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(Investigative Ophthalmology and Visual Science. 2004;45:2374-2383.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.03-1239
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Kainic Acid–Mediated Upregulation of Matrix Metalloproteinase-9 Promotes Retinal Degeneration

Xu Zhang, Mei Cheng, and Shravan K. Chintala

From the Eye Research Institute of Oakland University, Rochester, Michigan.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. Excitotoxicity has been proposed to play a pivotal role in retinal damage, but the mechanisms that underlie retinal damage are not clearly understood. In this study, the role of matrix metalloproteinases in excitotoxin-mediated retinal damage was investigated.

METHODS. KA, CNQX (6-cyano-7-nitroquinoxaline-2,3,-dione), NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline), MK801, or PBS was injected into the vitreous of CD-1 mice. MMP expression in the retina was analyzed by zymography, Western blot, and immunohistochemistry. Retinal ganglion cells (RGCs) were retrogradely labeled with aminostilbamidine methanesulfonate (Molecular Probes, Eugene, OR), and loss of fluorescently labeled RGCs in retinal flatmounts was quantified. Apoptotic cell death was assessed by TUNEL staining. Astrocyte activation was determined by immunohistochemistry, and laminin decrease was determined by immunohistochemistry and Western blot analysis.

RESULTS. Intravitreal injection of KA caused time- and dose-related MMP-9 upregulation in the retina. Increased MMP-9 activity and protein levels were associated with activation of astrocytes. Astrocyte-associated MMP-9 correlated with a decrease in laminin immunoreactivity in the ganglion cell layer and significant loss of retinal ganglion cells. KA-mediated upregulation of MMP-9 activity was associated with apoptosis of cells in the ganglion cell layer as early as 6 hours after injection, followed by apoptosis in cells in the inner nuclear layer by day 1. Intravitreal injection of the non-NMDA receptor antagonists, CNQX and NBQX decreased KA-induced MMP-9 activity and protein levels in the retina and attenuated retinal degeneration, whereas the NMDA receptor antagonist MK801 failed to offer protection. Further, a synthetic MMP inhibitor GM6001 decreased KA-mediated MMP-9 activity and offered significant protection against ganglion cell loss in the retina.

CONCLUSIONS. These results indicate that KA-mediated upregulation of MMP-9 activity promotes retinal degeneration and suggest that inhibition of KA-mediated MMP activity may offer protection against excitotoxin-induced retinal damage.

Although the mechanisms underlying ganglion cell loss after retinal ischemia are still under active investigation, it has been demonstrated that ischemic damage results in an acute decrease in adenosine triphosphate (ATP) production, activation of anaerobic glycolysis, and membrane depolarization.1 2 These complex events result in the release of excitatory amino acids (EAAs) such as glutamate into the extracellular space1 2 3 and mediate cellular effects through N-methyl-D-aspartate (NMDA), {alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid (KA)–type ionotropic receptors, and G-protein–coupled metabotropic receptors.4 5 6 Although glial cell–mediated compensatory reuptake mechanisms may decrease accumulation of the excitatory amino acid, increased levels exert neurotoxic and neurodegenerative effects on various cell types in the inner retina.5 6 7 8 9 10 11 Olney12 coined the term "excitotoxicity" to describe the toxic effect of increased glutamate in the retina. Previous studies have suggested the role of increased glutamate in glaucoma and in animal models of retinal ischemia and optic nerve crush.13 14 In addition, neurodegenerative effects similar to that observed with increased glutamate (excitotoxicity) have been documented in animal models involving intravitreal injections of the glutamate receptor agonist NMDA and the non-NMDA receptor agonist KA in chick, mouse, and rat species.9 10 11 15 16 17 18 19 20 Although excitotoxin-mediated activation of both NMDA and non-NMDA receptors, and the subsequent increase in intracellular calcium have been attributed to neuronal toxicity, the exact mechanisms that underlie excitotoxin-mediated cell death are still not clear at this time. We hypothesize that, in response to excitotoxicity, astrocytes present in the ganglion cell layer/vitreoretinal border may become active and express tissue-modulating proteases such as MMPs, which may then modulate the extracellular matrix (ECM) within the ganglion cell layer and predispose RGCs to detachment-induced apoptosis. In support of this, studies of the central nervous system have suggested that induction of matrix-modulating proteases such as tissue plasminogen activator (tPA) promotes neuronal cell loss.21 22 23 24 In addition, in an ischemia model, we have recently found that upregulation of matrix metalloproteinases (MMP)-9 promotes ganglion cell loss in the retina.25 In this context, recent years have seen an increase in literature suggesting the role of MMPs, not only in retinal development26 27 but also in several vitreoretinal diseases including proliferative vitreoretinopathy and age-related macular degeneration.28 Because MMPs are potent extracellular modulating proteases, their activity is highly regulated through gene transcription, proenzyme activation, and inhibition by tissue inhibitors of metalloproteinases (TIMPs).28

Based on the knowledge that ischemic retinal damage results in excitotoxicity,1 3 14 29 in the present study we injected a potent non-NMDA receptor agonist, KA,18 into mouse vitreous and tested whether KA induces retinal degeneration through upregulation of MMPs. The data in this study showed that intravitreal injection of KA alone can upregulate MMP-9 in the retina and can lead to apoptotic ganglion cell loss by, in part, modulating the ECM protein laminin. Furthermore, injection of non-NMDA receptor antagonists NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline) and CNQX (6-cyano-7-nitroquinoxaline-2,3,-dione), and a broad-spectrum MMP inhibitor GM6001, decreased MMP-9 activity in the retina and attenuated KA-induced retinal degeneration, suggesting that KA-mediated upregulation of MMP-9 exacerbates retinal damage.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Intravitreal Injections
All the experiments were performed in mice under general anesthesia according to institutional protocol guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Normal adult CD-1 mice (6–8 weeks old; Charles River Breeding Laboratories, Wilmington, MA) were anesthetized by an intraperitoneal injection of 1.25% Avertin (2,2,2-tribromoethanol in tert-amyl alcohol; 0.017 mL/g body weight; Aldrich, Milwaukee, WI). Intravitreal injection of KA was performed as previously described.10 Throughout this study, intravitreal injections were performed in a final volume of 2 µL. In control experiments, eyes (n = 4) were injected with 2 µL of 0.1 M phosphate-buffered saline (PBS, pH 7.4) alone and in treatment groups, eyes (n = 4) were injected with 2 µL of 0.5, 5, 10, and 20 mM (corresponding to 1, 10, 20, and 40 nmoles) KA (Sigma-Aldrich, St. Louis, MO) prepared in PBS. In separate experiments (n = 4 eyes), 2 µL of 10 mM KA (corresponding to 20 nmoles) plus 20 or 40 mM CNQX (corresponding to 40 and 80 nmoles; Tocris, Ellisville, MO), 10 mM KA plus 25 and 50 mM NBQX (corresponding to 50 and 100 nmoles; Tocris), or 10 mM KA plus 100 mM (+)-MK801 maleate (corresponding to 200 nmoles; Tocris) was injected into the vitreous.

Protein Extraction
At 3, 6, or 12 hours or 1, 2, or 4 days after intravitreal injection, animals were anesthetized with an overdose of tribromoethanol, and their eyes were enucleated. Enucleated eyes were cut in half at the equator, and the lenses were removed. Retinas were carefully peeled off with forceps and washed three times with phosphate-buffered saline (pH 7.4) to remove any vitreous that may have adhered to the retina. Three to four retinas each were placed in Eppendorf tubes containing 40 µL of extraction buffer (1% nonidet-P40, 20 mM Tris-HCl, 150 mM NaCl, and 1 mM Na3VO4 [pH 7.4]), and the tissues were homogenized. Tissue homogenates were centrifuged at 10,000 rpm for 5 minutes at 4°C and the supernatants were collected. Protein concentration in supernatants was determined using a protein assay (Bio-Rad Laboratories, Hercules, CA).

Gelatin Zymography
MMP activity was determined by zymography according to methods described previously.25 30 31 Briefly, retinal extracts containing equal amounts of protein (25 µg) were mixed with SDS gel-loading buffer32 and loaded without reduction or heating onto 10% SDS polyacrylamide gels containing 0.2% gelatin as a substrate for MMPs. After electrophoresis, the gels were washed three times with 2.5% Triton X-100 (15 minutes each time) and placed in calcium chloride buffer (10 mM CaCl2 [pH 7.4]) and incubated overnight at 37°C to allow proteolysis of the substrates in the gels. The gels were stained with 0.1% Coomassie brilliant blue-R250 and then destained with a solution containing 25% methanol and 10% acetic acid. Samples containing a mixture of murine MMP-9 and -2 were coelectrophoresed for comparison (data not shown). A reduced molecular weight size standard was also included on all gels (data not shown; Invitrogen-Life Technologies, Gaithersburg, MD). The area cleared by MMP-9 on gelatin zymograms was scanned by a densitometer. The data from three independent experiments are represented as mean arbitrary units ± SEM.

Immunohistochemistry
Eyes enucleated after KA injection were fixed with 4% paraformaldehyde for 1 hour at room temperature and embedded in optimal cutting temperature (OCT) compound (Sakura Finetek USA, Torrance, CA). Traverse, 10-µm-thick cryostat sections were cut and placed onto slides (Super-Frost Plus; Fisher Scientific, Pittsburgh, PA). Sections were subsequently processed for indirect immunofluorescence localization using antibodies against murine MMP-9 (1:1000 dilution, the kind gift of Robert Senior, Washington University School of Medicine, St. Louis, MO) and MMP-2 (1:100 dilution; Chemicon, Temecula, CA), microglial/macrophage marker, Mac-1 (1:100 dilution, CD11b/CD18; Chemicon; data not shown), glial fibrillary acidic protein (GFAP, 1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), glutamine synthetase (1:100 dilution; Chemicon; data not shown), and laminin (1:100 dilution; Sigma-Aldrich). Sections were incubated with appropriate AlexaFluor-conjugated secondary antibodies (Molecular Probes, Eugene, OR) for 1 hour at room temperature and mounted with a coverslip. Where indicated, Hoechst dye (0.1 mg/mL) was added to localize nuclei in the tissue. Sections were observed under a bright-field microscope (Nikon, Tokyo, Japan) equipped with epifluorescence, and digitized images were obtained with a digital camera (SPOT; Diagnostic Instruments, Sterling Heights, MI). Images were processed and compiled on computer (Photoshop vers. 5.5 and 7.0; Adobe Systems, Inc., Mountain View, CA).

MMP-9 immunohistochemistry in retinal flatmounts was then performed. Briefly, retinas from noninjected control or KA-injected eyes were fixed in 3% freshly prepared formaldehyde in 80 mM phosphate buffer (pH 7.4) containing 5% dimethyl sulfoxide, 8% sucrose, and 1% Triton X-100 (45 minutes, at room temperature). Retinas were washed with PBS and incubated with antibodies against mouse MMP-9 (2 days, at 4°C) diluted in 1% Triton X-100 and 1 mg/mL bovine serum albumin. Retinas were washed with PBS and incubated with appropriate AlexaFluor-conjugated secondary antibodies (Molecular Probes) for 1.5 days at 4°C. MMP-9 immunoreactivity was observed by epifluorescence microscope (Axiovert S100; Carl Zeiss, Thornwood, NY) equipped with a laser scanning confocal system (MRC-600; Bio-Rad).

Apoptosis Assay
Apoptosis in retinal cells was performed as described before.25 33 Briefly, 10-µm-thick cryostat sections were prepared as described earlier, and apoptotic cell death was detected by a TdT-mediated dUTP nick-end labeling (TUNEL) assay, using a kit (In Situ Cell Death Detection with Fluorescein; Roche Biochemicals, Mannheim, Germany) according to the protocol provided by the manufacturer. Tissue sections were examined by microscope (Nikon) equipped with epifluorescence, digital images were obtained (SPOT; Diagnostic Instruments), and images were compiled on computer (Photoshop vers. 5.5 and 7.0; Adobe Systems).

Western Blot Analysis
Aliquots containing an equal amount of protein (25 µg) were mixed with gel loading buffer and separated on 10% SDS-polyacrylamide gels. After electrophoresis, the proteins were transferred onto nylon membranes, and nonspecific binding was blocked with 10% nonfat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T). Membranes were then probed with antibodies against mouse MMP-9 (1:2500 dilution; Triple Point Biologics, Portland, OR), MMP-2 (1:5000 dilution; Laboratory Vision, Fremont, CA), or mouse laminin (1:1000; Sigma-Aldrich). After incubation with the primary antibodies, membranes were washed with TBS-T and incubated with appropriate peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Finally, the proteins on the membranes were detected by using a chemiluminescence kit (ECL; Amersham Pharmacia Biotech) and exposing the membranes to x-ray film. Purified MMP-9 (Triple Point Biologics) and MMP-2 (Laboratory Vision) were coelectrophoresed as positive standards (data not shown).

Morphometry
Morphologic changes in retinas after KA injection was determined according to a previously described procedure.33 Briefly, retinal cross sections were stained with hematoxylin and eosin (H&E), and loss of cells in the ganglion cell layer was quantified by counting the cells in a 40x field (representing ~15 µm2) at a distance of 1 to 2 mm from the optic disc. We did not attempt to differentiate between ganglion cells and displaced amacrine cells during counting. However, morphologically different glial and vascular endothelial cells were excluded from the cell counts. Data from four different eyes were evaluated by ANOVA, followed by a post hoc Tukey test (GB-Stat Software; Dynamic Microsystems, Houston, TX) and are expressed as the mean ± SEM.

Retrograde Labeling of Retinal Ganglion Cells
Retrograde labeling of ganglion cells was performed as previously described.25 34 Briefly, 1.5 µL of a 5% solution of aminostilbamidine methanesulfonate (Molecular Probes) in PBS was injected into the superior colliculi of anesthetized mice immobilized in a stereotaxic apparatus. KA was injected into the vitreous 1 week after application of the tracer. At various times after KA injection, the animals were euthanatized, and their eyes were enucleated and fixed in 4% paraformaldehyde for 1 hour at room temperature. After rinsing with PBS, retinas were detached from the eyecups and prepared as flattened wholemounts by mounting the vitreal side up. Retinal ganglion cells were counted in four microscope fields of identical size (155 µm2, 40x magnification) located approximately at the same distance from the optic disc, by using image analysis software (Scion Corp., Frederick, MD). Statistical significance was analyzed by ANOVA, followed by a post hoc Tukey test (GB-Stat Software; Dynamic Microsystems) and are expressed as the mean ± SEM.

Transmission Electron Microscopy
One day after KA injection, enucleated eyes were fixed for 15 minutes in 2% paraformaldehyde plus 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Eyes were cut in half at the equator, and the lenses were removed. Retinas were gently peeled off with forceps and fixed overnight in the same buffer. Retinas were washed with 7% sucrose in 0.1 M cacodylate buffer (pH 7.4) and incubated in 1% osmium tetroxide for 1 hour on ice. After washes with the same buffer, retinas were dehydrated, embedded in epoxy resin 812, and cut into 2-µm-thick sections with an ultramicrotome. Sections stained with uranyl acetate and lead citrate were examined under a transmission electron microscope (Morgagni 268; FEI, Hillsboro, OR) for the ultrastructural features of apoptosis.

MMP Inhibitor
A peptide, N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM6001; Chemicon), was used to inhibit MMP activity in KA-injected animals.35 The ability of this inhibitor to inhibit retinal MMP effectively was tested by determining MMP activity in retinal protein extracts. One hour before KA injection, mice were injected intraperitoneally (IP) with 100 mg/kg GM6001 dissolved in 1.5% carboxy methyl cellulose (CMC)/0.9% saline. Control mice were injected with 1.5% CMC/0.9% vehicle. Retinal extracts were prepared 1 day after KA injection, and MMP-9 activity was determined by zymography. The effectiveness of GM6001 in attenuating ganglion cell loss was determined by retrograde labeling studies. Ganglion cells were labeled with methanesulfonate, as described earlier. One hour before KA-injection, one group of animals (n = 3) received vehicle alone and the other group (n = 3) received GM6001 (100 mg/kg body weight) plus vehicle. One day after KA injection, animals were euthanatized, and their eyes were enucleated and fixed and the cells counted as described earlier.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Effect of KA on MMP Expression
To determine whether KA-induced retinal degeneration is mediated in part by MMPs, we first investigated the activity of MMPs in retinal extracts by gelatin zymography. Gelatin zymography indicated very low and constitutive levels of MMP-9 (105 kDa) in retinal extracts of noninjected control and PBS-injected eyes (Fig. 1) . In contrast, intravitreal injection of 20 nmoles of KA resulted in a time-related increase in MMP-9 activity in the retina. MMP-9 activity was increased as early as 6 hours, peaked around day 1, and returned to basal levels 4 days after KA injection (Fig. 1) . A very low level of MMP-2 activity (65 kDa) was also observed in retinal extracts of noninjected control and PBS-injected eyes, and no significant change in activity of this protease was observed after KA injection. Western blot analysis confirmed the transient increase in MMP-9 protein after KA injection. A slight increase in MMP-2 protein levels was also observed between 6 and 12 hours after injection (Fig. 1) . Gelatin zymography and Western blot analysis indicated that KA also caused a dose-related increase in MMP-9 gelatinolytic activity and protein levels in the retina (Fig. 2) . Although Western blot analysis indicated a slight increase in MMP-2 protein level in a time- and dose-related fashion (Figs. 1 2 , respectively), zymography assays indicate that this protein does not induce an increase in gelatinolytic activity in the retina, consistent with our previous observations.25 No other gelatinolytic bands were noticed after KA treatment, and no significant change in TIMP-1 and -2 was observed by Western blot analysis (data not shown). These results indicate that KA predominantly upregulates MMP-9 activity and protein levels in the retina.



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  		FIGURE 1. KA upregulated MMP-9 activity in the retina. Aliquots containing an equal amount of retinal protein (25 µg) from noninjected control or PBS and KA-injected (20 nmoles) eyes were subjected to gelatin zymography (top) and Western blot analysis (middle and bottom). The migration position of the activity bands representing MMP-2 and -9 are indicated. KA induced a transient upregulation of MMP-9 gelatinolytic activity and protein levels and also a slight increase in MMP-2 protein.

 


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  		FIGURE 2. Effect of various doses of KA on MMP activity in the retina. Aliquots containing an equal amount of retinal protein (25 µg) from noninjected control or PBS- or KA-injected (1, 10, 20, and 40 nmoles) eyes were subjected to gelatin zymography (top) and Western blot analysis (middle and bottom). The migration position of the activity bands representing MMP-2 and -9 are indicated. KA induced a dose-related upregulation of MMP-9 activity and protein levels in the retina and a slight increase in MMP-2 protein at higher doses.

 
Retinal cross sections were prepared 1 day after KA injection (a time point where increased MMP-9 activity and protein levels were observed, Fig. 1 ) and MMP-9 and -2 tissue localization was determined by immunohistochemistry. A very low level of MMP-9 immunoreactivity was observed in the ganglion cell layer in noninjected control and PBS-injected eyes (Fig. 3A , top, arrows). In contrast, KA-injected eyes showed increased MMP-9 immunoreactivity that was localized to astrocytes in the ganglion cell layer (Fig. 3A , top, arrows). MMP-2 immunoreactivity was constitutively observed in cells in the ganglion layer and also in Müller cells in noninjected control and PBS-injected eyes (Fig. 3A , bottom). Although no significant increase in MMP-2 immunoreactivity was observed after KA injection, the number of cells that expressed MMP-2 in the ganglion cell layer decreased at this time point, and a few remaining cells still showed positive immunostaining. The distribution pattern of MMP-9 and -2 in the retina indicate that astrocytes primarily express MMP-9, whereas several inner retinal cells express MMP-2.



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  		FIGURE 3. Distribution of KA-mediated MMPs in the retina. (A) Retinal cross sections prepared from noninjected control and PBS- or KA-injected eyes were immunostained with antibodies against MMP-9 and -2. Sections were incubated with Hoechst dye to stain the nuclei, and overlapping images of antibody staining and Hoechst dye are shown. Arrows: positive immunostaining. Compared with a low and constitutive level of MMP-9 in control and PBS-injected eyes, MMP-9 immunoreactivity was increased in KA-injected eyes. MMP-2 immunoreactivity was observed throughout the inner retina in ganglion cells, Müller cells, and astrocytes. (B) Retinal cross sections were double labeled with antibodies against GFAP and MMP-9. Arrows: MMP-9 positive immunostaining. Compared with a low level of GFAP immunoreactivity in astrocytes in control retinas, KA-injection resulted in increased GFAP immunoreactivity in the astrocytes and also in some Müller cells. Merged images of GFAP and MMP-9 show a greater overlap of MMP-9 immunoreactivity in astrocytes and not in Müller cells. (C) Immunostaining of flatmounted retinas with MMP-9 antibodies alone show constitutive levels of MMP-9 in cells characteristic of astrocytes and increased positive staining after KA injection. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.

 
To illustrate better the origin of MMP-9 by astrocytes, we immunostained retinal cross sections with antibodies against GFAP (a marker for astrocytes) and double labeled with antibodies against MMP-9. Data in Figure 3B (GFAP) indicate low levels of GFAP immunoreactivity in the ganglion cell layer in noninjected control retinas. KA injection resulted in increased GFAP immunostaining in the ganglion cell layer, suggesting the activation of astrocytes. Although most of the GFAP immunoreactivity was localized to astrocytes in the ganglion cell layer, a few Müller cells also showed GFAP immunoreactivity in radial fibers (Fig. 3B , GFAP). Compared with a low level of MMP-9 immunoreactivity in control retinas, KA-injected eyes showed increased MMP-9 immunoreactivity in astrocytes (Fig. 3B , MMP-9, arrows). Merged images of GFAP and MMP-9 indicate a greater overlap of GFAP immunoreactivity with MMP-9 in the ganglion cell layer and no significant positive staining in radial Müller cell processes, indicating that MMP-9 primarily originates from activated astrocytes (Fig. 3B , merged). Double labeling of retinal cross sections with antibodies against glutamine synthetase (a marker for Müller cells) and MMP-9 and antibodies against Mac-1 (a marker for microglial cells) and MMP-9 showed no overlapping (data not shown). Finally, we immunostained flatmounted retinas with antibodies against MMP-9 alone, and observed the retinas under a confocal microscope by mounting the specimens vitreous side up (Fig. 3C) . Observation of retinal flatmounts indicated a low and constitutive level of MMP-9 immunoreactivity in astrocytes. In contrast, increased MMP-9 immunoreactivity was observed in astrocytes after KA injection (Fig. 3C) . In addition, MMP-9 immunostaining was observed in the ECM in flatmounted retinas.

KA-Mediated Upregulation of MMP-9 Activity and Ganglion Cell Loss
To determine whether KA-mediated increase in MMP-9 activity contributes to ganglion cell loss, we retrogradely labeled ganglion cells with aminostilbamidine methanesulfonate (Molecular Probes) and their loss was determined in flatmounted retinas 12 and 24 hours after KA injection. Representative micrographs of fluorochrome-labeled cells indicate a clear loss of ganglion cells in KA-injected eyes compared with noninjected and PBS-injected eyes (Fig. 4A) . Quantification of remaining positive cells after KA injection showed a significant loss of retinal ganglion cells at both time points (Fig. 4B) . The mean number (± SE) of fluorochrome-positive cells at 12 and 24 hours after KA was 49.3 ± 3.77 (a 46% decrease from control and PBS) and 23.0 ± 6.06 (a 75% decrease from control and PBS), respectively (control versus PBS is not significant: control vs. 12 hours, *P < 0.05; control vs. 24 hours, *P < 0.05; ANOVA). In a separate set of experiments, retinal cross sections were prepared at various times after KA injection and stained with H&E, and the remaining cells in the ganglion cell layer were determined by morphometric analysis (Fig. 4C) . Cells remaining in the ganglion cell layer were then compared with increased MMP-9 gelatinolytic activity in the retina (Fig. 4D) . Observation of H&E-stained retinal cross sections indicate that cells in the ganglion cell layer became pyknotic as early as 6 hours and that most of these cells disappeared from the ganglion cell layer 1 day after KA injection. Data in Figure 4D indicate that progressive loss of cells in the ganglion cell layer correlates with an increase in MMP-9 activity. Further, the KA-mediated MMP-9 increase observed as early as 6 hours was associated with the appearance of TUNEL-positive cells in the ganglion cell layer (Fig. 5A) . At 6 hours only cells in the ganglion cell layer were TUNEL positive, and essentially no TUNEL-positive cells were observed in the rest of the retina. One day after KA injection TUNEL-positive cells were also noticed in the anterior portion of the inner nuclear layer. Electron microscopic images of retinal cross sections 1 day after KA injection clearly indicate nuclear condensation in ganglion cells, confirming that these cells undergo apoptosis (Fig. 5B) .



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  		FIGURE 4. KA-mediated MMP-9 upregulation was associated with retinal degeneration. (A) Retinal ganglion cells were retrograde-labeled 1 week before intravitreal injections, and their loss was observed in flatmounted retinas 12 and 24 hours after KA (20 nmoles) injection. (B) The remaining fluorochrome-labeled cells were quantified using image analysis and expressed as the mean number of cells ± SEM. Injection of 20 nmoles of KA induced a significant loss of RGCs at both the 12- and 24-hour time points (*P < 0.05). (C) Tissue morphology was assessed by H&E staining of retinal cryostat sections prepared 6 hours after KA injection and compared with noninjected and PBS-injected eyes. Cells present in the ganglion cell layer became pyknotic as early as 6 hours, and a significant number of these cells disappeared 2 days after KA injection. (D) At various times after KA injection, cells remaining in the ganglion cell layer were quantified and plotted against MMP-9 gelatinolytic activity (arbitrary densitometric units, mean ± SEM). Data indicate a correlation between MMP-9 increase and loss of cells in the ganglion cell layer. Abbreviations are as in Figure 3 . Magnification: (A, C) x40.

 


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  		FIGURE 5. KA induction of apoptotic cell death in the retina. (A) Retinal cross sections prepared 6 hours and 1 day after KA (20 nmoles) injection were subjected to TUNEL assay and compared with noninjected and PBS-injected eyes. (B) Ultrastructural changes in degenerating cells were observed by transmission electron microscopy. KA caused condensation of nuclei in ganglion cell layer as early as 6 hours and a TUNEL assay indicate that cell death was due to apoptosis. One day after KA injection, apoptotic cells were also noted in the inner nuclear layers. Transmission electron micrographs in (B) clearly indicate the nuclear condensation of ganglion cells in KA-injected eyes. Abbreviations are as in Figure 3 .

 
KA-Mediated MMP-9 Upregulation and Laminin Degradation
To determine whether the MMP-9 increase observed in retinal extracts (Fig. 1) , and its localization in the ganglion cell layer (Fig. 3) contributes to modulation of ECM, we immunostained retinal cross sections with antibodies against laminin (Fig. 6) . Immunofluorescence analysis indicated uniform and bright, sheetlike laminin immunostaining in the ganglion cell layer of noninjected control and PBS-injected eyes (Fig. 6A , arrows). Cells that were close to the boundary of the inner nuclear layer also showed positive immunostaining for laminin (Fig. 6A , small arrows). One day after KA-injection, laminin immunoreactivity was decreased in the ganglion cell layer, and a fragmented pattern of staining was observed (Fig. 6A , arrowheads). In addition, laminin immunoreactivity decreased in the inner nuclear layer one day after KA injection (Fig. 6A , small arrowheads). Comparison of the immunostaining of boxed areas shown in Figure 6B , enlarged from Figure 3 (top, MMP-9 immunostaining), and Figure 6A (laminin immunostaining) indicate a correlation between ganglion cell layer–associated MMP-9 increase and a decrease in laminin immunostaining (Fig. 6B , arrowheads). To confirm the decrease in laminin immunoreactivity after KA treatment, we performed Western blot analysis on whole retinal extracts (Fig. 6B , laminin Western blot). We found that the intensity of the laminin immunoreactive protein band of ~200 to 220 kDa (the size of ß/{gamma}chains of laminin) present in control retinal extracts was reduced 1 day after KA treatment. Further, appearance of TUNEL-positive cells in the ganglion cell layer and in the inner nuclear layer also correlated with a decrease in laminin immunostaining (Fig. 6B) .



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  		FIGURE 6. Effect of KA-mediated MMP-9 upregulation on laminin loss in the retina. (A) Retinal cross sections prepared from noninjected control or KA-injected (20 nmoles) eyes were immunostained with antibodies against laminin. Retinal sections were counterstained with Hoechst dye to stain the nuclei, and the images shown are the overlapping images of antibody staining and dye. The bright, sheetlike, laminin-positive immunostaining observed in control eyes (arrows) decreased after KA injection (arrowheads). (B) Comparison of enlarged images of boxed areas from Figure 3 (MMP-9 immunostaining) and from Figure 6A (laminin immunostaining) showed a correlation between MMP-9 increase and a decrease in laminin immunostaining in both the ganglion cell layer (arrowheads) and in the inner nuclear layer (arrowheads) after KA injection (B). Western blot analysis showed a correlation between KA-mediated MMP-9 upregulation and a decrease in laminin immunoreactivity (B, boxed areas). Arrow: laminin band. Abbreviations are as in Figure 3 .

 
Non-NMDA Receptor Antagonists and MMP-9 Activity in the Retina
To determine whether blocking of KA-induced excitotoxicity by the specific non-NMDA receptor antagonists CNQX and NBQX downregulates MMP-9, we injected 20 nmoles KA along with various amounts of CNQX (2, 20, 40, and 80 nmoles) and NBQX (5, 50, and 100 nmoles). One day after injection, retinal extracts were prepared, and MMP-9 expression was determined by both zymography and Western blot analysis (Fig. 7) . Gelatin zymography and Western blot data indicated that 20 nmoles of KA-alone resulted in MMP-9 increase in the retina (Fig. 7) , consistent with the data shown in Figures 1 and 2 . Very low concentrations of CNQX (2 and 20 nmoles) failed to inhibit MMP-9 activity (data not shown). However, 80 nmoles CNQX reduced MMP-9 activity and protein levels (Fig. 7) in the retina. Injection of 80 nmoles CNQX alone (without KA) showed no increase in MMP-9 activity over PBS-injected controls. In addition, compared with 50 nmoles of another non-NMDA receptor antagonist NBQX, injection of 100 nmoles of NBQX reduced MMP-9 activity and protein levels (Fig. 7) , whereas the NMDA receptor antagonist MK801 (200 nmoles) failed to inhibit KA-mediated upregulation of MMP-9 activity in the retina.



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  		FIGURE 7. Non-NMDA receptor antagonists inhibit MMP-9 in the retina. One day after intravitreal injection of KA (20 nmoles) with or without indicated concentrations of non-NMDA receptor antagonists, retinal extracts were prepared, aliquots containing an equal amount of protein (25 µg) were subjected to gelatin zymography (A), and MMP-9 protein was determined by Western blot analysis (B). Results show that higher concentrations of both CNQX and NBQX decreased KA-induced MMP-9 gelatinolytic activity and protein levels in the retina.

 
Effect of Non-NMDA Receptor Antagonists on Retinal Degeneration
To determine whether the CNQX- and NBQX-mediated inhibition of MMP-9 offers retinal protection, we prepared retinal cross sections 2 days after KA injection and stained them with H&E, and the cells remaining in the ganglion cell layer were quantified by morphometric analysis. Morphometric analysis indicated a significant loss of cells in the ganglion cell layer in eyes that had undergone intravitreal injection of KA alone (Fig. 8) . In eyes that received KA alone the mean number of cells (± SEM) in the ganglion cell layer were 9.33 ± 3.44 compared with 32.5± 2.07 in control eyes, with ~71% cell loss (control versus KA, *P < 0.05, ANOVA). Compared with control eyes, 40 nmoles of CNQX offered no protection against cell loss (32.5 ± 2.07 control vs. 10.66 ± 2.08 KA+40 nmoles CNQX, respectively, *P < 0.05, ANOVA), whereas 80 nmoles CNQX offered significant protection (32.5 ± 2.07 control vs. 28.66 ± 3.05 KA+80 nmoles CNQX, respectively, *P < 0.05). In addition, higher concentrations of NBQX offered significant protection against KA-induced cell loss (9.33 ± 3.44 KA vs. 32.5 ± 4.92 KA + 100 nmoles NBQX, respectively, *P < 0.05, ANOVA) and also attenuated cell loss in the inner nuclear layer (data not shown). The NMDA receptor antagonist MK801 failed to offer significant protection against KA-induced retinal degeneration (32.5 ± 2.07 control vs. 11.66 ± 2.51 KA+MK801, respectively, *P < 0.05, ANOVA).



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  		FIGURE 8. Non-NMDA receptor antagonists attenuated retinal degeneration. Two days after intravitreal injection of KA (20 nmoles), with or without indicated concentrations of non-NMDA receptor antagonists, retinal cross sections were prepared and stained with H&E, and loss of cells in the ganglion cell layer was quantified. Compared with noninjected control and PBS-injected eyes, KA injected showed a significant decrease in cells remaining in the ganglion cell layer (mean number of cells ± SEM). Injection of KA along with higher concentrations of both CNQX and NBQX showed significant protection against KA-induced cell loss whereas the NMDA antagonist, MK801, offered no protection (*P < 0.05; ANOVA). NS, not significant.

 
Effect of Synthetic MMP Inhibitor on Retinal Degeneration
We used a well-characterized synthetic broad-spectrum MMP inhibitor, GM6001,35 36 to determine whether inhibition of MMP activity would result in protection similar to that observed with non-NMDA antagonists. Before determining the effect of GM6001 on retinal protection, we first investigated its effect on MMP activity in the retina. One hour before KA-injection, animals (n = 3 for each group) were treated with vehicle alone or vehicle plus GM6001 or left untreated. Two microliters of KA (20 nmoles) were injected into untreated, vehicle-treated, and GM6001-treated animals, and retinal proteins were extracted 1 day after KA injection. Gelatin zymography and Western blot analysis showed an increase in MMP-9 activity and protein levels in vehicle-treated animals and in animals that left untreated (Fig. 9A) . In contrast, MMP-9 activity (but not protein level) was reduced in animals that received GM6001, compared with vehicle-treated animals, confirming that GM6001 inhibits MMP activity in the retina. In a separate set of experiments, the effect of GM6001 on retinal protection was determined by retrograde labeling studies. Retinal ganglion cells were retrogradely labeled 1 week before intravitreal injections, as described earlier. One hour before KA-injection, animals (n = 3 for each group) were treated with vehicle alone or vehicle plus GM6001 or left untreated. Two microliters of KA were injected into untreated, vehicle-treated, and GM6001-treated animals and 1 day after KA injection the remaining fluorochrome–positive cells were determined in retinal flatmounts (Fig. 9B) . Representative micrographs of gold-labeled cells indicate a clear loss of ganglion cells in untreated and vehicle-treated animals. In contrast, GM6001-treated animals showed significant protection against KA-induced ganglion cell loss compared with vehicle-treated animals (Fig. 9B) . The mean number of remaining fluorochrome-labeled cells were 90 ± 3.91, 23.0 ±6.06, 27 ± 8.98, and 63.7 ± 11.12 in noninjected control eyes and KA-injected, KA plus vehicle-injected, and KA plus GM6001-injected eyes, respectively (*P < 0.05, ANOVA, Fig. 9C ). Compared with vehicle-treated animals, which showed approximately 75% ganglion cell loss, KA+GM6001-treated animals showed only a 29% cell loss.



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  		FIGURE 9. GM6001, a synthetic MMP inhibitor, attenuated ganglion cell loss. (A) One day after intravitreal injection of KA (20 nmoles) into vehicle or vehicle+GM6001–treated animals, retinal extracts were prepared, and aliquots containing an equal amount of protein (25 µg) were subjected to gelatin zymography. Compared with noninjected controls, KA-injected eyes showed an increase in MMP-9 in the retina. Compared with untreated and vehicle treated animals, GM6001-treated animals showed a decrease in KA-mediated MMP-9 upregulation. (B, C) Retinal ganglion cells were retrogradely labeled 1 week before intravitreal injections, and their loss was quantified 24 hours after KA injection. Compared with noninjected controls, KA injected eyes showed a significant decrease in ganglion cells in the retina. In contrast, compared with untreated and vehicle treated eyes, eyes that received GM6001 showed significant protection against ganglion cell loss (*P < 0.05. NS, not significant).

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
Although several studies have suggested that excitotoxicity has a role in retinal damage, the mechanisms involved in excitotoxic retinal degeneration are still not clearly understood. Despite the fact that excessive accumulation of excitotoxins causing neuronal cell loss due to calcium overload is widely accepted,1 3 a direct relationship between increased excitotoxin and subsequent neuronal cell death has not been fully elucidated, and the damage may not be mediated solely by excitotoxins. In vitro studies have suggested that elevation of extracellular glutamate induced by ischemia and subsequent activation of glutamate receptors may not be the sole cause of neuronal injury but may initiate the process leading to neuronal cell loss.37 38

Based on the results presented in this study, our working hypothesis is that intravitreal injection of KA activates non-NMDA glutamate receptors in ganglion cells and astrocytes that are present in the immediate vicinity of the injection site at the vitreoretinal border. In this context, several cells in the retinal layers are known to express AMPA/KA receptors. Activation of the receptors may then lead to calcium overload and initiate neuronal cell loss in the presence of KA, as previously shown. At the same time, astrocytes present in the ganglion cell layer–vitreoretinal border may also be activated and respond to KA39 by expressing tissue-modulating protease MMP-9. Once synthesized, MMP-9 may then affect the ECM within the ganglion cell layer and also in the anterior portion of the inner nuclear layer. In the absence of a physiological matrix, ganglion cells may undergo apoptosis by a mechanism similar to detachment-induced apoptosis.40 41 In addition, accumulation of excitotoxins released from dying neurons may further exacerbate the loss of cells in the inner nuclear layer. In support of this hypothesis, the data presented in this study show a correlation between KA-mediated upregulation of MMP-9 and loss of cells initially in the ganglion cell layer and subsequently in the inner nuclear layer. Because MMP-9 was upregulated in the ganglion cell layer, we reasoned that this protease could modulate ECM proteins such as laminin, an abundantly expressed protein in the ganglion cell layer, and promote ganglion cell loss. The data presented in this study indeed show a correlation between MMP-9 upregulation and a decrease in laminin immunostaining in the ganglion cell layer. Laminins are the major ECM components of the inner limiting membrane of the ganglion cell layer,42 43 44 and studies of the central nervous system have suggested that injury-induced protease activity plays a role in modulating the laminins within their location of synthesis and may predispose neuronal cells to excitotoxic cell death21 in the absence of a physiological ECM.40 41 In support of this, in vitro studies have shown that neural cell attachment to laminin promotes cell survival and makes neuronal cells resistant to glutamate induced toxicity.45 In addition, we have recently shown that purified MMP-9 degrades the laminin present in uninjured control retinal extracts in vitro, and optic nerve ligation-induced MMP-9 correlates with a decrease in laminin immunostaining in vivo.25

The exact mechanisms that trigger KA-induced protease synthesis are not clear. However, a few possibilities can be postulated. First, because MMP-9 has an AP-1 transcriptional element in its promoter, upregulation of this protease may represent an attempt to accomplish tissue remodeling through activation of immediate-early genes.46 Second, excitotoxicity may result in induction of cytokines such as IL-1ß, which in turn may result in the upregulation of MMP-947 48 and may result in increased NO. NO may indirectly mediate IL-1ß–induced MMP-9 synthesis or may also exacerbate retinal damage by inhibiting uptake of excitotoxins by glial cells.49 Upregulation of endogenous retinal IL-1ß has been shown to occur after intravitreal injection of NMDA50 and KA,51 and our recent results indicate that IL-1ß plays a major role in the ischemia-induced MMP-9 increase and subsequent retinal damage.52 Third, although it is speculative at this time, upregulation of proteases may represent the tissues’ effort to accomplish synaptic remodeling by activating MMP-9.37 53 54 Although low levels of MMP-9 may aid in accomplishing this task, the presence of excessive protease levels may lead to loss of the neuronal matrix interactions and may exacerbate retinal damage.

Because cell death was associated with KA-mediated upregulation of MMP-9 in the retina, we reasoned that injection of non-NMDA receptor antagonists might block KA-mediated MMP-9 synthesis in the retina and offer retinal protection. Although very low amounts of CNQX (2 and 20 nmoles) failed to show a significant inhibition in MMP-9 activity (data not shown) higher concentrations of CNQX (80 nmoles) and NBQX (100 nmoles) decreased MMP-9 activity and protein levels and offered significant protection against retinal degeneration. The results presented herein are consistent with the finding of neuroprotective effects of non-NMDA receptor antagonists reported earlier and suggest that the neuroprotection observed with these antagonists may be due in part to a reduction in KA-mediated MMP upregulation in the retina. In addition, the synthetic broad-spectrum MMP inhibitor GM6001 decreased KA-mediated MMP-9 upregulation and offered significant protection against KA-induced retinal ganglion cell loss, suggesting that MMP-9 upregulation plays a major role in retinal degeneration. Although, the MMP inhibitor (GM6001) used in this study is not specific for individual members of the MMP family,36 we were not concerned with the lack of specificity, because we found increased levels of only MMP-9 and to some extent MMP-2, and no other proteases were observed in gelatin zymograms. At this time, we cannot completely rule out the inhibition of other as yet unidentified MMPs by GM6001 in the retina. Taken together, these results suggest that both non-NMDA receptor antagonists and the MMP-9 inhibitor GM6001 offer retinal protection by inhibiting MMP-9 activity in the retina. However, we also cannot rule out the role of tPA and the plasminogen activation system in excitotoxin mediated retinal degeneration.

In conclusion, these data provide the first evidence that KA alone can upregulate MMP-9 in the retina and promote retinal damage and suggest that inhibition of the proteolytic cascade may offer retinal protective strategies in conditions in which excitotoxins play a degenerative role.


   Acknowledgements
 
The authors thank Delore Semaan for quantification of ganglion cells; Fay Hansen Smith for confocal microscopy; Loan Dong for assistance with transmission electron microscopy; and Frank J. Giblin, Barry S. Winkler, and Ari Sitaramayya for critical reading of the manuscript.


   Footnotes
 
Supported by National Eye Institute project Grant EY13643 (SKC) and Vision Research Infrastructure Development Grant EY014803.

Submitted for publication November 13, 2003; revised February 16, 2004; accepted March 8, 2004.

Disclosure: X. Zhang, None; M. Cheng, None; S.K. Chintala, 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: Shravan K. Chintala, Eye Research Institute, 409 Dodge Hall, Oakland University, Rochester, MI 48309; chintala{at}oakland.edu.


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

  1. Lipton P. Ischemic cell death in brain neurons. Physiol Rev. 1999;79:1431–1568.[Abstract/Free Full Text]
  2. Nishizawa Y. Glutamate release and neuronal damage in ischemia. Life Sci. 2001;69:369–381.[CrossRef][ISI][Medline][Order article via Infotrieve]
  3. Lipton SA, Rosenberg PA. Excitatory amino acids as a final common pathway for neurologic disorders. N Engl J Med. 1994;330:613–622.[Free Full Text]
  4. Nakanishi S. Molecular diversity of glutamate receptors and implications for brain function. Science. 1992;258:597–603.[Abstract/Free Full Text]
  5. Osborne NN, Ugarte M, Chao M, et al. Neuroprotection in relation to retinal ischemia and relevance to glaucoma. Surv Ophthalmol. 1999;43(suppl 1)S102–S128.
  6. Osborne NN, Larsen A, Barnett NL. Influence of excitatory amino acids and ischemia on rat retinal choline acetyltransferase-containing cells. Invest Ophthalmol Vis Sci. 1995;36:1692–1700.[Abstract/Free Full Text]
  7. Lucas DR, Newhouse JP. The toxic effects os sodium L-glutamate on the inner layers of the retina. Arch Ophthalmol. 1957;58:193–201.[Abstract/Free Full Text]
  8. Matini P, Moroni F, Lombardi G, Faussone-Pellegrini MS. Ultrastructural and biochemical studies on the neuroprotective effects of excitatory amino acid antagonists in the ischemic rat retina. Exp Neurol. 1997;146:419–434.[CrossRef][ISI][Medline][Order article via Infotrieve]
  9. Siliprandi R, Canella R, Carmignoto G, et al. N-methyl-D-aspartate-induced neurotoxicity in the adult rat retina. Vis Neurosci. 1992;8:567–573.[ISI][Medline][Order article via Infotrieve]
  10. Li Y, Schlamp CL, Nickells RW. Experimental induction of retinal ganglion cell death in adult mice. Invest Ophthalmol Vis Sci. 1999;40:1004–1008.[Abstract]
  11. Manabe S, Lipton SA. Divergent NMDA signals leading to proapoptotic and antiapoptotic pathways in the rat retina. Invest Ophthalmol Vis Sci. 2003;44:385–392.[Abstract/Free Full Text]
  12. Olney JW. Glutamate-induced retinal degeneration in neonatal mice. electron microscopy of the acutely evolving lesion. J Neuropathol Exp Neurol. 1969;28:455–474.[ISI][Medline][Order article via Infotrieve]
  13. Yoles E, Schwartz M. Degeneration of spared axons following partial white matter lesion: implications for optic nerve neuropathies. Exp Neurol. 1998;153:1–7.[CrossRef][ISI][Medline][Order article via Infotrieve]
  14. Yoles E, Schwartz M. Elevation of intraocular glutamate levels in rats with partial lesion of the optic nerve. Arch Ophthalmol. 1998;116:906–910.[Abstract/Free Full Text]
  15. Morgan IG. AMPA is a powerful neurotoxin in the chicken retina. Neurosci Lett. 1987;79:267–271.[CrossRef][ISI][Medline][Order article via Infotrieve]
  16. Morgan IG, Ingham CA. Kainic acid affects both plexiform layers of chicken retina. Neurosci Lett. 1981;21:275–280.[CrossRef][ISI][Medline][Order article via Infotrieve]
  17. Zeevalk GD, Nicklas WJ. Chemically induced hypoglycemia and anoxia: relationship to glutamate receptor-mediated toxicity in retina. J Pharmacol Exp Ther. 1990;253:1285–1292.[Abstract/Free Full Text]
  18. Izumi Y, Benz AM, Kurby CO, et al. An ex vivo rat retinal preparation for excitotoxicity studies. J Neurosci Methods. 1995;60:219–225.[CrossRef][ISI][Medline][Order article via Infotrieve]
  19. Ehrlich D, Morgan IG. Kainic acid destroys displaced amacrine cells in post-hatch chicken retina. Neurosci Lett. 1980;17:43–48.[CrossRef][ISI][Medline][Order article via Infotrieve]
  20. Honjo M, Tanihara H, Kido N, Inatani M, Okazaki K, Honda Y. Expression of ciliary neurotrophic factor activated by retinal Müller cells in eyes with NMDA- and kainic acid-induced neuronal death. Invest Ophthalmol Vis Sci. 2000;41:552–560.[Abstract/Free Full Text]
  21. Chen ZL, Strickland S. Neuronal death in the hippocampus is promoted by plasmin-catalyzed degradation of laminin. Cell. 1997;91:917–925.[CrossRef][ISI][Medline][Order article via Infotrieve]
  22. Tsirka SE, Gualandris A, Amaral DG, Strickland S. Excitotoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator. Nature. 1995;377:340–344.[CrossRef][Medline][Order article via Infotrieve]
  23. Tsirka SE, Rogove AD, Bugge TH, Degen JL, Strickland S. An extracellular proteolytic cascade promotes neuronal degeneration in the mouse hippocampus. J Neurosci. 1997;17:543–552.[Abstract/Free Full Text]
  24. Masos T, Miskin R. mRNAs encoding urokinase-type plasminogen activator and plasminogen activator inhibitor-1 are elevated in the mouse brain following kainate-mediated excitation. Brain Res Mol Brain Res. 1997;47:157–169.[Medline][Order article via Infotrieve]
  25. Chintala SK, Zhang X, Austin JS, Fini ME. Deficiency in matrix metalloproteinase gelatinase B (MMP-9) protects against retinal ganglion cell death after optic nerve ligation. J Biol Chem. 2002;277:47461–47468.[Abstract/Free Full Text]
  26. Canete Soler R, Gui YH, Linask KK, Muschel RJ. MMP-9 (gelatinase B) mRNA is expressed during mouse neurogenesis and may be associated with vascularization. Brain Res Dev Brain Res. 1995;88:37–52.[CrossRef][Medline][Order article via Infotrieve]
  27. Sheffield JB, Graff D. Extracellular proteases in developing chick neural retina. Exp Eye Res. 1991;52:733–741.[CrossRef][ISI][Medline][Order article via Infotrieve]
  28. Sivak JM, Fini ME. MMPs in the eye: emerging roles for matrix metalloproteinases in ocular physiology. Prog Retin Eye Res. 2002;21:1–14.[CrossRef][ISI][Medline][Order article via Infotrieve]
  29. Quigley HA, Nickells RW, Kerrigan LA, Pease ME, Thibault DJ, Zack DJ. Retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis. Invest Ophthalmol Vis Sci. 1995;36:774–786.[Abstract/Free Full Text]
  30. Rosenberg GA, Dencoff JE, McGuire PG, Liotta LA, Stetler-Stevenson WG. Injury-induced 92-kilodalton gelatinase and urokinase expression in rat brain. Lab Invest. 1994;71:417–422.[ISI][Medline][Order article via Infotrieve]
  31. McGuire PG, Alexander SM. Urokinase expression during the epithelial-mesenchymal transformation of the avian somite. Dev Dyn. 1992;194:193–197.[ISI][Medline][Order article via Infotrieve]
  32. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685.[CrossRef][Medline][Order article via Infotrieve]
  33. Zhang X, Chaudhry A, Chintala SK. Inhibition of plasminogen activation protects against ganglion cell loss in a mouse model of retinal damage. Mol Vis. 2003;9:238–248.[ISI][Medline][Order article via Infotrieve]
  34. Kipnis J, Yoles E, Schori H, Hauben E, Shaked I, Schwartz M. Neuronal survival after CNS insult is determined by a genetically encoded autoimmune response. J Neurosci. 2001;21:4564–4571.[Abstract/Free Full Text]
  35. Winding B, NicAmhlaoibh R, Misander H, et al. Synthetic matrix metalloproteinase inhibitors inhibit growth of established breast cancer osteolytic lesions and prolong survival in mice. Clin Cancer Res. 2002;8:1932–1939.[Abstract/Free Full Text]
  36. Galardy RE, Cassabonne ME, Giese C, et al. Low molecular weight inhibitors in corneal ulceration. Ann N Y Acad Sci. 1994;732:315–323.[ISI][Medline][Order article via Infotrieve]
  37. Haddad GG, Jiang C. Mechanisms of anoxia-induced depolarization in brainstem neurons: in vitro current and voltage clamp studies in the adult rat. Brain Res. 1993;625:261–268.[CrossRef][ISI][Medline][Order article via Infotrieve]
  38. Calabresi P, Marfia GA, Amoroso S, Pisani A, Bernardi G. Pharmacological inhibition of the Na(+)/Ca(2+) exchanger enhances depolarizations induced by oxygen/glucose deprivation but not responses to excitatory amino acids in rat striatal neurons. Stroke. 1999;30:1687–1694.[Abstract/Free Full Text]
  39. Clark B, Mobbs P. Transmitter-operated channels in rabbit retinal astrocytes studied in situ by whole-cell patch clamping. J Neurosci. 1992;12:664–673.[Abstract]
  40. Grossmann J. Molecular mechanisms of "detachment-induced apoptosis-anoikis". Apoptosis. 2002;7:247–260.[CrossRef][ISI][Medline][Order article via Infotrieve]
  41. Meredith JE, Jr, Fazeli B, Schwartz MA. The extracellular matrix as a cell survival factor. Mol Biol Cell. 1993;4:953–961.[Abstract]
  42. Libby RT, Xu Y, Selfors LM, Brunken WJ, Hunter DD. Identification of the cellular source of laminin beta2 in adult and developing vertebrate retinae. J Comp Neurol. 1997;389:655–667.[CrossRef][ISI][Medline][Order article via Infotrieve]
  43. Sarthy PV, Fu M. Localization of laminin B1 mRNA in retinal ganglion cells by in situ hybridization. J Cell Biol. 1990;110:2099–2108.[Abstract/Free Full Text]
  44. Miner JH, Lewis RM, Sanes JR. Molecular cloning of a novel laminin chain, alpha 5, and widespread expression in adult mouse tissues. J Biol Chem. 1995;270:28523–28526.[Abstract/Free Full Text]
  45. Gary DS, Mattson MP. Integrin signaling via the PI3-kinase-Akt pathway increases neuronal resistance to glutamate-induced apoptosis. J Neurochem. 2001;76:1485–1496.[CrossRef][ISI][Medline][Order article via Infotrieve]
  46. Borden P, Heller RA. Transcriptional control of matrix metalloproteinases and the tissue inhibitors of matrix metalloproteinases. Crit Rev Eukaryot Gene Expr. 1997;7:159–178.[ISI][Medline][Order article via Infotrieve]
  47. Gottschall PE, Yu X. Cytokines regulate gelatinase A and B (matrix metalloproteinase 2 and 9) activity in cultured rat astrocytes. J Neurochem. 1995;64:1513–1520.[ISI][Medline][Order article via Infotrieve]
  48. Vecil GG, Larsen PH, Corley SM, et al. Interleukin-1 is a key regulator of matrix metalloproteinase-9 expression in human neurons in culture and following mouse brain trauma in vivo. J Neurosci Res. 2000;61:212–224.[CrossRef][ISI][Medline][Order article via Infotrieve]
  49. Hu S, Sheng WS, Ehrlich LC, Peterson PK, Chao CC. Cytokine effects on glutamate uptake by human astrocytes. Neuroimmunomodulation. 2000;7:153–159.[CrossRef][ISI][Medline][Order article via Infotrieve]
  50. Kido N, Inatani M, Honjo M, et al. Dual effects of interleukin-1beta on N-methyl-D-aspartate-induced retinal neuronal death in rat eyes. Brain Res. 2001;910:153–162.[CrossRef][ISI][Medline][Order article via Infotrieve]
  51. Yabuuchi K, Minami M, Katsumata S, Satoh M. In situ hybridization study of interleukin-1 beta mRNA induced by kainic acid in the rat brain. Brain Res Mol Brain Res. 1993;20:153–161.[Medline][Order article via Infotrieve]
  52. Zhang X, Chintala SK. Influence of interleukin-1 beta induction and mitogen-activated protein kinase phosphorylation on optic nerve ligation-induced matrix metalloproteinase-9 activation in the retina. Exp Eye Res. 2004;78:849–860.[CrossRef][ISI][Medline][Order article via Infotrieve]
  53. Yamada H, Fredette B, Shitara K, et al. The brain chondroitin sulfate proteoglycan brevican associates with astrocytes ensheathing cerebellar glomeruli and inhibits neurite outgrowth from granule neurons. J Neurosci. 1997;17:7784–7795.[Abstract/Free Full Text]
  54. Hsueh YP, Yang FC, Kharazia V, et al. Direct interaction of CASK/LIN-2 and syndecan heparan sulfate proteoglycan and their overlapping distribution in neuronal synapses. J Cell Biol. 1998;142:139–151.[Abstract/Free Full Text]



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