Our data provide several fundamental advances in understanding glaucoma. First, our gene expression data and subsequent functional tests showed that local stress in neural tissues, in response to high IOP, induces proinflammatory signaling and the activation of transendothelial migration. The transendothelial migration pathway is the first pathway to significantly change in glaucoma, being activated prior to changes in highly sensitive measures of neuronal dysfunction and neural damage. Second, we clearly showed that the entry of monocytes (and/or monocyte-derived cells) into the eye occurs very early in DBA/2J glaucoma and prior to neural damage. Third, we showed that a 1-time treatment with x-rays protects a treated eye from glaucoma. This treatment alters endothelial cell activation in the face of proinflammatory signaling and abrogates migration of monocytes into the eye. Adding a damaging molecule that is made by the monocytes to irradiated eyes, along with other glaucomatous stresses, restores neural damage, with a topography that is characteristic of glaucoma. Together, these data strongly imply that monocyte entry is required for neural damage to occur. However, further experiments that prevent monocyte entry independent of any radiation treatment are required to definitively prove if this is so. Although not directly assessed, a functional role of invading monocytes in glaucomatous RGC death may be supported by an independent study showing that a mutation in the Itgam
gene, which encodes CD11b, protects from RGC death subsequent to laser-induced IOP elevation (44
). Although increased expression of proinflammatory molecules was previously documented in glaucoma and animal models with experimentally raised IOP (26
), in most studies they were not timed in relation to disease stage, and it was not known that these changes were required for neural damage. In addition to endothelial cells and leukocytes, neuroinflammatory processes are likely to be controlled and mediated by both microglia and astrocytes (34
) in the ONH during glaucoma. For example, astrocytes may initially respond to high IOP and/or other glaucomatous stresses by activating early inflammatory signaling (reviewed in ref. 52
). It is likely that early inflammatory signaling protects against local tissue stresses (metabolic/biological stress and mechanical strain) or injury. A protective role of ocular inflammation against RGC degeneration has been shown after axon injury (53
However, after prolonged IOP elevation and/or exposure to other stresses, the initially protective response may develop into chronic and damaging inflammation. Immune dysfunction is reported to contribute to some forms of glaucoma (55
), and both protective and damaging roles are suggested for T cells (reviewed in ref. 56
). However, the roles of T cells in glaucoma require further clarification. In agreement with some published studies (57
), our current study did not detect or implicate T cells. In another study, experimental induction of high IOP upregulated the cytokine TNF. This induction was sequentially followed by microglial activation, loss of oligodendrocytes, and delayed loss of RGCs. It was suggested that microglial activation damages oligodendrocytes, resulting in RGC loss (44
). However, oligodendrocyte loss occurs later in the inherited DBA/2J glaucoma and appears to be a secondary response to damage (58
To our knowledge, the present study is the first to show the transendothelial migration of monocytes into neural tissue of the eye after IOP elevation and to suggest a primary importance of this monocyte entry for glaucomatous neural damage. Leukocyte migration into tissues is a highly regulated process, involving complex interactions between circulating leukocytes and the vascular endothelium. The selectins are key adhesion molecules displayed on leukocytes and endothelial cells. They mediate the first critical step in the leukocyte-endothelial cell adhesion cascade and are necessary for leukocyte tethering, rolling, and subsequent transendothelial migration from blood vessels into tissues (reviewed in ref. 59
). Unexpectedly, our data implicate the aberrant regulation of selectins and their ligands as an early pathogenic event in endothelial cells of the ONH that is required for the initiation of glaucomatous damage. Also unexpected, our data show that a 1-time radiation treatment prevents the display of activated L selectin ligands by endothelial cells at a much later age. Endothelial cells in radiation-treated eyes do not display functional L selectin ligands, as assessed by MECA-79, an antibody that specifically detects functional ligands and serves as a new and early biomarker for glaucoma. Expression of E selectin was previously reported as a biomarker in the drainage structures of human eyes with glaucoma, but it was not determined whether this was an early pathologic event or a secondary response to damage (60
). That report did not assess whether E selectin had a pathophysiological role in either IOP elevation or glaucomatous neurodegeneration.
It is not simple to envision how radiation protects in the long term, especially as early glaucomatous stresses are still present in radiation-treated eyes. One possibility is that radiation induces long-lasting epigenetic changes (61
) that alter gene expression and prevent glaucoma. Given the need to treat local cells for protection, it is likely that the radiation somehow alters a local cell population, or possibly populations of different cell types, that normally contribute to the early pathogenesis of glaucoma. Possibly affected cell types include stem cells, resident microglia, and astrocytes (16
). Stem cells have not been implicated in glaucoma pathogenesis, and we have not documented obvious changes in resident microglia in radiation-treated eyes, compared to untreated eyes, at these early stages. Under stress conditions, astrocytes can produce cytokines and other molecules and so are capable of initiating and controlling early inflammatory responses in glaucoma (reviewed in ref. 51
). Our gene expression data show that the early inflammatory responses are intact in radiation-treated eyes but that the entry of monocytes is prevented. Collectively, our data strongly support transendothelial migration of monocytes as a primary step for initiating or propagating neural damage during early stages of glaucoma, a step that is prevented by radiation treatment.
It is not clear how radiation suppresses invasion of monocytes in the long term, including still-unborn monocytes that are remotely located. Endothelial cells are a key cell type in locally controlling transendothelial migration of monocytes and the nature of inflammatory responses. Our data show that endothelial cells are somehow changed by the radiation treatment. In radiation-treated eyes, there is altered expression of various endothelial genes that control transendothelial migration. Additionally there is abrogated activation of L selectin ligands. Thus, endothelial cells may be directly impacted by the effects of irradiation, resulting in modulation of a neuroinflammatory process that is critical for glaucoma development. Expression of the endothelial gene Glycam1
is greater in radiation-protected eyes than in early glaucoma (Figure C). GLYCAM1 is one of the glycoprotein ligands for L selectin, which is secreted from endothelial cells and is present in normal serum (62
). Expression of Glycam1
is upregulated in 4.5-month-old mice (2 months after irradiation; data not shown), long before the onset of glaucoma in our colony of DBA/2J mice. Thus, the steady-state expression of Glycam1
is increased long term and may explain the radiation protection. Secreted GLYCAM1 is likely to modulate endothelial-leukocyte interactions, leukocyte-leukocyte interactions, and L selectin–mediated intracellular signaling that modulates transendothelial migration (63
). GLYCAM1 secretion was inversely related to leukocyte transendothelial migration from blood (64
). Thus, the radiation treatment induces a long-term increase in Glycam1
expression, which may be a key factor inhibiting entry of monocytes into the optic nerve during very early glaucoma. Further experiments will test this possibility. If correct, the manipulation of Glycam1
expression may prove effective against a variety of inflammatory conditions.
Our data support what we believe to be a new model in which monocytes are essential for glaucomatous damage and suggest that glaucoma is primarily a neuroinflammatory disease. Much of the field has focused on astrocytes, microglia, and RGCs themselves, with some interest in the endothelial control of blood flow (65
). Our demonstration that specific radiation of the eye can prevent glaucoma is an important finding. Since localizing tissue irradiation to the relevant tissue is therapeutically desirable, we developed an x-ray machine to specifically irradiate the mouse eye. Ocular radiation proved highly effective at preventing glaucoma, being much more effective than even the most robust of the other reported treatments in the same mouse model (e.g., refs. 26
), with the exception of whole body irradiation (16
). It will be important to extensively evaluate the efficacy and safety of this treatment in other mammalian and primate models. The potential dangers of radiation exposure are well documented. Side effects vary depending on the dose and length of exposure but can include cataracts and cancer (71
). It is difficult to explain why some individuals are detected with glaucoma subsequent to radiation treatment for ocular tumors. The different outcomes may result from parameters that differ to those in our experiments and may include age at treatment, radiation dose or exact timing and duration of treatment, and/or individual genetic makeup. On the other hand, it is possible that radiation treatment (or other treatments that alter transendothelial migration) may have prevented glaucoma in some individuals, but this is not known as it is not possible to predict glaucoma development prior to the onset of disease features. The great success of localized x-ray radiation in our study suggests that it may be possible to develop safe, radiation-based treatments of the eye or optic nerve that may be effective against human glaucoma. Potential protective effects of radiation have been considered for a long time (72
). Recently, radiation (0.65 Gy) was shown to protect against retinal degeneration in 2 mouse models (76
). Since the protection in that study diminished with time, and the dose was well below the protective dose we demonstrate here, the protective mechanisms are likely to be different from those studied here.
The control of transendothelial migration is very complex and highly context dependent. Therefore, it may not be straightforward to develop highly effective nonradiation-based therapies to lessen glaucoma by preventing monocyte entry into the ONH. Steroids are known to reduce transendothelial migration. In some individuals, steroid use can actually lead to glaucoma (77
). This appears contradictory to our findings, but IOP levels are often very high in steroid glaucoma, and this very severe insult may overwhelm any protective antiinflammatory effect. Alternatively, endothelial cells in different vascular beds may behave differently in response to steroids, with possibly a smaller effect on transendothelial migration in the ONH compared with that in other tissues. Statins are also known to reduce transendothelial migration. Interestingly, some studies have shown that long-term statin use appears to be associated with a reduced risk of open-angle glaucoma (78
). However, given both the robust and long-term efficacy of a single dose of x-ray radiation in preventing cellular entry into the optic nerve and retina, it will be important to further evaluate the use of x-rays for preventing glaucoma and other inflammatory conditions.