Seven to nine month old male and female IL-6-/- mice (B6;129S2-IL6^tm1kopf/J) and respective genomic controls (B6;129SF2/J) were used for all experiments. IL-6-/- mice contain a neomycin selection cassette in exon 2 of the IL-6 gene preventing transcription of the mRNA product (Kopf et al., 1994). Founder mice were obtained from Jackson Laboratories (Bar Harbor, ME) and experimental mice were bred and genotyped in-house using the following primers provided by Jackson Labs: 5′-TTC-CAT-CCA-GTT-GCC-TTC-TTG-G-3′, 5′-TTC-TCA-TTT-CCA-CGA-TTT-CCC-AG-3′ and 5′-CCG-GAG-AAC-CTG-CGT-GCA-ATC-C-3′. Mice were housed in accordance with NIH guidelines and maintained on a 12 h light/dark cycle with ad libitum access to standard mouse chow and water. This study was carried out in accordance with the ARVO statement for the use of animals in ophthalmic and vision research and was approved by the IACUC of Vanderbilt University Medical Center.

Acute IOP elevation was induced in WT and IL-6-/- mice using the microbead occlusion model, as previously described (Sappington et al., 2010). For anterograde axonal transport, axon density measurements, and retinal gliosis, mice from both genotypes received a unilateral injection of 1.5 μl (1 × 10⁶ microbeads/mL) of 15 μm polystyrene beads conjugated to an Alexa Fluor 488 chromophore. The contralateral eye served as a surgical control and was injected with an equal volume of saline. For experiments looking at visual acuity and corneal integrity, 7–11 mice from both genotypes received bilateral injections of 1.5 μl microbeads and a separate cohort of mice served as controls and received bilateral injections of an equivalent volume of saline. All mice received two microbead/saline injections 4 weeks apart to raise IOP for a total of 8 weeks. Following IOP elevation, mice were sacrificed by transcardial perfusion of 50 ml of 1X PBS followed by 100 ml of 4% paraformaldehyde. Eye and brain tissue were stored in 4% PFA at 4°C until use.

IOP was measured in awake, behaving mice, using a Tonolab rebound tonometer (TonoLab; Reichert, Depew, NY), as previously described (Echevarria et al., 2013; Formichella et al., 2014; Echevarria et al., 2016). Prior to initial injection, mean baseline IOP for each mouse was calculated from approximately 60 individual readings taken over a period of 6 days (10 measurements/day) within a 2 week timeline. Following microbead or saline injections, weekly IOP was determined as the mean of 20–30 measurements, taken over 2–3 days (10 measurements/day) each week for a total of 8 weeks. IOP measurements were taken at the same time of day to remove any effect of circadian rhythm on IOP measurements. To avoid corneal irritation and discomfort, 0.5% proparacaine anesthetic drops (Akorn Inc, Lake Forest, IL), and lubricating eye drops were applied to each eye before and after IOP measurements were taken respectively.

Immunohistochemistry of whole mount retinas was done as previously described (Sims et al., 2012; Echevarria et al., 2013, 2016). Primary antibodies against glial fibrillary acidic protein (GFAP, 1:500; Cat# Z033429-2; DAKO) to label astrocytes, ionized calcium-binding adapter molecule-1 (Iba-1, 1:250; Cat# 019-19741; WAKO) to label microglia, and β-Tubulin III (TUJ1, 1:500; Cat#845501; BioLegend) to label RGCs were used. Secondary antibodies were used at a concentration of 1:200 and consisted of donkey α-rabbit attached to either a Rhodamine Red-X (Cat# 711-295-152; Jackson Immuno Labs) or Alexa-647 (Cat# 711-605-152; Jackson Immuno Labs) fluorophore.

Generation of IL-6 probes and FISH in naïve WT and IL-6-/- whole mount retina were done as previously described (Crish et al., 2013). Probes were made against a nucleotide sequence encompassing exons 2–5 of IL-6 [nucleotides 107–651 of (NCBI Ref Seq: NM_031168.2)]. The transcript inserted into the pGEM-T Easy Vector (Promega, Madison WI) was generated by PCR using primers to IL-6 (forward 5′-ATCCAGTTGCCTTCTTGGGACTGA-3′ and reverse 5′TGGCTAAGGACCAAGACCATCCAA-3′). Following FISH, retinas underwent immunohistochemistry as described above to label RGCs.

Imaging of whole mount retinas was done on an inverted confocal microscope (Olympus FV-1000; Center Valley, PA) through the Vanderbilt University Medical Center Cell Imaging Shared Resource Core. IL-6 and β-Tubulin III labeling was imaged at 100X, while GFAP and Iba-1 was imaged at 60X. For both groups, 7–9 pseudo-random z-stack images in the mid central/mid-peripheral areas through the ganglion cell (GCL) and nerve fiber layers (NFL) of the retina were acquired using a digital camera and image analysis software (FV-100 ASW; Olympus). GFAP and Iba-1 percent area was calculated using NIS elements AR software (Nikon Instruments, Melville, NY), as previously described (Formichella et al., 2014). The area (mm²) of the image containing above background signal intensity of Iba-1 or GFAP (See Figures 6A,B; red labeling) was calculated and reported as a percentage of the total area of the image. Total area of each image and background signal threshold was equal among all images. Microglia cell density was calculated by counting the number of Iba-1 positive cell somas and dividing the counts by the area of the image.

Anterograde axonal transport capabilities of RGCs were assessed with cholera toxin beta-subunit (CTB) conjugated to a 488 fluorophore, as previously described (Crish et al., 2010; Formichella et al., 2014; Ward et al., 2014; Bond et al., 2016). Briefly, mice were given a 1.5–2 μl intravitreal injection of CTB (10 μg/μl in sterile ddH₂O; Cat# C-34775, Life Technologies) using a 33 gauge needle attached to a Hamilton syringe under 2.5% isoflurane anesthesia. Five days after CTB injection, mice were sacrificed by transcardial perfusion as described above. To quantify axon transport, whole brains were cryopreserved in 30% sucrose for 24–48 h at 4°C. Using a sliding microtome, 50 μm sections were obtained through the superior colliculus (SC). CTB signal in these sections was imaged en montage at 10X, using a Nikon Eclipse Ti inverted microscope (Nikon Instruments, Melville, NY). Anterograde axonal transport was quantified as previously described (Crish et al., 2010). Briefly, the SC from each image was outlined and CTB signal above background was divided by total pixel area to determine the volume of SC with CTB labeling. This value was used to create a colorimetric 2D retinotopic map of CTB transport ranging from 0% (blue) and 100% (red). Intact transport was defined as percent area with CTB signal >70% density (red/yellow).

Axon density was measured in semi-thin sections of optic nerve as previously described (Sappington et al., 2010; Ward et al., 2014). Briefly, optic nerves were post fixed at least 48 h in 2.5% glutaraldehyde and embedded in epon. Semi-thin (700 nm) cross-sections of optic nerve near the chiasm were stained with 1% p-Phenylenediamine (PPD) and 1% toluidine blue to highlight myelin and glia, respectively. Optic nerve cross-sections were imaged en montage at 100X magnification on an upright Olympus Provis AX (Olympus, Melville, NY) microscope. To calculate axon density, a 50 × 50 μm grid mask was placed on the montaged image using NIS elements AR software. The number of axons was manually counted by a blind-observer in 8–10 squares of the grid. Each square counted was equal in area (0.0025 mm²). To measure nerve area, the circumference of the nerve was traced in montaged images of optic nerve cross-sections. Nerve area was calculated as the area (mm²) within this outline using NIS elements software.

The optokinetic response is a naturally occurring reflex that serves as a functional tool for quantitative analyses of visual system function in mice (Douglas et al., 2005). Briefly, each mouse was placed on a platform surrounded by four LCD computer monitors. A sinusoidal grating of alternating white and black bars rotating in either a clock-wise or counter clock-wise fashion was projected on the monitors. Mice able to perceive the moving stimulus produced a reflexive movement of the head in the direction of the stimulus. The visual acuity of each mouse was measured by changing the spatial frequency of the black and white bars. The visual acuity threshold was determined as the highest spatial frequency for which reflexive tracking was noted. The presence of the reflexive head movement was recorded by an observer using a camera mounted above the mouse. Mice were tested for baseline visual acuity threshold 1–2 weeks before microbead/saline injection and 4 and 8 weeks post-initial microbead/saline injection.

Mice were anesthetized with a ketamine/xylazine cocktail (80/5 μg/gram of mouse), pupils were dilated with 0.5% Tropicamide, and eyes kept moist with lubricating eye drops. Live volumetric scans of the cornea were obtained using SD-OCT running the Bioptogen ultra-high resolution spectral domain OCT system with cornea bore (Bioptogen, Morrisville, NC). Quantification of injury area was performed using Image J software (National Institute of Health).

To confirm genetic ablation of IL-6, we conducted PCR to confirm the presence of the neomycin selection cassette in exon 2 of the IL-6 gene. In all IL-6-/- mice used in this study, PCR amplification of exon 2 revealed a 380 bp product in the IL-6-/- mouse compared to the 174 bp PCR product in the WT mouse (Figure 1A). Loss of IL-6 mRNA was corroborated using in situ hybridization. In a subset of WT and IL-6-/- whole mount retina co-immunolabeled with the RGC-specific marker β-Tubulin (TUJ1), labeling for IL-6 mRNA using an antisense fluorescent in situ hybridization (FISH) probe showed robust signal that co-localized to TUJ1+ positive RGCs in WT mice (Figure 1B; left). Conversely, anti-sense labeling for IL-6 mRNA was not detected in IL-6-/- mice (Figure 1C; left). Similarly, significant IL-6 mRNA labeling was not detected following incubation with the sense probe in either genotype (Figures 1B,C; right). To examine the impact of IL-6 deficiency on the progression of IOP-induced RGC neurodegeneration, we utilized the microbead occlusion model (Sappington et al., 2010) of glaucoma to elevate IOP for a total of 8 weeks in WT and IL-6-/- mice. Baseline IOP was 4% lower in IL-6-/- mice (16.4 +/− 0.79 mmHg), compared to WT mice (17.1 +/− 1.32 mmHg; p < 0.01; Figure 1D; left). Microbead injection increased IOP by ~ 20%, as compared to saline-injected controls for both genotypes (WT; p < 0.001, IL-6-/-; p < 0.001, Figure 1D; right and Figure 1E). In accordance with baseline IOP measurements, the mean IOP (mmHg) for both saline- (p < 0.05) and microbead-injected (p < 0.05) was lower in IL-6-/- mice than their WT counterparts (Figure 1E). However, with respect to baseline IOP, the magnitude of IOP elevation was similar (~2.5 mmHg) in microbead-injected WT and IL-6-/- mice (p > 0.05, Figure 1F).

In glaucoma, degeneration of the optic nerve starts at the distal end of the optic nerve and progresses in a distal to proximal fashion (Crish et al., 2010). Unlike the distal optic nerve of saline-injected WT mice (Figure 2A; top), the distal optic nerve of microbead-injected WT mice presented with signs of structural pathology, including increased glial infiltration and degenerating axon profiles (Figure 2A; bottom). This was accompanied by a slight enlargement in nerve area (Figure 2C; left) and a 15% decrease in axon density, as compared to saline-injected mice (p < 0.05, Figure 2D; left). In contrast, while distal optic nerves from microbead-injected IL-6-/- mice presented with some gliosis, no change in degenerating axon profiles were noted (Figure 2B). Similarly, there were no measurable changes either in nerve area (Figure 2C; right) or myelinated axon density (p > 0.05, Figure 2D; right), as compared to saline-injected IL-6-/- mice. However, independent of IOP, optic nerves from IL-6-/- mice contained approximately ~15% fewer myelinated RGC axons than those from WT mice (p < 0.05, Figure 2D).

Previous reports indicate that functional deficits in anterograde axon transport along the optic projection precede structural degeneration of RGC axons in glaucoma (Crish et al., 2010, 2013). To measure active anterograde transport in RGC axons, we injected the active uptake, active transport tracer cholera toxin beta subunit (CTB) into the vitreous of IL-6-/- and WT mice 8 weeks after the initial microbead or saline injection. We measured anterograde transport of CTB from RGC soma in the retina to RGC terminals in the superior colliculus (SC) by quantifying CTB labeling in serial sections of SC and generating 2D reconstructions of CTB labeling in the SC (Figure 3A). In WT mice, 8 weeks of elevated IOP led to a ~50% decrease in CTB transport to the SC, as compared to saline-injected mice (p < 0.001, Figure 3A; top, Figure 3B; left). Interestingly, in IL-6-/- mice, IOP elevation also resulted in a ~50% decrease in CTB transport (p < 0.001, Figure 3A; bottom, Figure 3B; right). Similar to previously published studies (Crish et al., 2010; Lambert et al., 2011; Ward et al., 2014), these deficits occurred in a sectoral manner, extending from the periphery toward the optic disc in WT and IL-6-/- mice (Figure 3A). No differences in axon transport were noted between genotype in saline-injected animals (p > 0.05, Figure 3B).

Loss of vision in glaucoma is irreversible and caused by degeneration of RGCs and their axons (Calkins, 2012). To ensure detection of any vision loss associated with microbead-induced glaucoma, we performed bilateral injections of microbeads in one cohort of WT and IL-6-/- mice. A second cohort received bilateral injections of saline. We measured visual acuity by optomotry every 4 weeks for the duration of the experiment. Over the course of 8 weeks, microbead-injected WT mice exhibited significant depreciation of visual acuity at each time point compared to baseline, resulting in an overall 22% decrease in visual acuity (p < 0.001 for all, Figure 4A; gray). However, in saline-injected WT mice, visual acuity did not significantly differ from baseline at either time point (p > 0.05, Figure 4A; white). Comparison of visual acuity between saline- and microbead-injected WT mice revealed a significant ~15% decrease in both raw visual acuity (p < 0.05, Figure 4C) and percent baseline visual acuity (p < 0.05, Figure 4D). In IL-6-/- mice, visual acuity dropped 8% with either saline (p < 0.01) or microbeads (p < 0.05), as compared to baseline acuity (Figure 4B). However, this initial reduction in visual acuity did not differ between saline- and microbead-injected IL-6-/- mice (p > 0.05, Figures 4C,D) and remained unchanged between 4 and 8 weeks for both groups (saline: p > 0.05; microbead: p > 0.05; Figure 4B). That this slight decrease in visual acuity was noted in both saline- and microbead-injected IL-6-/- mice and remained stable for the 8 week experiments suggest that it arises from an IOP-independent influence. No difference in visual acuity was noted between WT and IL-6-/- mice at any time point (p > 0.05, Figure 4C).

Previous studies indicate IL-6-/- mice exhibit deficits in wound healing (Lin et al., 2003; McFarland-Mancini et al., 2010). As the microbead/saline injections require puncturing of the cornea, we used spectral-domain optical coherence tomography (SD-OCT) imaging to examine whether perturbed healing of the cornea could underlie the reduction in visual acuity noted in both saline and microbead-injected IL-6-/- mice. Two weeks following intra-cameral injection of saline or microbeads in WT eyes, SD-OCT imaging revealed complete closure of the epithelial layer and approximately 2/3 closure of the stroma and endothelial layers at the injection site (Figure 5A). Quantification of the remaining corneal wound revealed no significant change over the remaining 6 weeks (p > 0.05; Figure 5C). In IL-6-/- mice, SD-OCT imaging revealed complete closure of the epithelial layer by 2 weeks. However, limited closure of the stroma and endothelial layers was noted (Figure 5B). This reduction in stroma and endothelial wound healing was noted throughout the 8 week experiment (Figure 5B). Quantification of corneal injury revealed that the area of corneal wound was 2-fold larger in IL-6-/- mice than in WT mice at all three time points (p < 0.05; Figure 5C). Like WT mice, the area of the corneal wound did not change over the 8 week experiment in IL-6-/- (p > 0.05; Figure 5C).

Recent studies suggest that changes in glial reactivity in the retina occur in response to IOP elevation, and impact RGC degeneration in both genomic and inducible models of glaucoma (Martin et al., 2003; Sappington and Calkins, 2006; Inman and Horner, 2007; Bosco et al., 2008; Johnson and Morrison, 2009; Johnson et al., 2011; Echevarria et al., 2013; Lye-Barthel et al., 2013; Formichella et al., 2014; Hines-Beard et al., 2016). To determine whether IL-6 deficiency alters glial reactivity associated with RGC axonopathy, we performed a morphological analysis of astrocyte and microglia reactivity in retina. We visualized astrocytes and microglia in whole-mount retina from saline- and microbead- injected WT and IL-6-/- mice with immunolabeling against the astrocyte- specific label glial fibrillary acidic protein (GFAP) and the microglia- specific marker ionized calcium binding adaptor molecule (Iba-1). While Iba-1 labels various types of myeloid cells, we selected this marker because (1) 100% of retinal microglia are Iba-1 positive (Bosco et al., 2011), (2) Iba-1 expression remains rather stable across activation states compared to other microglia/macrophage markers (Bosco et al., 2011) and (3) with the exception of amoeboid microglia, other myeloid cells and microglia can be readily distinguished by morphology. To account for changes in both glia density and hypertrophy/ramification, we quantified the percent of retinal area covered by GFAP+ astrocytes (Figure 6A) and Iba-1+ microglia (Figure 6B). Our previous work indicates that percent area coverage is a highly reliable measure of reactivity (Formichella et al., 2014). GFAP immunolabeling revealed no gross genotype- or IOP-dependent changes in astrocytic morphology (Figure 6C). Quantification of astrocyte coverage confirmed no significant change in astrocyte morphology with respect to either IOP or genotype (p > 0.05 for all; Figure 6E). In contrast, Iba-1 immunolabeling revealed qualitative changes in microglia that appeared to relate to both genotype and IOP (Figure 6D). Quantification revealed 32% more microglia coverage in saline-injected IL-6-/- mice vs. WT mice (p < 0.05; Figure 6F). IOP elevation decreased microglia coverage by 45% in WT retina (p < 0.05; Figure 6F), as compared to saline-injected controls (Figure 6F). While it appeared as if there was decreased microglial coverage in IL-6-/- retina following IOP elevation compared to saline-injected controls, it did not reach statistical significance (p > 0.05; Figure 6F). Additionally, microglia coverage remained higher in IL-6-/- mice than in WT mice following IOP elevation (p < 0.05, Figure 6F). Based on qualitative assessment, IOP-induced changes in percent area coverage appeared to arise from changes in microglia density (Figure 6D). To quantitatively test this observation, we measured the density of Iba-1+ microglia across all experimental groups. We found that microbead-induced IOP elevation decreased the density of microglia by 37% in WT retina (p < 0.05) and by 36% in IL-6-/- mice (p < 0.05), as compared to saline-injected controls (Figure 6G). There was no significant difference in the density of microglia between WT and IL-6-/- mice regardless of treatment (p > 0.05; Figure 6G).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.