Microglia activation is essential for BMP7-mediated retinal reactive gliosis

Mouse retinal astrocytes were isolated in the Sheibani lab and maintained as previously described in [11, 41]. Microglial cells were isolated from retinas of newborn (P0-P4) immortomouse back crossed into C57BL/6J as described in [42] with some modifications. Briefly, the retinas were placed in a solution of Trypsin/EDTA (5 ml; 0.25% trypsin and 1 mM EDTA; Thermo Scientific) and incubated at 37 °C for 5 min. Following incubation, the samples were triturated by pipette, and 5 ml of DMEM with 10% FBS was added to stop trypsin activity. The digested tissue was centrifuged for 5 min at 400 × g at room temperature, the supernatant was carefully aspirated, and the pellet was re-suspended in the microglia medium [a 1:1 mixture of DMEM: F12 (Thermo Scientific) containing 10% FBS and 44 U/ml of interferon-γ (R&D Systems, Minneapolis, MN)], plated on a single well of a 6-well plate, and incubated in a tissue culture incubator at 33 °C and 5% CO₂. The cells were allowed to grow for 1-2 weeks and fed every 3-4 days until nearly confluent. The medium was then removed from the plate and rinsed with PBS containing 0.04% EDTA. The plate was then incubated with 2 ml of PBS containing 0.04% EDTA and placed on a multipurpose rotator at 100 rpm at room temperature for 20–30 min. The supernatant was collected in a 15-ml tube containing 3 ml of DMEM with 10% FBS and centrifuged at 400 × g for 5 min. The detached cells were then re-plated in the microglia medium, allowed to reach confluence, and expanded into 60-mm dishes. The purity of the microglial cultures was inspected by immunocytochemical staining and flowcytometric analysis for F4/80 (eBiosciences; San Diego, CA) and keratin sulfate (Seikagaku Corporation; Jersey City, NJ). The purity of culture was nearly 95% using FACS and immunostaining analysis. Astrocytes and microglia were grown in tissue culture dishes (BD Falcon) in an incubator with 5% CO₂ at 33 °C and passaged every 5–7 days using trypsin-EDTA, and the medium changed every 3–4 days. Cells were treated with 1 μl/ml vehicle (4 mM HCL with 0.1% BSA), 100 ng/ml of mouse bone morphogenetic protein 7 (BMP7; R&D systems), 300 ng/ml mouse interferon-gamma (IFN-γ; R&D systems), or 100 ng/ml LPS (Sigma). Medium from microglial cells incubated with BMP7 or vehicle (conditioned medium) for 24 h was used to treat retinal astrocytes. The conditioned medium was added to the retinal astrocytes medium at 25% concentration in the presence of DMSO or 2.5 μM ALK2/ALK3/ALK6 inhibitor LDN193189 [43]. The retinal astrocytes were allowed to grow for 24 h; after which, cells were harvested for RNA isolation and RT-qPCR analysis.

Experiments were carried out in 4–8 weeks old male C57BL/6J. All procedures were in accordance with the guidelines set by the Institutional Animal Care and Use Committee (IACUC) at the school of science IUPUI (protocol number SC230R). For BMP7 injection studies, n = 8 mice were used with the left eye injected with the vehicle and the right eye injected with BMP7. For the PLX studies, two groups of mice were considered, the age-matched control chow group (n = 12) and the PLX group (n = 12), kept on PLX chow diet. For the PLX BMP7 injection studies, two groups of mice were considered, the age-matched control group (n = 12) and the PLX group (n = 12), kept on the PLX diet. Both the groups were injected with the vehicle control in the left eye and BMP7 in the right eye. P30 VE-YFP mice (n = 3), which express YFP in endothelial cells, generated by crossing a line of mice containing an enhanced yellow fluorescent protein (YFP) with a floxed stop sequence upstream of the YFP (B6.129X1-Gt(ROSA)26Sor^tm1(EYFP)Cos/J; strain number 006148 Jackson laboratory) [44] with the VE-cadherin-cre line (B6.FVB-Tg(Cdh5-cre)7Mlia/J; stock number 006137 Jackson laboratory) [45], were used for immunofluorescence experiments determining PU.1 co-localization in the retina.

Postnatal day 30 (P30), C57BL/6J mice were anesthetized with ketamine and xylazine cocktail. Mice were injected intravitreally with 1 μl of vehicle (4 mM HCL with 0.1%BSA) or 1 μl BMP7 (20 ng/μl) as previously stated in [11]. Intraocular injections were performed using a manual microsyringe (World Precision Instruments) and pulled glass micropipettes.

C57BL/6J mice were kept on chow feed containing 1200 ppm PLX5622 (PLX; Plexxikon Inc.) for up to 21 days, starting at P30. Eyes were harvested at 7, 14, and 21 days following start of the PLX diet for assessment of loss of microglia. The control mice were kept on the control chow supplied by Plexxikon Inc. To determine if loss of microglial cells affected BMP7-mediated gliosis, mice were maintained on PLX chow for the entirety of the experiment. In some animals, eyes were injected with 1 μl vehicle (4 mM HCL with 0.1%BSA) or 1 μl (20 ng/μl) BMP7 14 days following treatment with PLX, and eyes were harvested and processed 3 and 7 days postinjection.

Eyes from euthanized C57BL/6J mice were enucleated, washed in PBS, and either fixed in 4% paraformaldehyde (PFA) for immunofluorescence (IF) or dissected to isolate the retina for preparation of RNA and/or protein. For IF analysis, enucleated eyes were washed and fixed in 4% PFA, incubated in ascending series of sucrose, and frozen in a sucrose OCT solution as previously described [11]. Thick sections (12 μm) were cut using Leica CM3050S cryostat onto Superfrost Plus slides (ThermoScientific) and stored at −80 °C until use. Retinas from enucleated eyes were isolated as previously described [11]. Isolated retinas were immediately processed for RNA isolation using RNeasy kit (Qiagen).

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was performed to detect changes in markers associated with gliosis and inflammation as previously described [11]. The primers for RT-qPCR analysis are listed in Table 1 of [11]. Included in this table is the accession number of each gene, the sequence of each primer, product length, and calculated efficiency of each primer. Primers used for assessing changes in inflammation are listed in Table 1. RT-qPCR was performed using SYBR green master mix (Roche) with the reactions carried out in the LighCycler480 system (Roche). The change in RNA levels was measured using the 2 ^−ΔΔCt method, where Ct is the crossing threshold/crossing point (Cp) value. Relative RNA levels were calculated using the geometric means from the Ct value derived from three housekeeping genes: β- 2 Microglobulin (B2m), succinate dehydrogenase complex subunit A (Sdha), and signal recognition particle 14 kDa (Srp14). A no template control was also tested for each marker.

Frozen tissue sections were labeled as previously described [11]. Antigen retrieval was performed by using 1% sodium dodecyl sulfate (SDS) in 0.01 M PBS (5 min at room temperature) or by heat antigen retrieval method. Briefly, sections were washed with 1× PBS, postfixed with 4% PFA, and permeabilized with methanol. Sections were then incubated in 10 mM sodium citrate buffer at 65 °C for 45 min, allowed to cool at room temperature (RT) for 20 min, rinsed in deionized (DI) water 3×, and washed in PBS once. To reduce autofluorescence, slides were then incubated with 1% sodium borohydride in PBS for 2 mins at RT. Slides were then blocked with 10% serum (goat or donkey) in 1× PBS with 0.25% TritonX-100 for 1 h followed by primary antibody diluted in blocking buffer overnight at 4 °C. Slides were then incubated with Dylight conjugated secondary antibodies (1:800; Jackson Immunoresearch) or Alexa flour (1:500; Invitrogen) conjugated secondary antibodies for 1 h at RT in the dark, washed with 1× PBS, incubated with Hoechst staining solution (2 μg/ml in PBS), and then mounted with Aqua Polymount (Polysciences).

Biotin-streptavidin amplification was done by incubating slides with biotinylated antibody (1:500; Vector Labs) for 1 h at RT followed by Dylight conjugated to streptavidin (1:100; Vector Labs) for 1 h at RT, in lieu of Dylight or Alexa flour conjugated secondary antibodies. For co-labeling involving primary antibodies made in the same host, tyramide signal amplification was performed as per manufacturer’s protocol (Perkin Elmer). For primary antibodies made in mouse, reagents from the mouse on mouse kit (Vector Labs) were used for blocking and primary antibody dilution. Labeled slides were imaged using Olympus Fluoview FV 1000. Antibodies used for immunofluorescence are listed in Table 2. Cell counts were performed using the cell counter plugin of ImageJ. 40× images (n = 9) of retinal sections labeled with SOX9, CALBINDIN, CHX10, and BRN3A and 60× image of retinal flatmounts (n = 8) labeled for GαTRANSDUCIN were used for cell count analysis. Retinal thickness was measured on cross section of retina 200 μm away from the optic nerve.

Extraction of proteins from retinal tissue was performed using lysis buffer as previously described in [11]. Briefly, retinal tissue was homogenized in PBS and centrifuged at 13,000 rpm, 4 °C for 10 min. The supernatant was discarded, and the pellet incubated with lysis buffer (150 mM NaCl, 50mMTris pH 8.0, 2 mM EDTA, 5% TritonX-100; 100 mM PMSF and protease inhibitor cocktail, RPI corp.) for 20 min at 4 °C. The samples were centrifuged at 13,000 rpm, 4 °C for 10 min, and total protein was estimated using BCA protein assay kit (ThermoScientific).

Forty micrograms of protein was loaded onto 4–20% SDS precast gels (Expedeon), placed in a Biorad gel run apparatus, and run at 150 V for 1 h. Proteins were transferred onto a PVDF membrane, which was blocked with a 5% milk in tris buffered saline tween 20 (TBST) at RT for 1 h on a shaker. The blots were incubated with primary antibody-diluted TBST at 4 °C overnight on a shaker. The following day, the blots were washed in TBST and incubated with HRP conjugated secondary diluted 1:5000 in TBST for 1 h at RT. Blots were washed in TBST, incubated with super signal west femto chemiluminescent substrate (ThermoScientific), and visualized on x-ray films. β-TUBULIN was used as a loading control, and the concentrations of antibodies used are listed in Table 2.

Enzyme linked immunosorbent assay (ELISA) for IFN-γ was performed on media from treated cells in vitro or from whole mouse retina protein lysates using the mouse IFN-γ ELISA kit (Cat # ENEM1001, ThermoScientific) as per manufacturer’s protocol.

Preparation of retinal flatmounts and immunolabeling was done as described in [46]. Briefly, enucleated eyes were washed in 1× PBS, fixed in 4% PFA for 15 min, transferred to 2× PBS on ice for 10 min, and followed by retina isolation. Four to five radial incisions were made in the retina to create a petal shape. Excess PBS was absorbed, and retinas were transferred to cold methanol (−20 °C) for 20 min. The tissue was washed with 1× PBS and blocked in Perm/Block solution (1× PBS, 0.3% TritonX-100, 0.2% bovine serum albumin, and 5% donkey or goat serum). The tissue was then washed in PBSTX (1× PBS + 0.3% TritonX-100) and incubated with primary antibody (Table 2) overnight at 4 °C. On the following day, the tissue was washed in PBSTX, incubated with secondary antibody, washed, incubated with Hoechst solution, and mounted onto a slide with Aqua Polymount (Polysciences, Inc). Labeled slides were imaged using Olympus Fluoview FV 1000. Morphological analysis of labeled microglia (n = 4 per time point) for changes in area and number of branches was performed using the Scholl analysis plugin in Fiji image analysis software [47]. Briefly, the flatmount image was loaded on to the Fiji software and converted to binary. To calculate the area of the cell, the “Measure” plugin was selected from the “Analyze” options. To determine the number of branches, a center of analysis was defined via the straight line method. This line was drawn from the center of the cell to the end of the longest branch to define a valid “Startup ROI.” The program was run on the default parameters with the starting radius set at 10 pixels.

Statistical analysis was performed via unpaired Students’s t test using SPSS software (IBM) between control/vehicle and treated groups for RT-qPCR, cell counts, and microglia morphology. RT-qPCR and densitometries from PLX and control mice injected with vehicle or BMP7 were analyzed via one way ANOVA with Tukey’s test for post hoc analysis. p ≤ 0.05 were considered to be statistically significant.