Molecular Vision 2006; 12:1199-1210 <http://www.molvis.org/molvis/v12/a136/> Received 1 July 2005 | Accepted 11 August 2006 | Published 18 October 2006

Download Reprint

Retinal gene profiling in a hereditary rodent model of elevated intraocular pressure

Rita Naskar,¹ Solon Thanos^1,2
 
 

¹Department of Experimental Ophthalmology, School of Medicine, University Eye Hospital Muenster; ²Interdisciplinary Centre for Clinical Research (IZKF), Muenster Germany

Correspondence to: Dr. Rita Naskar, Interdisciplinary Centre for Clinical Research (IZKF) Domagkstrasse 3, 48149 Muenster Germany; Phone: +49 251 8352663; FAX: +49 251 8352946; email: Rita.Naskar@ukmuenster.de

Abstract

Purpose: To characterize the changes in retinal gene expression induced by elevated intraocular pressure (IOP) in a hereditary rodent model.

Methods: A rat model derived from the RCS-rdy^- strain develops IOP elevation spontaneously without experimental manipulation. Retinal gene expression after IOP elevation was compared with age-matched RCS-rdy^- retinas having normal IOP levels The MWG Rat 10k array, which comprises 9715 rat genes spotted onto one array was used. Quantitative real-time PCR (qRT-PCR) was used to verify the expression of heat shock protein-27 (Hsp-27), SA hypertension-associated gene, c-myc, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), vascular endothelial growth factor (VEGF), myocilin, interleukin-7 (IL-7), mitogen activated protein kinase 13 (MAPK-13) and crystallin beta-A1 (Cryba1). The cellular distribution of c-myc, glial fibrillary acidic protein (GFAP), VEGF, and SA was assessed using immunohistochemistry.

Results: Elevated IOP of 37.7±5.0 mmHg shifted the retina's program of gene expression, with 75 genes being upregulated (equal to or higher than 3.0 fold) and 45 genes being downregulated (equal to or lower than 0.3 fold). These genes mediate various cellular processes such as cell adhesion, cell structure, hypertension, immunity, protein sythesis, proteolysis, transcription, and signaling. The regulation pattern of SA, VEGF, c-myc, IL-7, and MAPK-13, which are uniquely regulated in our model were confirmed by qRT-PCR experiments. The regulation of Hsp-27, TIMP-1, myocilin, and Cryba1, which have previously been associated with elevated IOP were also confirmed with qRT-PCR. The protein products of c-myc, SA, and GFAP were localized to astrocytes and Müller cells. Neurons in the ganglion cell layer and inner nuclear layer were VEGF-immunopositive.

Conclusions: This study identified some of the genes that are differentially regulated, probably in response to long-term IOP exposure, in this animal model. The expression pattern of many genes is common to experimental models of elevated IOP and other retinal disorders such as diabetic retinopathy. However many genes are uniquely expressed in the retina of our model. This suggests that the mode of IOP elevation be it experimental or spontaneous could be relevant in determining which genes are regulated. Müller glia acquire a reactive phenotype as indicated by the upregulation of GFAP, c-myc, SA, and other Müller cell markers, emphasizing their relevance in pressure related- and other types of retinal injury. These data provide further evidence that IOP-mediated retinal injury is multifactorial and depends upon the interaction of different neuronal, glial, extracellular matrix, and vasogenic components.

Introduction

Elevated intraocular pressure (IOP) is the risk factor in glaucoma. In some patients, damage to the optic nerve and retinal ganglion cells (RGCs) continues despite significant reductions in intraocular pressure (IOP) [1]. It is possible that IOP inititates a self-propagating process of RGC [2,3] degeneration arising from the release of toxic substances by dying cells, such as glutamate [4] release of reactive oxygen species, changes in mitochondrial function, lack of trophic factors [5], and differential transcription of survival- or death-related genes [6]. Studies in humans and animal models suggest that RGC die at least in part by apoptosis in response to elevated IOP. Apoptosis has been observed in experimental glaucoma in rat [7] and monkey [8] as well as in humans with glaucoma [9]. Various stimuli can induce apoptosis, characterized by condensation and shrinkage of the nucleus, followed by complete fragmentation of the cell and subsequent phagocytosis of the debris by surrounding macrophages [10]. In addition to the widespread death of RGCs, other retinal neurons and glia [11,12] are also susceptible to pressure changes. Photoreceptors are affected [13-17], at least after long-term exposure to elevated pressure. Hence it is essential that the cascade of events leading to RGC death and gene expression in the entire retina are better understood.

Given the complexity of glaucomatous damage, animal models of glaucoma include the elevation of IOP by experimental manipulation [18-21], genetic models [22-25], as well as hereditary mutants [24,25]. Elevated IOP in all these models causes alterations at the histological level and leads to changes in gene expression. However, the mode of IOP elevation differs among the models, from the occlusion of aqueous humor efflux to injection of hypertonic substances and genetic alterations.

We have analyzed a substrain of the RCS-rdy^- rat that spontaneously develops IOP, RGC loss and optic nerve head cupping. A simple dose-response correlation between the magnitude and duration of IOP elevation and RGC loss exists in this hereditary rodent model, in which RGC cell loss is time dependent [25]. The present study was directed at examining retinal gene expression. We employed the oligonucleotide microarray technology to screen for changes in gene expression that develop as a consequence of long-term exposure to elevated IOP.

Methods

Animals

All experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and were carried out with the written permission of the relevant local institutional authorities. The animals were housed in a standard animal room with food and water provided ad libitum and a 12 h:12 h light-dark cycle. Experiments were carried out with the following three groups, all of which were 12 months old at the time of sacrifice.

Hereditary intraocular pressure evaluation

The mutant bred in our laboratory was derived from the Royal College of Surgeons (RCS-rdy^-) strain with animals having unilaterally enlarged globes. The first signs of asymmetric eye growth appear at two to three months, after which the affected eyes continue to increase in size. IOP values reach 35±7 mmHg by 1.5 years of age [25]. Concomitant with elevated IOP, the number of RGCs decreased with age, with 92±26 RGC/mm² remaining after 1.5 years, as compared to 1887±117 RGC/mm² in control wild type retinas. We have shown that RGC loss occurs in a time-dependent fashion in this model, thus allowing us to study the effect of gradual, chronic elevation of IOP on RGCs [25]. Elevated IOP also causes atrophy of the optic nerve head as determined by direct ophthalmoscopy. The pars plicata and pars planata of the affected ciliary body are hypertrophied and elongated, respectively. The iridocorneal angle is open in affected eyes [25]. Nine animals, aged twelve months, with elevated IOP were selected for this study. Three animals were used for the microarray study, three for the qRT-PCR experiments and three for the immunohistochemistry study.

Dystrophic retina with normal intraocular pressure

The animals with elevated IOP originated from the RCS-dystrophic strain, hence age-matched (12 months), pink-eyed, congenic RCS-rdy^- rats having inherited retinal dystrophy were used as controls for the microarray (n=3) and qRT-PCR (n=3) experiments. These animals had normal IOP levels (14.5±1.9 mmHg).

Control

Age-matched (12 months) rats of the Sprague-Dawley strain (n=9) were used as the common control against which the dystrophic and the hereditary-IOP retinas were compared. The IOP of these animals was normal 15.0±1.2 mmHg. Three animals were used as controls for each of the microarray experiments, the qRT-PCR experiments and the immunohistochemistry in order to detect possible changes in the distribution of the proteins of interest.

Intraocular pressure measurement

Intraocular pressure was measured while rats were lightly anesthetized by ether inhalation. The eyes were additionally anesthetized with a drop of topical 0.5% proparacaine (URSA-Pharm, Saarbruecken, Germany). All measurements were carried out weekly between 9:00 a.m and 12:00 p.m. using a tonometer (Tono-Pen XL, Mentor, Norwell, MA) that was calibrated before each session according to the manufacturer's instructions. On any given eye, ten tonometer readings taken directly from the display of the instrument were recorded and averaged. "Off" readings and instrument-generated averages were ignored.

Microarrays

Three rats with IOP elevation, three with retinal dystrophy but normal IOP, and three Sprague-Dawley rats (control) were used for the microarray analysis. Animals were killed in a CO₂ chamber, and their eyes were immediately enucleated and placed on ice. The retina was removed quickly and collected in RLT-Buffer, a component of the RNeasy kit (Qiagen, Hilden, Germany). A minimum of 10 μg of total RNA/retina was isolated using the RNeasy kit according to the manufacturer's instructions. Total RNA was then shipped on dry ice to MWG Biotech (Ebersberg, Germany), where an aliquot of the RNA underwent a quality analysis using the 2100 Bioanalyser system. The RNA was then amplified with T₇ polymerase following reverse transcription into cDNA during which fluorescence labeled nucleotides (Cy3/Cy5) were incorporated. The labeled probes were hybridized to 10k chips (MWG Biotech). Three separate hybridizations per group were carried out with cDNA derived from three separate animals.

The 10 k chip consists of 9715 rat genes (5535 Rat 5 k genes) spotted onto one array with an additional 4180 annotated open reading frames (ORFs) from an in-house MWG EST sequencing project. To design microarrays with optimal hybridization conditions, existing databases are filtered for redundant sequences and the oligonucleotides are designed with the Oligos-4-Array developed by MWG. This requires that non-target genes be less than 75% similar over a 50 base target region. Also, if the 50 base target region is marginally similar (50-75%), it must not include a stretch of complementary sequence >15 contiguous bases. The oligonucleotide design thus guarantees the exclusion of both dimer and secondary structure formation. Cross-hybridization is minimized by exhaustive BLAST and global Smith-Waterman searches.

The microarrays were scanned at a resolution of 10 μm at three photomultiplier gain settings in order to optimize the dynamic range. The resulting three images were integrated into one intensity value for each spot using the software packages ImaGene and GeneSight (MWG Biotech), and MAVI (MWG Biotech). The fluorescent signals were corrected and normalized for the difference between Cy3 and Cy5. Samples from each of the three cohybridizations were compared independently with each other. Using the signal values of probe sets that were reliably detected (Detection Call: "Present") in the majority of experiments per group, two-sample, two tailed t-tests between the "experimental" versus "control" groups (hereditary-IOP vs normal retina or dystrophic retina vs normal retinas) were computed. Probe sets with a t-test p<0.05 were selected as candidate genes and the ratio of means (fold change) between the two groups was calculated with "control" as denominator. The final fold-change is the average values of three independent experiments. The cut-off values for up- and downregulation were set at >3.0 and <0.3, respectively.

Genes differentially regulated in the dystrophic retina were compared with those regulated in hereditary-IOP retina in order to screen for genes regulated as a result of elevated IOP only. Most of the genes were not regulated between the two groups, whereas others were regulated in the same direction in both groups. However their description is beyond the scope of this study.

Biological function of differentially expressed genes with a greater than 3.0 fold or less than 0.3 fold change were modeled according to their biological process using the Protein ANalysis THrough Evolutionary Relationships (PANTHER) Classification System (Applied Biosystems, San Diego, CA). This allows high-throughput analysis of proteins (and their genes), which can be classified according to families and subfamilies, molecular functions, biological processes and pathways.

Quantative real-time PCR

Real-time PCR was performed on an ABI PRISM 7900 sequence detector (Applied Biosystems, San Diego, CA) in 384-well plates. For the qRT-PCR, total RNA was isolated from retinas of a second set of animals, because the RNA from the microarray experiments did not suffice for both experiments. Three rats with IOP elevation, three animals with retinal dystrophy but normal IOP, and three Sprague-Dawley rats (control) were used for qRT-PCR experiments. One microgram of total RNA was first reverse transcribed using the Omniscript Reverse Transcriptase (5 mM dNTPs, 10X RT Buffer, 10 units/μl RNase inhibitor, and 10 μM Oligo-dT primer; MWG Biotech) in a total volume of 20 μl for 1 h at 37 °C. The enzyme was inactivated by heating at 95 °C for 5 min. The cDNA was diluted twofold, of which 1 μl was used for each 20 μl PCR using the TaqMan Universal PCR Master Mix and Assays-on-Demand^TM (Applied Biosystems). Assays-on-Demand gene expression products consisted of a 20X mix of unlabeled PCR primers and TaqMan MGB probe (FAM-TAMRA-dye labeled) and were used to quantify the expression of nine genes listed as follows. Hsp-27 (Assay Rn00583001_g1; assay generates an amplicon of 136 base pairs at position 496 on the NM_031970.1 transcript); SA rat hypertension associated homolog (Assay Rn01506557_m1; assay generates an amplicon of 76 base pairs at position 1286 on the NM_03323.1 transcript); tissue inhibitor of matrix metalloproteinase-1 (TIMP-1, Assay Rn00587558_m1; assay generates an amplicon of 91 base pairs at position 157 on the NM_053819.1 transcript); C-myc (Assay Rn00561507_m1; assay generates an amplicon of 84 base pairs at position 567 on the NM_012603.2 transcript); vascular endothelial growth factor (VEGF, Assay Rn00586458_m1; assay generates an amplicon of 115 base pairs at position 776 on the NM_053653.1 transcript); myocilin (Assay Rn00578382_m1; assay generates an amplicon of 70 base pairs at position 680 on the NM_030865.1 transcript); interleukin-7 (IL-7, Assay Rn00681900_m1; assay generates an amplicon of 158 base pairs at position 158 on the NM_013110.2 transcript); mitogen-activated protein kinase 13 (MAPK-13, Assay Rn00570269_m1; assay generates an amplicon of 62 base pairs at position 314 on the NM_031970.1 transcript); crystallin β-A1 (Cryba1, Assay Rn01499960_m1; assay generates an amplicon of 72 base pairs at position 422 on the NM_012936.1 transcript). The 18S RNA (Assay Hs99999901_s1) gene served as an endogenous control. The assays are designed for the detection and quantitation of specific rat genetic sequences in RNA samples converted to cDNA. The reaction components consisted of 10 μl of TaqMan Universal PCR Master Mix, AmpErase UNG (2X), 1 μl of Assay-on-Demand (20X), and 1 μl of cDNA in a 20 μl reaction. The PCR conditions for all genes were as follows: UNG activation, 50 °C for 2 min; preheating, 95 °C for 10 min; followed by 40 cycles of denaturation (95 °C for 15 s) and annealing/elongation (60 °C for 1 min). Each sample was run in duplicate.

The data was analyzed using the SDS 2.2 Software (Applied Biosystems, San Diego, CA). 18S RNA served as the endogenous control to normalize the amount of cDNA added to each reaction (Δ C_t), and the mean Δ C_t of control samples was used as the calibrator to calculate ΔΔC_t. The comparative C_t method was employed, whereby the relative quantity of the respective target gene mRNA, normalized to the endogenous control and relative to the calibrator, is expressed as fold change=2^-ΔΔC_t.

Immunohistochemistry

Frozen sections (12 μ thick) of enucleated eyes from Sprague-Dawley and hereditary-IOP were fixed in cold acetone for 10 min. They were washed three times for 5 min each in phosphate-buffered saline (PBS) and blocked with 10% fetal calf serum (FCS) for 30 min. The primary antibodies were diluted in 10% FCS and the sections incubated overnight at 4 °C. The monoclonal anti-cmyc (Santa Cruz Biotechnology, CA) and anti-VEGF (Sigma, Taufkirchen, Germany) were diluted at 1:100. A rabbit anti-glial fibrillary acidic protein (anti-GFAP) diluted at 1:80 (Sigma) was used. The rabbit anti-SA antibody diluted at 1:350 (gift from Dr. Meseguer, Barcelona, Spain). The secondary antibodies were a goat antirabbit Cy2 or goat anti-rabbit TRITC diluted at 1:200. After rinsing the slides three times each in PBS for 5 min, the sections were incubated with a goat antimouse Cy2 antibody (Dianova, Hamburg, Germany; 1:200 in 10% FCS) for 30 min at room temperature and washed three times for 5 min each in PBS. Finally the slides were coverslipped using Mowiol (Hoechst, Frankfurt, Germany) and viewed under an epifluorescence microscope (Axiophot, Zeiss, Oberkochen, Germany) with the appropriate filters. Negative controls consisted of sections processed without addition of the primary antibodies. Control and experimental sections were stained simultaneously to avoid variations in immunohistochemical staining.

Results

Changes in intraocular pressure

The IOP of the hereditary-IOP rat eyes was 37.0±5.5 mmHg (n=9) at 12 months of age. The IOP of age-matched dystrophic animals with normal IOP (n=6) and that of Sprague-Dawley rats (n=9) was 14.5 ± 1.9 mmHg and (15.0±1.2 mm Hg), respectively. The contralateral eyes had normal IOP values but were not used in this study.

Microarray analysis

Microarrays were used to survey the change in gene expression of dystrophic retinas versus retinas with hereditary elevated IOP. Statistical analysis showed that 120 or 1.24% of the genes in eyes with IOP elevation were regulated differentially compared to the dystrophic retina. Seventy-five genes had equal to or greater than 3 fold change and 45 genes had equal to or lower than 0.3 fold change (Table 1). The data derived from three independent microarrays was considered statistically significant at p<0.05 or lower.

Based on the PANTHER classification, the regulated genes were grouped according to the following major biological processes: anion/cation transport, cell adhesion-mediated signaling, cell adhesion, cell structure, cytokine and chemokine-mediated signaling, immunity, G-protein mediated signaling, lipid and fatty acid transport, MAPKKK cascade, mRNA transcription regulation, protein biosynthesis and phosphorylation, proteolysis and vision. A few genes were involved in multiple biological processes.

Real-time PCR analysis

Quantative real-time PCR was used to examine the expression of seven genes (Hsp-27, SA, c-myc, TIMP-1, VEGF, myocilin, and IL-7) that were upregulated on the microarrays in order to confirm the data (Figure 1). The selection was based on the following criteria. TIMP-1, Hsp-27, and VEGF have been described to be regulated in other models of experimentally elevated IOP [26]. Myocilin is associated with the pathogenesis of juvenile open angle glaucoma [27]. The c-myc gene is of interest because transgenic c-myc mice exhibit progressive enlargement of the globes with ocular malformations also characteristic of our model [28]. The SA gene has never been described in the retina and is known to be associated with hypertension, which makes it an interesting candidate in the context of this study. IL-7 is a cytokine that has not been previously described in association with elevated IOP. The expression of two downregulated genes was also verified using qRT-PCR. MAPK-13 was selected as an example for a downregulated gene involved in protein phosphorylation and the MAPKKK cascade, and Cryba1 because of its involvement in vision.

All nine genes selected for qRT-PCR quantification were significantly (p<0.0001) up- or downregulated in the elevated IOP retinas, respectively, thereby confirming the microarray data. TIMP-1 (23.7 fold±0.95); c-myc (9.95 fold±0.74); SA (16.7 fold±2.2); Hsp-27 (3.6 fold±0.49); VEGF (3.6 fold±1.03), myocilin (3.2 fold±0.84), and IL-7 (4.2 fold±0.50) were upregulated. MAPK-13 (0.24 fold±0.10) and Cryba1 (0.29 fold±0.13) were downregulated on microarrays and confirmed by qRT-PCR. The results of the real-time PCR experiments were qualitatively the same as the microarray data in all cases, although there were quantitative variations. The two-tailed Student's t-test was used to determine whether the difference in gene expression between dystrophic retinas and retinas exposed to elevated IOP was significant.

Immunohistochemistry

Immunohistochemical analysis was performed on retinal sections of control and hereditary-IOP eyes to determine the localization of SA, c-Myc, GFAP, and VEGF. In control retinas (Figure 2A), immunostaining for c-myc was localized to the RGCs and neurons in the inner nuclear layer (INL). The pattern of c-Myc immunostaining altered in the hereditary-IOP retina with the protein being localized to astrocytes and Müller cells (Figure 2B). GFAP immunostaining was carried out to confirm that these c-myc positive processes were Müller cells. In the control retina, GFAP staining was restricted to the nerve fiber layer, as was to be expected (Figure 2C). IOP elevation in the hereditary model led to activation of GFAP in astrocytes and cells whose morphology was consistent with that of Müller cells, having a radial orientation and processes that extend across all retinal layers (Figure 2D). Strong VEGF immunoreactivity was seen in the ganglion cell layer (GCL) and INL of normal retinas (Figure 2E). In the hereditary-IOP retina, VEGF was markedly decreased in the GCL, although the few surviving RGCs were still VEGF-immunopositive, whereas the INL was still strongly VEGF-immunopositive (Figure 2F). Interestingly SA was not detectable in normal retinas (Figure 2G), whereas it was markedly increased in the Müller cells in the hereditary-IOP retinas (Figure 2H). Due to the massive photoreceptor degeneration in this strain of rats, only the INL, IPL, and GCL remain in the sections.

Discussion

Elevated IOP can cause the release of toxic substances and reactive oxygen species by dying cells in the retina, changes in mitochondrial function, trophic factor deprivation, and differential transcription of survival- or death-related genes [6]. Several pioneering studies using traditional mRNA-expression analysis techniques for single genes (RT-PCR and in situ hybridization) demonstrated changes in numerous genes in the retina and optic nerve following IOP elevation. Recently, three groups reported changes in gene regulation in the glaucomatous monkey retina [29], the rat retina after injection of an hypertonic salt solution into episcleral veins [26], and in cultured astrocytes derived from human glaucomatous optic nerves [30] using oligonucleotide microarrays. These studies represent an important first step in defining changes in gene expression and elucidating the biological pathways activated following IOP elevation.

The goal of this study was to identify genes that are differentially regulated in our hereditary model of elevated IOP, as compared to the dystrophic retina. Numerous genes previously described to be regulated in experimental glaucoma altered their expression in the dystrophic retina with and without elevated IOP. This suggests that their regulation is an event common to different retinal insults, rather than exclusively due to IOP elevation. Examples include lipocalin-2, Lgals3, GFAP, vimentin, ceruloplasmin, α-2-macroglobulin, heat-shock protein-27, TIMP-1, and fibrillin-1. All these genes are also markers for Müller cells in diabetic retinas [31]. Our study revealed that long term IOP elevation alone triggers a specific set of changes in the retina's gene expression profile. The corresponding biological functions mediated by these genes are in accord with the changes one would expect in this pathological state. The regulation of the large number of genes precludes their detailed functional description (Table 1). Instead, the following discussion will focus on a few interesting genes regulated in this unique model of elevated IOP.

It is notable that numerous genes involved in mRNA regulation, transcription and cell cycle control were regulated. Gene expression is controlled by transcription factors [32] with long-lasting effects. One such gene that aroused our interest was c- myc, known to be involved in cell-cycle entry, mRNA transcription, oncogenesis and the promotion of cell differentiation, and apoptosis induction in the absence of certain growth factors [32-35]. Treatment of cultured cortical neurons with glutamate increased c-Myc levels as early as six hours after treatment [33], and in various areas of the brain following middle cerebral artery occlusion [36,37]. Interestingly, transgenic mice carrying the c-myc gene display progressive unilateral or bilateral enlargement of the globes caused by elevated IOP, as well as a dramatic decrease in the number of RGCs and elongation of the ciliary body [28], features also characteristic of our hereditary-IOP model [25]. Overexpression of c-myc in the transgenic mice might induce hypertrophy of the iris and ciliary body, causing IOP elevation [28]. The c-myc gene is expressed in various ocular tissues [38-40] and is associated with active cell proliferation. Cell proliferation and apoptosis pathways may be coupled, and the aberrant re-entry of postmitotic neurons into the cell division cycle causes apoptosis in neurodegenerative diseases [39,40]. RGC loss is the hallmark of glaucoma, and RGCs die by apoptosis [8-10,21,41]. It was interesting to note that Brn-3b, marker for RGCs was also downregulated (fold change=0.4). We have previously documented that RGC loss in this model is time- and pressure-dependant [25]. c-myc may not be the actual trigger of apoptosis, but rather acts as a sensitizer to whichever trigger is present [35,42]. The c-myc protein was localized to the astrocytes and Müller cells of the hereditary-IOP retina. In healthy retinas glial cells were c-myc immunonegative, whereas RGCs were c-myc positive, suggesting a translocation of the protein from neurons to glial cells. Both astrocytes and Müller cells were also immunopositive for GFAP, a marker for reactive gliosis. Astrocytes and Müller cells in the normal retina weakly express GFAP, but this can be increased by a variety of injury stimuli such as trauma [43], inherited retinal degeneration [44], and elevated IOP as seen in glaucoma [25,35,42,45-49]. In neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's diseases, myc genes are strongly upregulated in reactive astrocytes that are also GFAP positive. This suggests that the myc oncogenes play a primary role in the response of glial cells to neuronal damage in these diseases, since neither n-myc nor c-myc overexpression was associated with the soma of vulnerable neurons [50,51].

Many genes responsible for anion and cation transport, G-protein-, cytokine- and chemokine-mediated signaling were regulated. Retinal neurons and their axons most likely require additional metabolic support under the continuous stress of elevated IOP in order to maintain membrane potential and general ion homeostasis. Hernandez and co-workers [48] have shown that glaucomatous ONH astrocytes regulate potassium channels and alpha-2-ATPase to compensate for the metabolic stress they are subjected to under raised pressure. Signal transduction genes may be regulated by environmental stress [52]. Especially interesting was the observation that genes involved in JAK-STAT signaling such as interleukin-7, interferon-β-1, interleukin receptor-2 and the interferon gamma inducing factor were upregulated. It has previously been shown by others [53] and in our model [25] that STAT-3 and -6 are upregulated in the retina following exposure to elevated IOP, indicating a possible neuroprotective role for STAT3. Hence the JAK-STAT cascade could be activated to protect retinal neurons from degeneration. Interleukin-7 (IL-7) and interferon-beta-1 are also mediators of T-cell-mediated immunity, and their regulation could reflect a local response to IOP elevation. In this regard, the observation that IL-7 is regulated as a response to IOP elevation is novel. Genes that mediate immunity are likely to have a dual role of surveillance and response to stressors.

Myocilin (MYOC) is a protein with multiple functions, mediating signal transduction, cell structure, cell motility, and vision. Mutations in the MYOC gene are associated with juvenile open-angle glaucoma [27]. MYOC is expressed in different tissues of the rat eye such as the retina [54] or astrocytes of the optic nerve [55]. An earlier study showed that myocilin is downregulated in the rat retina but upregulated in the optic nerve head (ONH) after induction of elevated IOP, and upregulated in the retina following optic nerve crush [54]. In human and monkey glaucoma eyes, myocilin is downregulated in the optic nerve and colocalized to myelin [55]. Our microarray and qRT-PCR results show clear upregulation of myocilin in the retina. The discrepancy in the results reported in the literature can be attributed to the mode of pressure elevation and the tissues involved. Ahmed et al. cauterized the episcleral veins, which leads to immediate IOP elevation, whereas in our model, IOP elevation develops gradually in a time-dependent manner. Various stimuli including mechanical stretch can induce myocilin expression for example in trabecular meshwork cells [56]. According to the mechanical theory for the pathogenesis of glaucoma, elevated IOP causes stretching of the laminar beams and damage to RGC axons [57]. The sclera and the entire posterior segment undergo stretching and thinning in primary open-angle glaucoma [58], and stretching and rearrangement of the cribriform plates of the lamina cribrosa occur in most patients as a response to elevated IOP [59]. Changes in IOP over an extended period of time could result in stretching of the ocular tissues, especially the retina and the supporting extracellular matrix (ECM), explaining the upregulation of myocilin in the retina.

Tissue stretching could also explain the regulation of the ECM TIMP-1 and -3 in the hereditary-IOP retina. Matrix metalloproteinases (MMPs) and their inhibitors, TIMPs, are crucial to the maintenance of the retinal ECMs such as the interphotoreceptor matrix and internal limiting membrane. Their degradation is controlled by homeostasis between the MMPs and TIMPs [60]. MMPs are a family of neutral zinc proteinases that degrade the major components of the ECM and play a role in embryogenesis, development, tumor metastasis, and wound healing [61]. Chick retinal cells that produce TIMP-1 and -2 in vitro in response to mechanical stretching were identified as Müller cells and a few astrocytes [61]. Mechanical stress associated with elevated IOP might be transmitted to the Müller cells since they span the entire width of the retina (from the internal limiting membrane to the photoreceptors). Similarly, retinal pigment epithelial cells [62] and trabecular cells [63,64] release TIMP-1 and -2 in response to mechanical stretching in vitro. Ahmed et al. [26] have also reported retinal upregulation of TIMP-1 mRNA in their rat glaucoma model. Increased levels of TIMP-1 and -2 can protect retinal cells by suppressing the degradation of the ECM. Agapova et al. [59] examined the patterns of MMP and TIMP expression in the optic nerve heads of monkeys following glaucoma induction and optic nerve transection, and found that astrocytes in the glaucomatous lamina cribrosa respond to elevated IOP [59] by expressing MT1-MMP and MMP1. However, this did not occur following acute loss of axons in the transection model. The authors suggest that elevated IOP is a major factor in remodeling the optic nerve in glaucoma [59,65]. In addition, neuroprotective effects have been attributed to TIMP-1, which is expressed by RGCs [66-68]. We postulate a dual role for TIMP-1 as it could be neuroprotective by both inhibiting MMPs and through antiapoptotic actions.

Two other genes with antiapoptotic actions that were upregulated are Hsp-27 and VEGF. Heat shock proteins (HSPs) have a protective role in response to a wide range of cellular stimuli including ischemic stress [69]. Hsp-27, a member of the family of small HSPs acts as a molecular chaperone for neuronal and glial cells and functions to prevent apoptosis. In the retina, it is localized to RGCs [70]. Tezel and coworkers have carried out numerous studies on the function of Hsp-27 and have also shown that serum antibodies to HSP-27 are elevated in glaucoma and cancer [71,72]. Exogenous application of Hsp-27 [73-75] and its overexpression [76] prevent RGC death caused by retinal ischemia and axotomy, respectively. There are also data suggesting that crystallins exert chaperone-like activity during the stress associated with glaucoma [77]. Further, these crystallins prevent apoptosis by inhibiting caspases [78]. Crystallins are traditionally believed to constitute the lens proteins for augmenting the refractive power of the transparent lens [79] and can be grouped into three classes: alpha, beta, and gamma crystallins. IOP elevation induced by the injection of hypertonic saline into the episcleral veins of the rat eye resulted in the downregulation of crystallin-βB2, crystallin-αA, and crystallin-αB eight days following IOP elevation [26] and nine crystallins were downregulated in the DBA/2J mouse model of glaucoma [80]. In our study, Cryba1 was the only one to be downregulated. Since a protective role has been postulated for crystallins, their modulation may have a therapeutic effect.

VEGF is a polypeptide that until recently was believed to be a specific mitogen for endothelial cells mediating angiogenesis [81]. It is present in the aqueous humor and vitreous, and its concentration in the eye increases in neovascular glaucoma and primary open-angle glaucoma [82]. However, the administration of VEGF to fetal ventral mesencephalic explants resulted in neurite outgrowth in addition to angiogenesis, suggesting a novel role for VEGF as a neurotrophic factor [83]. Other studies confirmed this observation [81]. In a spinal cord injury model, VEGF improved behavioral recovery, reduced the number of apoptotic cells and increased VEGF receptors [84] Immediately following retinal ischemia-reperfusion injury VEGF and its receptor KDR/Flk decreased at the mRNA and protein levels in the rat retina. This study revealed that RGCs are the major source of VEGF [85]. Our immunohistochemistry data showed strong VEGF immunoreactivity in the GCL and INL of normal retina. In the hereditary-IOP retina, immunoreactivity in the GCL was markedly decreased, although the few surviving RGCs were still VEGF-immunopositive, whereas INL cells remained strongly immunoreactive. VEGF mRNA levels were upregulated implying that the few surviving RGCs increase their synthesis of VEGF in an attempt to normalize retinal levels. On the other hand, the role played by vascular insufficiency at the optic nerve head in the pathogenesis of IOP-mediated RGC loss should not be ignored. Elevated pressure may compromise the blood supply to the eye. The presence of flame-shaped hemorrhages in human glaucomatous optic nerves provides evidence in support of a vascular role in glaucoma pathophysiology [86]. Hence VEGF may also mediate angiogenic effects in response to elevated IOP in the retina.

In this regard, it was interesting to find that a novel gene, the genetic hypertension component or SA gene, not previously related to any form of retinal injury, was upregulated 14.6 fold. This is the first study to report on the localization of SA to the retina. Iwai and Ignami were the first to describe the upregulation of SA in the kidneys of spontaneously hypertensive rats in comparison to normotensive Wistar-Kyoto rats. The SA gene can influence blood pressure [87], and is localized to rat chromosome-1 [88] and human chromosome-16p13.11 [89]. SA mRNA is also expressed in the epithelial cells of the choroid plexus in the rat brain, in neurons of the CA1-CA4 pyramidal cell layer, and the dentate gyrus of the hippocampus and the cerebellar Purkinje cell layer [90]. SA expression in the choroid plexus implies a role in water-electrolyte transport. We have demonstrated the expression of the SA gene in the retina and have localized its expression to the Müller cells. Future work will have to be carried out to elucidate its exact role in the eye. Based on its function in the kidney and the fact that arterial hypertension has been postulated to be associated with glaucoma [91].

The model used in this study differs from other experimental IOP elevation models in the mechanism of IOP induction and its intensity, because the mode of IOP elevation is gradual rather than acute similar to human glaucoma. The genetic background underlying the hereditary elevation of IOP remains to be elucidated. Future work will be aimed at determining the roles played by the induced and repressed genes in retinal injury and the role of agonists/antagonists for therapeutic neuroprotective strategies.

Acknowledgements

This study was supported by the Deutsche Forschungsgemeinschaft (D.F.G.; grant NA 425-1 to R.N. and TH 386-16/1 to S.T.) and the Interdisciplinary Clinical Research Center (IZKF; grant Tha3/005/04 to S.T). We thank Mechthild Wissing for performing the immunohistochemistry experiments. We are very grateful to Dr. Anna Meseguer for kindly providing us with anti-SA antibody.

References

1. Cockburn DM. Does reduction of intraocular pressure (IOP) prevent visual field loss in glaucoma? Am J Optom Physiol Opt 1983; 60:705-11.

2. Libby RT, Anderson MG, Pang IH, Robinson ZH, Savinova OV, Cosma IM, Snow A, Wilson LA, Smith RS, Clark AF, John SW. Inherited glaucoma in DBA/2J mice: pertinent disease features for studying the neurodegeneration. Vis. Neurosci. 2005;22:637-48.

3. Schwartz M, Yoles E. Self-destructive and self-protective processes in the damaged optic nerve: implications for glaucoma. Invest Ophthalmol Vis Sci 2000; 41:349-51.

4. Dreyer EB, Zurakowski D, Schumer RA, Podos SM, Lipton SA. Elevated glutamate levels in the vitreous body of humans and monkeys with glaucoma. Arch Ophthalmol 1996; 114:299-305.

5. Quigley HA, McKinnon SJ, Zack DJ, Pease ME, Kerrigan-Baumrind LA, Kerrigan DF, Mitchell RS. Retrograde axonal transport of BDNF in retinal ganglion cells is blocked by acute IOP elevation in rats. Invest Ophthalmol Vis Sci 2000; 41:3460-66.

6. Caprioli J, Kitano S, Morgan JE. Hyperthermia and hypoxia increase tolerance of retinal ganglion cells to anoxia and excitotoxicity. Invest Ophthalmol Vis Sci 1996; 37:2376-81.

7. Garcia-Valenzuela E, Shareef S, Walsh J, Sharma SC. Programmed cell death of retinal ganglion cells during experimental glaucoma. Exp Eye Res 1995; 61:33-44.

8. 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; 36:774-86.

9. Kerrigan LA, Zack DJ, Quigley HA, Smith SD, Pease ME. TUNEL-positive ganglion cells in human primary open-angle glaucoma. Arch Ophthalmol 1997; 115:1031-5.

10. Nickells RW, Zack DJ. Apoptosis in ocular disease: a molecular overview. Ophthalmic Genet 1996; 17:145-65.

11. Wang X, Tay SS, Ng YK. An electron microscopic study of neuronal degeneration and glial cell reaction in the retina of glaucomatous rats. Histol Histopathol 2002; 17:1043-52.

12. Lam TT, Abler AS, Tso MO. Apoptosis and caspases after ischemia-reperfusion injury in rat retina. Invest Ophthalmol Vis Sci 1999; 40:967-75.

13. Panda S, Jonas JB. Decreased photoreceptor count in human eyes with secondary angle-closure glaucoma. Invest Ophthalmol Vis Sci 1992; 33:2532-36.

14. Janssen P, Naskar R, Moore S, Thanos S, Thiel HJ. Evidence for glaucoma-induced horizontal cell alterations in the human retina. Ger J Ophthalmol 1996; 5:378-85.

15. Kendell KR, Quigley HA, Kerrigan LA, Pease ME, Quigley EN. Primary open-angle glaucoma is not associated with photoreceptor loss. Invest Ophthalmol Vis Sci 1995; 36:200-205.

16. Quigley HA, Green WR. The histology of human glaucoma cupping and optic nerve damage: clinicopathologic correlation in 21 eyes. Ophthalmology 1979; 86:1803-30.

17. Quigley HA, Dunkelberger GR, Green WR. Chronic human glaucoma causing selectively greater loss of large optic nerve fibers. Ophthalmology 1988; 95:357-63.

18. Shareef SR, Garcia-Valenzuela E, Salierno A, Walsh J, Sharma SC. Chronic ocular hypertension following episcleral venous occlusion in rats. Exp Eye Res 1995; 61:379-382.

19. Ueda J, Sawaguchi S, Hanyu T, Yaoeda K, Fukuchi T, Abe H, Ozawa H. Experimental glaucoma model in the rat induced by laser trabecular photocoagulation after an intracameral injection of India ink. Jpn J Ophthalmol 1998; 42:337-44.

20. Mittag TW, Danias J, Pohorenec G, Yuan HM, Burakgazi E, Chalmers-Redman R, Podos SM, Tatton WG. Retinal damage after 3 to 4 months of elevated intraocular pressure in a rat glaucoma model. Invest Ophthalmol Vis Sci 2000; 41:3451-59.

21. Naskar R, Wissing M, Thanos S. Detection of early neuron degeneration and accompanying microglial responses in the retina of a rat model of glaucoma. Invest Ophthalmol Vis Sci 2002; 43:2962-68.

22. John SW, Smith RS, Savinova OV, Hawes NL, Chang B, Turnbull D, Davisson M, Roderick TH, Heckenlively JR. Essential iris atrophy, pigment dispersion, and glaucoma in DBA/2J mice. Invest Ophthalmol Vis Sci. 1998; 39:951-62.

23. Chang B, Smith RS, Hawes NL, Anderson MG, Zabaleta A, Savinova O, Roderick TH, Heckenlively JR, Davisson MT, John SW. Interacting loci cause severe iris atrophy and glaucoma in DBA/2J mice. Nat Genet 1999; 21:405-409.

24. Heywood R. Glaucoma in the rat. Br Vet J. 1975; 31:213-21.

25. Thanos S, Naskar R. Correlation between retinal ganglion cell death and chronically developing inherited glaucoma in a new rat mutant. Exp Eye Res 2004; 79:119-29.

26. Ahmed F, Brown KM, Stephan DA, Morrison JC, Johnson EC, Tomarev SI. Microarray analysis of changes in mRNA levels in the rat retina after experimental elevation of intraocular pressure. Invest Ophthalmol Vis Sci 2004; 45:1247-58.

27. Taguchi M, Kanno H, Kubota R, Miwa S, Shishiba Y, Ozawa Y. Molecular cloning and expression profile of rat myocilin. Mol Genet Metab 2000; 70:75-80.

28. Ishibashi K, Yamamoto H, Hatano M, Koizumi T, Yamamoto M, Tokuhisa T. Enlargement of the globe with ocular malformations in c-Myc transgenic mice. Jpn J Ophthalmol 1999; 43:201-208.

29. Miyahara T, Kikuchi T, Akimoto M, Kurokawa T, Shibuki H, Yoshimura N. Gene microarray analysis of experimental glaucomatous retina from cynomologous monkey. Invest Ophthalmol Vis Sci 2003; 44:4347-56.

30. Yang P, Agapova O, Parker A, Shannon W, Pecen P, Duncan J, Salvador-Silva M, Hernandez MR. DNA microarray analysis of gene expression in human optic nerve head astrocytes in response to hydrostatic pressure. Physiol Genomics 2004; 17:157-69.

31. Gerhardinger C, Costa MB, Coulombe MC, Toth I, Hoehn T, Grosu P. Expression of acute-phase response proteins in retinal Muller cells in diabetes. Invest Ophthalmol Vis Sci 2005; 46:349-57.

32. Yoshimura N, Kikuchi T, Kuroiwa S, Gaun S. Differential temporal and spatial expression of immediate early genes in retinal neurons after ischemia-reperfusion injury. Invest Ophthalmol Vis Sci 2003; 44:2211-20.

33. Liu X, Zhu XZ. Roles of p53, c-Myc, Bcl-2, Bax and caspases in glutamate-induced neuronal apoptosis and the possible neuroprotective mechanism of basic fibroblast growth factor. Brain Res Mol Brain Res 1999; 71:210-16.

34. Battey J, Moulding C, Taub R, Murphy W, Stewart T, Potter H, Lenoir G, Leder P. The human c-myc oncogene: structural consequences of translocation into the IgH locus in Burkitt lymphoma. Cell 1983; 34:779-87.

35. Staunton MJ, Gaffney EF. Apoptosis: basic concepts and potential significance in human cancer. Arch Pathol Lab Med 1998; 122:310-19.

36. Nakagomi T, Asai A, Kanemitsu H, Narita K, Kuchino Y, Tamura A, Kirino T. Up-regulation of c-myc gene expression following focal ischemia in the rat brain. Neurol Res 1996; 18:559-63.

37. McGahan L, Hakim AM, Robertson GS. Hippocampal Myc and p53 expression following transient global ischemia. Brain Res Mol Brain Res 1998; 56:133-45.

38. Hirning U, Schmid P, Schulz WA, Rettenberger G, Hameister H. A comparative analysis of N-myc and c-myc expression and cellular proliferation in mouse organogenesis. Mech Dev 1991; 33:119-125.

39. McShea A, Wahl AF, Smith MA. Re-entry into the cell cycle: a mechanism for neurodegeneration in Alzheimer disease. Med Hypotheses 1999; 52:525-27.

40. Copani A, Uberti D, Sortino MA, Bruno V, Nicoletti F, Memo M. Activation of cell-cycle-associated proteins in neuronal death: a mandatory or dispensable path? Trends Neurosci 2001; 24:25-31.

41. McKinnon SJ. Glaucoma, apoptosis, and neuroprotection. Curr Opin Ophthalmol 1997; 8:28-37.

42. Evan G, Littlewood T. A matter of life and cell death. Science 1998; 281:1317-22.

43. Bignami A, Dahl D. The radial glia of Muller in the rat retina and their response to injury. An immunofluorescence study with antibodies to the glial fibrillary acidic (GFA) protein. Exp Eye Res 1979; 28:63-69.

44. Ekstrom P, Sanyal S, Narfstrom K, Chader GJ, van Veen T. Accumulation of glial fibrillary acidic protein in Muller radial glia during retinal degeneration. Invest Ophthalmol Vis Sci 1988; 29:1363-71.

45. Kim IB, Kim KY, Joo CK, Lee MY, Oh SJ, Chung JW, Chun MH. Reaction of Muller cells after increased intraocular pressure in the rat retina. Exp Brain Res 1998; 121:419-24.

46. Wang X, Tay SS, Ng YK. An immunohistochemical study of neuronal and glial cell reactions in retinae of rats with experimental glaucoma. Exp Brain Res 2000; 132:476-84.

47. Wang L, Cioffi GA, Cull G, Dong J, Fortune B. Immunohistologic evidence for retinal glial cell changes in human glaucoma. Invest Ophthalmol Vis Sci 2002; 43:1088-94.

48. Hernandez MR, Agapova OA, Yang P, Salvador-Silva M, Ricard CS, Aoi S. Differential gene expression in astrocytes from human normal and glaucomatous optic nerve head analyzed by cDNA microarray. Glia 2002; 38:45-64.

49. Tezel G, Chauhan BC, LeBlanc RP, Wax MB. Immunohistochemical assessment of the glial mitogen-activated protein kinase activation in glaucoma. Invest Ophthalmol Vis Sci 2003; 44:3025-33.

50. Ferrer I, Blanco R. N-myc and c-myc expression in Alzheimer disease, Huntington disease and Parkinson disease. Brain Res Mol Brain Res 2000; 77:270-76.

51. Hirvonen HE, Salonen R, Sandberg MM, Vuorio E, Vastrik I, Kotilainen E, Kalimo H. Differential expression of myc, max and RB1 genes in human gliomas and glioma cell lines. Br J Cancer 1994; 69:16-25.

52. Salvador-Silva M, Aoi S, Parker A, Yang P, Pecen P, Hernandez MR. Responses and signaling pathways in human optic nerve head astrocytes exposed to hydrostatic pressure in vitro. Glia. 2004;45:364-377.

53. Ji JZ, Elyaman W, Yip HK, Lee VW, Yick LW, Hugon J, So KF. CNTF promotes survival of retinal ganglion cells after induction of ocular hypertension in rats: the possible involvement of STAT3 pathway. Eur J Neurosci 2004; 19:265-72.

54. Ahmed F, Torrado M, Johnson E, Morrison J, Tomarev SI. Changes in mRNA levels of the Myoc/Tigr gene in the rat eye after experimental elevation of intraocular pressure or optic nerve transection. Invest Ophthalmol Vis Sci 2001; 42:3165-72.

55. Ricard CS, Agapova OA, Salvador-Silva M, Kaufman PL, Hernandez MR. Expression of myocilin/TIGR in normal and glaucomatous primate optic nerves. Exp Eye Res 2001; 73:433-47.

56. Tamm ER. Myocilin and glaucoma: facts and ideas. Prog Retin Eye Res 2002; 21:395-428.

57. Flammer J, Orgul S, Costa VP, Orzalesi N, Krieglstein GK, Serra LM, Renard JP, Stefansson E. The impact of ocular blood flow in glaucoma. Prog Retin Eye Res 2002; 21:359-93.

58. Zatulina NI, Panormova NV, Sennova LG, Mal'tsev VV. [Quantitative biochemical shifts in the connective tissue of the rear section of the eyeball in glaucoma and atherosclerosis]. Vestn Oftalmol 1989; 105:37-41.

59. Agapova OA, Kaufman PL, Lucarelli MJ, Gabelt BT, Hernandez MR. Differential expression of matrix metalloproteinases in monkey eyes with experimental glaucoma or optic nerve transection. Brain Res 2003; 967:132-43.

60. Namba M, Matsuo T, Shiraga F, Ohtsuki H. Retinal cells produce TIMP-1 and TIMP-2 in response to cyclic mechanical stretching. Ophthalmic Res 2001; 33:163-69.

61. Nagase H, Woessner JF Jr. Matrix metalloproteinases. J Biol Chem 1999; 274:21491-94.

62. Yamaguchi K, Matsuo T, Shiraga F, Ohtsuki H. TIMP-1 production by bovine retinal pigment epithelial cells increases in response to cyclic mechanical stretch. Jpn J Ophthalmol 2001; 45:470-74.

63. Okada Y, Matsuo T, Ohtsuki H. Bovine trabecular cells produce TIMP-1 and MMP-2 in response to mechanical stretching. Jpn J Ophthalmol 1998; 42:90-94.

64. WuDunn D. The effect of mechanical strain on matrix metalloproteinase production by bovine trabecular meshwork cells. Curr Eye Res 2001; 22:394-97.

65. Hernandez MR. The optic nerve head in glaucoma: role of astrocytes in tissue remodeling. Prog Retin Eye Res 2000; 19:297-21.

66. Agapova OA, Ricard CS, Salvador-Silva M, Hernandez MR. Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human optic nerve head astrocytes. Glia 2001; 33:205-16.

67. Guo L, Moss SE, Alexander RA, Ali RR, Fitzke FW, Cordeiro MF. Retinal ganglion cell apoptosis in glaucoma is related to intraocular pressure and IOP-induced effects on extracellular matrix. Invest Ophthalmol Vis Sci 2005; 46:175-82.

68. Maatta M, Tervahartiala T, Vesti I, Airaksinen J, Sorsa T. Levels and activation of matrix metalloproteinases in aqueous humor are elevated in uveitis-related secondary glaucoma. J. Glaucoma 2006;15:229-237.

69. Plumier JC, Krueger AM, Currie RW, Kontoyiannis D, Kollias G, Pagoulatos GN. Transgenic mice expressing the human inducible Hsp70 have hippocampal neurons resistant to ischemic injury. Cell Stress Chaperones 1997; 2:162-167.

70. Hawkes EL, Krueger-Naug AM, Nickerson PE, Myers TL, Currie RW, Clarke DB. Expression of Hsp27 in retinal ganglion cells of the rat during postnatal development. J Comp Neurol 2004; 478:143-8.

71. Tezel G, Seigel GM, Wax MB. Autoantibodies to small heat shock proteins in glaucoma. Invest Ophthalmol Vis Sci 1998; 39:2277-87.

72. Wax MB, Tezel G, Kawase K, Kitazawa Y. Serum autoantibodies to heat shock proteins in glaucoma patients from Japan and the United States. Ophthalmology 2001; 108:296-302.

73. Yokoyama A, Oshitari T, Negishi H, Dezawa M, Mizota A, Adachi-Usami E. Protection of retinal ganglion cells from ischemia-reperfusion injury by electrically applied Hsp27. Invest Ophthalmol Vis Sci 2001; 42:3283-6.

74. Whitlock NA, Agarwal N, Ma JX, Crosson CE. Hsp27 upregulation by HIF-1 signaling offers protection against retinal ischemia in rats. Invest Ophthalmol Vis Sci 2005; 46:1092-8.

75. Li Y, Roth S, Laser M, Ma JX, Crosson CE. Retinal preconditioning and the induction of heat-shock protein 27. Invest Ophthalmol Vis Sci 2003; 44:1299-304.

76. Kretz A, Schmeer C, Tausch S, Isenmann S. Simvastatin promotes heat shock protein 27 expression and Akt activation in the rat retina and protects axotomized retinal ganglion cells in vivo. Neurobiol Dis. 2006; 21:421-30.

77. Salvador-Silva M, Ricard CS, Agapova OA, Yang P, Hernandez MR. Expression of small heat shock proteins and intermediate filaments in the human optic nerve head astrocytes exposed to elevated hydrostatic pressure in vitro. J Neurosci Res 2001; 66:59-73.

78. Alge CS, Priglinger SG, Neubauer AS, Kampik A, Zillig M, Bloemendal H, Welge-Lussen U. Retinal pigment epithelium is protected against apoptosis by alphaB-crystallin. Invest Ophthalmol Vis Sci 2002; 43:3575-3582.

79. Wistow GJ, Piatigorsky J. Lens crystallins: the evolution and expression of proteins for a highly specialized tissue. Annu Rev Biochem 1988; 57:479-504.

80. Steele MR, Inman DM, Calkins DJ, Horner PJ, Vetter ML. Microarray analysis of retinal gene expression in the DBA/2J model of glaucoma. Invest Ophthalmol Vis Sci 2006; 47:977-985.

81. Rosenstein JM, Krum JM. New roles for VEGF in nervous tissue--beyond blood vessels. Exp Neurol 2004; 187:246-53.

82. Tripathi RC, Li J, Tripathi BJ, Chalam KV, Adamis AP. Increased level of vascular endothelial growth factor in aqueous humor of patients with neovascular glaucoma. Ophthalmology 1998; 105:232-7.

83. Silverman WF, Krum JM, Mani N, Rosenstein JM. Vascular, glial and neuronal effects of vascular endothelial growth factor in mesencephalic explant cultures. Neuroscience 1999; 90:1529-41.

84. Widenfalk J, Lipson A, Jubran M, Hofstetter C, Ebendal T, Cao Y, Olson L. Vascular endothelial growth factor improves functional outcome and decreases secondary degeneration in experimental spinal cord contusion injury. Neuroscience 2003; 120:951-60.

85. Ogata N, Yamanaka R, Yamamoto C, Miyashiro M, Kimoto T, Takahashi K, Maruyama K, Uyama M. Expression of vascular endothelial growth factor and its receptor, KDR, following retinal ischemia-reperfusion injury in the rat. Curr Eye Res. 1998;17:1087-96.

86. Jonas JB, Gusek GC, Naumann GO. [Qualitative morphologic characteristics of normal and glaucomatous optic papillae]. Klin Monatsbl Augenheilkd 1988; 193:481-8.

87. Iwai N, Kurtz TW, Inagami T. Further evidence of the SA gene as a candidate gene contributing to the hypertension in spontaneously hypertensive rat. Biochem Biophys Res Commun 1992; 188:64-9.

88. Lindpaintner K, Hilbert P, Ganten D, Nadal-Ginard B, Inagami T, Iwai N. Molecular genetics of the SA-gene: cosegregation with hypertension and mapping to rat chromosome 1. J Hypertens 1993; 11:19-23.

89. Szpirer C, Riviere M, Szpirer J, Levan G, Guo DF, Iwai N, Inagami T. Chromosomal assignment of human and rat hypertension candidate genes: type 1 angiotensin II receptor genes and the SA gene. J Hypertens 1993; 11:919-25.

90. Mishima A, Shigematsu K, Harada N, Himeno A, Taguchi T, Ishinaga Y, Nabika T. Strain differences in SA gene expression in brain and kidney of normotensive and hypertensive rats. Cell Mol Neurobiol 2000; 20:633-52.

91. Tielsch JM, Katz J, Sommer A, Quigley HA, Javitt JC. Hypertension, perfusion pressure, and primary open-angle glaucoma. A population-based assessment. Arch Ophthalmol 1995; 113:216-221.