Cell Proliferation and Interleukin-6–Type Cytokine
Signaling Are Implicated by Gene Expression Responses
in Early Optic Nerve Head Injury in Rat Glaucoma
Elaine C. Johnson, Thomas A. Doser, William O. Cepurna, Jennifer A. Dyck, Lijun Jia,
Ying Guo, Wendi S. Lambert, and John C. Morrison
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
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 deter-
mined by microarray and quantitative PCR (QPCR) analysis.
Significantly altered gene expression was determined by mul-
ticlass 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 catego-
ries 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 com-
ponents, including increased expression of IL-6 (1553%). In
contrast, astrocytic glial fibrillary acidic protein (Gfap) mes-
sage 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. (Invest Ophthalmol Vis
Sci. 2011;52:504–518) DOI:10.1167/iovs.10-5317
and a characteristic remodeling of the optic nerve head (ONH)
xperimentally elevated IOP in animals produces glaucoma-
like retinal ganglion cell (RGC) and optic nerve axonal loss
that includes the rearrangement of glial cells and the deposi-
tion of extracellular matrix (ECM) proteins. Although experi-
mental 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.1,2Recently, sophisticated
3-D histomorphometric and biomechanical studies have been
used to examine some of the earliest morphologic and struc-
tural ONH responses in experimental glaucoma attributed to
elevated IOP in primates.3–11However, 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
To examine the response to acute IOP elevation, the ante-
rior 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 avail-
able to obstruct aqueous outflow and produce glaucoma-like
optic nerve degeneration.18–23In 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.24Next, we used immunohistochemistry to study
the chronology of morphologic and protein distribution
changes in the ONH. At 3 days after IOP elevation, we ob-
served decreased labeling in ONH glial columns for gap junc-
tion protein alpha 1 (connexin 43, Gja1) and increases in a cell
proliferation marker.25These changes were followed at 1
week by decreased labeling for neurotrophins and Gfap, in-
creased 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 approxi-
mately the same duration of IOP elevation.26
More recently, we have used our glaucoma model to deter-
mine 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
From the Kenneth C. Swan Ocular Neurobiology Laboratory,
Casey Eye Institute, Oregon Health and Science University, Portland,
Supported by National Institutes of Health Grants EY016866 and
EY010145 and by an unrestricted grant from Research to Prevent
Submitted for publication February 3, 2010; revised June 23 and
August 10, 2010; accepted August 16, 2010.
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
Corresponding author: Elaine C. Johnson, 3375 SW Terwilliger
Boulevard, CERES, Portland, OR 97201; email@example.com.
Investigative Ophthalmology & Visual Science, January 2011, Vol. 52, No. 1
Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc.
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.
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.28Rats were housed in low-level con-
stant light to minimize circadian IOP fluctuation.29,30IOP was mea-
sured frequently in anesthetized animals, primarily using a tonometer
(Tono-PenXL; Medtronic, Minneapolis, MN).29,31For some glaucoma
model eyes included in the PCR studies, IOP history was collected
using a rebound tonometer (TonoLab; Colonial Medical Supply, Fran-
conia, NH), as described.32Except 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 micros-
copy 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
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 ?L
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 manufac-
turer’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 Technolo-
gies, Palo Alto, CA).
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
sent 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 se-
quence differences between closely related species (Morrison JC, et al.
IOVS 2005;46:ARVO E-Abstract 1250).33,34The probes correspond to
approximately 8000 individual Entrez gene IDs, 1000 additional tran-
scripts 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.35Expres-
sion patterns in each ONH were independently determined relative to an
ONH RNA reference standard, derived by pooling aliquots from all sam-
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 interpreta-
tion of the glaucoma model results. These samples were processed
simultaneously with the glaucoma model samples from initial RNA
amplification through microarray data preprocessing and normaliza-
tion 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.29,36Gene 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 rat37
and, therefore, represent noninjurious, physiological circadian re-
sponses. 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 (prin-
cipal 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)
IOVS, January 2011, Vol. 52, No. 1
Early ONH Gene Expression Responses to Elevated IOP505
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 signif-
icantly (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 manu-
script to refer to specific genes and their protein products.
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 in-
crease the final group sizes (12 control, 20 Early Injury, and 24 Ad-
vanced 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, ver-
sion 9.0; Sci Ed Software, Cary, NC). Primers used are listed in Supple-
mentary Table S2, http://www.iovs.org/lookup/suppl/doi:10.1167/
iovs.10-5317/-/DCSupplemental. 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 de-
signed 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.
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 in-
volvement becomes more widespread, and of many genes
reflecting all the processes secondary to ongoing axon loss.
Regression analysis showed that genes upregulated in propor-
Optic Nerve Injury Grade
Pattern Discovery Analysis
Significant Eigengene Patterns
Array 6600 Eigengene-1:
Array 8400 (dotted) Eigengene-1:
Array 6600 Eigengene-2:
4th Order Polynomial
Array 8400 (dotted) Eigengene-2:
4th Order Polynomial
Array 6600 Eigengene-3:
2nd Order Polynomial
Array 8400 (dotted) Eigengene-3 :
4th Order Polynomial
Array 6600 Eigengene-4:
Array 8400 (dotted) Eigengene-4:
3rd Order Polynomial
The Pattern Discovery module in SAM was used to identify the signif-
icant 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 sepa-
rately 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 signifi-
cantly 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).
Definition of the Early and Advanced ONH Injury groups.
506 Johnson et al.
IOVS, January 2011, Vol. 52, No. 1
tion to injury grade were associated with the gene ontology
(GO) categories of extracellular matrix, humoral immune re-
sponse, 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 doc-
umented 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 tonom-
eter (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 intermin-
gled with morphologically normal ones.
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, http://www.iovs.org/lookup/suppl/
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. Supplemen-
tary Tables S4 and S5, http://www.iovs.org/lookup/suppl/doi:
10.1167/iovs.10-5317/-/DCSupplemental, list genes significantly cha-
nged in expression in the other between-group comparisons.
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-trans-
forming 1 (securin, Pttg, 622%), and budding uninhibited by
benzimidazoles 1 (Bub1, 559%), three genes with specific
functions in chromosome separation during cytokinesis, sug-
gesting completion of the cell cycle. Note that the most up-
regulated gene in Table 2 was topoisomerase 2a (Top2a,
1424%). Although not a component of the cell cycle GO cate-
gory, this enzyme is essential for DNA replication and a com-
ponent of the related category of cell proliferation. The second
most upregulated GO category in Early Injury was the cytoskel-
eton. 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 cate-
gory was immune process genes. Among the most affected of
these genes were the cytokines chemokine (C-X-C motif) li-
gand 1 (Cxcl1, 271%) and Lif (223%), both of which reached
peak levels in the Early Injury group. The immune process
TABLE 1. Microarray Study: IOP Histories by Experimental Group
(Mean ? SD)
IOP mm Hg
(Mean ? SD)
(1 ? grade ? 2)
(2 ? grade ? 5)
1.0 ? 0.0
1.3 ? 0.369 ? 25
124.3 ? 0.8 251 ? 164
Typical IOP History: Early Injury Group
Glaucoma Model Eye
Optic Nerve Injury Grade 1.4
Typical IOP History: Early Injury Group
Glaucoma Model Eye
Optic Nerve Injury Grade 1.4 Optic Nerve Injury Grade 1.4Optic Nerve Injury Grade 1.4 Optic Nerve Injury Grade 1.4 Optic Nerve Injury Grade 1.4Optic Nerve Injury Grade 1.4
Typical IOP History: Early Injury Group
Glaucoma Model EyeGlaucoma Model Eye Glaucoma Model Eye Glaucoma Model EyeGlaucoma Model Eye
Optic Nerve Injury Grade 1.0Optic Nerve Injury Grade 1.0 Optic Nerve Injury Grade 1.0Optic Nerve Injury Grade 1.0 Optic Nerve Injury Grade 1.0Optic Nerve Injury Grade 1.0
IOP mm Hg
Typical IOP History: Early Injury Group
Control EyeControl Eye Control Eye Control Eye Control Eye
IOP mm Hg
Typical IOP History: Early Injury Group Typical IOP History: Early Injury Group Typical IOP History: Early Injury GroupTypical IOP History: Early Injury Group
IOP mm Hg
IOP mm Hg
Days Following Injection Days Following Injection
IOP mm Hg
group. On day 0, hypertonic saline was injected into an episcleral vein
to induce elevated IOP, which became apparent at day 20.
IOP history of a glaucoma model eye in the Early Injury
injury grade of 1.4. Dark condensed degenerating axons (white ar-
rows) 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 morphologi-
cally intact axons. The remaining optic nerve appears normal. Scale
bar, 50 ?m.
The area of lesion in a glaucoma model optic nerve with an
IOVS, January 2011, Vol. 52, No. 1
Early ONH Gene Expression Responses to Elevated IOP507
category also included components of the complement cas-
cade (C1qc, C1qa, Cfi, Cfh, and C1r) that were upregulated to
between 215% and 150% of control values in Early Injury.
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 gluta-
mate transporters Slc38a1 (38%) and Slc1a3 (48%) and the
potassium channels Kcnd2 (42%) and Kcnj12 (50%).
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 interme-
diate arrays, the probes identified as changed were 96% iden-
tical with those identified by the original analysis (Supplemen-
tary Table S3, http://www.iovs.org/lookup/suppl/doi:10.1167/
TABLE 2. Microarray Analysis Genes Most Upregulated in the Early ONH Injury Group Compared with Control Values
GeneGene Name*Gene Symbol
Kinesin family member 18A
5?-Nucleotidase domain containing 2
Protein regulator of cytokinesis 1
PDZ binding kinase
Kinesin family member 22
Pituitary tumor-transforming 1
Cytoskeleton associated protein 2
Ttk protein kinase
Nuclear receptor subfamily 5, A, 2
Budding uninhibited by benzimidazoles 1
Rac GTPase-activating protein 1
Fos-like antigen 2 (Fosl2), mRNA
RIKEN cDNA 2700099C18
Aldo-keto reductase family 1, member C14
Cell division cycle associated 8-Borealin
Kinesin family member 23
NIMA-related expressed kinase 2
Spindle pole body component 25 homolog
Diaphanous homolog 3
Suppressor of cytokine signaling 3
Tribbles homolog 1
TPX2, microtubule-associated protein
Insulin-like growth factor binding protein 3
Matrix metallopeptidase 7, matrylisn
Karyopherin (importin) ?2
Tumor necrosis factor receptor superfamily, member 12A
Potassium voltage-gated channel, delayed-rectifier, S, 3
Cyclin-dependent kinase inhibitor 2C
UDP-N-acetyl-?-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3
Nucleolar and spindle-associated protein 1
Dimethylarginine dimethylaminohydrolase 1
Centrosomal protein 55
Minichromosome maintenance deficient 6
Chemokine (C-X-C motif) ligand 1
Strawberry notch homolog 2
Immediate early response 3
Cold shock domain protein A
Phostensin RIKEN cDNA 2310014H01
Shc SH2-domain binding protein 1
Centromere protein E
CCR4 carbon catabolite repression 4-like
* Italic gene names indicate functions associated with cell proliferation and mitosis.
† Values indicated as percentage of control eye mean, the average of six biologically independent sample values from six separate,
508 Johnson et al.
IOVS, January 2011, Vol. 52, No. 1
TABLE 3. Microarray Analysis Genes Most Downregulated in the Early ONH Injury Group Compared with Control Values
GeneGene Name Gene Symbol
Aldehyde dehydrogenase family 1, subfamily A1
Inhibitor of DNA binding 2
HMG-box protein (Sox21)
Aldolase 3, C isoform-L
Monocyte to macrophage differentiation-associated 2-L
3-Hydroxy-3-methylglutaryl-Coenzyme A synthase 2
SPARC-like 1 (hevin)
Transforming growth factor, ?2
Nuclear factor I/A
KH domain containing 1B
Catenin, delta 1 transcript variant 1
Zinc finger, DHHC domain containing 23
Solute carrier family 38, member 1
Rho GTPase activating protein 20
Ectonucleotide pyrophosphatase/phosphodiesterase 5
Fatty acid desaturase 1-L
DNA segment, Chr 4, ERATO Doi 103
Abhydrolase domain containing 3
SET domain, bifurcated 1
Transmembrane protein 14A
Glutathione S-transferase, mu 5
Phosphoserine aminotransferase 1
Protein phosphatase 1, regulatory (inhibitor) subunit 1B
Protein tyrosine phosphatase-like-L
Diazepam binding inhibitor
Glutathione S-transferase, mu 1
Potassium voltage-gated channel, Shal-related family, member 2
Myeloid/lymphoid or mixed-lineage leukemia 3
Cystathionine beta-synthase L
TGFB-induced factor 2
Glutathione S-transferase, ?4
Peroxisomal biogenesis factor 11a
Transient receptor potential cation channel, subfamily M,
N-myc downstream regulated gene 4
Fasciculation and elongation protein zeta 1
A disintegrin and metallopeptidase domain 10
Inhibitor of DNA binding 3
Dickkopf homolog 3
RIKEN cDNA 1700019D03 gene
Chimerin (chimaerin) 1
Nuclear receptor binding protein 2
Diacylglycerol kinase, beta
UTP14, U3 small nucleolar ribonucleoprotein, homolog B
SLIT-ROBO Rho GTPase activating protein 3
Fibronectin type III domain containing 5
Solute carrier family 1 (glial high-affinity glutamate transporter)
Cysteine and histidine rich 1
Microtubule-associated protein tau
WD repeat domain 68
Rho guanine nucleotide exchange factor (GEF) 4
Acyl-CoA synthetase long-chain family member 1
Fibroblast growth factor receptor 2
Rn18s ribosomal RNA
Thyroid hormone receptor interactor 12
Mitogen activated protein kinase kinase 6
Potassium inwardly-rectifying channel, subfamily J, member 12
Serine/threonine kinase 11
* Values indicated as percentage of control eye mean, the average of six biologically independent sample values from six separate, independent
IOVS, January 2011, Vol. 52, No. 1
Early ONH Gene Expression Responses to Elevated IOP509
iovs.10-5317/-/DCSupplemental). With the exception of the
addition of Bcas1 as among the most downregulated (41%),
there was no change in the genes identified as most signifi-
cantly affected (Tables 2, 3) or significant GO categories (Ta-
bles 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 mes-
sages, growth-associated protein 43 (Gap43) and neurofila-
ment 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,38,39but 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
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 con-
trols 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 cyto-
kinesis. Extra spindle pole bodies homolog 1 (Espl1 or sepa-
rase) 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.
Increased DNA Content with Pressure-Induced
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 4. Upregulated Gene Ontology Categories in Early
Significantly Upregulated Gene
Cell component or biological function*
Immune system process
Blood vessel development
Generation of neurons
* To simplify the table, closely related subcategories are not listed.
For example, cell cycle includes 23 significantly affected subcategories
of the same genes, grouped by their relationship to different aspects of
TABLE 5. Downregulated Gene Ontology Categories in Early
Significantly Downregulated Gene
Cell component or biological function
Glucose metabolic process
Lipid metabolic process
Epithelial cell proliferation
Serine family amino acid
Intrinsic membrane protein
Positive regulation of progression
through cell cycle
associated genes in Early and Advanced ONH Injury groups by QPCR.
Top2a plays a role in DNA replication, whereas Prc1 and Espl1 func-
tion in cytokinesis. *P ? 0.05, ‡P ? 0.01, and †P ? 0.001 compared
with the control group.
Verification of upregulation of selected cell proliferation
TABLE 6. Total ONH DNA Content
DNA Content (Percentage of
100 ? 8
117 ? 6*
184 ? 12†
1.0 ? 0.0
1.2 ? 0.1
4.6 ? 0.2
* P ? 0.05, † P ? 0.00001, one-tailed t-test.
510 Johnson et al.
IOVS, January 2011, Vol. 52, No. 1
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.40–42Lif
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 pro-
tein, and its entry into the nucleus.43Lif 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 initi-
ate 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 impor-
tant roles in glial responses to injury, including glial progenitor
cell proliferation and differentiation to astrocytes,42,45,46we
used QPCR to determine the responses of all IL-6–type cyto-
kines 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 car-
diotrophin-like cytokine factor 1 (Clcf1, 325%) and downregu-
lation of ciliary neurotrophic factor (Cntf, 40%) in the Early
Injury group (Fig. 5).
Cellular receptor expression will determine the functional
responses to IL-6 family cytokines. We used both our microar-
ray data and QPCR to determine expression levels of the
components of all the IL-6–type cytokine receptor compo-
nents 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 compo-
nents (II6st and Cntfr, array data, II6ra by QPCR; Supplemen-
tary Table S6, http://www.iovs.org/lookup/suppl/doi:10.1167/
iovs.10-5317/-/DCSupplemental) 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, http://www.iovs.
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).47Socs4, anotherinhibitorofEgfrsignaling,
was significantly upregulated (175%) as well by array analysis. By
QPCR, Egfr levels were unchanged by Early Injury (95% ? 17%),
(Supplementary Table S6, http://www.iovs.org/lookup/suppl/doi:
Cytokine Cxcl1 in Early Injury
Cytokine Cxcl, also highly expressed in the Early Injury group
(273%), is expressed by injured astrocytes,48microglia, and
oligodendroglial precursors,49is a mitogen for both endothelial
cells and oligodendroglia,49,50and 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 ?2 (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.52–54However, our studies show that Tgfb2 is
downregulated in Early Injury ONH (34%, Table 3; QPCR,
Supplementary Table S6, http://www.iovs.org/lookup/suppl/
doi:10.1167/iovs.10-5317/-/DCSupplemental), confirming our
earlier observation.27In contrast, our QPCR analyses dem-
onstrated that Early Injury had no significant effect on mRNA
levels of brain-derived neurotrophic factor, neurotrophin
4/5, nerve growth factor, neurotrophic tyrosine kinase re-
ceptor Trkb, or p75 neurotrophin receptor (Supplementary Table
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.55,56Our previous studies demonstrate that changes in
and Socs3 expression in glaucomatous
ONH injury examined by QPCR. This
demonstrates the dramatic upregula-
tion 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 compo-
nent for Lif, Clcf1, and Cntf were sig-
nificantly downregulated in both in-
jury groups. Upregulation of a target of
Jak-Stat signaling, Socs3, in both injury
groups was significant, confirming ar-
ray results. Note that the scale of the
y-axis is log2and that Control group
variation is indicated by thicker SEM
bars. *P ? 0.05, ‡P ? 0.01, †P ? 0.001
compared with Controls.
IL-6–type cytokine, Lifr,
IOVS, January 2011, Vol. 52, No. 1
Early ONH Gene Expression Responses to Elevated IOP511
ECM gene expression and protein deposition also occur
early during IOP-induced injury in rat glaucoma model
ONH,24,25,27an 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.57Other
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 astro-
cyte migration.58Sparcl1 has antiadhesion, antiproliferation
properties and is present in differentiated astrocytes and
Increased collagen expression, a characteristic of human
and experimental glaucomatous ONH remodeling,24,62–64was
indicated in our array study by modest upregulation of colla-
gens 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 (?1, ?3, ?5, ?V, and ?9;
?1, ?3, ?4, ?5, and ?7), only integrins ?1 (Itgb1) and ?V
(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 cytoskel-
eton to fibronectin, laminins, tenascins, Spp1, vitronectin,
L1 cell adhesion molecule, Postn, and various RGD-contain-
ing proteins.65In neural tissues, Itgav is expressed by glia
and is often involved in the mediation of vascular interac-
tions.66QPCR confirmed Itgav downregulation in the Early
Injury group (Fig. 6).
In an earlier study of retinal gene expression changes in our
glaucoma model,67Timp1 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 expres-
sion 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,27QPCR 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 mes-
sages associated with mature ONH astrocytes were evalu-
ated 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
in ONH ECM gene expression verified
and determined by QPCR. Although
changes began in early injury, upregu-
lated gene expression of four matricel-
lular 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 metalloprotei-
nase inhibitor, was significantly up-
regulated in both injury groups. Note
that the scale of the y-axis is log2and
that the variation of the Control group
is indicated by thicker SEM bars. *P ?
0.05, ‡P ? 0.01, and †P ? 0.001 com-
pared with Control group. For Postn,
Fbln2, and Mgp, differences between
Early and Advanced injury group val-
ues are also significant (P ? 0.01).
in astrocyte-associated ONH messages
determined by QPCR. Levels of four
messages associated with mature astro-
cytes (Gfap, Aqp4, Gja1, and Megf10)
either were unchanged or were signifi-
cantly downregulated. Also significantly
downregulated was transcription factor
ative capacity. Nes and Vim are interme-
diate proteins expressed in immature as-
trocytes. Moderate Vim upregulation
was found in the Advanced Injury
group. *P ? 0.05, ‡P ? 0.01, †P ?
512Johnson et al.
IOVS, January 2011, Vol. 52, No. 1
channel associated with astrocytic endfeet and the blood
brain/cerebral spinal fluid barrier.68,69Gja1 is a key compo-
nent of astrocytic gap junctions.70Megf10 is associated with
astrocytic phagocytosis.40The transcription factor Pax2 is
specifically expressed by optic nerve astrocytes71–73and
associated with proliferative capacity.71,74,75Nestin (Nes)
and vimentin (Vim) are expressed in immature or prolifer-
ating astrocytes.76Vim levels were increased only in the
Advanced Injury group (145% ? 8%). Additionally, microar-
ray 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 (Supple-
mentary Table S6, http://www.iovs.org/lookup/suppl/doi:
Microglial-Specific Marker Expression in Early
IOP-Induced ONH Injury
Expression levels of some microglial activation markers not present
tory factor (Aif1, or IBA1), a calcium channel associated with micro-
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.78P2ry12 downregu-
and was significant in the Early ONH injury group (Fig. 8).
Other messages present on our arrays that are enriched in
microglia include cd34 antigen,80ferritin light chain (Ftl1),
translocator protein (peripheral benzodiazepine receptor,
Tspo), TGF?1 receptor 1 (Tgfb1r1), and milk fat globule EGF
factor 8 protein (Mfge8). Of these, only Cd34 was signifi-
cantly upregulated in the Early Injury group (Supplementary
Table S3, http://www.iovs.org/lookup/suppl/doi:10.1167/
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,81was not signifi-
cantly altered in expression in glaucoma model ONH (Supplemen-
taryTable S6, http://www.iovs.org/lookup/suppl/doi:10.1167/
iovs.10-5317/-/DCSupplemental). By array analysis, platelet-derived
growth factor ? (Pdgfa) was unchanged, and myelin basic protein
(Mbp) was significantly increased only in the Advanced Injury group
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 asso-
ciated 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, http://www.iovs.org/lookup/
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 angiogene-
sis and blood-brain barrier permeability. Thetranscriptionfactor
hypoxia inducible factor 1? (Hif1a) as well as erythropoietin
(Epo) and heme oxygenase 1 (Hmox1) are frequently upregu-
lated under hypoxic conditions (Supplementary Table S6, http://
DCSupplemental). Of these, the only significant change in the
Early Injury group was a downregulation of Vegfb to 61% ? 5%.
ONH Gene Expression Change with Physiological
The physiological IOP elevation associated with the dark phase
of the light cycle resulted in moderate, but significant, expres-
sion changes in many ONH genes. DAVID analysis indicated
that upregulated genes primarily affected signal transduction
and the cytoskeleton, whereas downregulated genes were pri-
marily 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 gen-
erally 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%.
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 num-
ber of factors, including the quality and relevance of the sam-
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.
Microglial marker message levels determined by QPCR.
IOVS, January 2011, Vol. 52, No. 1
Early ONH Gene Expression Responses to Elevated IOP 513
ples 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 ex-
pression 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 sup-
port these observations. These studies also confirm similar
observations made in our earlier study of longer (1-mm) seg-
ments 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),41,42,82–87and both can exert axonal neuroprotective,
regenerative,88–92and proliferative responses.45,93All three
cytokines can induce astrocytic differentiation from precursor
cells.42,94–96Lif may also promote microglial proliferation.97In
addition, these cytokines may act on ONH vascular compo-
nents because IL-6 can stimulate smooth muscle cell prolifer-
ation,98although Lif has been shown to inhibit retinal endo-
thelial cell proliferation.99Although less is known about Clcf1,
this cytokine does have both growth factor100and neuropro-
tective properties,101and its expression is associated with
response to stress.102In contrast to the other three, Cntf was
significantly downregulated in Early Injury, as has been ob-
served after nerve crush.103ONH astrocytes express Cntf and
its tripartite receptor components,104and, in retina, Cntf re-
presses glial proliferation.105Therefore, the downregulation of
Cntf may complement the proliferation-promoting actions of
the upregulated cytokines in this family. Together, these ob-
servations suggest that altered expression of these cytokines
may contribute to proliferative responses in the ONH to ele-
vated IOP exposure.
In Early Injury ONH, expression levels for the receptor
components of the IL-6-family cytokines were unchanged, ex-
cept for the downregulation of Lifr, the common receptor
component for Lif, Clcf1, and Cntf. Lifr is expressed by most
ONH cellular components106–112113and is important for astro-
cyte precursor differentiation, including Jak-Stat pathway-reg-
ulated Gfap expression.114–117Therefore, 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 pre-
conditioning-induced stress,118suggesting that Lifr downregu-
lation 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
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 in-
creased expression of this intermediate filament protein is
often considered necessary and sufficient evidence of an astro-
cytic reaction. In glaucoma model retinas and the RGC layer,
Gfap upregulation is readily observed.67,119–121However, in
the ONH, early injury from elevated IOP exposure is not
associated with the significant upregulation of Gfap or any
sage levels determined by QPCR.
Message levels for the vasoactive
compounds, End1 and Adm, were
not significantly altered, Vegf iso-
forms were not upregulated, and
message levels of Hif1a remained
unchanged. The smooth muscle
myosin Myh11 was significantly
downregulated in the Advanced In-
jury group. *P ? 0.05.
514 Johnson et al.
IOVS, January 2011, Vol. 52, No. 1
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 find-
ing also suggests an initial passive, rather than active, contri-
bution of astrocytes to further nerve injury because a reduction
in differentiated astrocytic functions is likely to include com-
promised 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 my-
elinated nerve122and individual astrocytes are in functional
contact with many axons,123–125such a compromise is likely to
contribute significantly to further axonal vulnerability to ele-
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.79,126,127Additionally, our array data
showed that Nrp1 was significantly upregulated by early injury,
and Nrp1 upregulation has been localized to activated micro-
glia.128Further evidence of microglial activation became ap-
parent in eyes with more advanced injury as Aif1 expression
was significantly increased. However, additional studies are
needed to determine specifically whether both microglial pro-
liferation 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 expres-
sion of some genes associated with the vascular component of
the ONH. Similar Myh9 and Myh11 responses are observed in
carotid artery injury,129and Tnfrsf12a upregulation is associ-
ated 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 down-
regulated in the ONH in early injury. Although the specific
functions of Vegfb protein are not well understood, it is abun-
dant 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 upregula-
tion 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.27Postn is expressed in cells subjected
to mechanical stress, particularly vascular smooth muscle
cells.134Postn and the other affected matricellular genes are
expressed by astrocytes39,59,135,136and are altered in expres-
sion 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 be-
tween astrocyte processes and the vasculature may be partic-
Currently, we are using immunohistochemistry to identify pro-
liferating ONH cells and those that express the IL-6–type cy-
tokines 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 re-
sponses, with the goal of understanding their relationship to
the development of glaucomatous axon loss.
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