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(Investigative Ophthalmology and Visual Science. 2004;45:4576-4582.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-0166
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Susceptibilities to and Mechanisms of Excitotoxic Cell Death of Adult Mouse Inner Retinal Neurons in Dissociated Culture

Xianmin Luo,1 Akemichi Baba,2 Toshio Matsuda,2 and Carmelo Romano1,3

1From the Departments of Ophthalmology and Visual Sciences and 3Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri; and the 2Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To explore the susceptibilities of adult retinal neurons in dissociated culture to treatments with excitotoxic agonists and the mechanisms of the resultant retinal cell death.

METHODS. C57B6 mice were used. Retinas were removed, dissociated, plated on a polylysine/laminin substrate, and maintained in vitro for 5 to 7 days. Excitotoxic agonists (glutamate, N-methyl-D-aspartate [NMDA], or kainic acid [KA]) were added for 30 minutes or 24 hours, sometimes in the presence of modified extracellular ion concentrations or potential blocking agents. The next day, cells were fixed and immunocytochemically stained to identify ganglion and amacrine cells. Surviving cells were counted.

RESULTS. Ganglion cells from adult mouse retinas were much less susceptible to excitotoxic death than those prepared from neonatal retinas. Adult amacrine cells were killed by KA, NMDA, or glutamate. Experiments with selective blockers demonstrated that KA killed through AMPA ({alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, whereas NMDA and glutamate exerted toxicity through a combination of AMPA and NMDA receptors. The KA-induced death of amacrine cells was not mediated by chloride ions. Removal of extracellular sodium, however, completely prevented the amacrine cell death, and removal of extracellular calcium prevented approximately 70% of the death. The path of calcium entry was investigated. Experiments with selective blockers indicated that the lethal calcium entry was via reverse operation of a sodium-calcium exchanger.

CONCLUSIONS. There is a profound developmental regulation in the sensitivity of retina ganglion cells to excitotoxic insults. Excessive intracellular sodium and calcium are the proximal causes of amacrine cell death. The pathologic calcium entry is dependent on the sodium overload, which then drives a sodium–calcium exchanger to take up calcium.

Excitotoxicity, that is, pathologic overactivation of excitatory glutamate receptors leading to neuronal death, is believed to play a role in many neurologic and neurodegenerative diseases, such as cerebral ischemia,1 trauma,2 Parkinson’s disease,3 and Huntington’s disease.4 A potential role for excitotoxicity has also been suspected in retinal diseases, such as retinal ischemia and glaucoma.5 There is little direct evidence for this. However, several lines of evidence suggest that excitotoxicity may be an important factor in retinal neuronal death in these conditions. Glutamatergic agonists delivered to retinal ganglion cells, either in vivo6 7 or in vitro,8 9 are toxic to ganglion cells. The ganglion cell death that results from optic nerve crush can be partially prevented by administration of an N-methyl-D-aspartate (NMDA) receptor antagonist.10 Elevated levels of glutamate have been measured in the vitreous of patients with glaucoma and from animals subjected to experimental ocular hypertension,11 12 but these data have not been confirmed.13 14 15

A difficulty with in vitro studies of the retina is that they have almost always been conducted in retinal cells obtained from embryonic or early postnatal animals. Glaucoma, except in rare cases, is an adult-onset disease. There are profound differences between developing and mature neurons. The job of immature neurons is to find their place (migration), find their partners (process extension and pathfinding), and initiate communication (synapse formation). Adult neurons, though retaining some plasticity, mostly process information. The cellular physiology subsuming these different functions is mediated by developmentally regulated expression of different genes. Therefore, to understand the mechanisms of glaucoma, we must study adult retina, ganglion cells in particular. Toward this end, we have been studying adult mammalian retinal neurons in dissociated cell culture, using the technique described by Luo et al.16 We have chosen to work in the mouse, as this is the mammalian species of choice for genetic manipulation.

In this report, we describe the establishment of dissociated cultures of adult retinal neurons from mice, demonstrate the insensitivity of adult ganglion cells to excitotoxic insults, and provide information relevant to the mechanism of the excitotoxic death of adult amacrine systems.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Tissue Collection and Cell Culture
All animal experimentation adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The tissue collection and culture method closely followed that described in our prior study.16 Adult (>6 weeks) or immature (postnatal day 7) C57B6 mice (Charles River, Wilmington, MA) were anesthetized with pentobarbital sodium and decapitated and the eyes enucleated. After the cornea and anterior structures were removed, the retinas were removed and placed in CO2-independent medium (catalog 18045-058; Invitrogen-Gibco, Grand Island, NY) on ice and minced with fine scissors. They were then washed in Ringer’s solution lacking Ca2+ and Mg2+ supplemented with EDTA (0.1 mM) and incubated in 0.5 mL of 0.2% papain for 20 minutes at 37°C. The papain was preactivated by adding 1 µL papain suspension (Worthington Biochemicals, Lakewood, NJ) to 24 µL buffer containing (in mM) 1.1 EDTA, 0.067 ß-mercaptoethanol, and 5.5 cysteine and incubating for 10 minutes at 37°C. Individual cells were dissociated by gentle trituration and washing in NeuroBasal A medium (Invitrogen-Gibco) supplemented with 2% B27 additives and 2% fetal bovine serum. Cells were seeded into 16-well microscope slide culture chambers at 200,000 per well. The glass surface had been coated with poly-L-lysine (2 µg/cm2, 2 hours) followed by laminin (1 µg/cm2, overnight). Cells were maintained in an atmosphere of 95% air and 5% CO2 at 37°C.

Treatment with Agonists
After 5 to 7 days in culture, the excitatory amino acid agonist, glutamate (10 µM–1 mM), kainic acid (KA; 10 µM–1 mM), or NMDA (32–320 µM) was added at the indicated concentrations. The drug was present for 30 minutes (after which time the cells were washed twice with fresh media), or for 24 hours. In some experiments, potential antagonists or modified extracellular ion concentrations were added and removed at the same time as the agonists.

Immunocytochemistry
At the end of the experimental procedures, cells were washed twice in phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 15 minutes. After two PBS washes, cells were permeabilized with 0.1% Triton X-100 in PBS for 5 minutes, then incubated with PBS containing 0.5% bovine serum albumin and 0.1% Tween-20 for 15 minutes. Primary antibody in the same buffer was then added and permitted to incubate for 2 hours at room temperature or at 4°C overnight. After several rinses with PBS, fluorescently labeled secondary antibodies were added in the same buffer as used for the primary antibody and incubated for an hour. For double labeling, the cultures were again rinsed in PBS several times, and another round of primary and secondary antibody incubations performed exactly as the first.

To identify ganglion cells, we stained cultures with an antibody against the light chain of neurofilament (anti-NF-70; Chemicon, Temecula, CA). To identify amacrine cells, an antibody against PGP 9.5 (ubiquitin hydroxylase; Chemicon), which stains both amacrine cells and ganglion cells, was used. Those cells positive for PGP and negative for NF70 cells were counted as amacrine cells. All the ganglion cells in a well were counted (usually 100–300), and the amacrine cells in 24 40x fields were counted. No cells showing pathologic nuclear staining were counted (as revealed by 4',6'-diamino-2-phenylindole [DAPI] staining). Statistical analysis (ANOVA and Bonferroni-corrected t-tests) was conducted on computer (Prism; GraphPad, San Diego, CA; or Excel; Microsoft, Redmond, WA).


   Results
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 Materials and Methods
 Results
Discussion
 References
 
Adult mouse retinal neurons were cultured successfully, with only minor modifications made to the the methods described for pig and rat retinas previously.16 17 NeuroBasal A (Invitrogen-Gibco) supplemented with 2% B27 additives18 (a mix of hormones, trace minerals, and antioxidants) gave the best survival of the several media tested. Virtually all classes of neural retinal cells were present in the cultures (not illustrated), including horizontal cells (labeled with anti-calbindin), bipolar cells (labeled with anti-PKC{alpha}), rods (labeled with anti-opsin), cones (labeled with anti-arrestin), astrocytes (labeled with anti-glial fibrillary protein [GFAP]), and Müller cells (labeled with anti-vimentin), but this report is concerned only with ganglion and amacrine cells. The survival of ganglion cells in particular was increased when fetal bovine serum (2%) was present instead of or in addition to the B27 additives. All experiments were performed on cells maintained in NeuroBasal A (Invitrogen-Gibco) containing both B27 and serum.

Experiments were performed on cultures maintained in vitro for 5 to 7 days. This duration permitted recovery from dissociation-induced mechanical/proteolytic damage, process outgrowth, and expression of distinct morphologic phenotypes.

Figure 1 illustrates staining for ganglion cells (NF-70-positive, green) and amacrine cells (PGP 9.5-positive, red; but NF-70 negative) in a 7-day-old culture. The ganglion cells had long, smooth, relatively unbranched processes, presumably axons, whereas the amacrine cells had relatively uneven processes with numerous branches sprouting along their lengths.



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  		FIGURE 1. Adult mouse ganglion and amacrine cells in dissociated culture for 7 days. Left: ganglion cells (green) labeled with an antibody to NF-70; Middle: amacrine and ganglion cells (red) labeled with antibody to PGP 9.5. Right: merged images.

 
To ascertain whether neonatal and adult ganglion cells differ in sensitivity to excitotoxic insults, we treated both types of cultures with excitotoxic agonists (Fig. 2) . Agonists were added for 30 minutes and washed away, and the cells were maintained for 24 hours, or the cells were maintained in agonist for 24 hours. Cells were then fixed and stained to demarcate the NF-70+ ganglion cells, which were counted. There was a remarkable difference, with neonatal cells subject to much more cell death than adult cells. Indeed, the agonists were nearly ineffective on adult ganglion cells.



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  		FIGURE 2. Adult and neonatal ganglion cells in dissociated culture had different sensitivities to excitotoxins. Cells were maintained in vitro for 7 days, and then treated with the excitotoxic agonists glutamate (1 mM), NMDA (1 mM), or KA (320 µM) for 30 minutes. The agonists were washed off and fresh media added. At 24 hours, the cells were fixed, stained, and counted. Counts normalized to vehicle control. Data are expressed as the mean ± SEM of results in three experiments, with duplicate cultures per experiment. *P < 0.05 different from control.

 
When the exposure times to agonists were increased to 24 hours, only KA provided significant killing of ganglion cells (Fig. 3A) . The excitotoxins were, however, toxic to adult amacrine cells (Fig. 3B) . KA was most effective, killing 75% and 85% of cells with 30-minute and 24-hour exposures, respectively. Both glutamate and NMDA were effective toxins for adult amacrine cells also, but required 24 hours’ exposure to kill most of the cells. Concentration–response curves are presented in Figure 4 . The excitotoxic cell loss caused by all agonists was dose dependent at the expected concentrations.



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  		FIGURE 3. Adult ganglion (A) and amacrine (B) cells in dissociated culture had different sensitivities to excitotoxins. Cells were maintained in vitro for 7 days and then treated with the excitotoxic agonists glutamate (1 mM), NMDA (1 mM), or KA (320 µM) for 30 minutes or 24 hours. For the 30-minute treatment, the agonists were washed off and fresh media added. At 24 hours, the cells were fixed, stained, and counted. Counts normalized to vehicle control. Data are expressed at the mean ± SEM of results in three experiments, with duplicate cultures per experiment. *P < 0.05 different from control.

 


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  		FIGURE 4. Concentration–response curves for effects of agonists on adult ganglion and amacrine cells. Agonists at the indicated concentrations were present for 30 minutes or 24 hours.

 
The ability of glutamate receptor antagonists to block KA-induced amacrine cell death is shown in Figure 5 . All the cell loss, after either 30 minutes or 24 hours of KA exposure, was mediated through AMPA ({alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, as it is wholly blocked by the selective noncompetitive AMPA receptor blocker GYKI53655. MK-801, the NMDA receptor blocker, was not able to block KA toxicity after 30 minutes or 24 hours. NMDA or the endogenous agonist glutamate were also lethal to dissociated adult retinal amacrine cells. This toxicity was mediated by a combination of NMDA and non-NMDA receptor activation, since full protection required a combination of subtype-selective antagonists.



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  		FIGURE 5. Antagonists of excitatory amino acids receptors block toxicity of agonists to amacrine cells. Concentrations of agonists are as in Figures 2 and 3 . Antagonists were GYKI 53655 (GYKI), 100 µM, and MK-801 (MK), 10 µM, present alone or in combination during the agonist incubation. Asterisks omitted for clarity. Effects were significant at P < 0.05.

 
The ability of KA and glutamate to kill amacrine cells rapidly is a positive control for the experiment with ganglion cells, and indicates that the sensitivity to excitotoxicity is a cell-type specific property, not some physical or other generic problem in the cultures.

What is the mechanism of excitotoxic amacrine cell death? We explored the ion sensitivity of this process since several distinct Na+- , Cl–-, and Ca2+-dependent mechanisms have been described (Fig. 6) . KA was added to cultures in solutions lacking one or more of the ions. Omission of any of the ions in the absence of agonist was without effect on amacrine cell survival. Chloride omission did not afford any protection, indicating that the process is distinct from the Cl- and inhibitory-transmitter–dependent cell death that we have described in chick retina and rat cerebellar granule cells.19 20 However omission of Ca2+ gave substantial protection and Na+ omission near complete protection.



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  		FIGURE 6. Ion substitution experiments for KA toxicity to amacrine cells. Cells were treated with KA at 32 µM for 30 minutes, in the presence of media that lacked sodium (equimolar choline replacement), chloride (methylsulfate replacement), or calcium (magnesium replacement). *Significantly different from the control; **significantly different from KA.

 
We explored the path for lethal Ca2+ entry. There are three possibilities: (1) It may be directly regulated through Ca2+-permeable glutamate receptors (i.e., AMPA receptors lacking the GluR2 subunit); (2) KA-induced depolarization may open voltage-gated calcium channels (VGCCs) to a pathologic extent; or (3) KA may cause directly extensive sodium loading because of the uncontrolled opening of the glutamate receptors, and the cell may extrude the ion via reverse-mode operation of the Na-Ca exchanger, leading to excessive Ca2+ entry. To determine which hypothesis is correct, we used pharmacological blockers of these three processes: N-acetylspermine (NAS) to block the Ca-permeable AMPA channels,21 Cd2+ to block VGCCs, and KB-R7943 to prevent operation of the Na-Ca exchanger.22 The results were clear: Only blockade of the Na-Ca exchanger provided protection (Fig. 7) . The extent of protection was similar to that obtained when Ca2+ was omitted from the extracellular medium. This also is consistent with protection via omission of Na+: Without the Na+ overload, there would be no lethal Ca2+ entry.



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  		FIGURE 7. Calcium antagonists. Cells were incubated with KA (32 µM) in the presence or absence of cadmium chloride (Cd, 30 µM), naphthyl-acetyl spermine (NAS, 10 µM), or KB-R7943 (KB, 10 µM). Only KB provided protection.

 
To explore further the hypothesis that the reverse mode of the Na-Ca exchanger is responsible for the Ca2+-dependent component of KA-induced death of amacrine cells, two additional experiments were performed. We tested lower concentrations of KB-R9743, since, at 10 µM, both forward and reverse modes would be inhibited.23 Significant protection was observed at 1 and 3 µM. We also tested another Na-Ca exchange inhibitor of a different chemical class, SEA0400.24 SEA0400 provided significant protection at 1 and 3 µM.


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Our previous work in embryonic chick retina indicated that the mechanisms of excitotoxic neuronal death in that tissue were independent of calcium and instead were dependent most critically on chloride.19 This does not appear to be the case in adult mammalian amacrine cells. Based on our pharmacological experiments, we propose that Na+ overload is the initial step in lethality, followed by excess Ca2+ entry due to Na+ extrusion/Ca2+ influx through reverse operation of the Na+-Ca2+ exchanger.

There are K+-independent and -dependent Na-Ca exchangers: the NCX and NCKX families, respectively.25 Because the extent of protection provided by KB-R7943, a selective inhibitor of the reverse (Ca2+ efflux) mode of NCX, was comparable to that caused by removal of extracellular Ca2+, it seems likely that NCKX is not involved. A Na-Ca exchanger has been demonstrated to be present in amacrine cell dendrites, where it is critically important in removing calcium after spiking and thereby shaping amacrine cell responses.26 27

Ca2+ entry via reverse operation of the exchanger after a sodium load is an important mechanism of disease after ischemia. This has been shown in both the spinal cord,28 29 and the cerebrum,24 30 31 in vivo and in vitro, as well as in non-neural tissues, such as the heart32 33 34 and the kidney.35 36

Three different Na-Ca exchangers have been cloned, NCX1, NCX2, and NCX3.37 38 39 Localization studies in the retina have not been performed, and so we cannot identify with certainty which one is responsible for mediating the lethal Ca2+ entry into amacrine cells. However, indirect evidence points to NCX2. First, NCX2 is the predominant form in adult rodent brain, with mRNA expressed at a level an order of magnitude higher than NCX1 or NCX340 41 (although all isoforms are present and differentially distributed in the brain42 ). Second, the pharmacology suggests NCX2. KB-R9743 has a similar, or slightly higher affinity for the NCX2 exchanger than the NCX1 exchanger expressed in BHK cells,43 44 whereas SEA0400 has a much higher affinity for NCX1 than KB-R7943 does.45 Because both SEA0400 and KB-R7943 are effective, but SEA0400 is not more potent than KB-R7943 (Figs. 8 9) , this is consistent with NCX2’s mediating lethal Ca2+ entry in adult mouse retinal amacrine cells after addition of KA.



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  		FIGURE 8. Concentration response of neuroprotective effect of KB-R9743 (KB) against KA (KA). Cells were incubated with KA (32 µM) in the presence or absence of cadmium chloride KB-R7943 at the indicated concentrations (in micromolar). Significant protection (*P < 0.02) was observed at three concentrations.

 


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  		FIGURE 9. Concentration response of neuroprotective effect of SEA0400 against KA. Cells were incubated as in Figure 8 , but with or without cadmium chloride SEA0400. Significant protection (*P < 0.02) was observed at two concentrations.

 
Adult mouse ganglion cells were insensitive to excitotoxic-agonist–induced death. This is an example of how cellular physiology and pathologic susceptibilities are developmentally regulated, and why neonatal cell culture is a poorly suited model for investigating the underlying bases of adult disease.

This insensitivity of adult mouse ganglion cells to excitotoxic insults is perhaps surprising but is consistent with results in adult pig cultures.16 Intravitreal injection of glutamate agonists has been described to kill at least some ganglion cells in chicks46 and rodents.6 7 47 48 We cannot exclude the possibility that this insensitivity is an artifact of the dissociation. Indeed, we have observed (Luo X, Romano C, unpublished results, 2003) that the transcription factor brn3a, present in the nuclei of most ganglion cells in vivo,49 is gradually lost from NF-positive ganglion cells in culture. An analogous loss of receptors, exchangers, or other critical molecules responsible for the in vivo sensitivity of ganglion cells to excitotoxins may occur and thereby alter the sensitivity of the cells to glutamatergic agents.

However, there is reason to believe that this developmental difference is not merely due to dissociation and culture. First, we observed this difference between identically prepared cultures from neonatal and adult retinas. If the observed difference were an artifact, this artifactual process would itself have to be subject to developmental regulation. Second, our findings are consistent with the results using intact explant cultures obtained by Izumi et al.50 In this study, rat retinas of various ages were acutely explanted and exposed to NMDA, and toxic effects were noted histologically. Little or no toxicity was seen in the youngest retinas examined (postnatal day 0), but the toxic effect increased with development, peaking near day 9. NMDA-induced damage and then its level declined, and adult retinas showed almost no response to NMDA. These data, taken along with ours, indicates that there is a profound developmental regulation of the direct toxic action of glutamate receptor overactivation on ganglion cells.

What then is the explanation for the effectiveness of excitotoxins in vivo? Perhaps the exposure time is longer than 24 hours in these cases. Alternatively, the agonists may be having an indirect action, requiring other ocular structures to participate in the toxic mechanism, structures not present in either explant or dissociated retina cultures. The cellular and molecular differences that account for the difference between adult and immature neurons and between amacrine and ganglion cells therefore remain unknown and are topics for future investigations.


   Footnotes
 
Supported in part by grants from The Glaucoma Foundation and Research to Prevent Blindness, Inc., and the RPB Lew Wasserman Merit Award (CR).

Submitted for publication February 17, 2004; revised August 9, 2004; accepted August 16, 2004.

Disclosure: X. Luo, None; A. Baba, None; T. Matsuda, None; C. Romano, None

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: Carmelo Romano, 660 South Euclid Avenue, Campus Box 8096, St. Louis, MO 63110; romano{at}vision.wustl.edu.


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 Results
 Discussion
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