April 2006
Volume 47, Issue 4
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Retinal Cell Biology  |   April 2006
Activation of Signaling Pathways and Stress-Response Genes in an Experimental Model of Retinal Detachment
Author Affiliations
  • David N. Zacks
    From the Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan.
  • Ying Han
    From the Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan.
  • Yong Zeng
    From the Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan.
  • Anand Swaroop
    From the Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan.
Investigative Ophthalmology & Visual Science April 2006, Vol.47, 1691-1695. doi:10.1167/iovs.05-1209
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      David N. Zacks, Ying Han, Yong Zeng, Anand Swaroop; Activation of Signaling Pathways and Stress-Response Genes in an Experimental Model of Retinal Detachment. Invest. Ophthalmol. Vis. Sci. 2006;47(4):1691-1695. doi: 10.1167/iovs.05-1209.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Despite the high metabolic demands of the neural retina, its detachment from the retinal pigment epithelium does not lead to immediate death for most of the cells. This study was undertaken to test the hypothesis that intrinsic protective mechanisms are activated in the neural retina during early stages of retinal detachment.

methods. Retinal detachments were created in Brown Norway rats by injection of 1% hyaluronic acid into the subretinal space. Gene expression profiles of retinas detached for 24 hours were generated with a gene microarray (rat U34 GeneChips; Affymetrix, Santa Clara, CA) and compared to the profiles from control attached retinas in a robust multiarray protocol and false-discovery-rate analysis. Changes in individual, differentially expressed genes were validated by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Additional qRT-PCR and immunoblot analyses were performed for additional selected genes.

results. Genome-wide expression profiling revealed 27 genes that are differentially expressed in retinas detached for 24 hours. In silico analysis and functional clustering suggested that most genes belonged to three signaling pathways: interleukin-6/STAT, transforming growth factor-β/Smad, and aryl hydrocarbon receptor oxidative stress response. Additional analyses of selected genes from these pathways demonstrated a time-dependent increase in their expression in detached retinas.

conclusions. Retinal detachment results in the early activation of stress-response genes and specific signaling pathways. This adaptive response may enable the photoreceptor cells to survive the acute phase of a retinal detachment, and it is the breakdown of these protective mechanisms in chronic disease that leads to the ultimate death of the cell.

Retinal detachment is defined as the separation of the neurosensory retina from the underlying retinal pigment epithelium (RPE). It can result in severe vision loss, either directly from the death of photoreceptor cells or because of alterations in other retinal cell types (both neuronal and glial elements). 1 It is expected that these changes are the direct consequence of the altered microenvironment of the retina. Molecular pathways implicated in photoreceptor death include the intrinsic apoptosis pathway 2 3 and the FAS/FAS-ligand–mediated pathway. 4 However, the precise molecular mechanisms underlying the adaptive cellular response and the pathways leading to apoptotic cell death have not been clearly delineated. 
Despite the high metabolic demand of the outer retina, its detachment from the RPE does not induce a massive, synchronized, and/or immediate death of the photoreceptors. Studies of experimental detachment in mice using cell counts and markers of apoptosis (TUNEL staining) show that the rate of death is relatively slow, with a peak of approximately 5% to 10% of cells exhibiting TUNEL-positive staining by 3 days after detachment. 5 Though measurable decreases in outer nuclear layer thickness may occur in as little as 1 to 3 days after detachment, a significant number of photoreceptors survive for many days or even weeks. 3 6 7 Outcome analyses of macula-involving rhegmatogenous retinal detachments in humans have shown that similar visual acuity results are obtained if retinal reattachment is achieved anytime within 1 week after the detachment occurs. 8 9 Though these analyses are based on a retrospective review of reattachment surgery outcomes and are limited by the fact that they primarily measure visual acuity and do not reflect more subtle aspects of visual function, such as contrast sensitivity, they confirm that enough photoreceptors and other retinal elements survive the detached state to allow for the presence of a clinical “window of opportunity” for recovery of significant visual function after retinal reattachment. 
The asynchronous and delayed photoreceptor death in experimental retinal detachments, as well as the clinically recognized “window of opportunity” for reattachment of rhegmatogenous detachments, suggests the possibility that intrinsic protective mechanisms may be activated in neural retina. Such protective mechanisms may permit photoreceptor survival for a period after detachment. Photoreceptor apoptosis and other detachment-associated sequelae would result only when these intrinsic protective mechanisms are overcome by the chronic detachment. Relatively little is known about the types of protective, molecular pathways that become activated in the early, postdetachment time frame. The purpose of this study was to test the hypothesis that activation of intrinsic protective mechanisms occurs in early stages of retinal detachment. 
Methods
Experimental Retinal Detachment
All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines established by the University Committee on Care and Use of Animals of the University of Michigan. Retinal detachments were created in adult male Brown Norway rats (300–400 g), as previously described. 3 Briefly, rats were anesthetized with a 50:50 mix of ketamine (100 mg/mL) and xylazine (20 mg/mL), and pupils were dilated with topical phenylephrine (2.5%) and tropicamide (1%). A sclerotomy was created approximately 2 mm posterior to the limbus with a 20-gauge microvitreoretinal blade (Walcott Scientific, Marmora, NJ), with special caution taken to avoid damaging the lens. A Glaser subretinal injector (32-gauge tip; BD Ophthalmic Systems, Sarasota, FL) connected to a syringe filled with 10 mg/mL sodium hyaluronate (Healon; Pharmacia and Upjohn Co., Kalamazoo, MI) was introduced through the sclerotomy into the vitreous cavity. The tip of the subretinal injector was introduced into the subretinal space through a peripheral retinotomy, and the sodium hyaluronate was slowly injected. In all experiments, approximately one third to one half of the neurosensory retina was detached from the underlying retinal pigment epithelium. Detachments were made in the left eye, with the right eye serving as the control. For control eyes, a sham surgery was performed in which all procedures were completed except introduction of the subretinal injector and creation of the detachment. In experimental eyes, only the detached portion of the retina was harvested for analysis. All samples were harvested 24 hours after the detachment. 
Microarray and Statistical Analysis
Total RNA was extracted (TRIzol; Invitrogen, Carlsbad, CA) and purified (RNeasy Mini Kit; Qiagen, Valencia, CA). RNA quality was checked by gel electrophoresis. The first strand of cDNA was synthesized with 10 μg of total RNA, 1 μL of T7-(dT)24 primer (100 picomoles/μL; Invitrogen) and a reverse transcriptase (Superscript II RNase H Reverse Transcriptase kit; Invitrogen), as per the manufacturer’s instructions. Second-strand cDNA synthesis was performed by adding the single-strand cDNA solution DEPC-H2O (91 μL), 5× second-strand buffer (30 μL; Invitrogen), 10 mM dNTP mix (3 μL; Invitrogen), 10 U/μL Escherichia coli DNA ligase (1 μL; Invitrogen), 10 U/μL E. coli DNA polymerase I (4 μL; Invitrogen), and 2 U/μL E. Coli RNase H (1 μL; Invitrogen) and incubated at 16°C for 2 hours. T4 DNA polymerase (2 μL; Invitrogen) was then added and the solution incubated in a 16°C water bath for 5 minutes. The reaction was then stopped by adding 10 μL of 0.5 M EDTA (Ambion, Austin, TX). The double-stranded cDNA was purified with a pre-spin phase-lock gel (PLG) tube (VWR Scientific; Chicago, IL) run for 30 seconds at 12,000g. Phenol-chloroform-isoamyl alcohol (25:24:1; 162 μL; Invitrogen) was added to the cDNA, vortexed briefly, and rerun through the PLG tubes for 2 minutes at 12,000g. The aqueous phase was transferred to a fresh 1.5 mL tube, and diluted 2:1 with 7.5 M NH4OAc (Sigma-Aldrich, St. Louis, MO) and this mixture diluted 1:2.5 with absolute ethanol. This mixture was vortexed thoroughly and centrifuged at room temperature for 20 minutes at 12,000g. The pellet was washed twice with 0.5 mL of 80% ethanol. After it was air dried, the pellet was resuspended in RNase-free water and quantified by gel electrophoresis. 
Biotin-labeled complementary RNA (cRNA) was synthesized with an RNA transcript labeling kit (BioArray High Yield RNA kit; Enzo Life Sciences, Farmingdale, NY) according to the manufacturer’s instructions. Product was purified and quantified as indicated above. Fragmentation of the cRNA was performed with a kit (GeneChip Sample Cleanup Module Kit; Affymetrix, Inc., Santa Clara, CA). 
Fragmented cRNA was hybridized to the Rat U34 GeneChip (Affymetrix, Inc.), according to the manufacturer’s instructions. After hybridization, the chips were run through a fluidics station and scanned through a spectrophotometer (both from Affymetrix). 
We then subjected the microarray data to a robust multiarray analysis protocol 10 and false-discovery-rate analysis, 11 as previously described. 12 13 This allowed probe level background adjustments and quantile normalization and assigned log-transformed perfect-match values to each gene. Genes were then categorized as having significant changes in transcript abundance with a false-discovery rate set at less than or equal to 50% (i.e., the probability that an identified gene is actually differentially expressed is 50%). 
Quantitative Real-Time Polymerase Chain Reaction Analysis
Quantitative real-time polymerase chain reaction (qRT-PCR) was performed as previously described, 4 with rat hypoxanthine phosphoribosyl transferase (rHPRT) used for internal normalization. All primers were designed to span intron–exon boundaries. Samples lacking reverse transcriptase or cDNA template served as the negative control. For each primer set, qRT-PCR was performed on samples derived from three different animals and repeated three times per sample. The average change in expression (x-fold) relative to the rHPRT transcript level was calculated. 
Immunoblot Analysis
Western blot analyses were performed on detached retinas from experimental eyes and attached retinas from control eyes, as previously described. 4 Antibodies against the following proteins were used: STAT-1 (Axxora, LLC, San Diego, CA), STAT-3 (Affinity BioReagents, Golden, CO), CNTF (Abcam, Inc., Cambridge, MA), and TGFβ-1 (R&D Systems, Inc, Minneapolis, MN). Equal loading was verified by ponceau S staining and densitometry analysis of a nonspecific band present across all lanes. 
Results
To determine the cellular response to a 24-hour detachment, we generated gene expression profiles with the rat U34 GeneChip (Affymetrix, Inc.). Four independent samples, each containing two detached retinas, were compared to the attached retinas of the contralateral eyes. After normalization and statistical analysis, we identified 27 genes that exhibited a significant change in expression in the detached versus the attached retinas (Table 1) . An additional 10 expressed sequence tags (ESTs) were identified but not analyzed further with qRT-PCR. Of the 27 known genes, 23 showed higher and 4 showed lower expression in the detached retinas. For each of these genes, the modulation of transcription was confirmed by qRT-PCR (Table 1) . The direction of transcriptional alteration was confirmed for 25 (93%) of the 27 genes. 
In silico analysis revealed that 10 of the differentially expressed and validated genes are transcription factors. Of these, nine exhibited higher and only one (nerve growth factor induced gene B) lower expression in the detached retina. Other genes encoded proteins involved in receptor-mediated apoptosis, ion channels, transport or binding proteins, cytokine, and growth factor receptors and cofactors. Enhanced expression of genes involved in receptor-mediated apoptosis is consistent with our previous results showing a time-dependent increase in the transcript level of FAS-pathway intermediates. 4  
Functional clustering of the differentially expressed genes based on known association with specific stress-response signaling pathways suggests the increased transcription of genes belonging to three families of stress-response pathways. These are the interleukin-6/STAT pathway, transforming growth factor-β/Smad pathway, and aryl hydrocarbon receptor (Ahr) oxidative stress response pathway. We predicted that additional components of these stress-response pathways may be transcriptionally altered at later time points. To test this, we performed quantitative real-time PCR and immunoblot analysis on select components of these pathways in retinas detached for 3 and 7 days as described in the following sections (Table 2 , Fig. 1 ). 
Interleukin-6 Pathway
Among the genes detected on the microarray, three (11%) correspond to interleukin-6 (IL-6), signal transducer and activator of transcription-1 (STAT-1), and nuclear factor IL-6 (NFIL-6). IL-6 exerts its effect through the binding of α- and β-receptors. The latter is also known as gp130. 14 IL-6 first binds the IL-6α-receptor, and this complex binds to a homodimer consisting of two IL-6β-receptors, resulting in the intracellular activation of one or more of the five members of the STAT family of transcription factors. We performed qRT-PCR to detect the transcriptional activation of IL-6, IL-6α-receptor, IL-6β-receptor, and STAT1 through -5. Retinas were analyzed at 3 and 7 days after detachment. 
All the components of the IL-6 pathway tested showed increased expression during retinal detachment. The STAT-1 gene demonstrated the largest increase, with an over 80-fold change in transcript levels by 7 days after detachment. IL-6, itself, also had an increased expression, with an almost 20- and 30-fold increase at 3 and 7 days after detachment, respectively. Of the genes selected for immunoblot analysis, there was a corresponding time-dependent increase in the protein level. 
The growth factor CNTF (ciliary neurotrophic factor) belongs to the IL-6-like family of cytokines and uses a signal transduction pathway similar to that of IL-6, with activation of STAT transcription factors. 14 CNTF transcript levels were increased approximately eightfold in detached retinas. Western blot analysis showed that there was also an increase in the level of the CNTF protein. Similar to IL-6, CNTF also binds to an α-receptor, but this complex then binds to a heterodimer, composed of one gp130 subunit and one LIF (leukemia inhibitory factor) receptor subunit. 14 In our model, the LIF receptor was marginally upregulated by only approximately 1.6-fold in both 3- and 7-day detachments (data not shown). 
TGF-β Pathway
One of the genes that showed downregulation on the microarray was the latent TGF-β binding protein-3 (LTBP-3). LTBP-3 plays a role in sequestering extracellular TGF-β, thus preventing it from binding its receptor. A decrease in LTBP-3 would effectively result in an increased level of free TGF-β available for receptor binding. We sought to determine whether the decrease in LTBP-3 coincides with an actual increase in TGF-β itself and/or its downstream signal transducers. Transcript levels for TGF-β1 and TGF-β3 were elevated by up to 40- and 50-fold by day 7 after detachment, respectively; but TGF-β2 showed only modest transcriptional upregulation. Protein expression was tested and confirmed for TGF-β1. After TGF binds to its surface receptor, the intracellular cascade is propagated through the Smad family of transcription cofactors. 15 Within this set of genes, Smads3, -5, and -8 showed the highest levels of increased transcription, albeit only approximately 2.3 to 2.5 times that present in the attached retinas. 
Ahr Oxidative Stress-Response Pathway
The final pathway extracted from the microarray analysis for further confirmation was the Ahr–mediated response to oxidative stress. The Ahr was first identified as being essential in the mediation of cellular responses to dioxin exposure and has subsequently been shown to help in cellular adaptation to oxidative stress. 16 In a normal cell, the Ahr is localized to the cytoplasm. The release of oxidative reactive species can result in their binding to Ahr. This complex then translocates to the nucleus where the activated Ahr can perform its function as a transcription factor. The Ahr receptor, when activated, results in the transcription of a “gene battery” consisting of five genes, whose products are important in mediating the cellular response to the oxidative stress. These genes are cytochrome P-450, NAD(P)H:quinone-oxidoreductase, aldehyde dehydrogenase 3, UDP:glucuronosyl-transferase, and glutathione transferase. 
At the 24-hour time point, our microarray and qRT-PCR analyses showed a threefold upregulation of the Ahr in the detached retina. We predicted that increased transcription of the individual components of the Ahr “gene battery” would be detected in 3- and 7-day-old detachments, and this prediction was confirmed with qRT-PCR. For some of these genes, the transcript levels peaked at 3 days, but for some, the elevated levels persisted up to the 7-day time point. 
Discussion
Separation of the retina from its source of nutritional and metabolic support can exist in a variety of diseases such as retinal detachment, diabetic retinopathy, and macular degeneration. Despite the recent advances in the treatment of these diseases, the associated damage to the neurosensory retina can limit the final visual outcome. Understanding the molecular responses of the retina to detachment provides a logical entry point for the development of targeted therapeutics for photoreceptor survival and improving visual outcomes. In the present study, we demonstrated for the first time the activation of signaling pathways, particularly those that control transcription, as a primary mechanism by which the retina responds to the severe trauma of detachment. This study forms the basis for defining the specific molecular mechanisms by which the complex array of detachment-induced changes in retinal cell arrangement, structure, and survival are initiated. 
We have used gene profiling and qRT-PCR technology to develop and validate expression changes at an early stage (24 hours) of retinal detachment. Based on this transcriptional profile, we identified three stress-response pathways for further investigation. We demonstrated the time-dependent increase in transcript levels as well as increased protein translation for multiple components of these three prototypical pathways. These cytokine, growth factor, and stress response pathways all have a known precedent for their role in cellular response to injury. Their upregulation in our experimental model of retinal detachment suggests that they contribute to the intraretinal response to this particular form of injury. 
IL-6 is a cytokine that can play an important role in regulating cell death. 17 18 On binding to its receptor, IL-6 induces the activation of the STAT family of transcription factors. These transcription factors have been associated with photoreceptor differentiation 19 20 as well as retinal survival to ischemia–reperfusion injury. 21 CNTF, also upregulated in our system, has been shown to be neuroprotective of photoreceptors in experimental models of retinal degenerations. 20 CNTF uses the same receptor-mediated activation of the STAT transcription factors. Our results show the transcriptional and translational upregulation of the IL-6/STAT pathway as well as of CNTF, suggesting that elements within the retina itself produce the very factors that might allow for its own survival after detachment. This is consistent with the previous report of fibroblast growth factor receptor 1 and STAT3 activation in detached feline retinas. 22  
Similarly, TGF-β is upregulated in our experimental system. On the gene microarray analysis, we detected a downregulation of the protein latent TGF-β binding protein 3 (LTBP-3). LTBP-3 is an extracellular protein involved in the sequestration of TGF-β. The downregulation of the sequestering protein, coupled with the increased transcription and translation of TGF-β itself, suggests a resultant net increase in the extracellular concentration of this growth factor. It has been shown that TGF-β immunostaining is increased in Müller cells of detached retinas. 23 Our results suggest that this is due in part to an increased level of TGF-β transcription. TGF-β may have a beneficial effect on photoreceptor survival, 24 but it has also been shown to play a role in detachment-induced retinal pigment epithelial (RPE) cell dedifferentiation, 25 as well as to contribute to glial proliferation in the proliferative vitreoretinopathy that occurs after retinal detachment. 23 TGF-β-induced RPE cell dedifferentiation occurs through the Smad3 pathway intermediate. 25 In our system, Smad3, -5, and -8 exhibited the greatest amount of upregulation compared with other Smad genes, consistent with their possible involvement in postdetachment TGF-β-mediated signaling. TGF-β may also exert an antiapoptotic effect in detached retinas through the transcription factor PEBP-2, which is a known regulator of the Bcl-2 apoptosis gene. 26 PEBP-2 levels are known to increase in response to TGF-β, 27 and we detected and confirmed increased PEBP-2 transcript levels with our microarray analysis and qRT-PCR. 
The final pathway revealed by microarray and further expanded on with qRT-PCR is the Ahr pathway. The Ahr was first described as being important in cellular response to dioxin exposure. 16 On stimulation, the Ahr induces the transcription of a set of five genes, known as the aryl hydrocarbon gene battery. This gene battery codes for oxidative stress response proteins, including cytochrome P450, NAD(P)H:quinone oxidoreductase, aldehyde dehydrogenase 3, UDP glucuronosyltransferase, and glutathione transferase. The Ahr is known to respond to increased levels of retinoic acid and synthetic retinoids, suggesting that it may play a role in mitigating the oxidative stress retinal detachment may create due to alteration and interference in the normal metabolism of the retinal chromophore. We also validated an increase in the transcript levels of the gene encoding for retinol binding protein type 4. This is consistent with the hypothesis that the retina is responding to the altered chromophore metabolism. 
Of interest was that some of the aryl hydrocarbon gene battery members exhibited lower transcript levels at 7 days after detachment compared with 3 days after detachment. This corresponds to the marked increase in TGF-β transcript at day 7. The Ahr can act as a negative regulator of the TGF-β transcription. 28 29 The significance of this relationship in retinal detachment remains to be determined, but our data indicate a possible cross-talk between these upregulated pathways. 
The three stress-response pathways we describe in this work require further delineation in terms of their specific role in postdetachment photoreceptor survival. Elucidation of downstream targets for these transcription factors and signaling proteins will permit a better understanding of photoreceptor survival and retinal reorganization (including neurosensory, glial, and vascular components of the retina) which is necessary for the targeted development of therapeutic interventions and for preserving retinal structure to improve visual outcomes after retinal detachment. 
 
Table 1.
 
Results of Gene Microarray and qRT-PCR Analyses for Retinal Detachments of 24-Hour Duration
Table 1.
 
Results of Gene Microarray and qRT-PCR Analyses for Retinal Detachments of 24-Hour Duration
Gene Name Chip PCR
Transcription factors
 Aryl hydrocarbon receptor 3.04 3.65
 Polyomavirus enhancer binding protein (PEBP2β) 2.19 2.10
 Interferon regulatory factor 1 2.54 3.81
 Activating transcription factor 3 5.05 30.93
 JunB 3.38 5.85
 Signal transducer and activator of transcription 1 (STAT 1) 8.40 18.83
 NF-E2-related factor 2 2.57 5.32
 Nuclear factor interleukin 6 (NF-IL6) 2.53 10.50
 Nerve growth factor-induced gene (NGFI) 0.41 0.60
 Kruppel-associated box (KRAB)-zinc finger protein 2 2.32 1.82
Apoptosis intermediates
 Tumor necrosis factor α 3.59 12.75
 Caspase 3 3.15 4.68
Transporters/membrane channels
 Solute carrier family 11, member 2 3.30 2.63
 Solute carrier family 20, member 2 2.48 1.84
 Retinol-binding protein 4 2.82 2.46
Receptors/Cofactors
 Interleukin 6 signal transducer 2.75 2.70
 Latent TGFβ binding protein (LTBP)-3 0.30 1.01
 Integrin-associated protein 2.60 3.44
Membrane proteins
 MHC class I antigen 2.78 9.73
 MHC class Ib antigen 2.71 8.45
 RT1 class I gene 2.70 5.35
 Synuclein 2.43 1.78
 Drosophila disc-large tumor suppressor homologue (synapse-associated protein) 2.86 2.69
Intracellular enzymes
 Guanine nucleotide binding protein β-4 0.36 6.74
 Protein tyrosine phosphatase, non–receptor-type 1 2.64 4.98
Paracrine agents
 Endothelin-2 5.72 14.98
Structural proteins
 Stathmin-1 0.45 0.90
Table 2.
 
Increase in Transcript Level in the Detached Retina Compared with That in the Attached Retina for Components of Stress-Response Pathways
Table 2.
 
Increase in Transcript Level in the Detached Retina Compared with That in the Attached Retina for Components of Stress-Response Pathways
Component 3 Days 7 Days
Interleukin-6/STAT pathway
 Interleukin-6 22.6 35.9
 Interleukin-6α receptor 8.2 6.5
 Interleukin-6β receptor (gp130) 4.1 7.6
 CNTF 7.8 9.0
 STAT1 14.6 87.1
 STAT2 1.6 13.9
 STAT3 2.5 5.3
 STAT4 14.7 6.9
 STAT5 1.0 1.5
Transforming growth factor-β pathway
 Transforming growth factor-β1 4.0 38.7
 Transforming growth factor-β2 4.9 1.0
 Transforming growth factor-β3 6.7 53.2
 Smad1 1.8 1.3
 Smad2 1.5 1.3
 Smad3 2.3 2.6
 Smad4 1.3 1.4
 Smad5 1.4 2.3
 Smad7 1.3 1.4
 Smad8 1.8 2.3
Aryl hydrocarbon receptor-oxidative stress response genes
 Cytochrome P450 6.3 17.1
 NAD(P)H:quinone oxidoreductase 1 14.6 9.1
 Aldehyde dehydrogenase 3 18.6 6.2
 UDP-glucuronosyltransferase 11.4 3.4
 Glutathione transferase 5.5 6.2
Figure 1.
 
Western blot analysis for select genes with detachment-induced increases in transcript levels. For each gene, there was a time-dependent increase in transcript level that had an associated increase in protein level. Lane 1: 3-day attached retina; lane 2: 3-day detached retina; lane 3: 7-day attached retina; and lane 4: 7-day detached retina.
Figure 1.
 
Western blot analysis for select genes with detachment-induced increases in transcript levels. For each gene, there was a time-dependent increase in transcript level that had an associated increase in protein level. Lane 1: 3-day attached retina; lane 2: 3-day detached retina; lane 3: 7-day attached retina; and lane 4: 7-day detached retina.
FisherSK, LewisGP, LinbergKA, VerardoMR. Cellular remodeling in mammalian retina: results from studies of experimental retinal detachment. Prog Retin Eye Res. 2005;24:395–431. [CrossRef] [PubMed]
HisatomiT, SakamotoT, MurataT, et al. Relocalization of apoptosis inducing factor in photoreceptor apoptosis induced by retinal detachment in vivo. Am J Pathol. 2001;158:1271–1278. [CrossRef] [PubMed]
ZacksDN, HanninenV, PantchevaM, EzraE, GrosskreutzC, MillerJW. Caspase activation in an experimental model of retinal detachment. Invest Ophthalmol Vis Sci. 2003;44:1262–1267. [CrossRef] [PubMed]
ZacksDN, ZhengQD, HanY, BakhruR, MillerJW. FAS-mediated apoptosis and its relation to intrinsic pathway activation in an experimental model of retinal detachment. Invest Ophthalmol Vis Sci. 2004;45:4563–4569. [CrossRef] [PubMed]
YangL, BulaD, ArroyoJG, ChenDF. Preventing retinal detachment-associated photoreceptor cell loss in Bax-deficient mice. Invest Ophthalmol Vis Sci. 2004;45:648–654. [CrossRef] [PubMed]
EricksonPA, FisherSK, AndersonDH, SternWH, BorgulaGA. Retinal detachment in the cat: the outer nuclear and outer plexiform layers. Invest Ophthalmol Vis Sci. 1983;24:927–942. [PubMed]
LewisGP, TalagaKC, LinbergKA, AveryRL, FisherSK. The efficacy of delayed oxygen therapy in the treatment of experimental retinal detachment. Am J Ophthalmol. 2004;137:1085–1095. [CrossRef] [PubMed]
RossWH, KozyDW. Visual recovery in macula-off rhegmatogenous retinal detachments. Ophthalmology. 1998;105:2149–2153. [CrossRef] [PubMed]
HassanTS, SarrafizadehR, RubyAJ, BarretsonBR, KuczynskiB, WilliamsGA. The effect of duration of macular detachment on results after the scleral buckle repair of primary, macula-off retinal detachments. Ophthalmology. 2002;109:146–152. [CrossRef] [PubMed]
IrizarryRA, BolstadBM, CollinF, CopeLM, HobbsB, SpeedTP. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 2003;31:e15. [CrossRef] [PubMed]
BenjaminiY, HochbergY. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Statist Soc B. 1995;57:289–300.
YoshidaS, MearsAJ, FriedmanJS, et al. Expression profiling of the developing and mature Nrl−/− mouse retina: identification of retinal disease candidates and transcriptional regulatory targets of Nrl. Hum Mol Genet. 2004;13:1487–1503. [CrossRef] [PubMed]
ZareparsiS, HeroA, ZackDJ, WilliamsRW, SwaroopA. Seeing the unseen: microarray-based gene expression profiling in vision. Invest Ophthalmol Vis Sci. 2004;45:2457–2462. [CrossRef] [PubMed]
HeinrichPC, BehrmannI, HaanS, HermannsHM, Muller-NewenG, SchaperF. Principles of interleukin (IL)-6-type cytokine signaling and its regulation. Biochem J. 2003;374:1–20. [CrossRef] [PubMed]
ten DijkeP, HillCS. New insights into TGF-β-Smad signaling. Trends Biochem Sci. 2004;29:265–273. [CrossRef] [PubMed]
NebertDW, RoeAL, DieterMZ, SolisWA, YangY, DaltonTP. Role of the aromatic hydrocarbon receptor and [Ah] gene battery in the oxidative stress response, cell cycle control and apoptosis. Biochem Pharmacol. 2000;59:68–85.
PengYP, QiuYh, LuJH, WangJJ. Interleukin-6 protects cultured cerebellar granule neurons against glutamate-induced neurotoxicity. Neurosci Lett. 2005;374:192–196. [CrossRef] [PubMed]
BansalMB, KovalovichK, GuptaR, et al. Interleukin-6 protects hepatocytes from CCl(4)-mediated necrosis and apoptosis in mice by reducing MMP-2 expression. J Hepatol. 20005;42:548–556.
RheeKD, GoureauO, ChenS, YangXJ. Cytokine-induced activation of signal transducer and activator of transcription in photoreceptor precursors regulates rod differentiation in the developing mouse retina. J Neurosci. 2004;24:9779–9788. [CrossRef] [PubMed]
PetersonWM, WangQ, TzekovaR, WiegandSJ. Ciliary neurotrophic factor and stress stimuli activate the Jak-STAT pathway in retinal neurons and glia. J Neurosci. 2000;20:4081–4090. [PubMed]
TeruiK, EnosawaS, HagaS, et al. STAT3 confers resistance against hypoxia/reoxygenation-induced oxidative injury in hepatocytes through upregulation of Mn-SOD. J Hepatol. 2004;41:957–965. [CrossRef] [PubMed]
GellerSF, LewisGP, FisherSK. FGFR1, signaling, and AP-1 expression after retinal detachment: reactive Müller and RPE cells. Invest Ophthalmol Vis Sci. 2001;42:1363–1369. [PubMed]
GuerinCJ, HuL, ScicliG, ScicliAG. Transforming growth factor beta in experimentally detached retina and periretinal membranes. Exp Eye Res. 2001;73:753–764. [CrossRef] [PubMed]
LenziL, CoassinM, LambiaseA, BoniniS, AmendolaT, AloeL. Effect of exogenous administration of nerve growth factor in the retina of rats with inherited retinitis pigmentosa. Vision Res. 2005;45:1491–1500. [CrossRef] [PubMed]
SaikaS, Kono-SaikaS, TanakaT, et al. Smad3 is required for dedifferentiation of retinal pigment epithelium following retinal detachment in mice. Lab Invest. 2004;84:1245–1258. [CrossRef] [PubMed]
ChenG, ZengWZ, YuanPX, et al. The mood-stabilizing agents lithium and valproate robustly increase the levels of the neuroprotective protein bcl-2 in the CNS. J Neurochem. 1999;72:879–882. [CrossRef] [PubMed]
BaeSC, LeeKS, ZhangYW, ItoY. Intimate relationship between TGF-beta/BMP signaling and runt domain transcription factor, PEBP2/CBF. J Bone Joint Surg Am. 2001;83(suppl 1)S48–S55. [PubMed]
ZaherH, Fernandez-SalgueroPM, LetterioJ, et al. The involvement of Ahr in the activation of transforming growth factor-β and apoptosis. Mol Pharmacol. 1998;54:313–321. [PubMed]
GuoJ, SartorM, KaryalaS, et al. Expression of genes in the TGF-β signaling pathway is significantly deregulated in smooth muscle cells from aorta of aryl hydrocarbon receptor knockout mice. Toxicol Appl Pharmacol. 2004;194:79–89. [CrossRef] [PubMed]
Figure 1.
 
Western blot analysis for select genes with detachment-induced increases in transcript levels. For each gene, there was a time-dependent increase in transcript level that had an associated increase in protein level. Lane 1: 3-day attached retina; lane 2: 3-day detached retina; lane 3: 7-day attached retina; and lane 4: 7-day detached retina.
Figure 1.
 
Western blot analysis for select genes with detachment-induced increases in transcript levels. For each gene, there was a time-dependent increase in transcript level that had an associated increase in protein level. Lane 1: 3-day attached retina; lane 2: 3-day detached retina; lane 3: 7-day attached retina; and lane 4: 7-day detached retina.
Table 1.
 
Results of Gene Microarray and qRT-PCR Analyses for Retinal Detachments of 24-Hour Duration
Table 1.
 
Results of Gene Microarray and qRT-PCR Analyses for Retinal Detachments of 24-Hour Duration
Gene Name Chip PCR
Transcription factors
 Aryl hydrocarbon receptor 3.04 3.65
 Polyomavirus enhancer binding protein (PEBP2β) 2.19 2.10
 Interferon regulatory factor 1 2.54 3.81
 Activating transcription factor 3 5.05 30.93
 JunB 3.38 5.85
 Signal transducer and activator of transcription 1 (STAT 1) 8.40 18.83
 NF-E2-related factor 2 2.57 5.32
 Nuclear factor interleukin 6 (NF-IL6) 2.53 10.50
 Nerve growth factor-induced gene (NGFI) 0.41 0.60
 Kruppel-associated box (KRAB)-zinc finger protein 2 2.32 1.82
Apoptosis intermediates
 Tumor necrosis factor α 3.59 12.75
 Caspase 3 3.15 4.68
Transporters/membrane channels
 Solute carrier family 11, member 2 3.30 2.63
 Solute carrier family 20, member 2 2.48 1.84
 Retinol-binding protein 4 2.82 2.46
Receptors/Cofactors
 Interleukin 6 signal transducer 2.75 2.70
 Latent TGFβ binding protein (LTBP)-3 0.30 1.01
 Integrin-associated protein 2.60 3.44
Membrane proteins
 MHC class I antigen 2.78 9.73
 MHC class Ib antigen 2.71 8.45
 RT1 class I gene 2.70 5.35
 Synuclein 2.43 1.78
 Drosophila disc-large tumor suppressor homologue (synapse-associated protein) 2.86 2.69
Intracellular enzymes
 Guanine nucleotide binding protein β-4 0.36 6.74
 Protein tyrosine phosphatase, non–receptor-type 1 2.64 4.98
Paracrine agents
 Endothelin-2 5.72 14.98
Structural proteins
 Stathmin-1 0.45 0.90
Table 2.
 
Increase in Transcript Level in the Detached Retina Compared with That in the Attached Retina for Components of Stress-Response Pathways
Table 2.
 
Increase in Transcript Level in the Detached Retina Compared with That in the Attached Retina for Components of Stress-Response Pathways
Component 3 Days 7 Days
Interleukin-6/STAT pathway
 Interleukin-6 22.6 35.9
 Interleukin-6α receptor 8.2 6.5
 Interleukin-6β receptor (gp130) 4.1 7.6
 CNTF 7.8 9.0
 STAT1 14.6 87.1
 STAT2 1.6 13.9
 STAT3 2.5 5.3
 STAT4 14.7 6.9
 STAT5 1.0 1.5
Transforming growth factor-β pathway
 Transforming growth factor-β1 4.0 38.7
 Transforming growth factor-β2 4.9 1.0
 Transforming growth factor-β3 6.7 53.2
 Smad1 1.8 1.3
 Smad2 1.5 1.3
 Smad3 2.3 2.6
 Smad4 1.3 1.4
 Smad5 1.4 2.3
 Smad7 1.3 1.4
 Smad8 1.8 2.3
Aryl hydrocarbon receptor-oxidative stress response genes
 Cytochrome P450 6.3 17.1
 NAD(P)H:quinone oxidoreductase 1 14.6 9.1
 Aldehyde dehydrogenase 3 18.6 6.2
 UDP-glucuronosyltransferase 11.4 3.4
 Glutathione transferase 5.5 6.2
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