Abstract
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.
Highlights
Retinal ganglion cells (RGCs) are the only neurons of the retina that send the visual information through the optic nerve to the retinorecipient areas of the brain
For the present studies, using an in vivo rat retinal model of axonal damage, we demonstrate for the first time that the systemic administration of DHF results in activation of the main tropomyosinrelated kinase B (TrkB) activated signaling pathways: (i) phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), and (ii) mitogen activated protein kinases extracellular signal regulated kinases 1 and 2 (MAPK/ERK; ERK1/2), suggesting their involvement in
Western blotting analysis showed an increase in the amount of total mitogen-activated protein kinase (MAPK) protein from 1 day after intraorbital optic nerve transection (IONT) (Figure 1A,B), with a strong phosphorylation from 1 to 3 days after IONT in vehicle-treated groups and from 3 days in DHF-treated groups
Summary
Retinal ganglion cells (RGCs) are the only neurons of the retina that send the visual information through the optic nerve to the retinorecipient areas of the brain. Among all experimental models to study neuronal degeneration and neuroprotection, intraorbital optic nerve transection (IONT) is widely used due to its high reproducibility and easy access to RGC axons compared to other central nervous system (CNS) tracts. This model has been used frequently both in rats [1,2,3,4,5] and mice [5,6,7,8,9,10], showing two phases of RGC degeneration: a first fast phase, from 1 to 9 days after IONT in mice [7,11] or 14 in rats [2,4,5], leaving 15% of the RGC population alive; and a second slower phase, from 9 to 45 days or longer, in which RGCs degenerate much slower [5]. MAPK/ERK pathways activated by BDNF seem to be relevant for local protein synthesis involved in prolonged increase in synaptic transmission, essential in neuronal plasticity and long-term potentiation [33], and they have an important role in neuronal growth and differentiation [34]
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