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(Investigative Ophthalmology and Visual Science. 2003;44:3025-3033.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.

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Immunohistochemical Assessment of the Glial Mitogen-Activated Protein Kinase Activation in Glaucoma

Gülgün Tezel,1 Balwantray C. Chauhan,2 Raymond P. LeBlanc,2 and Martin B. Wax3,4

1From the Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, Louisville, Kentucky; the 2Department of Ophthalmology, Dalhousie University, Halifax, Nova Scotia, Canada; 3Pharmacia Corporation, Chesterfield, Missouri; and the 4Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To determine whether retinal glial cells exhibit an activated phenotype in glaucomatous human eyes and whether the mitogen-activated protein kinases (MAPKs) are associated with glial activation in glaucoma.

METHODS. Activated phenotypes of retinal macroglia (astrocytes and Müller cells) and microglia were identified by morphologic assessment and immunostaining for the cell markers glial fibrillary acidic protein (GFAP) and HLA-DR, respectively, in 30 eyes obtained from glaucomatous donor eyes in comparison with normal control eyes from 20 age-matched donors. Cellular localization of the activated forms of MAPKs, including extracellular signal-regulated kinases (ERK), c-Jun amino(N)-terminal kinase (JNK), and p38, were studied in the retina of these eyes by immunoperoxidase staining and double immunofluorescence labeling with phosphorylation site-specific antibodies.

RESULTS. Retinal astrocytes and Müller cells exhibited a hypertrophic morphology and increased immunostaining for GFAP in the glaucomatous retina. Although an increase was detectable in the number and size of cells positive for HLA-DR immunostaining in the glaucomatous retina compared with the control retina, microglial activation was not as prominent or widespread as the macroglial activation detected in the same eyes. The intensity of immunostaining and the number of immunostained cells for the activated MAPKs were greater in retina sections from glaucomatous eyes than in control eyes, being most prominent for phospho-ERK. Double immunofluorescence labeling demonstrated that the increased retinal immunostaining for phospho-ERK was predominantly, but not exclusively, localized to glial cells, whereas, the immunostaining for phospho-JNK or phospho-p38 was mainly associated with nonglial cells.

CONCLUSIONS. These findings provide evidence that retinal glial cells undergo activation in the glaucomatous human retina. A prominent and persistent activation of ERK in activated glial cells suggests that this signaling pathway is probably associated with the induction and/or maintenance of the activated glial phenotype in glaucoma. Because MAPKs are involved in determination of ultimate cell fate, their differential activity in neuronal and activated glial cells in the glaucomatous retina may be associated, in part, with the differential susceptibility of these cell types to glaucomatous injury.

Glial cells located in the retina, as well as in the optic nerve head,1 undergo an activation process in glaucoma, as shown in different animal models2 3 and in studies with glaucomatous human donor eyes.4 Glial cells are known to support neuronal tissue by supplying metabolites and growth factors, by scavenging toxic agents,5 and by providing guidance to the axons.6 Therefore, glial activation in glaucomatous eyes may initially represent a cellular attempt to limit the extent of neuronal injury and to promote tissue repair. Consequently, activated glial cells may also have noxious effects on neuronal tissue by creating mechanical injury and/or changing the microenvironment of neurons.7 8 9 For example, activated glial cells in glaucomatous eyes produce neurotoxic substances, such as nitric oxide synthase10 and TNF-{alpha}.11 12 In addition, in vitro experiments have provided direct evidence that glial cells activated in response to glaucomatous stressors, such as elevated pressure and ischemia, are directly involved in facilitating the apoptosis of retinal ganglion cells due to increased production of apoptosis-promoting substances, including nitric oxide and TNF-{alpha}.13 These in vitro experiments demonstrate that glaucomatous stressors induce cell death in retinal ganglion cells, whereas cocultured glial cells survive the same stress conditions. This finding is in agreement with the common thought that glaucoma is a selective disease that results in the degeneration of retinal neurons, mostly the retinal ganglion cells and their axons, although the primary injury site remains elusive.14 15 16 17 However, the factors determining cellular susceptibility to glaucomatous injury, such that neurons die while glia are spared, are unclear.

Transfer of information for cell death or survival programs is hierarchically organized by the cascades of kinases, by which several adaptive/protective or pathogenic proteins are functionally activated by phosphorylation.18 Among the signal transduction pathways involved in cell fate, mitogen-activated protein kinases (MAPKs) occupy a central place.19 20 Two relatively well-characterized MAPK signaling pathways are the extracellular signal-regulated kinases (ERK; p44 MAPK/ERK1 and P42 MAPK/ERK2) and the stress-activated protein kinases (SAPK), including the c-Jun amino(N)-terminal kinase (JNK; SAPK1), and the p38 (SAPK2). These MAPKs are activated by dual phosphorylation on threonine and tyrosine residues.21 22 Activation of the ERK pathway, which is mostly initiated by mitogens and survival factors, results in the modulation of transcriptional activity leading to cell growth and differentiation.23 In contrast, the SAPK pathway is only weakly activated by mitogens, but is strongly activated by cytokines, such as TNF-{alpha}, as well as a diverse array of environmental stresses, such as UV radiation and osmotic shock,24 resulting in altered transcription, translation, and activation of factors involved in cell death.25 26 Therefore, a balance between the survival-promoting ERK pathway and the death-promoting JNK and p38 pathways is commonly accepted as regulating cell fate.27 It seems feasible that the differential responses and the ultimate fate of retinal cells during glaucomatous neurodegeneration may be similarly related to the functional activation of several proteins by phosphorylation, which may vary among different cell types, depending on kinase activity. Thus, a better understanding of the signaling cascades in glaucomatous eyes can provide information to explain the differential responses of retinal cell types to glaucomatous stress.

Although the MAPK signaling cascades have been evaluated in the brain, studies in the retina are limited, and the role of these kinase pathways in glaucoma is unknown. To identify MAPKs activated in the glaucomatous retina, using immunohistochemistry and phosphorylation site-specific antibodies, we studied the cellular distribution of the activated forms of ERK, JNK, and p38 in the retina of glaucomatous human donor eyes in comparison with normal eyes of age-matched donors. We particularly sought to determine MAPKs activated in retinal glial cells in glaucoma to obtain information about the signaling pathways associated with glial activation that occurs in these eyes. Our observations revealed a prominent and persistent activation of ERK in glial cells in the glaucomatous retina, which may be associated with the relative resistance of these cells to glaucomatous injury.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Human Donor Eyes
Thirty donor eyes with a diagnosis of glaucoma (ages, 56–94 years), and 20 normal eyes of age-matched donors (ages, 55–96 years) were obtained from the Glaucoma Research Foundation (San Francisco, CA), the Mid-America Eye Bank (St. Louis, MO), Douglas H. Johnson (Mayo Clinic, Rochester, MN), and the authors (MBW; Pharmacia Corporation and Washington University, St. Louis, MO; BCC and RPL; Dalhousie University, Halifax Nova Scotia, Canada). All the donor eyes were handled according to the tenets of the Declaration of Helsinki. Donors of normal eyes had no history of eye disease. There was no diabetes, collagen vascular disease, infection or sepsis in any of the donors. The cause of death of all the donors used in this study was acute myocardial infarction or cardiopulmonary failure. Clinical findings in donors with glaucoma were well documented and derived from intraocular pressure readings, optic disc assessments, and visual field tests (Table 1) .


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  		TABLE 1. Clinical Data for Glaucomatous Donor Eyes

 
All the donor eyes were enucleated within 2 to 4 hours after death and fixed within 6 to 12 hours. The posterior poles were dissected from the surrounding tissues, washed extensively in 0.2% glycine in phosphate-buffered saline (pH 7.4), embedded in paraffin, and oriented sagittally for cutting 6-µm sections.

Procedures
All the histologic slides subjected to immunohistochemistry were masked for the identity and diagnosis of donors. In addition, the protein examined (different cell markers or different MAPKs) was not indicated on the slides; however, all the histologic slides were numbered by a technician unfamiliar with the retina and optic nerve head disease, before their immunostaining. To control variations in the immunostaining, slides obtained from glaucomatous and control eyes, as well as the negative control slides, were simultaneously subjected to immunohistochemistry. The intensity of immunostaining was first qualitatively graded as negative (-), faint (+), moderate (++), and strong (+++) using at least five histologic sections from each donor eye. We then performed quantitative image analysis to obtain complementary information. For this purpose, the number and size of immunostained cells were determined on digitized images with the NIH Image program (http://rsb.info.nih.gov/nih-image/ available by ftp from zippy.nimh.nih.gov/ or from http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). In addition, chromogen quantity per pixel was measured using the TIFFalyzer program28 (http://www.uic.edu/com/dom/gastro/QIHC/imgs/tiffalyzer.osx.dmg/ developed by Randal Cox, Bioinformatics Group, Departments of Genetics and Medicine, University of Illinois, Chicago, IL), and the value obtained from the negative control slide was subtracted from the experimental slide to determine the intensity of immunostaining.

Donors with glaucoma whose eyes were used for immunohistochemistry were receiving antiglaucoma treatment, and their last available intraocular pressure readings were within normal limits. Because of this, and the retrospective nature of our data collection, we considered that the determination of a direct relationship between the intraocular pressure and retinal glial response would not be precisely informative. Therefore, we determined the relationship of glial response with the stage of glaucomatous damage and the type of glaucoma.

To learn whether there is a relationship between the retinal regions exhibiting glial activation and the location of visual field defects, we determined the correlation of the grading of GFAP immunostaining and visual field defects in glaucomatous eyes. Although immunoreactivity may vary between different individuals, we considered that a masked evaluation could be informative in determining the correlation of retinal GFAP immunostaining with visual field defects in corresponding retinal quadrants of individual eyes. This could be possible in 12 glaucomatous eyes, which were freshly obtained and were marked for nasal, temporal, superior, and inferior sites before their processing. Thus, it could be possible to know the retinal orientation of histologic sections and to correlate the pattern of immunostaining with functional damage in these eyes.

Visual field defects in four quadrants were also classified in these 12 eyes in a masked fashion. These classifications were based on the last available visual field test results (at most 2 years before death) in the patients’ clinical record. All the visual field test results evaluated had been obtained using a visual field perimeter (30-2 program, Humphrey Field Analyzer; Zeiss Humphrey Systems, Dublin, CA) and had met the reliability criteria of a fixation loss less than 20% and false-positive and -negative rates less than 30%. We calculated the mean of visual field indices within quadrants of the total deviation plot, and the quadrants were defined as having no, mild, moderate, or advanced visual field deficit, if the mean defect was more than -2 dB, -2 to -6 dB, -7 to -15 dB, or less than -15 dB.29 30

Immunohistochemistry
For immunoperoxidase staining, retinal sections from normal and glaucomatous eyes were deparaffinized, rehydrated, and pretreated with 3% hydrogen peroxide in methanol to decrease endogenous peroxidase activity. After the sections were washed with phosphate-buffered saline solution containing 0.1% bovine serum albumin, they were incubated with 20% inactivated normal donkey serum (Chemicon International, Inc., Temecula, CA) for 30 minutes at room temperature to block background staining. The sections were then incubated with monoclonal antibodies against glial fibrillary acidic protein (GFAP; 1:400, Sigma-Aldrich, St. Louis, MO), and HLA-DR (1:100; Accurate Chemical, Westbury, NY) to identify astrocytes and microglial cells, respectively. A mouse antibody against {alpha}-smooth muscle actin (1:800; Sigma-Aldrich) was used to identify pericytes. In addition, we used monoclonal antibodies against phospho-ERK1/ERK2 MAPK (recognizes dual phosphorylated protein at threonine 202 and tyrosine 204, 1:400) and phospho-SAPK/JNK (recognizes dual phosphorylated protein at threonine 183 and tyrosine 185, 1:1000) and a polyclonal antibody against phospho-p38 MAPK (recognizes dual phosphorylated protein at threonine 180 and tyrosine 182, 1:1000). All the phosphorylation site-specific MAPK antibodies were purchased from Cell Signaling Technology (Beverly, MA). After the sections were washed, they were incubated with the biotinylated secondary antibodies (1:400; Chemicon International, Inc.) for 1 hour at room temperature and then with avidin-biotin complex (ABC solution; Vector Laboratories, Burlingame, CA) for 1 hour at room temperature. After several washes, color was developed by incubation with 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich) used as a cosubstrate for 5 to 7 minutes. Sections were counterstained with hematoxylin and mounted (Permount; Fischer Scientific, Pittsburgh, PA). The primary antibody was eliminated from the incubation medium, or mouse serum (Sigma-Aldrich) was used to replace the primary antibody to serve as negative control. Slides were examined with a microscope (Nikon, Tokyo, Japan), and images were recorded by digital photomicrography (Optronics, Goleta, CA).

For double immunofluorescence labeling, sections were incubated with a mixture of mouse and rabbit antibodies at 1:400 dilution for 1 hour at room temperature. Primary antibodies used in double immunolabeling included the mouse antibodies against MAPKs described earlier and rabbit antibodies against GFAP (1:400), HLA-DR (1:100), {alpha}-smooth muscle actin (1:800), or Brn-3a (1:400, Chemicon International, Inc.). Brn-3a is a member of the POU-domain genes, which are known to be expressed by most ganglion cells across a variety of mammalian species.31 The sections were then incubated with a mixture of rhodamine-red and Oregon-green–labeled secondary antibodies (1:400; Molecular Probes, Eugene, OR) for another 1 hour at room temperature. Negative controls were performed by replacing the primary antibody with serum or by incubating sections with each primary antibody followed by the inappropriate secondary antibody to determine that each secondary antibody was specific to the species it was raised against. Slides were examined in a fluorescence microscope (Nikon), and images were recorded by digital photomicrography (Optronics).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
We first determined whether the retinal glial cells exhibit an activated phenotype in glaucomatous donor eyes compared with normal control eyes of age-matched donors. An activated glial phenotype was assessed by the presence of cellular hypertrophy. Increased immunostaining of glial intermediate filaments, mainly GFAP, was also considered to be an indicator of the activated macroglial phenotype. Immunoperoxidase staining with a monoclonal antibody against GFAP was therefore performed to identify the activated retinal macroglial cells: astrocytes and Müller cells. In addition, the cellular localization of HLA-DR, which is commonly accepted to be a marker for activated microglial cells,32 33 was studied by determining the immunostaining using a specific monoclonal antibody.

Our findings demonstrated that the macroglial cells exhibit a hypertrophic morphology in the glaucomatous retina, and their GFAP immunostaining is increased compared with the control retinas obtained from age-matched donors. In the control retina, immunostaining for GFAP was localized in the nerve fibers and ganglion cell layers (Fig. 1A) . This distribution pattern of GFAP immunostaining is consistent with the known localization of retinal astrocytes, which can be differentiated from the retinal ganglion cells by their characteristic darker, smaller, and irregular nucleus relative to that of ganglion cells, and by their proximity to the blood vessels of the inner retina.34 35 Another macroglial cell type in the retina, the Müller cell, is characterized by a radial orientation and processes that extend all through the retina.36 Although the cell bodies of Müller cells are located in the inner nuclear layer,34 35 in the control retina sections, no immunostaining for GFAP was detectable in this layer (Fig. 1A) . However, GFAP immunostaining in the inner retinal layer of the control eyes may also be associated with the Müller cells’ end feet, because similar to astrocytes, Müller cells contribute to the formation of the internal limiting membrane, the blood vessel glial limiting membranes, and the glial sheaths of the ganglion cells and nerve fibers.37



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  		FIGURE 1. Retinal immunoperoxidase staining for GFAP and HLA-DR. (A) Immunostaining for GFAP in control retina obtained from a normal donor eye. (B–D) Increased immunostaining for GFAP in the glaucomatous retina obtained from donor eyes with primary open-angle glaucoma with moderate damage. (B) Cell bodies of the Müller cells (black arrowhead) exhibiting immunostaining for GFAP. However, their GFAP immunostaining was negative in the control retina shown in (A). Notice the hypertrophic morphology of astrocytes and their increased GFAP immunostaining in higher-magnification image shown in (C). Another higher magnification image in (D) shows GFAP positive Müller cells. (D) Cell bodies of the Müller cells (black arrowhead) located in the inner nuclear layer exhibit immunostaining for GFAP, although GFAP immunostaining was negative in the control retina shown in (A). (E) Immunostaining of perivascular cells for HLA-DR (arrow) in a control eye. However, as shown in (F), in the glaucomatous retina from a donor with primary open-angle glaucoma with advanced damage, cells positive for HLA-DR immunostaining (arrows) were spread out within the inner retinal layers and outer plexiform layer, away from the vasculature. Double immunolabeling for (G) HLA-DR (red) and (H) {alpha}-smooth muscle actin (green), respectively. (I) HLA-DR-positive microglial cells negative for the pericyte marker {alpha}-smooth muscle actin. gc, ganglion cell; in, inner nuclear; on, outer nuclear layers; v, blood vessel. Chromogen, DAB; nuclear counterstain, Mayer’s hematoxylin. Magnification: (A, B, E, F) x120; (C, D, G, H, I) x500.

 
In the glaucomatous retina, the GFAP-positive astrocytes exhibited a hypertrophic morphology, and the intensity of their GFAP immunostaining was greater compared with the control retina (Figs. 1B 1C) . These alterations were widespread and detectable in all the slides examined, although the intensity of immunostaining and the number of cells positive for GFAP immunostaining exhibited individual or regional differences. Although no immunostaining for GFAP was detectable in the inner nuclear layer of the control retina (Fig. 1A) , in the glaucomatous retina, GFAP immunostaining was detectable in scattered cells in the inner nuclear layer, which likely correspond to the Müller cells (Fig. 1D) . Increased immunostaining for GFAP in the glaucomatous retina was also detectable through all the retinal layers in association with the processes of glial cells. Immunostaining for GFAP in the glaucomatous retina was qualitatively graded as moderate or strong. Digital image analysis showed that the size and number of glial cells exhibiting GFAP immunostaining were approximately 60% greater in the glaucomatous eyes than in the control eyes. The intensity of GFAP immunostaining (mean ± SD) was approximately five times greater (154 ± 16 vs. 33 ± 12 energy units/pixel) in the glaucomatous retina than in the control retina.

In the control retina, a small number of cells located mostly around the vasculature in the inner retinal layers exhibited immunostaining for HLA-DR (Fig. 1E) and should correspond to microglial cells, because they were negative for the pericyte marker, {alpha}-smooth muscle actin (Fig. 1G 1I) . In the glaucomatous eyes, HLA-DR–positive microglial cells were spread out within the inner retinal layers and outer plexiform layer, away from the vasculature (Fig. 1F) . In addition, the number and size of these cells, but not the intensity of their HLA-DR immunostaining, was approximately 20% greater in the glaucomatous retina than in the control retina. Thus, the alterations in the retinal microglial cells were not as prominent as that observed in the macroglial cells in the same eyes.

Increased retinal immunostaining for glial markers was similar between glaucomatous eyes with primary open-angle glaucoma and those with normal-pressure glaucoma. Increased immunostaining for GFAP in the glaucomatous retina was widespread, and despite interindividual differences, no prominent regional difference was detectable in any individual eyes. Table 2 documents the corresponding grading of visual field defects and GFAP immunostaining in 12 glaucomatous eyes. As shown in Table 2 , no association was detectable between the intensity of GFAP immunostaining and the functional damage in these eyes. Even in glaucomatous eyes with focal visual field defects, the intensity of retinal immunostaining for GFAP was mostly similar between retinal regions corresponding to decreased visual field sensitivity or normal visual field sensitivity.


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  		TABLE 2. Grading of Visual Field Damage and GFAP Immunostaining in Corresponding Quadrants

 
Immunoperoxidase staining was then performed using phosphorylation site-specific antibodies to MAPKs, including ERK, JNK, and p38, to determine the cellular localization of the activated MAPKs in glaucomatous retinas in comparison with control retinas obtained from age-matched donors. Among different MAPKs studied, most prominent immunostaining was detectable for phospho-ERK. Retinal immunostaining for phospho-ERK was widespread and detectable in all the glaucoma slides examined, although the intensity of immunostaining and the number of immunostained cells exhibited individual differences. An examination of retina sections obtained from normal donor eyes revealed faint immunostaining (24 ± 11 energy units/pixel) for phospho-ERK in scattered cells through the retina (Fig. 2A) . The intensity of immunostaining for phospho-ERK was approximately four times greater (94 ± 14 energy units/pixel) in retina sections obtained from glaucomatous donor eyes (Figs. 2B 2C) . An approximately 40% increase was detectable in the number of cells positive for phospho-ERK immunostaining in all the glaucomatous sections examined. As seen in Figure 2 , immunostaining for phospho-ERK in retina sections from glaucomatous donor eyes was detectable in cell bodies, processes, and nuclei. Based on the morphologic assessment of cell types, increased immunostaining for phospho-ERK in the glaucomatous retina was mostly, but not exclusively, associated with glial cells located in the nerve fibers and ganglion cell layers or in the inner nuclear layer. Increased immunostaining for phospho-ERK in the glaucomatous retina was also detectable through all the retinal layers, which likely corresponds to the processes of glial cells.



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  		FIGURE 2. Retinal immunoperoxidase staining for phospho-ERK. (A) Control retina; (B, C) glaucomatous retinas obtained from a donor eye with primary open-angle glaucoma with moderate damage. Although faint immunostaining for phospho-ERK was detectable in scattered cells in the control retina, there was increased immunostaining for phospho-ERK in the glaucomatous retina. Based on the morphologic assessment of cell types, increased immunostaining for phospho-ERK in the glaucomatous retina was mostly associated with astrocytes (A–C, arrows) located in the nerve fibers and ganglion cell layers, or with Müller cells (A, B, black arrowheads) located in the inner nuclear layer. However, retinal ganglion cells (A–C, white arrowheads) were negative for phospho-ERK immunostaining. Abbreviations, chromogen, and counter stain as in Figure 1 . Magnification: (A, B); x120; (C) x500.

 
Although immunostaining for phospho-ERK was widespread through the glaucomatous retina, immunostaining for phospho-JNK and phospho-p38 was detectable only in the scattered cells in these retinas. Figure 3 demonstrates retinal immunostaining for the activated ERK, JNK, and p38 in serial sections obtained from the same glaucomatous eye. Although retinal immunostaining for phospho-ERK was most prominent in glial cells (Fig. 3A) , immunostaining for phospho-JNK or phospho-p38 in the glaucomatous retina was mostly associated with the large cell bodies in the retinal ganglion cell layer, which likely correspond to the retinal ganglion cells (Figs. 3B 3C) . In addition to these cells, however, some faint immunostaining for phospho-p38 was also detectable in scattered glial cells or their processes (Fig. 3C) . Control slides in which the primary antibodies were omitted or replaced with serum were all negative for specific immunostaining for MAPKs.



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  		FIGURE 3. Immunoperoxidase staining of a glaucomatous retina for different MAPKs. Retinal immunostaining for phospho-ERK in a donor eye with primary open-angle glaucoma with advanced damage (A) was mostly associated with astrocytes (arrow) located in the nerve fibers and ganglion cell layers, or with Müller cells (black arrowhead) located in the inner nuclear layer. Retinal ganglion cells (white arrowheads) unlabeled for phospho-ERK. However, immunostaining for phospho-JNK (B) or phospho-p38 (C) in the same eye was mostly detectable in large cell bodies, probably corresponding to the retinal ganglion cells (B, C, black arrowheads). Some of the glial cells (C, arrow) also exhibit phospho-p38 immunostaining. Abbreviations, chromogen, and counterstain as in Figure 1 . Magnification, x120.

 
Double immunofluorescence labeling was performed to identify retinal cell types exhibiting immunostaining for the activated forms of MAPKs, and to determine the association of these MAPKs with the activated glial phenotype in glaucoma. Antibodies against GFAP and HLA-DR were used to identify retinal macroglial and microglial cells, respectively. Similar to the results of immunoperoxidase staining, immunofluorescence labeling for these markers was greater in glaucomatous retinas than in normal control retinas obtained from age-matched donors. Double immunolabeling demonstrated that the increased immunostaining for phospho-ERK in the glaucomatous retina was mostly associated with the activated glial cells. Phospho-ERK immunostaining was predominantly, not exclusively, colocalized with the immunostaining for GFAP (Figs. 4A 4C) or the immunostaining for HLA-DR (Figs. 4D 4F) in the glaucomatous retina. This colocalization was detectable in all the glaucoma slides examined, in which immunostaining for phospho-ERK was detectable in essentially all the activated glial cells. However, the scattered immunostaining for phospho-p38 in the glaucomatous retina was mostly localized to the cells positive for Brn-3a, a marker for retinal ganglion cells (Figs. 4G 4I) .



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  		FIGURE 4. Double immunofluorescence labeling in retina sections from glaucomatous donor eyes. (A–C) and (D–F) Double immunolabeling in retina sections obtained from two moderately damaged donor eyes with normal-pressure glaucoma. Immunostaining for (A) GFAP (green) and (B) for phospho-ERK (red); (C) colocalization (yellow) of GFAP and phospho-ERK in glial cells, astrocytes, or Müller cells (arrows). Immunostaining for (D) HLA-DR (green) and (E) phospho-ERK (red); (F) Colocalization (yellow) of HLA-DR and phospho-ERK in microglial cells (arrows). (G–I) Double immunolabeling in a moderately damaged donor eye with normal-pressure glaucoma. Immunostaining for (G) Brn-3a (green) and (H) phospho-p38 (red); (I) colocalization (yellow) of Brn-3a and phospho-p38 in some, but not all, of the retinal ganglion cells (arrowheads). Abbreviations as in Figure 1 . Magnification, x120.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Glial activation has been identified in the optic nerve head of glaucomatous human eyes1 and in the retina of experimental glaucoma models.2 3 More recently, macroglial activation has also been demonstrated in the glaucomatous human retina.4 Our findings further document glial activation in the retina of glaucomatous human donor eyes, which includes both the macroglial and the microglial components. In addition, our observations reveal prominent and persistent activation of ERK in activated glial cells in the glaucomatous retina. This finding suggests an association of ERK signaling with the induction and/or maintenance of the activated glial phenotype in glaucomatous eyes.

The classic hallmarks of glial activation are cellular hypertrophy and increased expression of glial intermediate filaments, most notably, GFAP. Although Müller cells do not express a significant amount of GFAP in normal retina, their GFAP expression has been shown to be upregulated in a wide variety of retinal pathologic states, including glaucoma.2 3 4 Thus, the hypertrophic morphology and increased GFAP immunostaining in retinal astrocytes and Müller cells in glaucomatous eyes compared with age-matched control eyes indicates that activation of retinal macroglial cells is a prominent feature of the glaucomatous retina. In addition to activation of macroglia, retinal microglial cells exhibited an increase in number and size in glaucomatous donor eyes. However, the microglial activation was not as prominent as macroglial activation detected in the same eyes. This differential activation of glial cell types has similarly been observed in the other types of neurodegeneration,38 39 in which microglial activation has occurred earlier and/or has been transient compared with macroglial activation.

Because multiple forms of injury can induce glial activation, diverse triggering factors are likely. Regarding glaucoma, two prominent stress factors identified in glaucomatous eyes, elevated pressure and ischemia, have been demonstrated to influence several aspects of the optic nerve head and retinal astrocytes, in vitro.13 40 41 42 However, whether the glial activation in glaucoma is induced by elevated intraocular pressure and/or ischemia or whether additional factors are also involved in the activation of glial cells in glaucomatous eyes is unclear. In our observations glial activation was widespread with no detectable association with intraocular pressure-dependency (primary open-angle glaucoma versus normal-pressure glaucoma) or the stage of glaucomatous damage. These suggest that even if elevated intraocular pressure is an important factor for the initiation of glial response in glaucoma, additional factors, as well as continuing intraocular pressure elevation, are probably involved in the spreading and/or persistence of glial activation in these eyes. From the perspective of neurotoxic influences of activated glial cells on retinal ganglion cells,13 the widespread and persistent nature of glial activation in glaucomatous eyes also supports the idea that the glial response may consequently contribute to spreading the damage by secondary degeneration of retinal ganglion cells, which is likely an important component of glaucomatous neurodegeneration.43

Our immunohistochemical detection of the phosphorylated (active) MAPKs demonstrated that essentially all hypertrophic active glial cells in the glaucomatous retina exhibit immunostaining for phospho-ERK. These findings suggest that ERK signaling is involved in the induction and/or maintenance of the glial activation in glaucoma. This is consistent with previous observations in the central nervous system that suggest that the activated phenotype of glial cells is, in part, under the control of ERK signaling. ERKs are widely expressed in the central nervous system44 and have been suggested to be associated with the activation of glial cells in response to a variety of injury. Both in vitro studies of brain astrocytes45 and experimental in vivo models of brain injury46 have demonstrated that the activated astrocytes in diverse pathologic lesions exhibit a chronic activation of ERK. Immunohistochemical detection of phospho-ERK in a series of human neurosurgical specimens using phosphorylation site-specific antibodies, as we used in the current study, has consistently revealed an intense immunoreactivity of the activated astrocytes in both subacute and chronic lesions, including infarction, mechanical trauma, chronic epilepsy, and progressive multifocal leukoencephalopathy. However, neurons, oligodendroglia, and most inflammatory cells have showed little or no detectable activation.47 These observations suggest that the activation of ERK signal may be a critical step for the triggering and/or persistence of glial activation. In addition, the ERK signaling pathway has been associated with p27(Kip1),48 49 which has recently been proposed to regulate the activation and proliferation of Müller cells after retinal injury.50

Immunohistochemistry in fixed specimens allows only a limited sampling of dynamic events occurring over time. However, our findings obtained from a diverse sample of glaucomatous donor eyes strongly suggest a chronic activation of the ERK pathway in chronically activated retinal glial cells in glaucoma. If the activation of glial cells and the ERK pathway were transient in glaucoma, we would have detected the GFAP and ERK immunostaining in only a small fraction of the glial cells and not consistently in the glaucomatous eyes. However, immunostaining for GFAP and phospho-ERK was detectable in all the glaucomatous sections examined.

Several mechanisms may explain the widespread and persistent nature of glial response and the prolonged ERK activation in glaucoma. First, the glial cells and the ERK pathway may be chronically activated in glaucomatous eyes because of the continuous presence of extracellular stimulatory factors, such as elevated intraocular pressure, ischemia, oxidative stress, or glutamate excitotoxicity. For example, ischemia51 and glutamate excitotoxicity,52 which are both implicated in glaucoma, have been associated with the activation of MAPKs in brain glial cells. Second, the ERK signaling pathway is activated in glial cells through an altered expression of pathway components in such a way that extracellular stimuli are not required for activation. Alternatively, autocrine stimulation may be involved in the glial response and ERK activity in glaucoma, in which several factors synthesized by activated glial cells may induce GFAP expression and ERK signaling. For example, TNF-{alpha}, the glial production of which is increased in the glaucomatous retina,53 is known to be an inducer of GFAP expression in astrocytes through ERK signaling.54 Last, a paracrine signal may mediate the spreading wave of glial activation and the activation of ERK signaling in glaucoma as elicited by brain injury. It has been demonstrated, in vitro, that the injury of approximately 5% of astrocytes is sufficient to activate ERK in the entire population of cells in a culture dish.55

Although double immunolabeling demonstrated a predominant localization of ERK signaling to activated glial cells in the glaucomatous retina, immunostaining for phospho-p38 was found to be mostly associated with nonglial cells. Activation of p38 has been detected in brain microglia and astrocytes after focal cerebral ischemia in vivo,46 56 as well as in cultured astrocytes exposed to hypoxia in vitro, which was accompanied by an induced expression of heat shock protein (Hsp)70.57 As in the ERK signaling pathway, the p38 pathway is involved in TNF-{alpha} production of glial cells,58 and in the induction of nitric oxide synthase by TNF-{alpha},59 both of which are implicated in glaucomatous neurodegeneration. Therefore, immunostaining for phospho-p38 in retinal glial cells in glaucomatous eyes, although not predominant, may signify the function of this signaling pathway in these cells in glaucoma. It is feasible to speculate that perhaps a rapid and transient activation of p38 is responsible for the characteristic subtle and localized immunostaining for phospho-p38 in retinal glial cells compared with the more diffuse immunostaining of the same cell type for phospho-ERK.

Regarding immunostaining for the activated forms of MAPKs in nonglial cells, the p38 pathway has been implicated in the death of axotomized retinal ganglion cells in chick embryos60 and in rats, in association with glutamate-related apoptosis.61 In addition to p38, we also detected, by double immunolabeling, limited immunostaining of the glaucomatous retina for phospho-JNK, which was predominantly associated with nonglial cells. This is in accordance with previous observations that JNK plays a major role in various forms of neuronal death, including axotomy-induced death.62 It should be noted that in contrast to diffuse immunostaining for phospho-ERK in glial cells throughout the glaucomatous retina, there was a more restricted pattern of immunostaining for the activated forms of JNK or p38 to scattered cells, mostly to the nonglial cells. This observation may be due to more transient activation of JNK and p38 in predominant association with dying cells.

Glial activation in glaucoma is accompanied by complex alterations in the expression of hundreds, if not thousands of genes,63 which remain uncharacterized. Differential cellular vulnerability of glial cells and retinal ganglion cells to glaucomatous damage is also unclear. In vitro studies demonstrate that glaucomatous stressors, such as elevated pressure or ischemia, induce apoptosis in retinal ganglion cells, whereas cocultured glial cells survive the same stress conditions.13 This is in agreement with observations in glaucomatous eyes that glaucoma is a relatively selective disease of retinal ganglion cells and/or their axons, whereas glial cells are preserved.14 15 16 17

Evidence suggests that the balance between the positive and negative regulators modulated by selective signaling pathways affects the survival or demise of cells in response to a noxious stimulus. The functional activation of several adaptive–protective or pathogenic proteins is known to require phosphorylation, and the cell fate (cell death or defensive-protective adaptations and survival) varies among cell types depending on kinase activity.18 Although ERK signaling has been implicated in the maintenance of neuronal cell survival after retinal injury,64 we found that the activation of this kinase pathway in neuronal cells is not predominant in the glaucomatous retina. Therefore, our observation of the activated ERK signaling predominantly in the activated glial cells in glaucomatous eyes suggests that the activity of this kinase pathway may account, in part, for the relative protection of glial cells against glaucomatous damage, whereas retinal ganglion cells undergo apoptosis. ERK signaling is indeed known to be protective against multiple noxious events,23 including those thought to be involved in glaucomatous neurodegeneration. The ERK pathway has been associated with cell survival of free radical injury,65 66 and ERKs have been demonstrated to have a dominant protecting effect over apoptotic signaling from death receptors, including TNF receptor-1.67 For example, phosphorylation of TNF receptor-1 by ERK may inhibit its apoptotic activity while preserving its ability to activate NF-KB through a bcl-2–dependent mechanism.68 69

In conclusion, in the present study retinal glial cells, including astrocytes, Müller cells, and microglia, underwent activation in glaucomatous human retina. Second, glial activation in glaucoma was associated with the activation of ERK signaling, whereas the activation of this signaling cascade was not prominent in the retinal ganglion cells. We propose that differential activity of signaling cascades determining ultimate cell fate in neuronal and activated glial cells in glaucomatous eyes may be associated, in part, with differential responses and susceptibility of these cell types to glaucomatous injury. Elucidation of specific signaling pathways involved in the triggering and maintenance of glial activation can enable the design of therapeutic tools to promote the beneficial and block the detrimental effects of glial activation and to modulate the survival of retinal ganglion cells in glaucoma.


   Acknowledgements
 
The authors thank Douglas H. Johnson (Mayo Clinic, Rochester, MN) for generously providing glaucomatous donor eyes, and Belinda McMahan for excellent technical assistance.


   Footnotes
 
Supported in part by the Glaucoma Foundation, New York, New York (GT); Glaucoma Research Foundation, San Francisco, California (GT); the American Health Assistance Foundation, Clarksburg, Maryland (GT); the University of Louisville (GT); an unrestricted grant to the Department of Ophthalmology and Visual Sciences, University of Louisville, from Research to Prevent Blindness Inc., New York, New York; and Grant MT-11357 from the Canadian Institutes of Health Research (BCC).

Submitted for publication November 7, 2002; revised February 6, 2003; accepted March 27, 2003.

Disclosure: G. Tezel, None; B.C. Chauhan, None; R.P. LeBlanc, None; M.B. Wax, Pharmacia Corporation (E)

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: Gülgün Tezel, MD, Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, Louisville, KY 40202; gulgun.tezel{at}louisville.edu.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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