been retrogradely labled with Texas Red dextran amine (TDA). <b>A.</b> Uncut normal retina. <b>B.</b> Retina from animal at 6 weeks after axotomy, eye injected with PBS and DMSO (vehicle). <b>C.</b> Retina from animal at 6 weeks after axotomy, eye injected with PBS and DMSO with the addition of the RALDH inhibitor disulfiram (RALDH inhib). <b>D.</b> Retina from animal at 6 weeks after axotomy, eye injected with PBS and DMSO with the addition of the pan-RAR antagonist Ro-41-5253 (RAR antag). <b>E.</b> Quantification of RGC percentage survival in the temporal region of the retina at 6 weeks after axotomy. Vehicle-treated retinas show a significant 40% decrease in RGC numbers. Treatment with RALDH inhibitor further reduces survival. Only the antagonist against RAR, not RARβ, further reduces survival. One-way ANOVA followed by <i>post-hoc</i> Tukey tests: * p < 0.05, ** p < 0.01, *** p < 0.001, N = 4 animals in all cases. Error bars represent the S.E.M. Scale bar in A = 50 μm.</p

Axotomy-induced AKT activation is dependent on RA.

<p><b>A</b>. Representative Western blots of phosphorylated-AKT and total AKT from extracts of retinas from uncut animals, and animals at 1 week after axotomy. Approximate molecular weight in kDa is indicated. The order of some of the bands was rearranged to correspond to the bar chart below. <b>B.</b> Quantification of Western blots, standardized to control values. pAKT levels are increased at 1 week after axotomy. Application of ATRA or other RAR agonists do not significantly increase the levels of pAKT further. Inhibition of RA synthesis, or application of RAR antagonists, prevents the axotomy-induced increase in pAKT. One-way ANOVA followed by <i>post-hoc</i> Tukey tests: * p<0.05, ** p<0.01, *** p<0.001, N = 3 pools in all cases, each of 2 animals and with duplicate measurements. Error bars represent the S.E.M.</p