The molecular basis of retinal ganglion cell death in glaucoma

Glaucoma is a group of diseases characterized by progressive optic nerve degeneration that results in visual field loss and irreversible blindness. A crucial element in the pathophysiology of all forms of glaucoma is the death of retinal ganglion cells (RGCs), a population of CNS neurons with their soma in the inner retina and axons in the optic nerve. Strategies that delay or halt RGC loss have been recognized as potentially beneficial to preserve vision in glaucoma; however, the success of these approaches depends on an in-depth understanding of the mechanisms that lead to RGC dysfunction and death. In recent years, there has been an exponential increase in valuable information regarding the molecular basis of RGC death stemming from animal models of acute and chronic optic nerve injury as well as experimental glaucoma. The emerging landscape is complex and points at a variety of molecular signals - acting alone or in cooperation - to promote RGC death. These include: axonal transport failure, neurotrophic factor deprivation, toxic pro-neurotrophins, activation of intrinsic and extrinsic apoptotic signals, mitochondrial dysfunction, excitotoxic damage, oxidative stress, misbehaving reactive glia and loss of synaptic connectivity. Collectively, this body of work has considerably updated and expanded our view of how RGCs might die in glaucoma and has revealed novel, potential targets for neuroprotection.     

Retinal ganglion cell death in experimental glaucoma

AIMS: To determine whether parasol retinal ganglion cells (magnocellular pathway) are selectively lost in the primate model of glaucoma. METHODS: Ocular hypertension was induced in one eye of six Macaca fascicularis monkeys for 6-14 weeks. The retinal ganglion cells in these eyes were labelled retrogradely with the tracer horseradish peroxidase (HRP) implanted into the optic nerve and subsequently examined in retinal whole mount preparations. The degree of retinal ganglion cell loss was estimated from Nissl stained tissue by comparison with the contralateral untreated control eye. RESULTS: In the three glaucomatous retinas with the best labelling 1282 cells could be classified, of which 182 were parasol cells and 1100 were midget cells. Linear regression analysis did not demonstrate a significant reduction in the proportion of parasol to midget cells with increasing cell loss (regression slope 0.023, 95% CI -0.7 to 0.11). Compared with the control eye the cell soma of the remaining retinal ganglion cells in glaucomatous eyes were reduced in size by 20% for parasol cells (p=0.003) and by 16% for midget cells (p <0.001). CONCLUSION: The results of this study do not support the hypothesis that selective loss of parasol retinal ganglion cells occurs in experimental glaucoma. In addition, the change in cell soma size distributions following ocular hypertension suggests that both parasol and midget retinal ganglion cells undergo shrinkage before cell death.     

Ganglion cell death in glaucoma: pathology recapitulates ontogeny

I review some improvements in our knowledge about the death of retinal ganglion cells in glaucoma during the last 20 years. These include the realisation that glaucoma damage precedes its detection by perimetry, the fact that the lamina cribrosa is a major site of axonal injury to ganglion cells, and the association between regional structure of optic nerve head connective tissue and the pattern of glaucoma damage. The selective susceptibility of larger retinal ganglion cells and its functional significance are described.
Apoptosis is the mode of cell death in at least some ganglion cells in experimental glaucoma. This supports a theory that retrograde axonal transport failure leads to loss of trophic factor influence on ganglion cells, causing them to initiate their own suicide. As a consequence of this theory, two therapeutic avenues are suggested for prevention of glaucoma injury and cell death: delivery of trophic factors and manipulation of ganglion cell genetic expression of controlling influences over programmed cell death.     

The mechanisms of neuronal death in glaucoma

The current therapy of glaucoma (drugs, LASER, surgery) acts mainly on the trabecular meshwork and on the ciliary body. The loss of the visual function in glaucoma is however determined by the disfunction and death of the ganglion cells. The main theories trying to explain the death of the ganglion cells in glaucoma are analyzed. Their practical value is represented by the finding of the ideal neuroprotective drug.     

Regulation of cell death and survival pathways in experimental glaucoma

This study investigates cell death and survival pathways in experimental glaucoma using the translimbal photocoagulation laser model. Glaucoma was induced unilaterally in 79 Wistar rats and all eyes developed elevated intraocular pressure. The involvement of caspase-3, p-AKT and members of the MAP kinase pathway was evaluated by immunohistochemistry and Western blotting. We found that protein levels of caspase-3 were elevated from day 15 to day 30 (p<0.05). All investigated members of the MAP kinase pathway were significantly activated. P-SAPK/JNK activation began on day 2, reaching a 6-fold elevation by day 30 (p<0.05). The p-P38 level was elevated on days 2 and 8 (p<0.05), followed by a decrease to baseline on day 15. The level of p-ATF-2, the substrate of P38, was significantly elevated at all time points tested, up to day 30 (p<0.05). P-ERK was detected early (p<0.05) on day 1, returning to normal on day 15. The pro-survival protein p-Akt, a member of the PI3-kinase survival pathway, was also detected early on day 1 (p<0.05) returning to baseline on day 8 and remaining unchanged up to 64days. We conclude that retinal ganglion cell death in glaucoma involves activation, at different time points, of multiple pro-apoptotic pathways (the MAP kinase pathway and the caspase family) and pro-survival (PI-3 Kinase/ Akt and p-ERK).     

Mouse models of retinal ganglion cell death and glaucoma

Once considered too difficult to use for glaucoma studies, mice are now becoming a powerful tool in the research of the molecular and pathological events associated with this disease. Often adapting technologies first developed in rats, ganglion cell death in mice can be induced using acute models and chronic models of experimental glaucoma. Similarly, elevated IOP has been reported in transgenic animals carrying defects in targeted genes. Also, one group of mice, from the DBA/2 line of inbred animals, develops a spontaneous optic neuropathy with many features of human glaucoma that is associated with IOP elevation caused by an anterior chamber pigmentary disease. The advent of mice for glaucoma research is already having a significant impact on our understanding of this disease, principally because of the access to genetic manipulation technology and genetics already well established for these animals.     

Neuronal death in glaucoma

Glaucoma is recognized to have its major detrimental effect upon the eye by killing retinal ganglion cells. The process of cell death appears to be initiated at the optic nerve head, though other sites of injury are possible but unsubstantiated. At present the injury at the nerve head seems related to the level of the eye pressure, but its detailed mechanism is as yet unexplained. There is a greater loss of ganglion cells from some areas of the eye, and this feature of glaucoma seems related to the regional structure of the supporting connective tissues of the optic nerve head. Larger retinal ganglion cells have been consistently shown to have somewhat greater susceptibility to injury in glaucoma, though all cells are injured, even early in the process. Ganglion cells die by apoptosis in human and experimental glaucoma, opening several potential areas for future therapies to protect them from dying. Neurotrophin deprivation is one possible cause of cell death and replacement therapy is a potential approach to treatment.     

Retinal ganglion cell death in glaucoma: mechanisms and neuroprotective strategies

Various cellular and molecular mechanisms that may lead to apoptotic cell death of retinal ganglion cells in glaucoma are discussed. These cellular mechanisms include neurotrophic factor deprivation, ischemia, glial cell activation, glutamate excitotoxicity, and abnormal immune response. Based on experimental and clinical evidence, the rationale for various neuroprotective strategies is described.     

Apoptotic retinal ganglion cell death in the DBA/2 mouse model of glaucoma

The DBA/2 mouse has been used as a model for spontaneous secondary glaucoma. We attempted to determine the in vivo time course and spatial distribution of retinal ganglion cells (RGCs) undergoing apoptotic death in DBA/2 mice. Female DBA/2 mice, 3, 9-10, 12, 15, and 18 months of age, received intravitreal injections of Annexin-V conjugated to AlexaFluor 1h prior to euthanasia. Retinas were fixed and flat-mounted. Annexin-V-positive RGCs in the hemiretina opposite the site of injection were counted, and their locations were recorded. Positive controls for detection of apoptotic RGCs by Annexin-V labeling included rats subjected to optic nerve ligation, and C57BL/6 mice subjected to either optic nerve ligation or intravitreal injection of NMDA. To verify that Annexin-V-labeled cells were RGCs, intravitreal Annexin-V injections were also performed on retinas pre-labeled retrogradely with FluoroGold or with DiI. Annexin-V-positive RGC locations were analyzed to determine possible clustering and areas of preferential loss. Annexin-V labeled apoptotic RGCs in eyes after optic nerve ligation, intravitreal NMDA injection, as well as in aged DBA/2 animals. In glaucomatous DBA/2 mice 95-100% of cells labeled with Annexin-V were also FluoroGold- and DiI-positive. This confirms that Annexin-V can be used to specifically detect apoptotic RGCs in rodent retinas. In DBA/2 mice, apoptotic RGC death is maximal from the 12th to the 15th month of age (ANOVA, p<0.001, Fisher's post hoc test) and occurs in clusters. These clusters are initially located in the midperipheral retina and progressively occur closer to the optic nerve head with increasing age. Retrograde axonal transport of FluoroGold in the glaucomatous mouse retina is functional until at least 2-3days prior to initiation of apoptotic RGC death.     

Mitochondria: Their role in ganglion cell death and survival in primary open angle glaucoma

Retinal ganglion cell axons within the globe are functionally specialised being richly provided with many mitochondria. Mitochondria produce the high energy that is required for nerve conduction in the unmylenated part of the ganglion cell axons and for the maintenance of optimum neuronal function. We proposed that in the initiation of glaucoma (POAG) an alteration in the quality of blood flow dynamics in the optic nerve head results in sustained or intermittent ischemia of a defined nature. This results in normal mitochondrial function being negatively affected and as a consequence retinal ganglion cell function is compromised. Ganglion cells in this state are now susceptible to secondary insults which they would normally tolerate. One secondary insult to ganglion cell mitochondria in such a state might be light entering the eye. Other insults to the ganglion cells might come from substances such as glutamate, prostaglandins and nitric oxide released from astrocytes and microglia in the optic nerve head region. Such cascades of events initiated by ischemia to the optic nerve head region ultimately cause ganglion cells to die at different rates.     

Correlation between retinal ganglion cell death and chronically developing inherited glaucoma in a new rat mutant

Glaucoma is a progressive optic neuropathy with characteristic optic disc changes, retinal ganglion cell loss and progressive visual field defects. Elevated intraocular pressure is considered to be a major risk factor in glaucomatous neuropathy. This study aimed to characterize and document a new chronic glaucoma model in the rat with respect to the effect of elevated intraocular pressure on overall retinal dysfunction and retinal ganglion cell loss, and to elucidate the possible mechanisms underlying this cell loss. Intraocular pressure (IOP) was measured in rats using a Tonopen. RGCs were retrogradely labeled with the fluorescent dye, 4-[didecylaminostyryl]-N-methyl-pyridinium-iodide (4-Di-10 ASP) and quantified on retinal flat mounts using fluorescence microscopy. The optic nerve head was examined fundoscopically. Changes in the histological appearance of the whole eyes was studied in paraffin sections, and immunohistochemistry was carried out on cryostat sections. The levels of mRNA for several genes were compared between control and glaucomatous retinae using semi-quantitative RT-PCR. Mutant animals are affected with either a unilateral or bilateral enlargement of the globes having an IOP that ranged from 25 to 45 mmHg, as compared to control values of 12-16 mmHg. The IOP of glaucomatous eyes increased significantly with age to attain a value of 35+/-7.3 at 1.5 years. Concomitant with the rise in IOP, the number of labeled RGCs continued to decrease in number with age. A total of 1887+/-117RGC mm(-2) could be labeled in wild-type control and juvenile mutant pre-glaucomatous retinas, whereas this number dropped to 92+/-26RGC mm(-2) at 1.5 years. Ophthalmoscopy revealed atrophied optic nerve heads in the affected eyes. The pars plicata and the pars plana of the ciliary body of glaucomatous eyes were hypertrophied and elongated, respectively. The anterior chamber was narrow and the irido-corneal angle open in glaucoma eyes. The mRNA of glial-fibrillary-acidic protein, endothelin-1, STAT-3 and STAT-6 increased in the retinas correlating with the severity and duration of the disease. Changes in the expression of GFAP and endothelin-1 could be confirmed using immunohistochemistry. This model may help to address several fundamental issues in the pathogenesis of glaucoma and aid in the development of neuroprotective strategies.