|Molecular Vision 2007;
Received 22 April 2007 | Accepted 30 August 2007 | Published 5 October 2007
Ischemic preconditioning alters the pattern of gene expression changes in response to full retinal ischemia
Frederike Dijk,1 Arthur A.B. Bergen1
1Department of Molecular Ophthalmogenetics, 2Department of Cellular Quality Control, Netherlands Institute for Neuroscience-an institute of the Royal Netherlands Academy of Arts and Sciences (KNAW), Amsterdam, The Netherlands
Correspondence to: W. Kamphuis, Department of Cellular Quality Control, Netherlands Institute of Neuroscience (NIN-KNAW), Meibergdreef 47, 1105 BA Amsterdam, The Netherlands; Phone: 31 20 5666101; FAX: 31 20 5665500; email: firstname.lastname@example.org
Purpose: Ischemic conditions in the retina have been implicated in several retinopathological conditions. Experimentally induced ischemia for 60 min followed by reperfusion leads to a loss of neurons in the inner retina. In contrast, a 5 min ischemic episode triggers a series of alterations that protect the retina against the damaging effects of a subsequent 60 min ischemic insult. This phenomenon is called ischemic preconditioning (IPC). To study the changes altered by IPC, we assessed the gene expression patterns in the rat retina after ischemia (60 min) followed by reperfusion (I/R) and compared these to the gene expression patterns after ischemia/reperfusion in preconditioned animals (IPC-I/R).
Methods: Changes in gene expression were studied, by means of microarrays, at 1, 2, 6, and 12 h after I/R in naíve and preconditioned animals. To identify functional pathways of interest, we used significantly regulated genes as input for gene ontology analysis. Microarray results were validated by real-time quantitative PCR.
Results: Most genes that were altered by I/R showed a comparable change in both naíve and preconditioned animals. Differential expression was found for a total of 1312 genes of the 20,280 features (6.4%) present on the array with a differential change of 1.7 fold or more. The list of genes with a differential change was characterized by a statistically significant overrepresentation of genes associated to the gene ontology terms tRNA aminoacylation (with a decreased expression due to preconditioning), immune response (with most genes upregulated), and apoptosis (mixed direction of changes). The results of quantitative PCR assays were in agreement with the microarray data.
Conclusions: The response of several functional groups of genes on ischemia was altered by a preconditioning stimulus. Most prominent differences were found for the group of genes encoding for aminoacyl-tRNA synthetases (ARSs), which is in line with the previously observed decreased expression of ARSs after induction of preconditioning. Our observations indicate that activation of translational activity may be a mediator of ischemia-associated damage in the retina, and IPC may prevent activation of this mechanism. An altered expression of genes implicated in immune response and in apoptosis may also be involved in effectuating IPC.
Retinal ischemia for a duration of 60 min leads to a selective loss of amacrine and ganglion cells via apoptosis [1-3]. Ischemia is one of the factors implicated in glaucoma-associated cell loss in the inner retina. Laser Doppler flow cytometry showed that optic nerve blood flow is already decreased in glaucoma suspects before visual field defects become manifest, suggesting an involvement of ischemic conditions in the early stages of glaucoma . Glaucomatous visual field defects have been reported to occur prior to structural changes of the optic nerve head or nerve fiber layer, and these typical structural aberrations may be a consequence of the loss of nerve fibers caused by retinal ischemia [5-9]. The loss of retinal ganglion cells in the glaucomatous retina corresponds to the pattern of neuronal degeneration, by apoptosis, after experimental ischemia [1,3]. However, a 5 min episode of retinal ischemia, does not inflict damage but rather triggers an endogenous form of neuroprotection, termed ischemic preconditioning (IPC) or tolerance. The retina of preconditioned animals is fully protected against the morphological and functional damage resulting from a full ischemic insult [10,11]. Insight in the underlying mechanisms of IPC may lead to a better understanding of how retinal cells deal with stressful conditions, mechanisms that may falter in retinopathological conditions. IPC induced protection is only transient with maximum protection achieved 24-72 h after the preconditioning stimulus. Although IPC has also been observed in brain, spinal cord, and other tissues, the protective effect of IPC in the retina is particularly robust because it offers full protection against functional impairment and cell loss while in other tissues cell loss is only partially prevented [12-15]. The involvement of Hif1 transcription factor [13,16], Hsp27 (Hspb1) upregulation , stimulation of adenosine A1 and A2α receptors [17,18], protein kinase C activation , leukocyte rolling inhibition , inducible nitric oxide synthase , and the opening of mitochondrial potassium-dependent ATP channels  have all been proposed as underlying mechanisms of preconditioning (for reviews see [10,22]).
In a previous study, we used a microarray approach to describe the alterations in retinal gene expression as a result of the induction of IPC . Changes were first dominated by altered expression levels of genes encoding transcription factors. At stages coinciding with the time window of effective protection, transcript levels of aminoacyl-tRNA synthetases (ARSs) genes and of genes involved in metabolism and transport of amino acids were decreased. These observations indicate that a reduced translational activity may contribute to the preconditioning-induced neuroprotection. In the present study, we addressed the question of how preconditioning alters the response to a full ischemic insult. By comparing the effects of a full ischemic insult on the gene expression pattern between naíve and preconditioned animals, we were able to identify genes modulated by the preconditioning stimulus. This approach may reveal molecular pathways that play a pivotal role in the induction/prevention of retinal damage. In line with our previous findings after IPC, additional evidence was found for a role of ARSs.
Animals and anesthetics
Animal handling and experimental procedures were reviewed and approved by the ethical committee for animal care and use of the Royal Netherlands Academy for Sciences, acting in accordance with the European Community Council directive of 24 November 1986 (86/609/EEC) and the ARVO statement for the use of animals in ophthalmic and vision research. The procedure to induce transient ischemia followed by reperfusion (ischemia/reperfusion: I/R) has been described in detail previously [24-27]. In short, adult male Wistar rats (Harlan Laboratories, Horst, the Netherlands) weighing 200-300 g were anesthetized by an intramuscular injection of hypnorm (fentanylcitrate and fluanisone; 0.5 ml/kg body weight; Janssen Pharmaceuticals, Beerse, Belgium) in combination with an intraperitoneal injection of valium (diazepam; 0.5 ml/kg body weight; Hoffman-La Roche, Basel, Switzerland). Neither hypnorm nor valium is known to have neuroprotective properties. A local anesthetic, Oxybuprocain (benzalkoniumchloride; 0.4% w/v; Smith and Nephew, Hoofddorp, the Netherlands) was applied to both eyes and mounted onto a stereotactic frame (Stoelting, Wood Dale, IL). A steel 30-gauge infusion needle (BD Biosciences, Alphen aan den Rijn, The Netherlands) connected to a saline reservoir was placed in the middle of the anterior chamber of the left eye. The other eye served as control and was either not-operated on or was sham-operated on by inserting a needle into the anterior chamber without elevating intraocular pressure. The reservoir was opened and lifted to 1.70 m. After 60 min of ischemia, the reservoir was lowered and reperfusion resumed immediately.
The first group of animals (I/R), was subjected to 60 min ischemia followed by reperfusion. The retinas were studied after the following reperfusion times: 1 h (n=5), 2 h (n=5), 6 h (n=4), and 12 h (n=4). The second group (IPC-I/R) was preconditioned by 5 min of ischemia followed by a reperfusion interval of 24 h, and then allowed to undergo a second period of ischemia lasting 60 min. Retinas were studied after the following reperfusion times: 1 h (n=7), 2 h (n=6), 6 h (n=6), and 12 h (n=7). This protocol leads to full protection against the deleterious effects of 60 min ischemia has been shown previously by histology . Animals were killed by the administration of an intraperitoneal overdose of sodium pentobarbital (0.8 ml; 60 mg/ml, Ceva Santa Animale, Maassluis, The Netherlands).
The isolation of total RNA from the retina has been described in detail [23,28]. In short, the whole retina free of vitreous was isolated, frozen on dry ice, and stored at -80 °C. Frozen retinas were thawed in Trizol (Invitrogen, Breda, The Netherlands) and homogenized immediately. Total RNA was isolated following the manufacturer's instructions. Precipitated RNA was dissolved in 8 μl RNase-free water. The RNA yield was around 10 μg/retina (ND-1000 spectrophotometer; NanoDrop Technologies, Wilmington, DE) and quality checks showed sharp ribosomal RNA bands with minimal degradation (2100 Bioanalyser, Agilent Technologies, Amstelveen, The Netherlands).
Experimental set-up and probe generation
Of each animal, 1 μg total RNA from each sample was used to make pooled samples for each experimental group (n=4-7/group) and one pooled sample for all contralateral control retinas (n=44) including 34 unoperated eyes and 10 sham-operated eyes. To test the validity of pooling the non-operated and sham-operated samples for microarray analysis, we determined by qPCR the transcript levels of reference genes and a selection of stress-induced genes (c-fos, c-jun, Il6, Hmox1, and Gfap). No differences were found between the unoperated and sham-operated retinas, which is in line with our previous findings [27,28]. Moreover, the statistical analysis of the complete quantitative PCR (qPCR) dataset for 41 different genes revealed no differences between the unoperated and sham-operated retinas.
Details of the amino allyl-UTP aRNA probe synthesis were described previously (Amino Allyl MessageAmp aRNA kit, Ambion, Nieuwerkerk a/d lJssel, The Netherlands) [23,29]. In short, or first strand cDNA synthesis, 1.5 μg RNA of the pooled sample was primed with a T7 Oligo(dT) primer. Following second strand cDNA synthesis, in vitro transcription generated amino allyl-UTP labeled aRNA. Electropherograms of the amplified aRNA showed a symmetrical length distribution with a peak around 1.5 kb with a maximum length of 5.5 kb and no differences in size distribution between the different samples. The aRNA was coupled to Cy3 or Cy5 monoreactive dyes (Amersham) and purified from excess dye over a Chromaspin-30 column (Clontech). Incorporation efficiency and yield were determined on a spectrophotometer (NanoDrop Technologies). The aRNa was labeled with Cy3 or Cy5 monoreactive dyes (Amersham, Eindhoven, The Netherlands) and purified. A common reference design was used for microarray hybridization. A 30 μg sample of aRNA of the control-group was labeled with Cy3. A 1 μg aliquot served as the identical common reference present on all arrays and was hybridized simultaneously with 1 μg Cy5-labeled aRNA from one of the experimental groups.
Oligonucleotide arrays were obtained from Agilent (22K catalog rat array, 60-mer oligonucleotides, product number G4130A). The complete design can be found at Agilent or GEO-NCBI. These arrays are designed for the simultaneous hybridization of two aRNA samples labeled with Cy5 (treatment-group) and Cy3 (control). After normalization, the dye ratio represented the transcript ratio. The details of this technique have been described before [23,30], and can be found at Agilent.
The array images were acquired using an Agilent microarray scanner set at 5 μm resolution. Images were loaded into Feature Extraction software (v8.5; Agilent) and combined with the latest array information (design-file) with default settings for all parameters including normalization. For details see Feature Extraction® Software User Manual v8, at Agilent. This results in the signal strength of the treatment (Cy5) and control (Cy3) channels, the relationship between the two channels in terms of log ratio, the associated p-value, and information concerning various quality control fields. Images and MAGE files were uploaded to Rosetta Biosoftware Resolver system, v5.0. Detailed information on the Rosetta ResolverTM system can be found at Rosetta Biosoftware (Rosetta Biosoftware, Seattle, WA), and in several published papers [31,32].
Our study design involved pooled-samples for the microarrays and must be considered as a fast screening tool to select features of interest, followed by gene ontology (GO) analysis and a series of confirmatory qPCRs on the individual animals. As previously described in detail , criteria to select features-of-interest were based on our extensive qPCR experience with the retinal ischemia model. Power analysis of our qPCR data shows that a >1.7 fold change in expression level is statistically significant (p<0.05; power 0.80) [27,28]. To eliminate features with a low signal intensity, we added a technical log ratio error of p<0.01, assigned by Feature Extraction® software, as second criterion. A set of 19 features were excluded because of dye bias. Furthermore, 56 features were excluded for reasons of signal saturation, and 25 features were excluded because examination by qPCR showed for these genes highly variable transcript levels caused by a transfer of small amounts of lens epithelium during retina isolation . Previous work has demonstrated the validity of these selection criteria .
The annotation of the genes on the array was improved using web-based annotation tools DAVID-2007 (National Institute of Allergy and Infectious Diseases)  and Source (Stanford University, Stanford, CA). Of the 20,280 features, 92% could be linked to a UniGene cluster ID; representing 14,127 unique UniGenes. GO analysis was used to identify overrepresented or depleted functional groups of genes and was performed with GOstat  and DAVID-2007 . Lists of selected genes-of-interest were compared with the total set of genes present on the array. Fisher's exact test was performed to rank GO terms in GOstat. The enrichment calculation performed in DAVID, called EASE score, is a conservative adjustment to the Fisher exact probability that weighs significance in favor of themes supported by more genes [33,35,36]. The derived p-values are a score system to organize the view, and facilitate exploration rather than a strict decision making line. A p<0.01 was considered to be of potential interest.
Real time quantitative polymerase chain reaction
For qPCR, 2 μg total RNA from each individual retina were dissolved in 4 μl H2O, (DNase I treated, 0.5 unit, amplification grade, Invitrogen). The RNA sample was reverse transcribed into first strand cDNA with 100 U Superscript III Reverse Transcriptase (Invitrogen) and 50 ng random hexamer primers. The resulting cDNA sample served as a template for SYBR green RT-qPCR analysis (ABI 7300 Real-time system; Applied Biosystems, Nieuwerkerk a/d lJssel, The Netherlands) using SYBR® Green PCR Master Mix (Applied Biosystems). The amplification efficiencies (E) had values close to 2 for all primer combinations. The resulting Ct values were converted to absolute amounts of cDNA present in the sample (E-Ct) . To correct for differences in cDNA load between the different samples, we normalized the target PCR to a set of reference PCRs. For this, qPCR data for a selection of candidate reference genes on experimental and control samples were used as input for a geNorm analysis [28,37]. Rho (rhodopsin), Hprt (hypoxanthine phosphoribosyl transferase), Gapdh (glyceraldehyde-3-phosphate dehydrogenase), Pde6b (phosphodiesterase 6B), Grm6 (glutamate receptor, metabotropic 6), and Prkcα (protein Kinase C, α) were most stable and were used for normalization. A series of qPCR assays on each of the individual cDNA samples were performed to confirm previously described changes in transcript levels [27,28]. Sequences of the used primers are available on request.
Identification of genes-of-interest
The number of selected features of interest for each experimental condition, based on the change relative to the common reference sample, composed of the unoperated and sham-operated retinas, is presented in Table 1. The complete list of genes corresponding to the features-of-interest, fold-change, assigned error, p-values, and UniGene IDs for each of the time points is presented in Appendix 1. Comparison of the changes after I/R in naíve animals with those in preconditioned animals (IPC-I/R) showed considerable overlap. For example, expression of c-fos was increased 29 fold 1 h after I/R and and 33 fold 1 h after IPC-I/R. The effect of the preconditioning stimulus on the c-fos transcript levels is, therefore, only a relative 1.1 fold effect. Of the 254 signatures 1 h after I/R, 38% showed a significant change in the same direction after IPC-I/R. This fraction increased with time: 52%, 62%, and 77%, in the 2, 6, and 12 h I/R groups, respectively. Agglomerative clustering confirmed the similar response to ischemia in naíve and conditioned retinas (data not shown). GO term analysis identified under both conditions the involvement of transcription regulation (1, 2 h), cell death (1, 2, 6, 12 h), immune response (6, 12 h), vascular development (6, 12 h), and cell motility (6, 12 h), as main categories.
We reasoned that genes with an identical response to ischemia in naíve and preconditioned animals were unlikely to be critically modulated by IPC and thus did not play a pivotal role. It was therefore decided to focus on those features with a differential change in response to IPC-I/R compared to the response after I/R only. The use of an identical common reference sample in the Cy3 channel of all arrays enabled the generation of virtual ratios from the original real ratio experiments, thus allowing the accumulation of information from all arrays in this series. In this way, gene transcripts that showed a differential response after IPC-I/R compared to I/R were selected. The number of features-of-interest identified after the re-ratio step for each reperfusion time is given in Table 1 and the complete list is presented in Appendix 1. The compiled list of all reperfusion times includes 1506 different features, reflecting 1282 unique UniGene cluster IDs. 246 UniGene IDs were altered at two time points, 109 at three, and 55 at all four reperfusion times. For further analysis; (i) for each time point, the list of features-of-interest was analyzed by GO term analysis, and (ii) a list was compiled of features with >4.0 fold change in combination with an assigned technical error of p<0.01. The latter analysis resulted in 35 upregulated and 59 downregulated features and the list is presented in Appendix 2. These genes were functionally annotated, and PubMed was surveyed for evidence supporting a possible role in cell survival as well as cell loss .
Gene ontology term analysis of differentially regulated genes after ischemia reperfusion between conditioned and naíve animals
For each time point, the list of features-of-interest (converted to UniGene cluster IDs) were used as input for GO term tools to identify functions/processes of interest [33,34]. The Functional Annotation Clustering tool of DAVID was used to gain insight into what biological processes were overrepresented. The results are summarized in Table 2. An extensive version can be found in Appendix 2. The clusters tRNA aminoacylation, immune response, and apoptosis were identified at three or four of the studied time points.
Genes involved in tRNA aminoacylation (ARSs) were overrepresented at all four reperfusion intervals: Cars, Farslb, Iars, Kars, Lars, Nars, Rarsl, Sars2, and Tars. Compared to controls, these genes were found to be upregulated after ischemia and decreased after ischemia in preconditioned animals. The average decrease after IPC-I/R relative to I/R was as follows: -55±5% at 1 h, -52±2% at 2 h, -40±3% at 6 h, and -45±2% at 12 h. On the array, specific probes were present for 12 other ARSs genes. Although the alterations for these genes did not meet the selection criteria, nearly all showed a similar trend of a relatively decreased expression of IPC-I/R versus I/R (Yars, Gars, Wars, Sars1, Qrsl1, Quars, Varsl2l, Hars2, and Rars). For Vars2, Farsla, and Dars no such trend was found. In summary, 18 out of the 21 different ARSs genes studied by microarray showed decreased transcript levels after IPC-I/R relative to I/R. The average decrease was -36±5% at 1 h, -35±5% at 2 h, -26±4% at 6 h, and -29±4% at 12 h (mean±SEM; n=18).
Transcript levels of Cars, Iars, Lars, Nars, Tars, and Yars were also assessed by qPCR. The results are presented in Table 3. After I/R, levels gradually increased on average by 7% at 1 h, by 15% at 2 h, by 82% (p<0.031; Wilcoxon Matched-Pairs Signed-Ranks test; n=6) at 6 h, and by 109% (p<0.031) at 12 h. After IPC-I/R, levels were first increased by 34% (p<0.031) at 1 h, but thereafter, no significant changes were found. Presenting the qPCR results as re-ratio values (IPC-I/R relative to I/R): +29±12% (p<0.05) at 1 h, -26±15% at 2 h (p<0.04), -18±7% at 6 h (p<0.03), and -37±3% at 12 h (p<0.01).
The GO term amino acid and derivative metabolism included, in addition to ARS genes, 13 genes that paralleled the changes described for the ARSs (see Appendix 2). One of these genes was Slc7a1 (solute carrier family 7 member A1), a cationic amino acid transporter. After induction of IPC, changes were found in the expression of amino acid transporter genes Slc7a1, Slc3a2, Slc6a6, and Slc38a2 . In the present study these four genes showed decreased transcript levels after IPC-I/R relative to I/R at all time points of around -30%. Two genes with a function in tRNA metabolism, Rg9mtd1 and Trnt1, were also relatively decreased at all time points.
An overrepresentation of genes engaged in the response of the immune system was disclosed at all stages but most pronounced at 6 and 12 h (Table 2 and Appendix 2). Genes implicated in immune response were overrepresented at 6 h (16 genes) and at 12 h (35 genes). In total, 44 genes were linked to immune response of which the majority (n=36) was upregulated after IPC-I/R relative to I/R at one or more time points. The average increase was +64±18% at 1 h, +109±22% at 2 h, +78±9% at 6 h, and +119±26% at 12 h (mean±SEM; n=36). The average decrease observed for the other eight genes was between -20% and -42%. From the list of features with the highest fold-change, two more genes with a link to immune response were identified, both with increased expression: Serpina3n (associated with inflammation) and Lcp1 (lymphocyte cytosolic protein 1).
A group of 40 different genes related to apoptosis or programmed cell death were differentially expressed at 2, 6, and 12 h (Table 2 and Appendix 2). A subgroup of 12 genes was linked to the GO term anti-apoptosis. Of the 40 genes, 25 were upregulated and 15 were downregulated. From the list of features with the highest ranking fold-change three more genes with a link to apoptosis were identified: Nupr1 (nuclear protein 1; up to 6.5 fold increase), Muc4 (mucin 4; up to 4.8 fold increase), and Ppp3ca (protein phosphatase 3, catalytic subunit, alpha isoform; up to 4.3 fold decrease).
Qualitative polymerase chain reaction validation
To validate the outcome of our array analysis, we performed qPCR assays on cDNAs of the individual retinas underlying the pooled samples used for microarray analysis. For a series of transcripts with a previously characterized alteration after I/R [27,28] together with a selection of genes from Table 2, the mean qPCR-derived change with respect to the controls was calculated as log10 experimental/control. These values were plotted against the corresponding log10 values derived from the microarrays. For the eight different experimental groups, the correlation coefficient R2 ranged between 0.74 and 0.83. For all data points combined (n=291), R2=0.75. The result of the linear regression is presented in Figure 1 and the outcome of the qPCR assays is presented in Appendix 3. Of the 291 data points studied, 95 represented features-of-interest selected on the microarray results. Of these 95 data points, 72 showed a statistically significant change in the same direction based qPCR (76%; p<0.05; student's t-test). Of the 291 data points that were studied by both techniques, 57 data points had a statistically significant alteration based on qPCR but were not selected according to the microarray selection criteria (β=0.19). These results are in line with the outcome of our previous studies showing a good correspondence between microarray and qPCR results [23,29].
Retinal ischemic preconditioning offers protection against a subsequent, otherwise damaging, ischemic insult [10,11,17,19,22,38,39]. Although IPC is also observed in brain, spinal cord, and other tissues [12-15], the protective effect of IPC is particularly robust in the retina, offering full protection against functional impairment and cell loss, while cell loss is only partially prevented in other tissues. We conducted a series of microarray experiments to acquire an inventory of the changes in gene expression profile, intending to find new leads for the molecular pathways underlying the preconditioned state, previously reported , and to identify pathways that were modulated by preconditioning when a retina was subjected to a full ischemic insult, reported here. We hypothesized that differentially expressed genes were relevant for the effectuation of IPC-induced neuroprotection. In both unconditioned and conditioned retinas, ischemia led to changes in an increasing number of genes involving 1-2% of the features on the array at 1 h, up to 6-10% at 12 h. In contrast, the number of differentially expressed genes showed an opposite development with the highest number of genes at 1 and 2 h. Surprisingly, the extensive pattern of changes at 6 and 12 h was similar between naíve and preconditioned retinas. This showed that many changes in gene expression had no specific relationship to the occurrence of cell loss that starts to build up after 4 h of reperfusion and peaks around 6 to 12 h after ischemia . However, the altered gene expression following ischemia in preconditioned retinas suggested that some, yet to be recognized, changes may occur despite the prevention of cell loss. For instance, GO term analysis showed a significant overrepresentation of genes implicated in vascular development or angiogenesis 6 and 12 h after I/R as well as IPC-I/R (e.g. Tnfa, Icam1, Il1b, and Cox2), suggesting that neovascularization may be promoted, a possible effect not addressed by morphological studies thus far [40-42].
We hypothesized that the group of differentially expressed genes were important for the protective effects induced by IPC. GO term analysis revealed an overrepresentation of genes involved in tRNA aminoacylation, immune response, and programmed cell death. GO term based selection and clustering of genes presumed that changes in a biological function were associated with concerted changes in transcript levels of the associated genes. The disadvantage of this approach was that genes encoding for key regulatory factors, annotated in functional context with only a limited number of other genes, did not emerge in our analysis as pathways of potential interest. For example, the observed relative decrease of Azin1 (ornithine decarboxylase antizyme inhibitor) at all time points by 1.9-4.5 fold may be of interest as Azin1 prevents the degradation of ornithine decarboxylase; its decrease may lead to a decreased production of harmful polyamines . Yet Azin1 was not identified within the GO term analysis.
The expression of ARS encoding genes was upregulated after ischemia but decreased after ischemia in preconditioned animals. At all reperfusion times a differential change was found for 18 of the 21 different ARS transcripts studied with a relative decrease of 25-35% of IPC-I/R compared to I/R. Examining the expression of several ARSs by qPCR revealed a significant increase after I/R compared to control levels, which is in line with the microarray findings. However, after IPC-I/R, the levels were not significantly different from control whereas the microarray showed decreased expression. Nevertheless, when comparing the qPCR determined ARS expression levels after IPC-I/R directly to those after I/R, a decrease of 20-35% is evident. This decrease is statistically significant at 2, 6, and 12 h reperfusion.
Previously, we showed that ARS mRNA levels decrease by 30-50% at 24-48 h after IPC induction . Combined with the results of the present study we conclude that in naíve animals I/R leads to increased ARS gene expression. Preconditioned retinas have lower ARS mRNA levels when subjected to I/R, levels remain decreased or only increase to levels comparable to those found in controls. The inference of these findings is that protein synthesis is necessary for the effectuation of ischemia-induced neurodegeneration and that IPC may avoid this [44-46]. Several studies have shown that the application of cycloheximide, a protein synthesis inhibitor, during the ischemic event or during subsequent reperfusion results in reduced apoptosis in brain [47-51]. Our findings suggest that suppression of protein synthesis by decreased ARSs levels may be an effective mechanism to prevent apoptosis-mediated cell loss .
Proliferation of resident immunocompetent microglia in the retina and infiltration of leukocytes from the bloodstream are triggered by injury or inflammation and may lead to proapoptotic events [53,54]. One day after ischemia the number of cells expressing microglia/macrophage-specific markers was increased . In line with their findings, we found no clear upregulation of these markers (OX42, ED1, and OX6) at 1, 2, 6, or 12 h post-ischemia. This shows that proliferation of microglia takes place after 12 h reperfusion. We were not able to conclude whether this process of proliferation was modulated by IPC.
The expression of several chemokines was shown to peak at 6 h reperfusion, which may be involved in the attraction and infiltration of leukocytes into the inner retina after ischemia at 12 h reperfusion . Our microarray results confirmed increased mRNA levels at 2 h and thereafter of Ccl2 (monocyte chemoattractant protein-1), Ccl3 (macrophage inflammatory protein-1a), and Cxcl10 (interferon inducible protein-10). The finding of reduced adhesion of leukocytes after IPC suggested that inhibition of immune cell infiltration could contribute to the prevention of leukocyte-mediated damage [19,57,58]. However, our microarray and qPCR results revealed that markers for immune cell activation are upregulated after I/R and after IPC-I/R, with similar fold-change and similar time course. In addition to the aforementioned chemokines, increased expression was found for Ccl5, Ccl12, Ccl20, and Cxcl12. Other immune cell components that increased were: Vegfa ; Icam1, an inflammatory marker that can be induced by VEGF [40,60]; Itgb2 (CD18), which together with Icam1 is essential for leukocyte adhesion in the retina ; Cox2 (Ptgs2), a proinflammatory mediator [40,61]; Ccl7, monocyte chemotactic protein 3 precursor ; Tyrobp, killer cell activating receptor associated protein, and Dap-12 . These results show that ischemia leads to an activation of the immune system components involved in the attraction and adhesion of leucocytes in both naíve and in preconditioned animals. However, the GO term immune response was still overrepresented at all time points in the group of differentially expressed genes, showing that the immune response to ischemia was modulated by prior IPC. Most differentially expressed genes were found to be upregulated in the preconditioned retinas compared to the naíve retinas. C1qb, C1s, C3, C4a, C5r1, and Serping are implicated in complement activation along classical and alternative pathways. Cxcl2, Egr1, Il1b, Il6, and Tlr2 are associated with pro-inflammatory roles. Sele plays a role in leukocyte adhesion, and Pttprc is a leukocyte surface glycoprotein. S100a8 is an abundant protein with pro-apoptotic activity in the cytosolic fraction of neutrophils and its release is a potential biomarker for inflammation [64,65]. S100a8 was upregulated under both conditions but stronger after IPC. Only a few genes within this cluster may be related to a potential neuroprotective role effectuated by a diminished immune response. Expression of Tnfrsf1a was increased at all four time points. Released Tnfrsf1a reduced the proapoptotic effect of TNFα . The observed downregulation of Tnfrsf6 prevented activation of the apoptosis effector caspase-8 . The twofold upregulation at 1, 2, and 6 h of Serping1, a serine protease inhibitor also known as C1 inhibiting factor, suppressed the activity of C1s. Transcript levels of another serine protease inhibitor, Serpina3n were upregulated by two-seven fold. This inhibitor may have a role in preventing degenerative proteolysis, which is induced by inflammatory stimuli .
In summary, although an activation of the immune response takes place after ischemia regardless of preceding IPC, the pattern is significantly different. Whether these differences underlie the reduced adhesion of leukocytes after IPC thus preventing infiltration and subsequent leukocyte-mediated damage, remains to be investigated [19,57,58].
A cluster of 40 genes associated with programmed cell death were identified as differentially expressed. Functional annotation revealed several genes that were modulated as a result of IPC and may be involved in the inhibition of apoptosis-mediated cell death after ischemia. To our knowledge, at least the changes observed for the following 10 genes were in line with a suppressed apoptosis. The upregulation of Anxa1 (inhibitor of phospholipase A2 activity, expressed by neutrophils and monocytes and protective in myocardial I/R ), Bdnf (a cell survival promoter), Igf1 (attenuates caspase activation and has neuroprotective effects in brain ), and Muc4 (a repressor of apoptosis ) are all linked to anti-apoptotic effects. The downregulation Bok1 (a pro-apoptotic Bcl-2 family member ), Dedd (directs procaspases to targets ), Cidec (apoptosis inducing activity ) and of Tnfrsf6 (prevents activation of the apoptosis effector caspase-8) are also in line with a supression of apoptosis by IPC. Also, noteworthy was the 1.7 to 4.3 fold decreased expression of Ppp3ca, the catalytic subunit of calcineurin. Increased intraocular pressure leads to the initiation of a calcineurin-mediated mitochondrial apoptotic retinal ganglion cell death pathway in glaucoma and blockers of calcineurin are neuroprotective . However, not all changes were in line with suppressed apoptosis. The decreased expression of Birc1b, Perp, Phlda1, and Ripk3 might have proapoptotic consequences.
In conclusion, our microarray study on the changes in gene expression profile after preconditioning and after ischemia identified many interesting new leads for genes involved in pathways leading to either neurodegeneration or neuroprotection. One of the more intriguing findings is the pattern of changes in ARS encoding genes. The tentative neuroprotective effect of limiting translation in retinal cell survival and cell loss merits further research on the therapeutic potential in the prevention of retinal degeneration in glaucoma, diabetes, and central retinal artery occlusion.
This work was supported by grants from: Rotterdamse Vereniging Blindenbelangen (R.V.B.B.), Stichting Blinden-Penning, Landelijke Stichting voor Blinden en Slechtzienden (L.S.B.S.), Algemene Nederlandse Vereniging ter Voorkoming van Blindheid (A.N.V.V.B.), and Stichting O.O.G.
1. Lam TT, Abler AS, Tso MO. Apoptosis and caspases after ischemia-reperfusion injury in rat retina. Invest Ophthalmol Vis Sci 1999; 40:967-75.
2. Osborne NN, Casson RJ, Wood JP, Chidlow G, Graham M, Melena J. Retinal ischemia: mechanisms of damage and potential therapeutic strategies. Prog Retin Eye Res 2004; 23:91-147.
3. Nickells RW. Apoptosis of retinal ganglion cells in glaucoma: an update of the molecular pathways involved in cell death. Surv Ophthalmol 1999; 43 Suppl 1:S151-61.
4. Piltz-seymour JR, Grunwald JE, Hariprasad SM, Dupont J. Optic nerve blood flow is diminished in eyes of primary open-angle glaucoma suspects. Am J Ophthalmol 2001; 132:63-9.
5. Robin AL, Barnebey HS, Harris A, Osborne N. Glaucoma management: beyond intraocular pressure. Ophtalmology Times 1997; 22 (suppl. 2):S1-23.
6. Quigley HA, Nickells RW, Kerrigan LA, Pease ME, Thibault DJ, Zack DJ. Retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis. Invest Ophthalmol Vis Sci 1995; 36:774-86.
7. Osborne NN, Melena J, Chidlow G, Wood JP. A hypothesis to explain ganglion cell death caused by vascular insults at the optic nerve head: possible implication for the treatment of glaucoma. Br J Ophthalmol 2001; 85:1252-9.
8. Sommer A, Katz J, Quigley HA, Miller NR, Robin AL, Richter RC, Witt KA. Clinically detectable nerve fiber atrophy precedes the onset of glaucomatous field loss. Arch Ophthalmol 1991; 109:77-83.
9. Quigley HA, Katz J, Derick RJ, Gilbert D, Sommer A. An evaluation of optic disc and nerve fiber layer examinations in monitoring progression of early glaucoma damage. Ophthalmology 1992; 99:19-28.
10. Roth S. Endogenous neuroprotection in the retina. Brain Res Bull 2004; 62:461-6.
11. Roth S, Li B, Rosenbaum PS, Gupta H, Goldstein IM, Maxwell KM, Gidday JM. Preconditioning provides complete protection against retinal ischemic injury in rats. Invest Ophthalmol Vis Sci 1998; 39:777-85.
12. Kawahara N, Wang Y, Mukasa A, Furuya K, Shimizu T, Hamakubo T, Aburatani H, Kodama T, Kirino T. Genome-wide gene expression analysis for induced ischemic tolerance and delayed neuronal death following transient global ischemia in rats. J Cereb Blood Flow Metab 2004; 24:212-23.
13. Tang Y, Pacary E, Freret T, Divoux D, Petit E, Schumann-Bard P, Bernaudin M. Effect of hypoxic preconditioning on brain genomic response before and following ischemia in the adult mouse: identification of potential neuroprotective candidates for stroke. Neurobiol Dis 2006; 21:18-28.
14. Carmel JB, Kakinohana O, Mestril R, Young W, Marsala M, Hart RP. Mediators of ischemic preconditioning identified by microarray analysis of rat spinal cord. Exp Neurol 2004; 185:81-96.
15. Stenzel-Poore MP, Stevens SL, Xiong Z, Lessov NS, Harrington CA, Mori M, Meller R, Rosenzweig HL, Tobar E, Shaw TE, Chu X, Simon RP. Effect of ischaemic preconditioning on genomic response to cerebral ischaemia: similarity to neuroprotective strategies in hibernation and hypoxia-tolerant states. Lancet 2003; 362:1028-37.
16. Whitlock NA, Agarwal N, Ma JX, Crosson CE. Hsp27 upregulation by HIF-1 signaling offers protection against retinal ischemia in rats. Invest Ophthalmol Vis Sci 2005; 46:1092-8.
17. Li B, Yang C, Rosenbaum DM, Roth S. Signal transduction mechanisms involved in ischemic preconditioning in the rat retina in vivo. Exp Eye Res 2000; 70:755-65.
18. Konno T, Sato A, Uchibori T, Nagai A, Kogi K, Nakahata N. Adenosine A2A receptor mediated protective effect of 2-(6-cyano-1-hexyn-1-yl)adenosine on retinal ischaemia/reperfusion damage in rats. Br J Ophthalmol 2006; 90:900-5.
19. Nonaka A, Kiryu J, Tsujikawa A, Yamashiro K, Nishijima K, Miyamoto K, Nishiwaki H, Honda Y, Ogura Y. Inhibitory effect of ischemic preconditioning on leukocyte participation in retinal ischemia-reperfusion injury. Invest Ophthalmol Vis Sci 2001; 42:2380-5.
20. Sakamoto K, Yonoki Y, Kubota Y, Kuwagata M, Saito M, Nakahara T, Ishii K. Inducible nitric oxide synthase inhibitors abolished histological protection by late ischemic preconditioning in rat retina. Exp Eye Res 2006; 82:512-8.
21. Roth S, Dreixler JC, Shaikh AR, Lee KH, Bindokas V. Mitochondrial potassium ATP channels and retinal ischemic preconditioning. Invest Ophthalmol Vis Sci 2006; 47:2114-24.
22. Gidday JM. Cerebral preconditioning and ischaemic tolerance. Nat Rev Neurosci 2006; 7:437-48.
23. Kamphuis W, Dijk F, Van Soest S, Bergen AAB. Global Gene Expression Profiling of Ischemic Preconditioning in the Rat Retina. Mol Vis 2007; 13:1020-30 <http://www.molvis.org/molvis/v13/a111/>.
24. Osborne NN, Larsen AK. Antigens associated with specific retinal cells are affected by ischaemia caused by raised intraocular pressure: effect of glutamate antagonists. Neurochem Int 1996; 29:263-70.
25. Osborne NN. Neuroprotection to the retina: relevance in glaucoma. In "Vascular Risk Factors and Neuroprotection in Glaucoma-Update 1996" (S. M. Drance, Ed.), Kugler Publications, Amsterdam/New York (1997). p. 139-155.
26. Dijk F, Kamphuis W. Ischemia-induced alterations of AMPA-type glutamate receptor subunit. Expression patterns in the rat retina--an immunocytochemical study. Brain Res 2004; 997:207-21.
27. Dijk F, van Leeuwen S, Kamphuis W. Differential effects of ischemia/reperfusion on amacrine cell subtype-specific transcript levels in the rat retina. Brain Res 2004; 1026:194-204.
28. Dijk F, Kraal-Muller E, Kamphuis W. Ischemia-induced changes of AMPA-type glutamate receptor subunit expression pattern in the rat retina: a real-time quantitative PCR study. Invest Ophthalmol Vis Sci 2004; 45:330-41.
29. Kamphuis W, Dijk F, Kraan W, Bergen AA. Transfer of lens-specific transcripts to retinal RNA samples may underlie observed changes in crystallin-gene transcript levels after ischemia. Mol Vis 2007; 13:220-8 <http://www.molvis.org/molvis/v13/a25/>.
30. Hughes TR, Mao M, Jones AR, Burchard J, Marton MJ, Shannon KW, Lefkowitz SM, Ziman M, Schelter JM, Meyer MR, Kobayashi S, Davis C, Dai H, He YD, Stephaniants SB, Cavet G, Walker WL, West A, Coffey E, Shoemaker DD, Stoughton R, Blanchard AP, Friend SH, Linsley PS. Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer. Nat Biotechnol 2001; 19:342-7.
31. Hughes TR, Marton MJ, Jones AR, Roberts CJ, Stoughton R, Armour CD, Bennett HA, Coffey E, Dai H, He YD, Kidd MJ, King AM, Meyer MR, Slade D, Lum PY, Stepaniants SB, Shoemaker DD, Gachotte D, Chakraburtty K, Simon J, Bard M, Friend SH. Functional discovery via a compendium of expression profiles. Cell 2000; 102:109-26.
32. Roberts CJ, Nelson B, Marton MJ, Stoughton R, Meyer MR, Bennett HA, He YD, Dai H, Walker WL, Hughes TR, Tyers M, Boone C, Friend SH. Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles. Science 2000; 287:873-80.
33. Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA. DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol 2003; 4:P3.
34. Beissbarth T, Speed TP. GOstat: find statistically overrepresented Gene Ontologies within a group of genes. Bioinformatics 2004; 20:1464-5.
35. Bluthgen N, Brand K, Cajavec B, Swat M, Herzel H, Beule D. Biological profiling of gene groups utilizing Gene Ontology. Genome Inform 2005; 16:106-15.
36. Hosack DA, Dennis G Jr, Sherman BT, Lane HC, Lempicki RA. Identifying biological themes within lists of genes with EASE. Genome Biol 2003; 4:R70.
37. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002; 3:RESEARCH0034.
38. Whitlock NA, Lindsey K, Agarwal N, Crosson CE, Ma JX. Heat shock protein 27 delays Ca2+-induced cell death in a caspase-dependent and -independent manner in rat retinal ganglion cells. Invest Ophthalmol Vis Sci 2005; 46:1085-91.
39. Li B, Roth S. Retinal ischemic preconditioning in the rat: requirement for adenosine and repetitive induction. Invest Ophthalmol Vis Sci 1999; 40:1200-16.
40. Zheng L, Gong B, Hatala DA, Kern TS. Retinal ischemia and reperfusion causes capillary degeneration: similarities to diabetes. Invest Ophthalmol Vis Sci 2007; 48:361-7.
41. Hayashi T, Deguchi K, Nagotani S, Zhang H, Sehara Y, Tsuchiya A, Abe K. Cerebral ischemia and angiogenesis. Curr Neurovasc Res 2006; 3:119-29.
42. del Zoppo GJ, Mabuchi T. Cerebral microvessel responses to focal ischemia. J Cereb Blood Flow Metab 2003; 23:879-94.
43. Takano K, Ogura M, Nakamura Y, Yoneda Y. Neuronal and glial responses to polyamines in the ischemic brain. Curr Neurovasc Res 2005; 2:213-23.
44. Garcia L, Burda J, Hrehorovska M, Burda R, Martin ME, Salinas M. Ischaemic preconditioning in the rat brain: effect on the activity of several initiation factors, Akt and extracellular signal-regulated protein kinase phosphorylation, and GRP78 and GADD34 expression. J Neurochem 2004; 88:136-47.
45. Koumenis C, Naczki C, Koritzinsky M, Rastani S, Diehl A, Sonenberg N, Koromilas A, Wouters BG. Regulation of protein synthesis by hypoxia via activation of the endoplasmic reticulum kinase PERK and phosphorylation of the translation initiation factor eIF2alpha. Mol Cell Biol 2002; 22:7405-16.
46. DeGracia DJ, Hu BR. Irreversible translation arrest in the reperfused brain. J Cereb Blood Flow Metab 2007; 27:875-93.
47. Snider BJ, Du C, Wei L, Choi DW. Cycloheximide reduces infarct volume when administered up to 6 h after mild focal ischemia in rats. Brain Res 2001; 917:147-57.
48. Linnik MD, Zobrist RH, Hatfield MD. Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats. Stroke 1993; 24:2002-8;discussion2008-9.
49. Tortosa A, Rivera R, Ferrer I. Dose-related effects of cycloheximide on delayed neuronal death in the gerbil hippocampus after bilateral transitory forebrain ischemia. J Neurol Sci 1994; 121:10-7.
50. Aronowski J, Strong R, Grotta JC. Reperfusion injury: demonstration of brain damage produced by reperfusion after transient focal ischemia in rats. J Cereb Blood Flow Metab 1997; 17:1048-56.
51. Park WS, Sung DK, Kang S, Koo SH, Kim YJ, Lee JH, Chang YS, Lee M. Therapeutic window for cycloheximide treatment after hypoxic-ischemic brain injury in neonatal rats. J Korean Med Sci 2006; 21:490-4.
52. Park SG, Ewalt KL, Kim S. Functional expansion of aminoacyl-tRNA synthetases and their interacting factors: new perspectives on housekeepers. Trends Biochem Sci 2005; 30:569-74.
53. Schuetz E, Thanos S. Microglia-targeted pharmacotherapy in retinal neurodegenerative diseases. Curr Drug Targets 2004; 5:619-27.
54. Langmann T. Microglia activation in retinal degeneration. J Leukoc Biol 2007; 81:1345-51.
55. Zhang C, Lam TT, Tso MO. Heterogeneous populations of microglia/macrophages in the retina and their activation after retinal ischemia and reperfusion injury. Exp Eye Res 2005; 81:700-9.
56. Jo N, Wu GS, Rao NA. Upregulation of chemokine expression in the retinal vasculature in ischemia-reperfusion injury. Invest Ophthalmol Vis Sci 2003; 44:4054-60.
57. Neufeld AH, Kawai S, Das S, Vora S, Gachie E, Connor JR, Manning PT. Loss of retinal ganglion cells following retinal ischemia: the role of inducible nitric oxide synthase. Exp Eye Res 2002; 75:521-8.
58. Tsujikawa A, Ogura Y, Hiroshiba N, Miyamoto K, Kiryu J, Tojo SJ, Miyasaka M, Honda Y. Retinal ischemia-reperfusion injury attenuated by blocking of adhesion molecules of vascular endothelium. Invest Ophthalmol Vis Sci 1999; 40:1183-90.
59. Ogata N, Yamanaka R, Yamamoto C, Miyashiro M, Kimoto T, Takahashi K, Maruyama K, Uyama M. Expression of vascular endothelial growth factor and its receptor, KDR, following retinal ischemia-reperfusion injury in the rat. Curr Eye Res 1998; 17:1087-96.
60. Joussen AM, Poulaki V, Qin W, Kirchhof B, Mitsiades N, Wiegand SJ, Rudge J, Yancopoulos GD, Adamis AP. Retinal vascular endothelial growth factor induces intercellular adhesion molecule-1 and endothelial nitric oxide synthase expression and initiates early diabetic retinal leukocyte adhesion in vivo. Am J Pathol 2002; 160:501-9.
61. Ju WK, Kim KY, Neufeld AH. Increased activity of cyclooxygenase-2 signals early neurodegenerative events in the rat retina following transient ischemia. Exp Eye Res 2003; 77:137-45.
62. Wang X, Li X, Yaish-Ohad S, Sarau HM, Barone FC, Feuerstein GZ. Molecular cloning and expression of the rat monocyte chemotactic protein-3 gene: a possible role in stroke. Brain Res Mol Brain Res 1999; 71:304-12.
63. Takaki R, Watson SR, Lanier LL. DAP12: an adapter protein with dual functionality. Immunol Rev 2006; 214:118-29.
64. Striz I, Trebichavska 2 I. Calprotectin-a pleiotropic molecule in acute and chronic inflammation. Physiol Res 2004; 53:245-53.
65. Yui S, Nakatani Y, Mikami M. Calprotectin (S100A8/S100A9), an inflammatory protein complex from neutrophils with a broad apoptosis-inducing activity. Biol Pharm Bull 2003; 26:753-60.
66. Harrington JF, Messier AA, Levine A, Szmydynger-Chodobska J, Chodobski A. Shedding of tumor necrosis factor type 1 receptor after experimental spinal cord injury. J Neurotrauma 2005; 22:919-28.
67. Kim HS. Park CK Retinal ganglion cell death is delayed by activation of retinal intrinsic cell survival program. Brain Res 2005.
68. Takamiya A, Takeda M, Yoshida A, Kiyama H. Expression of serine protease inhibitor 3 in ocular tissues in endotoxin-induced uveitis in rat. Invest Ophthalmol Vis Sci 2001; 42:2427-33.
69. La M, D'Amico M, Bandiera S, Di Filippo C, Oliani SM, Gavins FN, Flower RJ, Perretti M. Annexin 1 peptides protect against experimental myocardial ischemia-reperfusion: analysis of their mechanism of action. FASEB J 2001; 15:2247-56.
70. Guan J, Bennet L, Gluckman PD, Gunn AJ. Insulin-like growth factor-1 and post-ischemic brain injury. Prog Neurobiol 2003; 70:443-62.
71. Karg A, Dinc ZA, Baok O, Ucvet A. MUC4 expression and its relation to ErbB2 expression, apoptosis, proliferation, differentiation, and tumor stage in non-small cell lung cancer (NSCLC). Pathol Res Pract 2006; 202:577-83.
72. Rodriguez JM, Glozak MA, Ma Y, Cress WD. Bok, Bcl-2-related Ovarian Killer, Is Cell Cycle-regulated and Sensitizes to Stress-induced Apoptosis. J Biol Chem 2006; 281:22729-35.
73. Schutte B, Henfling M, Ramaekers FC. DEDD association with cytokeratin filaments correlates with sensitivity to apoptosis. Apoptosis 2006; 11:1561-72.
74. Liang L, Zhao M, Xu Z, Yokoyama KK, Li T. Molecular cloning and characterization of CIDE-3, a novel member of the cell-death-inducing DNA-fragmentation-factor (DFF45)-like effector family. Biochem J 2003; 370:195-203.
75. Huang W, Fileta JB, Dobberfuhl A, Filippopolous T, Guo Y, Kwon G, Grosskreutz CL. Calcineurin cleavage is triggered by elevated intraocular pressure, and calcineurin inhibition blocks retinal ganglion cell death in experimental glaucoma. Proc Natl Acad Sci U S A 2005; 102:12242-7.