A goat anti-rabbit IgG H&L (Alexa Fluor? 488; abcam, ab150077, RRID:AB_2630356) was utilized for a rabbit anti-LC3B polyclonal antibody or a rabbit anti-SQSTM1 polyclonal antibody
A goat anti-rabbit IgG H&L (Alexa Fluor? 488; abcam, ab150077, RRID:AB_2630356) was utilized for a rabbit anti-LC3B polyclonal antibody or a rabbit anti-SQSTM1 polyclonal antibody. Stained sections were imaged by confocal laser scanning microscopy (LSM510 Axiovert200?M; Carl Zeiss Angiotensin I (human, mouse, rat) Meditec, Goettingen, Germany). of AlloP. Injection of polystyrene microbeads into the anterior chamber improved intraocular pressure about 3-fold and induced RGC apoptosis. A single intravitreal injection of AlloP or autophagy activators prevented apoptosis and safeguarded RGCs with autophagy activation. We conclude that AlloP may serve as a potential restorative agent for the treatment of glaucoma via varied mechanisms. Abbreviations: 2HBCD: 2-Hydroxypropyl)–cyclodextrin; 3-MA: 3-methyladenine; AlloP: allopregnanolone; AP: autophagosome; AVd: degradative autophagic vacuoles; GCL: ganglion cell coating; INL: inner nuclear coating; IOP: intraocular pressure; IPL: inner plexiform coating; LC3B-I: cytosolic form of LC3B; LCB-II: lipidated form of LC3B; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; mPTP: mitochondrial permeability transition pore; NDS: neuronal damage score; NFL: nerve dietary fiber coating; OH: ocular hypertension; ON: optic nerve; ONL: outer nuclear coating; OPL: outer plexiform coating; p-STR: scotopic threshold response; RGC: retinal ganglion cells; RT-PCR: real-time reverse transcription polymerase chain reaction; SQSTM1: sequestosome 1; TUNEL: TdT-mediated dUTP Nick End Labeling glaucoma model with isolated rat retinas, we previously reported that AlloP attenuated pressure-induced Angiotensin I (human, mouse, rat) retinal injury [16C18]. Because a specific GABR antagonist inhibits neuroprotective effects of AlloP, GABAergic signaling likely mediates the neuroprotection by AlloP. However, neuroprotection by Angiotensin I (human, mouse, rat) AlloP may not specifically involve GABRs. AlloP was found to activate autophagy inside a mouse model of Niemann-Pick Type C disease  and main astrocyte cultures , suggesting that upregulation of autophagic flux may contribute to endogenous neuroprotective mechanisms . In the present study, we used a rat ocular hypertension (OH) model having a closed chamber incubation system (Fig. S2) and an OH model following injection of polystyrene microbeads into the anterior chamber to examine neuroprotective effects of AlloP, focusing on the part of autophagy. Results Neuroprotective effects of AlloP in an ex lover vivo glaucoma model Consistent with our earlier reports [17,18], retinas incubated at 10 mm Hg (Number 1A) exhibited normal appearance but those at 75 mm Hg showed axonal swelling in the nerve dietary fiber coating (NFL) (Number 1B); 1?M AlloP attenuated this damage (Number 1C). To confirm the neuroprotective effects of AlloP involve GABRs, we given 1?M picrotoxin, a GABR antagonist. As previously observed , picrotoxin overcame the neuroprotective effect of AlloP under hyperbaric conditions (Number 1D). Open in a separate window Number 1. The effects of autophagy activators and autophagy inhibitors on retinal morphology in glaucoma models. (A-H) Light micrographs of pressure-loaded retinas. (A) 10 mm Hg. (B) 75 mm Hg. Arrowheads, axonal swelling. (C) AlloP at 75 mm Hg. (D) Co-administration of AlloP and picrotoxin at 75 mm Hg. (E and F) Rapamycin (E) or torin 2 (F) at 75 mm Hg. (G and H) Administration of bafilomycin A1 (G) or SAR405 (H) induced severe degeneration in retinas incubated with AlloP at 75 mm Hg. Arrows, RGC degeneration. Level bars: 20?m. IKK-alpha (I-P) RGC survival and neuroprotection in pressure-loaded whole mounted retinas. (I) 10 mm Hg. (J) 75 mm Hg. (K) AlloP at 75 mm Hg. (L) Combination of AlloP and picrotoxin at 75 mm Hg. (M) Rapamycin at 75 mm Hg. (N) torin 2 at 75 mm Hg. (O) Combination of AlloP and bafilomycin A1 at 75 mm Hg. (P) Combination of AlloP and SAR405 at 75 mm Angiotensin I (human, mouse, rat) Hg. Level bars: 200?m (Q) The number of RBFOX3-positive cells in whole-mount retinas (n?=?5 per experiment, Tukey *p? ?0.05). (R-Y) TUNEL staining. (R) 10 mm Hg. (S) 75 mm Hg. Arrows show TUNEL-positive cells in the GCL. (T) AlloP significantly decreased the number of TUNEL-positive Angiotensin I (human, mouse, rat) cells at 75 mm Hg. (U) Combination of AlloP and picrotoxin at 75 mm Hg. (V) Rapamycin at 75 mm Hg. (W) torin 2 at 75 mm Hg. (X) Combination of AlloP and bafilomycin A1 at 75 mm Hg. (Y) Combination of AlloP and SAR405 at 75 mm Hg. Level bars: 30?m. (Z) The number of TUNEL-positive RGCs per 200?m of retina section (n?=?5 per experiment, Tukey *p? ?0.05) Next, we examined the involvement of autophagy in neuroprotection of pressure-induced retinal injury using autophagy activators (rapamycin, torin 2) and autophagy inhibitors (bafilomycin.