Scientific Papers

NRF2/ARE mediated antioxidant response to glaucoma: role of glia and retinal ganglion cells | Acta Neuropathologica Communications


AAV construct development and AAV production

pAAV.CMV.Nrf2 was purchased from Addgene (Watertown, MA; plasmid #67,636) and packaged into AAV2/2 at SignaGen (Fredrick, MD). pAAV.CMV.eGFP was purchased from Addgene (plasmid #67,634) and packaged into AAV2/2 at SignaGen. The AAV.ARE.tdTomato reporter virus was constructed as previously described [19]. The construct was tested in ARPE-19 cells prior to use in vivo (Supplemental Fig. 1). ARPE-19 cells were purchased from ATCC (Manassas, VA) and grown as previously described [20]. Cells in complete media were transfected with plasmid DNA mixed with FuGENE HD (Promega, Madison, WI; #E2311) at a ratio of 1:4 (1 mg DNA: 4 ml FuGENE). Eight-well chamber slides and 6-well plates were transfected with 200ng and 1 mg DNA per well, respectively. One day after transfection, the media was replaced with serum-free (SF) medium and incubated for 24 h. Cells were treated with SF medium containing either 5 mM sulforaphane, an NRF2 activator, (MedChemExpress, Monmouth Junction, NJ; #HY-13,755) or vehicle (0.025% DMSO) and incubated for an additional 24 h. Cells were preserved with Histochoice, washed with PBS, and coverslips mounted with ProLong Gold (Thermo Fisher). For immunoblots, 10 µg of PBS-soluble protein was analyzed per lane. Blots were probed with rabbit anti-RFP (Rockland Immunochemicals, Inc., Limerick, PA; #600-401-379) and mouse anti-HA (Cell Signaling Technologies, Danvers, MA; #2367). ARE activation, based on increased tdTomato (tdTom) fluorescence, was detected in transfected cells treated with sulforaphane, but not in those that did not receive sulforaphane nor those that were transfected with the plasmid carrying the inactive M4 ARE (Supplemental Fig. 1). Cells transfected with WT ARE and treated with either DMSO or sulforaphane produced both tdTom and ZsGreen, with more intense tdTom in the cells treated with sulforaphane (Supplemental Fig. 1). However, in the ARPE19 cells transfected with M4 ARE, there was no tdTom band (Supplemental Fig. 1).

AAVs were generated that express Cre recombinase and tdTom (see Table 1). The promoters included either the 1 kb human vimentin (Vim) promoter that was derived from Addgene plasmid #29,114 [21] or the 0.66 kb mouse gamma-synuclein (Sncg) promoter (Addgene plasmid #153,163) [22]. RGCs were targeted with the combination of Sncg promoter and AAV2/2 capsid, which has been widely used to efficiently infect RGCs after intravitreal injection. Additionally, the Sncg promoter has been characterized such that following intravitreal injection, more than 85% of the transduced RGCs also double-labeled with RBPMS [22]. While we aimed to primarily transduce astrocytes with the combination of a 1 kb Vim promoter and a modified AAV6 capsid (ShH10 with an additional Y455F mutation) [31], we have not excluded the possibility that some Müller glia were also transduced following intravitreal injection. Control AAVs contained tdTomato without Cre (see Table 1). All Cre and control AAVs were packaged in-house using triple transfection of HEK cells.

Table 1 Plasmids used to produce AAVs in this study

Mice

Control mice (C57Bl/6J), Nrf2 knockout mice (B6.129 × 1-Nfe2l2tm1Ywk/J) or Nrf2 floxed mice (C57BL/6-Nfe2l2tm1.Sred/SbisJ) (Jackson Labs, Bar Harbor, ME) were group-housed, maintained on a 12-h light-dark cycle, and provided food and water ad libitum. An equal distribution of 2–3 month old male and female mice were used for this project.

Microbead occlusion model (MOM)

IOP was bilaterally elevated using the well-characterized microbead occlusion model (MOM) of glaucoma [23, 24]. We injected 2 µl of 15-µm diameter FluoSpheres polystyrene microbeads into the anterior chamber of anesthetized mice (Thermo Fisher, Waltham, MA) as previously described [23,24,25]. Additional mice received bilateral injections of an equivalent volume of lactated Ringer’s saline solution as controls. Briefly, 1.5 mm outer diameter/1.12 mm inner diameter filamented capillary tubes (World Precision Instruments, Sarasota, FL) were pulled using a P-97 horizontal puller (Sutter Instrument Company, Novato, CA), and the resulting needles were broken using forceps to an inner diameter of ~ 100 μm. Microbeads were loaded and injected using a microinjection pump (World Precision Instruments, Sarasota, FL). Mice were anesthetized with isoflurane and dilated using topical 1% tropicamide ophthalmic solution (Patterson Veterinary, Devens, MA), and 2 µl (~ 2,000 microbeads) were injected. The needle was maintained in the injection site for 20 s before retraction to reduce microbead efflux. Mice were given topical 0.3% tobramycin ophthalmic solution (Patterson Veterinary, Devens, MA) following injection.

IOP measurements

IOP was measured immediately prior to microbead injection and biweekly thereafter using the Icare TonoLab rebound tonometer (Colonial Medical Supply, Franconia, NH) as previously described [23,24,25]. Mice were anesthetized using isoflurane, and 10 measurements were acquired from each eye within 2 min of induction of anesthesia.

AAV injections

For experiments with an endpoint of 5 weeks post-IOP elevation, viruses were intravitreally injected 1 wk prior to MOM injections. For experiments with an endpoint of 2 wks post-IOP elevation, viruses were intravitreally injected 2 weeks prior to MOM injections. All mice used in this study were injected with 1ul of virus at a concentration of 1 × 109 GC/ul.

In vivo electrophysiology

Mice were dark adapted overnight, dilated with 1% tropicamide for 10 min and anesthetized with 20/8/0.8 mg/kg ketamine/xylaxine/urethane according to previously published methodology [8, 19]. Mice were placed on the heated surface of the ERG system to maintain body temperature. Corneal electrodes with integrated stimulators (Celeris System, Diagnosys LLC, Lowell, MA) were placed on eyes that were lubricated with GenTeal drops. Subdermal platinum needle electrodes were placed in the snout and back of the head at the location of the visual cortex. A ground electrode was placed in the back of the mouse. For VEPs, mice were exposed to 50 flashes of 1 Hz, 0.05 cd.s/m2 white light with a pulse frequency of 1 flash. For flash ERGs and VEPs, mice were first exposed to flashes of 1 Hz, 1 cd.s/m2 white light with a pulse frequency of 1 following dark adaptation. Secondly, after the mice had already been exposed to the flash ERG/VEP, we recorded the photopic negative response (PhNR) of the ERG by exposing mice to 100 continuous flashes of white light on a green background with a pulse frequency of 2. Each experimental group had 12–16 eyes.

Dihydroethidum fluorescence

A dye that fluoresces in the presence of superoxide and, to a lesser extent, hydrogen peroxide, dihydroethidum (DHE), was utilized for these studies as previously described [31]. Mice were anesthetized with 2.5% isofluorane and intravitreally injected with 1 µl (0.5 μm) of DHE (ThermoFisher Scientific, Waltham, MA) diluted in phosphate-buffered saline (PBS) using a 30-gauge Hamilton syringe. Just prior to imaging, mice were anesthetized with ketamine/xylazine and eyes were dilated with 1% tropicamide. Thirty minutes after DHE injection, fluorescence was imaged on a Micron IV retinal imaging microscope (Phoenix Research Labs, Pleasanton, CA) using an FF02-475/50 nm excitation filter (Semrock, Inc. Rochester, NY) and ET620/60X emission filter (Chroma Technology Corp., Bellows Falls, VT). The average intensity of the fluorescence throughout the retina was quantified using ImageJ [32]. For each experimental group, 6–8 eyes were analyzed. Notably, the DHE fluorescence was measured for each group during the same imaging session.

Tissue collection

For western blots and qPCR, retinas were collected fresh and flash-frozen from mice euthanized by anesthetic overdose and cervical dislocation. For immunohistochemistry and optic nerve histology, tissue was fresh collected and post-fixed in 4% paraformaldehyde until use at 4oC.

Protein assay

Protein concentrations were determined from 10 µl of retina homogenates with the Pierce BCA Protein Assay Kit (cat#: 23,225, ThermoFisher Scientific, Waltham, MA). BSA was used as the protein standard. Absorbance was measured with the plate reader SpectraMax M2 (Molecular Devices, San Jose, CA).

Western blot

Single retinas were sonicated in lysis buffer (PBS, EDTA and Halt protease inhibitor) and centrifuged for 30 min at 4oC. 4x Laemmli buffer (Bio-rad, cat# 1,610,747) containing b-mercaptoethanol was added to the samples and heated for 5 min at 95oC. Known amounts of protein (10–20 µg/retina) or protein ladder (cat#1,610,375, Bio-rad, Hercules, CA) were loaded in 4–20% polyacrylamide gels (Bio-Rad #456–1095). Proteins were transferred onto nitrocellulose using the Bio-Rad trans blot turbo transfer system. Membranes were blocked in 2% BSA in TBS-T overnight at 4oC. Membranes were incubated in primary antibody (see Table 2) at room temperature with rocking for 2 h. Membranes were washed three times at 1x TBS-T for five min each. Membranes were incubated with secondary antibody (IRDye 800CW Donkey anti-rabbit, #926-32213 or IRDye 680CW Donkey anti-mouse, #926-68022,1:5000 in 1% BSA/TBS) at room temperature for 1 h protected from light. Membranes were washed again three times at 1x TBS-T for five min each. Following washing, blots were imaged with a Bio-Rad ChemiDoc system. Band density was quantified by scanning the blot using Adobe Photoshop. Each band was selected with the same frame and set measurements were used to obtain the gray mean value for each. Band intensity measurements from protein of interest were divided by band intensity measurements of loading control (b-actin). Each experimental group consisted of 5 retinas.

Quantitative PCR

Retinas were extracted from euthanized mice and placed immediately onto dry ice and stored at 80oC until homogenized by hand using 1.5ml-capacity pestles (cat#46C911, Grainger, Nashville, TN). RNA was extracted using a Qiagen RNeasy kit (Valencia, CA) according to manufacturer’s protocol. RNA concentration and purity were measured on a spectrophotometer. First-strand complementary DNA (cDNA) was synthesized from 250 ng of RNA from each sample using the Superscript III First-Strand synthesis system and oligo-dT20 primers (Invitrogen, Waltham, MA). Quantitative PCR (qPCR) was performed using Power SYBR green master mix (Applied Biosystems, Waltham, MA). All primer sequences were obtained from previous studies; we assessed the following: Prdx6, Gpx1, Ho-1 and Sod3 (see Table 3). All qPCR was performed in triplicate using an Applied Biosciences 7300 real-time PCR system (Waltham, MA). The amplification threshold was set using system software. To calculate the expression of genes, we first normalized to the CT of the housekeeping gene, GAPDH. Then, we calculated the negative delta delta CT and normalized the results from all transcripts data to reflect the fold change over the negative delta delta CT of the saline-injected control. Each experimental group had 4–5 retinas.

Immunohistochemistry

Eyes were embedded in paraffin and sectioned at 10 microns according to previously published methods using the Vanderbilt Vision Research Center histology core [8, 19]. Slides were then warmed on a slide warmer at a medium setting (about 40 oC) for 30 min. Slides were then placed in a rack and went through a series of deparaffinization steps: xylene (10 min), 100% ethanol (10 min), 100% ethanol (5 min), 95% ethanol (5 min), 80% ethanol (5 min), 60% ethanol (5 min), 40% ethanol (5 min). Slides were then placed in coplin jar covered with sodium citrate solution and boiled for 30 min (2.94 g of tri-sodium citrate dehydrate in 1 L of DI water, adjusted to pH of 6.0 and then added 0.5ml of Tween 20). Following boiling, slides were washed twice in 1x PBS for 5 min. Then, slides incubated in sodium borohydride solution (0.05 g sodium borohydride dissolved in 50ml DI water, made fresh every time) at room temperature. Slides were then placed in blocking buffer (500mL 1x PBS, 1.25mL Triton-X, 1.25mL Tween 20, 0.5 g sodium citrate, 11.25 g glycine, 5 g BSA) and 5% normal donkey serum (cat #: D9663, Millipore Sigma, Darmstadt, Germany) for 1 h at room temperature. Slides were washed once with 1xPBS and placed in primary antibody diluted in staining buffer (500mL 1x PBS, 1.25mL Triton X, 1.25 mL Tween 20, 5 g BSA) overnight at 4oC in a humidified chamber. The following day, slides were twice washed with 1x PBS for 5 min each. Secondary antibody was diluted in staining buffer and was added to the slides for 2 h at room temperature at 1:200 dilution after spinning for 10 min at 13,000 g. After 2 h, slides were washed twice in 1x PBS for 5 min each. Then, slides were coverslipped with Vectashield containing DAPI (cat#: H-1200-10, Vector Laboratories, Burlingame, CA) and sealed with nail polish. Slides were imaged on a Nikon Eclipse epifluorescence microscope (Nikon, Melville, NY). All images were collected from the same retinal region with identical magnification, gain and exposure settings. Fluorescence intensity was quantified via ImageJ as previously described [8]. A rectangle was selected around the region of interest, channels were split for multiple antibodies, threshold was adjusted, noise was de-speckled and fluorescence intensity was measured. Fluorescence intensity was normalized to saline-injected mice. Each experimental group included 5 eyes.

Optic nerve counts

Optic nerves were post-fixed in glutaraldehyde followed by Resin 812 embedding and Araldite 502 (cat#: 14,900 and 10,900 respectively, Electron Microscopy Sciences, Hatfield, PA) according to previously published protocols [8, 19, 26]. Leica EM-UC7 microtome was used to collect 1 μm thick sections of the optic nerves. Sections were then stained with 1% paraphenylenediamine and 1% toluidine blue and were imaged on a Nikon Eclipse Ni-E microscope using 100x oil immersion objective (Nikon Instruments, Melville, NY). The optic nerves were montaged into a 5 × 5 image using the Nikon Elements software to scan a large image. We used the Counting Array and Better Cell Counter plugins to ImageJ, which creates a grid of nine squares overtop the montaged optic nerve. We manually counted healthy and degenerating axons, which are color-coded by the plugins. Degenerative axon profiles were identified by dark paraphenylenediamine staining due to collapsed myelin or loose myelin (onioning) surrounding the axon. A grid was used to avoid bias, by always counting in the same squares, using a cross configuration. 20% of the optic nerve cross-sectional area was counted and the total was multiplied by five to estimate total and degenerating axons within the nerve. Each experimental group included 4–5 nerves.

Data Analysis

All statistical analyses were performed using GraphPad Prism software (La Jolla, CA). A one-way ANOVA with a Bonferroni or Tukey post hoc test (a = 0.05) was used to analyze western blot quantification, IHC fluorescence quantification, ON quantification data, and ERG/VEP latencies and amplitudes. A one-way ANOVA and Dunnett’s multiple comparisons post hoc test (a = 0.05) were used to analyze the qPCR results. Means and standard deviation were calculated for each data set.

Table 2 Antibodies used in this study
Table 3 qPCR primers used in this study



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