In glaucoma, harmful intraocular pressure often contributes to retinal ganglion cell death. It is not clear, however, if intraocular pressure directly insults the retinal ganglion cell axon, the soma, or both. The pathways that mediate pressure-induced retinal ganglion cell death are poorly defined, and no molecules are known to be required. DBA/2J mice deficient in the proapoptotic molecule BCL2-associated X protein (BAX) were used to investigate the roles of BAX-mediated cell death pathways in glaucoma. Both Bax+/− and Bax−/− mice were protected from retinal ganglion cell death. In contrast, axonal degeneration was not prevented in either Bax+/− or Bax−/− mice. While BAX deficiency did not prevent axonal degeneration, it did slow axonal loss. Additionally, we compared the effects of BAX deficiency on the glaucoma to its effects on retinal ganglion cell death due to two insults that are proposed to participate in glaucoma. As in the glaucoma, BAX deficiency protected retinal ganglion cells after axon injury by optic nerve crush. However, it did not protect retinal ganglion cells from N-methyl-D-aspartate (NMDA)-induced excitotoxicity. BAX is required for retinal ganglion cell death in an inherited glaucoma; however, it is not required for retinal ganglion cell axon degeneration. This indicates that distinct somal and axonal degeneration pathways are active in this glaucoma. Finally, our data support a role for optic nerve injury but not for NMDA receptor-mediated excitotoxicity in this glaucoma. These findings indicate a need to understand axon-specific degeneration pathways in glaucoma, and they suggest that distinct somal and axonal degeneration pathways may need to be targeted to save vision.
Glaucoma is a group of diseases whose unifying characteristic is death of nerve cells (retinal ganglion cells) that connect the eye to the brain. Glaucoma is often associated with a harmfully high pressure inside the eye (intraocular pressure) contributing to nerve cell death. Various treatments are used to lower eye pressure, but currently no commonly used treatments directly protect the nerve cells. DBA/2J mice develop elevated eye pressure with age, and this pressure kills retinal nerve cells. The authors use this mouse model to investigate how these nerve cells die in glaucoma. They show that there are distinct degeneration pathways activated in different parts of the retinal nerve cells. They found that the biochemical pathway in the nerve cell body, which resides in the retina, requires a molecule called BAX (BCL2-associated X protein). In contrast, pathways in the part of the cell (axon) that connects the cell body to the brain do not require BAX. Because degeneration pathways in the cell body and of the axon also may be molecularly different in human glaucoma, it will be important to consider them all when designing therapies. Their data also suggest that the BAX gene is a candidate to modulate glaucoma susceptibility.
Citation:Libby RT, Li Y, Savinova OV, Barter J, Smith RS, et al. (2005) Susceptibility to Neurodegeneration in a Glaucoma Is Modified by Bax Gene Dosage. PLoS Genet 1(1): e4. doi:10.1371/journal.pgen.0010004
Editor: David Valle, Johns Hopkins Institute, United States of America
Received: January 16, 2005; Accepted: April 6, 2005; Published: July 25, 2005
Copyright: © 2005 Libby et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: BAX, BCL2-associated X protein;IOP, intraocular pressure;NMDA, N-methyl-D-aspartate;RGC, retinal ganglion cell;SEM, standard error of the mean
Glaucoma is a common blinding disease affecting approximately 70 million people worldwide . Glaucoma is often associated with elevated intraocular pressure (IOP). IOP elevation and glaucoma are typically spontaneous, progressive, idiopathic processes and are most common in the elderly . Although IOP-lowering treatments slow the development and progression of glaucoma in many patients [3,4], it is not always possible to reduce IOP to a “safe” level . Vision loss in glaucoma is the result of retinal ganglion cell (RGC) death with accompanying optic nerve atrophy, so glaucoma is a neuropathy. IOP elevation is not detected in a significant subset of glaucomas [6,7]. Thus, the unifying characteristic of glaucoma is RGC death. While there are several hypotheses as to why elevated IOP kills RGCs, both the precise biochemical cascades that are triggered within RGCs and the nature of the proximal insult(s) that trigger these cascades remain superficially defined . No treatments that directly protect the neurons are in routine clinical use.
The complex nature of glaucoma makes studies of its pathogenesis difficult . Consequently, no specific molecules have been shown to be essential for RGC death in glaucoma. Standard glaucoma-relevant models include direct RGC trauma, direct optic nerve trauma, and suddenly induced IOP elevation [10–18]. Although these induced models have provided valuable information, the relevance of specific damaging mechanisms may differ significantly between spontaneous and experimentally induced glaucomas. Thus, studies using inherited glaucoma models are also necessary.
Apoptosis is known to contribute to RGC death following experimentally induced insults including axotomy and IOP elevation (e.g., [19,20]), and there is also some evidence that apoptosis is involved in human glaucoma [21,22]. A number of molecules that are known to affect apoptosis are reported to be important regulators of RGC death after various induced insults. These include X-linked inhibitor of apoptosis protein (XIAP) [23–25], p38 , several caspases [27–30], the B-cell lymphoma/leukemia 2 (BCL2) family of apoptotic regulators [20,31–34], and members of the c-Jun N-terminal kinase (JNK) [35,36] and tumor necrosis factor (TNF) [35,37] signaling pathways. One of these molecules, BCL2-associated X protein (BAX; a proapoptotic member of the BCL2 family), has a major role in mitochondrial-mediated apoptosis in different neuronal cell types [38,39]. In mice, BAX deficiency increases the number of RGCs in the adult retina by 220% by allowing more RGCs to survive during development . Genetic or induced BAX deficiency is also known to prevent RGC apoptosis after optic nerve crush and axotomy [13,18,40]. Thus, BAX-mediated apoptosis is clearly an important mechanism of stress-induced RGC death. Whether or not this pathway has a role in IOP-induced RGC death in either experimentally induced or inherited glaucomas is not known.
Understanding the pathophysiologic mechanisms of RGC death in glaucoma and the genetic susceptibility factors contributing to this process is important for the development of effective and individualized treatments. Here, we use the genetically uniform DBA/2J mouse model of glaucoma [41–43] to assess the importance of mitochondrially mediated apoptosis in an inherited glaucoma. Importantly, we show that in this model of inherited glaucoma there are distinct RGC death and axonal degeneration pathways. The RGC death pathway is BAX dependent and, therefore, apoptotic. The axonal degeneration pathway is BAX independent. Finally, our data suggest that reducing BAX levels in the retina may retard the rate of vision loss in glaucoma.
Apoptosis Is Physiologically Relevant for RGC Death in an Inherited Glaucoma
To determine if RGC apoptosis has a significant role in an inherited glaucoma, we assessed DNA fragmentation, chromatin condensation, and cellular ultrastructure in glaucomatous DBA/2J retinas. We identified hallmarks of apoptosis including the presence of TUNEL-positive cells that peaked between 10 and 13 mo of age (the period when the majority of RGC death occurs in this model) (Figure 1). These results confirm earlier suggestive studies that apoptotic pathways are important mediators of RGC death in spontaneous glaucoma [19–22].
(A–C) A double-labeling assay that identifies fragmented DNA using fluorescently labeled dUTP (A) and detects chromatin condensation by binding of the dye YOYO-1 (B) was used to assess the presence of these hallmarks of apoptosis in glaucomatous DBA/2J eyes at 10–11 mo of age (a time when many RGCs die). A cell in the retinal ganglion cell layer (GCL, arrowhead) has both of these features of apoptosis as indicated by double labeling (C). INL, inner nuclear layer.
(D–F) Electron microscopy provided further evidence for apoptosis. (D) An example of a healthy RGC. (E) Chromatin condensation (a hallmark of apoptosis) along the inner surface of the nuclear envelope in a ganglion cell (arrows). The internal limiting membrane of the retina is indicated by arrowheads. (F) An apoptotic body in the ganglion cell layer (arrows) containing a nuclear fragment with prominent condensed chromatin (asterisk) and other cell remnants.
(G) A TUNEL assay (see Materials and Methods) was used to assess the prevalence of cell death at different ages. TUNEL labeling was not detected at 7 mo (an age prior to glaucomatous cell death) and peaked at 10–13 mo, when most RGCs die. No TUNEL-positive cells were detected in nonglaucomatous, age-matched control mice. These results support an important role of apoptosis in RGC death in spontaneous glaucoma. Scale bar, 1 μm.
Homozygous but Not Heterozygous BAX Deficiency Alters RGC Number
To test the role of BAX in glaucomatous RGC death, we extensively backcrossed a previously characterized null allele of Bax (Baxtm1Sjk)  onto the inbred DBA/2J background. In mammals, approximately twice as many RGCs are produced during retinal development than survive into adulthood [45–47]. As expected from previous studies of retinal development on a different genetic background , complete BAX deficiency increased the number of RGC-layer somata in adult DBA/2J mice by 220% (average cell number per 40× field ± standard error of the mean [SEM], number of retinas analyzed: Bax+/+, 199 ± 5.6, n = 7; Bax−/−, 437 ± 15.3, n = 8). In agreement with this, Bax−/− mice had 217% more RGC axons than Bax+/+ mice (Bax+/+, 50,504 ± 1,988, n = 8; Bax−/−, 108,907 ± 10,322, n = 4; p < 0.001). Reflecting the increased number of RGC axons and the proportional increase in glial cell types , the cross sectional area of Bax−/− optic nerves was significantly increased (average ± SEM, number of optic nerves measured: Bax+/+, 0.157 ± 0.005 mm2, n = 13; Bax−/−, 0.278 ± 0.008 mm2, n = 16; p < 0.001). In heterozygous Bax+/− mice, RGC number (average per 40× field ± SEM, 212 ± 14.0, n =5) and the optic nerve area (0.171 ± 0.007 mm2, n = 13) was not different from Bax+/+ mice (p = 0.352 and p = 0.107, respectively). Thus, heterozygous levels of BAX are sufficient for death of the normal numbers of RGCs during retinal development.
BAX Ablation Preserves RGC Numbers but Does Not Prevent RGC Axonal Degeneration in Glaucoma
To determine the role of BAX in glaucomatous RGC death, we assessed the effects of BAX deficiency on RGC survival and on RGC axonal degeneration (see Materials and Methods). Our results show that BAX is not required for RGC axon degeneration. Bax−/− mice developed severe optic nerve damage, including essentially complete loss of axons (Figure 2). In contrast, our experiments show that BAX is required for RGC death in glaucoma (Figure 3). Despite severe axonal degeneration, the numbers of cell bodies in the RGC layer of Bax−/− mice were normal. As a stringent test of this observation, we counted RGC-layer cell bodies in the retinas of mice with severe (≥ 95% loss) axon degeneration. The number of RGC cell bodies was normal in Bax−/− mice with more than 95% axon loss (Figure 3). Importantly, Bax+/− mice were also protected against glaucomatous RGC death. Bax+/− mice with an axon loss of 95% or more also had substantially increased survival of RGC cell bodies as compared to Bax+/+ controls (Figure 3E). Thus, RGC death and axonal degeneration are clearly distinguished in these experiments.
To assess the effects of Bax deficiency on optic nerve degeneration, we analyzed PPD-stained optic nerve cross sections from Bax+/+ and Bax−/− mice (n > 49 for each genotype; see Materials and Methods).
(A and B) Before the DBA/2J glaucoma damages RGCs, the optic nerves of both Bax+/+ (A) and Bax−/− mice (B) had a normal organization. The axons appeared healthy with a clear axoplasm and darkly stained myelin sheath.
(C and D) BAX deficiency did not prevent glaucomatous optic nerve damage. Severe degeneration involving extensive to complete axon loss and scarring occurred in both Bax+/+ (C) and Bax−/− mice (D). The majority of mice of both genotypes had this severe degree of damage by 12 mo of age. These experiments show that BAX is not required for glaucomatous axon degeneration. Scale bar, 50 μm.
To determine the effects of BAX deficiency on RGC death in glaucoma, we analyzed RGC layer cells at stages with and without glaucomatous optic nerve damage (see Materials and Methods). All shown images are from a similar region of the superior, peripheral retina.
(A and B) In both Bax+/+ (A) and Bax−/− (B) mice without glaucomatous optic nerve damage, the retinas appear healthy. The retinas of both genotypes are similar except that Bax−/− mice have extra RGCs (since BAX is important in normal developmental RGC death ).
(C and D) In contrast, an obvious difference was evident between the retinas of Bax+/+ and Bax−/− mice that had all suffered severe glaucomatous damage with 95% or more axon degeneration. As expected, for Bax+/+ retinas (C) from eyes with 95% or more optic nerve axon loss, there was a noticeable decrease in RGC layer cells (compare [C] to [A]). In contrast, retinas from Bax−/− mice with correspondingly damaged optic nerves (D) had suffered no obvious loss of RGC layer cells (compare [D] to [B]). This suggests that BAX is required for RGC death in DBA/2J glaucoma. As is well established for both RGCs and other neurons, Bax−/− RGCs that survive without axons have a shrunken morphology [38,82]. This is clearly evident in the Bax−/− glaucomatous mice (D).
(E) RGC layer cell counts for eyes with 95% or more axon degeneration confirmed that BAX is necessary for RGC death in this glaucoma. To allow comparison between genotypes, the percent of surviving cells is shown (% soma in mice with 95% or more axon loss compared to mice of the same genotype without glaucomatous damage). At 12 mo of age, Bax+/+ mice had 61.4% ± 3.8% of their RGC layer cells remaining, Bax−/− mice had no appreciable cell loss (101% ± 5.3%). The RGC layer cells of Bax+/− mice were also protected (89.2% ± 5.7%). The p values comparing differences in cell counts between nonglaucomatous and very severely glaucomatous (≥ 95% axon loss) eyes of the same genotype were: Bax+/+, p < 0.001; Bax+/−, p = 0.207; Bax−/−, p = 0.426. No cell loss was seen in Bax−/− mice even out to 18 mo (94.8% ± 4.4% cells surviving, p = 0.524 compared to nonglaucomatous Bax−/− mice). These findings show that BAX gene dosage has an important effect on the susceptibility of RGCs to glaucomatous death. Scale bar, 50 μm.
Other Proapoptotic Molecules Do Not Compensate for BAX Deficiency
In some neuronal cell types, BAX deficiency delays but does not prevent apoptosis . This is because other proapoptotic molecules (e.g., another BCL2 family member, BAK) mediate cell death in the BAX-deficient neurons . To test this possibility in the DBA/2J model, we aged Bax−/− mice to18 mo. As expected in a complex age-related disease, the severity of glaucomatous damage varies between individual DBA/2J eyes at any age. Nevertheless, by 12 mo of age, the majority of eyes have severe optic nerve damage (see below). Therefore, 18 mo of age is 6 mo after the majority of eyes have severe axon loss. Despite this extensive axonal degeneration, there was no obvious reduction in RGC numbers in any of the 18-mo-old Bax−/− eyes (Figure 3E). This result indicates that other molecules do not substitute for BAX and that BAX is essential for RGC apoptosis in DBA/2J inherited glaucoma.
Homozygous BAX Deficiency Alters IOP
DBA/2J mice develop a form of pigmentary glaucoma that is secondary to a progressive iris disease. Iris pigment and cell debris enter the ocular drainage structures, resulting in subsequent IOP elevation [41,42]. The increase in IOP induces RGC death. Manipulations that alleviate the iris disease and prevent IOP elevation also prevent RGC death in this strain . To assess the effects of BAX deficiency, the clinical phenotypes and IOP profiles of mice of each Bax genotype were carefully examined at multiple ages.
Periodic assessment of the progression of iris abnormalities by slit-lamp examinations (approximately every 2 mo between 3 and 12 mo of age) revealed no differences between mice of each Bax genotype. Histologic analysis confirmed these observations (unpublished data). Thus, BAX-mediated processes are not necessary for the progression of the iris disease.
Although iris damage was similar in mice of all three genotypes, Bax genotype did have an effect on IOP. The peak period of IOP elevation in DBA/2J mice is from 9 to 12 mo of age, with the IOP distribution clearly shifting upward between 8 and 9 mo. We monitored IOP at key ages (9, 10.5, and 12 mo). Surprisingly, Bax−/− mice tended to have lower IOP than either Bax+/− or Bax+/+ mice at 9 mo, and the difference was statistically significant at 10.5 mo of age (Figure 4). In contrast, the IOPs of Bax+/− and Bax+/+ mice were not different. Because of the lower IOP in Bax−/− mice, we analyzed IOP in 4-mo-old mice of each genotype and found no differences (average ± SEM, number of eyes examined: Bax+/+, 13.54 ± 0.45 mm Hg, n = 22; Bax+/−, 13.77 ± 0.33, n = 22; Bax−/− 13.46 ± 0.34, n = 20; p > 0.5). This result indicates that BAX deficiency does not alter baseline IOP but does have an effect as the IOP increases to glaucomatous levels in older mice.
To assess the possibility that Bax deficiency may delay axon degeneration by lessening the glaucomatous insult to which RGCs are exposed, we analyzed IOP at key ages of IOP elevation. (A) The average IOP for each genotype (± SEM) and (B) the actual IOP values recorded. Bax deficiency did not prevent IOP elevation. At both 9 mo and 10.5 mo, the average IOP of both Bax+/− and Bax−/− mice was significantly elevated compared to preglaucomatous DBA/2J mice (p < 0.001). The width of the horizontal line (black in [A], gray in [B]) represents the mean IOP ± SEM of a group of wild-type preglaucomatous DBA/2J mice that were 3 mo old. The degree of IOP elevation, however, was altered in Bax−/− mice. In both 9- and 10.5-mo-old Bax−/− mice, the average IOP was less than that of Bax+/+ and Bax+/− mice. This reduction in IOP elevation was significant at 10.5 mo (p < 0.01). The IOP of Bax+/− mice did not differ from wild type at either 9 or 10.5 mo (p > 0.82). By 12 mo, there was no difference in IOP between mice of any genotype (p > 0.25).
The lower IOP insult in Bax−/− mice does not account for the survival of their RGCs. This conclusion is supported by the normal RGC numbers remaining in Bax+/− mice with indistinguishable IOP from Bax+/+ mice. Previous studies have shown that BAX deficiency allows RGC survival following axotomy or optic nerve crush . By contrast, even when neuroprotective treatments are administered, only a small number RGCs survive in the short term (4–6 wk) in Bax+/+ mice exposed to severe axon trauma [51,52]. Thus, there is no reasonable explanation for the finding of prolonged survival of RGCs that have no axons other than that BAX is a necessary RGC-intrinsic molecule for apoptosis in this glaucoma model.
Bax Deficiency Delays Axon Degeneration
Although we have shown that axon degeneration is not dependent upon BAX, our results clearly identify BAX as an endogenous susceptibility factor for both RGC death and axonal degeneration in DBA/2J glaucoma. As discussed above, complete or partial BAX deficiency had a profound rescuing effect on RGC cell bodies. Importantly, decreasing functional Bax gene dosage also decreased susceptibility to glaucoma by delaying the progression of axon damage (Figure 5). At 10.5 mo of age, the majority of Bax+/+ mice had moderate or severe optic nerve damage (see Materials and Methods), with only 20% being mildly affected. In contrast, 53% of Bax−/− and 44% of Bax+/− mice were only mildly affected at 10.5 mo of age. At 12.0 mo of age, the distribution of optic nerve damage was indistinguishable among mice of the three Bax genotypes (Figure 5). Since mice of each Bax genotype were littermates that were housed in the same cages throughout aging, these results provide compelling evidence that decreasing BAX levels delays optic nerve damage.
BAX deficiency did not prevent glaucomatous axon degeneration. Nevertheless, the beneficial effect on RGC survival raised the possibility that it may have a protective effect on the axon and delay optic nerve damage. To assess this, three investigators masked to genotype determined the severity of optic nerve damage for mice of each Bax genotype at each age (n = 49–71 per genotype at each age; see Materials and Methods). At 10.5 mo, both Bax+/− and Bax−/− mice had significantly less optic nerve damage than Bax+/+ mice. Mild damage was evident in 53% of Bax−/− and 44% of Bax+/− optic nerves, compared to only 20% of Bax+/+ (p < 0.001 for both Bax+/− and Bax−/− compared to Bax+/+; Chi2 test). With disease progression to 12 mo of age, the distribution of optic nerve damage became indistinguishable among mice of different Bax genotypes (p > 0.10). Quantitative assessment of a random subset of nerves assigned each damage level (more than eight of each) demonstrates that the number of axons that remain in optic nerves having each damage level are clearly different (see Materials and Methods).
The delay of optic nerve damage in Bax+/− mice (note: Bax+/− mice had similar IOP insults to Bax+/+ mice) suggests that partially decreasing BAX levels in RGCs protects RGC axons. However, since complete BAX deficiency limited IOP elevation, a further protective effect of BAX deficiency by lowering IOP is also possible and may explain the trend toward greater axonal protection in Bax−/− mice. Thus, it is possible that either low-expressing or low-activity alleles of BAX may affect glaucoma susceptibility both by limiting and/or delaying IOP elevation and by directly protecting RGCs from damaging effects of harmfully high IOP.
An Integrated Approach Supports a Role of Direct Optic Nerve Injury in Glaucoma
Comparing the specific pathways active in glaucomatous RGC death to the pathways induced by acute, experimental manipulations can provide information about the initial insult(s) to RGCs in glaucoma. N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxic injury and direct axon injury are two insults that have been proposed to kill RGCs in glaucoma. Acute experimental procedures can be used to mimic these insults. Intraocular NMDA injection is used to mimic excitotoxic RGC insult, and controlled optic nerve crush is used to mimic a direct axon insult [53,54]. To assess the likely roles of these insults in a spontaneous glaucoma, we subjected preglaucomatous DBA/2J mice of differing Bax genotypes to these procedures. This allowed direct comparison of RGC death induced by these distinct excitotoxic and axonal insults to the naturally progressing glaucoma (Figure 6) in a single genetic context. Bax genotype had absolutely no effect on RGC death initiated by intraocular injection of the excitotoxin NMDA. In contrast, the RGCs of both Bax+/− and Bax−/− mice were profoundly protected against optic nerve crush. Since RGC death in the DBA/2J glaucoma is also BAX dependent, these data support a role for axon injury, but not for excitotoxicity (at least through the NMDA receptor) in this glaucomatous RGC death.
To help distinguish between the likely roles of mechanical axon insult and excitotoxicity in cell death induction in spontaneous glaucoma, we subjected preglaucomatous DBA/2J mice of each Bax genotype to either controlled optic nerve crush or NMDA-mediated excitotoxicity. For controlled crush and NMDA, the percent RGC survival in the manipulated eye compared to the contralateral control eye is shown. For ease of comparison, the data for glaucomatous damage are the same as shown in Figure 3. In contrast to the spontaneous glaucoma, NMDA-mediated RGC death is not dependent on BAX, as evident by the complete lack of protection from death in Bax−/− mice. As for the spontaneous glaucoma, RGC death induced by controlled optic nerve crush was completely dependent on BAX and prevented in both Bax+/− and Bax−/− mice. Overall, the effects of BAX in the face of spontaneous glaucoma and controlled crush were remarkably similar.
BAX-Mediated Apoptosis Is Important in an Inherited Glaucoma
Our findings provide important new information about RGC injury and death in glaucoma. BAX deficiency completely prevents RGC death in DBA/2J mice. These results conclusively demonstrate that apoptosis plays a pivotal role in this inherited model of glaucoma. BAX is the first molecule shown to be completely necessary for RGC death in any glaucoma. Considering the protection we demonstrate in this mouse model, it is worth assessing BAX pathways as important targets for new treatments in human glaucoma.
Distinct Pathways Mediate RGC Death and Axonal Degeneration in Glaucoma
Intrinsic axonal degeneration pathways have recently been identified [55,56]. The molecular components of these pathways appear to be distinct from those active in classical somal apoptosis [57,58]. Thus, the different compartments of a neuron can degenerate by different molecular processes. In glaucoma, it is not clear whether the same or different degeneration pathway(s) are activated in the cell body and axon. Our study demonstrates that BAX is required for RGC death but not for RGC axonal degeneration in DBA/2J glaucoma. This indicates that the axonal degeneration pathway is distinct from apoptosis in this inherited glaucoma. Our findings clearly demonstrate that axon degeneration is not a consequence of RGC death, since severe axon degeneration occurred in Bax−/− mice without RGC death. It is not yet clear whether the RGC apoptosis and axonal degeneration pathways have some common features or are completely distinct. However, for the design of therapeutic strategies for human glaucoma, our studies suggest that both apoptotic and axonal degeneration pathways should be considered.
Alternative Glaucoma Hypotheses
The initial RGC compartments that are insulted in glaucoma, as well as the nature of the damaging insults that induce degeneration, are not completely clear. In the excitotoxic hypothesis of glaucoma, elevated IOP leads to elevated intraocular glutamate levels . The elevated glutamate levels are proposed to cause excessive stimulation of glutamate receptors (NMDA type), leading to increased intracellular calcium levels and RGC death. A different glaucoma hypothesis involves direct optic nerve injury. In this hypothesis, high pressure places stress on the optic nerve as the nerve exits the eye through the lamina cribrosa . Important studies report that the first damage to RGCs is evident in the axon segment near the lamina cribrosa in the optic nerve head [61,62], so it was suggested that this is the first site of IOP-induced insult (see Quigley ). Although it definitively shows local axonal dysfunction, the occurrence of initial damage in this region does not conclusively indicate that this is the first or only site of neuronal insult. Because of optic nerve head architecture and the stress at the lamina cribrosa, it is conceivable that the axon segment at the lamina cribrosa may take substantial resources to maintain, especially when IOP is elevated. Somal stress may decrease available resources for axon maintenance and repair. Therefore, somal stress or damage may contribute to the abnormalities observed in the optic nerve head. As a group, Bax+/− mice had an indistinguishable IOP insult compared to Bax+/+ mice, but their RGCs did not undergo pressure-induced cell death. Importantly, RGC axonal degeneration was delayed in these Bax+/− mice. Therefore, our data imply that shielding the RGC cell bodies has a protective effect against axon degeneration.
Direct Optic Nerve Damage Resembles Glaucoma
To provide insight to the nature and location of the damaging insults that occur in glaucoma, we compared the effects of BAX deficiency on RGC death in inherited glaucoma to RGC death induced by either direct optic nerve injury or excitotoxicity (all in the genetically uniform DBA/2J strain). Intraocular NMDA injection was used to model excitotoxic RGC death, and controlled optic nerve crush was used to mimic direct optic nerve damage [53,54]. Unlike the DBA/2J glaucoma, our experiments show that the excitotoxic insult does not require BAX to induce RGC death. Although these experiments cannot rule out the possibility of an intrinsic excitotoxic mechanism, these results do not support a role of NMDA receptor-mediated excitotoxicity as a primary cause of glaucomatous RGC death. Similar to the DBA/2J glaucoma, RGC death following optic nerve crush requires BAX, and both Bax+/− and Bax−/− mice are profoundly protected. Along with our demonstration of an axon intrinsic degeneration pathway, these results further support the hypothesis  that direct optic nerve and axon injury is an important pathogenic component leading to RGC death in glaucoma.
Bax Can Modulate Neuronal Susceptibility in Glaucoma
Individual patients have different levels of susceptibility to glaucomatous RGC death [2,63]. Our experiments clearly identify Bax as an important modulator of neuronal susceptibility in DBA/2J glaucoma. BAX deficiency prevented RGC death and delayed optic nerve degeneration in both Bax+/− and Bax−/− mice. These results suggest that the use of BAX inhibitors could potentially be used to delay glaucomatous vision loss. In situations where BAX is important, pharmacologically suppressing BAX activity may significantly slow the progression of glaucoma. Since RGCs were maintained for an extended period after axon degeneration in Bax−/− mice, treatments that inhibit BAX pathways may allow long-term preservation of RGC cell bodies. Such treatments may allow the RGCs of patients to be stored in their own retinas until future treatment strategies are developed that can stimulate axonal growth and restore vision.
Complete Bax Deficiency Limits IOP Elevation
In addition to implicating BAX as a target for direct neuroprotective treatments, the lower IOP of Bax−/− mice suggests that BAX inhibition may delay or limit IOP elevation. These results suggest that apoptotic death of cells affecting aqueous humor drainage contributes to IOP elevation, at least in secondary glaucomas where the drainage structures are insulted by pigment and cell debris. In a previous study assessing neuroprotection by an apoptosis inhibitor in a rat model of glaucoma, the treated rats had lower IOP than the other group . Although not a conclusion of this rat study, the IOP data support a role for apoptosis in IOP elevation. In humans, cell death has been speculated to contribute to common forms of glaucoma (due to loss of drainage structure cells in old individuals and at late stages of glaucoma [64,65]). However, a primary role for ocular drainage pathway cell death during IOP elevation is not clearly established. Importantly, a recent study convincingly demonstrated endoplasmic reticulum stress and subsequent cell death in primary cultures of drainage pathway cells expressing human glaucoma mutations . Together with our finding that complete BAX deficiency delays IOP elevation in a glaucoma setting, these results strongly support further investigation of apoptotic pathways and effects of antiapoptotic drugs on IOP in human glaucoma.
BAX Is a Candidate Human Glaucoma Susceptibility Gene
The profound protection against RGC death and the delay in axon degeneration in Bax+/− mice together suggest BAX as a candidate human glaucoma susceptibility gene. It is important to note that we considered the possibility that a closely linked gene that was transferred from the 129/SV strain (in which the Bax mutation was generated) hitchhiked into the DBA/2J background along with Bax and explains the protection in heterozygotes. We conclude that this possibility is remote on the basis of the following observations. First, the RGCs of wild-type mice of the parental 129/SV strain are not protected from optic nerve crush. Bax heterozygosity protected the animals from both optic nerve crush and glaucoma in our experiments. This strongly implies that the parental strain does not have a modifier gene that would account for the protection we observed. Second, almost all RGCs were saved in the Bax+/− mice despite complete axon degeneration. To our knowledge, only two genes have been documented that can save the cell when the axon is destroyed. Substantial overexpression of Bcl2 (a BAX antagonist) can do this, as can Bax deficiency. Thus, it is very unlikely that there is a similarly potent gene in the congenic interval, and scanning the flanking chromosome identifies no obvious candidates.
Complete BAX deficiency has developmental consequences  and is unlikely to be common in the human population. However, human BAX alleles that quantitatively affect the level of BAX are identified, and are reported to affect the development and progression of some but not other diseases [67–72]. Other factors that control BAX expression could also be important. Lower levels of BAX are associated with a worse prognosis for some types of cancer . Our findings in Bax+/− mice support the hypothesis that quantitative variation in the level of BAX gene product may alter the prognosis of glaucomatous damage in individuals with high IOP. Although further studies are needed to assess this possibility, quantitative variation of BAX activity among human patients may have a substantial effect on susceptibility and disease progression. It is possible that lower-activity alleles may result in slower or less severe damage, whereas high-activity alleles may be detrimental. Characterization of BAX alleles may have important predictive value for disease progression.
Materials and Methods
Animals and husbandry.
Mice were housed in a 14 h light to 10 h dark cycle under previously described conditions . The Jackson Laboratory (Bar Harbor, Maine, United States) pathogen surveillance program regularly screened for pathogens. All experiments were conducted in accordance with the Association for Research in Vision and Ophthalmology's statement on the use of animals in ophthalmic research and were approved by our institutional animal care and use committees. Both male and female mice were used. For each age group and genotype, approximately equal numbers of males and females were used. A Bax null allele (Baxtm1Sjk ; herein referred to as Bax−) was backcrossed from B6.129X1-Baxtm1SjK (obtained from The Jackson Laboratory) onto DBA/2J for more than 12 generations to generate the congenic strain D2.129X1(B6)-Baxtm1Sjk/Sj. Congenic DBA/2J Bax+/− mice were intercrossed to produce Bax+/+, Bax+/−, and Bax−/− littermates. All three genotypes were housed together and analyzed simultaneously. DBA/2J mice were from our colony (Sj) that was initiated with mice purchased from The Jackson Laboratory. DBA/1J mice were obtained from The Jackson Laboratory.
Cell death related assays.
Eyes from DBA/2J or control DBA/1J mice were fixed in 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.2) for 3 h, transferred to 0.4% paraformaldehyde in 0.1 M phosphate buffer for 48 h, and infiltrated with paraffin. Eyes from two 10- to 11-mo-old DBA/2J mice and two control mice were sectioned at 5 μm thickness and subjected to a modified double labeling protocol that involved in situ end-labeling (equivalent to a TUNEL assay) of fragmented DNA (using BODIPY fluorophores; Molecular Probes, Eugene, Oregon, United States) and detection of condensed chromatin (with the dimeric cyanine dye YOYO-1; Molecular Probes) as published . Samples were analyzed with a confocal microscope. Conventional TUNEL assays were performed as previously reported  and conducted on the following numbers of DBA/2J mice of each age group: 7 mo (six), 8–9 mo (ten), 10–11 mo (16), 12–13 mo (nine), and 15–18 mo (eight). Five 10- to 12-mo-old control DBA/1J mice and more than 15 control mice of mixed genetic background ranging from 10 to 14 mo old were also analyzed. Counts of TUNEL positive cells were done as previously reported . Briefly, the number of TUNEL-positive RGC layer cells was counted for eight to 12 sagittal sections from each eye, and average values for each age group are reported. Eyes used for electron microscopy and histology were processed as previously described , except that tissue blocks were oriented for en face retinal sectioning through the ganglion cell layer.
Clinical examination and intraocular pressure measurement.
DBA/2J mice develop a pigmentary form of glaucoma that follows a characteristic easily detectable clinical course. DBA/2J mice (all genotypes) used in the spontaneous glaucoma experiments were assessed with a slit lamp to ensure that the Bax mutation did not alter the course of the disease. Slit-lamp examination and evaluation criteria (including pigment dispersion and transillumination) were previously described [41,42]. Examination of at least 40 mice of each genotype at 6 and 9 mo of age and at the time of harvest (10.5 or 12 mo) was performed. Additionally, smaller groups of mice (12–20 of each genotype) were analyzed at other ages between 3 and 12 mo of age. IOP was recorded [77,78] for mice of each genotype. The number of mice of each genotype successfully assessed at each age were as follows. For 4 mo, Bax+/+ n = 22, Bax+/− n = 22, Bax−/− n = 20; for 9 mo, Bax+/+ n = 21, Bax+/− n = 25, Bax−/− n = 18; for 10.5 mo, Bax+/+ n = 50, Bax+/− n = 54, Bax−/− n = 52; and for 12 mo, Bax+/+ n = 42, Bax+/− n = 42, Bax−/− n = 37. Student's t-tests were used for statistical comparisons.
Optic nerve damage.
Optic nerves were dissected, processed, embedded in plastic, sectioned and stained with paraphenylenediamine (PPD) as previously described , except that the staining time was increased to 35 min and Embed 812 medium was used. PPD stains all myelin sheaths, but differentially stains the axoplasm of sick or dying axons darkly. Counts of normal-appearing axons were performed using established nonbiased counting methods. Prior to beginning axon counts, the optic nerve was outlined at 100× magnification, and its cross-sectional area was automatically calculated. Magnification of the same nerve section was increased to 1,000×, and a total of 20 fields at 1,000× were electronically collected. The fields were spaced in a regular fashion across the entire nerve, taking care to avoid field overlap so that the same area was not counted twice. The 20 collected pictures were stacked on the computer screen so that only the final picture was visible to the operator. For nerves with a large number of axons (mildly and moderately affected nerves), a rectangular box that contained a minimum of 200 axons was then drawn on the twentieth image. For nerves with severe axon loss, a larger box was drawn so that a significant proportion of the nerve could be counted. The software program then “cut” a rectangle centred at the same location in all 20 images. Since the operator could only see the top image, this removed the possibility of unconscious operator bias and made the selection of axons to be counted random. Axons were counted manually and marked using the computer. The program tracked the total area counted and the total axon count for all 20 images. The total counted area averaged 12.1%, 14.2%, and 20.5% of the total nerve area for mildly, moderately, and severly affected nerves, respectively. The final count was calculated and expressed as number of axons per optic nerve. With this approach, the nerves with 95% or more axon loss were selected for RGC counts by comparing the remaining axon number to the average for unaffected nerves of the same genotype.
Because of the large number of mice (approximately 50–70 mice of each genotype at each age), an optic nerve rating scale was used for the glaucoma progression study (see Figure 5). The indicated damage levels are readily distinguishable upon inspection of the nerve without counting. Nevertheless, axon counts were performed on at least eight randomly selected nerves of each damage grade to provide quantitative information about these distinct stages of disease (see below). Two investigators (masked to genotype, age, and the damage level assigned by the other investigator) assigned a damage level to each nerve. The two investigators assigned the same grade more than 90% of the time (321 out of 355 nerves). For the nerves on which the initial two investigators differed, a third (masked) investigator was utilized. The third investigator's grade always agreed with one of the initial grades, and the most common assigned grade was used. The number of nerves of each genotype assessed at each age were as follows. For 10.5 mo, Bax+/+ n = 49, Bax+/− n = 62, Bax−/− n = 58; for 12 mo, Bax+/+ n = 71, Bax+/− n = 50, Bax−/− n = 65.
The damage levels and typical numbers of normal axons present at each stage (determined through axon counts by an investigator masked to damage grade) follow. The representative axon counts were determined for randomly selected nerves of each grade using the counting procedure described above. In mildly affected nerves, there was very mild or no damage, with healthy axons having a clear axoplasm and intact myelin sheath (average number of axons ± SEM: 50,504 ± 1,988; n = 8). In moderately affected nerves, darkly stained, degenerating axons were readily detectable, but the vast majority of axons appeared completely normal (average number of axons ± SEM: 31,410 ± 2,199; n = 8 ). In severely affected nerves, there was extensive axon damage throughout the optic nerve with obvious axon loss (average number of axons ± SEM: 7,970 ± 2,150; n = 17). The axon number was significantly different between optic nerves of each damage level (p < 0.001 for all comparisons, t-tests).
Ganglion cell death.
Eyes were fixed and retinas were flat-mounted and Nissl-stained with cresyl violet using a modification of the technique reported by Stone . Retinal ganglion cells make up approximately 40%–60% of the neurons in the ganglion cell layer of the mouse retina, and all RGC subtypes cannot be reliably distinguished from the other resident neuron in the ganglion cell layer (the displaced amacrine cell) based on cellular morphology [53,81]. This is especially true during disease, when morphology and marker expression can change dramatically. Consequently, cell loss was measured as a function of the change in total cell number compared to control eyes (strain and genotype matched nonglaucomatous eyes for the spontaneous glaucoma experiments and the contralateral nonmanipulated eye for the controlled crush and excitotoxic experiments). RGC density varies greatly with respect to retinal location. Therefore, two 40× fields were counted in each retinal quadrant and care was taken to ensure that the fields were the same distance from the periphery. For each individual eye, the eight counts for each retina were averaged. To assess RGC survival in the spontaneous glaucoma, retinas from eyes with very severely affected nerves that had fewer than 5% surviving axons were compared to retinas from unaffected eyes without glaucomatous nerve damage. RGC number was counted in approximately eight severely affected eyes and eight unaffected eyes of each genotype, except for unaffected control Bax+/− mice (five eyes) and 18 mo unaffected Bax−/− mice (four eyes).
NMDA injections and controlled optic nerve crush.
These experiments were performed as described previously . For NMDA injections, 2 μl of an 80 mM solution of NMDA in balanced saline solution was injected intravitreally into one eye of each mouse using a glass micropipet. After 4 d the eyes were harvested and cells counted as described above. Data were collected from ten Bax+/+ and eight Bax−/− mice. For optic nerve crush, the nerve of one eye was exposed and clamped approximately 0.5 mm from the globe with self-closing jeweler's forceps for 4 s. Eyes were harvested 21 d after surgery and cells counted. Data were collected from nine Bax+/+, nine Bax+/−, and seven Bax−/− mice. In each paradigm, cell loss was measured relative to the cell number present in the control eye of each mouse examined.
The GenBank (http://www.ncbi.nlm.nih.gov/) accession number for Bax is 12028.
We thank G. Cox, R. Burgess, and Edward Leiter for critical reading of the manuscript, and Amy Snow, Larry Wilson, Adriana Zabaleta, and Mihai Cosma for technical assistance with the experiments. Scientific support services at The Jackson Laboratory are subsidized by a core grant from the National Cancer Institute (CA34196). This work was supported in part by R29EY12223 (RWN) and F32EY014515 (RTL). SWMJ is an Investigator of The Howard Hughes Medical Institute.
RTL, RWN, and SWMJ conceived and designed the experiments. RTL, YL, OVS, JB, RSS, RWN, and SWMJ performed the experiments. RTL, YL, OVS, JB, RSS, RWN, and SWMJ analyzed the data. RTL and SWMJ wrote the paper.
- 1. Quigley HA (1996) Number of people with glaucoma worldwide. Br J Ophthalmol 80: 389–393.
- 2. Leske MC (1983) The epidemiology of open-angle glaucoma: A review. Am J Epidemiol 118: 166–191.
- 3. Kass MA,Heuer DK,Higginbotham EJ,Johnson CA,Keltner JL,et al. (2002) The ocular hypertension treatment study: A randomized trial determines that topical ocular hypotensive medication delays or prevents the onset of primary open-angle glaucoma. Arch Ophthalmol 120: 701–713. Discussion: 829-830.
- 4. The Advanced Glaucoma Intervention Study investigators (2000) The Advanced Glaucoma Intervention Study: 7. The relationship between control of intraocular pressure and visual field deterioration. The AGIS Investigators. Am J Ophthalmol 130: 429–440.
- 5. The Advanced Glaucoma Intervention Study investigators (2002) The Advanced Glaucoma Intervention Study: 11. Risk factors for failure of trabeculectomy and argon laser trabeculoplasty. Am J Ophthalmol 134: 481–498.
- 6. Klein BE,Klein R,Sponsel WE,Franke T,Cantor LB,et al. (1992) Prevalence of glaucoma. The Beaver Dam eye study. Ophthalmology 99: 1499–1504.
- 7. Kamal D,Hitchings R (1998) Normal tension glaucoma—A practical approach. Br J Ophthalmol 82: 835–840.
- 8. Osborne NN,Wood JP,Chidlow G,Bae JH,Melena J,et al. (1999) Ganglion cell death in glaucoma: What do we really know? Br J Ophthalmol 83: 980–986.
- 9. John SW,Anderson MG,Smith RS (1999) Mouse genetics: A tool to help unlock the mechanisms of glaucoma. J Glaucoma 8: 400–412.
- 10. Grozdanic SD,Betts DM,Sakaguchi DS,Allbaugh RA,Kwon YH,et al. (2003) Laser-induced mouse model of chronic ocular hypertension. Invest Ophthalmol Vis Sci 44: 4337–4346.
- 11. Mabuchi F,Aihara M,Mackey MR,Lindsey JD,Weinreb RN (2003) Optic nerve damage in experimental mouse ocular hypertension. Invest Ophthalmol Vis Sci 44: 4321–4330.
- 12. Ueda J,Sawaguchi S,Hanyu T,Yaoeda K,Fukuchi T,et al. (1998) Experimental glaucoma model in the rat induced by laser trabecular photocoagulation after an intracameral injection of India ink. Jpn J Ophthalmol 42: 337–344.
- 13. Li Y,Schlamp CL,Poulsen KP,Nickells RW (2000) Bax-dependent and independent pathways of retinal ganglion cell death induced by different damaging stimuli. Exp Eye Res 71: 209–213.
- 14. Johnson EC,Morrison JC,Farrell S,Deppmeier L,Moore CG,et al. (1996) The effect of chronically elevated intraocular pressure on the rat optic nerve head extracellular matrix. Exp Eye Res 62: 663–674.
- 15. Aihara M,Lindsey JD,Weinreb RN (2003) Experimental mouse ocular hypertension: Establishment of the model. Invest Ophthalmol Vis Sci 44: 4314–4320.
- 16. Levkovitch-Verbin H,Quigley HA,Martin KR,Valenta D,Baumrind LA,et al. (2002) Translimbal laser photocoagulation to the trabecular meshwork as a model of glaucoma in rats. Invest Ophthalmol Vis Sci 43: 402–410.
- 17. Garcia-Valenzuela E,Shareef S,Walsh J,Sharma SC (1995) Programmed cell death of retinal ganglion cells during experimental glaucoma. Exp Eye Res 61: 33–44.
- 18. Isenmann S,Engel S,Gillardon F,Bahr M (1999) Bax antisense oligonucleotides reduce axotomy-induced retinal ganglion cell death in vivo by reduction of Bax protein expression. Cell Death Differ 6: 673–682.
- 19. Quigley HA,Nickells RW,Kerrigan LA,Pease ME,Thibault DJ,et al. (1995) Retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis. Invest Ophthalmol Vis Sci 36: 774–786.
- 20. Nickells RW (1999) Apoptosis of retinal ganglion cells in glaucoma: An update of the molecular pathways involved in cell death. Surv Ophthalmol 43(Supp 1): S151–S161.
- 21. Tatton NA,Tezel G,Insolia SA,Nandor SA,Edward PD,et al. (2001) In situ detection of apoptosis in normal pressure glaucoma. A preliminary examination. Surv Ophthalmol 45(Suppl 3): S268–S272. Discussion: S273-S276 .
- 22. Kerrigan LA,Zack DJ,Quigley HA,Smith SD,Pease ME (1997) TUNEL-positive ganglion cells in human primary open-angle glaucoma. Arch Ophthalmol 115: 1031–1035.
- 23. Kugler S,Straten G,Kreppel F,Isenmann S,Liston P,et al. (2000) The X-linked inhibitor of apoptosis (XIAP) prevents cell death in axotomized CNS neurons in vivo. Cell Death Differ 7: 815–824.
- 24. Straten G,Schmeer C,Kretz A,Gerhardt E,Kugler S,et al. (2002) Potential synergistic protection of retinal ganglion cells from axotomy-induced apoptosis by adenoviral administration of glial cell line-derived neurotrophic factor and X-chromosome-linked inhibitor of apoptosis. Neurobiol Dis 11: 123–133.
- 25. McKinnon SJ,Lehman DM,Tahzib NG,Ransom NL,Reitsamer HA,et al. (2002) Baculoviral IAP repeat-containing-4 protects optic nerve axons in a rat glaucoma model. Mol Ther 5: 780–787.
- 26. Kikuchi M,Tenneti L,Lipton SA (2000) Role of p38 mitogen-activated protein kinase in axotomy-induced apoptosis of rat retinal ganglion cells. J Neurosci 20: 5037–5044.
- 27. Kermer P,Klocker N,Labes M,Thomsen S,Srinivasan A,et al. (1999) Activation of caspase-3 in axotomized rat retinal ganglion cells in vivo. FEBS Lett 453: 361–364.
- 28. Kermer P,Ankerhold R,Klocker N,Krajewski S,Reed JC,et al. (2000) Caspase-9: Involvement in secondary death of axotomized rat retinal ganglion cells in vivo. Brain Res Mol Brain Res 85: 144–150.
- 29. McKinnon SJ,Lehman DM,Kerrigan-Baumrind LA,Merges CA,Pease ME,et al. (2002) Caspase activation and amyloid precursor protein cleavage in rat ocular hypertension. Invest Ophthalmol Vis Sci 43: 1077–1087.
- 30. Hanninen VA,Pantcheva MB,Freeman EE,Poulin NR,Grosskreutz CL (2002) Activation of caspase 9 in a rat model of experimental glaucoma. Curr Eye Res 25: 389–395.
- 31. Tatton WG,Chalmers-Redman RM,Tatton NA (2001) Apoptosis and anti-apoptosis signalling in glaucomatous retinopathy. Eur J Ophthalmol 11(Suppl 2): S12–S22.
- 32. Wakabayashi T,Kosaka J,Hommura S (2002) Up-regulation of Hrk, a regulator of cell death, in retinal ganglion cells of axotomized rat retina. Neurosci Lett 318: 77–80.
- 33. Napankangas U,Lindqvist N,Lindholm D,Hallbook F (2003) Rat retinal ganglion cells upregulate the pro-apoptotic BH3-only protein Bim after optic nerve transection. Brain Res Mol Brain Res 120: 30–37.
- 34. Bonfanti L,Strettoi E,Chierzi S,Cenni MC,Liu XH,et al. (1996) Protection of retinal ganglion cells from natural and axotomy-induced cell death in neonatal transgenic mice overexpressing bcl-2. J Neurosci 16: 4186–4194.
- 35. Tezel G,Yang X,Yang J,Wax MB (2004) Role of tumor necrosis factor receptor-1 in the death of retinal ganglion cells following optic nerve crush injury in mice. Brain Res 996: 202–212.
- 36. Yoshida K,Behrens A,Le-Niculescu H,Wagner EF,Harada T,et al. (2002) Amino-terminal phosphorylation of c-Jun regulates apoptosis in the retinal ganglion cells by optic nerve transection. Invest Ophthalmol Vis Sci 43: 1631–1635.
- 37. Tezel G,Yang X (2004) Caspase-independent component of retinal ganglion cell death, in vitro. Invest Ophthalmol Vis Sci 45: 4049–4059.
- 38. Deckwerth TL,Elliott JL,Knudson CM,Johnson EM Jr,Snider WD,et al. (1996) BAX is required for neuronal death after trophic factor deprivation and during development. Neuron 17: 401–411.
- 39. Mosinger Ogilvie J,Deckwerth TL,Knudson CM,Korsmeyer SJ (1998) Suppression of developmental retinal cell death but not of photoreceptor degeneration in Bax-deficient mice. Invest Ophthalmol Vis Sci 39: 1713–1720.
- 40. Qin Q,Patil K,Sharma SC (2004) The role of Bax-inhibiting peptide in retinal ganglion cell apoptosis after optic nerve transection. Neurosci Lett 372: 17–21.
- 41. Anderson MG,Smith RS,Hawes NL,Zabaleta A,Chang B,et al. (2002) Mutations in genes encoding melanosomal proteins cause pigmentary glaucoma in DBA/2J mice. Nat Genet 30: 81–85.
- 42. John SW,Smith RS,Savinova OV,Hawes NL,Chang B,et al. (1998) Essential iris atrophy, pigment dispersion, and glaucoma in DBA/2J mice. Invest Ophthalmol Vis Sci 39: 951–962.
- 43. Chang B,Smith RS,Hawes NL,Anderson MG,Zabaleta A,et al. (1999) Interacting loci cause severe iris atrophy and glaucoma in DBA/2J mice. Nat Genet 21: 405–409.
- 44. Knudson CM,Tung KS,Tourtellotte WG,Brown GA,Korsmeyer SJ (1995) Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Science 270: 96–99.
- 45. Strom RC,Williams RW (1998) Cell production and cell death in the generation of variation in neuron number. J Neurosci 18: 9948–9953.
- 46. Potts RA,Dreher B,Bennett MR (1982) The loss of ganglion cells in the developing retina of the rat. Brain Res 255: 481–486.
- 47. Young RW (1984) Cell death during differentiation of the retina in the mouse. J Comp Neurol 229: 362–373.
- 48. Burne JF,Staple JK,Raff MC (1996) Glial cells are increased proportionally in transgenic optic nerves with increased numbers of axons. J Neurosci 16: 2064–2073.
- 49. Doughty ML,De Jager PL,Korsmeyer SJ,Heintz N (2000) Neurodegeneration in Lurcher mice occurs via multiple cell death pathways. J Neurosci 20: 3687–3694.
- 50. White FA,Keller-Peck CR,Knudson CM,Korsmeyer SJ,Snider WD (1998) Widespread elimination of naturally occurring neuronal death in Bax-deficient mice. J Neurosci 18: 1428–1439.
- 51. Cheng L,Sapieha P,Kittlerova P,Hauswirth WW,Di Polo A (2002) TrkB gene transfer protects retinal ganglion cells from axotomy-induced death in vivo. J Neurosci 22: 3977–3986.
- 52. Mo X,Yokoyama A,Oshitari T,Negishi H,Dezawa M,et al. (2002) Rescue of axotomized retinal ganglion cells by BDNF gene electroporation in adult rats. Invest Ophthalmol Vis Sci 43: 2401–2405.
- 53. Li Y,Schlamp CL,Nickells RW (1999) Experimental induction of retinal ganglion cell death in adult mice. Invest Ophthalmol Vis Sci 40: 1004–1008.
- 54. Levin LA (2001) Animal and culture models of glaucoma for studying neuroprotection. Eur J Ophthalmol 11(Suppl 2): S23–S29.
- 55. Raff MC,Whitmore AV,Finn JT (2002) Axonal self-destruction and neurodegeneration. Science 296: 868–871.
- 56. Coleman MP,Perry VH (2002) Axon pathology in neurological disease: A neglected therapeutic target. Trends Neurosci 25: 532–537.
- 57. Finn JT,Weil M,Archer F,Siman R,Srinivasan A,et al. (2000) Evidence that Wallerian degeneration and localized axon degeneration induced by local neurotrophin deprivation do not involve caspases. J Neurosci 20: 1333–1341.
- 58. Whitmore AV,Lindsten T,Raff MC,Thompson CB (2003) The proapoptotic proteins Bax and Bak are not involved in Wallerian degeneration. Cell Death Differ 10: 260–261.
- 59. Vorwerk CK,Gorla MS,Dreyer EB (1999) An experimental basis for implicating excitotoxicity in glaucomatous optic neuropathy. Surv Ophthalmol 43(Suppl 1): S142–S150.
- 60. Quigley HA (1999) Neuronal death in glaucoma. Prog Retin Eye Res 18: 39–57.
- 61. Quigley HA,Hohman RM,Addicks EM,Massof RW,Green WR (1983) Morphologic changes in the lamina cribrosa correlated with neural loss in open-angle glaucoma. Am J Ophthalmol 95: 673–691.
- 62. Quigley HA,Addicks EM,Green WR,Maumenee AE (1981) Optic nerve damage in human glaucoma. II. The site of injury and susceptibility to damage. Arch Ophthalmol 99: 635–649.
- 63. Libby RT,Gould DG,Anderson MG,Smith RS,John SWM (2005) Complex genetics of glaucoma susceptibility. Annu Rev Genomics Hum Genet. In press.
- 64. Alvarado J,Murphy C,Polansky J,Juster R (1981) Age-related changes in trabecular meshwork cellularity. Invest Ophthalmol Vis Sci 21: 714–727.
- 65. McMenamin PG,Lee WR,Aitken DA (1986) Age-related changes in the human outflow apparatus. Ophthalmology 93: 194–209.
- 66. Liu Y,Vollrath D (2004) Reversal of mutant myocilin non-secretion and cell killing: Implications for glaucoma. Hum Mol Genet 13: 1193–1204.
- 67. Moshynska O,Moshynskyy I,Misra V,Saxena A (2005) G125A single-nucleotide polymorphism in the human BAX promoter affects gene expression. Oncogene 24: 2042–2049.
- 68. Starczynski J,Pepper C,Pratt G,Hooper L,Thomas A,et al. (2003) The P2X7 receptor gene polymorphism 1513 A→C has no effect on clinical prognostic markers, in vitro sensitivity to fludarabine, Bcl-2 family protein expression or survival in B-cell chronic lymphocytic leukaemia. Br J Haematol 123: 66–71.
- 69. Moshynska O,Sankaran K,Saxena A (2003) Molecular detection of the G(-248)A BAX promoter nucleotide change in B cell chronic lymphocytic leukaemia. Mol Pathol 56: 205–209.
- 70. Zeng SM,Yankowitz J,Widness JA,Strauss RG (2003) Sequence-based polymorphisms in members of the apoptosis Bcl-2 gene family and their association with hematocrit level. J Gend Specif Med 6: 36–42.
- 71. Kuhlmann T,Glas M,zum Bruch C,Mueller W,Weber A,et al. (2002) Investigation of bax, bcl-2, bcl-x and p53 gene polymorphisms in multiple sclerosis. J Neuroimmunol 129: 154–160.
- 72. Saxena A,Moshynska O,Sankaran K,Viswanathan S,Sheridan DP (2002) Association of a novel single nucleotide polymorphism, G(-248)A, in the 5′-UTR of BAX gene in chronic lymphocytic leukemia with disease progression and treatment resistance. Cancer Lett 187: 199–205.
- 73. Sturm I,Kohne CH,Wolff G,Petrowsky H,Hillebrand T,et al. (1999) Analysis of the p53/BAX pathway in colorectal cancer: Low BAX is a negative prognostic factor in patients with resected liver metastases. J Clin Oncol 17: 1364–1374.
- 74. Smith RS,Zabaleta A,Kume T,Savinova OV,Kidson SH,et al. (2000) Haploinsufficiency of the transcription factors FOXC1 and FOXC2 results in aberrant ocular development. Hum Mol Genet 9: 1021–1032.
- 75. Tatton NA,Maclean-Fraser A,Tatton WG,Perl DP,Olanow CW (1998) A fluorescent double-labeling method to detect and confirm apoptotic nuclei in Parkinson's disease. Ann Neurol 44: S142–S148.
- 76. Smith RS,Zabaleta A,John SW,Bechtold LS,Ikeda S,et al. (2002) General and specific histopathology. In: Smith RS , editor. Systemic evaluation of the mouse eye. New York: CRC Press. pp. 265–297. pp.
- 77. Savinova OV,Sugiyama F,Martin JE,Tomarev SI,Paigen BJ,et al. (2001) Intraocular pressure in genetically distinct mice: an update and strain survey. BMC Genet 2: 12.
- 78. John SWM,Hagaman JR,MacTaggart TE,Peng L,Smithes O (1997) Intraocular pressure in inbred mouse strains. Invest Ophthalmol Vis Sci 38: 249–253.
- 79. Anderson MG,Libby RT,Gould DB,Smith RS,John SWM (2005) High-dose radiation with bone marrow transfer prevents neurodegeneration in an inherited glaucoma. Proc Natl Acad Sci U S A 102: 4566–4571.
- 80. Stone J (1981) The wholemount handbook. Sydney: Maitland Publishing. pp. 3–23. pp.
- 81. Jeon CJ,Strettoi E,Masland RH (1998) The major cell populations of the mouse retina. J Neurosci 18: 8936–8946.
- 82. Sun W,Oppenheim RW (2003) Response of motoneurons to neonatal sciatic nerve axotomy in Bax-knockout mice. Mol Cell Neurosci 24: 875–886.