Systemic administration of 7,8‐dihydroxyflavone (DHF) after intraorbital optic nerve section in the rat produces neuroprotection of the Brn3a+retinal ganglion cell population
Authors: Vidal‐Villegas Beatriz , Gallego‐Ortega A. , Di Pierdomenico J. , Galindo‐Romero C. , García‐Feijoo J. , de la Casa J. M. Martínez , Villegas‐Pérez M. P. , Vidal‐Sanz M. 
Published: December 2022

Objective: To investigate the short‐ and long‐term responses of different retinal ganglion cell (RGC) populations to intraorbital optic nerve transection (IONT) and their protection with systemic administration of 7,8‐dihydroxyflavone (DHF), a potent selective agonist of the tropomyosin related kinase B (TrkB) receptor,

Methods: Adult anaesthetized Sprague–Dawley rats received an IONT of their left eye and daily intraperitoneal (ip) injections of vehicle (0.9% NaCl containing 1% DMSO) or of DHF (5 mg/kg in the same vehicle). One group of animals was used to investigate long term protection of DHF and was processed 7, 10, 14, 21, 30 or 60 days (n = 8 for each survival interval) after the lesion. Another group of animals was used to investigate the responses of different RGC types to DHF treatment and was processed 7, 14 or 21 days (n = 5–8 for each survival interval) after the lesion. The retinas were prepared as wholemounts and immunolabelled for Brn3a, melanopsin (m), Osteopontin (OPN) and the T‐box transcription factor T‐brain‐2 (Tbr2) to identify the following retinal ganglion cells (RGCs) populations: Brn3a+, melanopsin+, α‐ like (OPN+), α‐OFF like (OPN+Brn3a+) and M4‐like/α‐ON sustained RGCs (OPN+Tbr2+). The labelled RGCs were quantified automatically (Brn3a) or dotted manually and quantified with a graphic software, and distribution of the different populations represented on topographical logical maps.

Results: IONT resulted in the loss of 83% or 59% of the Brn3a+ or m+RGC population, respectively, 14 days after vehicle treatment. The Brn3a+RGCs further diminished an additional 9% from 14 to 60 days whereas m+RGCs did not, thus at 60 days the proportion of surviving m+RGCs was almost five times greater than Brn3a+RGCs, thus showing greater resistance to IONT. Systemic administration of DHF resulted in prevention of Brn3a+RGCs loss during 21 days and permanent protection of m+RGCs. Different RGC populations showed different losses. In vehicle treated rats, IONT resulted in the rapid and massive loss of all OPN+Brn3a+RGCS, progressive and massive loss of Brn3a+RGCs and large but not progressive loss of OPN+RGCs or OPN+Tbr2+RGCs, indicating a certain resistance of the later types to IONT. DHF treatment resulted in significant prevention of the IONT‐induced loss of Brn3a+, OPN+ and OPN+‐Tbr2+ RGCs, but not of the OPN+‐Brn3a+.

Conclusions: Our in vivo studies on adult rats document that IONT and vehicle treatment results in characteristic patterns of loss of the different types of RGCs including Brn3a, melanopsin, alpha and alpha‐ON sustained RGCs, although melanopsin, alpha and alpha‐ON sustained RGCs show certain resistance to the injury. DHF treatment affords in vivo protection of axotomized RGCs that lasts 21 days for the Brn3a+, m+, alpha and alpha‐ON sustained, but not for alpha‐OFF RGCs, which were not responsive to treatment.