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(Investigative Ophthalmology and Visual Science. 2004;45:1247-1258.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.03-1123
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Microarray Analysis of Changes in mRNA Levels in the Rat Retina after Experimental Elevation of Intraocular Pressure

Farid Ahmed,1,2 Kevin M. Brown,3,4 Dietrich A. Stephan,4 John C. Morrison,5 Elaine C. Johnson,5 and Stanislav I. Tomarev1

1From the Laboratory of Molecular and Developmental Biology, National Eye Institute, Bethesda, Maryland; 3Children’s National Medical Center, Washington, DC; the 4Neurogenomics Division, The Translational Genomics Research Institute, Phoenix, Arizona; and 5The Kenneth C. Swan Ocular Neurobiology Laboratory, Casey Eye Institute, Oregon Health Science University, Portland, Oregon.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. The goal of this study was to identify altered patterns of retinal mRNA expression after experimental elevation of intraocular pressure (IOP) in a rat glaucoma model.

METHODS. Brown Norway rats (N = 16) received unilateral episcleral vein injection of hypertonic saline to elevate IOP. IOP was monitored daily by handheld tonometer, and retinas were collected 8 days and 5 weeks after surgery. Comparison of mRNA levels between experimental and fellow retinas was made using gene microarrays (rat U34A rat arrays; Affymetrix, Santa Clara, CA). Semiquantitative RT-PCR was used to confirm selected results from array analysis and to compare with alterations after optic nerve transection.

RESULTS. IOP elevation for 5 weeks resulted in reproducible changes in levels of 81 mRNAs. Of these, 74 increased, whereas only 7 decreased. The expression levels of 27 of these same messages were changed after 8 days of IOP elevation. In addition, four other genes demonstrated altered expression after the shorter period of elevated IOP exposure. Approximately half of the mRNAs with altered expression were associated with either neuroinflammatory responses or apoptosis. For 25 of the selected functionally relevant messages altered by array analysis, the alterations were confirmed by semiquantitative RT-PCR. The levels of 24 of 25 selected messages were also changed after optic nerve transection.

CONCLUSIONS. The activation of glia and the complement system after IOP elevation, which is similar to that described in several neurodegenerative diseases and after optic nerve transection, suggests that this rat glaucoma model could be used to evaluate the neuroprotective potential of therapeutic agents that target these processes.

Glaucoma is a term defining a group of optic neuropathies characterized by the death of retinal ganglion cells (RGCs) accompanied by excavation and degeneration of the optic nerve head. Risk factors in glaucoma include elevated intraocular pressure (IOP), age, race, family history, myopia, and diabetes. RGC loss and optic nerve (ON) degeneration in glaucoma are typical hallmarks of a neurodegenerative disease. RGC loss may also be induced by ON transection, which leads to molecular changes in the retina that partially overlap those induced by elevated IOP.1

There is a growing body of evidence that pathologic cascades leading to different neurodegenerative disorders, such as age-related macular degeneration2 and Alzheimer’s3 4 and Parkinson’s5 6 diseases involve a neuroinflammatory response. This response is characterized by activation of microglial cells and astrocytes and an increase in the levels of major histocompatibility complex (MHC) class I and II antigens, cytokines, and cell adhesion molecules and complement activation.2 3 4 6 7 Although progression of glaucoma in human or experimental animal models has been documented by morphologic changes in the retina and ON, data describing the molecular changes in the eye with progression of glaucoma are still limited.1 8 9 10 11 12 13 14 15 16 17 18 19 20 Elucidation of the molecular changes in the different tissues of the eye affected by glaucoma may lead to a better understanding of improved treatment for this blinding disease.

High-throughput cDNA and oligonucleotide microarray hybridization methods allow a rapid and comprehensive approach for identifying changes in gene expression patterns in glaucoma. These methods have been successfully used to identify changes in gene expression associated with different physiological and pathologic states, including aging,21 22 tumors,23 neurodegenerative diseases,24 25 and psychiatric disorders.26 Microarray analysis has been recently applied to the study of glaucoma. Changes in the astrocytes cultured from glaucomatous and normal optic nerve heads27 and in perfused, intact human trabecular meshwork in response to elevated intraocular pressure28 have been described. It is difficult to study changes in the gene expression pattern in the human retina during the course of glaucoma, because only postmortem human retinal samples can be obtained. Therefore, appropriate animal models may provide valuable information about the molecular events in the retina and the ON in the course of glaucoma.

Animal models for glaucoma rely on elevation of IOP, the dominant glaucoma risk factor. Although the monkey model may provide the best insight into the processes in the human glaucomatous retina and ON,29 30 the cost and limited availability of monkeys make them difficult to use for pilot studies. Several rat models of elevated-pressure–induced ON damage have been developed. In these models, IOP elevation has been achieved by injection of concentrated saline solution into the episcleral vein,31 cauterization of veins,32 trabecular laser photocoagulation after injection of India ink into the anterior chamber,33 or a laser injury to the trabecular meshwork.34 Chronic IOP elevation is accompanied by RGC loss, ON degeneration, and optic nerve head remodeling similar to that observed in human glaucoma.31 33 35 36 37 38 Chronic IOP elevation alters the levels and changes the distribution of selected mRNAs and proteins in the retina.1 16 17 19 20 37 39 40 41 Many of these changes are associated with altered axonal transport, glial activation, neurotrophin depletion, apoptosis, and RGCs loss.

In this study, we used oligonucleotide arrays to study changes in retinal mRNA levels in rats after experimental elevation of IOP by hypertonic saline injected into the aqueous humor outflow pathway.31 Our data demonstrate that there were reproducible changes in mRNA levels for 85 genes after 8 days or 5 weeks of elevated IOP. Of these mRNAs, 4 were altered only at the early time point, 27 were altered at both time points, and 81 were altered at the late time point. Functionally, the most abundant group of genes exhibiting modified expression after elevation of IOP has been associated with a neuroinflammatory response. Other abundant groups of genes with modified expression encode cytoskeletal and extracellular proteins, secreted glycoproteins, transcription factors, and proteases and their inhibitors. Changes in mRNA levels of 25 genes with modified expression after IOP elevation were also estimated after ON transection, by using semiquantitative RT-PCR. The expression of 24 of 25 mRNAs tested was changed in the same direction in both models of RGC degeneration.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Experimental Animals
All experiments complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Sixteen male Brown Norway rats weighing 300 to 400 g were used. Elevation of IOP in one eye of each animal was induced by injection of 50 µL of 1.75 M hypertonic saline solution through the episcleral vein to produce scarring and obstruction of aqueous humor outflow, as described previously.31 A handheld tonometer (TonoPen XL; Mentor, Norwood, OH) was used to measure IOP daily in awake animals, as described.31 42 Mean and peak IOP levels are presented as tonometer readings. Rats were killed 8 days (n = 4) or 5 weeks (n = 12) after the surgery, and the degree of ON damage was then estimated independently by five masked observers, as described.43 A grade scale from 1 (normal) to 5 (degeneration affecting the entire nerve area) was used, based on prior observations of a stereotyped pattern of injury in this model.31

ON transection was performed in six additional rats, as described.1 The left ON was cut in each animal, and the right eye served as a sham-operation control in which the surgery was performed without cutting the ON. Animals were euthanatized 10, 16, and 21 days after the surgery.

High-Density Oligonucleotide Microarray Analysis
Total RNA was isolated from the dissected retina (Total RNA Miniprep kit; Stratagene, La Jolla, CA). Total RNA (0.5 µg) was separated by electrophoresis on a 1.2% agarose, 2.2 M formaldehyde gel to evaluate the quality of RNA samples. Only undegraded samples of comparable quality were used for cRNA synthesis. For each reverse transcription reaction (SuperScript II; Invitrogen, Carlsbad, CA), 16 µg of total RNA was pooled from two experimental or two control retinas. Five independent pools of control and experimental retinas were used in these experiments.

Pooled RNA was processed for use on commercially available gene microarrays (U34A rat arrays; Affymetrix, Santa Clara, CA), according to the manufacturer’s protocol. Raw fluorescence intensity data were used to calculate signal intensities for each oligonucleotide probe set by the accompanying software (Microarray Suite 5.0; Affymetrix). The fluorescence intensity of each chip was linearly scaled to an average target intensity of 800, permitting reproducible interarray comparisons. Probe set hybridization performance identified signal intensities that were reliably detected as present and eliminated most nonspecific cross-hybridization signals, as previously described.44

Each microarray underwent a stringent quality control evaluation, including cRNA amplification more than fourfold, scaling and normalization factor of 0.5 to 5, percentage of probe sets reliably detecting more than 30% present call, 3'-5' ratio of GAPDH gene less than 3, and correlation coefficient R < 0.90 of mean signal intensities for each transcript between microarrays at the same experimental time point. In addition, quality control plots were generated using scaling factor and the percentage of present probe sets to identify systematic errors in the expression the profiling process (data not shown).

Semiquantitative RT-PCR
For cDNA synthesis, 1 µg of total RNA was reverse transcribed (SuperScript II; Invitrogen) and oligo(dT)-primer. The amount of synthesized cDNA was normalized by PCR using primers specific for cyclophilin and ribosomal protein L19 (Rpl 19; Table 1 ). PCR reactions were performed in a thermal cycler (PTC-200; MJ Research, Watertown, MA) using Taq polymerase (AmpliTaq; Applied Biosystems, Foster City, CA). Each PCR reaction was repeated at least twice. The thermal cycling parameters were as follows: 1 minute 30 seconds at 94°C, followed by 30 cycles of 30 seconds at 94°C, 1 minute 30 seconds at 59°C, and 1 minute at 72°C, and final incubation for 5 minutes at 72°C. PCR reaction products were analyzed by agarose gel electrophoresis. After adjustment of cDNA concentration in each pair of samples from the control and experimental eyes of the same animal, the relative abundance of mRNA for different genes was quantitated. Primers for each gene were located in different exons when possible. Different dilutions of cDNA samples were used to provide a linear range for the PCR reactions. The intensity of DNA bands was estimated using gel-digitizing software (UN-SCAN-IT; Silk Scientific Inc., Orem, UT).


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  		TABLE 1. Oligonucleotides Used in the Study

 
Immunohistochemical Staining
Rats (n = 15) with unilateral IOP elevation were perfused with paraformaldehyde, the experimental and fellow eyes embedded in paraffin, and the globe sections prepared and immunostained with antibodies to glial fibrillary acidic protein (GFAP) diluted to 2 µg/mL (Dako, Carpinteria, CA), as previously described.37


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Experimental Strategy
In the experimental glaucoma array studies, one eye served as the control while the second eye was experimentally treated. To detect the most common variations in mRNA levels between control and experimental retinas, we used RNA pools from two control or two experimental samples from animals with the same duration of IOP elevation for all array hybridizations. It has been demonstrated that sample pooling can reduce noise, while still allowing identification of most of the most significant gene expression changes that would have been detected by larger numbers of individual profiles.44

Table 2 provides data on average IOP as well as injury grade in the eyes after saline injection. Animals were killed 8 (n = 4) or 35 days (n = 12) after the surgery to detect early and late changes in the gene expression profile, respectively. The degree of the ON damage was less pronounced as a rule in eyes exposed to elevated IOP for a short period. Eyes exposed to elevated IOP for 35 days demonstrated different degrees of ON damage. Only eyes with grade 5 nerve damage at 35 days were used for microarray analysis at this time point (Table 2) . Validation of the array hybridization results for 25 functionally relevant genes was achieved by semiquantitative RT-PCR analysis of individual RNA pairs. One additional animal (experimental eye 591) was also used in the RT-PCR experiments.


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  		TABLE 2. Average IOP, Duration, Degree of Optic Nerve Damage, and Peak IOP in the Experimental Eyes after Injection of Hypertonic Saline Solution

 
Changes in the expression pattern of these selected genes were also analyzed after ON transection, by semiquantitative RT-PCR, to compare molecular changes in the retina induced by elevated IOP with another form of insult to the ON.

Elevated IOP-Induced Changes in the Retina
Pools of sample RNA were labeled and hybridized to the rat microarrays (U34A Rat Arrays; Affymetrix), containing oligonucleotides corresponding to approximately 8000 expressed genes. Therefore, the array represented approximately 20% to 25% of the estimated number of genes in the rat genome. Approximately 40% of the probe sets gave detectable signals after hybridization with retinal cRNA. We considered genes to be consistently changed in expression only if the average multiple of change between control and experimental pools was higher than 2 in at least two hybridization experiments. After 5 weeks of exposure to elevated IOP, 81 genes demonstrated reproducible changes in their expression levels in the retina (Table 3 , late changes). Of these mRNAs, 74 were increased and only 7 were decreased after elevation of IOP. In the retinas of eyes exposed to elevated IOP for 8 days, only 27 of the mRNAs changes seen in the 5-week specimens were found (Table 3 , early changes). Levels of four additional mRNAs, MHC class II antigen RT1-B alpha chain (Btnl2), ßB2-, {alpha}A-, and {alpha}B-crystallins, were reproducibly changed in these short-exposure retinas, but not in the group exposed to elevated IOP for 5 weeks. In general, early changes in the retina were quantitatively smaller than late changes.


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  		TABLE 3. Functional Classification of Genes that Show Differential Expression of Two-fold or Greater in at Least Two Independent Microarray Hybridization Experiments

 
Target Verification by Semiquantitative RT-PCR
Semiquantitative RT-PCR was used to verify changes in mRNA levels observed by the microarray analysis. We have demonstrated that under conditions used in our experiments, semiquantitative RT-PCR may be used to confirm changes in the levels of analyzed mRNAs.1 Eleven pairs of control and experimental retinas from eyes that were exposed to elevated IOP for 8 (n = 4) or 35 (n = 7) days were tested in these RT-PCR experiments. Twenty-five genes (see Table 3 ) were selected for these analyses. The results of semiquantitative RT-PCR experiments confirmed the results of array hybridization in all cases, although there were quantitative differences between the results of array hybridization and semiquantitative RT-PCR, as well as quantitative variations between individual pairs of control and experimental eyes. Figures 1 and 2 illustrate this point for seven genes tested with nine pairs of control and experimental retinas that were exposed to elevated IOP for 8 (n = 4; Fig. 1 ) or 35 (n = 5; Fig. 2 ) days. Quantitative differences in relative mRNA levels are often observed when different methods, such as array hybridization, Northern blot, semiquantitative RT-PCR, or real-time PCR, are used.1 27 45 46 47 48 Different reactions of individual animals to surgery may also contribute to differences observed between pairs of control and experimental animals. In our subsequent experiments with 18 genes (Fig. 3 , for example) we did not try to obtain quantitative estimates for semiquantitative RT-PCR reactions with individual pairs of control and experimental eyes but rather used semiquantitative RT-PCR for confirmation of general trends detected by array hybridization.



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  		FIGURE 1. Estimation by semiquantitative RT-PCR of mRNA levels of seven genes in total retina after a hypertonic saline injection. Control (C) and experimental (E) samples from eyes after an 8-day exposure to elevated IOP (early changes). Cyclophilin mRNA levels were used for normalization. Numbers below each panel show the calculated differences between control and experimental samples. Numbers on the right show average differences between control and experimental samples, as judged by semiquantitative RT-PCR and array hybridization.

 


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  		FIGURE 2. Estimation, semiquantitative RT-PCR, of mRNA levels of seven genes in total retina after a hypertonic saline injection. Control (C) and experimental (E) samples from eyes after 35 days’ exposure to elevated IOP (late changes). Cyclophilin mRNA levels were used for normalization. Description of numbers is provided in Figure 1 .

 


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  		FIGURE 3. Estimation, by semiquantitative RT-PCR, of mRNA levels of eight genes in total retina after a hypertonic saline injection. Control (C) and experimental (E) samples from eyes after 35 days’ exposure to elevated IOP (late changes).

 
Similarities between Changes Induced by ON Transection and Hypertonic Saline Injection
To compare molecular changes in the retina induced by elevated IOP and ON transection, we used semiquantitative RT-PCR to determine whether the 25 genes differentially regulated in the IOP model and tested by semiquantitative RT-PCR show changed expression after ON transection. Twenty-four of 25 mRNAs tested changed their levels in the same direction in both models. The only exception was mRNA for Egr1, also known as Krox24, whose level did not change after ON transection. Figure 4 shows the results of semiquantitative RT-PCR for 16 tested genes. On the basis of these results we concluded that elevated IOP and ON transection may induce overlapping molecular changes in the rat retina.



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  		FIGURE 4. Estimation, by semiquantitative RT-PCR, of mRNA levels of 16 genes in total retina after ON transection. C, control; E, experimental. Numbers show days after transection.

 
Activation of Glial Cells in the Retina after IOP Elevation
The level of GFAP mRNA was reproducibly increased in the retinas from eyes exposed to elevated IOP for both 8 and 35 days (Table 3) . Previous experiments established that the elevated levels of GFAP mRNA in the experimental retinas correlated with the elevated levels of GFAP protein, as judged by ELISA assay (Jia, et al. IOVS 2002;43:ARVO E-Abstract 4047). Elevation of the GFAP level may reflect the activation of astrocytes and Müller cells which are, together with microglial cells, the major glial cells in the retina. Immunohistochemical staining of retinal sections demonstrated a clear increase in GFAP immunoreactivity in the nerve fiber layer of experimental retinas from the eyes with a severe degree of ON damage compared with control retinas (Fig. 5) . Moreover, staining of the glial processes in the inner plexiform layer was evident in some parts of the retina (Fig. 5B) . We concluded that elevated IOP itself or RGC injury may lead to glial activation.



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  		FIGURE 5. GFAP immunohistochemical staining of the rat retina. Sections of a control (A) and a grade-5 experimental (B, C) retina were stained with GFAP antibodies using 3,3'-diaminobenzidine (DAB; brown) as the chromogen. Nuclei are counterstained with hematoxylin (blue). (B, arrows) Radial glial processes in the inner nuclear layer.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Glaucoma is a neurodegenerative disease that is defined by specific morphologic changes in the retina and optic nerve head. Rat models of pressure-induced ON and retinal damage reproduce many of the morphologic and molecular changes observed in glaucoma in humans. These models allow the investigation of the dynamics of morphologic and molecular changes during progressive damage. Although there are several papers describing molecular changes in the glaucomatous retina in humans or in animal models at the level of individual specific genes and proteins,8 13 14 9 1 39 41 this is the first report to provide a general survey of early and late changes in the retina in response to elevated IOP.

To study global changes in the gene expression patterns in the retina after elevation of IOP, we used oligonucleotide microarray expression profiling. Available data suggest that Affymetrix oligonucleotide microarrays give a more accurate and comprehensive picture of gene expression patterns than data from long cDNA microarrays.49 Besides monitoring changes in the expression patterns of individual genes, microarrays may be also helpful in identification of specific pathways activated in the pathologic course of glaucoma.

The experimental strategy that we used to select mRNAs with modified levels of expression after IOP elevation (sample pooling, reproducibility in at least two hybridization experiments, and the cutoff value of twofold for the multiple of change) produced reliable results. Changes in the mRNA levels for all 25 selected genes were confirmed by semiquantitative RT-PCR. Under the conditions used in our experiments, semiquantitative RT-PCR was a quick and nonexpensive method to confirm the direction of changes detected by array hybridization. However, semiquantitative RT-PCR is not perfect for a quantitative presentation of the observed changes, and we did not try to tabulate all changes in the gene expression levels observed by this method, but present only some quantitative estimates as an illustration (see Figs. 1 2 ).

Death of the RGCs is a hallmark of glaucoma, whereas morphologic changes in other retinal cell layers are much less pronounced.50 Death of RGCs, as judged by TUNEL staining37 and caspase activation,20 51 has been documented after experimental elevation of IOP by hypertonic saline injection into the aqueous humor outflow pathways. It is possible that molecular and morphologic changes in the RGCs may lead to molecular changes in other retinal cell layers, even in the absence of pronounced morphologic changes in these retinal layers. There are several reports describing changes in the photoreceptor layer52 and electroretinograms in glaucomatous retina.53 54 55 Studies of animal models of glaucoma suggest that amacrine cells may also be affected in glaucoma.39 In humans, the process of glaucoma may be associated with potential glial cell proliferation throughout most of the retina and increased immunostaining for GFAP.56 Several studies in animal models of glaucoma have suggested an activation of glial cells in the retina shortly after elevation of IOP39 40 57 or even after sham operations.39

Using array hybridization, we demonstrated that 85 genes, or approximately 1% of the genes on the arrays, showed consistent changes in expression after exposure of rat eye to elevated IOP. However, because only approximately 40% of the 8000 genes on the arrays gave hybridization signals, the amount of genes that changed their expression levels is probably higher. For example, we have previously demonstrated that the level of myocilin mRNA decreased in the retina after experimental IOP elevation.1 Although the oligonucleotide probe set for the myocilin gene was present on the rat U34A array, the hybridization signals were too low for this set to evaluate changes in the myocilin mRNA levels. Among the 85 genes with modified expression, 75 were upregulated, and only 10 were downregulated after elevation of IOP. In general, there was a correlation between the duration of IOP elevation and degree of retinal damage, as judged by the ON injury grade on one side and changes in mRNA levels on another side. We detected changes in the expression levels of 31 and 81 genes at early and late stages of retinal damage, respectively; 27 of these genes were the same. For these common genes, the magnitude of change was greater at late stages for 23 genes, with 4 genes (complement component C3, ß-2-microglobulin, Trmp-2, and lysozyme) showing similar changes at early and late stages. Three mRNAs were highly reduced at early stages and were not significantly changed at late stages. These mRNAs encoded {alpha}A-, {alpha}B-, and ßB2-crystallins. The reduced levels of mRNA encoding crystallins have been reported recently in the retina of mouse with diabetic retinopathy (Farjo, et al. IOVS 2003;44:ARVO E-Abstract 3297). At the same time, photoreceptor damaging exposure to intense light increased levels of several crystallin proteins in two to three times in light-exposed retinas compared with control retinas.58 The significance of these observations is not clear at present.

A specific deformation of the optic nerve head is a characteristic feature of glaucoma and is not observed in other forms of retinal degeneration associated with death of RGCs. For example, transection of the ON leads to a fast degeneration of the RGC layer without cupping of the ON.59 Therefore, we were interested in comparing molecular changes in the retina induced by elevated IOP and by ON transection to find specific markers that may distinguish IOP induced retinal damage from other forms of retinal degeneration. First, we tested the same 25 genes that were used for confirmation of the array data by semiquantitative RT-PCR. With one exception, mRNAs with changed levels after IOP elevation also had changes in expression after ON transection, suggesting that the two insults may activate overlapping molecular pathways and that the elevated IOP model is primarily an inner retinal injury. One mRNA that was upregulated after elevation of IOP and did not show detectable changes after ON transection encoded Egr1 protein. Egr1 transcription factor is a critical regulator of proliferation, differentiation, and apoptosis.60 It has been demonstrated by immunohistochemical methods that Egr1/Krox24 are upregulated in rat RGCs 24 hours after ON crush, reach maximum expression after 5 days, and return to the normal low level after 8 days.61 Because 10 days was the earliest time point after ON transection that was used in this work, our mRNA measurements are consistent with previous observations at protein levels. The elevated mRNA level for Krox24 in glaucomatous eyes is consistent with the ongoing RGC damage due to elevated IOP.

It is now well documented that neurodegenerative diseases, including Alzheimer’s,4 3 Parkinson’s,5 age-related macula degeneration,2 Tay-Sachs, and Sandhoff,62 are associated with the expression of characteristic proteins that are involved in inflammatory responses that characterize normal aging,22 hypertension,63 cardiovascular diseases,64 cancer,65 and many other diseases.66 Our data (Table 3) demonstrate that a significant fraction of the genes with modified expression in the retina are associated with inflammatory and immune response. These results cannot be explained by the overrepresentation of the genes involved in inflammation and immune response on the arrays, because such genes represent only a small percentage of the genes on the rat U34A arrays.

In glaucoma and other central nervous system (CNS) injuries, the inflammatory responses are likely to stem from glial activation, leading to the expression and release of inflammatory mediators, such as acute-phase proteins, proteases, complement, and cytokines.67 Our data (see Fig. 5 , Table 3 , and the following discussion) indicate that elevated IOP or RGC degeneration may activate glial cells in the experimental retinas. As has been shown in other model systems, activated glial cells may rapidly react to neuronal damage and play an important role in the protection of neural cells by destruction of pathogens and promotion of tissue repair. At the same time, they may kill cocultured neurons in vitro through the release of nitric oxide, reactive oxygen species, and cytokines, which may occur in vivo.68 Therefore activated glial cells potentially have both neuroprotective and pathogenic roles.

mRNA for Cebpd, complement components and contrapsin, which were increased in response to elevated IOP (see Table 3 ), are all induced by IL-1ß.69 Cytokines IL-1{alpha} and -1ß are expressed at relatively low levels in the retina and we did not detect them by array hybridization. Despite this, preliminary semiquantitative RT-PCR experiments demonstrated that the mRNA levels of IL-1ß and its receptor IL-1ß were increased after elevation of IOP (not shown). In addition, although we did not detect changes in the expression pattern of the TNF-{alpha} gene, array hybridization experiments demonstrated activation of Litaf, which has been implicated in the upregulation of the TNF-{alpha} gene.70

Recent data suggest that neurons express a significant number of molecules that were originally thought to mediate cell–cell interactions exclusively in immune function. In particular, class I MHC molecules, which were increased in our array studies, may play an essential role in neuronal signaling, activity-dependent changes in synaptic connectivity, and structural remodeling in the developing and mature CNS.71 72 Class I MHC is present in a specific subset of CNS neurons where it colocalizes with ß-2-microglobulin, a cosubunit of class I MHC. In adult rat brain, class I MHC and ß-2-microglobulin are highly expressed in brain stem and spinal motorneurons, as well as in nigral dopaminergic neurons.73 Neurons expressing these molecules are most vulnerable to neurodegeneration in diseases such as Parkinson’s. In the mouse retina, class I MHC mRNA is expressed in RGCs.74 Its increased expression in our elevated IOP rat retinas suggests a role for MHC in glaucoma.

Morphologic changes in the retina in the course of glaucoma may involve a remodeling of the tissue and changes in the extracellular matrix.75 Increased expression of neural cell adhesion molecule and tenascin have been reported in the glaucomatous human optic nerve head compared with the normal one.10 12 Several mRNAs encoding extracellular matrix proteins were upregulated in the surgically treated eyes in our experiments (Table 3) . SPARC1 mRNA demonstrated the most pronounced upregulation. It has been previously demonstrated that SPARC1 is expressed in reactive astrocytes and is activated subsequent to different neural traumas, including neurodegenerative diseases and acute neural damage.63 76 There are other similarities with human glaucoma observed with the model we used. Endothelin-1 mRNA is detected in the inner plexiform, ganglion, and nerve fiber layers in the human retina,77 and endothelin-1 peptide is elevated in aqueous humor in some patients with primary open-angle glaucoma,78 and may cause proliferation of astrocytes in the optic nerve head.79 TIMP-1 protein appears to be increased in microglia and other types of cells from the nerve fiber layer to the ON bundles in the glaucomatous optic nerve head.80 GFAP is increased in glaucomatous human retina,56 and Hsp27 and vimentin are increased in cultured human optic nerve head astrocytes after elevation of IOP.81 All these genes were upregulated in our model. Decreased amounts of neurofilament proteins have been reported in a monkey model of glaucoma.82 mRNA levels for NF-H, NF-M, and NF-L were also decreased in the model used in this study.

It must be remembered that in this study we evaluated retinal responses to elevated IOP, not the responses of the optic nerve head, which is the most likely site of early glaucomatous injury. Therefore, potentially unique and critical responses to both pressure and axonal damage in the nerve head itself remain to be evaluated.

In conclusion, the general pattern of genes activated in the retinas from eyes with elevated IOP is similar to the spectrum of genes activated in typical neurodegenerative diseases. These findings may provide new avenues for potential treatments of glaucoma. It has been suggested that inhibition of microglial activation and the complement system may be an attractive target for therapeutic intervention in Alzheimer’s disease.83 Our data suggest that a similar approach may be used in search for new glaucoma drugs.


   Acknowledgements
 
The authors thank Mario Torrado for help with isolation of several RNA samples and Joram Piatigorsky and Eric Wawrousek for critically reading the manuscript.


   Footnotes
 
2 Present affiliation: Department of Neuroscience, Georgetown University Medical Center, Washington, DC. Back

Supported by the National Institutes of Health Intramural Program, Research to Prevent Blindness, and the Glaucoma Research Foundation.

Submitted for publication October 10, 2003; revised December 15, 2003; accepted December 17, 2003.

Disclosure: F. Ahmed, None; K.M. Brown, None; D.A. Stephan, None; J.C. Morrison, None; E.C. Johnson, None; S.I. Tomarev, 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: Stanislav I. Tomarev, Section of Molecular Mechanisms of Glaucoma, Laboratory of Molecular and Developmental Biology, National Eye Institute, NIH, DHHS, Building 7, Room 103, Bethesda, MD 20814-9692; tomarevs{at}nei.nih.gov.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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