Background
Glaucoma is a complex, multifactorial disease characterized by the progressive dysfunction and loss of retinal ganglion cells. It is a leading cause of vision loss affecting approximately 80 million people worldwide [
1]. The earliest neurodegenerative events that injure retinal ganglion cell axons during glaucoma are unknown but multiple lines of inquiry have suggested a complex neuroinflammatory process involving multiple cell types [
2‐
12].
The DBA/2J (D2) mouse is widely used as a model of age-related chronic glaucoma as it shares hallmark features with the human disease. In D2 mice, by 12 months of age, glaucomatous damage is present in the majority of eyes in our colony [
13]. D2 glaucoma is characterized by a loss of retinal ganglion cells and axonal degeneration in the optic nerve. This axonal damage likely initiates at the optic nerve head (ONH) where the axons are insulted [
14]. Early during D2 glaucoma, a class of pro-inflammatory monocyte-like cells exit the vasculature and enter the ONH [
5]. Current experiments support a damaging role for these monocyte-like cells in early retinal ganglion cell axon injury [
5]. Our lab has previously shown that this cell entry can be prevented by a single, sub-lethal dose of γ-radiation or by a local (eye-only) dose of X-ray radiation [
5,
15]. This treatment subsequently provides life-long protection against glaucoma and has been confirmed by others [
16]. The protection is profound with up to 96% of eyes developing no glaucoma [
5,
15].
In order to mechanistically understand this radiation-based protection, Howell et al. performed gene expression profiling on optic nerve heads of pre-diseased and diseased mice. They compared expression in retinas and optic nerve heads from untreated D2 mice, strain-matched control D2-
Gpnmb
+ mice that do not develop elevated intraocular pressure (IOP) or glaucoma, and γ-radiated mice at different ages post-radiation therapy [
5,
6]. These data identified four genes that were differentially expressed following radiation therapy at a young age (4 months of age) and continued to be differentially expressed at ages up to 12 months of age (
Gfap,
Glycam1,
Npr3, and
Stat3) [
5,
6]. Of these, increased
Glycam1 expression was consistent between the retina and the tissue punch of the optic nerve head (that included adjacent choroidal tissue), making GlyCAM1 an excellent candidate to participate in the radiation-induced protection.
GlyCAM1 (
Glycosylation-dependent
cell
adhesion
molecule
1) is a proteoglycan ligand for L-selectin, modulating transendothelial migration of leukocytes [
17‐
21]. GlyCAM1 is an unusual L-selectin ligand in that it lacks a transmembrane domain and acts as a peripheral membrane-associated protein on the vascular endothelium [
18,
19]. GlyCAM1 is also secreted into the blood and may be a regulatory molecule that can directly bind to free leukocytes in the blood (as may other soluble L-selectin ligands) [
17,
22‐
25]. Existing evidence supports a role for GlyCAM1 as a negative regulator of extravasation. GlyCAM1 levels (protein and mRNA) decrease during acute antigen-primed inflammation and depletion of soluble L-selectin ligands (using L-selectin-IgG affinity columns) decreases the inhibitory effect of mouse plasma on leukocyte binding to endothelial cells of high endothelial venules in Stamper-Woodruff assays [
17,
23,
26]. In addition, selectin ligands that carry sialyl Lewis
X glycans, as does GlyCAM1, inhibit leukocyte binding to lymph nodes in an in vitro assay [
21].
Glycam1 is stably up-regulated following radiation treatment in glaucoma and monocyte entry into the optic nerve head is inhibited [
5]. This is consistent with a role for GlyCAM1 as a negative regulator of monocyte extravasation with the increased GlyCAM1 levels inhibiting extravasation in radiation-treated eyes. However, and to our knowledge, a definitive physiologic role for GlyCAM1 as a negative regulator of extravasation has not been experimentally demonstrated by directly and specifically removing
Glycam1 in vivo. Similarly, whether specifically increasing GlyCAM1 levels beyond those typical for non-inflamed tissues can negatively modulate extravasation is not reported (GlyCAM1 levels decrease in HEVs during acute inflammation). Here, we generated a D2 substrain lacking
Glycam1 to determine if GlyCAM1 is important for the radiation-induced neuroprotection and to determine if increased levels of GlyCAM1 impede extravasation in glaucoma.
Methods
Mouse strain, breeding, and husbandry
The mice were housed and fed in a 14-h light/10-h dark cycle with food and water available ad libitum. All breeding and experimental procedures were undertaken in accordance with the Association for Research for Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Visual Research. The Institutional Biosafety Committee (IBC) and the Animal Care and Use Committee (ACUC) at The Jackson Laboratory approved this study. The DBA/2J (D2) strain was utilized and has been described in detail elsewhere [
13]. D2.B6N-
Glycam1
tm1(KOMP)Vlcg (D2.
Glycam1
−/−
) mice were generated by backcrossing C57BL/6JN.
Glycam1
tm1(KOMP)Vlcg mice, heterozygous for the
Glycam1
tm1(KOMP)Vlcg allele, to DBA/2J a minimum of six times (>N6) before intercrossing to generate mice homozygous for the
Glycam1
tm1(KOMP)Vlcg allele (>N6F1). The presence of the allele was confirmed by standard PCR genotyping, and
Glycam1 levels were confirmed by quantitative RT-PCR. Equal numbers of male and female mice were used.
γ-Radiation therapy
A sub-lethal dose of γ-radiation (7.5 Gy) was administered using a
137Cesium source in a single dose at 10–12 weeks of age. The mice were placed on a rotating platform to ensure uniform administration of the treatment. The mice were monitored follow radiation treatment. This level of treatment does not result in any adverse conditions, and the mice do not require bone marrow reconstitution [
5,
15].
Axon labelling with PPD and assessment of glaucomatous damage
The processing of optic nerves and staining with paraphenylenediamine (PPD) was as published [
27]. PPD stains the myelin sheath of all axons but darkly stains the axoplasm of only damaged axons. It is well established to provide a very sensitive measure of optic nerve damage [
5,
6,
13]. Briefly, intracranial portions of optic nerves were fixed in 4% PFA at RT for 48 h, processed, and embedded in plastic. A retro-orbital segment of optic nerve from within a region up to 1 mm from the posterior surface of the sclera was sectioned (1-μm-thick sections) and stained with PPD. Typically, 30–50 sections are taken from each nerve. Multiple sections of each nerve were considered when determining damage level. The optic nerve was analysed by at least two individuals blinded to genotype and determined to have one of three damage levels:
1.
No or early damage (NOE)—less than 5% axons damaged and no gliosis. This level of damage is seen in age- and sex-matched non-glaucomatous mice and is not due to glaucoma. Although none of these eyes exhibit glaucomatous nerve damage, this damage level is called no or early glaucoma as some of these eyes have early molecular changes that precede neurodegeneration. These molecular changes can be detected by gene expression studies [
6]. The eyes with these early molecular changes but no degeneration are considered to have early glaucoma when discussing changes to monocyte extravasation in this paper.
2.
Moderate damage (MOD)—average of 30% axon loss and early gliosis
3.
Severe damage (SEV)—>50% axonal loss and damage with prominent gliosis
Flow cytometry
The mice were euthanized, and the eyes were enucleated and placed immediately into ice-cold HBSS. The optic nerve heads were dissected from the eyes in HBSS on ice and placed directly into a dispase and DNase solution. The samples were incubated for 30 min at 37 °C and shaken at 350 RPM. Following this, the samples were blocked in 2% BSA in HBSS and stained with antibodies against CD11b (PE), CD11c (FITC), and CD45.2 (APC). The samples were stained with propidium iodide (PI) for 5 min before being run on the flow cytometer. Four-colour flow cytometry was performed on a FACSCaliber (BD Bioscience), and the samples were run to completion. Cell populations were analysed using FloJo. For whole-blood leukocyte counts, the blood was collected from restrained mice via cheek bleed before being stained with PI and LDS751. The samples were run on a Guava flow cytometer (EMD-Millipore).
Immunofluorescent staining of retinal whole mounts and sections
The mice were euthanized, and their eyes were enucleated and placed in 4% PFA overnight (ON). For immunofluorescent staining retinas for cell counts, the retinas were dissected and flatmounted onto slides, permeabilized with 0.1% Triton-X for 15 min, blocked with 2% BSA in PBS, and stained ON at room temperature (RT) in primary antibody (1:500 with PBS; rabbit anti-RBPMS; Novus; NBP2-20112). After primary antibody incubation, the retinas were washed five times in PBS, stained for 4 h at RT with secondary antibody (AF594, ThermoFisher). The slides were then washed a further five times with PBS, stained with DAPI for 15 min, mounted with fluoromount, and coverslipped and sealed with nail-polish. For retinal sections, the eyes were cryoprotected in 30% sucrose ON, frozen in OCT, and cryosectioned at 18 μm. The slides were warmed to room temperature and the procedure above was followed. For GlyCAM1 staining, crude rabbit serum was used 1:50 in PBS (crude serum was prepared in rabbits after immunization against the peptide P2 (CKEPSIFREELISKD) coupled to KLH). GlyCAM1 rabbit serum was a kind gift from Jean-Philippe Girard and Nathalie Ortega (Institut de Pharmacologie et de Biologie Structurale, France) and generated in the lab of Steve Rosen (UCSF). For MECA-79 staining, a conjugated anti-MECA-79 AF488 antibody (eBiosciences; 53-6036-80) was used. The retinas were imaged on a Zeiss Axio Observer for low resolution counts, and the counting paradigm outlined in [
28] was followed. Retinal sections were imaged on a Leica SP8 for higher resolution images and processed using Imaris (Bitplane).
Nissl staining of frozen retinal sections
The mice were euthanized, and their eyes were enucleated and placed in 4% PFA ON. Following this, the eyes were cryoprotected in 30% sucrose, frozen in OCT, and cryosectioned at 18 μm. The slides were warmed to RT, placed in 1:1 alcohol:chloroform ON, and rehydrated through serial alcohol gradient. The slides were washed once in distilled water and stained for 15 min in 0.1% cresyl violet in distilled water before being differentiated in 95% alcohol, dehydrated in 100% alcohol, and cleared in xylene. The slides were left to dry at RT, mounted with fluoromount, and coverslipped and sealed with nail-polish. The sections were imaged using a Nikon Eclipse E200.
Clinical examination and intraocular pressure measurements
D2 mice develop elevated intraocular pressure and glaucoma subsequent to an iris disease [
13,
29]. In all D2 glaucoma experiments, the progression of the iris disease and intraocular pressure in mutant or drug-treated mice were compared to control D2 mice using previously described methods [
30,
31]. In each experiment, iris disease and intraocular pressure were assessed in ≥40 eyes per genotype or treatment. Iris disease was assessed at 2-month intervals starting at 6 months of age until experiment completion. Intraocular pressure was measured at 45-day intervals beginning at 8.5–9 months of age until experiment completion. Statistical analysis was performed using Student’s
t tests.
Quantitative RT-PCR
Total RNA (40 ng) from retinal or choroidal tissue was reverse transcribed using the MessageSensor Reverse Transcriptase kit (Ambion) with random decamer priming (Ambion) according to the manufacturer’s protocols. A portion of the cDNA was then used in a PCR reaction containing Taqman Univeral PCR Master Mix (Applied Biosystems). The gene-specific primers and probe sets were obtained from Applied Biosystems and used according to the manufacturer’s protocols (Glycam1; Mm00801716_m1). Real-Time PCR was performed on a ViiA 7 system (Applied Biosystems) with the standard protocol of 95 °C for 10 min to activate the DNA polymerase followed by 40 cycles of amplification. The threshold cycle (Ct) was determined using the QuantStudio Real-Time PCR software (version 1.2).
Statistical analysis
The sample size (number of eyes, n) is shown in each figure legend. Graphing and statistical analysis was performed in R. Student’s t test was used for pairwise analysis in quantitative plots, and Fisher’s exact test was used for nerve grade comparisons. Error bars refer to standard error of the mean unless otherwise stated. *P < 0.05, **P < 0.01, and ***P < 0.001.
Discussion
Glaucoma is a complex age-related disease. Although molecular mechanisms that drive glaucomatous neurodegeneration remain to be fully elucidated, our work and other’s have identified early axon damage within the optic nerve head, as well as other varied intrinsic and extrinsic factors, as early drivers of damage following periods of elevated IOP [
4,
6,
14,
41‐
44]. An increasing body of literature suggests that neuroinflammation is likely to be a key process in glaucoma [
42,
45‐
54]. The optic nerve head contains a putative blood-brain barrier controlling the passage of cells and molecules from the blood to the tissue local to the axons of retinal ganglion cells. The optic nerve head contains differing cell types (astrocytes, microglia, vascular endothelial cells, and pericytes) that all may play a role in regulating immune cell movement from the blood to the tissue local to the retinal ganglion cell axons. At this location, retinal ganglion cell axons remain mainly unmyelinated and thus may be particularly vulnerable to damage from immune-derived cells.
We previously discovered that monocyte-like cells enter the optic nerve head very early during DBA/2J glaucoma, and we proposed that the radiation-induced neuroprotection is at least partially mediated by preventing the extravasation of these cells [
5]. Evidence for extravasation of these cells from vessels included cell labelling by CFDA within the spleen with subsequent homing of labelled cells to the optic nerve head and flow cytometry demonstrating that these cells had high expression of CD45 (CD45
hi), a well-established marker for bone marrow-derived cells that circulate in the blood [
5,
39,
40,
55]. The data did not support their derivation from resident cells within the eye; resident microglial cells have low levels of CD45 (CD45
lo), and the number of CD45
lo cells was not detected to increase during glaucoma in these experiments [
5]. The monocyte-like cells that enter the eye produce various molecules that are known to be damaging in glaucoma, including endothelin 2 and complement component C1 [
3,
5]. The neuroprotective radiation treatment altered the activation of endothelial transmigration pathways and prevented the disease-dependent entry of these cells into the optic nerve head [
5]. Adding endothelin 2, a damaging molecule that is made by these cells, induced a glaucoma-like phenotype in radiation-treated eyes that had high IOP, while combined treatment to inhibit the effects of two damaging mediators that are made by these cells (complement C1 and endothelin 2) was profoundly protective against glaucoma [
5]. Together, these data argue for an important role of these infiltrated cells as damaging in DBA/2J glaucoma.
Others have confirmed the radiation-induced protection using DBA/2J mice [
16], while another study of survivors of the Hiroshima and Nagasaki atomic bombs suggests that radiation exposure protects from human glaucoma [
56]. However, when tested in an experimentally induced rat model of glaucoma, radiation was not found to be protective (however, there were many experimental differences and so the studies are not directly comparable) [
57]. Thus, and although there is evidence for infiltrating immune cells and immune cell activation in multiple models of glaucoma [
3,
5,
58‐
62], additional work is required to definitively establish their function and to extend these studies to other models of glaucoma and human glaucoma [
54]. Examining the role of these cells and the intricacies of the molecular mechanisms by which radiation therapy inhibits cell entry may lead to important human therapeutics.
GlyCAM1 is a receptor for leukocytes binding to L-selectin (primarily expressed by endothelial cells) [
24].
Glycam1 is constitutively up-regulated following radiation therapy and may protect from glaucoma by negatively modulating extravasation. To definitively test this hypothesis, we used a complete knockout of
Glycam1 in D2 mice. We found that GlyCAM1 is an important negative modulator of extravasation and is partially responsible for radiation-based protection. To our knowledge, this is the first clear evidence of a physiological impact for increased GlyCAM1 in inhibiting extravasation in neural tissues. Despite the substantial impact of
Glycam1 deficiency on extravasation, its impact on the radiation-induced neuroprotection was small (although still significant). Thus,
Glycam1 contributes to the neuroprotection but is not alone a major determinant.
The profound effect of
Glycam1 deficiency on cell entry into the eye but small effect on radiation protection may argue that preventing cellular infiltration is not a substantial contributor to the neuroprotection. Along with our current demonstration of a partial role of
Glycam1 in mediating the neuroprotection, the presence of monocyte-like cells (at levels comparable to those in untreated wild-type eyes) in largely protected
Glycam1-deficient irradiated eyes suggests that infiltrating cells are not the only contributors to glaucomatous damage. Alternatively or additionally, and although
Glycam1 deficiency overall promotes extravasation, its deficiency may largely prevent extravasation of a specific unidentified cell type that is highly potent in mediating glaucomatous damage. Another possibility is that the cells entering irradiated
Glycam1 deficient eyes do not have the same properties as those entering wild-type D2 eyes and that they are less capable of inducing glaucoma. This is a possibility as the molecular interactions during extravasation are an important determinant of the cell types that exit the vasculature and of their subsequent cellular phenotype [
63‐
66]. Further experiments are needed to resolve the role of infiltrating cells in glaucoma. Important experiments will include evaluating the effects of
Glycam1 deficiency on the infiltrating cells (detailed characterization of their molecular phenotype) and evaluating the importance of other molecules that participate in extravasation in both glaucoma and the radiation-induced neuroprotection.
It is important to note that cell types other than infiltrating monocyte-like cells may be critical in the radiation protection [
5,
15,
16]. Bosco and colleagues have reported increasing numbers of resident microglia in DBA/2J eyes at very early ages and show that early minocycline treatment protects from glaucoma (aimed at inhibiting damaging microglial activation) [
11,
67]. In these experiments, they reported that the degree of early microgliosis correlates with the degree of glaucoma severity at older ages [
11], that radiation treatment inhibits early microgliosis [
16], and that inhibition of microgliosis may underlie the radiation-induced neuroprotection. On the other hand, and despite confirming microglial activation, our experiments have provided no evidence for increased numbers of resident microglia (CD11b
+ CD45
lo) at 10.5 months of age or differential activation of ramified microglia between radiation-treated and untreated eyes (using CD68 as a marker of activated microglia) [
5]. Further, we have found that minocycline administration (started at older ages) actually made the glaucoma worse [
68]. The reasons for these differences likely reflect substantial differences in the nature (assays) and timing (ages assessed and stage of disease when treatments were initiated) of experiments, as well as potential environmental differences between institutions, which can substantially modify the disease phenotype. Clearly, further experiments are needed to resolve the roles of resident microglia and infiltrating monocyte-like cells in glaucoma and the radiation-induced protection.
Acknowledgements
The authors would like to thank Flow Cytometry, Electron Microscopy, Histology, and Gene Expression Services at The Jackson Laboratory; Gareth Howell, Krish Kizhatil, and Jeffrey Harder for discussion and experiment design; Mimi de Vries for assistance with organizing and the mouse colonies; Brynn Cardozo for colony maintenance; Nick Tolman and Amy Bell for intraocular pressure measurements; and Steven Rosen, Jean-Philippe Girard, and Nathalie Ortega for helpful discussion and the gift of GlyCAM1 sera.