Astrocyte remodeling without gliosis precedes optic nerve axonopathy
© The Author(s). 2018
Received: 19 March 2018
Accepted: 26 April 2018
Published: 10 May 2018
Astroyctes serve myriad functions but are especially critical in white matter tracts, where energy-demanding axons propagate action potentials great distances between neurons. Axonal dependence on astrocytes for even normal function accentuates the critical role astrocytes serve during disease. In glaucoma, the most common optic neuropathy, sensitivity to intraocular pressure (IOP) challenges RGC axons early, including degradation of anterograde transport to the superior colliculus (SC). Astrocyte remodeling presages overt axon degeneration in glaucoma and thus may present a therapeutic opportunity. Here we developed a novel metric to quantify organization of astrocyte processes in the optic nerve relative to axon degeneration in the DBA/2 J hereditary mouse model of glaucoma. In early progression, as axons expand prior to loss, astrocyte processes become more parallel with migration to the nerve’s edge without a change in overall coverage of the nerve. As axons degenerate, astrocyte parallelism diminishes with increased glial coverage and reinvasion of the nerve. In longitudinal sections through aged DBA/2 J nerve, increased astrocyte parallelism reflected elevated levels of the astrocyte gap-junction protein connexin 43 (Cx43). In the distal nerve, increased Cx43 also indicated with a higher level of intact anterograde transport from retina to SC. Our results suggest that progression of axonopathy in the optic nerve involves astrocyte remodeling in two phases. In an early phase, astrocyte processes organize in parallel, likely through gap-junction coupling, while a later phase involves deterioration of organization as glial coverage increases and axons are lost.
Astrocytes serve myriad functions in the mammalian central nervous system; aside from providing structural support, they additionally maintain the extracellular environment, optimize neuronal signaling, and release neurotransmitters themselves [39, 57]. Astrocytes are especially critical in white matter tracts, where axons propagate energy demanding action potentials great distances from their nuclei [29, 45]. Due to this relationship, astrocyte-axon interactions are increasingly recognized as important both in neural homeostasis and in pathology. Astrocytes exhibit multiple phases of remodeling due to neurodegenerative stress, demonstrating nuanced responsiveness and providing depth to a previously binary ‘reactive or quiescent’ classification system [33, 45, 46, 51]. Intriguingly, early astrocyte responses to neurodegeneration are often highly beneficial to injured neurons [16, 27, 32, 50]. In the optic nerve, astrocytes provide biochemical support and maintain extracellular ion balance for retinal ganglion cell (RGC) axons, which transmit retinal signals to visual structures in the brain [1, 53]. Glaucomatous optic neuropathy (or simply glaucoma), a neurodegenerative disease projected to affect some 11 million people by 2020 , selectively targets RGCs and their axons. Although age is the greatest risk factor, intraocular pressure (IOP) remains the only modifiable risk factor [19, 54]. However, many patients continue to lose RGC axons despite IOP-lowering treatments . RGC axons and the glia that support them are particularly sensitive to age-related stressors, such as deficits in metabolism and loss of anterograde axonal transport, both of which are common aspects of age-related neurodegenerative disease [2, 5, 7–9, 40]. Recent work has focused on early phases of axonal pathology in order to abate axon susceptibility prior to overt loss [12, 35].
In neurodegenerative disease, a glial scar generally fills volume previously occupied by axons . Such reactive gliosis contributes to remodeling within the optic nerve during glaucomatous progression, as in the DBA/2 J mouse model of hereditary glaucoma [3, 4, 22, 30, 43, 48]. However, earlier components of astrocyte remodeling presage overt axonal degeneration . Early RGC axonopathy in animal models of glaucoma is characterized by enlargement of the optic nerve concurrent with expansion of individual axons, accumulation of hyperphosphorylated neurofilaments, and loss of anterograde transport from the retina to central brain targets [11, 13, 36]. In the DBA/2 J mouse, astrocyte processes retract from local axon bundles and redistribute from the center of the nerve to the edge before reclaiming additional coverage later in progression . Early morphological remodeling may be reversible without leading to discernible axonal damage [33, 51]. Thus, understanding how astrocyte remodeling progresses early and its relationship to axonopathy may present novel therapeutic opportunities.
Here we measured defining features of astrocyte morphological remodeling that presage overt axonal degeneration in the optic nerve. We developed a novel method to quantify organization of astrocyte processes relative to their orientation during axonal degenerative progression [11, 30]. We find that astrocyte processes lose parallel orientation as axons are lost, coincident with changes in overall glial area and the distribution of astrocytes in the nerve. Diminished organization is closely related to axonal properties, rather than to changes in age or IOP. Parallelism closely relates both to astrocyte connectivity through gap junctions and maintenance of axonal anterograde transport.
Materials and methods
Animals and tissue preparation
Nerves used for cross-sectional analysis
All mice were transcardially perfused with PBS followed by 4% paraformaldehyde in PBS. A 1–3 mm section of optic nerve proximal to the globe was isolated and post-fixed for 1 h in 4% paraformaldehyde. Nerves were prepared for embedding in Epon resin and semi-thin (1–2 μm) cross-sectioning as described previously [11, 14, 21, 25, 41, 42]. Cross-sections were imaged using an Olympus Provis AX70 microscope equipped with a motorized X-Y-Z stage, a digital video camera, and 100× oil-immersion, differential interference contrast optics. Photomicrographs were obtained en montage to represent the entire cross-section.
A subset of eyes and optic nerves from DBA/2 J and D2 control mice were enucleated and dissected from the optic chiasm for longitudinal sectioning and immunohistochemical staining. Tissue was cryoprotected in a sequence of 10, 20, and 30% sucrose/PBS overnight and subsequently embedded and frozen in Tissue-Plus O.C.T. Compound (Fisher Healthcare, Houston, TX). We labeled 10 μm cryosections with the following antibodies: anti-glial fibrillary acidic protein (GFAP; EMD Millipore, Billerica, MA, 1:500), and anti-Connexin-43 (Cx43; Alomone Labs, Jerusalem, Israel, 1:250). Immunolabeling was visualized using appropriate DyLight-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA 1:200). Fluorescent montages were captured using an Olympus Provis AX70 microscope as described above. Confocal images were captured using an Olympus FV-1000 inverted microscope. Settings were kept constant for all sections so that comparisons in label intensity could be made.
Nerve and axon quantification
We measured cross-sectional nerve area in a total of 110 DBA/2 J optic nerves and axon density (axons/mm2) in a subset of 46 DBA/2 J optic nerves as described previously [3, 6, 21]. Briefly, we randomly selected 25–30 non-overlapping frames representing a known area of nerve. Previously developed routines were used to identify and count each axon for which a single, intact myelin sheath could be identified. Nerves with pathology so severe that intact myelin sheaths were difficult for the program to detect were excluded. The mean axon density relationship for all frames was taken as the representative axon density for the nerve. In these same 46 nerves, we additionally determined the cross-sectional area for each identified axon within a myelin sheath. A range of 6000–40,000 measurements were used to calculate mean axon area (in um2). We calculated correlation coefficients from best-fitting regression via Pearson’s coefficient.
Quantification of glial area and process organization
Optic nerve sections were filtered using a suite of MATLAB routines (MathWorks, Natick, MA) to generate a binary image that highlighted glial processes (described in ). Glial area and center of mass (CoM) were calculated as described previously. Briefly, the sum of all identified processes was used to determine the percentage of the nerve occupied by glia; the CoM was obtained by dividing the binary image into 20 concentric rings from the edge to the center of the nerve, measuring the percent glial area within each, and determining which division delineated 50% of total glial area on either side. Thus, a nerve with an even distribution of glial area between edge and center has a CoM of 10; nerves with glial area biased towards the edge have a lower CoM, while nerves with glial area biased towards the center have a higher CoM.
Tracing of anterograde axonal transport
Forty-eight hours prior to perfusion, a subset of animals were anesthetized with 2.5% isoflurane and bilaterally injected intravitreally with 2 μl of 0.5 mg cholera toxin subunit B (CTB) conjugated to Alexa Fluor 488 (Invitrogen) as previously described [13, 14]. Two days post-injection, animals were transcardially perfused with PBS followed by 4% paraformaldehyde in PBS. Brains were cryoprotected in 30% sucrose/PBS overnight, and 50 μm coronal midbrain sections were cut on a freezing sliding microtome. Serial superior colliculus sections were imaged using a Nikon Eclipse TI microscope (Nikon Instruments) and the intensity of the fluorescent CTB signal was quantified using ImagePro custom routines (Media Cybernetics) as previously described [13, 56]. CTB signal was normalized to background and alternating sections were analyzed for intensity. Intensity from each section was calculated to reconstruct a retinotopic map of intact anterograde transport across the superior colliculus. Intact transport for each map was defined as any region with an intensity ≥ 70% of the maximum CTB signal for that tissue.
Data for IOP and glial area analysis are presented as mean ± standard error of the mean (SEM) for each treatment. Statistical analysis and p-values for comparing means were obtained using Kruskal-Wallis one-way ANOVA or two-sided t-tests, and all data met criteria for normalcy as confirmed using the Shapiro-Wilk normality test; all datasets compared passed with p ≥ 0.22. Correlations were calculated using the two-tailed Pearson product moment test and verified by linear regression analysis. Statistical tests were considered significant if p < 0.05. All statistical tests were performed with SigmaPlot 12.5 (Systat Software Inc., San Jose, CA). Numbers of samples and measurements along with actual p values of significance are indicated where appropriate in the text or figure legends.
Optic nerve remodeling involves diminished astrocyte organization
Parallelism reflects early axonal changes
Astrocyte cytoskeletal reorganization depends on location in the nerve
Parallelism reflects both astrocyte connectivity and RGC axonal function
Our results demonstrate several key features of astrocyte organization and its alterations through pathology. In cross-sections of healthy DBA/2 J optic nerve (Fig. 1), astrocyte processes organize in a similar direction, i.e., exhibit a high degree of parallelism as indicated by our metric (Figs. 2 and 3). As axon pathology increases with a commensurate increase in glial coverage of the nerve, parallelism diminishes and astrocytes distribute more evenly across the nerve (Fig. 4). Accordingly, axon density, total number of axons, and axon expansion all predict changes in astrocyte parallelism more accurately than independent measures of glaucoma progression in the DBA/2 J (i.e., IOP and age; Fig. 5). Earlier we reported that axons expand in mean cross-sectional area up to a threshold of about 0.5 mm2 before their number begins to decline . Concomitantly, astrocyte processes retreat towards the edge of the nerve, meaning center-of-mass (or CoM) diminishes. In these pre-degenerative nerves, as mean axon area increases towards the threshold for loss, astrocyte parallelism increases without a change in overall glial coverage of the nerve (Fig. 6). Once axon loss begins and density diminishes, parallelism decreases in kind (Fig. 5e), presumably as astrocyte processes re-invade axon bundles . In total, these results from the DBA/2 J nerve indicate that astrocytes remodel biphasically. During early axon expansion prior to axonal loss, astrocyte processes increase in parallelism as CoM diminishes independent from gliosis (Fig. 6). Once axons are lost, gliosis involves diminished parallelism and uniform astrocyte distribution across the nerve (Fig. 4).
In longitudinal sections of nerve, we found that both GFAP and Cx43 - which marks astrocyte gap junctions  – are higher in proximal vs. distal segments (Figs. 7c, 8d). This is consistent with a more even distribution of astrocytes, as indicated by the higher CoM in the proximal vs. the distal segment (Fig. 7e). In earlier work, we demonstrated that anterograde axonal transport from retina to brain is an early harbinger of axon pathology in the DBA/2 J and degrades in distal-to-proximal fashion . We found here that parallelism of GFAP-labeled astrocyte processes was higher in distal segments of our 10-month old DBA/2 J nerves (Fig. 7f). As parallelism increased in the distal segment, astrocytes redistributed towards the nerve edge, as indicated by a lower CoM (Fig. 7g). This pattern of remodeling reflects that seen in early stages of axonal pathology, indicating that astrocytes in the distal nerve may be reacting to pathology prior to those in the proximal nerve.
In the distal segment, nerves containing elevated Cx43 also demonstrated higher levels of intact transport, but did not show a relationship with parallelism (Fig. 9b). In contrast, levels of Cx43 in the proximal segment predicted parallelism with no clear relationship to transport (Fig. 9a). Thus, these results indicate that in the distal segment, where axonopathy has already begun , Cx43 in astrocytes better reflects axon function than astrocyte organization. This is consistent with findings showing that gap-junction coupling of the astrocyte network is tightly modulated by axonal function and neuronal activity . Similarly, increased parallelism in the distal vs. proximal segment is reminiscent of our results from cross-sections of proximal nerve, where increased parallelism correlated with early expansion of axons prior to overt loss (Fig. 6b). Through gap-junction coupling, astrocytes may be able to more evenly distribute resources to those axons undergoing the highest degree of stress [17, 20, 36, 47].
Our results suggest that early progression in the optic nerve involves remodeling of astrocyte processes, first to a higher state of organization with increased parallelism (Fig. 6) and gap-junction coupling (Fig. 9) and then to gradual deterioration of organization as coverage increases and axons are lost (Figs. 4 and 5). Perhaps well-organized astrocyte processes lend structural stability to the nerve. In the optic nerve head, astrocytes form a continuously remodeling network that adapts to IOP exposure , with astrocyte actin and tubulin filaments gradually re-orienting as IOP increases . With short exposures to elevated IOP, astrocyte processes retract towards the cell body ; astrocyte processes also may also fortify the edge of the nerve, as coverage in the center diminishes (Fig. 3 of Sun et al., 2013). As astrocyte processes re-invade the nerve center with longer IOP exposure, their organization diminishes . With acute injury (nerve crush), astrocyte processes appear to detach from the edge , but this likely reflects the more severe nature of the injury. Similar to our results in the myelinated nerve, astrocyte remodeling following induced short-term elevations in pressure is most extensive distal from the sclera . Portions of the optic nerve beyond the sclera itself are unlikely to be impacted directly by elevated IOP, and secondary changes could involve different structural mechanisms. This may be why we did not note a simple linear relationship between IOP and loss of parallelism (Fig. 5b), but did find strong correlations between measures of axonal degeneration and loss of parallelism (Figs. 5 and 6).
Perhaps the most important question is whether early astrocyte remodeling is protective or pathogenic. Cx43 is elevated in the optic nerve head and the retina in human glaucomatous tissue , and here we find that astrocyte remodeling is associated with increased Cx43 (Fig. 9). Intriguingly, we additionally find that elevated Cx43 is also associated with intact axonal anterograde transport (Fig. 9d). This suggests that the early phases of astrocyte remodeling involving increased connectivity may be a component of an endogenously protective mechanism, wherein these alterations within astrocytes positively influence the health of axons within the optic nerve. In late stages of neurodegeneration Cx43-mediated coupling helps disperse inflammatory cytokines through gap junctions [10, 15]. The data presented herein leads us to believe that Cx43 exhibits a multitude of functions, like astrocytes themselves. In these early stages of pathology, gap junctions allow astrocytes to couple with neighboring cells and share signals that may contribute to the redistribution of resources to those regions most at risk.
Early optic nerve pathology involves astrocyte process remodeling, first to a higher state of organization and gap-junction coupling and then to gradual deterioration of organization as coverage increases and axons are lost. Morphologically, therefore, astrocytes exhibit multiple phases of remodeling due to neurodegenerative stress. We find that these early astrocyte responses to neurodegeneration are associated with beneficial functional outcomes for neurons. This responsiveness includes elevation in the gap-junction protein Cx43. Together, these data provide evidence for an endogenously protective mechanism wherein astrocytes increase connectivity to positively influence optic nerve axonal health during neurodegenerative stress.
Supported by National Institutes of Health grants R01 EY17427, R01 EY024997, T32 EY021453, T32 EY007135, and P30 EY008126, Departmental Unrestricted and Senior Investigator Awards from Research to Prevent Blindness, Inc., the Melza M. and Frank Theodore Barr Foundation through the Glaucoma Research Foundation, and the Stanley Cohen Innovation Fund through Vanderbilt University Medical Center. Imaging supported through the Vanderbilt University Medical Center Cell Imaging Shared Resource core facility (Clinical and Translational Science Award Grant UL1 RR024975 from National Center for Research Resources). The authors would like to thank Victoria Vest for her programming expertise along with Wendi Lambert for her assistance with animal husbandry, tissue processing, and for editing the manuscript.
MC carried out each experiment, performed statistical analysis, and drafted the manuscript. JC participated in coding and experimental design. DC participated in the design of the study and reviewed the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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