We analyze the 7,8-dihydroxyflavone (DHF)/TrkB signaling activation of two main intracellular pathways, mitogen-activated protein kinase (MAPK)/ERK and phosphatidylinositol 3 kinase (PI3K)/AKT, in the neuroprotection of axotomized retinal ganglion cells (RGCs). Methods: Adult albino Sprague-Dawley rats received left intraorbital optic nerve transection (IONT) and were divided in two groups. One group received daily intraperitoneal DHF (5 mg/kg) and another vehicle (1%DMSO in 0.9%NaCl) from one day before IONT until processing. Additional intact rats were employed as control (n = 4). At 1, 3 or 7 days (d) after IONT, phosphorylated (p)AKT, p-MAPK, and non-phosphorylated AKT and MAPK expression levels were analyzed in the retina by Western blotting (n = 4/group). Radial sections were also immunodetected for the above-mentioned proteins, and for Brn3a and vimentin to identify RGCs and Müller cells (MCs), respectively (n = 3/group). Results: IONT induced increased levels of p-MAPK and MAPK at 3d in DHF- or vehicle-treated retinas and at 7d in DHF-treated retinas. IONT induced a fast decrease in AKT in retinas treated with DHF or vehicle, with higher levels of phosphorylation in DHF-treated retinas at 7d. In intact retinas and vehicle-treated groups, no p-MAPK or MAPK expression in RGCs was observed. In DHF- treated retinas p-MAPK and MAPK were expressed in the ganglion cell layer and in the RGC nuclei 3 and 7d after IONT. AKT was observed in intact and axotomized RGCs, but the signal intensity of p-AKT was stronger in DHF-treated retinas. Finally, MCs expressed higher quantities of both MAPK and AKT at 3d in both DHF- and vehicle-treated retinas, and at 7d the phosphorylation of p-MAPK was higher in DHF-treated groups. Conclusions: Phosphorylation and increased levels of AKT and MAPK through MCs and RGCs in retinas after DHF-treatment may be responsible for the increased and long-lasting RGC protection afforded by DHF after IONT.

Purpose: To evaluate the optimal therapeutic window of 7,8‐Dihydroxyflavone (DHF), an agonist of TrkB receptor, as a neuroprotectant for degenerative retinal ganglion cells (RGCs) due to an excitotoxic damage with N‐methyl‐D‐aspartic acid (NMDA). Methods: In adult albino Sprague–Dawley rats, NMDA (5 μl of 100 nM) was intravitreally injected into the left eye (day 0) and the animals were sacrificed after 7 days. In these animals, the treatment regimen of systemically administered DHF (5 mg/kg) was evaluated in four groups of increasing dose range (n = 8/group): a single injection of DHF (day −1), two injections (days −1 and 0), four injections (days −1, 0, 1 and 2) and seven injections (day −1 and one daily injection until processing). The number of Brn3a+RGCs was automatically quantified in whole‐mounted retinas. Once the optimal dose of DHF was stablished, the time course of Brn3a+RGCs degeneration was analysed at 3, 7 and 14 days after NMDA administration (n = 8/time‐point) and their treatment with 4 injections of DHF or vehicle (0.9% NaCl containing 1% DMSO). The number of Brn3a+RGCs in injured retinas was quantified and the spatial distribution was assessed with isodensity maps. As control, intact retinas were used. Results: Seven days after NMDA injury, DHF treatment increases the percentage of surviving Brn3a+RGCs from 27% to 68% when retinas were treated with one injection to four injections, respectively (p < 0.05). No significant changes were found between four or seven DHF injections, indicating that the optimal treatment regimen for DHF is four injections and showing a rescue of Brn3a+RGCs over the entire retinal surface. Using this DHF dose at different time‐points after NMDA injury, DHF treatment rescues most Brn3a+RGCs degeneration at 3 days and it decreases up to 56% at 14 days. Conclusions: DHF prevents RGC death following NMDA excitotoxic damage.