Strain-Dependent Increases in Retinal Inflammatory Proteins And

Strain-Dependent Increases in Retinal Inﬂammatory Proteins and Photoreceptor FGF-2 Expression in Streptozotocin-Induced Diabetic Rats

Stefanie J. Kirwin,1 Suzanne T. Kanaly,2 Noelle A. Linke,2 and Jeffrey L. Edelman1

1 PURPOSE. Inflammation is thought to play a role in disease progression is by tightly controlling blood glucose levels. progression and vision loss in diabetic retinopathy (DR). How- Despite significant morbidity and effect on quality of life, there ever, the level of inflammation and the role of cytokines and is currently no approved pharmacotherapy for this sight-threat- growth factors in the early stages of this disease are poorly ening disease.1,3 understood. Streptozotocin (STZ)-induced hyperglycemia in In the past, DR was considered primarily a vascular disease. rats is widely used as a model of diabetic retinopathy, and It has been described as and is still classified by abnormalities therefore this model was used to better define the inflamma- of the retinal vasculature that progress from hyperpermeability tory response and the impact of the genetic background. of the vessels, to capillary nonperfusion and hemorrhaging, METHODS. The expression of a panel of 57 inflammatory pro- followed by neovascularization of the retina. Macular edema teins and growth factors in the retina of three rat strains was can develop independently of other changes and poses a seri- compared by using a highly sensitive flow cytometry–based ous threat to central vision.1,2 However, there is mounting assay. Hyperglycemia was induced in Brown Norway (BN), evidence that neuropathy develops before and independent of Long-Evans (LE), and Sprague-Dawley (SD) rats, and protein vascular complications and contributes to the disease.4 Inflam- expression in the retina was measured 4 weeks and 3 months matory molecules such as cytokines can be found in the retina later. of diabetic patients,5 although it is presently unclear whether inflammation contributes to DR or is secondary to it. RESULTS. The data revealed a subtle, but reproducible, inflammatory response in the retina of SD, but not in those of BN or Streptozotocin (STZ)-induced diabetes in the rat is com- LE, rats. Upregulation of fibroblast growth factor (FGF)-2 in the monly used as an experimental model of DR. The STZ rat photoreceptor nuclear layer coincided with the inflammatory model mimics the human disease by inducing hyperglycemia ␤ 6 response in SD rats and may constitute a neuroprotective after destruction of the -cells in the pancreas. Although there mechanism. Reduced expression of genes involved in the pho- are vascular changes in this model, the vasculopathy does not totransduction pathway indicates altered photoreceptor func- progress to neovascularization as is observed in humans. The tion. length of time that passes between the beginning of the hyperglycemic insult and the development of symptoms is also a ONCLUSIONS Taken together, these data show that inflamma- C . challenge as are the number of different mechanisms that may tory changes in the diabetic rat retina are highly strain depen- cause damage.7 In addition, inflammatory changes are subtle dent, and SD rats exhibit low-level inflammation similar to that and therefore sometimes difficult to demonstrate reproducibly. observed in diabetic patients. Therefore, SD rats may be a good Further complicating matters is the fact that different rat strains model for the study of early inflammatory changes in human show different responses to hyperglycemia and other insults to diabetic retinopathy. (Invest Ophthalmol Vis Sci. 2009;50: the retina.8–10 5396–5404) DOI:10.1167/iovs.09-3474 In this study, we characterized the inflammatory changes in iabetic retinopathy (DR) is the number one cause of blind- the retina of diabetic rats, while exploring the effect of genetic Dness in working-age people in the United States today, and background, including pigmentation, on such changes. A its prevalence is expected to rise, with a projected increase in highly sensitive flow cytometry-based assay (Luminex Corp., the number of patients with type II diabetes. Currently, the Austin, TX) was used to screen for a large number of cytokines only available treatment for DR is laser photocoagulation, and and growth factors in the retina of diabetic Brown Norway although it is highly effective in preserving sight, it can have (BN), Long-Evans (LE), and Sprague-Dawley (SD) rats. These rat serious side effects.1 Furthermore, this treatment is efficacious strains were chosen due to their documented use in the STZ- only in proliferative DR and cannot restore vision already lost induced DR model and their various levels of pigmentation, to the disease.2 Most patients with diabetes develop retinopa- with SD being the least pigmented and BN the most pigmented thy at some point, and the only proven method of slowing its rat strain. To date LE and BN rats have been used mostly to study vascular abnormalities in diabetes such as increased leu- kostasis11–16 and blood–retina barrier breakdown.9,14,15,17–21 However, electrophysiological studies indicate functional def- From the Departments of 1Biological Sciences and 2Pathology, icits in the neural retina22–24 and results from studies on ␥-ami- Allergan, Inc., Irvine, California. 24,25 26 Supported by Allergan, Inc. nobutyric acid signaling and microarray data in LE as 27 Submitted for publication January 28, 2009; revised March 27 and well as Mu¨ller cell abnormalities in BN rats suggest that other May 6, 2009; accepted July 28, 2009. hallmarks of experimental DR occur and that these strains Disclosure: S.J. Kirwin, Allergan, Inc. (E, F); S.T. Kanaly, Aller- merit further investigation. gan, Inc. (E, F); N.A. Linke, Allergan, Inc. (E, F); J.L. Edelman, The data show that despite the absence of gross inflamma- Allergan, Inc. (E, F) tory infiltrates in the retina, markers of inflammation are The publication costs of this article were defrayed in part by page present in SD rats, but not the other two strains. Furthermore, charge payment. This article must therefore be marked “advertise- ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. the data show that the cytokine response coincides with up- Corresponding author: Stefanie J. Kirwin, Allergan, Inc., Biological regulation of fibroblast growth factor (FGF-2) in photorecep- Science, 2525 Dupont Drive, Irvine, CA 92612; tors, and gene expression analysis suggests impairment of [email protected]. photoreceptor function.

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MATERIALS AND METHODS lerica, CA) at 1:200 dilution for 60 minutes. After they were washed with PBST, the tissues were reacted with biotinylated donkey anti- Animals and Induction of Diabetes mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) for 20 minutes, washed with PBST, and incubated for 20 minutes Diabetes was induced in male BN, LE, and SD rats (Charles River with horseradish-peroxidase streptavidin (Covance, Dedham, MA) and Laboratories, Wilmington, MA) weighing 250 to 300 g (BN: 244 Ϯ 19 g; rinsed in PBST. Signal was detected with AEC chromogen (Romulin; LE: 298 Ϯ 25 g; SD: 282 Ϯ 26 g) by intraperitoneal injection of 65 BioCare Medical, Concord, CA), then washed with PBST. Hematoxylin mg/kg bodyweight STZ in 0.09 M citrate buffer (pH 4.8; both Sigma- was applied for 5 minutes followed by a rinse with distilled water. Aldrich, St. Louis, MO). Age-matched control rats were injected with buffer only. At the time of injection, BN rats were moderately, but RNA Extraction and Real-Time PCR significantly (P Ͻ 0.05) smaller then LE or SD rats. Blood glucose was Retinas were removed and placed individually into tubes containing measured (Ascencia control system; Bayer, Tarrytown, NY), and rats 200 ␮L RNA stabilizer (RNAlater; Ambion, Austin, TX). After 24 hours with blood glucose levels above 220 mg/dL after 48 hours were at 4°C, excess fluid was removed and the samples were stored at deemed diabetic. Nondiabetic STZ-injected rats were reinjected with a Ϫ70°C until analysis. Frozen samples were placed into 1 mL lysis second dose on day 2.28–30 There was no significant difference in reagent (Qiazol; Qiagen, Valencia, CA) and homogenized (Tissue- weight or blood glucose levels 6 days after the initial injection between Tearor; Biospec, Bartlesville, OK). RNA was extracted (RNeasy Mini rats injected once and those receiving a second dose. No insulin was Kit; Qiagen) according to the manufacturers’ instructions. RNA quality supplied at any time. Occasionally, glucose levels exceeded the upper was then assessed (RNA 6000 Nano Kit; Agilent Technologies, Wald- limit of detection, and this unknown value was replaced by 600 mg/dL. bronn, Germany) and quantity determined by spectrophotometry. Re- Glucose spikes to these high levels were relatively rare and not limited verse transcription was performed on 1 ␮g of RNA using 25 mM MgCl , to any particular rat strain. Intraocular blood glucose was measured by 2 10 mM dNTP mixture, 25 U RNasin, and 15 U AMV reverse transcrip- making a 4- to 6-mm diagonal incision across the cornea, removing the tase in reverse transcription buffer (all Promega, Madison, WI) in 20 ␮L lens and then sampling the liquid in the eye cup using the capillary for 20 minutes at 42°C, followed by a 5-minute denaturing step at 95°C action of the control test stick (Ascencia; Bayer). The sample most and 5 minutes at 4°C. After reverse transcription, ddH O was added to likely contains a mixture of aqueous and vitreous humor. Animals were 2 a final volume of 200 ␮L. killed 4 weeks and 3 months after induction of diabetes. They were Real-time PCR was performed (7900HT Real-Time PCR System; Applied housed in plastic box cages in a specific pathogen-free, AALAC certi- Biosystems [ABI], Foster City, CA), with probe and primer sets (Taqman; ABI) fied facility on a 12-hour light/dark cycle. Room temperature was for Actb (ID: Rn00667869_m1), Arr3 (ID: Rn01424383_m1), Gnb3 maintained between 16°C and 23°C, average daily humidity between (Rn00516381_m1), Grk1 (ID: Rn00579470_m1), and Opn1mw (ID: 30% and 70%, and airflow between 10 and 30 air changes per hour. All Rn00585560_m1). All primers span exon junctions and therefore do animals were fed standard chow and treated according to the ARVO not recognize genomic DNA. Gene expression was normalized and Statement for the Use of Animals in Ophthalmic and Vision Research. converted to a linearized value by the following formula: unit ϭ Protein Analysis. Isolated neural retinas were snap frozen in ٙ (Ct Ϫ Ct ) ϫ 100.33 1.8 liquid nitrogen and samples were stored at Ϫ70°C until processing. actb gene x One retina each from three individual rats was combined for protein Statistical Analysis analysis. Protein was extracted by sonication of three pooled retinas in 300 ␮L Tris HCl (pH 7.5; Mediatech, Inc. Herndon, VA) containing Experiments analyzing retinal proteins were repeated at least three Complete, Mini protease inhibitor cocktail (Roche, Mannheim, Ger- times, each group containing three rats, making data the average of at many). Serum was collected by letting blood clot for 1 hour at room least nine rats from at least three independent experiments. Signifi- temperature, followed by a 10-minute centrifugation at 10,000g. Pro- cance was determined by Student’s t-test, unless otherwise noted. P Ͻ tein concentration was then determined (RodentMA Antigen 1.6 panel; 0.05 was considered significant. Rules Based Medicine Austin, TX). The assay uses mouse antibodies that cross-react with rat cytokines.31,32 Briefly, samples are incubated with microsphere multiplexes, which contain covalently linked assay RESULTS specific capture reagents, for 1 hour at room temperature before STZ-Induced Hyperglycemia in BN, LE, biotinylated reporter antibodies are added for an additional hour at room temperature. After incubation for 1 hour with streptavidin-phy- and SD Rats coerythrin solution and vacuum filtration, the samples are diluted in Diabetes was induced with STZ in three rat strains: BN, LE, and matrix buffer and analyzed with a multiplex bio-assay analyzer (100 IS SD. Over the 3-month observation period, average blood glu- System; Luminex Corp.). cose levels ranged from 330 to 542 mg/dL in the three rat Histopathology and Immunohistochemistry. Rats were strains (Fig. 1A), similar to previously published results in this killed after 3 months of diabetes, and the eyes were immediately fixed model.34–36 In contrast, vehicle-treated rats exhibited tightly in Davidson’s solution (BBC Biochemical, Mount Vernon, WA) for 24 controlled blood glucose levels in all strains (63–89 mg/dL; Fig. hours. After fixation, the eyes were transferred to 70% alcohol, pro- 1A). Intraocular levels were also measured to determine glu- cessed routinely for paraffin embedding, and sectioned at 4 ␮m. For cose concentration adjacent to the neural retina. Intraocular routine histopathologic evaluation the sections were stained with levels were similar to those in blood in diabetic and control hematoxylin and eosin. For immunohistochemistry, paraffin-embedded animals (Fig. 1B), suggesting that ocular humor is a major route sections were placed on charged slides and dried overnight at room of retinal glucose exposure in diabetes. Of note, SD rats temperature. After deparaffination and rehydration, slides were placed showed moderately higher levels of intraocular glucose than in citrate plus buffer (Scytek, Logan, UT) and brought to 125°C and 10 did the other two strains, a difference that reached statistical to 15 psi for 30 seconds in a Pascal pressure cooker, for antigen significance (P Ͻ 0.05). Glucose levels in the blood of diabetic retrieval, followed by washing in phosphate-buffered saline Tween animals, however, did not show consistent significant differ- (PBST) buffer (Scytek). Immunohistochemistry staining was performed ences over time between the three strains. Control BN, LE, and at room temperature with an autostainer (Dako, Carpinteria, CA). SD rats gained weight during the 3 months of observation, Tissue slides were treated for 10 minutes with 3% hydrogen peroxide however, the BN strain showed significantly less weight gain. (VWR, West Chester, PA), washed briefly with distilled water, treated In comparison, hyperglycemic rats of all strains failed to gain with a blocking agent (SuperBlock; Scytek) for 10 minutes, then re- weight over the same period (Fig. 1C). These data show that acted with mouse-anti-FGF-2 antibody (Clone bFM-2; Millipore, Bil- STZ induces hyperglycemia in blood and the eye in all three rat

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strains, and therefore indicate that any strain-specific molecular or functional changes in the retina are not due to large differences in glucose exposure. Protein Measurements in Normal and Diabetic Rat Retinas A modified bio-assay (Luminex) was used to measure the levels of 57 proteins of interest in retinas of normal and STZ-treated BN, LE, and SD rats 1 month and 3 months after diabetes induction. Of these 57 proteins, 24 were below detection limits in normal and STZ-treated retinas, and the concentration of 22 proteins, though present at detectable levels, was not significantly changed by STZ treatment (Table 1). Hyperglycemia-Induced Retinal Inflammation in SD Rats Several markers of inflammation have been reported in the neural retina in experimental models of diabetes37,38 and in diabetic human vitreous39–41 and tissues.42–44 Although there was no evidence of a measurable inflammatory response in diabetic LE and BN rats, SD rats did show a time-dependent increase in several inflammatory proteins. The chemokine eotaxin (CCL11) was significantly increased in diabetic SD retina after 1 month to 1.6 times the level in control retina. Although the same trend could be observed at 3 months, this change was not statistically significant (P ϭ 0.052; Fig. 2A). Eotaxin was also significantly upregulated in serum after 3 months of diabetes (Table 2), as well as at 4 weeks (data not shown) in SD rats. Macrophage colony-stimulating factor (M- CSF), monocyte chemoattractant protein-1 (MCP-1; CCL-2), and MCP-3 (CCL-7) which act on monocytes and macrophages, were significantly upregulated after 3 months of diabetes in SD rats (Figs. 2B–D). Although protein levels of these cytokines appeared to be low compared with their levels in overtly

TABLE 1. Proteins Not Signiﬁcantly Changed in Diabetic Retina

Detectable (but unchanged) Undetectable

Apolipoprotein A1 CD40,CD40L Calbindin EGF Endothelin-1 GCP-2 Factor VII GM-CSF GST␮ Growth Hormone IgA GST␣ ILϪ1␣,Ϫ1␤,Ϫ2,Ϫ5,Ϫ10 InterferonϪ␥ Leukemia inhibitory factor ILϪ3,Ϫ4,Ϫ7,Ϫ11,Ϫ12,Ϫ17 MIPϪ1␣,Ϫ3␤ KC/Gro␣ Myeloperoxidase Leptin Myoglobin Lymphotactin NGAL MCP-5 Stem Cell Factor MIPϪ1␤,Ϫ1␥,Ϫ2 SGOT MMP-9 Tissue Factor Oncostatin M Thrombopoietin RANTES VCAM

Proteins in the retina were not significantly affected by hyperglycemia. Complete panel of proteins tested in all strains at 4 weeks and three months,which did not show changes or were below the detectable level in the retina. FIGURE 1. Body weight, blood glucose, and vitreous glucose levels in Abbreviations: GST, glutathione transferase; Ig, immunoglobulin; BN, LE, and SD rats. (A) Blood glucose levels in rats of different strains IL, interleukin; MIP, macrophage inflammatory protein; NGAL, neutro- injected with STZ (solid line, filled symbols) or buffer only (broken phil gelatinase-associated lipocalin; SGOT, serum glutamic oxaloacetic line, open symbols) at multiple time points after injection. (B) Ocular transaminase; VCAM, vascular cell adhesion molecule; EGF, epidermal glucose levels at the time of death measured in the eye cup after lens growth factor; GCP, granulocyte chemotactic protein; GM-CSF, granu- removal. (C) Body weight of rats with induced diabetes and control locyte macrophage colony-stimulating factor; MMP, matrix metallopro- rats. Data are the average of results in at least three experiments with teinase; RANTES, regulated upon activation, normal T-cell expressed three rats per group (n Ͼ 9). Error bars, SD. and secreted.

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FIGURE 2. Retinal cytokine protein expression at 28 and 84 days of diabetes and in age-matched control rats. The concentration of eotaxin (A) was increased in SD rats after 4 weeks of diabetes. Protein levels of M-CSF (B), MCP-1 (C), and MCP-3 (D) were increased in SD rats after 3 months of diabetes. Protein expression was measured in three independent experiments. Each symbol represents data from three pooled diabetic retinas of three individual diabetic or control rats. Statistical signiﬁcance was determined with a Student’s t-test, and signiﬁcant prob- abilities are noted.

inflammatory diseases, the increase is statistically significant inflammatory response. Clusterin and vWF also showed signif- and highly reproducible. icantly increased levels in the serum of SD rats at 3 months, Further evidence of inflammation in the retina of SD rats whereas none of the other proteins that were significantly was the upregulation of clusterin after 4 weeks and 3 months changed in the retina were altered in the serum at this time of diabetes (Fig. 3A). These results confirm published reports (Table 2). These data indicate subtle, but highly reproducible of increased clusterin expression in the diabetic SD rat,45 but and strain-dependent, inflammatory protein increases in the show the observation to be highly strain dependent. Tissue retinas of diabetic rats. inhibitor of metalloproteinase (TIMP)-1 showed a similarly moderate, but significant upregulation after 4 weeks of diabe- Changes in Growth Factor Expression in the tes and more robust changes after 3 months (Fig. 3B). Although Retinas of Diabetic Rats this protein was increased in all SD diabetic samples compared Growth factors, particularly those with angiogenic effects, are with the control, the concentration in diabetic retinas varied of interest because of their role in proliferative diabetic reti- widely between different experiments. Therefore, the Mann- nopathy.46 In BN rats, there was a small but significant increase Whitney U test was used to compare the median values be- in vascular endothelial growth factor (VEGF) protein expres- tween the diabetic and control group, showing a significant ␤ sion in the retina after 4 weeks of diabetes. However, a signif- increase. In addition, -2 microglobulin (B2M; Fig. 3C) and von icant increase was not observed after 3 months. Although there Willebrand factor (vWF; Fig. 3D) were significantly upregu- was a trend toward increased VEGF levels in LE and SD rats at lated after 3 months of diabetes, further indicating an ongoing 4 weeks, it did not reach statistical significance (P ϭ 0.115 and P ϭ 0.057 respectively; Fig. 4A). FGF-9 exhibited the same temporal pattern as VEGF, though induction of this growth TABLE 2. Serum Protein Levels factor was of a greater magnitude in BN rats. By 3 months of Diabetic Control diabetes, FGF-9 levels in the retina of diabetic rats were similar to that in control rats (Fig. 4B). In contrast, and confirming B2M (␮g/mL) 69 (Ϯ14) 65 (Ϯ11) previously published data,47 FGF-2 was significantly increased Clusterin (␮g/mL) 183 (Ϯ37)* 118 (Ϯ30) in the retina of diabetic SD rats after 3 months. This increase in Eotaxin (pg/mL) 1401 (Ϯ343)* 811 (Ϯ126) FGF-2 protein was observed only in SD rats and, although FGF-9 (ng/mL) 0.3 (Ϯ0.7) 0.4 (Ϯ0.5) Ϯ Ϯ measurable, FGF-2 levels were unchanged in BN or LE rats FGF-2 (ng/mL) 2.1 ( 0.1) 2.3 ( 0.2) (Fig. 4C). MCP-1 (pg/mL) 696 (Ϯ290) 846 (Ϯ91) MCP-3 (pg/mL) 386 (Ϯ128) 468 (Ϯ61) M-CSF (ng/mL) 0.8 (Ϯ0.1) 0.8 (Ϯ0.1) Histopathologic Changes after 3 Months TIMP-1 (ng/mL) 4.4 (Ϯ4.7) 3.3 (Ϯ3.5) of Diabetes Ϯ Ϯ VEGF (pg/mL) 238 ( 44) 211 ( 70) No gross disease was found in any of the rat strains by histo- vWF (ng/mL) 532 (Ϯ165)* 195 (Ϯ44) logic examination (Figs. 5A–F and Ref. 48), and there was no Levels of proteins (ϮSD) significantly changed in the retina of change in retinal thickness after 3 months of diabetes (data not diabetic rats. Serum was collected from SD rats after 3 months of shown). Despite an increase in inflammatory proteins in SD diabetes and protein levels determined. Results are average of three rats, no inflammatory infiltrate was detected in the retinas of separate samples each from two independent experiments (n ϭ 6). any strain (Figs. 5A–F). Staining with specific antibodies also * P Ͻ 0.05 compared with control. revealed no evidence of macrophages, CD4 T cells, CD8 T

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FIGURE 3. Expression of inflammatory proteins in the diabetic rat retina. Concentrations of proteins upregulated during inflammation were measured in the retina as in Fig. 2. In SD rats, both clusterin (A) and TIMP-1 (B) were upregulated 4 weeks and 3 months after the onset of diabetes. B2M (C) and vWF (D) were increased after 3 months of diabetes in SD rats. Each symbol represents data from three pooled diabetic or control rats. A Student’s t-test was used to determine statistical significance. *Mann-Whitney U test.

cells, or B cells in either control or diabetic retinas (data not strains in the study of diabetic retinopathy and demonstrates shown). Immunohistochemistry for FGF-2 localized the protein the highly strain-dependent nature of this animal model. to the inner nuclear layer (INL) in the retina of control and STZ-induced diabetes in rats and mice has been used exten- diabetic SD rats, and showed a dramatic increase in FGF-2 sively to study DR. In the rat model, SD rats are a commonly expression in the photoreceptor nuclear layer (PNL) after 3 used strain. However, the lack of pigmentation in these albino months of diabetes. These data suggest subtle inflammatory rats clearly interferes with the function of the visual system,50 changes in the absence of peripheral immune cells in the retina and is therefore of limited use in some studies, particularly of diabetic SD rats that coincide with FGF-2 upregulation in those examining visual function. photoreceptors. In the present study, measurement of a large number of cytokines and growth factors as well as other inflammatory Effect of Hyperglycemia on Gene Expression markers showed that most proteins were either below detect- in Photoreceptors able limits or unchanged in the eyes of diabetic rats. These Gene expression in the retina was normalized to ␤-actin and results match prior published data that failed to show a large compared in diabetic and control BN, LE, and SD rats, to scale inflammatory response in this model. In fact, in BN and LE evaluate the effect of hyperglycemia on photoreceptor func- rats, there is no evidence of cytokine upregulation through tion. After 3 months of diabetes, probe arrays (TaqMan; ABI) three months of diabetes. However, SD rats show signs of a revealed a decrease in photoreceptor-specific opsin (Opn1mw) subtle, but reproducible inflammatory response. Four weeks mRNA in all three strains, suggesting reduced photoreceptor after the induction of diabetes, eotaxin, a basophile and eosin- function.49 Arrestin (Arr3) and rhodopsin kinase (Grk1) ex- ophil chemoattractant, was upregulated in the diabetic retina pression are reduced in BN and SD, but not LE rats, further of SD rats compared with their nondiabetic counterparts. Al- indicating strain-dependent differences in the diabetic retina though increased eotaxin expression in the retina was tran- beyond the inflammatory response. Transducin (Gnb3) mRNA sient, significantly elevated levels were present in the serum of was increased in the retina of diabetic SD rats only (Fig. 6). The diabetic rats after 3 months of diabetes. This excludes serum as decrease of inhibitory molecules arrestin and rhodopsin kinase the source of eotaxin in the retina, since the levels in this tissue mRNA contemporaneous with an upregulation of transducin are decreased at a time when serum levels hit their peak. gene interaction may indicate a compensatory mechanism in Increased protein levels of M-CSF, which act on monocytes and SD rat phototransduction. stimulate their growth, further indicate a response tailored to early immune mediators. This upregulation in SD rats occurred at the same time as increased MCP-1 and -3 protein expression, DISCUSSION cytokines that attract early inflammatory cells such as mono- In this study, we report inflammatory responses in the retina of cytes.51,52 Although MCP-3 is undetectable in vitreous or diabetic SD rats, but not LE or BN strains. Over 50 inflammatory serum of diabetic patients,53 MCP-1 has been found to be proteins and growth factors in the retina of diabetic BN, LE, increased in the vitreous of patients with diabetic retinopa- and SD rats were analyzed, and the data showed a subclinical thy41,53–55 and its level correlates with the presence of reti- inflammatory response in the retina of diabetic SD rats that nopathy.56 These inflammatory changes are subtle, and con- coincided temporally with the upregulation of FGF-2 in pho- centrations of all four cytokines are generally low, but the toreceptors as well as evidence of photoreceptor dysfunction. results are highly reproducible and clearly restricted to only It is the first direct comparison of three commonly used rat one rat strain. Despite the lack of overt inflammation, there are

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clusterin was overexpressed through 3 months of hyperglycemia and indicated early retinal damage. Of interest, though control levels of clusterin were similar between rat strains, there was no increase in either BN or LE rats. Like clusterin, TIMP-1 also showed a small but significant upregulation after 4 weeks of diabetes followed by a more robust change after 3 months in SD rats. Protein levels in the other two rat strains were low in both control and diabetic rats. Increased mRNA of the TIMP-1 ligand MMP-9 have been reported in SD rats,57 and although the proteins can be found in humans in normal interphotoreceptor matrix58 an increase correlates with vitreous hemorrhage in diabetic patients.59 The upregulation of B2M was another indicator of an ongoing immune response in SD rats, as was the increase in vWF after 3 months. Although some of the proteins upregulated in the retina were also increased in serum, these phenomena were most likely independent of each other. Only 3 of the 11 proteins that changed in the retina were also upregulated in serum and more important, eotaxin levels did not reach their peak in serum until 3 months of diabetes, a time at which retina levels had already decreased. The flow cytometry-based technique (IS 100 system; Lumi- nex) used in this study did not measure a significant increase in total VEGF in the diabetic retina of SD or LE rats at the times tested, but did show an increase in BN retina. This difference may be due to a less robust VEGF upregulation in SD than in BN rats, which has been demonstrated in retinal ischemia.8,9 Fur- thermore, vascular permeability in STZ-induced diabetes has been reported to be an early and transient phenomenon in SD rats, whereas the effect is prolonged in BN rats.10 Similar to VEGF expression, FGF-9 protein was upregulated in BN rat retinas, and this effect was transient and only observed at 4 weeks. FGF-2, on the other hand, was upregulated after 3 months in SD rats, confirming published reports.47 Immunohistochemis- try showed no inflammatory infiltrates in the retina, suggesting low-grade inflammation most likely originating in the retina. The amount of cytokines produced is evidently not high enough to induce transmigration of leukocytes into the retina. This result is in agreement with published data in both animals and patients, where increased leukostasis12,60 by neutrophils and macrophages42,60 in retinal vessels is reported without convincing evidence of extravasation of leukocytes into the retina. Although Miyamoto et al.61 report transmigration, the data presented do not lend strong support to such a claim. Immunohistochemistry further showed that FGF-2 is consti- tutively expressed in the INL, and the increase in FGF-2 was due to upregulation restricted to the PNL. Studies in other animal models have shown that FGF-2 is present in Mu¨ller cells and astrocytes,62 but is upregulated in response to injury in the retina only in photoreceptors,62,63 and this upregulation has a protective effect on these cells.64 Although there is a significant increase in apoptotic photoreceptors in diabetic SD rats, most of the photoreceptors appear unaffected.65 To compare photoreceptor viability between diabetic and control rats, genes expressed exclusively in photoreceptors were assessed. This gene expression has been shown to correlate to photore- FIGURE 4. Changes in growth factor expression in the hyperglycemic ceptor condition.66 Opn1mw mRNA was decreased in photo- rat retina. (A) A small, but highly reproducible increase in VEGF receptors of all strains, suggesting degeneration of these cells. expression was found in BN rats after 4 weeks of diabetes. (B) FGF-9 Furthermore, rhodopsin kinase and arrestin3 mRNA, which are was also increased in BN after 1 month, but not significantly changed also expressed exclusively in the photoreceptors in the ret- in any other strain or time point. (C) Protein levels of FGF-2 were 67,68 elevated in SD rats after 3 months of diabetes. Each symbol represents ina, were downregulated in BN and SD rats, but not in LE data from three pooled diabetic or control retinas. Statistical signifi- rats. Finally, Gnb3 expression increased in diabetic retina of cance was determined using Student’s t-test. SD, but not in BN or LE rats. These data suggest a negative effect of high glucose on activation of the phototransduction pathway as demonstrated by reduced opsin mRNA while rein- other indicators that hyperglycemia activates an immune re- forcing the notion of strain-dependent differences in the retina sponse in SD rats as early as 4 weeks after the induction of as seen in rhodopsin kinase, arrestin3, and Gnb3 expression. diabetes. Similar to an earlier report in diabetic SD rats,45 The reduced opsin expression may explain the reported reduc-

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FIGURE 5. H&E stain of the retina and localization of FGF-2. H&E staining of retinas from diabetic (A, C, E) and control (B, D, F) rats after 3 months of diabetes in BN (A, B), LE (C, D), and SD (E, F) showed no inﬂammatory inﬁltrates or changes in retinal thickness in any of the sections. FGF-2 immunostaining reveals an increase in this protein localized to photoreceptor nuclear layer (PNL) in diabetic SD rats (H) compared with control animals (G). Re- sults are representative of two independent experiments with three rats per group.

tion in photoreceptor activation with high flash intensity ERG The results of the present study clearly show that genetic in diabetic SD rats.69 However, the same study did not find an background plays a significant role in the response to hyper- effect on photoreceptor deactivation69 suggesting that the re- glycemia in the retina and is consistent with findings in other duction in rhodopsin kinase and arrestin3, although statistically studies of hyperglycemic and hypoxic stress in the retina.8–10 significant, does not affect function after 3 months of diabetes. This strain-dependent response is not due to differences in Of interest, mRNA of Gnb3 kinase which is part of the trans- glucose exposure in either blood or the area adjacent to the ducin complex is increased in SD rats. This increase may retina, as all three strains show similar levels of hyperglycemia. indicate a compensatory mechanism in damaged photorecep- A possible explanation of the lack of inflammation in LE and BN tors of this strain. rats is the presence of melanin in the two pigmented strains as It is tempting to speculate that hyperglycemia activates opposed to the lack of it in the albino SD rats. Melanin has been retinal glial cells either directly or through the binding of shown to have antioxidant properties by acting as a scavenger advanced glycation endproduct (AGE)–modified proteins to 70,71 AGE receptors on these cells. This activation may lead to for free radicals and reactive oxygen species. Nonpig- mented RPE cells show significantly more oxidative changes production of low levels of inflammatory cytokines. Although 72 their concentration is not high enough to induce infiltration of than pigmented ones and high-dose exposure to light dam- peripheral immune cells into the retina they may play a role in ages photoreceptor outer segments to a much larger extent in 73 retinal vessel leukostasis.61 The cytokines may also constitute a albino Wistar than LE rats. It is possible that hyperglycemia danger signal to photoreceptors, inducing upregulation of by itself is not enough to induce inflammatory changes in the FGF-2 and causing decreased function of photoreceptors. Al- rat eye, but that in nonpigmented rats the increase in oxidative ternatively, the ongoing inflammatory response and high glu- stress provides a secondary stimulus that results in upregula- cose levels may influence photoreceptor gene expression di- tion of inflammatory proteins in the retina and increase of rectly. FGF-2 expression in photoreceptors in SD rats.

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