Neuroprotection By Autophagy-Inducing Peptide Tat-beclin1 Against Acute Ocular Hypertension Induced Retinal Damage In Rat

Purpose:To determine whether intravitreal administration of autophagy-inducing peptide,tat-beclin 1,protects against retinal ganglion cells(RGCs)death after acute ocular hypertension injury.In addition,to investigate whether the autophagy pathway has a role in the apoptosis of RGCs.Methods:(1)Cell viability of peptide-pretreated primary cultured retinal neurons was measured using Cell Counting Kit-8(CCK-8)following exposure to oxygen and glucose deprivation/reperfusion(OGD/R)insult.(2)In Sprague-Dawley rats,the anterior chamber of the right eye was cannulated with a needle connected to a saline container elevated 1.5 meters above the eye for 60 minutes followed by intravitreal injection of 2.5μg tat-beclin 1,tat-scrambled or vehicle.Eyes were dissected 6h,12h,24h,48h,3d,7d after acute ocular hypertension injury.Change of retinal morphology was determined by histochemistry using Hematoxylin and Eosin(H&E)stained sections and quantified by inner retinal thickness.Retinal cell apoptosis was detected by TUNEL assay 24h after acute ocular hypertension injury.Expression of cleaved caspase-3,Beclin 1,LC3 and p62 were determined using western blot analyses.Retrograde labeling of RGCs by application of Fluoro-Gold on superior colliculus was used to identify the surviving RGCs after acute ocular hypertension injury.Results:(1)The CCK-8 results showed that the cell viability was significantly higher in the OGD1h/Rlh + tat-beclin 1 group when compared to the cell viability in the OGDlh/Rlh group(P<0.001).(2)3 days after acute ocular hypertension injury,inner retinal thickness decreased by approximately 30%as shown by images of H&E stained retinal sections.TUNEL staining showed that the number of necrotic cells increased following acute ocular hypertension.However,the number of necrotic cells in tat-beclin 1 group were lower than that of the vehicle-treated group.Acute ocular hypertension resulted in a progressive loss of FG-labeled RGCs in the vehicle-treated retinas,approximately 50%,whereas intravitreal injection of tat-beclin 1 increased the survival rate of RGCs to 73%at the same time.Western blot analysis showed that the expression of cleaved caspase-3 was downregulated in tat-beclin 1 treatment group when compared to the vehicle-treated retinas 24h after acute ocular hypertension injury.The expression of LC3Ⅱ/Ⅰ and beclin 1 were downregulated in the vehicle-treated retinas 24h after acute ocular hypertension injury,and intravitreal injection of tat-beclin 1 upregulated the expression of LC3Ⅱ/Ⅰ and beclin 1.Conclusions:This study supports that autophagy has a protective role in primary cultured retinal cells after OGD/R injury and in retinas after acute ocular hypertension injury.Autophagy-inducing peptide has a potential efficacy in new therapeutic strategies to ameliorate various retinal diseases.