Effect of Autophagy Modulators on Vascular, Glial, and Neuronal Alterations in the Oxygen-Induced Retinopathy Mouse Model

Abstract

Hypoxia is one of the main insults in proliferative retinopathies, leading to neovascularization and neurodegeneration. To maintain homeostasis, neurons require efficient degradation and recycling systems. Autophagy participates in retinal cell death, but it is also a cell survival mechanism. Here, we analyzed the role of autophagy at the three characteristic time periods in the oxygen-induced retinopathy (OIR) mouse model and determined if its modulation can improve vascular and non-vascular alterations. Experiments were performed with chloroquine (CQ) in order to monitor autophagosome accumulation by lysosomal blockade. Post natal day (P)17 OIR mouse retinas showed a significant increase in autophagy flux. In particular, an intense LC3B and p62 staining was observed in inner layers of the retina, mainly proliferating endothelial cells. After a single intraocular injection of Rapamycin at P12 OIR, a decreased neovascular area and vascular endothelial growth factor (VEGF) protein expression were observed at P17 OIR. In addition, whereas the increased expression of glial fibrillary acidic protein (GFAP) was reversed at P26 OIR, the functional alterations persisted. Using a similar therapeutic schedule, we analyzed the effect of anti-VEGF therapy on autophagy flux. Like Rapamycin, VEGF inhibitor treatment not only reduced the amount of neovascular tufts, but also activated autophagy flux at P17 OIR, mainly in ganglion cell layer and inner nuclear layer. Finally, the effects of the disruption of autophagy by Spautin-1, were evaluated at vascular, glial, and neuronal levels. After a single dose of Spautin-1, Western blot analysis showed a significant decrease in LC3B II and p62 protein expression at P13 OIR, returning both autophagy markers to OIR control levels at P17. In addition, neither gliosis nor functional alterations were attenuated. In line with these results, TUNEL staining showed a slight increase in the number of positive cells in the outer nuclear layer at P17 OIR. Overall, our results demonstrate that all treatments of induction or inhibition of the autophagic flux reduced neovascular area but were unable to completely reverse the neuronal damage. Besides, compared to current treatments, rapamycin provides a more promising therapeutic strategy as it reduces both neovascular tufts and persistent gliosis.

Metabolic Syndrome Triggered by Fructose Diet Impairs Neuronal Function and Vascular Integrity in ApoE-KO Mouse Retinas: Implications of Autophagy Deficient Activation

“…Recently, autophagy has demonstrated an important role in the eye diseases such as retinopathies as either a pro-survival or pro-death mechanism ( Arroba et al, 2016 ; Boya et al, 2016 ; Cammalleri et al, 2016 ; Lopes de Faria et al, 2016 ; Dehdashtian et al, 2018 ; Subirada et al, 2019 ). In this sense, changes in the autophagy flux have been described in conditions of hyperglycemia and hyperlipidemia ( Meng et al, 2017 ).…”

“…Electroretinographic activity was recorded after 2, 4, and 6 months of feeding according to previously described procedures ( Ridano et al, 2017 ; Lorenc et al, 2018 ; Subirada et al, 2019 ). Briefly, after overnight dark adaptation and under dim red illumination, mice were anesthetized with ketamine/xylazine (i.p.…”

“…For cryosection, eyes after 4 months of feeding were enucleated, fixed during 2 h with 4% PFA at room temperature, and incubated overnight in 10, 20, and 30% of sucrose in PBS at 4°C. Then, they were embedded in optimum cutting temperature (OCT, Tissue-TEK, Sakura) compound, and 10-μm-thick radial sections were obtained by using a cryostat, according to general methods ( Subirada et al, 2019 ). Retinal cryosections were then stored at −20°C under dry conditions until immunohistochemical analysis.…”

“…Immunostaining was performed as described previously ( Ridano et al, 2017 ; Lorenc et al, 2018 ; Subirada et al, 2019 ). Briefly, mouse cryosections were washed in PBS, blocked with 2% of BSA in PBS containing 0.1% Tween 20, for 1 h and then incubated overnight at 4°C with the following primary antibodies: rabbit polyclonal Anti-GFAP antibody from Dako (1/100; Carpinteria, CA, United States), rabbit polyclonal anti-LC3B (1/100; L7543, Sigma Aldrich) and mouse monoclonal anti-p62 (1/100; ab56416, Abcam).…”

“…Total RNA was extracted from neural retinas using Trizol (Invitrogen), according to the manufacturer’s instructions and was processed as previously reported ( Ridano et al, 2017 ; Subirada et al, 2019 ). Briefly, 1 μg of total RNA was reverse-transcribed in a total volume of 20 μL using random primers (Invitrogen, Buenos Aires, Argentina) and 50 U of M-MLV reverse transcriptase (Promega Corp.).…”