Retinal proteomic changes following unilateral optic nerve transection and early experimental glaucoma in non-human primate eyes

Experimental Eye Research 93 (2011) 13e28

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Retinal proteomic changes following unilateral optic nerve transection and early experimental glaucoma in non-human primate eyesq C. Stowell a, b, B. Arbogast c, G. Cioffi a, C. Burgoyne a, **, A. Zhou b, * a

Optic Nerve Head Research Laboratory, Discoveries in Sight Research Laboratories, Devers Eye Institute, Legacy Health System, Portland, OR, USA Robert S. Dow Neurobiology Laboratories, Legacy Health System, 1225 NE Second Avenue, Portland, OR 97232, USA c Oregon State University, Corvallis, OR, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 December 2010 Accepted in revised form 30 March 2011 Available online 23 April 2011

In this work we compared proteomic changes in non-human primate (NHP) retinas at the onset of early experimental glaucoma (EEG) and 3 weeks after optic nerve transection (ONT), as a means to identify regulators in the retina’s response to EEG and ONT. Both eyes of 7 NHPs with either unilateral EEG (n ¼ 4) or ONT (n ¼ 3) were enucleated. Proteins were analyzed by a label-free quantitative mass spectrometry system and the abundance of identified retinal proteins was compared between the treated eye and its contralateral control for each NHP. Cellular processes associated with regulated proteins were identified using the MetaCore program. As a result, a total of 209 and 200 proteins were identified in EEG and ONT retinas, respectively. The EEG eyes exhibited two distinguishable levels of maximum IOP: the highest IOP <27 mmHg (n ¼ 2) and >45 mmHg (n ¼ 2), termed mild IOP EEG and high IOP EEG eyes, respectively. A limited overlap of proteins regulated in the same direction was seen between the high IOP EEG and either the mild IOP EEG eyes or ONT eyes. Most of the proteins that were up regulated in the high IOP EEG eyes were down regulated in the mild IOP EEG eyes; the latter showed an overall down regulation that was not seen in the other two conditions. An association with cytoskeleton regulation was recognized for up-regulated proteins in the high IOP EEG eyes. We conclude that mild IOP EEG, high IOP EEG and ONT retinas exhibited condition-specific proteomic changes with little overlap between conditions. Cytoarchitecture regulation appears to be a component of the early retinal response to chronic experimental IOP elevation. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: early experimental glaucoma retina proteomics bioinformatics non-human primate

1. Introduction Early glaucomatous damage to the visual system may include pathophysiologic changes in the retina, optic nerve head (ONH), sclera, orbital optic nerve and lateral geniculate body. While strong evidence suggests that a multifactorial insult to the retinal ganglion cell (RGC) axons within the lamina cribrosa of the ONH (Bellezza et al., 2003; Downs et al., 2007; Howell et al., 2007; Yang et al., 2007a, 2007b) and apoptotic RGC death (Quigley et al., 1995) are central pathophysiologies, the molecular mechanisms that underlie early glaucomatous damage at all sites of injury remain poorly

q Grant Information: Legacy Good Samaritan Foundation, Sears Medical Trust and American Glaucoma Society Mid-Career Clinician Scientist Award. * Corresponding author. Current address: Neuroscience Institute, Morehouse School of Medicine, 720 Westview Dr. SW, Atlanta, GA 30310, USA. ** Corresponding author. E-mail addresses: [email protected] (C. Burgoyne), [email protected] (A. Zhou). 0014-4835/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2011.03.020

understood. Systematic characterization of each component of early glaucomatous vision loss is therefore indicated but difficult to model. Human ocular hypertension, the clinical presence of elevated intraocular pressure (IOP) without evidence for structural or functional damage to the visual system is a common condition in which patients are closely followed for the onset of clinically detectable ONH or visual field damage (Kass et al., 2002). Chronic, laser-induced IOP elevation in the non-human primate (NHP) eye, when carried to the onset of ONH surface changes (a condition termed Early Experimental Glaucoma (EEG)) as detected by Confocal Scanning Laser Tomography (CSLT), attempts to experimentally model human ocular hypertension and glaucoma at the point of clinically detectable ONH damage (Yang et al., 2011, 2007b, 2009, 2009; Bellezza et al., 2003; Burgoyne et al., 2004; Downs et al., 2007; Yang et al., 2007a). Surgical optic nerve transection (ONT) produces a primary, traumatic insult to the RGC axon within the orbit, providing a model of how the retina responds to RGC axonal trauma that is

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C. Stowell et al. / Experimental Eye Research 93 (2011) 13e28

not IOP-related. In the NHP eye, surgical ONT leads to the death of the majority of RGC within 6e10 weeks by apoptosis (Quigley et al., 1977; Villegas-Perez et al., 1993; Berkelaar et al., 1994). A molecular characterization and comparison of NHP retinas from EEG and ONT eyes should provide insight into the retinal response to IOP-related and non-IOP-related forms of RGC axon damage. In addition, any primary retinal response to elevated IOP should be present in the EEG but not the ONT eyes. The present experiment was designed to utilize a label-free quantitative mass spectrometry (MS) system to characterize retinal proteomic changes in NHP eyes that resulted from either laser-induced unilateral EEG (n ¼ 4 NHP) or unilateral surgical ONT (n ¼ 3 NHP), and compare changes under these two different conditions. Because the four EEG animals exhibited two different, clearly distinguishable ranges of IOP in response to lasering, the four EEG animals were retrospectively divided into mild IOP EEG and high IOP EEG groups (n ¼ 2 each), with the two EEG NHPs within each group closely matched for height and duration of IOP elevation. It is our impression that in the NHP experimental glaucoma model the height of IOP elevation far exceeds the duration of IOP elevation as a risk factor for both the onset and the progression of the neuropathy. Data to support this notion in the NHP (Burgoyne, 2011; Gardiner et al., submitted for publication) and rat have (Chauhan et al., 2002) been reported. In the present study, it was reasonable to expect that IOP was much higher in the high IOP treated eyes compared to the mild IOP treated eyes. We therefore believed that it was reasonable to argue that this was an important determinant of the retinal proteomes contained therein. We report that proteomic changes were substantially different within the mild IOP EEG, high IOP EEG and ONT retinas, that down regulation was the principal response within the mild IOP eyes and that cytoskeletal remodeling may play a role in the early retinal response to chronic IOP elevation. 2. Methods 2.1. Animals All animal treatments were in accordance with the ARVO statement for the use of animals in ophthalmic and vision research and were approved by the local Institutional Animal Care and Use Committee (IACUC). Adult NHP rhesus macaques (Macaca mulatta)

were housed in a temperature- and humidity-controlled room with a 12 h light: 12 h dark cycle and provided with food and water ad libitum. 2.2. Early Experimental Glaucoma (EEG) and Optic Nerve Transection (ONT) EEG was defined to be the onset of ONH surface change as revealed by longitudinal Heidelberg Retinal Tomography (HRT) images (Heidelberg Engineering, Heidelberg, Germany) acquired during standard imaging sessions on 3e5 occasions prior to and every 1e2 weeks following the onset of laser-induced IOP elevation (Bellezza et al., 2003; Downs et al., 2007; Yang et al., 2007a, 2007b). IOP was measured at the start of each imaging or laser session by Tonopen XL (Reichert Inc., Depew NY) in both eyes of each animal (mean of n ¼ 3 measures per eye) following an induction dose of ketamine (15 mg/kg IM) and either xylazine (0.8e1.5 mg/kg IM) or midazolam (0.2 mg/kg IM). For imaging sessions, isoflurane gas (1e2%; typically 1.25%) was also administered via endotracheal tube. Thus, IOP measurements were acquired under varied anesthetic agents and at variable times after administration of anesthetic agents, though typically within 30 min of anesthesia induction. Laser treatments were performed under ketamine only, ketamine and xylazine, or ketamine and dexmedetomidine anesthesia initially in two separate treatment sessions (180 of the trabecular meshwork in each session separated by 2 weeks). Laser treatments were repeated in subsequent weeks (but limited to a 90 sector) until an IOP elevation was first noted or if post-laser IOP had returned to normal levels. The onset of detected changes in HRT parameters required 2 subsequent confirmations. Because imaging was performed at actual IOP rather than manometrically controlled IOPs of 10 mm HG (our usual protocol, but not performed in this study to avoid the mild anterior chamber inflammation that can follow needle placement and removal), the onset of ONH surface change reflected both “permanent” or “fixed” deformation as well as any “acute” or “reversible” component of deformation due to the elevated IOP at the time of imaging (Burgoyne et al., 2004). Unilateral surgical ONT at 6e8 mm behind the globe was performed under isoflurane anesthesia and direct visualization via lateral orbitotomy as previously described (Burgoyne et al., 1995). Patency of the central retinal vasculature of the transected eye was

Table 1 Test subjects. Number Duration of time Cumulative IOP at time Range of mean of laser from 1st laser IOP insult of sacrifice arterial pressure Normal Maximum treatments treatments (days) (IOP  days) (mmHg) during enucleationa (Pre-laser) (Post-laser) (mm Hg)

Body temp during enucleation ( C)

15.5 14.5 13.15 9.3 13 16.2 18 17.8 12.85 11.8 16.5 16.15 16.3 15.2

50.3e74.3

31.3e33.7

27.3e39.0

33.8e35.2

55.3e90.3

N/A

66.0e108.0

31.6e38.7

64.3e99.3

34.2e35.6

70.0e167.7

32.3e35.9

73.7e103.0

32.8e36.5

Study ID

Animal Weight Sex Age Eye ID (kg) (yr)

Treatment IOP (mm Hg)

Mild IOP EEG 1 Mild IOP EEG 2b High IOP EEG 1 High IOP EEG 2 ONT 1

22877 5.6

F

9

23453 5.7

F

11

20031 5.2

F

9

21322 6.5

F

13

23498 5.0

F

10

ONT 2

22267 6.5

M

11

ONT 3

23511 9.5

F

6

EEG Normal EEG Normal EEG Normal EEG Normal ONT Normal ONT Normal ONT Normal

a b

Right Left Right Left Right Left Right Left Right Left Right Left Right Left

24 e 26 e 45.7 e 65.7 e e e e e e e

6 e 8 e 2 e 3 e e e e e e e

209 e 295 e 43 e 51 e e e e e e e

Blood pressure measured by BP cuff; Arterial Pressure ¼ Diastolic þ 1/3(Systolic  Diastolic). The ipsilateral treated eye was enucleated first in all of the test subjects except this one.

66 437 321 661 e3 54 10

24 14 19.9 19.9 45.7 17.7 10.3 11 9.7 7.7 8.3 14.3 7.3 4.3

C. Stowell et al. / Experimental Eye Research 93 (2011) 13e28

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Fig. 1. Highest IOP, Cumulative IOP exposures and pre-sacrifice TCA maps for both eyes of each animal. Left panel: IOP graphics. red e treated eyes; blue e contralateral control eyes; green e the cumulative IOP exposure (the difference between the areas under the red and blue curves). Dotted vertical line is the onset of lasering in the EEG eyes and ONT surgery in the ONT eyes. For each animal, the treated eye highest IOP (upper number) and cumulative IOP insult (lower number) are indicated. Right panel: Pre-sacrifice HRT TCA maps in right eye configuration for the treated (left column) and contralateral normal (right column) eyes of each animal. The TCA map for each eye conveys HRT detected optic nerve head surface change at the time of sacrifice (red superpixels depict statistically-significant posterior deformation and green superpixels depict statistically-significant anterior deformation) relative to that eye’s normal (baseline) HRT appearance. Note that only minimal change is present in the contralateral normal and ONT eyes. However, mild (mild IOP EEG #1 greater than mild IOP EEG #2) and moderate (high IOP EEG #2 greater than high IOP EEG #1) changes are present in the mild IOP and high IOP EEG eyes respectively. Because TCA imaging was performed at actual IOPs, rather than 30 min after manometer controlled IOPs of 10 mm Hg, these maps may over-emphasize the amount of permanent or “fixed” ONH surface deformation at the time of sacrifice (see methods). These TCA maps were retrospectively generated and were not available at the time of sacrifice. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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C. Stowell et al. / Experimental Eye Research 93 (2011) 13e28

2.3. Enucleation and sacrifice

Table 2 Numbers of identified proteins within individual animals. Study ID

Treated eye Control eye Detected in both eyes Total for both eyes

Mild IOP EEG

High IOP EEG

ONT

1

1

1

2

2

2

3

119 (20) 108 (14) 108 (26) 124 (42) 135 (34) 123 (23) 127 (14) 114 (15) 118 (24) 123 (41) 127 (45) 139 (38) 123 (23) 140 (27) 99 94 82 82 101 100 113 134

132

149

169

174

146

154

Top section: Numbers of proteins found in either the treated or control eye and numbers of proteins found only in that eye (in parentheses).

documented by fluorescein angiography within the first postoperative week. As a crude measurement of the magnitude and duration of IOP exposure, the cumulative IOP exposure (the difference between the area under the IOP curve of the treated eye and the area under that of the contralateral control eye) was calculated for all EEG and ONT subjects (Table 1 and Fig. 1).

All animals were sacrificed either at the onset of reproducible ONH surface change in the EEG eyes (described above), or 3 weeks post transaction in the ONT eyes. Under isoflurane anesthesia, eyes were enucleated at actual IOPs, which had been variably lowered from pre-anesthesia levels by the isoflurane, but not manometrically as we have previously published. Unless stated otherwise, the treated eye was enucleated first, followed by immediate enucleation of the contralateral control eye. After enucleation, each NHP was euthanized by exsanguanation. The enucleated eyes were individually placed in a petri dish embedded in ice and orbital tissues were removed. Whole retinas were separated from the choroids as much as possible, flash frozen on dry ice, and stored at 80  C until further processing. 2.4. Protein extraction and tryptic digestion All retinal tissues for this study were processed on the same day and one-at-a-time to ensure consistent processing. After being

Table 3 Regulated proteins in mild IOP eyes. SwissProt ID

UniProt ID

Gene name

Protein name

Up-regulated proteins Q2PFW6 SYUG_MACFA Q4R712 STMN1_MACFA

SNCG STMN1

Gamma-synuclein Stathmin

Down-regulated proteins P61604 CH10_HUMAN P05387 RLA2_HUMAN P62736 ACTA_HUMAN P63267 ACTH_HUMAN P68032 ACTC_HUMAN P68133 ACTS_HUMAN P63261 ACTG_HUMAN Q4R561 ACTB_MACFA P06733 ENOA_HUMAN Q4R5L2 ENOA_MACFA Q6S8J3 A26CA_HUMAN A5A3E0 A26CB_HUMAN P13929 ENOB_HUMAN P12277 KCRB_HUMAN P09104 ENOG_HUMAN P04406 G3P_HUMAN

HSPE1 RPLP2 ACTA2 ACTG2 ACTC1 ACTA1 ACTG1 ACTB ENO1 ENO1 A26C1A A26C1B ENO3 CKB ENO2 GAPDH

P63211

GBG1_HUMAN

GNGT1

P68871 P68223 P02042 P22626

HBB_HUMAN HBB_MACFA HBD_HUMAN ROA2_HUMAN

HBB HBB HBD HNRNPA2B1

P16402 P10412 P11137 P41219 P14618 P30613 P35243 Q60HC9 Q71U36 Q4R538 Q9BQE3 Q13748 Q6PEY2 P68371 Q4R4X8 P08670 Q4R4X4

H13_HUMAN H14_HUMAN MAP2_HUMAN PERI_HUMAN KPYM_HUMAN KPYR_HUMAN RECO_HUMAN TPIS_MACFA TBA1A_HUMAN TBA1B_MACFA TBA1C_HUMAN TBA3C_HUMAN TBA3E_HUMAN TBB2C_HUMAN TBB4_MACFA VIME_HUMAN VIME_MACFA

HIST1H1D HIST1H1E MAP2 PRPH PKM2 PKLR RCVRN TPI1 TUBA1A TUBA1B TUBA1C TUBA3C TUBA3E TUBB2C TUBB4 VIM VIM

10 kDa heat shock protein 60S acidic ribosomal protein P2 Actin Actin Actin Actin Actin Actin Alpha-enolase Alpha-enolase ANKRD26-like family C member 1A ANKRD26-like family C member 1B Beta-enolase Creatine kinase B-type Gamma-enolase Glyceraldehyde-3-phosphate dehydrogenase Guanine nucleotide-binding protein G(T) subunit gamma-T1 Hemoglobin subunit beta Hemoglobin subunit beta Hemoglobin subunit delta Heterogeneous nuclear ribonucleoproteins A2/B1 Histone H1.3 Histone H1.4 Microtubule-associated protein 2 Peripherin Pyruvate kinase isozymes M1/M2 Pyruvate kinase isozymes R/L Recoverin Triosephosphate isomerase Tubulin alpha-1A chain Tubulin alpha-1B chain Tubulin alpha-1C chain Tubulin alpha-3C/D chain Tubulin alpha-3E chain Tubulin beta-2C chain Tubulin beta-4 chain Vimentin Vimentin

*p-values for all proteins are 0.01.

Calculated MW

Average protein score

Average sequence coverage (%)

Ratio of treated to control*

13295 17302

215.36 175.19

54.36 19.34

1.15 1.12

10931 11664 42009 41877 42019 42051 41792 41736 47169 47125 121363 121370 46986 42644 47268 36053

191.74 161.86 191.36 190.98 194.00 193.40 253.61 254.42 602.23 601.86 188.88 196.99 184.54 424.53 306.83 314.43

40.32 46.41 10.73 10.49 11.44 12.72 18.87 17.03 26.85 27.22 4.08 5.01 7.05 27.80 29.53 18.66

1.32 1.34 1.72 1.72 1.72 1.72 1.88 1.88 1.81 1.81 2.01 2.12 1.72 1.64 1.70 1.57

8495

189.46

35.25

1.55

15998 16114 16055 37429

305.25 476.24 188.00 146.16

28.76 56.69 26.04 14.17

1.38 1.43 1.15 2.11

22349 21865 199539 53650 57937 61830 23130 26710 50135 50151 49895 49959 49916 49831 49585 53651 53708

157.98 163.55 408.45 161.62 337.73 105.68 160.53 157.57 422.56 479.89 464.06 298.10 290.29 264.48 171.49 1206.85 1244.13

14.07 15.33 9.69 10.97 23.60 3.67 22.04 29.51 19.45 21.71 22.16 17.24 15.92 18.54 16.77 40.88 41.12

1.92 1.92 1.42 1.90 1.83 1.87 2.82 1.51 1.96 1.96 1.96 2.02 2.03 1.83 1.64 1.74 1.74

C. Stowell et al. / Experimental Eye Research 93 (2011) 13e28

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Table 4 Proteins found only in the 2 treated or 2 control eyes of the mild IOP EEG animals. SwissProt ID

UniProt ID

Gene name

Protein name

P09496 P18669

CLCA_HUMAN PGAM1_HUMAN

CLTA PGAM1

Clathrin light chain A Phosphoglycerate mutase 1

Q4R572 O43423

1433B_MACFA AN32C_HUMAN

YWHAB ANP32C

P17066 Q4R4P1 Q4R4T5 P07197 P62937 P48741

HSP76_HUMAN HS90A_MACFA HS90B_MACFA NFM_HUMAN PPIA_HUMAN HSP77_HUMAN

HSPA6 HSP90AA1 Hsp90ab1 NEFM PPIA HSPA7

14-3-3 protein beta/alpha Acidic leucine-rich nuclear phosphoprotein 32 family member C Heat shock 70 kDa protein 6 Heat shock protein HSP 90-alpha Heat shock protein HSP 90-beta Neurofilament medium polypeptide Peptidyleprolyl cis-trans isomerase A Putative heat shock 70 kDa protein 7

thawed on ice, each retina was boiled directly into 500 mL ddH2O for 10 min, chilled on ice, and homogenized with a hand-held homogenizer (Kontes Microtube Pellet Pestle Rod with Motor). An additional 500 mL of chilled ddH2O was added and homogenates were centrifuged at 16,000 g for 30 min, and cleared supernatants were frozen again until the next step of analysis. Protein concentrations in cleared supernatants were determined by Bradford Assay. For each sample, 20 mg protein was lyophilized and resuspended with 100 mM ammonium bicarbonate, pH 8.0, to a final protein concentration of 1 mg/mL. Proteins were then denatured by incubation at 95  C for 10 min with 0.05% RapiGest SF (Waters Corp, Mildford, MA), followed by incubation at 60  C for 30 min with 20 mM dithiothreitol, incubation with 20 mM iodoacetamide for 10 min in the dark, and digestion with sequencing-grade trypsin (2.25  106 unit/mL; Promega, Madison, WI) at 37  C overnight. After digestion, RapiGest SF in the incubation was precipitated by the addition of trifluoroacetic acid to pH 2e2.5. 2.5. Mass spectrometry (MS) settings, protein identification and quantification The tryptic digests from the retinal specimen of each eye were analyzed by MS as previously described, with at least 3 technical

Calculated MW

Average protein score

Average sequence coverage (%)

Treated or control

27076 28803

129.77 126.70

11.74 25.95

Treated only Treated only

28082 26761

95.92 103.87

17.99 9.19

Control only Control only

71028 84789 83237 102448 18012 40244

139.75 198.03 148.19 215.12 131.53 108.74

7.35 7.27 6.99 10.97 18.03 6.42

Control Control Control Control Control Control

only only only only only only

replications (MS runs) for the retinal specimen (Stowell et al., 2010) from each eye. Briefly, the quantitative MS system consisted of an autosampler, a nanoAcquity Ultra Performance Liquid Chromatography (UPLC) system coupled in-line to a Micromass Ultima Global Quadrupoletime-of-flight mass spectrometer (Waters Corp, Millford, MA) (Silva et al., 2006; Li et al., 2009). The UPLC included a 5-mL sample loop and a trapping (desalting) column preceding a bridged-ethyl hybrid (BEH) C18 analytical column. Using the autosampler, replicate MS analyses of each sample were performed consecutively, and MS analyses for all samples of a treatment group were completed within 3 days. The separation of tryptic peptides by UPLC and the subsequent dual-energy MS identification and quantification of detected peptides, as managed by ProteinLynx Global Server version 2.3 (Waters) and with the use of a custom database of annotated, nonredundant macaca and human proteins from The Universal Protein Resource (Unitprot, www.UniProt.org) were performed as previously described (Stapels et al., 2010; Stowell et al., 2010). The data collection was done in real time and then the data were processed in the offline mode. The fmol amounts for each identified protein in a sample were determined by comparison to an internal standard, 100 fmol pre-digested glycogen phosphorylase. The ratios of total

Table 5 Regulated proteins in high IOP EEG eyes. SwissProt ID

UniProt ID

Gene name

Protein name

Calculated MW

Average protein score

Average sequence coverage (%)

Up-regulated proteins P06733 ENOA_HUMAN Q4R5L2 ENOA_MACFA Q6S8J3 A26CA_HUMAN A5A3E0 A26CB_HUMAN P13929 ENOB_HUMAN Q71U36 TBA1A_HUMAN Q4R538 TBA1B_MACFA Q9BQE3 TBA1C_HUMAN Q13748 TBA3C_HUMAN Q6PEY2 TBA3E_HUMAN Q9NY65 TBA8_HUMAN P68371 TBB2C_HUMAN

ENO1 ENO1 A26C1A A26C1B ENO3 TUBA1A TUBA1B TUBA1C TUBA3C TUBA3E TUBA8 TUBB2C

Alpha-enolase Alpha-enolase ANKRD26-like family C member 1A ANKRD26-like family C member 1B Beta-enolase Tubulin alpha-1A chain Tubulin alpha-1B chain Tubulin alpha-1C chain Tubulin alpha-3C/D chain Tubulin alpha-3E chain Tubulin alpha-8 chain Tubulin beta-2C chain

47169 47125 121363 121370 46986 50135 50151 49895 49959 49916 50093 49831

476.72 479.61 204.38 200.48 142.50 259.63 301.80 290.43 183.76 185.39 148.49 246.10

24.28 25.60 5.59 4.86 10.06 15.26 17.53 20.22 14.77 14.50 10.72 19.62

1.50 1.50 1.47 1.42 2.02 2.27 2.39 2.39 4.06 4.27 4.99 1.87

Down-regulated proteins P61604 CH10_HUMAN P68871 HBB_HUMAN P68223 HBB_MACFA P69891 HBG1_HUMAN P69892 HBG2_HUMAN P16402 H13_HUMAN Q9BZV3 IMPG2_HUMAN Q8HXQ1 SODC_MACFA

HSPE1 HBB HBB HBG1 HBG2 HIST1H1D IMPG2 SOD1

10 kDa heat shock protein Hemoglobin subunit beta Hemoglobin subunit beta Hemoglobin subunit gamma-1 Hemoglobin subunit gamma-2 Histone H1.3 Interphotoreceptor matrix proteoglycan 2 Superoxide dismutase [CueZn]

10931 15998 16114 16140 16126 22349 138593 15982

191.53 289.57 555.57 139.11 144.62 205.68 207.07 395.83

38.50 27.03 59.00 18.68 19.69 16.54 8.30 32.77

2.20 1.29 1.33 1.64 1.63 1.13 1.64 2.47

*p-values for all proteins are <0.01.

Ratio of treated to control*

18

C. Stowell et al. / Experimental Eye Research 93 (2011) 13e28

Table 6 Proteins found only in the 2 treated or 2 control eyes of the high IOP EEG animals. SwissProt ID

UniProt ID

Gene name

Protein name

Calculated MW

Average protein score

Average sequence coverage (%)

Treated or control

P14136 P08107 P34931 P17066 Q4R362 P04264 Q60HD8 P48741 P07437 Q4R5B3 Q60HC2 Q4R4X8 Q9BUF5 Q3ZCM7

GFAP_HUMAN HSP71_HUMAN HS71L_HUMAN HSP76_HUMAN H4_MACFA K2C1_HUMAN PGK1_MACFA HSP77_HUMAN TBB5_HUMAN TBB2A_MACFA TBB3_MACFA TBB4_MACFA TBB6_HUMAN TBB8_HUMAN

GFAP HSPA1A HSPA1L HSPA6 QtsA-19327 KRT1 PGK1 HSPA7 TUBB TUBB2A TUBB3 TUBB4 TUBB6 TUBB8

Glial fibrillary acidic protein Heat shock 70 kDa protein 1 Heat shock 70 kDa protein 1L Heat shock 70 kDa protein 6 Histone H4 Keratin Phosphoglycerate kinase 1 Putative heat shock 70 kDa protein 7 Tubulin beta chain Tubulin beta-2A chain Tubulin beta-3 chain Tubulin beta-4 chain Tubulin beta-6 chain Tubulin beta-8 chain

49880 70052 70375 71028 11367 66017 44586 40244 49670 49907 50432 49585 49857 49776

369.94 181.98 178.71 155.81 114.17 335.80 104.59 111.60 278.61 251.34 191.33 248.94 151.00 138.95

23.60 13.01 11.37 4.96 25.25 19.98 16.25 7.97 16.73 15.30 10.96 16.70 8.52 10.42

Treated Treated Treated Treated Treated Treated Treated Treated Treated Treated Treated Treated Treated Treated

only only only only only only only only only only only only only only

Q8SPH6 Q9H444 P14927 Q2PFW6 P61952

ATP5J_MACFA CHM4B_HUMAN QCR7_HUMAN SYUG_MACFA GBG11_HUMAN

ATP5J CHMP4B UQCRB SNCG GNG11

12586 24950 13530 13295 8480

163.35 86.64 118.70 310.47 99.07

37.35 14.07 29.28 64.33 24.09

Control Control Control Control Control

only only only only only

Q99878 Q16777 Q7L7L0 Q15293 Q2PFW2 P55854 Q6EEV6 Q16629 P07951 P54727

H2A1J_HUMAN H2A2C_HUMAN H2A3_HUMAN RCN1_HUMAN SUMO2_MACFA SUMO3_HUMAN SUMO4_HUMAN SFRS7_HUMAN TPM2_HUMAN RD23B_HUMAN

HIST1H2AJ HIST2H2AC HIST3H2A RCN1 SUMO2 SUMO3 SUMO4 SFRS7 TPM2 RAD23B

ATP synthase-coupling factor 6 Charged multivesicular body protein 4b Cytochrome b-c1 complex subunit 7 Gamma-synuclein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-11 Histone H2A type 1-J Histone H2A type 2-C Histone H2A type 3 Reticulocalbin-1 Small ubiquitin-related modifier 2 Small ubiquitin-related modifier 3 Small ubiquitin-related modifier 4 Splicing factor Tropomyosin beta chain UV excision repair protein RAD23 homolog

13936 13988 14121 38890 10871 11637 10685 27366 32850 43171

140.10 140.10 140.10 148.34 104.62 103.43 106.66 135.87 132.25 178.96

21.88 21.71 21.54 14.68 14.21 14.81 18.42 16.84 12.32 13.16

Control Control Control Control Control Control Control Control Control Control

only only only only only only only only only only

fmol to total nanograms in an analytic run were used to normalize each run. Proteins that were found in at least two of the analytic runs for each retinal sample were accepted as valid entries (Table 2). For each accepted protein, a fmol ratio was established between the treated eye and its contralateral control eye, and a p-value was determined (see next). Proteins that were found in both mild IOP EEG retinas, both high IOP EEG retinas, or at least 2 of 3 ONT retinas and showed a statistically-significant change compared to their contralateral normal eye retinas were termed “up” or “down regulated” (Tables 3, 5 and 7). Proteins that were identified in both mild IOP EEG, or both high IOP EEG or 2 of 3 ONT eyes without being identified in the appropriate contralateral control eyes were reported separately (Tables 4, 6 and 8). Proteins that were identified in both control eyes of the mild IOP EEG animals, or both control eyes of the high IOP EEG animals or 2 of 3 ONT animal control eyes without being identified in the appropriate contralateral treated eyes were also reported separately (Tables 4, 6 and 8). Since the use of a combined Macaca and Human database resulted in duplicate identifications of some proteins, for bioinformatic analyses, only proteins that could be distinctly identified by unique peptides were included. If no unique peptides were identified for a protein to distinguish between macaca and human proteins, the macaca protein was included in bioinformatic analyses, and the human protein was removed from the list. 2.6. Statistical analyses All statistical analyses were performed using the open source R software package version 2.11.0 (downloaded May 2010). Significance testing was performed using the generalized estimating

equations (GEE) technique (Yan, 2002; Yan and Fine, 2004; Højsgaard et al., 2005), which accounts for the correlation between data collected from the same eyes/animals. Three analyses were performed 1) the 4 EEG treated eyes were compared to the 4 EEG control eyes; 2) the 2 mild IOP EEG treated eyes were compared to the 2 high IOP EEG treated eyes; and 3) the 2 (EEG) or 3 (ONT) treated eyes for each group were compared to the control eyes for each group. 2.7. Bioinformatic analyses All regulated proteins (Tables 3, 5 and 7) as well as those that only appeared in the control or treated eyes (Tables 4, 6 and 8, Section 2.5, above) were imported into the MetaCore program (GeneGo, Inc. West Lothian, UK) and an association of these proteins with particular biological processes, metabolic and signaling pathways was analyzed. The overlaps between or among different treatment conditions for particular pathways were also analyzed. 2.8. Western blot analyses Aliquots of each sample were run on 10% SDS PAGE gels and transferred to PVDF membranes. The blots were then cut in half and the upper half of each was incubated with anti-vimentin antibody (Santa Cruz, sc-7558, goat polyclonal) and the bottom half of each was incubated with anti-gamma-synuclein antibody (Santa Cruz, sc-10698, goat polyclonal) overnight at 4  C. Next day, the blots were incubated with an HRP-conjugated rabbit anti-goat secondary antibody and analyzed with the enzyme-catalyzed chemiluminescence (ECL) method, using a Visualizer Western Blot Detection Kit

C. Stowell et al. / Experimental Eye Research 93 (2011) 13e28

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Table 7 Regulated proteins in ONT eyes. SwissProt ID

Gene name

Protein name

Calculated MW

Up-regulated proteins P68032 ACTC_HUMAN P68133 ACTS_HUMAN A5A3E0 A26CB_HUMAN P12277 KCRB_HUMAN P08754 GNAI3_HUMAN

ACTC1 ACTA1 A26C1B CKB GNAI3

42019 42051 121370 42644 40532

277.95 282.54 438.52 739.05 136.90

11.38 18.42 9.99 40.42 8.88

1.18 1.18 1.22 1.14 1.22

P63092

GNAS2_HUMAN

GNAS

45664

131.28

10.02

1.22

Q4R4M6 P07437 Q4R5B3 Q9BVA1 P68371 Q60HC2 Q4R4X8 Q9BUF5

HNRPK_MACFA TBB5_HUMAN TBB2A_MACFA TBB2B_HUMAN TBB2C_HUMAN TBB3_MACFA TBB4_MACFA TBB6_HUMAN

HNRNPK TUBB TUBB2A TUBB2B TUBB2C TUBB3 TUBB4 TUBB6

Actin Actin ANKRD26-like family C member 1B Creatine kinase B-type Guanine nucleotide-binding protein G(k) subunit alpha Guanine nucleotide-binding protein G(s) subunit alpha isoforms short Heterogeneous nuclear ribonucleoprotein K Tubulin beta chain Tubulin beta-2A chain Tubulin beta-2B chain Tubulin beta-2C chain Tubulin beta-3 chain Tubulin beta-4 chain Tubulin beta-6 chain

51028 49670 49907 49953 49831 50432 49585 49857

217.35 732.90 733.36 754.16 849.20 429.15 625.67 214.01

24.68 28.53 28.77 29.04 34.94 16.01 31.55 14.71

1.17 1.29 1.28 1.28 1.28 1.48 1.27 1.41

proteins 1433E_HUMAN 1433G_HUMAN RLA1_HUMAN GRP78_HUMAN ALDOA_HUMAN HSP74_HUMAN IMPG1_HUMAN IRBP_HUMAN LDHB_MACFA MDHM_MACFA MDHM_HUMAN ARRS_HUMAN TPIS_HUMAN UBIQ_MACFA

YWHAE YWHAG RPLP1 HSPA5 ALDOA HSPA4 IMPG1 RBP3 LDHB MDH2 MDH2 SAG TPI1 UBA52

14-3-3 protein epsilon 14-3-3 protein gamma 60S acidic ribosomal protein P1 78 kDa glucose-regulated protein Fructose-bisphosphate aldolase A Heat shock 70 kDa protein 4 Interphotoreceptor matrix proteoglycan 1 Interphotoreceptor retinoid-binding protein L-lactate dehydrogenase B chain Malate dehydrogenase Malate dehydrogenase S-arrestin Triosephosphate isomerase Ubiquitin

29173 28302 11513 72333 39420 94300 89387 135362 36638 35565 35531 45119 26669 8564

331.85 169.26 106.94 308.51 290.06 309.48 342.16 842.03 296.40 196.51 190.60 1105.63 274.81 243.94

39.65 23.75 47.27 20.57 30.96 24.02 15.44 22.36 19.86 27.69 23.51 44.35 47.22 57.02

1.17 1.35 1.34 1.20 1.22 1.42 1.66 1.24 1.10 1.32 1.32 1.09 1.35 1.35

Down-regulated P62258 P61981 P05386 P11021 P04075 P34932 Q17R60 P10745 Q4R5B6 Q4R568 P40926 P10523 P60174 P0C274

UniProt ID

Average protein score

Average sequence coverage (%)

Ratio of Treated to control*

*p-values for all proteins are <0.05.

(Millipore, Billerica, MA). The top half of each blot was then stripped and then incubated with anti-HSP70 antibody (Santa Cruz, sc-66048, mouse monoclonal), followed by incubation with an HRPconjugated goat anti-mouse secondary, and detected as above. Image documentation and semi-quantification of band density was

performed on a Kodak Image Station 2000RT system (Kodak, Rochester, NY) and reported as the mean þ standard deviation for either n ¼ 2 (EEG) or n ¼ 3 (ONT) densitometry readings. Representative images from an individual animal for each protein were chosen for the figures.

Table 8 Proteins found only in at least 2 treated or 2 control eyes of the ONT animals. SwissProt ID

UniProt ID

Gene name

Protein name

Calculated MW

Average protein score

Average sequence coverage (%)

Treated or control

P14136 P62873

GFAP_HUMAN GBB1_HUMAN

GFAP GNB1

49880 37377

388.13 329.22

31.57 34.02

Treated only Treated only

P62879

GBB2_HUMAN

GNB2

Glial fibrillary acidic protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2

37331

236.41

21.62

Treated only

Q8SPH6 Q4R562 Q4R374

ATP5J_MACFA ARRB1_MACFA CX6B1_MACFA

ATP5J ARRB1 COX6B1

12586 46322 10318

116.68 158.68 65.61

33.33 18.11 31.61

Control only Control only Control only

P14854

CX6B1_HUMAN

COX6B1

10192

62.74

24.42

Control only

Q13561 P06737 P29966

DCTN2_HUMAN PYGL_HUMAN MARCS_HUMAN

DCTN2 PYGL MARCKS

44231 97148 31554

157.82 382.66 223.85

29.03 16.12 27.26

Control only Control only Control only

P67809

YBOX1_HUMAN

YBX1

35924

130.92

21.60

Control only

Q4R4J7

NUCL_MACFA

NCL

ATP synthase-coupling factor 6 Beta-arrestin-1 Cytochrome c oxidase subunit VIb isoform 1 Cytochrome c oxidase subunit VIb isoform 1 Dynactin subunit 2 Glycogen phosphorylase Myristoylated alanine-rich C-kinase substrate Nuclease-sensitive element-binding protein 1 Nucleolin

76741

192.94

7.11

Control only

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C. Stowell et al. / Experimental Eye Research 93 (2011) 13e28

Table 9 Bioinformatic Analyses for mild IOP EEG eyes. Up-regulated processes Excluding treated only proteinsa

p-value

Excluding control only proteins Microtubule-based process Cellular carbohydrate catabolic process Response to lithium ion Cellular component biogenesis e including cytoskeleton organization and myofibril assembly Alcohol catabolic process Vascular process in circulatory system Skeletal muscle adaptation Pyruvate metabolic process Developmental process Regulation of eIF2 alpha phosphorylation by heme Gluconeogenesis

p-value 8.48E-09 1.63E-09 6.02E-09 5.86E-08

p-value 6.81E-03 9.E-03 2.15E-02

Excluding treated only proteinsa

p-value

p-value 9.03E-15 1.15E-10 1.36E-07 3.28E-06 4.28E-06

Excluding control only proteins Cytoskeleton remodeling_neurofilaments Glycolysis and gluconeogenesis Cytoskeleton remodeling_keratin filaments Cell adhesion_gap junctions Role of Nek in cell cycle regulation

p-value 6.30E-13 5.41E-11 7.62E-08 2.08E-06 2.72E-06

Including treated only proteins

p-value

Regulation of cellular amide metabolic process Regulation of cofactor metabolic process Monosaccharide metabolic process Carbohydrate catabolic process Vesicle coating Small molecule catabolic process Carbohydrate metabolic process Membrane organization Vesicle-mediated transport Respiratory burst Cellular macromolecule localization Gluconeogenesis Nicotinamide nucleotide metabolic process Cellular aldehyde metabolic process Regulation of carbohydrate catabolic process

3.79E-04 5.69E-04 8.06E-04 9.00E-04 1.70E-03 2.13E-03 2.66E-03 2.99E-03 3.49E-03 3.98E-03 4.01E-03 5.49E-03 5.73E-03 6.62E-03 7.18E-03

Down-regulated processes Including control only proteins Microtubule-based process Cellular carbohydrate catabolic process Response to lithium ion Cellular component biogenesis e including cytoskeleton organization and myofibril assembly

p-value 1.34E-09 4.44E-09 1.05E-08 3.12E-08

Alcohol catabolic process Vascular process in circulatory system Skeletal muscle adaptation Pyruvate metabolic process Developmental process Regulation of eIF2 alpha phosphorylation by heme

6.38E-08 7.72E-08 1.12E-07 1.17E-07 1.40E-07 2.34E-07

Response to biotic stimulus

4.15E-09

Up-regulated pathways Including treated only proteins Glycolysis and gluconeogenesis GABA-A receptor life cycle Triacylglycerol metabolism Down-regulated pathways Including control only proteins Cytoskeleton remodeling_neurofilaments Glycolysis and gluconeogenesis Cytoskeleton remodeling_keratin filaments Cell adhesion_gap junctions Role of Nek in cell cycle regulation a

2.37E-08 5.50E-08 1.63E-07 8.42E-08 2.24E-07 1.68E-07 2.69E-07

There were no processes or pathways that matched to the two proteins that were up-regulated in the mild IOP EEG eyes.

3. Results 3.1. Subject conditions For each NHP, the maximum post-laser IOP, the duration of time from first laser treatment to sacrifice, the cumulative IOP exposures and pre-sacrifice HRT topographic change analysis (TCA) maps for each of the treated eyes relative to its contralateral control eye are reported in Fig. 1 and Table 1. While we intended to deliver similar levels of IOP insult to the EEG eyes of all four EEG animals, two distinct levels and durations of IOP elevation were retrospectively apparent within the four EEG eyes (Fig. 1). Following a comparison of the two mild IOP EEG eyes to the high IOP EEG eyes, which suggested that 70% of the proteins were significantly different from each other (outlined below), the EEG animals were retrospectively separated into two treatment groups: 1) Mild IOP EEG NHPs 1 and 2 demonstrated detected IOPs less than 27 mm Hg and the period of post-laser observation to the onset of detected ONH surface change was long (209 and 295 days, respectively); and 2) High IOP EEG

NHPs 1 and 2 demonstrated detected IOPs that at times exceeded 45 mm Hg and the period of post-laser observation to the onset of detected ONH surface change was much shorter (43 and 51 days, respectively). Unlike the EEG animals, all ONT animals were sacrificed exactly 3 weeks post unilateral surgical optic nerve transection.

3.2. Changes in protein expression by individual animals and treatment groups Table 2 lists the total number of identified retinal proteins within the treated and contralateral control eye of each animal. On average, 123 proteins were identified and quantified for each eye, with average sequence coverage of 19% (7.3 peptides/protein). Among the proteins quantified for each eye, there was on average a 300-fold difference between the least and the most abundant protein (quantities of identified proteins in fmol are provided in Table S1).

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Table 10 Bioinformatic Analyses for high IOP EEG eyes. Up-regulated processes Including treated only proteins

p-value

Excluding treated only proteins

p-value

Microtubule-based process Cell killing Response to stress Cellular component biogenesis e including spindles Immune effector process Cellular component organization e including telomeres Negative regulation of peptidase activity Regulation of cell growth Response to protein stimulus Regulation of inclusion body assembly Protein refolding Negative regulation of vasoconstriction Response to biotic stimulus Regulation of myeloid cell apoptosis

6.92E-07 6.05E-06 2.85E-06 3.00E-07 1.06E-05 2.97E-06 1.51E-05 1.09E-05 5.44E-08 4.25E-08 1.54E-07 2.58E-08 6.06E-07 1.65E-06

Cytoskeleton organization Cell killing Innate immune response Cellular component biogenesis e including spindles Immune effector process Cellular component organization e including telomeres Induction of apoptosis Regulation of cell growth Regeneration Small molecule catabolic process Axon guidance Carbohydrate catabolic process

6.86E-06 1.77E-06 1.12E-04 1.51E-04 1.73E-04 1.36E-03 2.20E-03 3.53E-03 2.11E-03 2.12E-03 3.72E-03 6.11E-04

Down-regulated processes Including control only proteins Oxygen transport Nitric oxide transport Protein complex assembly Cellular component organization e including nucleosomes Regulation of response to stress Regulation of hydrolase activity Regulation of blood pressure Recombinational repair Regulation of ATPase activity M phase of meiotic cell cycle Cell aging Regulation of heart rate by chemical signal DNA integrity checkpoint Regulation of anatomical structure size Male gamete generation

p-value 1.47E-10 1.96E-03 3.73E-04 3.23E-06 1.79E-03 3.12E-03 2.34E-03 5.65E-04 5.65E-04 1.47E-03 2.45E-03 3.67E-03 5.22E-03 5.46E-03 5.66E-03

Excluding control only proteins Oxygen transport Nitric oxide transport Protein complex assembly Cellular component organization e including nucleosomes Regulation of response to stress Regulation of hydrolase activity Regulation of blood pressure Cell-cell recognition Erythrocyte development Female pregnancy Hemopoiesis Regulation of protein amino acid phosphorylation

p-value 2.22E-13 1.13E-04 8.08E-04 9.61E-04 1.87E-03 2.26E-03 3.73E-03 1.04E-02 1.04E-02 5.37E-03 1.67E-02 8.68E-03

Up-regulated pathways Including treated only proteins Cytoskeleton remodeling_neurofilaments Cytoskeleton remodeling_keratin filaments Glycolysis and gluconeogenesis Role of parkin in the ubiquitin-proteasomal pathway Cell adhesion_gap junctions

p-value 4.35E-11 6.52E-08 2.63E-06 2.63E-06 5.26E-06

Excluding treated only proteins Cytoskeleton remodeling_neurofilaments Glycolysis and gluconeogenesis Cell adhesion_gap junctions Role of Nek in cell cycle regulation Cytoskeleton remodeling_keratin filaments

p-value 2.36E-09 7.71E-07 1.54E-06 1.89E-06 2.71E-06

p-value 7.41E-04 1.84E-02

Excluding control only proteins EPO-induced PI3K/AKT pathway and Ca(2þ) influx Chromosome condensation in prometaphase

p-value 8.08E-05 7.69E-03

2.04E-02 2.13E-02 2.23E-02

Cell cycle_sister chromatid cohesion Cell cycle_initiation of mitosis Apoptosis and survival_granzyme A signaling

8.05E-03 9.14E-03 1.10E-02

Down-regulated pathways Including control only proteins EPO-induced PI3K/AKT pathway and Ca(2þ) influx Membrane trafficking and signal transduction of G-alpha (i) heterotrimeric G-protein Chromosome condensation in prometaphase Cell cycle_sister chromatid cohesion Delta508-CFTR traffic/sorting endosome formation in CF

Approximately 70% of the identified proteins were detected in both the treated and contralateral control eyes in each of the mild IOP EEG and ONT subjects, whereas in the high IOP EEG subjects, there was only 50% overlap between the treated and contralateral control eyes (Table 2). Within the 2 mild IOP EEG eyes, 2 of the detected retinal proteins showed significant up regulation, and 38 proteins were significantly down regulated. For the 2 high IOP EEG eyes, 12 proteins were significantly up regulated and 8 proteins were significantly down regulated. Of the detected retinal proteins in the ONT eyes, 14 were significantly up and 14 were significantly down regulated. Venn diagrams (Fig. 2) demonstrate little overlap in proteins regulated in the same direction among the three treatment groups, with the exception of two up-regulated proteins in both the high IOP EEG and ONT eyes, and five down-regulated proteins e four in both the mild IOP EEG and high IOP EEG eyes and one in both the mild IOP EEG and ONT eyes. However, an additional group of proteins were common to at least two treatment groups but

demonstrated opposing directions of up and down regulation (see the right panel of Fig. 2, Tables 3, 5 and 7 and the sections that follow). As noted, above, before formally treating the mild IOP and high IOP EEG animals as separate groups, we performed two analyses. First, we compared the 4 EEG eyes to the 4 control eyes and found no statistically-significant differences for any of the detected proteins. To test the hypothesis that this first finding was due to profound differences in the response of the mild and high IOP eyes, we then compared the 2 mild IOP EEG eyes to the 2 high IOP EEG eyes and found that of the proteins that were found in both treated eyes of both the mild and high IOP EEG animals, 70 percent of the proteins showed a statistically-significant difference (Table S1). 3.3. Mild IOP EEG-associated changes (Tables 3 and 4) Two proteins were up regulated and 38 proteins were down regulated in the mild IOP EEG eyes (Table 3). Two additional

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C. Stowell et al. / Experimental Eye Research 93 (2011) 13e28

Mild IOP EEG

2

High IOP EEG

0 0

10

0 2

Mild IOP EEG

33

High IOP EEG

4

4

0 1

0

Mild IOP EEG

26

High IOP EEG

14 4

24

3 6 25

12 13

ONT Up regulated

ONT

ONT Down regulated

All potentially regulated

Fig. 2. Venn diagrams of up-regulated, down-regulated, and all potentially regulated retinal proteins by treatment group. Numbers are proteins that were either statistically up (left diagram) or down (middle diagram) regulated in both of the mild IOP eyes, both of the high IOP eyes, or at least 2 of 3 of the ONT eyes, or all of the potentially regulated proteins (statistically-significantly changed plus treated- and control-only proteins (right diagram) in each treatment group).

proteins were found only in the treated eyes and 8 were found only in the control eyes (Table 4). The proteins that showed an increase in expression were gamma-synuclein and stathmin, whereas phosphoglycerate mutase 1 and Clathrin light chain A were found in the treated eye only. Among the 38 down-regulated mild IOP EEG proteins, fifteen were up regulated in the high IOP EEG eyes (Table 5), the ONT eyes (Table 7) or both. Among the downregulated proteins, there are six actin isoforms and seven tubulin isoforms. Also down regulated in the mild IOP EEG group were hemoglobin subunit beta, histone H1 and 10 kDa heat shock protein; these are the only proteins regulated in the same direction in both the mild IOP and high IOP EEG eyes. Triosephosphate isomerase (macaca) was down regulated in both the mild IOP EEG and ONT eyes. HSPA6 and HSPA7 were found in the control eyes only and were both found in the treated eyes only of the high IOP EEG animals. 3.4. High IOP EEG-associated changes (Tables 5 and 6) Twelve proteins were up regulated and 8 proteins were down regulated in the high IOP EEG eyes (Table 5). An additional 14 proteins were found only in the treated eyes and 15 were found only in the control eyes (Table 6). Among the proteins showing increased expression, the dominant groups are the tubulin protein isoforms most of which were down regulated in the mild IOP EEG eyes (Table 3). One of the tubulin isoforms and ANKRD26-like family C member 1B were up regulated in both the high IOP EEG and the ONT eyes. Gamma synuclein, which was slightly up regulated in the mild IOP EEG, was only found in the control eyes in the high IOP EEG. Among the proteins showing decreased expression, are four hemoglobin subunits and superoxide dismutase 1 (SOD1, with diverse functions including free radical scavenging, DNA fragmentation during apoptosis, actin binding and negative regulation of cell migration). ATP synthase-coupling factor 6 was found only in the control eyes in both the high IOP EEG and ONT NHPs. 3.5. ONT-associated changes in protein expression (Tables 7 and 8) Fourteen proteins were up regulated and 14 proteins were down regulated in the ONT eyes (Table 7). An additional 3 proteins were found only in the treated eyes and 9 were found only in the control eyes (Table 8). Among the proteins showing increased expression, there was a marked enrichment in tubulin beta isoforms (Table 7). As mentioned above, one of the tubulin isoforms along with ANKRD26-like family C

member 1B protein were up regulated in the high IOP EEG eyes as well as the ONT eyes. Glial fibrillary acidic protein was found only in the treated ONT eyes. It was also found only in the treated eyes in the high IOP EEG. Among the proteins showing decreased expression are a number of proteins that would be involved in glucose metabolism and energy production. Of note in the control-only proteins was dynactin subunit 2, which binds to both dynein and kinesin and helps facilitate transport. 3.6. Biological processes associated with regulated proteins (Tables 9e11) For each treatment group, the MetaCore program was used to perform bioinformatic analysis for the regulated proteins first including and then excluding the proteins that were found in the treated or control eyes, only. With the exception of the downregulated proteins in the mild IOP EEG eyes, Table 9, the addition of the treated- or control-only proteins increased the number of associated biological processes. Table 9 shows that there were no significant processes that were identified with the two proteins that were up regulated in the mild IOP EEG eyes. However, the addition of the 2 treated-only proteins generated a list with mostly metabolic processes. The first 10 processes listed for the down-regulated proteins in the mild IOP EEG were the same whether or not the control-only proteins were included, while the last process was different between the two lists. The high IOP EEG eyes demonstrated the largest difference in associated biological processes with inclusion or exclusion of the treated- or control-only proteins (Table 10). Eight of the processes for the up-regulated proteins and seven of the processes for the down-regulated proteins were the same with or without the treated- or control-only proteins. Of interest in these processes are the up regulation of cytoskeleton organization, or microtubulebased process, and the down regulation of transport in the high IOP EEG eyes. In Table 11, the addition of the treated- or controlonly proteins only added 1 and 2 processes to the lists for the upand down-regulated proteins respectively. Of note in these lists are the up regulation of natural killer cell mediated immunity and cell killing and the down regulation of catabolic processes suggesting the presence of apoptosis within the high IOP EEG retinas. 3.7. Western blot confirmation of proteomic quantifications Fig. 3 demonstrates results of Western blot analyses of selected proteins that were identified and quantified by MS analyses, as a means to validate proteomic quantifications.

C. Stowell et al. / Experimental Eye Research 93 (2011) 13e28

3.7.1. Vimentin By MS analysis vimentin was down regulated in the mild IOP EEG eyes (Table 3) and unchanged in the high IOP EEG and ONT eyes. The top row in Fig. 3 demonstrates down regulation in the mild IOP EEG treated eyes and little change in the abundance of vimentin in the high IOP EEG and ONT eyes when compared to their contralateral control eyes by semi-quantitation of western blot bands. 3.7.2. Gamma-synuclein By MS analyses, gamma-synuclein demonstrated up regulation in the mild IOP EEG eyes (Table 3), was only found in the control eyes in the high IOP EEG (Table 6) and was not detected within the ONT eyes. The middle row in Fig. 3, demonstrates modest up regulation in the mild IOP EEG eyes, substantial down regulation in the high IOP eyes, and down regulation in the ONT eyes by western blot and densitometry. While these data support the MS results for the mild and high IOP EEG eyes, for the ONT eyes they suggest that western blotting was more sensitive to both the presence and down regulation of this protein in the ONT eyes.

23

3.7.3. HSP70 By MS analysis, HSP70 was found in the control eyes only of the mild IOP EEG NHPs, treated eyes only in the high IOP EEG NHPs and was down regulated in the ONT eyes. The bottom row of Fig. 3 demonstrates that the western blot and densitometry data strongly support the direction of change of this protein in all three treatment groups. However, in the case of both the mild and high IOP NHPs, western blot detected this protein in all samples (rather than just the control eyes (mild IOP EEG) or treated eyes (high IOP EEG)) and was therefore more consistently sensitive to the presence of this protein than our MS technique. 4. Discussion This study compares proteomic changes in retinas from NHP eyes subjected to two forms of insult to the RGC axon and soma: 1) chronic IOP elevation (mild and high), followed to the onset of detectable ONH surface change; and 2) surgical transection of the orbital optic nerve (ONT) followed for three weeks post-transection.

Table 11 Bioinformatic analyses for ONT eyes. Up-regulated processes Including treated only proteins

p-value

Excluding treated only proteins

p-value

Natural killer cell mediated immunity Muscle system process Organelle organization e including cytoskeleton Microtubule-based process Response to stimulus Cellular component biogenesis e including myofibrils Cell killing Heart process Phosphocreatine metabolic process System development Muscle tissue morphogenesis Cellular chloride ion homeostasis

1.89E-07 6.57E-07 3.26E-06 3.33E-06 3.75E-06 4.76E-06 5.00E-06 6.84E-06 6.86E-06 8.73E-06 1.56E-05 3.08E-05

Natural killer cell mediated immunity Muscle system process Organelle organization e including cytoskeleton Microtubule-based process Response to stimulus Cellular component biogenesis e including myofibrils Cell killing Heart process Phosphocreatine metabolic process Multicellular organismal development Muscle tissue morphogenesis

8.42E-08 1.83E-07 5.31E-07 1.11E-06 7.86E-06 1.75E-06 3.32E-06 3.07E-06 4.08E-06 4.24E-06 7.02E-06

Down-regulated processes Including control only proteins Positive regulation of embryonic development Regulation of nuclease activity Outer mitochondrial membrane organization Cellular response to glucose starvation Chaperone-mediated protein complex assembly Protein targeting Alcohol catabolic process Negative regulation of hydrolase activity Catabolic process e including glucose and ribose Regulation of protein amino acid dephosphorylation Response to heat Anatomical structure arrangement e including brain development ER overload response Response to unfolded protein Cellular carbohydrate biosynthetic process Alcohol biosynthetic process

p-value 4.60E-06 1.38E-05 1.88E-05 2.14E-05 2.75E-05 1.09E-04 9.47E-05 2.36E-05 2.28E-05 2.45E-05 1.16E-04 4.23E-05 6.94E-05 2.49E-05 1.83E-05 2.99E-05

Excluding control only proteins Positive regulation of embryonic development Regulation of nuclease activity Outer mitochondrial membrane organization Cellular response to glucose starvation Chaperone-mediated protein complex assembly Protein targeting Alcohol catabolic process Negative regulation of hydrolase activity Catabolic process e including glucose Regulation of protein amino acid dephosphorylation Response to heat Anatomical structure arrangement e including brain development ER overload response Response to unfolded protein

p-value 2.10E-06 6.30E-06 8.57E-06 9.79E-06 1.26E-05 2.15E-05 2.85E-05 4.49E-05 4.57E-05 7.24E-05 8.55E-05 8.76E-05 9.65E-05 9.99E-05

Up-regulated pathways Including treated only proteins Regulation of cAMP levels by ACM Alpha-2 adrenergic receptor regulation of ion channels Muscle contraction_relaxin signaling pathway NF-AT signaling in cardiac hypertrophy G-protein signaling_rap2A regulation pathway

p-value 1.28E-05 1.46E-05 1.56E-05 3.89E-05 1.45E-04

Without treated only proteins Regulation of cAMP levels by ACM Alpha-2 adrenergic receptor regulation of ion channels Muscle contraction_relaxin signaling pathway NF-AT signaling in cardiac hypertrophy G-protein signaling_rap2A regulation pathway

p-value 5.37E-06 6.13E-06 6.53E-06 1.64E-05 8.49E-05

Down-regulated pathways Including control only proteins Glycolysis and gluconeogenesis Role of 14-3-3 proteins in cell cycle regulation Role of Akt in hypoxia induced HIF1 activation Galactose metabolism CFTR folding and maturation (norm and CF)

p-value 9.78E-04 5.29E-04 8.01E-04 4.18E-03 2.20E-02

Excluding control only proteins glycolysis and gluconeogenesis Role of 14-3-3 proteins in cell cycle regulation Role of Akt in hypoxia induced HIF1 activation CFTR folding and maturation (norm and CF) Role of heterochromatin protein 1 (HP1) family in transcriptional silencing

p-value 4.50E-04 2.46E-04 3.73E-04 1.53E-02 2.40E-02

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Fig. 3. Western blotting and densitometry analyses. Western Blots and densitometry analyses for Vimentin (upper row, black arrow points to the correct molecular weight), Gamma Synuclein (middle row) and heat shock protein 70 (bottom row) are reported. For each protein, blots (from representative individual animals) and densitometry measurements (pooled for all control and treated eyes) for the mild IOP (MIOP e left column), high IOP (HIOP e middle column) and ONT (ONT e right column) are shown. Densitometry readings are the mean  SD of the n ¼ 2 mild IOP EEG, n ¼ 2 high IOP EEG or n ¼ 3 ONT animals, pooled together. Densitometry readings (pooled control eyes e white, pooled treated eyes e black) and blots are arranged control eye data left, treated eye data right. Because the blots show the control and treated eyes of individual animals and the densitometry data is pooled from multiple measures of all control and treated eyes in each group, exact correspondence between the blots and the densitometry data should not be expected. Note that for HSP70, the pooled densitometry readings for both control and treated eyes from the high IOP EEG animals appear to overstate the difference seen in the blots.

The chosen experimental paradigms are important for the following reasons. First, EEG in NHP may be our best model of the conversion of human ocular hypertension to clinically detectable glaucomatous damage. Second, while our study’s inclusion of EEG conditions at both mild and high IOP elevation levels was inadvertent, and reduces the number of animals in each experimental group, it

mimics the diverse susceptibility of human ocular hypertensive patients and provides preliminary insight into mechanisms of damage that are IOP-related but only invoked at high IOP. Third, our inclusion of both EEG and the ONT retinas provides preliminary insight into non-IOP related mechanisms of glaucomatous retinal injury such as secondary degeneration. Finally, our study benefited

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from the use of an MS system that allowed absolute quantification of tissue proteins with multiple technical replicates. The principal findings of this report are as follows. First, while the mild and high IOP EEG eyes were enucleated at a similar early stage of ONH damage, and while the high IOP EEG eyes endured a duration of detected IOP elevation that was similar to the posttransection time in the ONT eyes, retinal proteomic changes under mild IOP EEG, high IOP EEG, and ONT conditions were substantially different, with very limited overlap among different treatment conditions. Second, a prominent alteration in cytoskeletal proteins was seen in the EEG and the ONT retinas. Third, the MS identified proteome of the mild IOP EEG eyes showed a general trend of down rather than up regulation. The current results include identification and quantitation of approximately 120 proteins per retina using a one-step protein extraction protocol. While this is a very limited number compared with the possible numbers of translated proteins in eukaryotic cells, it is in the same range as what is reported in a limited number of studies on retinas of different species with similar or even more comprehensive pre-MS preparation steps (Sloley et al., 2007; Decanini et al., 2008; Finnegan et al., 2008; Kwok et al., 2008; Gao et al., 2009; Kanamoto et al., 2009; Kim et al., 2009). In a few studies on retinas or immortalized RGC cells that employed a 2-D gel fractionation step prior to MS analyses, the numbers of unique protein spots ranged from a few hundred to over a thousand (Decanini et al., 2008; Finnegan et al., 2008; Kanamoto et al., 2009; Kim et al., 2009). It is worth noting, however, that only a small number (15e30) of gel spots showed a change under designed experimental conditions and were actually identified by MS. The current proteomic datasets, with their limitations in protein numbers, demonstrate consistent proteomic characteristics of the seven control retinas (Fig S1 and Table S2) and clear validated differences among different treatment conditions (Figs. 2 and 3; Tables 2e8). However, we consider these findings to be preliminary and sample preparations that include extraction enrichment and fractionation steps prior to MS analyses are planned for future studies. The differences in regulated proteins in the mild IOP EEG, high IOP EEG and ONT eyes may reflect, at least partially, differences in both the extent and mechanism of retinal injury in these animals. By differences in extent, we mean the number of retinal ganglion cells whose axons and soma (along with the outer retina) are experiencing physiologic and pathophysiologic levels of stress. In this regard, we have previously reported 16e30% orbital optic nerve axon loss at this EEG stage of damage in the NHP eye (Yang et al., 2007a). Because all retinal and orbital optic nerve tissues were processed for proteomic characterization, we do not have direct evidence for the extent of RGC axon injury at the time of sacrifice for the 7 animals in this study. While we currently follow our animals with longitudinal SDOCT RNFLT imaging, we did not have that capability at the time these animals were studied. However, a previous study by Quigley and colleagues (Quigley et al., 1995) reported the rate of RGC apoptosis to range from 1 to 13% and the magnitude of orbital optic nerve axon loss to be mild to moderate (approximately 20e40% for mild and 40e60% for moderate (personal communication)) in a group of chronic high IOP and ONTtreated NHP eyes that were sacrificed at post-treatment time points that were similar to those of this study. We estimate that at the time of enucleation, the rate of RGC apoptosis and the extent of RGC axonal damage in the mild IOP EEG eyes were likely modest as compared to that of the high IOP EEG and ONT eyes. It is also reasonable to suppose that the high IOP EEG and ONT eyes were at a similar extent of RGC axon and somal involvement. By differences in mechanism, we mean the principle components of the pathophysiologic insult may have been different in the

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mild IOP, high IOP and ONT eyes. The mechanisms of glaucomatous damage to the visual system remain controversial (Fechtner and Weinreb, 1994; Burgoyne et al., 2005; Burgoyne and Downs, 2008; Burgoyne, 2010). It is, however, reasonable to propose that while there may be primary retinal responses to even modest levels of IOP elevation, the primary insult to RGC axon transport is within the optic nerve head, is multifactorial in nature, and leads to the death of the RGC soma due to the loss of trophic signals or the incitement of other pro-apoptotic events (Quigley et al., 1995; Burgoyne, 2010). Surgical optic nerve transection (ONT), on the other hand, produces a primary, traumatic insult to the RGC axon within the orbit, providing a model of how the retina responds to RGC axonal trauma that is not IOP-related. In the NHP eye, surgical ONT leads to the death of the majority of RGC within 6e10 weeks by apoptosis (Quigley et al., 1977; Villegas-Perez et al., 1993; Berkelaar et al., 1994). In addition to these primary insults, both ONT and EEG are assumed to include secondary insults to otherwise healthy RGC axons and soma, in response to their damaged and/or dying neighbors. With regard to the fact that the mild and high IOP EEG eyes demonstrate substantial differences in their proteomes, it is reasonable to propose that there is a continuum of biological insult that occurs as IOP rises and that the predominant barriers to homeostasis are different at IOPs below 27 and above 45 mmHg. First there is strong evidence to suggest that IOP is a continuous risk factor (at all levels of IOP), and that the rate of structural and functional progression is principally influenced by the mean or maximum IOP level (Quigley et al., 1994; Agis, 2000; Gordon et al., 2002; Leske et al., 2003; Gardiner et al., submitted for publication). Second, there is also strong evidence to suggest that as IOP becomes elevated close to or beyond the point that ocular perfusion pressure begins to fall, hypoxia and ischemia may increasingly contribute both within the optic nerve head and retina (Alm and Bill, 1972, 1973; Liang et al., 2009; Stowell et al., 2010). With this issue in mind, we recently performed a retrospective analysis of IOP data from 47 NHPs involved in longitudinal studies of experimental glaucoma at the Devers Eye Institute (Burgoyne, 2011; Gardiner et al., submitted for publication). The rates of change of the Mean Position of the Disc (MPD) from confocal scanning laser tomography, and Retinal Nerve Fiber Layer Thickness (RNFLT) from spectral domain ocular coherence tomography, for both eyes of each animal were calculated over windows of 10 IOP measurements. Mixed effects models were formed to predict the rate of structural progression within a given window based on various characterizations of IOP including the cumulative insult up to the start of the window; the mean, maximum and variability of IOP within the window; and the interactions between cumulative IOP insult and each of these three IOP measures. After using a stepwise backwards elimination technique, the only significant predictor of the rate of change was IOP maximum. While these relationships may change over the course of the neuropathy, this analysis strongly suggests that for a given window of time, the maximum level of IOP most strongly influences optic nerve head structural progression in the NHP experimental glaucoma model as practiced in our hands (Gardiner et al., submitted for publication). Chauhan, reported a threshold IOP elevation level of approximately 15 mm Hg in the rat experimental glaucoma model beyond which the rate of structural progression within the ONH and peripapillary retina was obviously pronounced (Chauhan et al., 2002). They concluded: “Structural and functional changes in this model are best correlated to peak IOP change and not to duration of IOP elevation, suggesting the existence of an IOP-related damage threshold.” While retinal proteomic changes may not directly reflect ONH structural change in either of these models, it is reasonable to propose that they are related in both the rat and NHP eye.

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With specific regard for the fact that the mild IOP eyes primarily demonstrate down regulation, and the changes do not overlap with that in high IOP eyes, we speculate that down regulation may be a principal early response to environmental stress within these cells. Quill et al. recently demonstrated gene expression down regulation in human lamina cribrosa cells in cell culture at low levels of engineering strain (microscopic stretch) that transitioned to up regulation as the levels of strain increased (Quill et al., 2010). While the levels of engineering strain in the retina are not likely to be the principal pathophysiologic driver of the proteomic changes we report, this finding in optic nerve head astrocytes, may parallel the general down regulation seen here in the mild IOP EEG as compared to the high IOP EEG and ONT retinas. A more definite conclusion will await more expanded proteomic analyses. A lack of overlap in protein changes between mild IOP and high IOP eyes given the likely differences in extent and mechanism of pathophysiologic insult is therefore not surprising. Differential genomic and proteomic changes of neuronal tissues to mild and severe insults have been well documented, as a manifestation of cellular responses involving different pathways and processes in a series of previous studies (Stenzel-Poore et al., 2003; Stapels et al., 2010; Stowell et al., 2010). In contrast to the idea that initial down regulation is the principle response to early or mild challenges to retinal homeostasis, gammasynuclein in our study was mildly up-regulated in the mild IOP eyes, was found in the control eye only in the high IOP EEG eyes and was not identified by MS in the ONT eyes but was down regulated by western blot and densitometry (Fig. 3). Soto et al. have recently reported down regulation of retinal gamma-synuclein gene expression in 9-month-old DBA/2J mice when compared to 3 month old mice and concluded that RGC down regulation of gammasynuclein gene expression was a primary response to chronic IOP elevation in this model. This study also proposed that in the mouse eye gamma-synuclein was a highly selective marker of RGC soma. If this is also true in the NHP, this marker may provide insight into RGC specific alterations in NHP EEG. In the Soto study, the 9 month time point contained animals with a range of damage from unaffected to severe, graded by the extent of affected RGCs within retinal preparations; and at all stages of damage gamma-synuclein expression was down regulated by in situ hybridization (Soto et al., 2008). A more definite determination of the relationship between the current proteomic findings and the extent of retinal injury relies on future spatial and temporal analyses of the retinal cells of origin in these three experimental conditions. Taken together, apart from differences in the anatomic extent of involvement, the conditionspecific changes support the presence of a more advanced pathophysiological stage of damage in the high IOP EEG and ONT retinas. These data further suggest a regulatory role for these proteins in the retina’s response to this stage of insult. The overlap in retinal proteins that were regulated in the same direction between treatment conditions was limited to two upregulated proteins (high IOP EEG and ONT groups) and five down regulated proteins (four shared by the mild IOP and high IOP EEG groups and one shared by the ONT and mild IOP EEG groups). Bioinformatic analyses of the regulated proteins under each condition also revealed little similarity in the cellular processes that were associated with these proteins (Tables 9e11). As noted above, previous studies have characterized genomic or proteomic changes in the rat retina under either acute or chronic IOP elevation or ONT conditions (Ahmed et al., 2004; Tezel et al., 2005; Yang et al., 2007c; Miyara et al., 2008). Specifically, Ahmed et al. and Steele et al. have shown an increase in GFAP in glaucoma, which agrees with our high IOP EEG and ONT results. Ahmed et al. also showed an increase in alpha tubulin. Piri et al. also saw an increase in vision related proteins and G-coupled receptor protein

signaling after ONT as was indicated in our ONT data. The study by Miyahara et al. described genomic changes in retina of glaucomatous NHP eyes with moderate to severe glaucomatous damage (Miyahara et al., 2003). Their results suggested the involvement of a number of cellular processes including, but not limited to, signal transduction, immune response, cytoskeleton and the regulation of apoptosis. It is not clear, however, to what extent each of the processes was regulated. The use of the MetaCore program in the present study allowed for the ranking of significance of biological processes based on the number of proteins associated with particular processes in a given dataset. For each treatment condition, the bioinformatic analyses were run both with and without the control and treated eye-only proteins. Because these proteins were only found in either the treated or the control eyes, where present, they suggest up and down regulation in the treated eye of each animal (respectively), but we cannot be certain whether their expression is actually regulated or the protein was simply not detected in the other sample for some other reason. Our requirement that they be present consistently in both mild IOP EEG, both high IOP EEG and 2:3 ONT animals supports their regulation. However, their inclusion or exclusion did not make much difference in the biological processes that were returned. It is interesting to note that in the high IOP EEG eyes, the most significantly up-regulated processes are those of cytoskeleton organization, largely due to the increased expression of several tubulin proteins. The implication of such a phenomenon, if validated by other biochemical and neuroanatomical measurements, could be broad, in that a change in protein maturation processes and cytoskeletal organization affects essentially all aspects of cellular function. Future spatial and temporal studies will demonstrate whether changes in these proteins precede detectable ONH and optic nerve damage. Finally, our western blots, as presented, are representative in nature being from individual animals, whereas the densitometry results are based on readings from all eyes in each group. They therefore support but do not directly validate our MS findings. It should also be noted that for two of the proteins analyzed, western blots were more sensitive at detecting the presence of proteins, and in the case of gamma-synuclein in the ONT eyes were able to detect down regulation when the protein was not detected by MS. In summary, the present study provides a quantitative characterization of the NHP retinal proteome at the onset of EEG and 3 weeks following ONT. While they should be considered preliminary due to the limited number of animals and modest number of extracted proteins, our results suggest condition-specific regulation of retinal processes in both treatment groups and a marked difference among the EEG subjects based on the magnitude of elevated IOP. We are currently characterizing proteomic changes in the orbital optic nerve, optic nerve head and scleral tissues from these same eyes. Future studies will seek to identify proteomic change at the earliest stage of damage and the cell population of origin for such changes, with the ultimate goal being to foster the development of novel therapeutic strategies that are specific to each tissue of the glaucomatous eyes. Commercial disclosure None. Acknowledgements The authors thank Dr. Hongli Yang for MATLab graph generation, Jonathan Grimm for software support, Juan Reynaud for software and hardware support, Dr. Brad Fortune for critical reading of and

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commenting on the manuscript, Drs. Stuart Gardiner and Shaban Demirel for help with statistical analyses, and Galen Williams for his dedicated assistance during surgery, imaging and sacrifice procedures. It is with sadness that we acknowledge our co-author Brian Arbogast’s death after his contribution to the mass spectrometry portion of this analysis. Appendix. Supplementary data Supplementary information associated with this article can be found in the online version, at doi:10.1016/j.exer.2011.03.020. References Agis, 2000. The Advanced Glaucoma Intervention Study (AGIS): 7. The relationship between control of intraocular pressure and visual field deterioration. The AGIS investigators. Am. J. Ophthalmol. 130, 429e440. Ahmed, F., Brown, K.M., Stephan, D.A., Morrison, J.C., Johnson, E.C., Tomarev, S.I., 2004. Microarray analysis of changes in mRNA levels in the rat retina after experimental elevation of intraocular pressure. Invest. Ophthalmol. Vis. Sci. 45, 1247e1258. 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