January 2011
Volume 52, Issue 1
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Glaucoma  |   January 2011
Cell Proliferation and Interleukin-6–Type Cytokine Signaling Are Implicated by Gene Expression Responses in Early Optic Nerve Head Injury in Rat Glaucoma
Author Affiliations & Notes
  • Elaine C. Johnson
    From the Kenneth C. Swan Ocular Neurobiology Laboratory, Casey Eye Institute, Oregon Health and Science University, Portland, Oregon.
  • Thomas A. Doser
    From the Kenneth C. Swan Ocular Neurobiology Laboratory, Casey Eye Institute, Oregon Health and Science University, Portland, Oregon.
  • William O. Cepurna
    From the Kenneth C. Swan Ocular Neurobiology Laboratory, Casey Eye Institute, Oregon Health and Science University, Portland, Oregon.
  • Jennifer A. Dyck
    From the Kenneth C. Swan Ocular Neurobiology Laboratory, Casey Eye Institute, Oregon Health and Science University, Portland, Oregon.
  • Lijun Jia
    From the Kenneth C. Swan Ocular Neurobiology Laboratory, Casey Eye Institute, Oregon Health and Science University, Portland, Oregon.
  • Ying Guo
    From the Kenneth C. Swan Ocular Neurobiology Laboratory, Casey Eye Institute, Oregon Health and Science University, Portland, Oregon.
  • Wendi S. Lambert
    From the Kenneth C. Swan Ocular Neurobiology Laboratory, Casey Eye Institute, Oregon Health and Science University, Portland, Oregon.
  • John C. Morrison
    From the Kenneth C. Swan Ocular Neurobiology Laboratory, Casey Eye Institute, Oregon Health and Science University, Portland, Oregon.
  • Corresponding author: Elaine C. Johnson, 3375 SW Terwilliger Boulevard, CERES, Portland, OR 97201; johnsoel@ohsu.edu
Investigative Ophthalmology & Visual Science January 2011, Vol.52, 504-518. doi:10.1167/iovs.10-5317
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      Elaine C. Johnson, Thomas A. Doser, William O. Cepurna, Jennifer A. Dyck, Lijun Jia, Ying Guo, Wendi S. Lambert, John C. Morrison; Cell Proliferation and Interleukin-6–Type Cytokine Signaling Are Implicated by Gene Expression Responses in Early Optic Nerve Head Injury in Rat Glaucoma. Invest. Ophthalmol. Vis. Sci. 2011;52(1):504-518. doi: 10.1167/iovs.10-5317.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose.: In glaucoma, the optic nerve head (ONH) is the principal site of initial axonal injury, and elevated intraocular pressure (IOP) is the predominant risk factor. However, the initial responses of the ONH to elevated IOP are unknown. Here the authors use a rat glaucoma model to characterize ONH gene expression changes associated with early optic nerve injury.

Methods.: Unilateral IOP elevation was produced in rats by episcleral vein injection of hypertonic saline. ONH mRNA was extracted, and retrobulbar optic nerve cross-sections were graded for axonal degeneration. Gene expression was determined by microarray and quantitative PCR (QPCR) analysis. Significantly altered gene expression was determined by multiclass analysis and ANOVA. DAVID gene ontology determined the functional categories of significantly affected genes.

Results.: The Early Injury group consisted of ONH from eyes with <15% axon degeneration. By array analysis, 877 genes were significantly regulated in this group. The most significant upregulated gene categories were cell cycle, cytoskeleton, and immune system process, whereas the downregulated categories included glucose and lipid metabolism. QPCR confirmed the upregulation of cell cycle-associated genes and leukemia inhibitory factor (Lif) and revealed alterations in expression of other IL-6–type cytokines and Jak-Stat signaling pathway components, including increased expression of IL-6 (1553%). In contrast, astrocytic glial fibrillary acidic protein (Gfap) message levels were unaltered, and other astrocytic markers were significantly downregulated. Microglial activation and vascular-associated gene responses were identified.

Conclusions.: Cell proliferation and IL-6–type cytokine gene expression, rather than astrocyte hypertrophy, characterize early pressure-induced ONH injury.

Experimentally elevated IOP in animals produces glaucoma-like retinal ganglion cell (RGC) and optic nerve axonal loss and a characteristic remodeling of the optic nerve head (ONH) that includes the rearrangement of glial cells and the deposition of extracellular matrix (ECM) proteins. Although experimental glaucoma models have been produced in a number of species, primate and rodent models are the most extensively used. In both human and experimental glaucoma, there is a general consensus that the ONH is the primary site of initial glaucomatous injury to RGC axons. 1,2 Recently, sophisticated 3-D histomorphometric and biomechanical studies have been used to examine some of the earliest morphologic and structural ONH responses in experimental glaucoma attributed to elevated IOP in primates. 3 11 However, relatively few studies have examined the early cellular responses of the ONH to elevated IOP exposure. Such studies should help us understand how the ONH cells respond to an environment altered by elevated IOP and how these responses might affect axonal integrity. 
To examine the response to acute IOP elevation, the anterior chamber can be cannulated to allow IOP to be elevated for up to a few hours without altering retinal perfusion. After these acute elevations, abnormalities in axonal transport, including the accumulation of mitochondria, dynein, neurotrophins and their receptors, and the distribution of axonal and astrocytic proteins have been observed. 12 17  
For longer IOP elevation, a number of techniques are available to obstruct aqueous outflow and produce glaucoma-like optic nerve degeneration. 18 23 In initial studies using our chronic, hypertonic, saline-induced rat glaucoma model and immunohistochemistry, we found characteristic glaucoma-like changes in the rat ONH ECM. As early as 11 days after IOP elevation, we observed depositions of collagen IV, collagen VI, and laminin. 24 Next, we used immunohistochemistry to study the chronology of morphologic and protein distribution changes in the ONH. At 3 days after IOP elevation, we observed decreased labeling in ONH glial columns for gap junction protein alpha 1 (connexin 43, Gja1) and increases in a cell proliferation marker. 25 These changes were followed at 1 week by decreased labeling for neurotrophins and Gfap, increased vascular labeling of collagen VI, and the appearance of swollen, degenerating axons in the ONH and focal lesions in optic nerve cross-sections. In a separate study using laser-induced IOP elevation, an accumulation of the retrograde transport motor dynein was observed in the ONH at approximately the same duration of IOP elevation. 26  
More recently, we have used our glaucoma model to determine gene expression changes in extensively injured ONH. 27 As part of that study, we also reported expression changes in a few specific genes in ONH from eyes with focal optic nerve injuries (<50% axon degeneration). These genes had all been found to be highly regulated in ONH with extensive nerve injury. Among these, we found significant upregulation of tissue inhibitor of metalloproteinases 1 (Timp1), fibulin 2 (Fbln2), and tenascin c (Tnc) as well as downregulation of aquaporin 4 (Aqp4) in these focally injured ONH. In the study presented here, we greatly expand these initial findings by using microarray analysis and quantitative polymerase chain reaction (QPCR) to identify the gene expression changes in glaucoma model ONH from eyes with <15% optic nerve axon degeneration attributed to elevated IOP exposure. 
Methods
Glaucoma Model
All animal experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Oregon Health and Sciences University (OHSU) Animal Care and Use Committee. Unilateral IOP elevation was produced in 8-month-old, male Brown Norway rats by episcleral vein injection of hypertonic saline. 28 Rats were housed in low-level constant light to minimize circadian IOP fluctuation. 29,30 IOP was measured frequently in anesthetized animals, primarily using a tonometer (Tono-PenXL; Medtronic, Minneapolis, MN). 29,31 For some glaucoma model eyes included in the PCR studies, IOP history was collected using a rebound tonometer (TonoLab; Colonial Medical Supply, Franconia, NH), as described. 32 Except where noted, tissues were collected at 5 weeks after injection. 
Optic Nerve Grading
Retrobulbar optic nerves were collected, postfixed, embedded in Spurr's resin, and cross-sectioned for light microscopic evaluation. 27 Each nerve was graded for injury, on a scale from 1 (normal nerve) to 5 (extensive degeneration affecting nearly all axons), by at least five independent observers, and the mean was defined as the injury grade for that nerve. Previous studies using transmission electron microscopy have demonstrated that each unit increase in the injury grade is equivalent to degeneration affecting approximately 12% to 15% of the optic nerve axons. 30  
ONH Dissection
Enucleated globes were rapidly chilled in cold phosphate-buffered saline, and the anterior segment, lens, and retina were removed. Using a trephine, the ONH, including the initial portion of myelinated optic nerve, was dissected from the posterior pole of the globe. The ONH was then shortened to a uniform 0.4 mm in length using a razor blade and custom-designed matrix. This initial, largely unmyelinated ONH segment included the pia and pigmented peripapillary border tissue. The dissected ONH was then frozen in liquid nitrogen and stored at −80°C before RNA extraction. 
RNA Purification and Amplification
RNA was isolated by first sonicating the frozen nerve heads in 30 μL extraction buffer (Pico-Pure RNA Isolation Kit; Molecular Devices, Sunnyvale, CA) using an MS 0.5 probe (UP50H Ultrasonic Processor; Hielscher USA., Inc., Ringwood, NJ) in accordance with the manufacturer's directions, including DNase treatment. The purified RNA was quantified (RiboGreen RNA Quantitation Kit; Invitrogen, Carlsbad, CA), and 25-ng aliquots underwent two rounds of linear amplification (MessageAmp aRNA Amplification Kit; Ambion, Austin, TX) yielding ample aRNA for both microarray analysis and QPCR. The integrity of the amplified RNA was verified (2100 Bioanalyzer; Agilent Technologies, Palo Alto, CA). 
cDNA Microarrays
Aliquots of amplified RNA were reverse transcribed and dye-labeled at the OHSU Gene Microarray Shared Resource (http://www.ohsu.edu/gmsr/), which also prepared and processed the cDNA arrays and compiled the data. For each sample, two cDNA arrays (SMCmou8400A and SMCmou6600A) were used, containing a combined total of 15,400 probes. These are listed in Supplementary Table S1, and represent the entire initial release of the National Institute on Aging mouse library (http://lgsun.grc.nia.nih.gov/cDNA/15k.html). The length of the cDNA probes on these arrays makes them relatively insensitive to sequence differences between closely related species (Morrison JC, et al. IOVS 2005;46:ARVO E-Abstract 1250). 33,34 The probes correspond to approximately 8000 individual Entrez gene IDs, 1000 additional transcripts with Unigene designations, and 4000 expressed sequence tags (http://www.ncbi.nlm.nih.gov/sites/entrez). Each data point is the mean of four technical replicates. Gene expression data from each array were normalized at the facility using a modified Lowess procedure. 35 Expression patterns in each ONH were independently determined relative to an ONH RNA reference standard, derived by pooling aliquots from all samples. 
In addition to six control (uninjected, fellow eye) ONH, ONH was collected from 21 eyes with cumulative IOP above the control eye ranging from 34 mm Hg to 490 mm Hg. Mean IOP in control eyes, as measured by tonometer (Tono-PenXL; Medtronic), was 28.6 mm Hg. Although most of the glaucoma model ONH was collected at 5 weeks after episcleral vein injection, the analysis included eight ONH from eyes collected at 10 days after the first IOP reading of 35 mm Hg or more, so that gene expression changes that occurred early in the injury process would be enriched in the analysis. 
Physiological IOP Elevation Group
In an additional microarray analysis, gene expression changes with physiological IOP elevation were evaluated to enhance the interpretation of the glaucoma model results. These samples were processed simultaneously with the glaucoma model samples from initial RNA amplification through microarray data preprocessing and normalization in a single experiment at the OHSU Microarray Resource. For this group, animals were housed in standard 12-hour light/12-hour dark conditions. Tissues were collected from normal eyes during the dark-phase peak in circadian IOP elevation, when IOP is approximately 10 mm Hg above the light-phase IOP measurement of approximately 21 mm Hg and 2 to 3 mm Hg above the mean IOP of eyes in animals housed in constant, low-level light. 29,36 Gene expression alterations in these ONH were independently compared with the control ONH data, described here using the Two Class Unpaired Module of Significance Analysis of Microarrays (SAM, version 3.02; http://www-stat.stanford.edu/∼tibs/SAM/ provided in the public domain by Stanford University, Stanford, CA). Potential gene expression changes in these eyes do not result in significant axon loss over the lifespan of the rat 37 and, therefore, represent noninjurious, physiological circadian responses. Significant changes in expression attributed to physiological IOP elevation (1.3-fold change; 2% false discovery rate [FDR]) were noted and used to assist in interpreting the gene expression changes seen in our glaucoma model. 
Definition of the Early ONH Injury Group
We reasoned that the ONH from glaucoma model eyes with minimal optic nerve axon degeneration would most consistently display changes in gene expression associated with the earliest phases of axonal injury. Rather than arbitrarily dividing the experimental ONH into injury groups, we used the following visualization strategy to identify a group of ONH with early injury. First, we ordered the microarray data sets by increasing optic nerve injury grade, including all data from the 6 control and 21 glaucoma model ONH, with nerve injury grades ranging from 1.025 to 5. Then we used the Pattern Discovery module in SAM to identify the significant eigengenes (principal components). The SMCmou8400A and SMCmou6600A array data sets were separately analyzed and significant eigengenes (P < 0.05) from both were plotted against ONH injury grade. Curve fitting and regression analysis were used to determine the statistically significant best fit line for each eigengene. From this analysis, the Early and Advanced Injury groups of arrays were defined (see Results and Fig. 1
Figure 1.
 
Definition of the Early and Advanced ONH Injury groups. The Pattern Discovery module in SAM was used to identify the significant eigengenes (principal components) in the microarray data sets arrayed by increasing optic nerve injury grade (6 controls, 21 glaucoma model ONH from eyes with optic nerve injury grades from 1.025 to 5.) Each array data set (SMCmou6600A and SMCmou8400A) was separately analyzed, and significant eigengenes (P < 0.05) from both were plotted. Curve-fitting and regression analysis were used to determine the statistically significant best-fit line for each eigengene, as plotted here. The two array sets yielded nearly identical eigengene patterns for the first two eigengenes. The most significant pattern (Eigengene 1, red lines) on both arrays was a linear response, with maximal change in the most severely injured ONH. The second most significant pattern (Eigengene 2) for both array sets was a fourth-order polynomial curve, with peak changes below injury grade 2 (approximately 15% axons degenerating). This pattern indicated that a large number of significantly affected ONH genes responded maximally to early pressure-induced damage. Based on nerve injury grade and these two eigengene patterns, data from each sample was assigned to 1 of 3 groups for further analysis: Control ONH (untreated eyes), Early ONH Injury (glaucoma model ONH with optic nerve injury grades <2.0, n = 9), and Advanced ONH Injury (glaucoma model ONH with optic nerve injury grades >2.0, n = 12).
Figure 1.
 
Definition of the Early and Advanced ONH Injury groups. The Pattern Discovery module in SAM was used to identify the significant eigengenes (principal components) in the microarray data sets arrayed by increasing optic nerve injury grade (6 controls, 21 glaucoma model ONH from eyes with optic nerve injury grades from 1.025 to 5.) Each array data set (SMCmou6600A and SMCmou8400A) was separately analyzed, and significant eigengenes (P < 0.05) from both were plotted. Curve-fitting and regression analysis were used to determine the statistically significant best-fit line for each eigengene, as plotted here. The two array sets yielded nearly identical eigengene patterns for the first two eigengenes. The most significant pattern (Eigengene 1, red lines) on both arrays was a linear response, with maximal change in the most severely injured ONH. The second most significant pattern (Eigengene 2) for both array sets was a fourth-order polynomial curve, with peak changes below injury grade 2 (approximately 15% axons degenerating). This pattern indicated that a large number of significantly affected ONH genes responded maximally to early pressure-induced damage. Based on nerve injury grade and these two eigengene patterns, data from each sample was assigned to 1 of 3 groups for further analysis: Control ONH (untreated eyes), Early ONH Injury (glaucoma model ONH with optic nerve injury grades <2.0, n = 9), and Advanced ONH Injury (glaucoma model ONH with optic nerve injury grades >2.0, n = 12).
Determination of Genes Significantly Changed in Expression by ONH Injury Group
Next the Multiclass Analysis module of SAM (2% FDR) was used to compare the data from the Early Injury group to both the Control and the Advanced Injury group. This analysis identified all genes that were significantly altered in any group comparison. Then ANOVA and Tukey-Kramer posttest were used to identify genes that differed significantly (P < 0.05 corrected for multiple comparisons) and were changed in expression by 1.3-fold between groups so that our early injury group analysis identified all genes significantly regulated in early injury compared with the control group. Finally, DAVID Bioinformatics Resources 2008 (http://david.abcc.ncifcrf.gov/) was used to determine significantly regulated functional classes of these genes, using the array probes as background. Official Entrez and Rat genome database (http://rgd.mcw.edu/) gene symbols are used throughout the manuscript to refer to specific genes and their protein products. 
QPCR Analysis
QPCR was used to verify microarray analysis findings and to examine the expression of genes not included on our microarrays. In addition to aRNA from all the samples analyzed by microarray, aRNA was also prepared from additional control and glaucoma model ONH to increase the final group sizes (12 control, 20 Early Injury, and 24 Advanced Injury). Mean injury grades for these groups were comparable to those in the microarray study (1.0 ± 0.0, 1.4 ± 0.1, and 3.9 ± 0.3, respectively). ONH aRNA (25 ng) from each experimental sample, along with a standard curve ranging from 1.5 to 200 ng pooled control and experimental ONH aRNA, was reverse transcribed to cDNA for QPCR using a kit (SuperScript III Reverse Transcriptase; Invitrogen) according to the manufacturer's protocol. Primers for the 60 messages measured by QPCR were designed from rat nucleotide sequences (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/sites/entrez and Ensembl http://www.ensembl.org/index.html) for products within 350 bases of the target mRNA 3′ polyadenlyation site using appropriate software (Clone Manager, version 9.0; Sci Ed Software, Cary, NC). Primers used are listed in Supplementary Table S2. QPCR was performed (LightCycler DNA Master SYBR Green 1 Kit; Roche Applied Sciences, Indianapolis, IN), and products were verified by sequencing. Each target message was normalized to the average of three glyceraldehyde phosphate dehydrogenase (Gapdh) measurements on each sample. Gapdh was chosen for normalization because the levels of this message did not differ among groups and there was no significant correlation between ONH Gapdh level and IOP level or optic nerve injury grade. ONH gene expression was compared among three groups: controls (n = 12), Early Injury (optic nerve injury grade <2, n = 20), and Advanced Injury (optic nerve injury grade >2, n = 24). This analysis was designed to specifically examine gene expression changes associated with the earliest injury. Statistical analyses were performed using statistical software packages (Excel [Microsoft, Redmond WA] and Prism [GraphPad, San Diego, CA]). 
ONH DNA Quantitation
To determine whether injury from elevated IOP resulted in increased total ONH DNA content, DNA was extracted from comparable whole control and glaucoma model ONH with injury grades in a range similar to those used in the gene expression studies (PicoPure DNA Extraction Kit; Molecular Devices, Sunnyvale, CA) and was quantitated using an assay kit (Quant-iT PicoGreen dsDNA Assay Kit; Invitrogen, Carlsbad, CA) in accordance with the manufacturer's protocols. 
Results
The Early ONH Injury Group
Optic nerve injury grades were obtained for all samples, and eigengene analysis was used for data visualization to aid in the separation of glaucoma model sample array data, ordered by injury grade, into Early Injury and Advanced Injury groups for further analysis. The results of this eigengene analysis are shown in Figure 1. Note that the two array sets yielded nearly identical eigengene patterns for the first two eigengenes and that four significant patterns were found for each set. The most significant pattern (Eigengene 1) on both arrays was a linear response, with maximal change in the most severely affected ONH. We found that 52% of array probes were significantly correlated to Eigengene 1 (FDR <2%). This eigengene pattern is representative of genes that are regulated, either up or down in proportion to increasing optic nerve injury, likely reflecting changes related to early injury responses as optic nerve involvement becomes more widespread, and of many genes reflecting all the processes secondary to ongoing axon loss. Regression analysis showed that genes upregulated in proportion to injury grade were associated with the gene ontology (GO) categories of extracellular matrix, humoral immune response, lysosome, ribosome, cytoskeleton, cell differentiation, and signaling cascades. Genes that were downregulated in proportion to nerve injury grade included those regulating lipid biosynthesis, glucose metabolism, and mitochondria. 
The second most significant pattern (Eigengene 2) for both array sets was a fourth-order polynomial curve, with peak changes in expression below injury grade 2. Fifteen percent of array probes were significantly correlated to this Eigengene pattern, suggesting that a large group of genes demonstrated near maximal changes in expression in ONH from eyes with pressure-induced optic nerve damage of less than grade 2. 
Therefore, each sample was assigned to 1 of 3 groups for further analysis: Control ONH (untreated eyes, n = 6) and, based on injury grade and the two most significant eigengene patterns, Early ONH Injury (glaucoma model ONH, injury grades <2.0, n = 9) and Advanced ONH Injury (glaucoma model ONH, injury grade >2.0, n = 12). Table 1 summarizes the injury grade and the cumulative IOP elevation data for the two glaucoma model groups. In the Early Injury group, the mean interval between hypertonic saline injection and tissue collection was 21 ± 9 days. The mean interval between documented IOP elevation (5 mm above mean control eye IOP) and tissue collection was 12 ± 8 days. This group included six of the eyes that were collected at 10 days after the first tonometer (Tono-PenXL; Medtronic) IOP reading of 35 or greater. The peak IOP in the Early Injury group averaged 38.3 ± 1.9 mm Hg. Figure 2 illustrates a typical IOP history for a glaucoma model in the Early Injury group. Figure 3 illustrates an Early Injury group optic nerve lesion. These very early lesions are restricted to, at most, one or a few small, focal areas of the nerve cross-section, with a few degenerating axons intermingled with morphologically normal ones. 
Table 1.
 
Microarray Study: IOP Histories by Experimental Group
Table 1.
 
Microarray Study: IOP Histories by Experimental Group
Group n Optic Nerve Injury Grade (Mean ± SD) Cumulative Elevated IOP mm Hg (Mean ± SD)
Controls 6 1.0 ± 0.0
Early injury (1 < grade < 2) 9 1.3 ± 0.3 69 ± 25
Advanced injury (2 < grade ≤ 5) 12 4.3 ± 0.8 251 ± 164
Figure 2.
 
IOP history of a glaucoma model eye in the Early Injury group. On day 0, hypertonic saline was injected into an episcleral vein to induce elevated IOP, which became apparent at day 20.
Figure 2.
 
IOP history of a glaucoma model eye in the Early Injury group. On day 0, hypertonic saline was injected into an episcleral vein to induce elevated IOP, which became apparent at day 20.
Figure 3.
 
The area of lesion in a glaucoma model optic nerve with an injury grade of 1.4. Dark condensed degenerating axons (white arrows) occur in a few small, focal areas of the superior portion of the nerve cross-section, and a swollen axon (white arrowhead) is present. These degenerating axons are intermingled with many morphologically intact axons. The remaining optic nerve appears normal. Scale bar, 50 μm.
Figure 3.
 
The area of lesion in a glaucoma model optic nerve with an injury grade of 1.4. Dark condensed degenerating axons (white arrows) occur in a few small, focal areas of the superior portion of the nerve cross-section, and a swollen axon (white arrowhead) is present. These degenerating axons are intermingled with many morphologically intact axons. The remaining optic nerve appears normal. Scale bar, 50 μm.
Genes Significantly Regulated by Early Injury
The SAM Multiclass and ANOVA analysis yielded a combined total of 877 significantly regulated genes with unique Entrez Gene IDs in the Early Injury group, with 596 upregulated and 281 downregulated genes compared with the control group (Supplementary Table S3). Fifty-four genes were upregulated to greater than 250% of control ONH levels (Table 2), two-thirds of which have functions associated with the cell cycle and cell proliferation. Table 3 lists genes decreased in expression by at least 50% in the Early Injury group. Supplementary Tables S4 and S5, list genes significantly changed in expression in the other between-group comparisons. 
Table 2.
 
Microarray Analysis Genes Most Upregulated in the Early ONH Injury Group Compared with Control Values
Table 2.
 
Microarray Analysis Genes Most Upregulated in the Early ONH Injury Group Compared with Control Values
Accession No. Entrez Gene Gene Name* Gene Symbol Percentage of Control Mean†
BG087390 21973 Topoisomerase IIA Top2a 1424
BG069098 228421 Kinesin family member 18A Kif18a 970
AW547246 70021 5′-Nucleotidase domain containing 2 Nt5dc2 808
AW550270 21923 Tenascin C Tnc 762
BG088644 233406 Protein regulator of cytokinesis 1 Prc1 720
BG063624 52033 PDZ binding kinase Pbk 698
BG074248 110033 Kinesin family member 22 Kif22 651
BG069168 30939 Pituitary tumor-transforming 1 Pttg1 622
BG073145 80986 Cytoskeleton associated protein 2 Ckap2 608
AW553739 22137 Ttk protein kinase Ttk 598
BG065692 11504 Adamts1 Adamts1 593
AU018403 51945 Nuclear receptor subfamily 5, A, 2 Nr5a2 591
BG086805 12235 Budding uninhibited by benzimidazoles 1 Bub1 559
BG088110 26934 Rac GTPase-activating protein 1 Racgap1 555
BG072398 14284 Fos-like antigen 2 (Fosl2), mRNA Fosl2 530
AW553287 50706 Periostin Postn 521
BG070106 16819 Lipocalin 2 Lcn2 509
BG079289 77022 RIKEN cDNA 2700099C18 2700099C18Rik 499
BG070105 105387 Aldo-keto reductase family 1, member C14 Akr1c14 451
BG072904 19361 RAD51 homolog Rad51 436
BG078299 52276 Cell division cycle associated 8-Borealin Cdca8 429
BG068666 71819 Kinesin family member 23 Kif23 407
BG065826 18005 NIMA-related expressed kinase 2 Nek2 405
BG068948 66442 Spindle pole body component 25 homolog Spc25 385
BG086343 56419 Diaphanous homolog 3 Diap3 376
BG076991 12702 Suppressor of cytokine signaling 3 Socs3 371
BG073234 211770 Tribbles homolog 1 Trib1 360
BG073047 72119 TPX2, microtubule-associated protein Tpx2 356
BG088567 16009 Insulin-like growth factor binding protein 3 Igfbp3 354
BG076937 16906 Lamin B1 Lmnb1 349
BG071031 17393 Matrix metallopeptidase 7, matrylisn Mmp7 336
BG066442 16647 Karyopherin (importin) α2 Kpna2 334
BG080700 27279 Tumor necrosis factor receptor superfamily, member 12A Tnfrsf12a 329
BG079311 238076 Potassium voltage-gated channel, delayed-rectifier, S, 3 Kcns3 328
BG085472 12580 Cyclin-dependent kinase inhibitor 2C Cdkn2c 318
BG073518 12428 Cyclin A2 Ccna2 307
BG068045 14425 UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 Galnt3 304
BG066443 11806 Apolipoprotein A-I Apoa1 303
BG072979 108907 Nucleolar and spindle-associated protein 1 Nusap1 292
BG085966 14115 Fibulin 2 Fbln2 288
BG075693 69219 Dimethylarginine dimethylaminohydrolase 1 Ddah1 284
BG067382 74107 Centrosomal protein 55 Cep55 282
BG075357 17219 Minichromosome maintenance deficient 6 Mcm6 281
BG080268 14825 Chemokine (C-X-C motif) ligand 1 Cxcl1 271
BG069808 73710 Tubulin, β2B Tubb2b 269
BG085649 216161 Strawberry notch homolog 2 Sbno2 261
BG080666 15937 Immediate early response 3 Ier3 260
AW537534 326618 Tropomyosin 4 Tpm4 259
BG086929 56449 Cold shock domain protein A Csda 255
BG063171 76448 Phostensin RIKEN cDNA 2310014H01 2310014H01Rik 253
BG080690 20419 Shc SH2-domain binding protein 1 Shcbp1 252
BG081419 229841 Centromere protein E Cenpe 251
BG071607 12457 CCR4 carbon catabolite repression 4-like Ccrn4l 251
BG084556 17919 Myosin Vb Myo5b 250
Table 3.
 
Microarray Analysis Genes Most Downregulated in the Early ONH Injury Group Compared with Control Values
Table 3.
 
Microarray Analysis Genes Most Downregulated in the Early ONH Injury Group Compared with Control Values
Accession No. Entrez Gene Gene Name Gene Symbol Percentage of Control Mean*
BG086656 11668 Aldehyde dehydrogenase family 1, subfamily A1 Aldh1a1 15
BG073126 15902 Inhibitor of DNA binding 2 Id2 21
BG071519 223227 HMG-box protein (Sox21) Sox21 22
BG080229 11676 Aldolase 3, C isoform-L Aldoc 22
BG067929 75104 Monocyte to macrophage differentiation-associated 2-L Mmd2 25
BG087678 15360 3-Hydroxy-3-methylglutaryl-Coenzyme A synthase 2 Hmgcs2 29
BG074398 13602 SPARC-like 1 (hevin) Sparcl1 33
BG080206 20255 Secretogranin III Scg3 34
BG067564 21808 Transforming growth factor, β2 Tgfb2 34
BG073115 18027 Nuclear factor I/A Nfia 34
BG080743 54418 Formin 2 Fmn2 35
C85331 98582 KH domain containing 1B Khdc1b 35
C77281 12388 Catenin, delta 1 transcript variant 1 Ctnnd1 36
BG069709 332175 Zinc finger, DHHC domain containing 23 Zdhhc23 36
AW543487 105727 Solute carrier family 38, member 1 Slc38a1 38
BG080988 244867 Rho GTPase activating protein 20 Arhgap20 38
BG087491 83965 Ectonucleotide pyrophosphatase/phosphodiesterase 5 Enpp5 39
BG078755 76267 Fatty acid desaturase 1-L Fads1 39
BG065883 52225 DNA segment, Chr 4, ERATO Doi 103 D4Ertd103e 39
BG082257 106861 Abhydrolase domain containing 3 Abhd3 39
BG086781 84505 SET domain, bifurcated 1 Setdb1 39
BG080351 75712 Transmembrane protein 14A Tmem14a 40
BG070174 14866 Glutathione S-transferase, mu 5 Gstm5 41
BG084015 107272 Phosphoserine aminotransferase 1 Psat1 41
BG084178 19049 Protein phosphatase 1, regulatory (inhibitor) subunit 1B Ppp1r1b 41
BG067863 70757 Protein tyrosine phosphatase-like-L Ptplb 42
BG073601 13167 Diazepam binding inhibitor Dbi 42
BG074397 14862 Glutathione S-transferase, mu 1 Gstm1 42
BG087831 16508 Potassium voltage-gated channel, Shal-related family, member 2 Kcnd2 42
BG066209 231051 Myeloid/lymphoid or mixed-lineage leukemia 3 MII3 42
BG082752 12411 Cystathionine beta-synthase L Cbs 42
BG073940 228839 TGFB-induced factor 2 Tgif2 43
BG073190 14860 Glutathione S-transferase, α4 Gsta4 43
BG064512 18631 Peroxisomal biogenesis factor 11a Pex11a 44
AU018835 58187 Claudin 10A Cldn10a 44
BG072429 226025 Transient receptor potential cation channel, subfamily M, member 3 Trpm3 44
BG069624 234593 N-myc downstream regulated gene 4 Ndrg4 44
BG088903 235180 Fasciculation and elongation protein zeta 1 Fez1 45
BG069040 215821 D10Bwg1379e D10Bwg1379e 45
BG086971 11487 A disintegrin and metallopeptidase domain 10 Adam 10 45
AW557873 15903 Inhibitor of DNA binding 3 Id3 45
BG088107 50781 Dickkopf homolog 3 Dkk3 46
BG062997 67080 RIKEN cDNA 1700019D03 gene 1700019D03Rik 46
BG076141 108699 Chimerin (chimaerin) 1 Chn1 46
BG087940 223649 Nuclear receptor binding protein 2 Nrbp2 46
BG079962 236539 3-Phosphoglycerate dehydrogenase Phgdh 47
BG075920 217480 Diacylglycerol kinase, beta Dgkb 47
BG069750 17769 5,10-Methylenetetrahydrofolate reductase Mthfr 47
BG073197 195434 UTP14, U3 small nucleolar ribonucleoprotein, homolog B (yeast) Utp14b 47
BG075567 259302 SLIT-ROBO Rho GTPase activating protein 3 Srgap3 48
BG075594 384061 Fibronectin type III domain containing 5 Fndc5 48
BG070737 20512 Solute carrier family 1 (glial high-affinity glutamate transporter) Slc1a3 48
BG087565 54151 Cysteine and histidine rich 1 Cyhr1 48
BG066372 17762 Microtubule-associated protein tau Mapt 48
BG088467 71833 WD repeat domain 68 Wdr68 48
BG087239 226970 Rho guanine nucleotide exchange factor (GEF) 4 Arhgef4 49
BG074754 14081 Acyl-CoA synthetase long-chain family member 1 Acsl1 49
BG087975 14183 Fibroblast growth factor receptor 2 Fgfr2 49
BG081752 19791 Rn18s ribosomal RNA Rn18s 49
BG064101 14897 Thyroid hormone receptor interactor 12 Trip12 49
BG072359 14085 Fumarylacetoacetate hydrolase Fah 49
BG065128 26399 Mitogen activated protein kinase kinase 6 Map2k6 49
BG073314 16515 Potassium inwardly-rectifying channel, subfamily J, member 12 Kcnj12 49
C87546 20869 Serine/threonine kinase 11 Stk11 49
Identification of Functional Gene Categories Significantly Regulated in Early ONH Injury
The most significantly upregulated gene categories by DAVID analysis in the Early Injury group are listed in Table 4. The cell cycle was the most significantly upregulated category and included approximately 14% of all upregulated genes. Among the most upregulated genes in this category were protein regulator of cytokinesis 1 (Prc1, 720%), pituitary tumor-transforming 1 (securin, Pttg, 622%), and budding uninhibited by benzimidazoles 1 (Bub1, 559%), three genes with specific functions in chromosome separation during cytokinesis, suggesting completion of the cell cycle. Note that the most upregulated gene in Table 2 was topoisomerase 2a (Top2a, 1424%). Although not a component of the cell cycle GO category, this enzyme is essential for DNA replication and a component of the related category of cell proliferation. The second most upregulated GO category in Early Injury was the cytoskeleton. Several of the most upregulated genes in this category were kinesins with specific mitotic and spindle-associated functions (Kif18a, Kif22, Kif23, Nek2, Tpx2, and Nusap1; 970%–292%), also suggesting cell proliferation. The third category was immune process genes. Among the most affected of these genes were the cytokines chemokine (C-X-C motif) ligand 1 (Cxcl1, 271%) and Lif (223%), both of which reached peak levels in the Early Injury group. The immune process category also included components of the complement cascade (C1qc, C1qa, Cfi, Cfh, and C1r) that were upregulated to between 215% and 150% of control values in Early Injury. 
Table 4.
 
Upregulated Gene Ontology Categories in Early ONH Injury
Table 4.
 
Upregulated Gene Ontology Categories in Early ONH Injury
Significantly Upregulated Gene Categories Genes (n) P
Cell component or biological function*
    Cell cycle 82 <0.00001
    Cytoskeleton 77 <0.00001
    Immune system process 42 <0.00005
    Cell motility 25 <0.005
    Extracellular matrix 19 <0.005
    Lysosome 16 <0.005
    Plasma membrane 65 <0.001
    Cell adhesion 32 <0.05
    Proteolysis 35 <0.05
    Cell differentiation 80 <0.05
    Blood vessel development 16 <0.05
    Generation of neurons 19 <0.05
    Phosphorylation 42 <0.05
Table 5 summarizes the significant GO categories of the 271 genes downregulated in the Early Injury group. Most affected were GO categories related to glucose and lipid metabolism. However, the largest category was that for integral membrane proteins. Among the most downregulated genes with known functions in this class were the astrocyte glutamine and glutamate transporters Slc38a1 (38%) and Slc1a3 (48%) and the potassium channels Kcnd2 (42%) and Kcnj12 (50%). 
Table 5.
 
Downregulated Gene Ontology Categories in Early ONH Injury
Table 5.
 
Downregulated Gene Ontology Categories in Early ONH Injury
Significantly Downregulated Gene Categories Genes (n) P
Cell component or biological function
    Glucose metabolic process 9 <0.0005
    Peroxisome 7 <0.005
    Lipid metabolic process 20 <0.005
    Epithelial cell proliferation 4 <0.01
    Serine family amino acid biosynthetic process 3 <0.05
    Intrinsic membrane protein 64 <0.05
    Positive regulation of progression through cell cycle 3 <0.05
Inclusion of the few arrays available in the range of 2 to 4 did not have a substantial effect on the identification of genes significantly affected in the Early Injury Group. When we repeated our analysis excluding the data from these intermediate arrays, the probes identified as changed were 96% identical with those identified by the original analysis (Supplementary Table S3). With the exception of the addition of Bcas1 as among the most downregulated (41%), there was no change in the genes identified as most significantly affected (Tables 2, 3) or significant GO categories (Tables 4, 5). 
Changes in Axonal mRNA Levels as Indicators of Early ONH Injury
In our previous array analysis of extensively injured ONH, we found significant downregulation of several retinal ganglion cell–specific messages that are present in optic nerve axons. 27 In the present study, we examined two of these axonal messages, growth-associated protein 43 (Gap43) and neurofilament L (Nefl) by QPCR. Although both were significantly downregulated in the Advanced Injury group (to 26% ± 5% and 22% ± 5% of control values, respectively), only Gap43 was significantly reduced in Early Injury (65% ± 10%). 
The brain-specific isoform of aldolase, Aldoc, was among the most downregulated genes in Early Injury by array analysis (Table 3). Aldoc is a component of axonal mRNA, 38,39 but it is also expressed by astrocytes. By QPCR, Aldoc was confirmed to be significantly reduced in both Early (50% ± 9%) and Advanced (24% ± 4%) injury groups. In our arrays, the only distinctly axonal mRNA that was significantly downregulated in the Early Injury group was microtubule-associated protein tau (Mapt, 49%). Therefore, as expected based on the small amount of optic nerve degeneration in the Early Injury group, the axonal mRNA changes in Early Injury ONH were not dramatic. 
Confirmation of Upregulation of Cell Proliferation Genes in Glaucoma Model ONH
Our microarray studies identified the cell cycle as the most upregulated biological process in Early Injury. To confirm these observations, we used QPCR to examine expression levels for several genes associated with cell proliferation (Fig. 4). During DNA replication and transcription, Top2a controls the topological states of DNA by transient breakage and subsequent rejoining of DNA strands. By QPCR, Early Injury group Top2a values were 610% ± 153% of control. Prc1 regulates the mitotic spindle midzone formation during cytokinesis. Extra spindle pole bodies homolog 1 (Espl1 or separase) acts together with Pttg1 (622% in Early Injury on arrays) to regulate chromatid separation at anaphase. By QPCR, Prc1 and Espl1 levels were found to be 838% ± 213% and 438% ± 120% of control values in the Early Injury group. 
Figure 4.
 
Verification of upregulation of selected cell proliferation associated genes in Early and Advanced ONH Injury groups by QPCR. Top2a plays a role in DNA replication, whereas Prc1 and Espl1 function in cytokinesis. *P < 0.05, ‡P < 0.01, and †P < 0.001 compared with the control group.
Figure 4.
 
Verification of upregulation of selected cell proliferation associated genes in Early and Advanced ONH Injury groups by QPCR. Top2a plays a role in DNA replication, whereas Prc1 and Espl1 function in cytokinesis. *P < 0.05, ‡P < 0.01, and †P < 0.001 compared with the control group.
Increased DNA Content with Pressure-Induced ONH Injury
To confirm that cell cycle progression occurs early in ONH injury, total DNA content was measured in groups of glaucoma model ONH with optic nerve injury grades similar to those in the microarray and QPCR studies. ONH DNA content in the Early Injury group was increased to 117% (P < 0.05, one way t-test) compared with controls, whereas the DNA content was nearly doubled in ONH with Advanced Injury (Table 6). 
Table 6.
 
Total ONH DNA Content
Table 6.
 
Total ONH DNA Content
Group DNA Content (Percentage of Control Mean) Grade n
Control 100 ± 8 1.0 ± 0.0 10
Early injury 117 ± 6* 1.2 ± 0.1 11
Advanced injury 184 ± 12† 4.6 ± 0.2 13
Gene Expression Analysis of IL-6–type Cytokines and Janus Kinase-Signal Transducer and Activator of Transcription Signaling in Early ONH Injury
Our array analysis had demonstrated the upregulation of the cytokine Lif (223%) in Early Injury. Lif is known to promote glial cell proliferation and astrocytic differentiation. 40 42 Lif signaling by way of the Janus kinase-signal transducer and activator of transcription (Jak-Stat) pathway can lead to the upregulation of the feedback inhibitor suppressor of cytokine signaling 3 (Socs3), which was increased to 373% in the Early Injury group (Table 2). Another gene highly upregulated in this group is Racgap1 (556%), a gene associated with cytokinesis. Interestingly, Racgap1 has recently been shown to be essential for the Jak phosphorylation of transcription factor, Stat3 protein, and its entry into the nucleus. 43 Lif is a member of the IL-6–type cytokine family, all which share a common receptor component (IL-6 signal transducer, IL-6st, aka Gp130) to initiate signaling, primarily through Jak-Stat signaling pathways. 44  
Observation of Lif, Socs3, and Racgap1 among the most upregulated genes suggested Jak-Stat pathway activation in early ONH injury. Because members of this family play important roles in glial responses to injury, including glial progenitor cell proliferation and differentiation to astrocytes, 42,45,46 we used QPCR to determine the responses of all IL-6–type cytokines in glaucoma model ONH, many of which were not on our arrays. These studies not only confirmed the upregulation of Lif, they also revealed a significant and dramatic upregulation of IL-6 (1553%), coupled with significant responses by cardiotrophin-like cytokine factor 1 (Clcf1, 325%) and downregulation of ciliary neurotrophic factor (Cntf, 40%) in the Early Injury group (Fig. 5). 
Figure 5.
 
IL-6–type cytokine, Lifr, and Socs3 expression in glaucomatous ONH injury examined by QPCR. This demonstrates the dramatic upregulation of IL-6 in early ONH injury. In addition, it confirms the upregulation of Lif identified by array analysis and reveals significant regulation of 2 of 4 other members of this family that are expressed in the ONH, Clcf1 and Cntf. Lifr, the common receptor component for Lif, Clcf1, and Cntf were significantly downregulated in both injury groups. Upregulation of a target of Jak-Stat signaling, Socs3, in both injury groups was significant, confirming array results. Note that the scale of the y-axis is log2 and that Control group variation is indicated by thicker SEM bars. *P < 0.05, ‡P < 0.01, †P < 0.001 compared with Controls.
Figure 5.
 
IL-6–type cytokine, Lifr, and Socs3 expression in glaucomatous ONH injury examined by QPCR. This demonstrates the dramatic upregulation of IL-6 in early ONH injury. In addition, it confirms the upregulation of Lif identified by array analysis and reveals significant regulation of 2 of 4 other members of this family that are expressed in the ONH, Clcf1 and Cntf. Lifr, the common receptor component for Lif, Clcf1, and Cntf were significantly downregulated in both injury groups. Upregulation of a target of Jak-Stat signaling, Socs3, in both injury groups was significant, confirming array results. Note that the scale of the y-axis is log2 and that Control group variation is indicated by thicker SEM bars. *P < 0.05, ‡P < 0.01, †P < 0.001 compared with Controls.
Cellular receptor expression will determine the functional responses to IL-6 family cytokines. We used both our microarray data and QPCR to determine expression levels of the components of all the IL-6–type cytokine receptor components in glaucoma model ONH. By QPCR, we found that Lif receptor (Lifr) was downregulated to 35% ± 11% in Early Injury (Fig. 5), whereas the other IL-6 family receptor components (II6st and Cntfr, array data, II6ra by QPCR; Supplementary Table S6) were not significantly altered in expression in any experimental group. Lifr is the common receptor for Lif, Clcf1, Cntf, and two other cytokines that were unchanged in ONH mRNA expression, oncostatin M (Osm) and cardiotrophin 1 (Ctf 1; Supplementary Table S6). 
Among the Jak-Stat pathway components on our arrays, we found moderate, but significant, upregulation of Jak2 (146%) and Stat3 (147%) in Early Injury, whereas Jak1, Stat1, and Stat2 levels were not significantly affected. We also used QPCR to confirm the upregulation of Socs3, finding an increase to 402% ± 96% in the Early Injury group (Fig. 5). Socs3 can inhibit signaling through other receptors, such as epidermal growth factor receptor (Egfr). 47 Socs4, another inhibitor of Egfr signaling, was significantly upregulated (175%) as well by array analysis. By QPCR, Egfr levels were unchanged by Early Injury (95% ± 17%), whereas epidermal growth factor levels fell significantly to 38% ± 7% (Supplementary Table S6). 
Cytokine Cxcl1 in Early Injury
Cytokine Cxcl, also highly expressed in the Early Injury group (273%), is expressed by injured astrocytes, 48 microglia, and oligodendroglial precursors, 49 is a mitogen for both endothelial cells and oligodendroglia, 49,50 and may recruit progenitor cells to areas of injury. 51  
Other Growth Factors in Early Injury
A number of growth factors have been suggested to play important roles in ONH responses to IOP-induced injury. Transforming growth factor β2 (Tgfb2) is upregulated in the aqueous humor in glaucoma, exerts significant effects in the anterior segment, and may play a role in ONH ECM remodeling in glaucoma. 52 54 However, our studies show that Tgfb2 is downregulated in Early Injury ONH (34%, Table 3; QPCR, Supplementary Table S6), confirming our earlier observation. 27 In contrast, our QPCR analyses demonstrated that Early Injury had no significant effect on mRNA levels of brain-derived neurotrophic factor, neurotrophin 4/5, nerve growth factor, neurotrophic tyrosine kinase receptor Trkb, or p75 neurotrophin receptor (Supplementary Table S6). 
ECM Gene Expression Changes in Glaucoma Model ONH Injury
Human glaucomatous optic neuropathy involves extensive remodeling of the ECM of the lamina cribrosa, the network of connective, glial, and vascular tissue that spans the scleral opening and supports optic nerve axons as they exit the globe. 55,56 Our previous studies demonstrate that changes in ECM gene expression and protein deposition also occur early during IOP-induced injury in rat glaucoma model ONH, 24,25,27 an observation further supported by the DAVID analysis used in this study (Table 4). By QPCR, we confirmed increased expression levels of four of these ECM genes: Postn, Tnc, Fbln2, and matrix gla protein (Mgp; Fig. 6). All four are matricellular proteins, proteins that modulate cell-to-cell and cell-to-matrix interactions and are implicated in processes such as cell proliferation and migration. 57 Other matricellular proteins significantly affected in early injury were secreted phosphoprotein 1(osteopontin, Spp1 204%) and SPARC-like 1 (hevin, Sparcl1 (33%). Spp1 upregulation is associated with microglial activation and stimulates astrocyte migration. 58 Sparcl1 has antiadhesion, antiproliferation properties and is present in differentiated astrocytes and endothelial venules. 59 61  
Figure 6.
 
Pressure-induced changes in ONH ECM gene expression verified and determined by QPCR. Although changes began in early injury, upregulated gene expression of four matricellular ECM proteins (Postn, Tnc, Fbln2, and Mgp) became significant in the Advanced Injury group. In contrast, the integrin component Itgav was downregulated in the Early Injury group. Timp1, the ECM metalloproteinase inhibitor, was significantly upregulated in both injury groups. Note that the scale of the y-axis is log2 and that the variation of the Control group is indicated by thicker SEM bars. *P < 0.05, ‡P < 0.01, and †P < 0.001 compared with Control group. For Postn, Fbln2, and Mgp, differences between Early and Advanced injury group values are also significant (P < 0.01).
Figure 6.
 
Pressure-induced changes in ONH ECM gene expression verified and determined by QPCR. Although changes began in early injury, upregulated gene expression of four matricellular ECM proteins (Postn, Tnc, Fbln2, and Mgp) became significant in the Advanced Injury group. In contrast, the integrin component Itgav was downregulated in the Early Injury group. Timp1, the ECM metalloproteinase inhibitor, was significantly upregulated in both injury groups. Note that the scale of the y-axis is log2 and that the variation of the Control group is indicated by thicker SEM bars. *P < 0.05, ‡P < 0.01, and †P < 0.001 compared with Control group. For Postn, Fbln2, and Mgp, differences between Early and Advanced injury group values are also significant (P < 0.01).
Increased collagen expression, a characteristic of human and experimental glaucomatous ONH remodeling, 24,62 64 was indicated in our array study by modest upregulation of collagens Col4a1 (231%), Col6a2 (169%), and Col5a2 (168%) in the Early Injury group. 
Integrins are important mediators of cell ECM adhesion. 56 Among integrin probes on our arrays (α1, α3, α5, αV, and α9; β1, β3, β4, β5, and β7), only integrins β1 (Itgb1) and αV (Itgav) were significantly altered by Early Injury to 197% and 64% of controls, respectively. Intgb1 is a common receptor component for many ECM proteins, whereas Itgav receptors are somewhat more selective, linking the cellular cytoskeleton to fibronectin, laminins, tenascins, Spp1, vitronectin, L1 cell adhesion molecule, Postn, and various RGD-containing proteins. 65 In neural tissues, Itgav is expressed by glia and is often involved in the mediation of vascular interactions. 66 QPCR confirmed Itgav downregulation in the Early Injury group (Fig. 6). 
In an earlier study of retinal gene expression changes in our glaucoma model, 67 Timp1 was the most upregulated gene. Because probes for this protease inhibitor were not present on the arrays used in the present study, we determined its expression by QPCR, finding it significantly upregulated in the Early (437%) and Advanced ONH Injury group (Fig. 6). 
Astrocyte-Specific Marker Expression in Early IOP-Induced ONH Injury
As in our previous report, 27 QPCR demonstrated that the expression of the common marker for activated astrocytes, Gfap, was not increased in glaucoma model ONH (Fig. 7). Additionally, levels of five other astrocyte-associated messages associated with mature ONH astrocytes were evaluated and found to be either decreased or not significantly changed by QPCR: Aqp4, Gja1, multiple EGF-like-domains 10 (Megf10), and paired box 2 (Pax2). Aqp4 is a water channel associated with astrocytic endfeet and the blood brain/cerebral spinal fluid barrier. 68,69 Gja1 is a key component of astrocytic gap junctions. 70 Megf10 is associated with astrocytic phagocytosis. 40 The transcription factor Pax2 is specifically expressed by optic nerve astrocytes 71 73 and associated with proliferative capacity. 71,74,75 Nestin (Nes) and vimentin (Vim) are expressed in immature or proliferating astrocytes. 76 Vim levels were increased only in the Advanced Injury group (145% ± 8%). Additionally, microarray analysis showed that expression of the glial glutamate transporter Slc1a3 was significantly reduced in both injury groups (Table 3). Finally, S100b, a macroglial marker, was significantly downregulated in both injury groups (Supplementary Table S6). 
Figure 7.
 
Pressure-induced changes in astrocyte-associated ONH messages determined by QPCR. Levels of four messages associated with mature astrocytes (Gfap, Aqp4, Gja1, and Megf10) either were unchanged or were significantly downregulated. Also significantly downregulated was transcription factor Pax2, associated with astrocytic proliferative capacity. Nes and Vim are intermediate proteins expressed in immature astrocytes. Moderate Vim upregulation was found in the Advanced Injury group. *P < 0.05, ‡P < 0.01, †P < 0.001.
Figure 7.
 
Pressure-induced changes in astrocyte-associated ONH messages determined by QPCR. Levels of four messages associated with mature astrocytes (Gfap, Aqp4, Gja1, and Megf10) either were unchanged or were significantly downregulated. Also significantly downregulated was transcription factor Pax2, associated with astrocytic proliferative capacity. Nes and Vim are intermediate proteins expressed in immature astrocytes. Moderate Vim upregulation was found in the Advanced Injury group. *P < 0.05, ‡P < 0.01, †P < 0.001.
Microglial-Specific Marker Expression in Early IOP-Induced ONH Injury
Expression levels of some microglial activation markers not present on our arrays were determined by QPCR (Fig. 8). Allograft inflammatory factor (Aif1, or IBA1), a calcium channel associated with microglial hypertrophy and proliferation, was significantly upregulated, but only in the Advanced Injury group (298% ± 45%). Elevation of Colony stimulating factor 1 receptor (Csf1r), which plays a key role in microglial activation, in the Advanced Injury group did not reach statistical significance. Purinergic receptor P2Y12 (P2ry12) is an adenine nucleotide receptor abundant in microglia in the brain, 77 although it is also present in endothelial cells. 78 P2ry12 downregulation is associated with microglial activation and process retraction 79 and was significant in the Early ONH injury group (Fig. 8). 
Figure 8.
 
Microglial marker message levels determined by QPCR. Upregulation of calcium channel Aif1 and growth factor receptor Csf1r and downregulation of purine receptor P2ry12 are all associated with microglial activation. Of these messages, only the downregulation of P2ry12 was significant in the Early Injury group, whereas Aif1 levels reached significance only in the Advanced Injury group. *P < 0.05; ‡P < 0.01.
Figure 8.
 
Microglial marker message levels determined by QPCR. Upregulation of calcium channel Aif1 and growth factor receptor Csf1r and downregulation of purine receptor P2ry12 are all associated with microglial activation. Of these messages, only the downregulation of P2ry12 was significant in the Early Injury group, whereas Aif1 levels reached significance only in the Advanced Injury group. *P < 0.05; ‡P < 0.01.
Other messages present on our arrays that are enriched in microglia include cd34 antigen, 80 ferritin light chain (Ftl1), translocator protein (peripheral benzodiazepine receptor, Tspo), TGFβ1 receptor 1 (Tgfb1r1), and milk fat globule EGF factor 8 protein (Mfge8). Of these, only Cd34 was significantly upregulated in the Early Injury group (Supplementary Table S3). 
Oligodendroglia-Specific Marker Expression in Early IOP-Induced ONH Injury
In this study, though we limited our analysis to the initial 0.4-mm portion of the optic nerve, some axons in the distal portion of this segment were partially myelinated; therefore, our analysis includes a population of oligodendroglia. By QPCR, we found that chondroitin sulfate proteoglycan 4 (Cspg4, or NG2), an oligodendroglial precursor marker, 81 was not significantly altered in expression in glaucoma model ONH (Supplementary Table S6). By array analysis, platelet-derived growth factor α (Pdgfa) was unchanged, and myelin basic protein (Mbp) was significantly increased only in the Advanced Injury group (150%, Supplementary Table S4). 
Vascular-Specific Marker Expression in Early IOP-Induced ONH Injury
In addition to neural tissues, vascular components of the ONH are likely to play critical roles in the tissue response to elevated IOP, as confirmed by our DAVID analysis of upregulated gene categories (Table 4). The upregulated genes specifically associated by array analysis with the vasculature in the early ONH injury group most frequently were Adamts1 (593%), tweak receptor (Tnfrsf12a, 329%), non-muscle myosin Myh9 (244%), and the Vegfb binding protein neuropilin1 (Nrp1, 171%). In addition, among significantly downregulated genes were the endothelin b receptor (Ednrb, 55%) and vascular-associated Itgav (Supplementary Table S3). 
To better evaluate the potential contribution of vascular cells to the dramatic gene expression in early pressure-induced ONH injury, we examined some vascular-associated messages by QPCR (Fig. 9). Endothelin 1 (Edn1) and adrenomedullin (Adm) are potent vasoconstrictor and vasodilator proteins, respectively. Heavy chain myosin 11 (Myh11) is a marker for vascular smooth muscle. Vegfa and Vegfb regulate angiogenesis and blood-brain barrier permeability. The transcription factor hypoxia inducible factor 1α (Hif1a) as well as erythropoietin (Epo) and heme oxygenase 1 (Hmox1) are frequently upregulated under hypoxic conditions (Supplementary Table S6). Of these, the only significant change in the Early Injury group was a downregulation of Vegfb to 61% ± 5%. 
Figure 9.
 
Vascular-associated message levels determined by QPCR. Message levels for the vasoactive compounds, End1 and Adm, were not significantly altered, Vegf isoforms were not upregulated, and message levels of Hif1a remained unchanged. The smooth muscle myosin Myh11 was significantly downregulated in the Advanced Injury group. *P < 0.05.
Figure 9.
 
Vascular-associated message levels determined by QPCR. Message levels for the vasoactive compounds, End1 and Adm, were not significantly altered, Vegf isoforms were not upregulated, and message levels of Hif1a remained unchanged. The smooth muscle myosin Myh11 was significantly downregulated in the Advanced Injury group. *P < 0.05.
ONH Gene Expression Change with Physiological IOP Elevation
The physiological IOP elevation associated with the dark phase of the light cycle resulted in moderate, but significant, expression changes in many ONH genes. DAVID analysis indicated that upregulated genes primarily affected signal transduction and the cytoskeleton, whereas downregulated genes were primarily associated with ribosomal protein synthesis. Of the genes significantly regulated by physiological IOP elevation, 170 genes were significantly regulated in the same direction as in the Early Injury group. The magnitude of change was generally greater in Early Injury. For example, of the genes listed in Table 2, only five genes (Fosl2, Trib1, Ddah1, Tubb2b, and Tpm4) were also upregulated by physiological IOP elevation. For these five, the magnitude was <50% of the level reached in the Early Injury group. Similarly, for the genes downregulated by Early Injury listed in Table 3, Physiological IOP elevation downregulated only D4Ertd103e and Ctnnd1 by >50%. 
Discussion
Microarray analysis of gene expression is a powerful tool for evaluating tissue responses to injury, such as elevated IOP in the eye. However, the contribution of array analysis to the understanding of disease processes may be limited by a number of factors, including the quality and relevance of the samples analyzed, the adequacy of the study design, the array platform and the probe spectrum it contains, the quality of the technical procedure, and the statistical analysis. Additionally, key messages or pathway components may not be present at a level of abundance that allows their detection, or, perhaps more important, pathways may be regulated at the protein level rather than the gene expression level. With these caveats, the microarray analysis and supportive studies reported here lead to the following observations about the earliest ONH responses to elevated IOP exposure. 
The ONH Genomic Response to Minimal Pressure-Induced Injury Is Dominated by the Upregulation of Cell Proliferation Genes
Minimal exposure to elevated IOP (cumulative exposure of <100 mm Hg above controls over a 2-week period) results in the significant regulation of a large number of ONH genes. These Early Injury genes are dominated by the increased expression of genes associated with all phases of the cell cycle, indicating that cell cycle progression and mitosis are early and significant events in ONH injury. This study also shows that for most of these cell cycle genes, peak ONH levels are reached when optic nerve axon degeneration is <15%. Our findings of increased DNA content in these minimally injured ONH support these observations. These studies also confirm similar observations made in our earlier study of longer (1-mm) segments of glaucoma model ONH from eyes with extensive optic nerve axon degeneration. 27  
Altered IL-6–type Cytokine Signaling Is Implicated in the ONH Response to Early Elevated IOP-Induced Injury
This study demonstrates significant upregulation of three IL-6–type cytokines (IL-6, Lif, and Clcf1) and implicates altered Jak-Stat pathway signaling, as evidenced by Socs3 upregulation, as early ONH responses to elevated IOP exposure. IL-6 and Lif can be produced by all the major cellular components of the ONH (Lambert WS, et al. IOVS 2009;50:E-Abstract 2792), 41,42,82 87 and both can exert axonal neuroprotective, regenerative, 88 92 and proliferative responses. 45,93 All three cytokines can induce astrocytic differentiation from precursor cells. 42,94 96 Lif may also promote microglial proliferation. 97 In addition, these cytokines may act on ONH vascular components because IL-6 can stimulate smooth muscle cell proliferation, 98 although Lif has been shown to inhibit retinal endothelial cell proliferation. 99 Although less is known about Clcf1, this cytokine does have both growth factor 100 and neuroprotective properties, 101 and its expression is associated with response to stress. 102 In contrast to the other three, Cntf was significantly downregulated in Early Injury, as has been observed after nerve crush. 103 ONH astrocytes express Cntf and its tripartite receptor components, 104 and, in retina, Cntf represses glial proliferation. 105 Therefore, the downregulation of Cntf may complement the proliferation-promoting actions of the upregulated cytokines in this family. Together, these observations suggest that altered expression of these cytokines may contribute to proliferative responses in the ONH to elevated IOP exposure. 
In Early Injury ONH, expression levels for the receptor components of the IL-6-family cytokines were unchanged, except for the downregulation of Lifr, the common receptor component for Lif, Clcf1, and Cntf. Lifr is expressed by most ONH cellular components 106 112,113 and is important for astrocyte precursor differentiation, including Jak-Stat pathway-regulated Gfap expression. 114 117 Therefore, the downregulation of this receptor is consistent with the lack of increased Gfap expression. In addition, in the retina, Lifr plays an essential role in endogenous neuroprotective mechanisms triggered by preconditioning-induced stress, 118 suggesting that Lifr downregulation in the ONH may decrease the effectiveness of potential protective signaling by this receptor. 
These observations suggest the involvement of IL-6–type cytokines in early ONH injury; however, we fully realize that the cell-specific locations and impact on ONH integrity of potential activated receptor pathway signaling remains to be determined. 
Upregulation of GFAP or Other Astrocyte-Specific Genes Is Not a Characteristic of Early ONH Responses to Elevated IOP Exposure
Upregulation of Gfap is such a common marker for astrocytes responding to, or “activated” by, neural injury that the increased expression of this intermediate filament protein is often considered necessary and sufficient evidence of an astrocytic reaction. In glaucoma model retinas and the RGC layer, Gfap upregulation is readily observed. 67,119 121 However, in the ONH, early injury from elevated IOP exposure is not associated with the significant upregulation of Gfap or any other astrocyte-specific gene we examined. Rather, a number of unique astrocytic genes were significantly downregulated. This finding is consistent with the expected downregulation of differentiated cellular functions that would be anticipated if astrocytic mitosis were a major component of the proliferative responses observed in these minimally injured ONH. This finding also suggests an initial passive, rather than active, contribution of astrocytes to further nerve injury because a reduction in differentiated astrocytic functions is likely to include compromised astrocyte-mediated metabolic and functional support for axons. In the ONH, where the metabolic demands of unmyelinated axons are much greater than those of the myelinated nerve 122 and individual astrocytes are in functional contact with many axons, 123 125 such a compromise is likely to contribute significantly to further axonal vulnerability to elevated IOP. 
Activation and Potential Proliferation of Other Glia Cell Types
In early injury, a purine receptor abundant in brain microglia, 77 P2ry12, was significantly downregulated, a characteristic of microglial activation. 79,126,127 Additionally, our array data showed that Nrp1 was significantly upregulated by early injury, and Nrp1 upregulation has been localized to activated microglia. 128 Further evidence of microglial activation became apparent in eyes with more advanced injury as Aif1 expression was significantly increased. However, additional studies are needed to determine specifically whether both microglial proliferation and activation occur early in the time course of pressure-induced ONH injury. 
Early Injury and Vasculature-Associated Genes
Our array GO analysis and our QPCR studies indicate that early pressure-induced ONH injury is associated with altered expression of some genes associated with the vascular component of the ONH. Similar Myh9 and Myh11 responses are observed in carotid artery injury, 129 and Tnfrsf12a upregulation is associated with endothelial cell proliferation and migration. Nrp1 is localized to the perivascular region of injured neural tissues, 130 in proximity to its binding partner, Vegfb, which was downregulated in the ONH in early injury. Although the specific functions of Vegfb protein are not well understood, it is abundant in brain and retina and is thought to play a critical role in the survival, rather than growth, of vascular cells. 131 133  
ECM Matricellular Protein Gene Expression
Expression levels of a number of ECM matricellular proteins were significantly altered in early ONH injury. The upregulation of one of these messages, Postn, begins in early injury and is one of the most dramatically upregulated genes in advanced injury. In our study of extensively injured ONH, Postn was the most upregulated gene. 27 Postn is expressed in cells subjected to mechanical stress, particularly vascular smooth muscle cells. 134 Postn and the other affected matricellular genes are expressed by astrocytes 39,59,135,136 and are altered in expression during injury-induced vascular remodeling and repair. 137 142 These observations suggest that modification of astrocyte-ECM, and possibly vascular cell-ECM, interactions begins early in pressure-induced ONH injury, implying that the interface between astrocyte processes and the vasculature may be particularly vulnerable. 
Further Directions
Currently, we are using immunohistochemistry to identify proliferating ONH cells and those that express the IL-6–type cytokines as well as the protein products of other affected genes identified in this study. In addition, we are in the process of developing a model of glaucomatous ONH injury in which the level and duration of elevated IOP exposure can be precisely controlled. Such a model will allow us to determine the exact onset and time course of these ONH gene expression responses, with the goal of understanding their relationship to the development of glaucomatous axon loss. 
Supplementary Materials
Table st01, XLS - Table st01, XLS 
Footnotes
 Supported by National Institutes of Health Grants EY016866 and EY010145 and by an unrestricted grant from Research to Prevent Blindness, Inc.
Footnotes
 Disclosure: E.C. Johnson, None; T.A. Doser, None; W.O. Cepurna, None; J.A. Dyck, None; L. Jia, None; Y. Guo, None; W.S. Lambert, None; J.C. Morrison, None
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Figure 1.
 
Definition of the Early and Advanced ONH Injury groups. The Pattern Discovery module in SAM was used to identify the significant eigengenes (principal components) in the microarray data sets arrayed by increasing optic nerve injury grade (6 controls, 21 glaucoma model ONH from eyes with optic nerve injury grades from 1.025 to 5.) Each array data set (SMCmou6600A and SMCmou8400A) was separately analyzed, and significant eigengenes (P < 0.05) from both were plotted. Curve-fitting and regression analysis were used to determine the statistically significant best-fit line for each eigengene, as plotted here. The two array sets yielded nearly identical eigengene patterns for the first two eigengenes. The most significant pattern (Eigengene 1, red lines) on both arrays was a linear response, with maximal change in the most severely injured ONH. The second most significant pattern (Eigengene 2) for both array sets was a fourth-order polynomial curve, with peak changes below injury grade 2 (approximately 15% axons degenerating). This pattern indicated that a large number of significantly affected ONH genes responded maximally to early pressure-induced damage. Based on nerve injury grade and these two eigengene patterns, data from each sample was assigned to 1 of 3 groups for further analysis: Control ONH (untreated eyes), Early ONH Injury (glaucoma model ONH with optic nerve injury grades <2.0, n = 9), and Advanced ONH Injury (glaucoma model ONH with optic nerve injury grades >2.0, n = 12).
Figure 1.
 
Definition of the Early and Advanced ONH Injury groups. The Pattern Discovery module in SAM was used to identify the significant eigengenes (principal components) in the microarray data sets arrayed by increasing optic nerve injury grade (6 controls, 21 glaucoma model ONH from eyes with optic nerve injury grades from 1.025 to 5.) Each array data set (SMCmou6600A and SMCmou8400A) was separately analyzed, and significant eigengenes (P < 0.05) from both were plotted. Curve-fitting and regression analysis were used to determine the statistically significant best-fit line for each eigengene, as plotted here. The two array sets yielded nearly identical eigengene patterns for the first two eigengenes. The most significant pattern (Eigengene 1, red lines) on both arrays was a linear response, with maximal change in the most severely injured ONH. The second most significant pattern (Eigengene 2) for both array sets was a fourth-order polynomial curve, with peak changes below injury grade 2 (approximately 15% axons degenerating). This pattern indicated that a large number of significantly affected ONH genes responded maximally to early pressure-induced damage. Based on nerve injury grade and these two eigengene patterns, data from each sample was assigned to 1 of 3 groups for further analysis: Control ONH (untreated eyes), Early ONH Injury (glaucoma model ONH with optic nerve injury grades <2.0, n = 9), and Advanced ONH Injury (glaucoma model ONH with optic nerve injury grades >2.0, n = 12).
Figure 2.
 
IOP history of a glaucoma model eye in the Early Injury group. On day 0, hypertonic saline was injected into an episcleral vein to induce elevated IOP, which became apparent at day 20.
Figure 2.
 
IOP history of a glaucoma model eye in the Early Injury group. On day 0, hypertonic saline was injected into an episcleral vein to induce elevated IOP, which became apparent at day 20.
Figure 3.
 
The area of lesion in a glaucoma model optic nerve with an injury grade of 1.4. Dark condensed degenerating axons (white arrows) occur in a few small, focal areas of the superior portion of the nerve cross-section, and a swollen axon (white arrowhead) is present. These degenerating axons are intermingled with many morphologically intact axons. The remaining optic nerve appears normal. Scale bar, 50 μm.
Figure 3.
 
The area of lesion in a glaucoma model optic nerve with an injury grade of 1.4. Dark condensed degenerating axons (white arrows) occur in a few small, focal areas of the superior portion of the nerve cross-section, and a swollen axon (white arrowhead) is present. These degenerating axons are intermingled with many morphologically intact axons. The remaining optic nerve appears normal. Scale bar, 50 μm.
Figure 4.
 
Verification of upregulation of selected cell proliferation associated genes in Early and Advanced ONH Injury groups by QPCR. Top2a plays a role in DNA replication, whereas Prc1 and Espl1 function in cytokinesis. *P < 0.05, ‡P < 0.01, and †P < 0.001 compared with the control group.
Figure 4.
 
Verification of upregulation of selected cell proliferation associated genes in Early and Advanced ONH Injury groups by QPCR. Top2a plays a role in DNA replication, whereas Prc1 and Espl1 function in cytokinesis. *P < 0.05, ‡P < 0.01, and †P < 0.001 compared with the control group.
Figure 5.
 
IL-6–type cytokine, Lifr, and Socs3 expression in glaucomatous ONH injury examined by QPCR. This demonstrates the dramatic upregulation of IL-6 in early ONH injury. In addition, it confirms the upregulation of Lif identified by array analysis and reveals significant regulation of 2 of 4 other members of this family that are expressed in the ONH, Clcf1 and Cntf. Lifr, the common receptor component for Lif, Clcf1, and Cntf were significantly downregulated in both injury groups. Upregulation of a target of Jak-Stat signaling, Socs3, in both injury groups was significant, confirming array results. Note that the scale of the y-axis is log2 and that Control group variation is indicated by thicker SEM bars. *P < 0.05, ‡P < 0.01, †P < 0.001 compared with Controls.
Figure 5.
 
IL-6–type cytokine, Lifr, and Socs3 expression in glaucomatous ONH injury examined by QPCR. This demonstrates the dramatic upregulation of IL-6 in early ONH injury. In addition, it confirms the upregulation of Lif identified by array analysis and reveals significant regulation of 2 of 4 other members of this family that are expressed in the ONH, Clcf1 and Cntf. Lifr, the common receptor component for Lif, Clcf1, and Cntf were significantly downregulated in both injury groups. Upregulation of a target of Jak-Stat signaling, Socs3, in both injury groups was significant, confirming array results. Note that the scale of the y-axis is log2 and that Control group variation is indicated by thicker SEM bars. *P < 0.05, ‡P < 0.01, †P < 0.001 compared with Controls.
Figure 6.
 
Pressure-induced changes in ONH ECM gene expression verified and determined by QPCR. Although changes began in early injury, upregulated gene expression of four matricellular ECM proteins (Postn, Tnc, Fbln2, and Mgp) became significant in the Advanced Injury group. In contrast, the integrin component Itgav was downregulated in the Early Injury group. Timp1, the ECM metalloproteinase inhibitor, was significantly upregulated in both injury groups. Note that the scale of the y-axis is log2 and that the variation of the Control group is indicated by thicker SEM bars. *P < 0.05, ‡P < 0.01, and †P < 0.001 compared with Control group. For Postn, Fbln2, and Mgp, differences between Early and Advanced injury group values are also significant (P < 0.01).
Figure 6.
 
Pressure-induced changes in ONH ECM gene expression verified and determined by QPCR. Although changes began in early injury, upregulated gene expression of four matricellular ECM proteins (Postn, Tnc, Fbln2, and Mgp) became significant in the Advanced Injury group. In contrast, the integrin component Itgav was downregulated in the Early Injury group. Timp1, the ECM metalloproteinase inhibitor, was significantly upregulated in both injury groups. Note that the scale of the y-axis is log2 and that the variation of the Control group is indicated by thicker SEM bars. *P < 0.05, ‡P < 0.01, and †P < 0.001 compared with Control group. For Postn, Fbln2, and Mgp, differences between Early and Advanced injury group values are also significant (P < 0.01).
Figure 7.
 
Pressure-induced changes in astrocyte-associated ONH messages determined by QPCR. Levels of four messages associated with mature astrocytes (Gfap, Aqp4, Gja1, and Megf10) either were unchanged or were significantly downregulated. Also significantly downregulated was transcription factor Pax2, associated with astrocytic proliferative capacity. Nes and Vim are intermediate proteins expressed in immature astrocytes. Moderate Vim upregulation was found in the Advanced Injury group. *P < 0.05, ‡P < 0.01, †P < 0.001.
Figure 7.
 
Pressure-induced changes in astrocyte-associated ONH messages determined by QPCR. Levels of four messages associated with mature astrocytes (Gfap, Aqp4, Gja1, and Megf10) either were unchanged or were significantly downregulated. Also significantly downregulated was transcription factor Pax2, associated with astrocytic proliferative capacity. Nes and Vim are intermediate proteins expressed in immature astrocytes. Moderate Vim upregulation was found in the Advanced Injury group. *P < 0.05, ‡P < 0.01, †P < 0.001.
Figure 8.
 
Microglial marker message levels determined by QPCR. Upregulation of calcium channel Aif1 and growth factor receptor Csf1r and downregulation of purine receptor P2ry12 are all associated with microglial activation. Of these messages, only the downregulation of P2ry12 was significant in the Early Injury group, whereas Aif1 levels reached significance only in the Advanced Injury group. *P < 0.05; ‡P < 0.01.
Figure 8.
 
Microglial marker message levels determined by QPCR. Upregulation of calcium channel Aif1 and growth factor receptor Csf1r and downregulation of purine receptor P2ry12 are all associated with microglial activation. Of these messages, only the downregulation of P2ry12 was significant in the Early Injury group, whereas Aif1 levels reached significance only in the Advanced Injury group. *P < 0.05; ‡P < 0.01.
Figure 9.
 
Vascular-associated message levels determined by QPCR. Message levels for the vasoactive compounds, End1 and Adm, were not significantly altered, Vegf isoforms were not upregulated, and message levels of Hif1a remained unchanged. The smooth muscle myosin Myh11 was significantly downregulated in the Advanced Injury group. *P < 0.05.
Figure 9.
 
Vascular-associated message levels determined by QPCR. Message levels for the vasoactive compounds, End1 and Adm, were not significantly altered, Vegf isoforms were not upregulated, and message levels of Hif1a remained unchanged. The smooth muscle myosin Myh11 was significantly downregulated in the Advanced Injury group. *P < 0.05.
Table 1.
 
Microarray Study: IOP Histories by Experimental Group
Table 1.
 
Microarray Study: IOP Histories by Experimental Group
Group n Optic Nerve Injury Grade (Mean ± SD) Cumulative Elevated IOP mm Hg (Mean ± SD)
Controls 6 1.0 ± 0.0
Early injury (1 < grade < 2) 9 1.3 ± 0.3 69 ± 25
Advanced injury (2 < grade ≤ 5) 12 4.3 ± 0.8 251 ± 164
Table 2.
 
Microarray Analysis Genes Most Upregulated in the Early ONH Injury Group Compared with Control Values
Table 2.
 
Microarray Analysis Genes Most Upregulated in the Early ONH Injury Group Compared with Control Values
Accession No. Entrez Gene Gene Name* Gene Symbol Percentage of Control Mean†
BG087390 21973 Topoisomerase IIA Top2a 1424
BG069098 228421 Kinesin family member 18A Kif18a 970
AW547246 70021 5′-Nucleotidase domain containing 2 Nt5dc2 808
AW550270 21923 Tenascin C Tnc 762
BG088644 233406 Protein regulator of cytokinesis 1 Prc1 720
BG063624 52033 PDZ binding kinase Pbk 698
BG074248 110033 Kinesin family member 22 Kif22 651
BG069168 30939 Pituitary tumor-transforming 1 Pttg1 622
BG073145 80986 Cytoskeleton associated protein 2 Ckap2 608
AW553739 22137 Ttk protein kinase Ttk 598
BG065692 11504 Adamts1 Adamts1 593
AU018403 51945 Nuclear receptor subfamily 5, A, 2 Nr5a2 591
BG086805 12235 Budding uninhibited by benzimidazoles 1 Bub1 559
BG088110 26934 Rac GTPase-activating protein 1 Racgap1 555
BG072398 14284 Fos-like antigen 2 (Fosl2), mRNA Fosl2 530
AW553287 50706 Periostin Postn 521
BG070106 16819 Lipocalin 2 Lcn2 509
BG079289 77022 RIKEN cDNA 2700099C18 2700099C18Rik 499
BG070105 105387 Aldo-keto reductase family 1, member C14 Akr1c14 451
BG072904 19361 RAD51 homolog Rad51 436
BG078299 52276 Cell division cycle associated 8-Borealin Cdca8 429
BG068666 71819 Kinesin family member 23 Kif23 407
BG065826 18005 NIMA-related expressed kinase 2 Nek2 405
BG068948 66442 Spindle pole body component 25 homolog Spc25 385
BG086343 56419 Diaphanous homolog 3 Diap3 376
BG076991 12702 Suppressor of cytokine signaling 3 Socs3 371
BG073234 211770 Tribbles homolog 1 Trib1 360
BG073047 72119 TPX2, microtubule-associated protein Tpx2 356
BG088567 16009 Insulin-like growth factor binding protein 3 Igfbp3 354
BG076937 16906 Lamin B1 Lmnb1 349
BG071031 17393 Matrix metallopeptidase 7, matrylisn Mmp7 336
BG066442 16647 Karyopherin (importin) α2 Kpna2 334
BG080700 27279 Tumor necrosis factor receptor superfamily, member 12A Tnfrsf12a 329
BG079311 238076 Potassium voltage-gated channel, delayed-rectifier, S, 3 Kcns3 328
BG085472 12580 Cyclin-dependent kinase inhibitor 2C Cdkn2c 318
BG073518 12428 Cyclin A2 Ccna2 307
BG068045 14425 UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 Galnt3 304
BG066443 11806 Apolipoprotein A-I Apoa1 303
BG072979 108907 Nucleolar and spindle-associated protein 1 Nusap1 292
BG085966 14115 Fibulin 2 Fbln2 288
BG075693 69219 Dimethylarginine dimethylaminohydrolase 1 Ddah1 284
BG067382 74107 Centrosomal protein 55 Cep55 282
BG075357 17219 Minichromosome maintenance deficient 6 Mcm6 281
BG080268 14825 Chemokine (C-X-C motif) ligand 1 Cxcl1 271
BG069808 73710 Tubulin, β2B Tubb2b 269
BG085649 216161 Strawberry notch homolog 2 Sbno2 261
BG080666 15937 Immediate early response 3 Ier3 260
AW537534 326618 Tropomyosin 4 Tpm4 259
BG086929 56449 Cold shock domain protein A Csda 255
BG063171 76448 Phostensin RIKEN cDNA 2310014H01 2310014H01Rik 253
BG080690 20419 Shc SH2-domain binding protein 1 Shcbp1 252
BG081419 229841 Centromere protein E Cenpe 251
BG071607 12457 CCR4 carbon catabolite repression 4-like Ccrn4l 251
BG084556 17919 Myosin Vb Myo5b 250
Table 3.
 
Microarray Analysis Genes Most Downregulated in the Early ONH Injury Group Compared with Control Values
Table 3.
 
Microarray Analysis Genes Most Downregulated in the Early ONH Injury Group Compared with Control Values
Accession No. Entrez Gene Gene Name Gene Symbol Percentage of Control Mean*
BG086656 11668 Aldehyde dehydrogenase family 1, subfamily A1 Aldh1a1 15
BG073126 15902 Inhibitor of DNA binding 2 Id2 21
BG071519 223227 HMG-box protein (Sox21) Sox21 22
BG080229 11676 Aldolase 3, C isoform-L Aldoc 22
BG067929 75104 Monocyte to macrophage differentiation-associated 2-L Mmd2 25
BG087678 15360 3-Hydroxy-3-methylglutaryl-Coenzyme A synthase 2 Hmgcs2 29
BG074398 13602 SPARC-like 1 (hevin) Sparcl1 33
BG080206 20255 Secretogranin III Scg3 34
BG067564 21808 Transforming growth factor, β2 Tgfb2 34
BG073115 18027 Nuclear factor I/A Nfia 34
BG080743 54418 Formin 2 Fmn2 35
C85331 98582 KH domain containing 1B Khdc1b 35
C77281 12388 Catenin, delta 1 transcript variant 1 Ctnnd1 36
BG069709 332175 Zinc finger, DHHC domain containing 23 Zdhhc23 36
AW543487 105727 Solute carrier family 38, member 1 Slc38a1 38
BG080988 244867 Rho GTPase activating protein 20 Arhgap20 38
BG087491 83965 Ectonucleotide pyrophosphatase/phosphodiesterase 5 Enpp5 39
BG078755 76267 Fatty acid desaturase 1-L Fads1 39
BG065883 52225 DNA segment, Chr 4, ERATO Doi 103 D4Ertd103e 39
BG082257 106861 Abhydrolase domain containing 3 Abhd3 39
BG086781 84505 SET domain, bifurcated 1 Setdb1 39
BG080351 75712 Transmembrane protein 14A Tmem14a 40
BG070174 14866 Glutathione S-transferase, mu 5 Gstm5 41
BG084015 107272 Phosphoserine aminotransferase 1 Psat1 41
BG084178 19049 Protein phosphatase 1, regulatory (inhibitor) subunit 1B Ppp1r1b 41
BG067863 70757 Protein tyrosine phosphatase-like-L Ptplb 42
BG073601 13167 Diazepam binding inhibitor Dbi 42
BG074397 14862 Glutathione S-transferase, mu 1 Gstm1 42
BG087831 16508 Potassium voltage-gated channel, Shal-related family, member 2 Kcnd2 42
BG066209 231051 Myeloid/lymphoid or mixed-lineage leukemia 3 MII3 42
BG082752 12411 Cystathionine beta-synthase L Cbs 42
BG073940 228839 TGFB-induced factor 2 Tgif2 43
BG073190 14860 Glutathione S-transferase, α4 Gsta4 43
BG064512 18631 Peroxisomal biogenesis factor 11a Pex11a 44
AU018835 58187 Claudin 10A Cldn10a 44
BG072429 226025 Transient receptor potential cation channel, subfamily M, member 3 Trpm3 44
BG069624 234593 N-myc downstream regulated gene 4 Ndrg4 44
BG088903 235180 Fasciculation and elongation protein zeta 1 Fez1 45
BG069040 215821 D10Bwg1379e D10Bwg1379e 45
BG086971 11487 A disintegrin and metallopeptidase domain 10 Adam 10 45
AW557873 15903 Inhibitor of DNA binding 3 Id3 45
BG088107 50781 Dickkopf homolog 3 Dkk3 46
BG062997 67080 RIKEN cDNA 1700019D03 gene 1700019D03Rik 46
BG076141 108699 Chimerin (chimaerin) 1 Chn1 46
BG087940 223649 Nuclear receptor binding protein 2 Nrbp2 46
BG079962 236539 3-Phosphoglycerate dehydrogenase Phgdh 47
BG075920 217480 Diacylglycerol kinase, beta Dgkb 47
BG069750 17769 5,10-Methylenetetrahydrofolate reductase Mthfr 47
BG073197 195434 UTP14, U3 small nucleolar ribonucleoprotein, homolog B (yeast) Utp14b 47
BG075567 259302 SLIT-ROBO Rho GTPase activating protein 3 Srgap3 48
BG075594 384061 Fibronectin type III domain containing 5 Fndc5 48
BG070737 20512 Solute carrier family 1 (glial high-affinity glutamate transporter) Slc1a3 48
BG087565 54151 Cysteine and histidine rich 1 Cyhr1 48
BG066372 17762 Microtubule-associated protein tau Mapt 48
BG088467 71833 WD repeat domain 68 Wdr68 48
BG087239 226970 Rho guanine nucleotide exchange factor (GEF) 4 Arhgef4 49
BG074754 14081 Acyl-CoA synthetase long-chain family member 1 Acsl1 49
BG087975 14183 Fibroblast growth factor receptor 2 Fgfr2 49
BG081752 19791 Rn18s ribosomal RNA Rn18s 49
BG064101 14897 Thyroid hormone receptor interactor 12 Trip12 49
BG072359 14085 Fumarylacetoacetate hydrolase Fah 49
BG065128 26399 Mitogen activated protein kinase kinase 6 Map2k6 49
BG073314 16515 Potassium inwardly-rectifying channel, subfamily J, member 12 Kcnj12 49
C87546 20869 Serine/threonine kinase 11 Stk11 49
Table 4.
 
Upregulated Gene Ontology Categories in Early ONH Injury
Table 4.
 
Upregulated Gene Ontology Categories in Early ONH Injury
Significantly Upregulated Gene Categories Genes (n) P
Cell component or biological function*
    Cell cycle 82 <0.00001
    Cytoskeleton 77 <0.00001
    Immune system process 42 <0.00005
    Cell motility 25 <0.005
    Extracellular matrix 19 <0.005
    Lysosome 16 <0.005
    Plasma membrane 65 <0.001
    Cell adhesion 32 <0.05
    Proteolysis 35 <0.05
    Cell differentiation 80 <0.05
    Blood vessel development 16 <0.05
    Generation of neurons 19 <0.05
    Phosphorylation 42 <0.05
Table 5.
 
Downregulated Gene Ontology Categories in Early ONH Injury
Table 5.
 
Downregulated Gene Ontology Categories in Early ONH Injury
Significantly Downregulated Gene Categories Genes (n) P
Cell component or biological function
    Glucose metabolic process 9 <0.0005
    Peroxisome 7 <0.005
    Lipid metabolic process 20 <0.005
    Epithelial cell proliferation 4 <0.01
    Serine family amino acid biosynthetic process 3 <0.05
    Intrinsic membrane protein 64 <0.05
    Positive regulation of progression through cell cycle 3 <0.05
Table 6.
 
Total ONH DNA Content
Table 6.
 
Total ONH DNA Content
Group DNA Content (Percentage of Control Mean) Grade n
Control 100 ± 8 1.0 ± 0.0 10
Early injury 117 ± 6* 1.2 ± 0.1 11
Advanced injury 184 ± 12† 4.6 ± 0.2 13
Table st01, XLS
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