This study used mixed-sex DBA/2J (n = 27; 6–9 per age group), DBA/2J-Gpnmb⁺ (n = 15), and C57BL/6J (n = 7) mice of different ages. The DBA/2J mouse has two loss of function mutations that produce iris atrophy resulting in age-related elevation of intraocular pressure (IOP) and a course of degeneration of visual structures (John et al., 1998). Together, these characteristics resemble ocular hypertension-induced structural changes to the eye as seen in some forms of human glaucoma. DBA/2J-Gpnmb⁺ mice have the same background as the DBA/2J, however, they express a functioning wild type Gpnmb⁺ allele that prevents them from developing elevated IOP or glaucomatous pathology (Howell et al., 2007). Given the recent development of DBA/2J-Gpnmb⁺ mice as controls for glaucoma (Howell et al., 2007; Porciatti et al., 2010), we also used C57BL/6J mice as a comparison since the C57 strain has been commonly used as a control strain in the visual system (Crish et al., 2010; Porciatti et al., 2010). All animals were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and then housed and aged in the Comparative Medicine Unit at Northeast Ohio Medical University to the following time points. For the DBA/2J model, we used 3 month old mice representing the pre-glaucomatous ages, 9–10 month old mice representing ages at the onset of glaucomatous pathology where anterograde transport deficits and mild axonopathy are evident, 11–12 month old mice representing increasing transport deficits and axonopathy, and 13 months where retrograde transport deficits along with axon and cell body loss is evident (Buckingham et al., 2008; Crish et al., 2010). For controls, we used C57BL/6J mice from a convenience sample that ranged in age from 4–17 months, and 9–13 month old DBA/2J-Gpnmb⁺ mice—ages targeted for representing the onset of significant glaucomatous pathology in the DBA/2J mice. Mice were maintained in a 12 h light/dark cycle with standard rodent chow available ad libitum. All mice were housed in the same room under the same environmental conditions. The Northeast Ohio Medical University Institutional Animal Care and Use Committee approved all experimental procedures.

Mice were placed prone in a stereotaxic device (Stoelting, Wood Dale, IL) equipped with a nose cone set to deliver 2.5% isoflurane at 0.8 ml/min. Injections (1.5 μ l) of 1% cholera toxin subunit B (CTB) conjugated to Alexa Fluor-488 (Invitrogen, Carlsbad CA) in sterile phosphate buffered saline (PBS) were administered into the vitreal chamber of each eye using a 33-ga needle attached to a 25 μ l Hamilton syringe. Animals remained anesthetized in the stereotaxic device for immediate surgical preparation for the retrograde labeling procedure. CTB was used because it is selectively taken up by RGCs (except in the cases of advanced retinal pathology; Crish et al., 2010; Inman et al., 2012)—allowing us to determine uptake and transport in the retina. Furthermore, CTB has significant advantages over other commonly used tracers for anterograde transport in that it travels along all axons of the retinofugal projection and is visible in each component (Angelucci et al., 1996).

Surgical sites on the dorsal surface of the skull for mice were cleaned and prepared for the craniotomy procedure. Craniotomies (each 1 mm²) to expose the surface of the brain were drilled into each side of the skull using a 0.5 mm burr attached to a micro-motor drill. Two injection sites per hemisphere were used to administer FluoroGold to the entire rostro-caudal span of the SC and were delivered at the following stereotaxic coordinates (each relative to bregma and sagittal suture skull landmarks): −4.0 mm anterior-posterior, ±0.5 mm medial lateral; and −4.4 mm anterior-posterior, ±0.5 mm medial lateral. At each craniotomy site, a 25 μ l Hamilton syringe equipped with a 33-ga needle was lowered (via stereotaxic device) at a 45° angle to a depth of 0.5 mm below the skull surface. This approach differs from previous studies (e.g., Buckingham et al., 2008) in that it minimizes damage to the injection site within the SC, allowing analysis of intact anterograde transport from the retina and RGC marker labeling to determine structural persistence. Once at injection depth, 1 μ l of 3% FluoroGold (FG) (Fluoro-Gold, Fluorochrome; Denver, CO) in 10% dimethyl sulfoxide (DMSO)/PBS was injected. After each injection was complete, the needle was retracted over 30 s. Sterile bone wax was used to seal each of the skull openings and the incision site was closed with tissue adhesive (Vetbond; 3M, USA). Animals were allowed to recover for 72 h before sacrifice. FG was selected as our retrograde tracer because it is the most commonly used tracer for retrograde labeling of RGCs and it is taken up and packaged passively (Wessendorf, 1991). As long as RGC structure is present, FG is able to get inside the cell and into its vesicles. Any lack of FG-positive cell bodies seen in an intact projection would be due to issues such as transport blockade or similar deficits.

Seventy-two hours after injections, subjects received a single intraperitoneal injection of 120 mg/kg sodium pentobarbital and were transcardially perfused with 4% paraformaldehyde in PBS solution. This time frame was selected to allow both tracer applications to be performed at the same time—longer survival times typically used for FG tracing have been reported to result in loss of anterograde tracer in the retinofugal projection in a similarly sized mammal (Kahn and Krubitzer, 2002). While retrograde transport is slower than the fast anterograde transport CTB uses, this is still an adequate amount of time to cover the distances needed. The brains, optic nerve, and eyes were harvested and retinas were dissected from the eyes. Before histological preparation, the cortex overlying SC was removed and the brain was photographed to determine FG placement. Failed FG injections were identified by lack of tracer coverage of the majority of SC surface and these projections were then excluded from analysis (Abbott et al., 2014). Failed CTB injections were identified by lack of tracer in the retina and these projections were also excluded from analysis. Retinas were prepared as flattened whole-mounts: four radial cuts were made in the surface, vitreous humor was removed, and retinas were mounted nerve fiber layer side up on slides. Slides were then covered with Fluoromount-G mounting media (Southern Biotech; Birmingham, AL) and cover-slipped.

In order to assess whether axon loss was driving deficiencies in transport from the SC, we stained for estrogen-related receptor beta (ERRβ) which labels all components of retinal ganglion cells including their projection to the SC (Real et al., 2008; Crish et al., 2010). Fifty μm coronal serial slices through the SC were taken using a freezing sliding microtome. Every third section of the serial SC slices were added to a 96-well plate containing 200 μ l (per well) block solution (5% donkey serum, 1% Triton-X 100 in 1× PBS) for 2 h at room temperature. Sections were then incubated in primary antibody solution containing rabbit polyclonal antibody ERRβ (1:500, Sigma-Aldrich, E0156; St. Louis, MO) in 3% donkey serum, 1% Triton-X 100 in PBS for 72 h at 4°C, removed and washed 3× for 10 min, and then incubated in Alexa Fluor-594 conjugated secondary antibody (1:200, JacksonImmunoresearch; West Grove, PA) overnight at 4°C. Sections were rinsed 3 × 10 min in PBS before being mounted on microscope slides, and cover slipped with Fluoromount-G. Twenty μm sections of optic nerves were taken parallel to their long axis and stained for the axonal cytoskeletal proteins β-Tubulin (Covance, PRB-425P, 1:5000; Greenfield, IN) and superphosphorylated heavy chain neurofilament (pNF, Covance, SMI-310; Greenfield, IN). Secondary antibodies used for these assays were donkey anti-rabbit Alexa Fluor-594 conjugated (for β-Tubulin) or donkey anti-mouse Alexa Fluor-647 conjugated (for pNF; Jackson ImmunoResearch, West Grove, PA).

Retina and sections containing SC were photographed with a Zeiss AxioZoom V16 epifluorescent microscope equipped with a digital high-resolution camera (AxioCam MRm Rev.3; Zeiss, Jena, Germany) and a computer guided motorized Z and X-Y stage. Retinal whole-mount reconstructions were obtained by multi-frame acquisitions captured side-by-side with 10% overlap with a 3.6×/0.50 objective (Plan-Neofluar, Zeiss, Jena, Germany) at a final magnification of 160×. Each frame was Z-stacked at an interval of 4–8 μm, producing a stack between 5–10 images. Multi-frame tiles were defined to cover the whole retina completely. Tiled images were aligned to eliminate overlap and create a montage of the entire retinal surface using stitching software in Zen Pro (Zeiss; Jena, Germany). Superior colliculi from each animal were imaged at 80× under multiple channels to capture label from CTB and ERRβ. Optic nerves were imaged on an Olympus FV-1000 confocal microscope (Olympus, Cedar Valley, PA).

For analysis, Z-stack retina images were flattened into a single layer TIFF using extended depth of focus (EDF) software in Zen Pro. ImagePro (Media Cybernetics; Rockville, MD) software was used to count FG-labeled RGCs in the retina using subroutines similar to ones published by Salinas-Navarro et al. (2009a). In brief, we applied a sequence of 2-D filters to each retinal image that (a) defined and separated individual cells and (b) established criteria for cell size boundaries and roundness and (c) removed artifacts/noise. In order to calculate density of FG-labeled RGCs in each retina, we used the ImagePro software to measure the area of each retina. We divided the total number of FG-positive RGCs counted in each retina by the total area of that retina to yield a density measure of cells/mm². To quantify sectorial loss of FG labeling in retina characteristic of glaucomatous pathology, we measured the area of sections within the retina where FG-positive cells were absent. We subtracted these areas from the total area of the retina to produce a value that represented total area of intact FG and then divided the total area of intact FG by the total retinal area to create the variable “percent area intact” for analysis of intact retrograde transport. This variable was created to allow direct comparison with deficits in the retinotopic collicular map.

We quantified CTB and ERRβ signal density in multiple slices of the SC using a custom-written macro for NIH ImageJ (Rasband, 1997–2014). This macro performed the same function as the techniques described in Crish et al. (2010) for quantifying signal density in the SC. We set background intensity for each SC section by selecting a region of nonretinorecipient SC (layers IV-VII) or periaqueductal gray for comparison with the retinorecipient SC. We outlined the retinorecipient layers of the SC using layer IV as the ventral border and created bins of pixels running from medial to lateral SC. The number of pixels within bins with CTB or ERRβ signal brighter than background were divided by the total number of pixels in the bin to provide either CTB or ERRβ signal density at that location. We were able to visualize anterograde transport deficits and structural loss in the collicular retinotopic map by constructing colorimetric representations of CTB or ERRβ density [ranging from 0% (blue) to 100% (red)] at each mediolateral location in the SC section and aligning them using Origin Pro software (OriginLab; Northampton, MA). The methods used to create these representations are adapted from Crish et al. (2010) and the code for the ImageJ macro is freely available upon request from the authors.

We used two-way between-subjects factorial analyses of variance (ANOVA) with post-hoc Fisher's Least Significant Difference (LSD) tests to determine differences in transport between strains/ages. To compare differences in magnitude between anterograde, retrograde, and structural label within subjects for each age group, we used paired t-tests to delineate the effects of these planned comparisons. We also used one-way ANOVA to assess control data and Pearson's correlations for additional descriptive measures. Retrograde transport was defined as either FG-positive cell density in retina or percent area of intact FG staining in retina. Anterograde transport was defined as percent area fraction of intact CTB label in the SC contralateral to the retina analyzed for retrograde transport. Additionally, the structural intactness of each SC was measured by percent area fraction of intact ERRβ label. Retrograde and anterograde transport were compared within the same projection. Each retina and corresponding contralateral SC set were analyzed separately within each animal, as glaucomatous pathology differentially affects each eye thus necessitating the analysis of each projection as an independent measure (Schlamp et al., 2006; Crish et al., 2010).

FG-positive cell density in retina from C57BL/6J (C57) and DBA/2J-Gpnmb (D2G) control strains were compared across age groups to determine if the FG distribution pattern in D2G mice matched that of C57s and if there was similarity between younger and older controls. We compared the density of FG-positive cells in the retina across C57 mice (age range 4–17 months) and D2G mice (age range 9–13 months). Results of an ANOVA indicated no significant differences between ages or strains, F_{(6, 22)} = 1.18, P = 0.365, ns (Figure 1). This validated our use of D2G control mice as a comparable (and more background relevant) substitute for the C57 control strain. Additionally, as these numbers are similar to other studies using FG to label RGCs (e.g., Buckingham et al., 2008), this validated our modified method of FG application. Furthermore, this is the first reported analysis of FG transport in D2G mice. For the remaining analyses, we pooled the D2G control data across ages where we typically see glaucomatous pathology in the DBA/2J (9–12 months old) to form a single control group for this strain.

The following abbreviations will be used to label the DBA/2J age groups represented by our data: D3 (3-month old DBA/2J), D9-10 (9–10 month old DBA/2J), D11-12 (11–12 month old DBA/2J), and D13 (13-month DBA/2J). Figure 2 shows examples of tracing in whole mount brain (cortex removed) and retina. The left panel in row A shows clear anterogradely-transported CTB label in the major retinal targets (lateral geniculate nucleus, pretectum, and SC) and the right panel in row A shows good coverage of the SC with FG. Row B (left) shows a near complete loss of CTB in the left SC and nearly no loss on the right (right panel, row B). FG covers the SC completely. Row C illustrates the range of retrograde transport of FG we found, from complete coverage in the leftmost panel, to sectorial loss in the middle panel, and a near complete loss in the rightmost panel.

In D2G (Figures 3A,B) and young DBA/2J (D3) mice (data not shown), uniform CTB and ERRβ were evident in superficial SC. Colorimetric reconstructions of the entire SC demonstrated a uniformly dense labeling (indicated by red/orange/yellow colors) across the entire SC (Figures 3C,D). In aged DBA/2J mice, reductions in CTB label were often found (Figures 3E,I,M,O). Deficits in the SC occurred in sectorial patterns, with some areas devoid of CTB (indicated by blue/green color) and some with strong label (Figure 3G). In DBA/2J mice up to 12 months of age, RGC structural marker ERRβ appeared normal even in colliculi with severe CTB loss (compare Figures 3F,H with E,G; also J,L with I,K). In 13-month DBA/2J mice we saw significant loss of ERRβ label as well (Figures 3N,P).

As seen previously (e.g., Crish et al., 2010), lack of CTB label in the SC is not due to loss of RGC somata or axons. CTB-positive RGCs and axons are readily visible in whole mount retina—even in projections with complete absence of collicular CTB (Figure 4, first column). Dense concentrations of FG-positive RGC somata (FG is not typically visible in axons) are also obvious in all but some of the oldest animals studied here (Figure 4, second column). Examination of the optic nerves show persistence of CTB-positive axons in the distal-most portions of this structure (Figure 4, third column). Interestingly, these axons appear to have altered levels of cytoskeletal proteins. In contrast to D2G control mice, β-tubulin levels in the optic nerve of DBA/2J mice appear reduced whereas pNF is increased in mice with significant transport deficits (Figure 4, Tub & pNF columns). At advanced pathological stages where ERRβ loss is seen, β-tubulin and pNF are substantially reduced, indicating axon degeneration (Figure 4, bottom row).

In order to quantitatively compare intact retrograde transport (FG), anterograde transport (CTB), and structure (ERRβ) for each group of mice as well as compare how these values changed as a function of age in the DBA/2J mice, we used a 3 (CTB, FG, ERRβ) by 5 (D2G, D3, D9-10, D11-12, D13) factorial ANOVA for analysis. Significant main effects for label [F_{(2, 104)} = 29.98, P < 0.001] and strain/age [F_{(4, 104)} = 91.57, P < 0.001] were shown. A significant interaction between type of label and strain/age group qualified these main effects [F_{(8, 104)} = 8.64, P < 0.001]. Post-hoc Fisher's LSD tests were used to define the significant pairwise comparisons as described below.

As anticipated, there were no significant differences in intact anterograde (P = 0.808, ns), retrograde (P = 0.8310, ns), or structural label (P = 0.943, ns) between D2G controls and D3 (pre-glaucomatous) mice (Figures 5A–C). Therefore, we only report post-hoc pairwise comparisons between the DBA/2J age groups.

Glaucomatous DBA/2J (D9 and older groups) showed a marked reduction in CTB label in the SC compared to the D3 group (Figure 5A). The D3 group had significantly more intact label than D9-10, D11-12, and D13 (Fisher's LSD P < 0.001 in all three comparisons). Fisher's LSD tests indicated that there were no differences in CTB label between the three oldest DBA/2J groups (D9-10 vs. D11-12: P = 0.545, ns; D9-10 vs. D13: P = 0.098, ns; D11-12 vs. D13: P = 0.219, ns).

The persistence of RGC axon terminals (percent intact ERRβ) in the SC did not differ between D3 and D9-10 (Fishers LSD; P = 0.978, ns), D3 and D11-12 groups (P = 0.088, ns), or between D9-10 and D11-12 groups (P = 0.094, ns). ERRβ was significantly reduced in the 13-month old DBA/2J mice in contrast to D3, D9-10, and D11-12 groups (Fisher's LSD; P < 0.001 in all comparisons; Figure 5B).

Other studies of retrograde transport use FG-positive cell density in the retina as the primary measure of transport (see for example Buckingham et al., 2008). However, to perform within-animal comparisons between retrograde and anterograde transport, we established a variable that allowed direct comparison of FG in the retina with percent area fraction of CTB and ERRβ in the SC. Using montaged image tiles of the entire retinal surface (See Figure 2, bottom row), we quantified the percent area of intact FG label as well as FG-positive cell density in the same retina for all our mice and found that these variables were highly correlated (Pearson's r = 0.80, P < 0.0001; Figures 5C,D). Therefore, we used percent area intact FG in the retina to compare retrograde label with anterograde and structural labels.

In contrast to our pre-glaucomatous D3 mice, the percent area of intact FG was significantly reduced in the D9-10 group (Figure 5C) (Fisher's LSD; P = 0.02), D11-12 group (P < 0.001), and D13 group (P < 0.001). Intact retrograde transport levels were similar between the D9-10 and D11-12 groups (P = 0.312, ns), but showed a further reduction in the D13 age group (Fisher's LSD; P < 0.001 in both sets of comparisons).

We used paired t-tests to assess within-subject differences in the magnitude of CTB, FG, and ERRβ label for each age group of animals tested. In both the D2G control and D3 groups, there were no measurable differences in the magnitude of label between anterograde, retrograde, and structural markers. However, in the D9-10 group, the amount of intact FG was significantly greater than CTB, t₍₅₎ = 2.35, P = 0.03 (Figure 6). This difference was maintained in the D11-12 group [t₍₈₎ = 3.059, P = 0.02] before dropping to comparable levels in the D13 group.

Normal collicular ERRβ label persisted through 12 months of age as shown in Figure 5B and was significantly greater than retinal FG label in the D9-10 [t₍₅₎ = −3.41, P = 0.02] and D11-12 [t₍₈₎ = 3.85, P = 0.005] groups (Figure 6). Along with the previous data on CTB label magnitude, this finding indicates that the structure of the SC and some components of the RGC projection remained intact after both forms of transport showed significant deficits. In the oldest age group (D13), both forms of transport showed over 90% deficits in intact label with a corresponding 86% reduction in ERRβ. Although structural label was not maximally lost, ERRβ and the transport labels did not differ significantly at D13 (Figure 6).

Differential reductions in transport by individual projections are summarized in Figure 7. The top plot compares anterograde (CTB) and retrograde (FG) transport in the same projection. We have plotted a unity line where we would expect the points to fall if both forms of transport were similarly affected. Supporting our group observations, we have a significant cluster of cases below the unity line, indicating that we saw much larger anterograde deficits than retrograde deficits at younger ages. As shown in the bottom plot of Figure 7, when we compared retrograde transport to our RGC projection structural marker in the SC (ERRβ) we found a significant cluster of cases above the unity line, indicating FG transport deficits precede loss of ERRβ immunoreactivity in the SC.

Impaired movement of cargo along the axon initiates several degenerative mechanisms including distal Wallerian degeneration and apoptosis due to neurotrophin deprivation (see Coleman, 2005). There are a large number of possibilities that can negatively affect proper transport function, including modification of molecular motors, cytoskeletal disruption, and metabolic dysfunction (Coleman, 2005; Shea and Chan, 2008; Morfini et al., 2009a; Crish and Calkins, 2011). These changes can often affect very specific parts of transport such as different compartments of the axon or the direction of cargo movement away from the cell body or toward the cell body (Shea and Beaty, 2007). Due to the complexity of the nervous system, most of the evidence reporting differential transport effects in pathology are ex vivo (see for example, Brady et al., 1990; Morfini et al., 2007; Motil et al., 2007; Lee et al., 2011; LaPointe et al., 2013).

Differential transport loss in glaucoma may be due to greater sensitivity of anterograde transport to an intra-axonal blockade (Wang et al., 1995; Ross et al., 2006; see discussion in Shea and Beaty, 2007). These intra-axonal blockades may result from the disorganization and breakdown of the cytoskeleton with the consequential accumulation of protein cargo (Shea et al., 2004; Coleman, 2005; Crish et al., 2010). Dynein, the molecular motor that mediates retrograde transport, has greater lateral and bidirectional movement along microtubules within the axon. Therefore, dynein may more easily navigate around these blockades by translocating to other, intact microtubule tracks at that location. In contrast, the molecular motor that drives anterograde transport, kinesin, does not have this capability, therefore making it vulnerable to axonal blockade (Wang et al., 1995; Ross et al., 2006).

Additionally, the differential effects we have shown on axonal transport may result from regulation of the molecular motors themselves. Kinesin can be dysregulated or inhibited by kinases such as GSK-3β, p38, and JNK, and activity of these kinases is notably increased in neurodegenerative disorders (see Brady and Morfini, 2010; Wang et al., 2014 for discussions). Notably, p38 has recently been implicated in axonal transport deficits and distal axonopathy in DBA/2J mice (Dapper et al., 2013), JNK is rapidly upregulated after RGC injury (Fernandes et al., 2014), and investigations into GSK3β signaling pathways are ongoing in our laboratories.

A common hypothesis in glaucoma is that axonal transport is blocked by mechanical compression of the optic nerve head (ONH) where the RGC axons leave the eye. If this were the case then we would expect anterograde and retrograde transport to be similarly affected, but here in the DBA/2J mouse they are not. Furthermore, our observations of reduced levels of β-Tubulin and increased levels of pNF support the idea that axon transport is blocked by intra-axonal changes far removed from the ONH. Both microtubule breakdown and abnormal phosphorylation of neurofilaments have profound effects on axonal transport and changes to both these proteins may play a large role in the deficits seen here (Shea and Beaty, 2007). These intra-axonal changes may also result from disruptions in kinase signaling pathways (Wang et al., 2014). Even in extreme cases of CTB and ERRβ loss in the SC, CTB-positive axons, varicosities, or end bulbs are evident in the optic nerve and tract as shown in Figure 4 as well as in previous studies (Crish et al., 2010). This indicates that these axons (even one undergoing distal axonopathy as shown in Figure 4, bottom row) are still connected to their cell bodies in the retina. It is important to note that this idea does not detract from the role of the ONH in the development and progression of glaucoma—critical observations regarding the onset of pathological change have been noted in the ONH and the retina—and pathology in neurons (e.g., blocked axon transport and axonopathy in this region) is often far-removed from the site of the stressor (Conforti et al., 2007; Cuchillo-Ibanez et al., 2008).

Structural persistence of the RGC projection to the SC (as measured by intact ERRβ) was maintained in glaucomatous mice from 9 to 12 months of age and significant decreases in ERRβ label were only first measured at 13-months of age. Contrasting with both anterograde and retrograde labels that first showed significant reductions in the 9–10 month age group, our findings suggest that transport deficits are likely due to physiological or functional abnormalities as opposed to overt structural loss. Our results did differ from previous reports of Crish et al. (2010) in that our ERRβ label for intact SC was significantly reduced at 13-months, whereas significant reductions in ERRβ did not appear in those studies until 15–22 months. However, there is a significant amount of variability in structural persistence beyond 12 months of age (Crish et al., 2010) and it is possible that our smaller sample size of dual-traced animals captured more extreme deficit in ERRβ at 13-months. However, this does not affect our overall conclusions.

Breakdown or modification of cytoskeletal elements or motor proteins are common pathologies in neurological disorders such as Alzheimer's Disease and amyotrophic lateral sclerosis and can negatively affect neuron structure and function in terms of cellular integrity, axonal transport, and signaling (Coleman, 2005; Conforti et al., 2007; Cuchillo-Ibanez et al., 2008; Shea and Chan, 2008; Morfini et al., 2009a; Shea et al., 2009). It is possible that disruptions in axonal transport shown in our current studies result from intra-axonal blockades caused by aberrant cytoskeletal organization/breakdown or dysregulation of molecular motors that may reflect changes occurring in glaucoma occurring before overt degeneration of axons or cell bodies. A major question this research raises is what is the connection between these changes and other early pathologies seen in glaucoma? While anterograde transport interruptions can drive axonal degeneration distal to the blockage (see Coleman, 2005), the relationship between these deficits and early dendritic remodeling in the retina (Jakobs et al., 2005; Della Santina et al., 2013), inflammation (Bosco et al., 2011), and physiological deficits in RGC activity (Della Santina et al., 2013; see Saleh et al., 2007 for DBA/2J; Banitt et al., 2013 for humans) or action potential conduction (Baltan et al., 2010) remain to be determined. Inflammation as measured by increased microglial number and activation occurs quite early in the DBA/2J inner retina and ONH, peaking by 3 months (Bosco et al., 2011). Since microglial release of NO can halt anterograde axon transport (Stagi et al., 2005), there may be a negative impact of these cells on axon transport quite early, though undetected at 3 months in our study. The significant decreases in pattern electroretinogram (PERG) amplitude that affect the DBA/2J by 6 months of age (Saleh et al., 2007) suggest changes in inner retina activity that may be carried over into optic nerve as decreases in compound action potential amplitude, an observation made in DBA/2J mice with high IOP by 6 months of age (Baltan et al., 2010). Alternatively, decreases in axon transport of mitochondria in the DBA/2J optic nerve, for example, could also explain loss of compound action potential amplitude. Our study focused on transport changes very early (3 months) and late (9 months and beyond), so additional analysis of anterograde axon transport in between may be able to distinguish among the competing mechanisms for the transport pathology observed. Such information may provide exciting new avenues for treatments targeted at restoring function and substructure in glaucomatous projections that still remain largely intact.

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.