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(Investigative Ophthalmology and Visual Science. 2007;48:4785-4794.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.07-0343
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Prevention of Excitotoxicity in Primary Retinal Ganglion Cells by (+)-Pentazocine, a Sigma Receptor-1–Specific Ligand

Ying Dun,1 Muthusamy Thangaraju,2 Puttur Prasad,2 Vadivel Ganapathy,2 and Sylvia B. Smith1,3

1From the Departments of Cellular Biology and Anatomy, 2Biochemistry and Molecular Biology, and 3Ophthalmology, Medical College of Georgia, Augusta, Georgia.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Sigma receptors ({sigma}Rs) are nonopioid, nonphencyclidine binding sites with robust neuroprotective properties. Previously, the authors induced death in the RGC-5 cell line using very high concentrations (1 mM) of the excitatory amino acids glutamate (Glu) and homocysteine (Hcy) and demonstrated that the {sigma}R1 ligand (+)-pentazocine ((+)-PTZ) could protect against cell death. The purpose of the present study was to establish a physiologically relevant paradigm for testing the neuroprotective effect of (+)-PTZ in retinal ganglion cells (RGCs).

METHODS. Primary ganglion cells (GCs) were isolated by immunopanning from retinas of 1-day-old mice, maintained in culture for 3 days, and exposed to 10, 20, 25, or 50 µM Glu or 10, 25, 50, or 100 µM Hcy for 6 or 18 hours in the presence or absence of (+)-PTZ (0.5, 1, 3 µM). Cell viability was measured using the viability and apoptosis detection fluorescein in situ assays. Expression of {sigma}R1 was assessed by immunocytochemistry, RT-PCR, and Western blotting. Morphologic appearance of live ganglion cells and their processes was examined over time (0, 3, 6, 18 hours) by differential interference contrast (DIC) microscopy after exposure to excitotoxins in the presence or absence of (+)-PTZ.

RESULTS. Primary GCs showed robust {sigma}R1 expression. The cells were exquisitely sensitive to Glu or Hcy toxicity (6-hour treatment with 25 or 50 µM Glu or 50 or 100 µM Hcy induced marked cell death). Primary GCs pretreated for 1 hour with (+)-PTZ followed by 18-hour cotreatment with 25 µM Glu and (+)-PTZ showed a marked decrease in cell death: 25 µM Glu alone, 50%; 25 µM Glu/0.5 µM (+)-PTZ, 38%; 25 µM Glu/1 µM (+)-PTZ, 20%; 25 µM Glu/3 µM (+)-PTZ, 18%. Similar results were obtained with Hcy. {sigma}R1 mRNA and protein levels did not change in the presence of the excitotoxins. DIC examination of cells exposed to excitotoxins revealed substantial disruption of neuronal processes; cotreatment with (+)-PTZ revealed marked preservation of these processes. The stereoselective effect of (+)-PTZ for {sigma}R1 was established in experiments in which (–)-PTZ, the levo-isomer form of pentazocine, had no neuroprotective effect on excitotoxin-induced ganglion cell death.

CONCLUSIONS. Primary GCs express {sigma}R1; their marked sensitivity to Glu and Hcy toxicity mimics the sensitivity observed in vivo, making them a highly relevant model for testing neuroprotection. Pretreatment of cells with 1 to 3 µM (+)-PTZ, but not (–)-PTZ, affords significant protection against Glu- and Hcy-induced cell death. {sigma}R1 ligands may be useful therapeutic agents in retinal diseases in which ganglion cells die.

Sigma receptors ({sigma}Rs) represent unique nonopiate, nonphencyclidine binding sites in mammalian brain and peripheral organs, distinct from other known receptors.1 To date, two types of sigma receptors, distinguishable by biochemical and pharmacologic means, have been identified.2 The cDNA encoding type 1 sigma receptor ({sigma}R1), the better characterized of the two subtypes, was cloned initially from guinea pig3 and subsequently from human, mouse, and rat.4 5 6 7 The {sigma}R1 cDNA predicts a protein of 223 amino acids (Mr 25–28 kDa).3 Ligands for {sigma}R1 demonstrate robust neuroprotective properties, particularly against excitotoxic insults, including decreased neuronal responsivity to N-methyl-D-aspartate receptor stimulation,8 9 attenuation of postsynaptic glutamate (Glu)-evoked calcium influx,10 11 inhibition of ischemia-induced Glu release,12 13 and reduced NO production.9 14

{sigma}R1 expression has been demonstrated in ocular tissues including lacrimal gland,15 iris–ciliary body,16 17 lens,17 18 and retina.17 19 Using molecular and biochemical methods, we have studied {sigma}R1 in mouse retina.17 20 21 RT-PCR analysis amplified {sigma}R1 in neural retina and the RPE–choroid complex. In situ hybridization studies revealed abundant expression of {sigma}R1 in the ganglion cell layer, inner nuclear layer, inner segments of photoreceptor cells, and RPE cells. Immunohistochemical analysis confirmed these observations. Recent studies using primary cultures of mouse Müller cells localized {sigma}R1 to the endoplasmic reticulum and nuclear membranes.21 These cells and other retinal cell types demonstrate robust {sigma}R1 binding activity with an apparent Kd value of approximately 25 nM.21

Additional studies from our laboratory have focused on retinal ganglion cells, the second-order neurons of the visual system. Ganglion cells die in several retinal diseases, including glaucoma and diabetic retinopathy.22 23 Our earlier work showed that {sigma}R1 continues to be expressed in neural retina under hyperglycemic conditions and during diabetic retinopathy,20 making it a promising target for neuroprotection against cell death in these diseases. To test the neuroprotective properties of {sigma}R1 ligands in ganglion cells, we first used the rat ganglion cell line RGC-5 to determine whether (+)-pentazocine ((+)-PTZ), a highly selective benzomorphan-based {sigma}R ligand,24 can block RGC-5 cell death induced by excitotoxins such as Glu.25 The results were promising. Concentrations of Glu required to induce death in this cell line, however, were extremely high (millimolar range), despite the fact that in vivo ganglion cells are sensitive to micromolar (greater than 15 µM) concentrations of Glu.26 In addition, RGC-5 cells, like many transformed cell lines, replicate in culture, which is not a characteristic feature of neurons in vivo. Finally, the RGC-5 cell line does not form extensive processes characteristic of neurons, making it impossible to analyze effects of excitotoxins on neuronal processes. For these reasons, it was essential to determine whether our earlier promising results of neuroprotective effects of (+)-PTZ observed in the RGC-5 cell line could be replicated in a physiologically relevant system. Recently, we established the culture of primary ganglion cells from the mouse retina. Using the immunopanning procedures initially described for rats, we optimized the isolation and purification of these cells from neonatal mouse retina.27 The cells are positive for neuronal markers and for Thy1, which is considered a specific marker of retinal ganglion cells. In the present study, we found that the primary ganglion cells, like ganglion cells in the intact retina, form extensive neuronal processes and are exquisitely sensitive to glutamate; hence, the model is highly relevant to studies of neuroprotection.

In addition to assessing the efficacy of {sigma}R1 ligands in preventing Glu-induced ganglion cell death, we wanted to explore more fully the toxic effects of homocysteine (Hcy) on primary ganglion cells and to determine whether deleterious effects of exposure to Hcy were reversible using (+)-PTZ. Hcy, a nonprotein sulfur amino acid, is a metabolite of the essential amino acid methionine. Modest plasma elevation of Hcy is a risk factor in cardiovascular and neurodegenerative diseases.28 29 30 Less is known about the effects of Hcy on retinal function, though several clinical studies implicate Hcy in maculopathy, retinal vein occlusion, open-angle glaucoma, pseudoexfoliation glaucoma, and diabetic retinopathy.31 32 33 34 35 36 37 38 39 A recent report of a child with severe hyperhomocysteinemia caused by methionine synthase deficiency demonstrated decreased rod response and RGC loss as analyzed by ERG and visual evoked potential.40 We have attempted to understand the consequences of elevated levels of Hcy on retinal function using in vitro and in vivo models. In a study using mice, we injected micromolar concentrations of Hcy intravitreally and observed apoptotic RGC death,26 thus providing the first report of Hcy-induced retinal ganglion cell death in vivo. The present study explored the sensitivity of primary ganglion cells to micromolar concentrations of Hcy and analyzed the role of {sigma}R1 in preventing this excitotoxin-induced cell death. The data showed that primary ganglion cells are exquisitely sensitive to the toxic effects of Glu and Hcy; (+)-PTZ can prevent that cell death.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Reagents used were as follows: medium and reagent (Neurobasal/B27 medium; TRIzol reagent; Gibco-Life Technologies, Rockville, MD); trypsin inhibitor (Roche Applied Science, Indianapolis, IN); papain (Worthington Biochemical, Lakewood, NJ); brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and basic fibroblast growth factor (bFGF; PeproTech, Rocky Hill, NJ); goat anti-rabbit IgG and goat anti-rat IgG (both H+L; Affinipure; Jackson ImmunoResearch Laboratories, West Grove, PA); goat anti-mouse IgG-horseradish peroxidase (HRP), goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA); enhanced chemiluminescence (ECL) detection kit (Pierce Biotechnology, Rockford, IL); RNA PCR kit (GeneAmp; Applied Biosystems, Branchburg, NJ); Taq PCR kit (TaKaRa; Takara Bio Inc., Otsu, Shiga, Japan); sense and antisense primers for {sigma}R1 (Integrated DNA Technologies, Coralville, IA); viability assay (Live/Dead Assay; Molecular Probes, Eugene, OR); apoptosis detection kit (ApopTag Fluorescein In Situ Apoptosis Detection Kit; Chemicon International, Temecula, CA); D,L-homocysteine thiolactone hydrochloride (MP Biomedical, Inc., Solon, OH); L-glutamate, (+)-pentazocine, (–)-pentazocine, monoclonal antibodies to ß-actin and neurofilament 160 (NF-160), and all other chemicals (Sigma-Aldrich Chemical, St. Louis, MO).

Isolation and Culture of Primary Ganglion Cells
Primary ganglion cells (GCs) were isolated from the retinas of 1- to 2-day-old C57BL/6J mice that were the offspring of breeding pairs purchased from Harlan Sprague–Dawley Inc. (Indianapolis, IN). Care and use of the mice adhered to the principles set forth in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Immunopanning procedures and verification of purity of the cells have been described in detail.27 Cells were cultured on poly-D lysine and laminin-coated coverslips in medium (Neurobasal; Gibco-Life Technologies) containing 5 µg/mL insulin, 1 mM sodium pyruvate, 0.1 mg/mL transferrin, 60 ng/mL progesterone, 16 µg/mL putrescine, 40 ng/mL sodium selenite, 40 ng/mL triiodothyronine, 1 mM L-glutamine, 60 µg/mL N-acetyl cysteine, 2% B27, 50 ng/mL BDNF, 10 ng/mL CNTF, 10 ng/mL forskolin, 10 ng/mL bFGF, and 0.1 mg/mL BSA.

Immunocytochemical Analysis of {sigma}R1
To establish the presence of {sigma}R1 in primary GCs, cells were cultured on coverslips, fixed in methanol, and incubated for 3 hours at room temperature with antibody against {sigma}R125 or antibody against NF 160, a known neuronal cell marker. Proteins were detected using fluorescent dye (Alexa Fluor-488; Invitrogen, Carlsbad, CA)-conjugated anti-rabbit IgG for {sigma}R1 and fluorescent dye (Alexa Fluor-488; Invitrogen)-conjugated anti-mouse IgG for NF 160. Samples were washed with PBS, and slides were covered with mounting medium (Vectashield; Vector Laboratories, Burlingame, CA) containing 4,6-diamidino-2-phenylindole (DAPI) to stain nuclei. {sigma}R1 and NF-160 were detected by epifluorescence using a microscope (Axioplan 2; Carl Zeiss, Thornwood, NY) equipped with viewer software (AxioVision; Carl Zeiss) and an HRM camera.

Sensitivity of Primary GCs to Excitotoxic Amino Acids and Treatment with (+)-Pentazocine
To determine the concentration of the excitotoxin that induced 50% cell death, primary GCs, cultured on coverslips in 24-well plates, were exposed to varying concentrations of Glu (10, 20, 25, or 50 µM) and subjected to viability assay (Live/Dead Assay) that uses calcein-acetoxymethyl ester to detect living cells and ethidium homodimer-1 to detect dead cells, according to the manufacturer’s instructions. Cells were viewed by epifluorescence using different filters to detect cells with green fluorescence (live) or red fluorescence (dead). Cell viability was confirmed using the apoptosis detection (ApopTag; Chemicon) kit according to the directions of the manufacturer. The kit is based on the detection of DNA strand breaks through terminal dUTP nick-end labeling (TUNEL) analysis. In experiments testing the effects of Hcy (10, 25, 50, 100 µM) on GC viability, TUNEL analysis was used.

The effects of exposure of excitotoxin-treated cells to (+)-PTZ were assessed by treating cells with 25 µM Glu or 50 µM Hcy in the presence of varying concentrations of (+)-PTZ (0.5, 1, or 3 µM). Cell viability was assessed using the apoptosis detection (ApopTag; Chemicon) kit. Treatment paradigms included coculture of cells with excitotoxin and (+)-PTZ for 6 hours or pretreatment of cells for 1 hour with (+)-PTZ, followed by coculture with the excitotoxin and (+)-PTZ for 6 or 18 hours. Cells were examined by epifluorescence using standard fluorescein excitation and emission filters, and data were captured for image analysis. Each field was examined systematically for the presence of green fluorescence, indicative of apoptosis, and data were expressed per total number of cells in the field. In these experiments, at least three coverslips were prepared. Per coverslip, images were captured from at least four fields with the use of a software system (AxioVision; Carl Zeiss). Data were analyzed by one-way analysis of variance using a statistical package (SPSS version 15.0; SAS Institute, Cary, NC). Least significant difference (LSD) was the post hoc test. P < 0.05 was considered significant. Additional experiments were performed in which primary GCs were pretreated for 1 hour with (–)-PTZ (the levo-isomer of pentazocine) or (+)-PTZ (the dextro-isomer of pentazocine) and were then coincubated with 25 µM Glu for 18 hours. In further experiments, live ganglion cells were subjected to differential interference contrast (DIC) microscopy using an inverted microscope (Eclipse TE300; Nikon, Tokyo, Japan). Cell bodies and fibers radiating from them were photographed using a camera at 0, 3, 6, and 18 hours (CoolSnap; Photometrics, Tucson, AZ) after treatment with Glu or Hcy in the presence or absence of (+)-PTZ.

Semiquantitative and Real-Time RT-PCR Analysis of {sigma}R1 mRNA
To determine whether {sigma}R1 gene expression was altered in primary GCs treated with excitotoxins in the presence or absence of (+)-PTZ, total RNA was prepared from the cells using reagent (TRIzol; Gibco-Life Technologies). RT-PCR was carried out using primer pairs specific for mouse {sigma}R1.41 Sense primer (5'-TAT CGC AGT GCT GAT CCA-3') and antisense primer (5'-TAC TCC ACC ATC CAC GTG TT-3') corresponded to nucleotide positions 75 to 92 and 520 to 539, respectively, in the cloned mouse {sigma}R1 cDNA (GenBank accession no. AF030198). The expected PCR product size was 465 bp; 18S RNA was the internal standard. RT-PCR was performed in 35 cycles, with a denaturing phase of 1 minute at 94°C, annealing phase of 1 minute at 60°C, and an extension of 2 minutes at 75°C. Twenty microliters of the PCR products were gel electrophoresed and stained with ethidium bromide. Signals were quantified using a phosphorimaging system (Storm; Amersham Biosciences, Uppsala, Sweden), as described.25

For real-time RT-PCR, total RNA was extracted using reagent (TRIzol; Gibco-Life Technologies) and was quantified. RNA (5 µg) was reverse transcribed using the RNA PCR Kit (GeneAmp; Applied Biosystems). cDNAs were amplified for 45 cycles (iCycler; Bio-Rad, Hercules, CA) using microliter reaction (iQ SYBR Green Supermix; Bio-Rad Laboratories) and sequence-specific primers for mouse {sigma}R1 (sense primer, 5'-CTC GCT GTC TGA GTA CGT G-3'; antisense primer, 5'-AAG AAA GTG TCG GCT AGT GCA A-3'). The internal reference was hypoxanthine phosphoribosyltransferase 1 (HPRT1), for which the primers were sense primer (5'-GCG TCG TGA TTA GCG ATG ATG AAC-3') and antisense primer (5'-CCT CCC ATC TCC TTC ATG ACA TCT-3'). Expression of {sigma}R1 relative to HPRT1 was calculated by comparison of Ct values ({delta}-delta Ct).

Immunoblot Analysis of {sigma}R1
Western blotting of {sigma}R1 protein in primary retinal ganglion cells followed our published method.21 After protein samples were subjected to SDS-PAGE and transferred to nitrocellulose membranes, the membranes were incubated with anti-{sigma}R1 antibody (1:500) followed by incubation with HRP-conjugated goat anti-rabbit IgG antibody (1:3000). Proteins were visualized using the ECL Western blot detection system. Membranes were reprobed with mouse monoclonal anti-ß-actin antibody (1:5000) as a loading control. The films were analyzed using a digital imaging system (AlphaIMager 2200; Genetic Technologies, Miami, FL) as described.20


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
{sigma}R1 has been detected in intact retinal tissue including cells of the GC layer.17 To determine whether the primary GCs used in this study expressed {sigma}R1, we performed immunocytochemistry and immunoblotting. The primary GCs developed extensive processes and variably sized cell bodies (Fig. 1A) , which are characteristic of GCs in vivo. They are positive for the neuronal cell marker NF-160 (Fig. 1B) and the ganglion cell-specific marker Thy-1.27 {sigma}R1 is detected in primary GCs by immunocytochemistry (Fig. 1C) and by immunoblotting (Fig. 1D) . Thus, the cells are a useful and relevant model for studies of the protective role of {sigma}R1 ligands.


Figure 1
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  		FIGURE 1. Detection of {sigma}R1 in primary GCs isolated from mouse retina. (A) Phase-contrast images of mouse primary GCs after 3 days in culture. The cells have long processes extending from the variably sized cell bodies that are characteristic of GCs. (B) Immunolabeling of primary GCs with an antibody against NF-160, a neuronal marker detected with fluorescent dye (green); nuclei stained with DAPI (blue). (C) Immunolabeling of primary GCs with an affinity-purified polyclonal antibody against {sigma}R1, detected with fluorescent dye (green); nuclei stained with DAPI (blue). (D) Two separate preparations (left and right lanes) of primary GCs used for immunoblotting with an affinity-purified antibody against {sigma}R1 (Mr approximately 27 kDa) and an antibody against ß-actin (Mr approximately 45 kDa, internal loading control). Magnification bar, 15 µm.

 
In vivo, the intracellular concentration of Glu in neurons can be as high as 10 mM; however, the extracellular concentration of Glu must be maintained in the micromolar range to avoid toxicity.42 To determine whether the primary GCs used in this study exhibited similar sensitivity to extracellular Glu as they did in vivo, the cells were cultured in supplemented medium (Neurobasal; Gibco-Life Technologies) for 3 days and then 6 hours in medium containing increasing concentrations of Glu. Cell viability was assessed using a viability assay (Live/Dead Assay; Molecular Probes; Fig. 2A ); living cells were green and dead/dying cells were red. Cells incubated in medium with a final concentration of 10 µM Glu had excellent viability, but in the presence of 25 µM or 50 µM Glu, cell viability decreased by approximately 50% and approximately 80%, respectively (Fig. 2B) . Subsequent experiments used 25 µM Glu to induce cell death in the primary GCs. With the use of the viability assay (Live/Dead Assay; Molecular Probes), the number of living cells was determined in primary GCs exposed to 25 µM Glu alone or in primary GCs pretreated for 1 hour with varying concentrations of (+)-PTZ and then coincubated for 16 hours with (+)-PTZ and 25 µM Glu. Pretreatment of cells with 0.5, 1, or 3 µM (+)-PTZ resulted in markedly fewer dead cells than in cells exposed to Glu in the absence of (+)-PTZ (Fig. 2C) .


Figure 2
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  		FIGURE 2. Neuroprotective effect of (+)-PTZ on Glu-induced primary GC death. (A) Photomicrographs of the viability assay in primary GCs exposed 6 hours to medium with no additional Glu (control) or to medium containing 10 µM, 25 µM, and 50 µM Glu. Living cells fluoresce green; dead cells fluoresce red. (B) Quantitation of live primary GCs after exposure to Glu (10, 25, and 50 µM) for 6 hours. *Significantly different from control and 10 µM Glu; P < 0.001. (C) Primary GCs were pretreated with (+)-PTZ (0.5, 1, 3 µM) for 1 hour and then were coexposed to 25 µM Glu and (+)-PTZ (0.5, 1, 3 µM) for an additional 16 hours. Cells were subjected to the viability assay to determine viability. **Significantly different from control cells [not treated with Glu] and from cells exposed to Glu and (+)-PTZ; P < 0.001. (B, C) Data are expressed as the mean ± SE of the ratio of living cells to the total number of cells; data were normalized to the control value, which was considered 100% (n = 10).

 
To confirm and extend the findings described in Figure 2 , a second cell viability measure, the TUNEL assay, which detects DNA strand breaks in cells and is an indicator of apoptosis, was used. We sought to determine whether pretreating the cells for 1 hour with varying concentrations of (+)-PTZ followed by a 6-hour cotreatment with 25 µM Glu and (+)-PTZ would protect the cells against death. Figure 3 shows that control cells and cells incubated with 0.01% DMSO (vehicle control) had approximately 10% TUNEL-positive cells. Exposure of cells for 6 hours to 25 µM Glu led to approximately 50% TUNEL-positive cells, consistent with data obtained using the viability assay (Live/Dead assay; Molecular Probes; Fig. 2B ). Pretreatment of primary GCs with (+)-PTZ and followed by 6-hour cotreatment with Glu and (+)-PTZ reduced the number of TUNEL-positive cells slightly, but significantly, compared with cells treated with Glu alone. We next sought to determine whether a longer cotreatment time period with Glu and (+)-PTZ would alter the incidence of cell death (Fig. 4) . Cells were pretreated with (+)-PTZ (0.5, 1, or 3 µM) for 1 hour, followed by 18-hour coincubation with these concentrations of (+)-PTZ and 25 µM Glu. Figure 4A shows representative photomicrographs of the TUNEL assay when cells received no treatment or were treated with vehicle (0.01% DMSO), Glu (25 µM), or Glu plus (+)-PTZ (0.5, 1, or 3 µM). Green fluorescence reflected cells undergoing apoptosis. Cells treated with increasing dosages of (+)-PTZ showed significantly fewer TUNEL-positive cells. These results were quantified (Fig. 4B) , and the data showed that the longer cotreatment time (18 hours) led to a highly significant decrease in the number of TUNEL-positive cells.


Figure 3
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  		FIGURE 3. Assessment of the number of TUNEL-positive primary GCs after 6-hour incubation with Glu in the absence or presence of (+)-pentazocine ((+)-PTZ). Primary GCs were pretreated for 1 hour with or without (+)-PTZ (0.5 or 1 µM), followed by coincubation for 6 hours with Glu (25 µM) and (+)-PTZ (0.5 or 1 µM); the number of TUNEL-positive cells was determined using the apoptosis detection fluorescein method. Data are expressed as the mean ± SE of the ratio of dead/dying (TUNEL-positive) cells to the total number of cells (n = 10). *Significantly different from untreated control or vehicle (DMSO) control at P < 0.001. **Significantly different from treatment with 25 µM Glu alone (without (–)-(+)-PTZ).

 

Figure 4
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  		FIGURE 4. Assessment of the number of TUNEL-positive primary GCs when pretreated with (+)-PTZ and then coincubated for 18 hours with Glu and (+)-PTZ. (A) Representative photomicrographs showing results of the TUNEL assay. Cells that fluoresce green are TUNEL positive, indicative of apoptosis. All samples were labeled also with DAPI (blue) to stain nuclei. For each pair of photomicrographs, the left image shows the merged (DAPI plus TUNEL staining), and the right image shows only the TUNEL staining. Top: representative images of cells that received no Glu exposure (control, DMSO vehicle control). Middle: marked increase in TUNEL-positive cells after 18-hour exposure to 25 µM glutamate. Remaining three panels show fewer TUNEL-positive cells when pretreated and coincubated with (+)-PTZ (0.5, 1, or 3 µM). (B) Quantification of the data from TUNEL analysis shown in (A). The number of TUNEL-positive cells was determined per 100 cells counted. Data are expressed as the mean and SE of the ratio of dead/dying cells to the total number of cells (n = 10). *Significantly different from control; P < 0.001. **Significantly different from treatment with 25 µM Glu alone (without (–)-(+)-PTZ).

 
To determine whether the protective effects of (+)-PTZ against Glu-induced cell death were associated with alterations in {sigma}R1 gene expression, primary GCs were cultured in the presence of 25 µM Glu for 6 or 18 hours in the presence or absence of 3 µM (+)-PTZ (1-hour pretreatment plus cotreatment). Semiquantitative RT-PCR was performed with RNA isolated from control and treated cells. 18S mRNA was analyzed as an internal control. Data showing RT-PCR amplification of the two bands (465 and 315 bp) representing {sigma}R1 and 18S, respectively, are presented in Figure 5A (6-hour cotreatment) and 5B (18-hour cotreatment). Quantitation of the band densities showed that there were negligible differences in the expression of {sigma}R1 in primary GCs, whether they were exposed to 25 µM Glu for 6 or 18 hours in the presence or absence of (+)-PTZ (data not shown). Although the {sigma}R1 mRNA levels were similar in primary GCs exposed to Glu in the presence or absence of (+)-PTZ, it was not certain that protein levels were comparable. To investigate this, primary GCs were cultured in medium containing 25 µM Glu for 6 or 18 hours in the presence or absence of 3 µM (+)-PTZ (pretreatment 1 hour, plus cotreatment), and immunoblotting was performed. Immunoblotting experiments, using an antibody generated against {sigma}R1,20 detected a band of the appropriate molecularweight (Mr approximately 26–27 kDa) in control cells and those treated with 25 µM Glu in the presence or absence of (+)-PTZ and in cells treated with (+)-PTZ alone (Figs. 5C 5D) . Blots were stripped and reprobed with ß-actin (Mr approximately 45 kDa) as an internal control. Quantification of the band intensities revealed no significant differences in {sigma}R1 protein levels between Glu-treated cells and controls or those treated with (+)-PTZ (data not shown).


Figure 5
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  		FIGURE 5. {sigma}R1 mRNA and protein levels in primary GC after treatment with Glu and (+)-PTZ. Primary GCs were incubated for 6 hours (A, C) or 18 hours (B, D) in the absence or presence of 25 µM Glu and in the absence or presence of 3 µM (+)-PTZ. (A, B) Total RNA was isolated and used for semiquantitative RT-PCR. Primer pairs specific for mouse {sigma}R1 mRNA (465 bp) were used. 18S RNA (315 bp) was analyzed in the same RNA samples as the internal control. RT-PCR products were run on a gel and stained with ethidium bromide. (C, D) Proteins were extracted from cells and subjected to SDS-PAGE, followed by immunoblotting using an affinity-purified antibody against {sigma}R1 (Mr approximately 27 kDa) or ß-actin (Mr approximately 45 kDa; internal loading control). M, DNA marker; Con, control; Glu, 25 µM glutamate-treated cells; P + G, (+)-PTZ 3 µM plus 25 µM glutamate; (+)-PTZ, 3 µM (+)-PTZ incubation alone.

 
In addition to analyzing the effects of Glu on primary GCs, we examined the effects of increasing concentrations of Hcy on primary GC survival and investigated whether (+)-PTZ could alter these effects. Figure 6 shows the dose-response data for primary GCs cultured in the supplemented medium (Neurobasal) for 3 days and then incubated for 6 hours in medium containing increasing concentrations of Hcy (10, 25, 50, and 100 µM). Although earlier studies with the RGC-5 cell line required millimolar concentrations of Hcy to induce cell death,25 the present experiments using the primary GCs showed that exposure of cells to 10 or 25 µM Hcy led to a slight elevation (approximately 20%) in the number of TUNEL-positive cells compared with untreated controls. When the cells were exposed to 50 µM or 100 µM Hcy, 60% and 80% of the cells were TUNEL positive, respectively. This marked sensitivity of the primary GCs is in keeping with the evidence that micromolar quantities of Hcy can induce apoptotic ganglion cell death in vivo26 and supports the relevance of the primary GCs in studying Hcy-induced toxicity. Based on these data, subsequent experiments used 50 µM Hcy to induce cell death.


Figure 6
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  		FIGURE 6. Dose-dependent increase in TUNEL-positive primary GCs after exposure to Hcy. The number of TUNEL-positive cells was determined using the apoptosis detection fluorescein method in primary GCs exposed to Hcy (10, 25, 50, and 100 µM) for 6 hours. Data are expressed as the mean ± SE of the ratio of dead/dying cells to the total number of cells (n = 10). *Significantly different from control; P < 0.001.

 
The efficacy of (+)-PTZ as a neuroprotectant in the Hcy-induced primary GC death was tested (Fig. 7) . When cells received 1-hour pretreatment with (+)-PTZ, followed by 6-hour cotreatment with 50 µM Hcy and 0.5 or 1 µM (+)-PTZ, there was a slight decrease in the number of TUNEL-positive cells, but the difference did not reach statistical significance (Fig. 7A) . However, pretreatment/cotreatment of cells for 6 hours with 3 µM (+)-PTZ led to a significant decrease in the number of dying cells (Fig. 7A) . When cells were pretreated 1 hour with (+)-PTZ followed by 18-hour cotreatment with 50 µM Hcy in the presence of (+)-PTZ, there was a dose-dependent decrease in the incidence of cell death (Fig. 7B) . Primary GCs treated with 1 and 3 µM (+)-PTZ showed a highly significant decrease in the number of TUNEL-positive cells (28% and 25%, respectively, compared with 66% cell death in the Hcy-treated cells). Thus, (+)-PTZ treatment afforded marked protection against neuronal cell death induced by this excitotoxin and Glu.


Figure 7
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  		FIGURE 7. Assessment of the number of TUNEL-positive primary GCs after 6- or 18-hour incubation with Hcy and (+)-PTZ. Primary GCs were pretreated for 1 hour with (+)-PTZ (0.5, 1, or 3 µM) followed by coincubation for 6 hours (A) or 18 hours (B) with Hcy (50 µM) and (+)-PTZ (0.5, 1, or 3 µM). The number of TUNEL-positive cells was determined using the apoptosis detection fluorescein method. Data are expressed as the mean ± SE of the ratio of dead/dying cells to the total number of cells (n = 10). *Significantly different from control; P < 0.001. **Significantly different from cells treated with 50 µM Hcy; P < 0.001.

 
As in studies analyzing the effects of excess Glu on {sigma}R1 expression (Fig. 5) , we examined {sigma}R1 expression after Hcy treatment of cells. RT-PCR showed no difference in the expression of {sigma}R1 mRNA levels, regardless of whether cells were exposed to Hcy for 6 or 18 hours or whether they received (+)-PTZ treatment before Hcy exposure (data not shown). These data were confirmed by real-time RT-PCR. Similarly, there was no difference in {sigma}R1 protein levels after the same duration of Hcy exposure in the presence or absence of (+)-PTZ, as examined by Western blot. The data suggest that the neuroprotective effects of (+)-PTZ involved {sigma}R1 activation rather than altered {sigma}R1 expression.

An advantage of using primary GCs is that the cells develop extensive processes reminiscent of neurons in vivo. This feature can be monitored, and the consequence of exposure to excitotoxins can be evaluated. With the use of DIC microscopy, primary GCs were examined over a period of 18 hours after exposure to 25 µM Glu or 50 µM Hcy (in the absence or presence of (+)-PTZ), and images were captured. A panel of representative photomicrographs is provided in Figure 8 . Control cells, which received no excitotoxin treatment, had plump cell bodies typical of healthy neurons that measured approximately 7 to 8 µm in diameter. Intact processes radiated from many cell bodies. Typically, three to five of these processes per cell body extended approximately 10 to 15 µm before they branched into more complex networks. Figure 8 (top row) shows control cells viewed over the 18-hour time period. When cells were treated with Glu, there was progressive loss of the processes. Minimal disruption was evident 3 hours after treatment; however, by 6 hours, the cell bodies were shrunken, the processes were shortened, and the complex extensions from the processes were no longer present. By 18 hours, cells treated with Glu were markedly altered in appearance compared with nontreated controls. A few cells retained short processes, but many had no processes at all. Cell bodies were often shriveled and had condensed nuclei. Similarly, when cells were treated with Hcy, disruption of cell fibers was clear, especially after 6-hour treatment; the processes were stubby and appeared clipped. After 18-hour incubation with Hcy, the fibers were either no longer present or had contracted significantly, forming a small network around the cell body. Interestingly, cells pretreated with (+)-PTZ for 1 hour and then cotreated with Glu or Hcy showed marked preservation of neuronal processes. Cell bodies were similar to the nontreated control cells, and the fibers emanated in complex arrays. These morphologic studies supported the TUNEL assays regarding the effects of Glu or Hcy on cell viability. They also demonstrated the profound neuroprotective effects of (+)-PTZ on the cells.


Figure 8
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  		FIGURE 8. Differential interference contrast microscopic analysis of cells exposed to Glu or Hcy and (+)-PTZ. Primary GCs were isolated and cultured as described. Control cells (Con) were not exposed to excitotoxins; the row of images labeled Glu shows cells that were incubated with 25 µM Glu over a period of 18 hours; photomicrographs were acquired at 0, 3, 6, and 18 hours after incubation. The row of images labeled Hcy shows cells that were incubated with 50 µM Hcy over an 18-hour period and photographed at 0, 3, 6, and 18 hours after incubation. In additional experiments, cells were pretreated with (+)-PTZ for 1 hour and then coincubated with (+)-PTZ and the excitotoxin for 18 hours. Cell bodies and processes of cells cotreated with Glu or Hcy and (+)-PTZ were similar in appearance to control cells. Top left: control, 0 time. Arrow points to a process extending from the cell body. Magnification bar, 15 µm (all photomicrographs are the same magnification).

 
We chose (+)-PTZ because of its potency and its high selectivity as a ligand for {sigma}R1.24 43 Compared with (+)-PTZ, (–)-PTZ has several orders of magnitude less affinity toward {sigma}R1. To provide further support that the observed protective effects of (+)-PTZ in primary GCs against excitotoxicity are mediated by {sigma}R1, we compared the efficacy of neuroprotection of (+)-PTZ to (–)-PTZ. Primary GCs were pretreated with (+)-PTZ or (–)-PTZ for 1 hour, followed by coincubation with these compounds in the presence of 25 µM Glu. After 18 hours, the cells were subjected to TUNEL assay, and the number of TUNEL-positive cells was determined. As expected, treatment of cells with (+)-PTZ decreased the incidence of TUNEL-positive cells significantly; treatment of cells with (–)-PTZ had no effect on the incidence of apoptosis induced by Glu (Fig. 9) . The data demonstrate the selective role of {sigma}R1 activation in the observed protective effects.


Figure 9
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  		FIGURE 9. Assessment of the number of TUNEL-positive primary GCs pretreated with either (+)-PTZ or (–)-PTZ followed by 18-hour coincubation with Glu. Primary GCs were incubated for 1 hour with (+)-PTZ or (–)-PTZ (0.5, 1 µM) followed by coincubation with PTZ and 25 µM Glu for 18 hours. TUNEL-positive cells were determined using the apoptosis detection kit. The number of TUNEL-positive cells was determined per 100 cells counted. Data are expressed as the mean ± SE of the ratio of dead/dying cells to the total number of cells (n = 10). *Significantly different from treatment with 25 µM Glu alone; P < 0.001.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The purpose of this study was to determine whether ligands for {sigma}R1 afforded neuroprotection in an excitotoxic model of retinal neuronal death. Earlier studies from our laboratory suggested that the {sigma}R1 ligand (+)-PTZ could prevent cell death induced by Glu and Hcy in the RGC-5 (ganglion) cell line. The concentration of the excitotoxins required to induce death in this cell line was high (millimolar range). Although it was considered a first step in assessing neuroprotection by the {sigma}R1 ligand, it was not likely to be physiologically relevant because considerably lower concentrations of Glu and Hcy induced ganglion cell apoptosis in vivo.26 Using methods adapted from rats, we optimized the isolation and maintenance of ganglion cells from neonatal mice27 and used these cells for the present study. The cells are notable for their expression of neuronal and ganglion cell-specific markers and their extensive processes characteristic of ganglion cells.

Using primary GCs, we first established that {sigma}R1 was present in these cells,as it was in the intact mouse retina. Our immunocytochemical and immunoblotting studies showed robust expression of {sigma}R1 in these cells. We next established that these cells are exquisitely sensitive to the toxic effects of Glu and Hcy. Approximately 50% of the cells died when exposed to micromolar quantities of Glu (25 µM) or Hcy (50 µM). The active form of Hcy is thought to be the L-isomer; however, Hcy can only be purchased commercially as D,L-homocysteine thiolactone hydrochloride. It is likely that the 50 µM D,L-Hcy used in this study reflected approximately 25 µM of the active (L-isomer form) of the Hcy, underscoring how sensitive the primary ganglion cells are to this excitotoxin. In addition to quantifying the incidence of apoptosis, we were able to visualize the effects of these excitotoxins on cell morphology over the course of the experiment and observed that the cell bodies shrank and the neuronal processes retracted markedly as a result of exposure to these excitotoxins.

The {sigma}R1 ligand (+)-PTZ showed marked neuroprotective effects, particularly when the cells were preincubated for 1 hour before exposure to the excitotoxins. Within 6 hours, cell viability improved. Over the 18-hour exposure to (+)-PTZ, the effects were considerable. Interestingly, microscopic examination of the cells revealed marked preservation of the plump cell body and extensive neuronal processes emanating from the cells that had been treated with (+)-PTZ. Pretreatment followed by cotreatment yielded a better result than did cotreatment alone.

We also determined whether the neuroprotective effects of (+)-PTZ were mediated by an alteration of {sigma}R1 gene or protein expression. RT-PCR analysis and immunoblotting data suggested that they were not, regardless of whether Glu or Hcy was used to induce cell death. It is logical to assume then that the neuroprotective effects were related to activation of {sigma}R1 through (+)-PTZ binding. The concentration of (+)-PTZ (500 nM), at which it served as a neuroprotectant in the present study, correlated well with the known affinity of (+)-PTZ for the {sigma}R1 (Kd approximately 25 nM).21 42 Furthermore, the neuroprotective effects of (+)-PTZ were shown to be mediated by {sigma}R1 because experiments using (–)-PTZ, the levo-isomer of pentazocine, showed no neuroprotection. Similar findings confirming the specificity of (+)-PTZ for {sigma}R1 have been reported in brain.14

We showed in earlier studies using primary Müller cells cultured from mouse retina that {sigma}R1 binding activity increased when cells were exposed to oxidative stress.21 Such studies were feasible using the primary Müller cells because, on harvesting, the cells proliferate. This is not the case with the primary GCs. In keeping with their phenotype as a neuronal cell, they do not proliferate. Therefore, it is difficult, if not impossible, to obtain sufficient primary GCs to monitor (+)-PTZ binding unless extraordinary numbers of mice are used. This technical limitation is also a hindrance in studying the signaling events that follow (+)-PTZ-induced {sigma}R1 activation. Studies from other laboratories using different model systems showed activation of protein kinase C after (+)-PTZ-induced {sigma}R1 activation.43 Whether this signaling pathway plays a role in (+)-PTZ-mediated protection of primary GCs observed in the present study remains to be seen. There is also evidence for protein–protein interaction between {sigma}R1 and specific ion channels in the plasma membrane.44 Binding of ligands to {sigma}R1 induces receptor translocation from intracellular sites to the plasma membrane, where protein–protein interaction leads to alterations in the activity of specific ion channels. This would influence intracellular calcium signaling. Thus, potentially diverse mechanisms could mediate the protection of primary GCs from excitotoxicity after (+)-PTZ–induced {sigma}R1 activation. We plan to focus in future studies on the signaling events related to {sigma}R1 activation in these cells.

The results of this study bring us closer to a physiologically relevant model for exploring the neuroprotective effects of {sigma}R1 ligands for retinopathy, particularly ganglion cell death. It is imperative to determine whether the results obtained in this in vitro system are relevant in vivo. Hence, the time is ripe to test {sigma}R1 ligands in an animal model in which these cells die, such as is observed in diabetic retinopathy or glaucoma.


   Acknowledgements
 
The authors thank Barbara Mysona and Angeline Martin-Studdard for helpful discussions regarding these experiments.


   Footnotes
 
Supported by National Institutes of Health Grants R01 EY014560 and EY012830.

Submitted for publication March 21, 2007; revised May 17, 2007; accepted July 23, 2007.

Disclosure: Y. Dun, None; M. Thangaraju, None; P. Prasad, None; V. Ganapathy, None; S.B. Smith, 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: Sylvia B. Smith, Department of Cellular Biology and Anatomy, Medical College of Georgia, 1459 Laney-Walker Boulevard, CB 2820, Augusta, GA 30912-2000; sbsmith{at}mail.mcg.edu.


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