Neurodegeneration severity can be predicted from early microglia alterations monitored in vivo in a mouse model of chronic glaucoma

1The ability to detect and monitor a neurodegenerative disease soon after its onset and to anticipate its future progression is a fundamental step towards uncovering early pathogenic mechanisms and developing targeted therapies. Such an early diagnostic and prognostic strategy depends on the detection of cellular and/or molecular markers dynamically linked with the pathogenic process of neurodegeneration. Growing evidence indicates that a wide range of neurodegenerative diseases are associated with innate immune responses from microglia, and in certain contexts, from recruitment of blood-derived monocytes or macrophages.

2Microglia are long-lived myeloid cells that stably inhabit the adult CNS within parenchymal and perivascular niches. Functionally, they constantly interact with surrounding neurons, blood-brain barrier cells and other glia. Stress or damage to surrounding cells causes rapid microglial activation, as identified by complex molecular, functional and cellular changes, as well as microgliosis, which refers to the expansion of microglial cell numbers by local self-renewal and/or recruitment of monocytes and/or macrophages from the blood-stream or, potentially, from latent progenitors. Thus, microglia, as ubiquitous, dynamic sensors of CNS damage and dyshomeostasis, are ideally suited to detect and indicate the progression of pathogenic processes.

3Microgliosis and microglial activation mirror the course of neurodegeneration in both clinical and animal model studies of multiple diseases, such as Alzheimer's, Parkinson's and Huntington's disease. Moreover, live imaging studies that have monitored microglial alterations have found evidence for their involvement at preclinical disease stages. Thus, CNS-resident microglia and infiltrating monocytes and macrophages are emerging as promising sensitive indicators of neuronal decline; however, their ability to predict later disease is not well defined, particularly at early stages of disease. Detection of microglial distribution and activation by molecular imaging of the brain using positron emission tomography has underscored the relevance of microglial activation as a relevant biomarker of disease in multiple neurological and psychiatric conditions. The actual behavior of microglia at the cellular level during health, acute injury and chronic neurodegeneration has been studied in animal models over the course of minutes, and for up to 4 months, by direct observation of fluorescently labeled cells using two-photon confocal imaging of the brain, retina and spinal cord, and by confocal scanning laser ophthalmoscopy (cSLO) of the retina. Given that in vivo imaging of the retina using cSLO is non-invasive, this technique has been used to directly visualize GFP-labeled retinal microglia in the mouse eye, and in some cases to monitor changes in a single animal over a limited period of time. Although these previous studies have detected changes in microgliosis in response to acute injury, what remains to be determined is whether this approach can be used in a chronic, progressive, age-related model of retinal disease to detect and quantify early changes in microglia that would predict the later course of neurodegeneration. Clinical issueSpecific neuronal populations progressively deteriorate in neurodegenerative diseases. Early detection is key for slowing or halting neuronal decline and loss, underscoring the importance of defining reliable indicators of disease onset and progression. The course of neurodegeneration is mirrored by innate immune responses of CNS-resident microglia and infiltrating monocytes. Live imaging studies have detected early microglial alterations in animal models and humans, suggesting that they have potential value as sensitive indicators of disease progression. However, whether the early changes of innate immune cells are related to and can predict later neurodegeneration is unknown. Glaucoma results in progressive neurodegeneration of retinal ganglion cells (RGCs) and their axons in the optic nerve and retina. The disease is mostly asymptomatic at early stages and is detectable only once vision is irreversibly compromised. Despite this, the possibility of live monitoring retinal microglia to predict future neurodegeneration severity in glaucoma has not yet been explored. Confocal ophthalmoscopy (cSLO) allows for direct and repeated visualization and tracking of the microglial alterations that precede neurodegeneration in the intact retinal niche.ResultsHere, the authors use a mouse model of inherited glaucoma that, as in the human condition, develops age-dependent and variably severe neurodegeneration, coupled with innate immune responses. To assess the kinetics of early microgliosis (accumulation of microglia at the site of injury) and microglia activation, they used a DBA/2J reporter substrain in which microglia and infiltrating monocytes express green fluorescent protein (GFP) under the control of the fractalkine receptor locus (Cx3cr1), and imaged young mice at 1 to 5 months of age by cSLO to monitor early microglial changes. The authors then aged the mice to 10 months and analyzed optic nerve pathology ex vivo. Across individual eyes, live images collected at pre-pathological ages revealed dynamic patterns of retinal microglial activation and an unexpected diversity in the levels of microgliosis at the optic nerve head (ONH), which was attenuated by minocycline. Notably, they identified a significant correlation between early microgliosis at the ONH and the late severity of nerve pathology.Implications and future directionsThis study establishes microglia as indicators and early predictors of late optic nerve degeneration in a model of chronic glaucoma. This live imaging approach is the first to visualize, quantify and monitor microglial cellular changes during early stages of a neurodegenerative disease. The findings are broadly relevant to the field of neurodegeneration because they link microglial changes to future disease progression. Moreover, this study developed a non-invasive strategy to monitor visual biomarkers that is applicable to tracking and managing glaucoma and other chronic CNS diseases that affect RGCs.

4Clinical issue

5Specific neuronal populations progressively deteriorate in neurodegenerative diseases. Early detection is key for slowing or halting neuronal decline and loss, underscoring the importance of defining reliable indicators of disease onset and progression. The course of neurodegeneration is mirrored by innate immune responses of CNS-resident microglia and infiltrating monocytes. Live imaging studies have detected early microglial alterations in animal models and humans, suggesting that they have potential value as sensitive indicators of disease progression. However, whether the early changes of innate immune cells are related to and can predict later neurodegeneration is unknown. Glaucoma results in progressive neurodegeneration of retinal ganglion cells (RGCs) and their axons in the optic nerve and retina. The disease is mostly asymptomatic at early stages and is detectable only once vision is irreversibly compromised. Despite this, the possibility of live monitoring retinal microglia to predict future neurodegeneration severity in glaucoma has not yet been explored. Confocal ophthalmoscopy (cSLO) allows for direct and repeated visualization and tracking of the microglial alterations that precede neurodegeneration in the intact retinal niche.

6Results

7Here, the authors use a mouse model of inherited glaucoma that, as in the human condition, develops age-dependent and variably severe neurodegeneration, coupled with innate immune responses. To assess the kinetics of early microgliosis (accumulation of microglia at the site of injury) and microglia activation, they used a DBA/2J reporter substrain in which microglia and infiltrating monocytes express green fluorescent protein (GFP) under the control of the fractalkine receptor locus (Cx3cr1), and imaged young mice at 1 to 5 months of age by cSLO to monitor early microglial changes. The authors then aged the mice to 10 months and analyzed optic nerve pathology ex vivo. Across individual eyes, live images collected at pre-pathological ages revealed dynamic patterns of retinal microglial activation and an unexpected diversity in the levels of microgliosis at the optic nerve head (ONH), which was attenuated by minocycline. Notably, they identified a significant correlation between early microgliosis at the ONH and the late severity of nerve pathology.

8Implications and future directions

9This study establishes microglia as indicators and early predictors of late optic nerve degeneration in a model of chronic glaucoma. This live imaging approach is the first to visualize, quantify and monitor microglial cellular changes during early stages of a neurodegenerative disease. The findings are broadly relevant to the field of neurodegeneration because they link microglial changes to future disease progression. Moreover, this study developed a non-invasive strategy to monitor visual biomarkers that is applicable to tracking and managing glaucoma and other chronic CNS diseases that affect RGCs.

10A relevant neurodegenerative disease of the retina with unresolved pathogenesis is glaucoma. The ability to detect and monitor glaucoma at early stages would offer the potential for early intervention in this disease. As a model of inherited, age-dependent glaucoma, we use DBA/2J mice, which like human patients undergo slow, asynchronous and chronic decline and loss of retinal ganglion cells (RGCs) and their axons in the optic nerve and retina. Axonopathy first manifests at the optic nerve head (ONH), where unmyelinated axons exit the eye, and progressively expands across asymmetrical areas of the retina, offering remarkable spatial resolution to track disease progression. Microglia activation has been associated with human glaucoma, and early alterations in retinal microglia, and potentially monocyte infiltration, precede detectable neuronal pathology in DBA/2J mice. However, a predictive link between early microglia and/or peripheral monocyte alterations and later disease progression has not been defined.

11To address this, our current approach took advantage of the remarkable variability of neurodegeneration severity across DBA/2J eyes. To track retinal and ONH microglia and recruited monocytes in vivo with single-cell resolution, we generated DBA/2J mice carrying the microglia and monocyte reporter CX3CR1-GFP and applied recently optimized cSLO imaging and automated morphometric cell analysis to quantitatively monitor microgliosis and microglia activation from 1 to 5 months of age in a large cohort of young mice. After allowing these same mice to age, we analyzed optic nerve pathology ex vivo. We provide evidence that microglial alterations are selectively detectable in eyes that progress to glaucoma at later stages, and establish that early microgliosis has a strong correlation with the severity of optic nerve degeneration. Taken together, these findings provide the first evidence that in vivo monitoring of the time course and dynamics of early microglia might serve as sensitive predictors of late chronic neurodegeneration.

12To allow the long-term monitoring and quantification of the kinetics of microgliosis and microglia activation during chronic glaucoma in live DBA/2J mice, we performed backcrossing to generate a substrain carrying a knock-in allele that expresses GFP under the control of the fractalkine receptor promoter (Cx3cr1; ). This reporter not only labels resident microglia but also peripheral monocytes and macrophages that can infiltrate the diseased or injured CNS. Using cSLO in Cx3cr1GFP/+ DBA/2J mice aged 1 to 5 months, we imaged individual eyes to track GFP+ cells localized to the ONH and central retina. At monthly intervals, we collected single and multiple xy-point confocal images centered on the optic disc in fundus images, and spanning approximately the central 1.5 mm or 1.5×3 to 4 mm of each retina, respectively, and its inner 30 to 40 µm in depth (Fig. 1A,B; supplementary material Fig. S1A). These live confocal images provided enough cellular resolution to visualize cell somata, and in some cases arbor dimensions (Fig. 1C), and to recognize three distinct subsets of GFP+ cells of microglial or macrophage origin: (1) a large population of parenchymal cells tiling the retina (200 to 300 cells), (2) a central cluster of microglia and/or blood-derived monocytes or macrophages localized to the ONH area, and (3) perivascular cells radiating from the ONH (Fig. 1B). We compared live and ex vivo confocal images of individual eyes collected less than 3 days apart, and confirmed that cSLO detects individual GFP+ cells throughout the inner retinal layers (supplementary material Fig. S1B-E). Therefore, cSLO live imaging allows the tracking of cellular changes in retinal microglia and infiltrating monocytes that are localized adjacent to RGC somata, dendrites and unmyelinated axons across the inner retinal surface and ONH.

13We applied cell segmentation and morphometry to cSLO images of the central 1.5-1.7 mm2 of retina to identify activated parenchymal microglia. Briefly, using intensity-threshold-based morphometric analysis, we identified activated parenchymal microglia as cells with large somal areas (>50-60 µm2) and a few short processes, and non-activated parenchymal and perivascular microglia as cells with somata that were 2-3 times smaller in area and had visibly extensive and complex arbors. Microglial soma size has been shown to increase with Iba1 upregulation, and provides a metric to determine the activation status of individual GFP+ cells in live image analysis. In sequential images of the same retinas collected at 3 and 4 months of age, we mapped and quantified numbers of activated and non-activated microglia across eight sectors radiating from the optic disc (supplementary material Fig. S2A,B), and at each age detected variable numbers of activated microglia between sectors and retinas, which were uncoupled from the concurrent changes in total GFP+ cell density (supplementary material Fig. S2B,C). For most of the individual retinas monitored over 2 months, numbers and distribution of GFP+ cells and activated microglia were dynamic (supplementary material Fig. S2C, left), although some retinas maintained relatively static levels and/or patterns of activation (supplementary material Fig. S2C, right).

14Previous live imaging studies of retinal GFP+ microglia and macrophages have tracked changes in cell numbers in response to acute optic nerve injury. To determine the sensitivity of our methods to detect longitudinal changes in microgliosis in a chronic model, we induced a subtle reduction in microgliosis during early disease progression by treating young DBA/2J mice with minocycline, which we have previously shown decreases both the number and activated phenotype of retinal microglia and/or peripheral monocytes. First, we identified a subset of 2-month-old mice with relatively high ONH microgliosis by cSLO imaging, then administered systemic minocycline (120 mg/kg body weight for 6 weeks), and collected cSLO images after treatment at 4 months of age (Fig. 2A). The quantification of GFP+ cell numbers within the ONH area showed that there were significant decreases in cell clustering in the treated eyes (P<0.05, n=9 eyes, Student's t-test; Fig. 2B). The ONH GFP+ cells also displayed a manifest reduction in Iba1 expression relative to untreated eyes (Fig. 2C), consistent with the microglial deactivation reported for minocycline in this model. Thus, live retinal imaging can track an induced attenuation in early microgliosis.

15Previous studies have established that DBA/2J mice typically lack histologically detectable retinal and optic nerve pathology in the first 6 months of age; however, young mice show clustering, activation and proliferation of Iba1+ microglia and/or peripheral monocytes at the ONH in some eyes. Given that the age of glaucoma onset and progression are diverse in this model, it was unclear whether these early microglia and infiltrating monocyte changes were linked to later neurodegeneration. To track the presence and levels of early ONH microgliosis prior to RGC degeneration, we analyzed cSLO retinal images collected between 1 and 5 months of age with preferential imaging at 3 months of age (Fig. 3A; supplementary material Fig. S1), when microgliosis in the DBA/2J retina peaks. We detected a significant increase in the mean number of GFP+ cells localized to the ONH at 3 months (P<0.01, n=19 to 59 eyes; Student's t-test), which persisted through 5 months (Fig. 3B), in agreement with ex vivo studies. Furthermore, statistical analysis controlling for the potential effect of repeated imaging on individual eyes confirmed this result (linear mixed model; http://CRAN.R-project.org/package=lme4; P<0.0001), demonstrating that the effect of repeated observations on individual eyes is negligible (P=0.0942). Notably, we observed that eyes of the same age displayed widely variable GFP+ cell density, ranging from 5 to 30 cells per 0.05 mm2.

16The diversity in levels of ONH microgliosis raised the question as to whether it reflects early, differential progression towards disease in a subset of eyes. To address this possibility, we assessed optic nerve pathology ex vivo in the same eyes at 10 months of age, when glaucomatous degeneration is evident in many DBA/2J mice (Fig. 4A-C). Glaucoma onset and the severity of neurodegeneration are highly variable in DBA/2J mice, thus axonal pathology can manifest with variable levels and distribution in individual optic nerves. Using an established damage scoring system, high-resolution light microscopy images of 1-µm-thick cross-sections of individual nerves were categorized at one of three levels of damage based on qualitative parameters of pathology (Fig. 4C; see Materials and Methods). Next, the numbers of GFP+ cells per ONH at 1 through to 5 months of age were plotted for nerves in each damage category (Fig. 4D). All 1-month-old mice showed similarly low GFP+ cell densities in the ONH, but this analysis revealed a distinct trend to increase from 2 to 5 months of age, where nerves with severe late damage showed the highest levels of microgliosis, whereas nerves with moderate damage were preceded by more intermediate and variable levels of microgliosis. A Kruskal–Wallis rank ordered test showed that the median GFP+ cell density per ONH for each level of severity of optic nerve pathology was significantly different at 3 months (χ2=07.84, P=0.0199) and 4 months of age (χ2=16.27, P=0.0003), and marginally significant at 5 months of age (Fig. 4E). This initial analysis shows a significant relationship between the early number of GFP-labeled cells at the ONH and the extent of late optic nerve degeneration.

17To thoroughly define the correlation between ONH microgliosis at a young age and the susceptibility to develop glaucoma at an older age, we established a quantitative measure of optic nerve pathology based on the magnitude of gliosis and gliotic scaring associated with nerve degeneration. It is well documented that as axons are lost in the optic nerve, the area is replaced by reactive glia (hypertrophic and proliferative), and by a glial scar at the end stages of neurodegeneration. Therefore, the reduction of the non-axonal area at the expense of expanding glial territories in late stages of glaucoma was used as readout of nerve pathology severity. For this, we applied threshold-based segmentation of glial cells and scar tissue to high-resolution light images of entire optic nerve sections from 10-month-old mice (Fig. 5A, see Materials and Methods). Consistent with the variability in glaucoma onset and progression in DBA/2J mice, individual optic nerves showed large variability in the relative area occupied by glial cells or scar, which was consistently increased in nerves with widespread axonal damage and loss. According to the quantification of non-axonal area, individual Cx3cr1GFP/+ DBA/2J nerves were classified as healthy if their total glial territory occupied less than 20% of the nerve, which is comparable to Gpnmb+/SjJ DBA/2J nerves (A.B., K.T.B., S.R.A., M.R.S., D.J.C. and M.L.V., unpublished data), the congenic control strain that carry a wild-type Gpnmb allele and do not undergo RGC degeneration. Glaucomatous nerves with increased glial coverage were classified as having medium (20-40%) or high (>40%) gliosis (Fig. 5B). To confirm this classification, we compared the average glial area in each category and observed a significant increase from low to medium and then to high gliosis (P<0.001, n=41 eyes; Student's t-test). We thus used the expansion of gliosis and gliotic scar throughout areas devoid of RGC axons as a novel and objective measure of nerve degeneration severity.

18Plotting the levels of early ONH microgliosis versus the magnitude of late nerve gliosis for each individual eye (Fig. 5C) revealed that nerves with low glial area or mild damage at 10 months showed low ONH microgliosis at 3 months. In contrast, eyes with moderate or severe nerve degeneration, evident by extensive gliosis or glial scaring, were preceded by intermediate and high levels of early ONH microgliosis, respectively. The comparison of the mean ONH microgliosis for eyes grouped by late nerve pathology revealed significant increases across the three levels of gliosis (P<0.001 and P<0.05, n=41 eyes; Student's t-test). A Spearman's rank-ordered correlation showed that nerve glial area at 10 months was significantly correlated with the number of microglial cells observed at 3 or 4 months (rho=0.56, P=0.0001; Fig. 5D). These analyses establish a predictive link between early microglia and/or peripheral monocyte alterations and later disease progression.

19The selective increase in microgliosis detected prior to optic nerve degeneration in DBA/2J mice could involve resident microglia and/or proinflammatory monocyte entry to the ONH, as recently suggested. To address whether the early microgliosis detected by cSLO can be attributed to infiltrating cells, we assessed ex vivo the expression of sialoadhesin (Siglec-1 or CD169), which identifies infiltrating monocytes and macrophages, as well as vitreal hyalocytes and dendritic cells, but is absent in resident microglia. Sialoadhesin-expressing cells are absent in the brain and retina under physiological conditions, where the blood-brain or blood-retina barrier is intact. Immunostaining of retinal whole mounts at 4 months of age detected only scarce sialoadhesin-expressing cells, and their numbers were independent of the relative levels of ONH microgliosis detected by live imaging at 3 months (Fig. 6A,B). Furthermore, quantification of cells expressing sialoadhesin in the ONH area at 4 months of age (n=10 retinas; Fig. 6C) detected no sialoadhesin in 86.60% of CX3CR1-GFP+ cells, colocalization of GFP and sialoadhesin in 7.39% of cells, and 0.58% of cells expressing sialoadhesin but not GFP (in only two out of ten retinas). Consistent with previous reports of monocyte infiltration, we detected an increased presence of cells that were positive for both sialoadhesin and GFP throughout the central retina in 17-month-old DBA/2J mice (n=4); these cells were localized to the ONH, the retinal parenchyma and along blood vessels; however, such cells were infrequent at 4 months of age (Fig. 6D,E). Overall, these results reveal the presence of a small subset of peripheral monocytes confined to the ONH area in young DBA/2J mice, and point to resident microglia as the main cells underlying microgliosis during early stages of glaucoma progression.

20Activation of microglia and innate immunity responses have been linked to the early stages of neurodegeneration. In the retina, microglia activation is also associated with early stages of disease in animal models of chronic glaucoma, as well as age-related macular degeneration. However, it has been unclear whether these early responses are indicative of later severity or patterns of neurodegeneration. Here, we addressed this using the DBA/2J model of glaucoma, a chronic model of retinal neurodegeneration that is variable in onset and progression. Building upon previous studies of direct in vivo imaging of CX3CR1-GFP+ retinal microglia and infiltrating monocytes using cSLO in models of acute injury, and morphometric quantification of microglia activation in live confocal images, we performed live imaging at early disease stages, then allowed the animals to age and assessed late RGC pathology in the optic nerve.

21We found that early microgliosis within the ONH is strongly correlated with later severity of optic nerve degeneration. The detection of early microgliosis is consistent with previous ex vivo studies that have reported microglia and/or peripheral monocyte clustering, activation and proliferation at the ONH prior to detectable neuronal pathology in this model, as well as early infiltration of proinflammatory monocytes. Gene expression studies have also identified the activation of pathways related to innate immune responses and monocyte recruitment during early progression of chronic glaucoma. Previous studies tracking GFP+ microglia by cSLO after induction of acute damage to RGCs, by optic nerve crush or intraocular pressure elevation, have detected increases in retinal microgliosis that were associated with reduced survival of RGCs. Here, using a chronic model of glaucoma, we found early microgliosis clustered at the ONH, and were able to link this to the later severity of optic nerve degeneration. Our findings suggest that ONH microgliosis might be an early indicator of RGC stress or damage, consistent with the localization of the earliest axonal damage to the retina-nerve interface.

22Proinflammatory, circulating monocytes infiltrate the DBA/2J ONH and have been observed in 10.5-month-old animals. At young ages, our preliminary analysis suggests that microglia represent the majority of the CX3CR1-GFP+ cell population contributing to early microgliosis within the ONH and central retina, because there are few cells that express sialoadhesin (Siglec-1 or CD169), a marker of peripheral monocytes and/or macrophages, as well as of dendritic cells, although it is possible that sialoadhesin expression only identifies a subset of retinal and ONH peripheral monocytes. Interestingly, we find in 17-month-old DBA/2J retinas that sialoadhesin is expressed in the majority of amoeboid monocyte-lineage cells that are abundant within the ONH and throughout the retina, and which have been attributed to monocyte infiltration in previous studies. Thus, we suggest that early microgliosis is mainly driven by changes in resident microglia, both in the ONH and retina. Given the importance of proinflammatory monocyte infiltration in neurodegeneration, it will be important to explore the crosstalk between microglia and infiltrating monocytes and/or macrophages during early versus late disease progression in glaucoma.

23The pigmentary form of glaucoma in DBA/2J mice depends upon mutations in the two melanosomal proteins TYRP1 (tyrosinase-related protein 1) and GPNMB (glycoprotein nmb). We have previously shown that early clustering and activation of microglia was not evident in mice of the congenic substrain DBA/2J Gpnmb+/SjJ. Notably, GPNMB is also expressed in cells of the macrophage and microglia lineage, and functions as a repressor of inflammation. Thus, the early microglial changes documented in the DBA/2J mouse could reflect intrinsic immune components, or immune responses to early stress or damage in RGCs that might contribute to glaucoma pathogenesis. Nevertheless, ONH and central retinal microglial alterations represent the earliest retinal change evident in the DBA/2J mouse, and are strongly correlated with later disease. Although intraocular pressure is not consistently elevated at young ages in DBA/2J mice, progressive functional decreases have been detected after 3 months of age. Thus early changes in RGC function are apparent, consistent with the early changes that we see in microglia at these ages.

24We conclude that monitoring microglia activation and microgliosis at prepathological stages might be a powerful tool for detecting the onset and tracking progression of neurodegenerative disease. The retina is an ideal region of the CNS to achieve this because specific cell populations can be directly and repeatedly visualized in the intact organism by the use of cSLO and cell-selective transgenic labels. Previous live imaging studies have used the Cx3cr1GFP/+ transgenic label for cSLO imaging in acute models of induced RGC injury. Here, we applied this to a chronic progressive neurodegenerative disease, and provided quantitative analysis of cell changes in the context of chronic neurodegeneration, by extending the period of tracking for microglial and/or peripheral monocytic changes over 5 months, and then analyzing disease progression at 10 months. The tracking of early microglial changes in young DBA/2J mice offers the possibility of optimizing the use of this chronic model by allowing the selection of young animals in which one or both eyes will likely progress to severe glaucoma at advanced ages, thus reducing the inter-individual variability in disease progression within experimental samples. This strategy should advance the already intense studies aimed at elucidating the complex cellular and molecular mechanisms that underlie neurodegeneration in chronic glaucoma, and identify initiating events associated with disease onset or early progression potentially relevant to glaucoma patients.

25Other imaging modalities allow quantitative detection of regional microglia activation in vivo in chronic CNS pathologies, including positron emission tomography of radioligands for microglia and astrocyte translocator protein (TSPO) receptors, as well as magnetic resonance and bioluminescence imaging of neuroinflammation. However, in vivo visualization of microglia by cSLO provides cellular resolution, which enables quantitative analysis of microglia activation based on somal size, similar to analysis of brain microglial activation in two-photon confocal images. We observed highly dynamic and sectorial patterns of microglia activation within the retinal parenchyma, suggesting that microglia might be responsive to local changes within the retina during early disease stages. Given that these changes are complex, this possibility will be addressed in future studies using early markers of neuronal stress or dysfunction. Our assessment of microglial behavior near glaucoma onset demonstrates the power of in vivo retinal imaging to detect early progression of neurodegeneration at the cell level, with high spatial and temporal resolution.

26We also establish the feasibility of visually tracking induced changes in retinal microglia activation to assess therapies, in this case by administering minocycline to decrease activation. With the development of non-genetic labels for microglia, this could be a potential tactic applicable to both glaucoma management and treatment evaluation. Future studies could also evaluate the possibility of detecting retinal microglia and/or peripheral monocyte changes resulting from diverse pathologies because the retina and optic nerve are targeted for neurodegeneration in diseases such as Alzheimer's, Parkinson's and multiple sclerosis. In line with this, the use of live detection of retina and ONH pathology for early Alzheimer's disease management is under intense study. Overall, our findings suggest that retinal microglia might serve as sensitive neuroimaging biomarkers to detect early and/or unidentified stages of disease onset and progression in glaucoma, and potentially in other neurodegenerative diseases that impact the retina and optic nerve.

27Heterozygote Cx3cr1GFP/+ DBA/2J mice were derived by backcrossing homozygous C57BL/6.129P-Cx3cr1tm1Litt/J mice, which express GFP under the control of the fractalkine receptor locus, for more than ten generations with DBA/2J mice (Jackson Laboratories, Bar Harbor, ME). All mice were bred in-house, introducing new breeders every 3 to 4 generations. This study used females only. Mice were maintained and imaged in pathogen-free facilities, using protocols approved by the Institutional Animal Care and Use Committee at the University of Utah.

28Retinal and ONH GFP+ microglial and/or peripheral monocyte cells were monitored using confocal scanning laser ophthalmoscope (cSLO) images collected at monthly intervals in mice aged 1 to 5 months (±1 week), following a recently reported protocol. Eyes with corneal or iris defects detectable by fundus imaging were excluded from this study, and imaging was discontinued after 5 months of age, when corneal and/or iris defects prevented effective pupil dilation and reliable imaging. Briefly, mice anesthetized by intraperitoneal injection of Avertin (1.3% 2,2,2-tribromoethanol and 0.8% tert-amyl alcohol, Sigma-Aldrich, St Louis, MO) and fitted with PMMA contact lenses (Cantor & Nissel Ltd., Northants, UK), were imaged unrestrained with a cSLO system equipped with a 55° wide-field lens and real-time eye tracker (Spectralis HRA+OCT, Heidelberg Engineering). The central retina was visualized in single-point images spanning an area of ∼1.5 mm in diameter, or in composite images spanning 40 to 60% of the retina (∼1.5×3-4 mm), both across the inner planes of the retina and ONH (30-40 µm axial depth; 55-60° focus), using by 820-nm or 488-nm laser excitation (460-490 nm barrier filter set) to produce fundus and fluorescence images, respectively.

29To analyze ONH microgliosis, we manually identified and counted total GFP+ cells within a ∼250-µm-diameter circle centered on the optic disc in cSLO images. In the surrounding central retina, we performed automated cell and somal segmentation followed by morphometric analysis using intensity-based thresholding in cSLO images of the central retina (∼1.5 mm2). Briefly, individual somal areas were automatically measured, sorted into groups of activated microglia (>50-60 µm2), non-activated microglia (10-50 µm2) and manually identified cells <10 µm2) and mapped to eight radial sectors (∼200 µm2) that subdivided the central retina and excluded the ONH.

30A subset of mice that showed high ONH microgliosis by cSLO imaging at 2 months old (n=9), was administered systemic minocycline (120 mg/kg body weight, Sigma-Aldrich) on weekdays (Monday-Friday) for 6 weeks, from 2.5 to 4 months of age, by oral gavage.

31Mouse eyes were collected as previously described. Briefly, under full anesthesia induced by inhalation of 2% (vol) Isoflurane in 2 liter/min oxygen, mice were transcardially perfused with 5 ml PBS followed by 20 ml 4% paraformaldehyde (PFA; Electron Microscopy Sciences, Hatfield, PA) in 0.1 M PBS circulated with a peristaltic pump (Dynamax, Rainin, Oakland, CA). Eyes with optic nerves were isolated from the brain and post-fixed for 2 h in PFA at 4°C, and nerves were dissected and further fixed overnight in 1.2% PFA and 0.8% glutaraldehyde in PB (Electron Microscopy Sciences).

32Retinas, with the dorsal pole of the eyes marked with a cauterizer, were processed as previously described. Whole-mount retinas were immunostained using mouse anti human phospho-neurofilament (pNF, Dako, Carpinteria, CA), rabbit anti-human Iba1 (Wako, Richmond, VA), goat anti-GFP (Abcam, Cambridge, MA), and rat anti-mouse CD169 (sialoadhesin) conjugated to Alexa Fluor 647 (MOMA-1 clone, AbD Serotec, Bio-Rad, Raleigh, NC) primary antibodies, which were detected with Alexa-Fluor-conjugated donkey anti-IgG secondary antibodies (Invitrogen, La Jolla, CA).

33Confocal images spanning entire retinal flat mounts were generated as previously described using a confocal imaging system equipped with a 20× lens and a resonant scanner (A1R confocal, Eclipse Ti inverted microscope and NIS-Elements C, Nikon). Multipoint images (625 xy positions) were acquired at high resolution (0.41 µm/px), then stitched and projected as maximal intensity of the inner 30-40 µm of retina (0.8-µm step). To allow image analysis and quantification, the parameters of image acquisition were maintained constant between retinal samples, and, for illustration, images had their brightness and contrast minimally edited. Counts of ONH cells expressing GFP and/or sialoadhesin where manually performed in the central 200×200 µm around the optic disc, visualizing each channel independently in maximum-intensity projections, and verifying colocalization in slice view, through the z plane.

34Nerves from 10-month-old Cx3cr1GFP/+ DBA/2J mice including the retro-orbital, myelinated segment (1-1.5 mm post-lamina), were prepared as 1-µm-thick plastic cross-sections, and stained with a modified paraphenylenediamine (PPD) protocol and Toluidine Blue. High-resolution multipoint (36 xy) images were generated using a compound light-microscope and a 60× lens (BX51 and cellSens software, Olympus, Center Valley, PA).

35By direct visual inspection of high-resolution multipoint images obtained from entire optic nerve cross-sections, we identified glial cells and the gliotic scar as areas lacking dystrophic or intact axons, as well as meninges and vascular lumen. To segment glial areas and vascular spaces, we applied automatic thresholds to the red RGB channel to generate a binary overlay representing axon-free nerve areas (FluoRender, University of Utah). To quantify the relative area occupied by glial cells and/or the gliotic scar, we measured the cross-sectional area of the nerve and subtracted the area corresponding to blood vessels and meninges. Random samples independently segmented and measured by two investigators showed <5% variation in measured glial area (n=10).

36Statistical significance was calculated by a Student's post-hoc t-test (unpaired, two tailed; Fig. 2B, Fig. 3B and Fig. 5B,C), by a linear mixed model (http://CRAN.R-project.org/package=lme4; Fig. 3B) or Kruskal–Wallis rank-ordered test (Fig. 4E), and is indicated in the graphs as: *P<0.05, **P<0.01 and ***P<0.001. Correlation analysis was performed by Spearman's rank-ordered test (Fig. 5D). Analyses were conducted in R: A language and environment for statistical computing 2014 (R Core Team, R Foundation for Statistical Computing, Vienna, Austria) or Excel v. 14.4.6 (Microsoft, Redmond, WA).