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From the Glaucoma Research Laboratory, Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland.
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Abstract |
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METHODS. Glaucoma was induced in one eye of 35 adult Wistar rats by translimbal diode laser treatment to the trabecular meshwork. Twenty-five more rats underwent unilateral optic nerve transection. Two glutamate transporters, GLAST (EAAT-1) and GLT-1 (EAAT-2), were studied by immunohistochemistry and quantitative Western blot analysis. Treated and control eyes were compared 3 days and 1, 4, and 6 weeks after injury. Optic nerve damage was assessed semiquantitatively in epoxy-embedded optic nerve cross sections.
RESULTS. Trabecular laser treatment resulted in moderate intraocular pressure (IOP) elevation in all animals. After 1 to 6 weeks of experimental glaucoma, all treated eyes had significant optic nerve damage. Glutamate transporter changes were not detected by immunohistochemistry. Western blot analysis demonstrated significantly reduced GLT-1 in glaucomatous eyes compared with control eyes at 3 days (29.3% ± 6.7%, P = 0.01), 1 week (55.5% ± 13.6%, P = 0.02), 4 weeks (27.2% ± 10.1%, P = 0.05), and 6 weeks (38.1% ± 7.9%, P = 0.01; mean reduction ± SEM, paired t-tests, n = 5 animals per group, four duplicate Western blot analyses per eye). The magnitude of the reduction in GLT-1 correlated significantly with mean IOP in the glaucomatous eye (r2 = 0.31, P = 0.01, linear regression). GLAST was significantly reduced (33.8% ± 8.1%, mean ± SEM) after 4 weeks of elevated IOP (P = 0.01, paired t-test, n = 5 animals per group). In contrast to glaucoma, optic nerve transection resulted in an increase in GLT-1 compared with the control eye (P = 0.01, paired t-test, n = 15 animals). There was no significant change in GLAST after transection.
CONCLUSIONS. GLT-1 and GLAST were significantly reduced in an experimental rat glaucoma model, a response that was not found after optic nerve transection. Reductions in GLT-1 and GLAST may increase the potential for glutamate-induced injury to RGC in glaucoma.
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Introduction |
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Glutamate levels may become dangerously elevated in disease states by overloading the normal glutamate reuptake mechanisms. Alternatively, these mechanisms may malfunction and be unable to handle the normal amount of glutamate. Glutamate transporters (GTs) are high-affinity, sodium-dependent transporters responsible for the rapid reuptake of synaptically released glutamate throughout the central nervous system (CNS).12 Five GTs have been identified, and four of these are found in the retina. In both the eye and the brain, glial cells are responsible for the bulk of glutamate reuptake. The major glial GT in the brain is GLT-1 (EAAT-2), whereas in the retina, the major glial GT is GLAST (EAAT-1), which is localized exclusively to Müller cells.13 14 15 GLT-1 is also found in the retina, mainly in cone bipolar cells, particularly near their synapses with RGCs. This suggests that GLT-1 may be particularly essential in regulating the glutamate concentration in the immediate vicinity of RGC dendrites.16
In the brain, GT abnormalities have been implicated in a number of neurologic diseases, including amyotrophic lateral sclerosis (ALS) and some forms of epilepsy.17 In the sporadic form of ALS, 70% of patients have been demonstrated to have a moderate to severe loss of GLT-1 in both motor cortex and spinal cord.18 Patients with ALS have also been shown to have elevated glutamate levels in the cerebrospinal fluid compared with control subjects.19
We wanted to know whether changes in GTs occur in glaucoma. Previous immunohistochemical studies have hinted at a reduction in EAAT-1 (GLAST) in a rat glaucoma model20 and human glaucoma,21 although only three human subjects with glaucoma were studied, and limited clinical information was available on the experimental and control subjects. Vorwerk et al.22 demonstrated that exogenous treatment with subtype-specific antisense oligonucleotides to GLT-1 (EAAC-1) was associated with increased vitreous glutamate and RGC death in rats.22
Our hypotheses were that experimental IOP elevation may be associated with changes in the number and distribution of retinal GT and that GT changes may contribute to RGC death in glaucoma. We also wanted to know whether retinal GT responses to acute, massive RGC death after optic nerve transection differed from those induced by chronic, moderate elevation of IOP. Thus, we studied GLAST and GLT-1 immunohistochemically and by quantitative Western blot analysis in a rat model of glaucoma and after optic nerve transection. We report a reduction in both GLAST and GLT-1 after the induction of glaucoma, which did not occur after optic nerve transection, and provide preliminary data on the time course of these changes.
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Methods |
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Experimental Glaucoma
Elevated intraocular pressure (IOP) was induced in one eye of 35 animals by treating the aqueous outflow area by an external approach with a 532-nm diode laser using a protocol developed by Levkovitch-Verbin et al.23
Briefly, animals were anesthetized with intraperitoneal ketamine (50 mg/kg) and xylazine (5 mg/kg) and topical proparacaine 1% eye drops. Laser energy was delivered to the trabecular meshwork at the slit lamp without the use of additional lenses. The laser beam was directed perpendicular to the trabeculum and parallel to the iris. Initial treatment was 40 to 50 spots of 50-µm size, 0.4-W power and 0.6-second duration. Treatment was repeated at 1 week if the difference in IOP between the two eyes was less than 6 mm Hg. IOP was measured under anesthesia as the average of 10 readings with a tonometer (Tonopen XL; Mentor Ophthalmics, Norwell, MA). IOP measurements were taken immediately before, and 1 day after and 3 days after each treatment and then weekly for the duration of the experiment. Rats were killed at several time points after the first laser treatment. For Western blot studies, the time points were 3 days and 1, 4, and 6 weeks (n = 5 rats in each group). For immunohistochemical analysis, a further 15 rats were studied at 1, 4, and 6 weeks after the first laser treatment (n = 5 rats in each group).
Optic Nerve Transection
Optic nerve transection was performed unilaterally in 25 rats under anesthesia with intraperitoneal ketamine (50 mg/kg) and xylazine (5 mg/kg) and topical proparacaine 1% eye drops. With the use of a binocular operating microscope, the superior conjunctiva was incised, the muscles and connective tissue were separated, and the intraorbital optic nerve was exposed. With a diamond knife, the optic nerve was transected 1 to 2 mm behind the globe, with care taken not to interfere with the blood supply. The eye was dressed with antibiotic ointment. The retinas were examined ophthalmoscopically to assure blood vessel patency. For Western blot analysis, 15 rats were killed 3 days, 1 week, or 6 weeks after transection (n = 5 rats in each group). For immunohistochemical studies, 10 more rats were killed 1 or 6 weeks after transection (n = 5 rats per group).
Immunohistochemistry
Polyclonal rabbit anti-GLT-1 antibodies were kindly donated by Jeffrey Rothstein, The Johns Hopkins University. Polyclonal rabbit anti-GLAST antibodies were obtained from Alpha Diagnostics International (San Antonio, TX). Polyclonal goat anti-vimentin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rats were killed by exsanguination while under deep ketamine/xylazine anesthesia, before intracardiac perfusion with 4% paraformaldehyde in 0.1% phosphate buffer (pH 7.2) at a rate of 20 mL/min for 20 minutes. Both eyes were enucleated, and optic nerves were postfixed in 4% paraformaldehyde until processing for grading of axonal loss. After removal of the anterior segments, eyes were cryopreserved in sucrose and optimal cutting temperature compound (OCT; Sakura Finetek USA. Inc., Torrance, CA). Cryosections 8 to 16 µm thick were collected onto slides (Superfrost Plus; Fisher Scientific; Pittsburgh, PA) and stored at -80°C before immunolabeling by the streptavidin-biotin peroxidase technique of Lutty et al.24
GLT-1 and GLAST antibodies were used at dilutions of 1:100. Negative control experiments included nonimmune serum of the same species as the primary antibody at the same protein concentration and incubation buffer alone. Labeled sections were mounted in Kaiser’s glycerol jelly and viewed by Nomarski optics. Images of all slides were captured digitally with standardized microscope and camera settings (Axioskop and Axiocam with Axiovision ver. 3 software; Carl Zeiss, Inc., Thornwood, NY). Specific transporter labeling of each slide was graded by two experienced, masked observers on a semiquantitative scale at one of five levels by comparison with digital grading standard slides for each GT. Duplicate slide gradings were averaged to obtain a final grade for each eye. The correlation between the gradings of the two observers was strong (r = 0.88 for GLAST and r = 0.90 for GLT-1). The immunohistochemical grades were compared between treated and control eyes by a number of statistical tests including the Wilcoxon rank sum test and Student’s paired t-test.
Western Blot Analysis
The same antibodies to GLAST and GLT-1 as used for immunohistochemistry were used for Western blot analysis of retinas from separate cohorts of animals. Eyes were enucleated with rats under deep ketamine-xylazine anesthesia, anterior segments were removed, and retinal wholemounts were isolated and shock frozen at -80°C within approximately 2 minutes of enucleation. Retinas were later homogenized into 300 µL of a solution containing 20 mM Tris (pH 7.4), 10% sucrose, 1 mM EDTA and protease inhibitors at 4°C. Whole-cell homogenates were prepared ultrasonically, and protein concentration was assayed with a kit (Bio-Rad, Hercules, CA).25
Proteins were separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with 30 µg of protein loaded in each lane and four duplicate lanes per eye, to allow assessment of variability. Proteins were transferred to nitrocellulose membrane. Effective protein transfer was verified by Coomassie protein staining. The membranes were blocked and probed overnight at 4°C with anti-GLAST or anti-GLT-1 antibodies at concentrations of 1:500 and 1:1000, respectively. A peroxidase-conjugated donkey anti-rabbit secondary antibody (Amersham Pharmacia Biotech UK, Ltd., Amersham, UK) was used at a concentration of 1:5000. Immunoblots were visualized by chemiluminescence (ECL Plus; Amersham Pharmacia Biotech UK, Ltd.), with the exposure time to autoradiograph film (X-OMAT AR; Eastman Kodak, Rochester, NY) adjusted to avoid over- or undersaturation. Image-analysis software (OptiQuant version 3.1, Packard Co., Meriden, CT) was used to quantify the intensity of the specific bands. Briefly, the developed photographic films were scanned on a flatbed scanner, using standard settings. The images were converted to gray-scale negatives with image-analysis software (Photoshop ver. 5.0; Adobe, San Diego, CA) and Optiquant was then used to estimate the intensity of each band. After subtraction of background, readings for the four duplicate bands for each eye were averaged. Treated and control eyes were compared by Student’s paired t-test.
Optic Nerve Evaluation
Optic nerve cross sections from 1.5 mm posterior to the globe were postfixed in 1% osmium tetroxide in phosphate buffer. Nerves were embedded in epoxy resin, and 1-µm sections were stained with 1% toluidine blue and examined in a masked fashion by two experienced observers. Neuronal damage was classified semiquantitatively as grade 0 (0%–25% damage), grade 1 (26%–50% damage), grade 2 (51%–75% damage), or grade 3 (76%–100%), according to a previously described and verified method.26
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In epoxy-embedded, toluidine blue-stained optic nerve cross sections, the myelin sheath surrounding normal axons appears as a single, clearly delineated ring. When the optic nerve is damaged by elevated IOP, visible axonal swelling and fragmentation of the myelin ring occur. Axons are lost as pressure-induced degeneration continues. We have found that experienced observers can assess the degree of optic nerve axonal loss in histologic sections and that the grade correlates well with the axon loss calculated by formal counting of remaining normal axons in sampled microscopy fields, using a computer image analysis system (MetaMorph-based; Universal Imaging Corp., West Chester, PA).23
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Discussion |
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The decrease in measured GLT-1 and GLAST in the experimental glaucoma group was of substantial magnitude, from one third to one half in the Western blot analyses. Furthermore, optic nerve transection caused a different reaction. This indicates that the response in experimental glaucoma was specific to some aspect of the injury caused by abnormal IOP in this model and was not simply a generalized reaction to ganglion cell damage. Conceivably, trabecular laser treatment could cause retinal or optic nerve damage independent of, or in addition to, elevated IOP. However, extensive histologic analysis of the retinas of rats with trabecular laser-induced glaucoma has shown no signs of inflammation or damage to cells other than RGCs.23 We have also found RGC loss correlate with IOP but not with the number of laser burns applied, further evidence that RGC death was caused by IOP in our model.
Before concluding that the alterations in GT in the experimental glaucoma setting were important pathogenetically, we should consider carefully the potential meaning of these findings. It is important to realize that our studies immunolocalized and quantified GT protein but did not assess possible changes in the functional capacity of the transporters. Conceivably, there may be sufficient reserve in GT capability to absorb loss of one half of the transporter molecules without excess glutamate exposure. However, it seems possible that the alterations that we measured would be associated with dysfunction.
There are several possible explanations for decreases in measured GT levels. An initial hypothesis may be that elevated IOP directly killed the cells on which GT reside—namely, Müller cells (GLAST) and bipolar cells (GLT-1).15 29 Glaucoma is known to cause primary death of RGCs30 but, there is no evidence of death in other cell types, either in rat chronic glaucoma models23 31 or in human open-angle glaucomatous eyes.26 There is one report of swelling of photoreceptors in experimental glaucoma in monkeys,32 but no alterations in Müller or bipolar cells were shown, and this finding was not observed by others studying similar material.33 We also did not find changes in the distribution or amount of vimentin labeling in our rat glaucomatous tissues, suggesting that Müller cells were, at the least, not substantially reduced in number. In addition, we noted that there was a decline in GT levels that partially recovered. This is more compatible with reversible injury to Müller or bipolar cells than it is with cell death.
A second hypothesis is that cells decrease their GT levels in experimental glaucoma as a physiological response to the altered conditions of the model. As ganglion cells die, extracellular glutamate levels could increase (by release from dying neurons) or decrease (if synaptic activity involving glutamate as a neurotransmitter declined as ganglion cells disappeared). If the amount of GT protein were responsive to the extracellular glutamate concentration, an increase in glutamate might elicit an increase in amount or functional capacity of GT to preserve homeostasis, and vice versa. For example, GLAST mRNA expression increases in the penumbra of focal cerebral infarcts in animal models,34 a situation in which increased glutamate and its toxicity are known to occur. Hence, if the decrease in GT that we observed is an appropriate physiological response, it suggests that glutamate levels were lower, not higher, as sensed by these cells.
Third, the decrease in GT with elevated IOP may reflect a primary injury to Müller and bipolar cells that does not reflect a homeostatic physiological response. Such an injury could be related to ischemia or even perhaps to a direct effect of pressure on retinal cells other than RGCs. In the rat brain, transient cerebral ischemia led to changes in the presence of GLT-1 in astrocytes.35 In some areas of the hippocampus, GLT-1 protein levels decreased significantly, whereas in other areas, there was an increase with reperfusion. In zones with a decrease in GLT-1, the reduction in astrocytic GLT-1 correlated with the loss of pyramidal neurons. Cerebral astrocytes express GLT-1 in vitro only in the presence of neurons.36 37 Thus, the decrease in GLT-1 after ischemic neuronal death could be related to the loss of a neuronal trophic factor that is necessary for GLT-1 expression. If ganglion cells have a similar relationship with Müller glia, this would provide a speculative linkage between ganglion cell death and alteration of GT levels in the retina. Of course, the fact that ischemia—reperfusion can cause either increase or decrease in GT indicates that we cannot attribute any consistent relationship between failure of blood flow and its consequences on the one hand and alteration in GT on the other.
The observation that retinal GT responded differently to elevated IOP and transection emphasizes that not all models of optic nerve injury cause the same disease at the level of the retina, a distinction that should be considered when the results obtained in different animal models are evaluated. Elevated IOP is an intraocular insult to which all cells of the retina and optic nerve head are exposed, whereas optic nerve transection or crush is, by definition, an extraocular injury that can be expected to cause primary damage to RGCs alone. One unifying hypothesis to explain the differences in GT responses we observed would be that transection causes rapid, massive RGC death with consequent acute retinal glutamate release and GT upregulation, whereas IOP elevation may primarily affect GT, with any effect on glutamate levels in the retina or vitreous occurring as a secondary phenomenon. If GT changes in experimental glaucoma are a contributor to, rather than an effect of, RGC death, then GT responses might be expected to occur before significant RGC death. This is indeed what we observed for GLT-1, which decreased by 29% after only 3 days of glaucoma and was maximally reduced by 55% after 1 week. In contrast, RGC loss in our glaucoma model continues to increase for at least the first 9 weeks after induction of elevated IOP.23
By whatever mechanism GT levels decrease in experimental glaucoma, the consequences for the retina are predictable. Glutamate is an important neurotransmitter in the retina, and its levels are regulated by a variety of mechanisms.16 Excess glutamate or stimulation of its membrane-associate receptors causes neuronal injury and death.1 2 3 4 5 6 7 Autoradiography of intact and dissociated rat retinas indicates that glutamate uptake by Müller cells dominates total retinal glutamate uptake, and the only GT present on Müller cells is GLAST.14 15 Furthermore, Müller cells have been shown to protect RGCs in culture from glutamate-induced neurotoxicity.38 A decrease in GLAST after the induction of glaucoma in the rat eye would have the potential to increase the threat of neurotoxicity to RGCs, if it were associated with a concomitant decrease in glutamate clearance. The impact of a decline in neuronal GT (GLT-1) is more difficult to interpret, because it may occupy a lesser role in glutamate homeostasis. Although the bulk of glutamate uptake is by GLAST into Müller cells, some glutamate is presented by Müller cells to neurons expressing GLT-1, EAAC-1, and EAAT-5.16 Therefore, a decrease in GLT-1 may also potentiate glutamate neurotoxicity in the immediate vicinity of RGC dendrites.
The relationship of our findings to human glaucoma are also not simple. The rat model causes loss of ganglion cells by increasing the IOP with laser treatment of the outflow channels. Clearly, this is potentially analogous to injury to human eyes from IOP substantially above the normal range, such as in a minority of persons with primary open-angle glaucoma and in those with secondary glaucoma. In one immunohistochemical study of glaucomatous tissue from three human donors, investigators reported a qualitative decrease in labeled GLAST, but did not detect any difference from control in GLT-1 labeling.21 Their study did not use Western blot analysis or other methods to quantify GT presence or function. The suggestion that there is a linkage between glaucomatous injury and glutamate toxicity was raised by a report that glutamate (alone) among the amino acids was found in higher than normal concentration in the vitreous of humans with various degrees of glaucoma, in the eyes of a small number of monkeys with experimental glaucoma9 and in dogs with a form of primary glaucoma.10 With other optic nerve or retinal injury, there are various outcomes of intravitreal measurement of glutamate levels. With optic nerve crush in rats, an elevation of glutamate occurred in the aqueous humor, but not in the vitreous.39 With ischemic infarction of the rabbit retina, high vitreous glutamate levels were detected.40 In extensive studies of rat eyes with experimental IOP elevation, modest increases in a variety of amino acids were detected, including some increase in glutamate (Levkovitch-Verbin et al., unpublished observations, 2001). However, the increases were not specific for glutamate, they did not reach the magnitude reported in human and monkey eyes, and they were associated with increases in intravitreal protein levels. This suggests that there may have been simply a general breakdown of the blood-retinal barrier, rather than a specific increase in glutamate. The issue of whether intravitreal glutamate levels are an essential element in experimental or human glaucoma injury deserves more careful scrutiny.
However, it is too limited a viewpoint to assume that high levels of glutamate in the vitreous are a necessary condition for excitotoxicity to be involved in glaucomatous neuropathy. The local concentration of glutamate at the membrane receptors of ganglion cells is the important issue for toxicity. This could be very different from the level in samples of vitreous. Vitreous humor must be removed for experimental measurement by a process that inevitably disturbs its state before removal. These manipulations could themselves alter the measured amount of glutamate.
The present study provides evidence for reduction in the levels of the GTs GLAST and GLT-1 in a rat model of experimental glaucoma. Because another experimental optic nerve injury, complete transection, resulted in a different response (no change in GLAST and an increase in GLT-1 expression), the change in experimental glaucoma seems to represent a specific effect of elevated IOP. With simultaneous, massive injury to all RGC axons by transection, there was an increase in one GT. As discussed, this may indicate a homeostatic response that attempts to mitigate a putative increase in extracellular glutamate concentration. The findings in the glaucoma model, which clearly caused major RGC loss, point to a failure to generate this GT response. This failure could have a potentially detrimental influence on the ability of the retina to maintain appropriate glutamate levels.
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Acknowledgements |
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Footnotes |
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Submitted for publication November 6, 2001; revised February 12, 2002; accepted March 12, 2002.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Harry A. Quigley, Wilmer 122, Wilmer Eye Institute, Johns Hopkins Hospital, 600 North Wolfe Street, Baltimore, MD 21287; hquigley{at}jhmi.edu.
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