Polyethylene glycol induced mouse model of retinal degeneration

Experimental Eye Research 127 (2014) 143e152

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Polyethylene glycol induced mouse model of retinal degeneration Valeriy V. Lyzogubov, Nalini S. Bora, Ruslana G. Tytarenko, Puran S. Bora* Department of Ophthalmology, Jones Eye Institute, Pat & Willard Walker Eye Research Center, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 April 2014 Accepted in revised form 23 July 2014 Available online 1 August 2014

Age-related macular degeneration (AMD) is a leading cause of irreversible blindness. This study was done to characterize dry AMD-like changes in mouse retinal pigment epithelium (RPE) and retina after polyethylene glycol (PEG) treatment. We injected male C57BL/6 mice subretinally with PBS, 0.025, 0.25, 0.5 and 1.0 mg of PEG-400 and the animals were sacrificed on day 5. Eyes were harvested and processed for histological analysis. In all other experiments 0.5 mg PEG was injected and animals were sacrificed on days 1, 3, 5 or 14. Paraffin, 5 mm and plastic, 1 mm and 80 nm sections were used for further analysis. Subretinal injection of 0.5 mg PEG induced a 32% reduction of outer nuclear layer (ONL) thickness, 61% decrease of photoreceptor outer and inner segment length, 49% decrease of nuclear density in the ONL and 31% increase of RPE cell density by day 5 after injection. The maximum level of TUNEL positive nuclei in the ONL (6.8 þ 1.99%) was detected at day 5 after PEG injection and co-localized with Casp3act. Histological signs of apoptosis were observed in the ONL by light or electron microscopy. Degeneration of RPE cells was found in PEG injected eyes. Gene expression data identified several genes reported to be involved in human AMD. C3, Cfi, Serping1, Mmp9, Htra1 and Lpl were up-regulated in PEG injected eyes compared to PBS controls. PEG leads to morphological and gene expression changes in RPE and retina consistent with dry AMD. This model will be useful to investigate dry AMD pathogenesis and treatment. © 2014 Elsevier Ltd. All rights reserved.

Keywords: age-related macular degeneration retinal degeneration mouse model pathology

1. Introduction Age-related macular degeneration (AMD) is the leading cause of blindness in the United States as well as around the world. AMD accounts for more than 50% of blindness in Caucasian Americans (Friedman et al., 2004; Congdon et al., 2004). AMD is characterized by drusen deposits, RPE cell changes, geographic atrophy of the RPE cells and choroid capillaries and neovascular maculopathy. There are two clinical subtypes of AMD e dry and wet. These subtypes also represent the pathological stages of AMD. Features of dry AMD can be seen in the “early stage” of AMD that is characterized by accumulation of lipofuscin in RPE, decrease in number of RPE cells Abbreviations: AMD, Age-related macular degeneration; RPE, retinal pigment epithelium; Casp3act, active Caspase 3; PCNA, proliferating cell nuclear antigen; ONL, outer nuclear layer; IACUC, Institutional Animal Care and Use Committee; IHC, immunohistochemistry; PIS&POS, photoreceptor inner and outer segment; OPL, outer plexiform layer; OLM, outer limiting membrane; CK18, cytokeratin 18; MMP9, matrix metalloproteinase 9; HTRA1, high-temperature requirement A serine peptidase 1; ONC, optic nerve crush; ChC, choroidal capillaries. * Corresponding author. Department of Ophthalmology, Jones Eye Institute, Pat & Willard Walker Eye Research Center, University of Arkansas for Medical Sciences, 4301 West Markham Street, Slot#523-7, Little Rock, AR 72205, USA. Tel.: þ1 501 686 8293; fax: þ1 501 686 8316. E-mail address: [email protected] (P.S. Bora). http://dx.doi.org/10.1016/j.exer.2014.07.021 0014-4835/© 2014 Elsevier Ltd. All rights reserved.

and thickening and loss of structure of Bruch's membrane (Coleman et al., 2008; Sarks et al., 1999; Zarbin, 2004). The hallmark of dry AMD is the formation of drusen. Studies have shown that drusen contain acute phase proteins, components of the complement system (C3, C5, C5b-9, CFH and others), lipids, sialic acid and other biochemical components (Coleman et al., 2008; Klein et al., 2005; Nozaki et al., 2006). The “advanced stage” of dry AMD (also known as geographic atrophy) is characterized by hypertrophied RPE cells and calcified Bruch's membrane. Formation of new blood vessels originating from choroid (neovascularization) is the hallmark of wet AMD. Although several studies have focused on treating wet AMD, few studies have reported on treatment for dry AMD (Gehrs et al., 2010; Tan et al., 2008; Yehoshua et al., 2011). Understanding the mechanisms responsible for dry AMD during its initial phases will be highly beneficial in designing new therapeutic strategies for the treatment of dry AMD. For this purpose, availability of a clinically relevant animal model is highly desirable. We have previously shown that PEG can induce choroidal neovascularization in mouse (Lyzogubov et al., 2011). In the present communication we will describe effects of subretinal injection of PEG (0.5 mg) on retina and choroid. The aim of this study was to investigate PEG-induced RPE and retinal degeneration and to develop a simple and effective animal model of dry AMD.

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2. Material and methods 2.1. Animals Male C57BL/6 mice (6e8 weeks old) were purchased from the Jackson Laboratory (Bar Harbor, ME). Animals were kept under 12h dark/12-h light conditions. This study was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Arkansas for Medical Sciences, Little Rock, AR. 2.2. Subretinal injection of PEG Subretinal injection of PEG was performed as previously described (Lyzogubov et al., 2011). Details are given in Supplemental material. To test the dose dependent effect of PEG400 (Polyethylene glycol with mean molecular weight 400, Spectrum Chemicals and Laboratory Products, Gardena, CA) we divided animals into 5 groups (n ¼ 5 animals in each group). We injected PBS, 0.025, 0.25, 0.5 or 1.0 mg of PEG (PEG diluted in PBS). Volume of each injection was 2 mL. Subretinal bleb formation was considered as successful subretinal injection. About 20% retinal detachment was observed immediately after injection however this detachment was further reduced after 24 h of the injection. We have observed that (while injecting) the injected PEG is diffused (spill over) into untreated space and affects photoreceptors and RPE i.e. akin to geographical atrophy (GA). Eyes were harvested at day 5 post-injection. In another experiment we injected 0.5 mg of PEG (n ¼ 5 mice) or PBS (n ¼ 5 mice) and sacrificed animals at days 1, 3, 5 and 14 post-injection. At each time point we fixed 5 eyes in formalin and 5 eyes were processed for electron microscopy. 2.3. Tissue processing for light and electron microscopy We prepared formalin fixed paraffin embedded section and plastic sections for light and electron microscopy using routine methods described in details in the Supplemental materials. We stained paraffin sections with hematoxylin and eosin (H&E) or used the sections for immunohistochemistry (IHC). Semi-thin sections were stained using epoxy tissue stain containing toluidine blue and basic fuchsin (Electron Microscopy Sciences, Hatfield, PA). Thin sections were counterstained with uranyl acetate and lead citrate (both from Polysciences Inc., Warrington, PA) and examined using a FEI Tecnai G2 TF20 transmission electron microscope (FEI Worldwide Corporate Headquarters, Hillsboro, Oregon).

We stained paraffin sections using a mouse monoclonal antiPCNA antibody (Ab) (Cell Signaling, Danvers, MA), a rabbit antiCaspase 3 active (Biovision, Milpitas, CA), rabbit anti-Atg12 (Cell Signaling, Danvers, MA), mouse monoclonal anti-Cytokeratin 18 (CK18) Ab (ABR, Golden, CO), sheep anti-Rhodopsin (Abcam, Eugene, OR); AF488-conjugated goat anti-mouse IgG (H þ L); AF594-conjugated donkey anti-rabbit IgG (H þ L), AF594conjugated donkey anti-sheep IgG (H þ L) (all from Molecular Probes, Eugene, OR). The mouse on mouse (M.O.M.) Kit (Invitrogen, Valencia, CA) was used to block possible non-specific binding of mouse Ab to mouse IgGs localized in mouse tissue. Negative control sections were stained with isotype matched control Ab at identical concentrations to those of the primary Ab or without primary or secondary Ab. Nuclei were stained using ProLong antifade reagent with DAPI (Invitrogen, Grand Island, NY). The Olympus Vanox-S AH-2 fluorescent microscope (Olympus Optical, Japan) was used to count TUNEL-positive nuclei in the ONL. Representative images were captured using the laser confocal microscope LSM510. 2.6. Total RNA extraction Mouse eyes, injected with PBS (n ¼ 5) or 0.5 mg PEG (n ¼ 5) were collected on day 5 after injection. Total RNA was purified separately from retina and RPE-choroid using RNasy protect minikit (Qiagen, Valencia, CA). The RNA from retina and RPE-choroid was shipped to the DNA Facility Core Lab, University of Iowa, IA for gene expression analysis. 2.7. Statistical analysis Data were analyzed and compared using ANOVA or Student ttest, and differences were considered statistically significant with P < 0.05. Data are presented as mean value (M) ± standard error (SE). 3. Results 3.1. Dose- and time-dependent effect of PEG on the thickness of ONL

Three H&E stained paraffin sections of each eye were chosen for analysis. We captured 3 images from each eye close to site of the injection between optic nerve and ciliary body. Site of the injection was identified by the presence of tiny retinal damage caused by the needle. We used the ImageJ program (NIH, Bethesda, MD) to measure thickness of the outer nuclear layer (ONL), length of photoreceptor inner and outer segment (PIS&POS), density of nuclei in the ONL and number of RPE cells in the RPE layer. Three measurements were performed for each image. All measurements were performed in a blinded manner by two investigators.

In a first experiment we injected PBS, 0.025, 0.25, 0.5 and 1.0 mg of PEG subretinally to investigate the dose-dependent effect of PEG on thickness of the ONL of the retina. The mice were sacrificed on day 5 post-PEG injection (n ¼ 5 mice/group). Eyes were harvested and paraffin (5 mm) sections were stained with H&E. We measured ONL thickness and found that 0.5 and 1.0 mg of PEG significantly (ANOVA test, p < 0.05) reduced thickness of the ONL (Fig. 1AeF). In a second experiment, the minimal effective dose (0.5 mg) of PEG or PBS was injected (subretinal) to investigate the time-dependent effect of PEG on thickness of the retinal ONL. The PEG and PBS injected animals (n ¼ 5 mice/group) were sacrificed at days 1, 3, 5 and 14 after injection. We measured ONL thickness in paraffin sections stained with H&E and found significant (ANOVA test, p < 0.05) reduction of ONL thickness on days 5 and 14 in PEG injected animals compared to PBS treated controls (Fig. 1G). Based on these findings we used subretinal injection of 0.5 mg of PEG for further experiments, and all subsequent animals were sacrificed on day 5 after injection.

2.5. TUNEL assay and immunohistochemistry

3.2. PEG-induced caspase-dependent apoptosis of photoreceptors

The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method was used to detect apoptotic cells. The paraffin sections were stained with the TdT in Situ Apoptosis Detection Kit e Fluorescein TUNEL-based Apoptosis Detection Assay (R&D Systems, Minneapolis, MN).

Photoreceptor death is a characteristic of dry AMD (Zarbin, 2004). We hypothesized that reduction of ONL thickness after subretinal injection of PEG was caused by death of photoreceptors. This photoreceptor cell death may be caused by apoptosis. To test this hypothesis we performed TUNEL assays and IHC for Casp3act

2.4. Morphometry

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Fig. 1. Dose and time-dependent degenerative changes in mouse retina after subretinal injection of PEG. Mouse eyes were injected with PBS, 0.025, 0.25, 0.5 or 1.0 mg of PEG and sacrificed 5 days after injection. Paraffin sections (5 mm) of the eyes were stained with H&E, and images of the sections were captured. Thickness of the outer nuclear layer (ONL) was measured using the ImageJ program. Representative images of (AeE) demonstrate reduction of ONL thickness after injection of 0.5 and 1.0 mg of PEG. A graph (F) shows statistically significant (ANOVA, *p < 0.05) differences of ONL thickness after injection of 0.5 and 1.0 mg of PEG compared to PBS injected controls. Mouse eyes were injected with PBS or 0.5 mg of PEG, and animals were sacrificed on days 1, 3, 5 and 14 after injection. ONL thickness was measured. A graph (G) shows a statistically significant (ANOVA, *p < 0.05) difference of ONL thickness after injection of 0.5 mg PEG compared to PBS injected controls on days 5 and 14.

using paraffin sections. We found TUNEL positive nuclei in the ONL of the retina in PBS and PEG injected eyes (Fig. 2AeH). Quantification of TUNEL positive nuclei showed a significant (ANOVA test, p < 0.05) increase of % of TUNEL positive nuclei in PEG treated eyes compared to PBS controls on days 3 and 5 after injection (Fig. 2I). Co-localization experiments of TUNEL staining and IHC staining were also performed for Casp3act using PEG injected eyes on day 5 after injection. A strong co-localization of TUNEL positive nuclei was observed with cytoplasmic and nuclear staining for Casp3act in the ONL (Fig. 2JeR). We observed condensation of chromatin, fragmentation of nuclei and formation of apoptotic bodies in the ONL of PEG treated eyes by light (Fig. 3B) and electron microscopy (Fig. 3E). These signs of apoptosis were not found in PBS treated eyes (Fig. 3A and D). Our results also showed that POS in PBS treated eyes exhibited a striated pattern because of numerous lamellae (Fig. 3A). The microscopic examination of PEG injected eyes demonstrated a reduction of PIS and POS length (Fig. 3B). The total length of PIS and POS (PIS&POS) was measured using paraffin sections stained with H&E in PEG and PBS treated animals. A statistically significant (t-test, p < 0.05) decrease (61%) of PIS&POS length in PEG injected eyes was observed compared to PBS controls (Fig. 3C). The density of nuclei in ONL (number of nuclei per 1 mm of outer limiting membrane length) in paraffin sections stained with H&E in PEG and PBS treated animals was also investigated. We observed a statistically significant (t-test, p < 0.05) reduction (49%) of nuclear density in PEG injected eyes compared to PBS controls (Fig. 3F).

3.3. PEG-induced degeneration of RPE and activation of autophagy We investigated plastic (1 mm) sections of mouse eyes stained with toluidine blue and basic fuchsin using light microscopy. Microscopic examination revealed that in the eyes of mice injected with PBS (control group) RPE cells form a single layer of uniform flat cells attached to Bruch's membrane (Fig. 4A). Nuclei of RPE cells were oval and oriented parallel to Bruch's membrane. We found areas of RPE degeneration: reduction of RPE thickness, depigmentation, and deposition of “drusen”-like structures in eyes injected with PEG (Fig. 4B). The paraffin (5 mm) sections of PBS and PEG injected eyes were stained for a molecule involved in autophagy, Atg12, and we found a local increase of Atg12 expression in the RPE of PEG treated eyes (Fig. 4D) compared to PBS control eyes (Fig. 4C). No positive staining was found in negative control sections (Fig. 4E, F). We investigated thin (80 nm) plastic sections of PBS (Fig. 4G) and PEG (Fig. 4HeJ) treated eyes using electron microscopy and found macroautophagosomes located in RPE (Fig. 4H, I) in PEG injected eyes. Macroautophagosomes contained vesicles of different size, mitochondria, pigmented granules and membranous material (Fig. 4HeJ). Autophagosomes were separated from cytoplasm by double layered membranes (Fig. 4I). 3.4. PEG-induced proliferation of RPE We examined paraffin sections stained using the TUNEL method and, unlike in the ONL, did not find any TUNEL positive nuclei in

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Fig. 2. Caspase dependent apoptosis in ONL. Mouse eyes were injected with PBS or 0.5 mg of PEG, and animals were sacrificed on days 1, 3, 5 and 14 after injection. Paraffin sections (5 mm) of the eyes were stained using the TUNEL method. Sections were captured using an LSM510 laser confocal microscope. Images of green channel (TUNEL) and differential interference contrast (DIC) were merged. Representative images of mouse retina demonstrate TUNEL positive nuclei (green color) in PBS (AeD) and PEG (EeH) injected groups at different time points. We counted TUNEL positive nuclei and total number of nuclei in the ONL and calculated the fraction of TUNEL-positive nuclei in the ONL. A graph (I) shows a statistically significant (ANOVA, *p < 0.05) increase of the fraction of TUNEL-positive nuclei in the ONL in PEG injected eyes compared to PBS injected control group at days 3 and 5 after injection. Paraffin sections of PEG injected eyes (day 5 after injection) were stained using the TUNEL method and immunohistochemistry for Casp3act. Images of red channel (J, M, P; Casp3act), green channel (K, N, Q; TUNEL), and merged (L, O, R) red, green and blue channel (DAPI staining of nuclei) were captured using the LSM510 laser confocal microscope in the ONL. Panel L shows that TUNEL-positive nuclei (green color) were co-localized with positive staining for Casp3act (red color). TUNEL-negative nuclei (blue color) did not stain for Casp3act. Negative controls for Casp3act (M and 0) and TUNEL (P and R) showed no red staining. IPL e inner plexiform layer; INL e inner nuclear layer; OPL e outer plexiform layer; ONL e outer nuclear layer; PIS e inner segment of photoreceptors.

RPE after PBS or PEG injection at any time points described above (data not shown). At the same time we found areas of RPE cell proliferation in plastic (Fig. 5B) and paraffin sections (Fig. 5 I and K). RPE formed two or more layers; some RPE cells were round shaped, depigmented and full of phagosomes of shredded POS (Fig. 5B). IHC for rhodopsin in areas of RPE proliferation showed numerous rhodopsin positive granules located in cytokeratin 18 positive pigmented cells (RPE cells) in eyes of PEG-treated group (Fig. 5E). Proliferation of RPE and overload of cells with shredded rhodopsin positive material was a consistent finding in PEG injected eyes and was absent in all PBS treated eyes investigated (Fig. 5A, D). No positive staining was found in negative control sections (Fig. 5F, G). We counted the number of RPE cells and calculated density of RPE cells (number of RPE cells per mm of RPE layer) and found a statistically significant (t-test, p < 0.05) increase (31%) in density of RPE cells in PEG treated eyes compared to PBS controls (Fig. 5C).

The paraffin sections of PEG and PBS injected eyes were stained for PCNA (a proliferation marker) using IHC. We did not observe positive staining in RPE of PBS injected eyes (Fig. 5H). No positive staining was found in negative control sections (Fig. 5J, K). We found intense PCNA positive staining of nuclei and cytoplasm of RPE cells exclusively in PEG injected eyes (Fig. 5I). 3.5. Gene expression analysis We investigated effect of PEG on genes expression in RPEchoroid and retina separately. We dissected eyes at day 5 after injections. At this time point retina can be easily separated from RPE and choroid. Most of the affected genes were up-regulated, and only few genes (Slc35d3 and Rgr) were down-regulated (Table 2, Table 3, Supplemental) in the PEG treated group compared to PBS control. We have only included genes which were 2 or more fold

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Fig. 3. Photoreceptor degeneration in PEG injected mouse eyes. Mouse eyes were injected subretinally with PBS (A, D) or 0.5 mg of PEG (B, E). Animals were sacrificed on day 5 after injection. Plastic sections (1 mm) were stained with toluidine blue and basic fuschin and observed under light microscopy (A, B). Plastic sections (80 mm) were observed under electron microscopy (D, E). Nuclei with condensed chromatin (arrows), fragmented nuclei (f) and apoptotic bodies (ab) were found in the ONL of PEG injected eyes (B, E) but not in PBS injected controls (A, D). Combined PIS&POS length was measured. A graph (C) showed a significant (t-test, *p < 0.05) reduction of PIS&POS length in PEG treated eyes. Numbers of nuclei in the ONL were counted, and length of the OLM was measured. Ratio of nuclei in the ONL per mm of OLM was significantly (t-test, *p < 0.05) decreased in PEG treated eyes (F).

up-regulated or down-regulated and p < 0.05 in the PEG treated group compared to PBS control (Table 2, Table 3, Supplemental). There was a big difference between responses of RPE-choroid and retina to PEG-induced damage. We found that only 7 genes, Akr1b8, C3, Casp1, Fcgr4, Lyz2, Serpina3n and Timp1, were up-regulated in both RPE-choroid and retina after PEG treatment. However, we observed that several other genes were up regulated in PEG injected mouse RPE-choroid (C3, Mmp9 and Htra1) or retina (C3, Cfi, Serping1 and lpl) compared to PBS injected mice (Table 1; Tables 2 and 3 Supplemental). These up-regulated genes were previously described as having a role in human AMD (Chen et al., 2010; Cipriani et al., 2012; DeWan et al., 2007; Gehrs et al., 2010; Holliday et al., 2013; Hussain et al., 2011; Lee et al., 2010; Merle et al., 2013; Seddon et al., 2013; Tian et al., 2012; Wang, 2014). We also detected up-regulation of genes involved in apoptosis and proliferation, immunoregulation and inflammation, autophagy and phagocytosis (Table 1; Tables 2 and 3 Supplemental) in PEG injected mouse RPE-choroid or retina compared to PBS injected mice. 4. Discussion Mouse models are very useful for studying various pathological mechanisms and pathways for different ocular diseases (Chang

et al., 2005; Hafezi et al., 2000; Ramkumar et al., 2010). One of the main reasons for the usefulness of mouse models is the availability of various transgenes and knockout strains. There are several mouse models for retinal degeneration including wet AMD that have helped in investigating therapeutic options for various retinal disorders (Chang et al., 2005; Davis et al., 2012; Hafezi et al., 2000; Lyzogubov et al., 2009, 2010; 2011; Ramkumar et al., 2010). Key pathological evens of human dry AMD (lipofuschin accumulation, deposit and drusen formation, RPE and retinal degeneration) were reproduced in different genetically modified mice: Cp/  Heph/Y mice (Hadziahmetovic et al., 2008); Cfh/ mice (Coffey et al., 2007); Cfh/ mice with expression of chimeric CFH proteins (CfhTg/mCfh) (Ufret-Vincenty et al., 2010); Ccl2/ and Ccr2/ mice (Ambati et al., 2003); Cx3crI/ mice (Zeiss, 2010); Ccl2/ Cx3crI/ double KO mice (Tuo et al., 2007; , Ross et al., 2008); Sod/  mice (Imamura et al., 2006). Bright continuous light exposition to SpragueeDawley rats induced increased expression of the complement components (C1s,C2,C3, C4), complement receptors CR3 and CR4, C3ar1 and C1qr1 genes in retinas (Rutar et al., 2011). Mutations in fibulin-3 gene in mice caused AMD-like phenotype and activation of the complement system in area of sub-RPE deposits formation (Fu et al., 2007). Immunization of mice with mouse serum albumin adducted with carboxyethylpyrrole

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Fig. 4. RPE degeneration and autophagy activation in PEG injected mouse eyes. Mouse eyes were injected subretinally with PBS or 0.5 mg PEG. Animals were sacrificed 5 days after injection. Plastic sections (1 mm) were stained with toluidine blue and basic fuschin and observed under light microscopy (A, B). Panel A represents normal structures of RPE-choroid after PBS injection. Reduction of RPE thickness, depigmentation and accumulation of “drusen”-like deposits between RPE and Bruch's membrane (arrows) were found in PEG treated eyes (B). Paraffin (5 mm) sections were stained for autophagy molecule Atg12 using IHC (C, D) and observed under a fluorescent microscope. Atg12 positive staining (red) was increased in PEG treated eyes (D) compared to PBS controls (C). No positive staining was observed in negative control (without primary Ab) sections of PBS (E) and PEG (F) treated eyes. Plastic sections (80 mm) were observed under electron microscope (GeJ). Panel G represents normal structures of RPE-choroid after PBS injection. Macroautophagosomes (star) were observed in PEG treated eyes (HeJ). Image I is an enlarged part (square) of image H. These structures were located in the cytoplasm or close to Bruch's membrane (BM). Mitochondria (m), pigmented granules (p), and vesicular structures (v) were found in macroautophagosomes (I, J). Autophagosomes were separated from the cell cytoplasm by double layered membrane (I, arrows). ChC e choroidal capillaries.

induces production of antibodies to this hapten, accumulation of C3d in Bruch's membrane, formation of drusen (Salomon et al., 2011). “Smoking mice” were suggested as a good model for investigation of accumulation of drusen-leke proteins in Bruch's

membrane (Wang and Neufeld, 2010). Most of these models require expensive animals and long time to develop signs of dry AMD. We used wild type mice, inexpensive agent (PEG) to induce retinal degeneration; and only in 5 days atrophy and

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Fig. 5. RPE proliferation in PEG injected mouse eyes. Mouse eyes were injected subretinally with PBS (A) or 0.5 mg of PEG (B). Animals were sacrificed 5 days after injection. Plastic sections (1 mm) were stained with toluidine blue and basic fuschin and observed under light microscopy (A, B). Panel A represents normal structures of RPE-choroid after PBS injection. The RPE had two or more layers in PEG treated eyes (B). Number of RPE cells per mm of RPE layer was significantly (*p < 0.05) increased in the PEG treated group compared to PBS controls (C). Immunohistochemical staining for rhodopsin (red color), RPE cell marker cytokeratin 18 (CK18, green color) and DIC (black and white) showed on images D and E. Rhodopsin was detected in photoreceptor's outer segment (POS) of both PBS and PEG treated groups. Rhodopsin was detected within RPE cells in PEG treated group only except a rhodopsin positive granules was located in pigmented CK18 positive RPE cells in PBS injected eyes (arrow, D). Numerous rhodopsin positive granules were found in PEG treated eyes (arrows, E). Rhodopsin positive granules were located in basal area of RPE cells close to Bruch's membrane (BM). No positive red staining was observed in negative control sections of PBS (F) or PEG (G) treated eyes. Immunohistochemical staining for the proliferation marker PCNA (green staining) showed increased expression of PCNA in PEG treated eyes (I) compared to PBS controls (H). No positive green staining was observed in negative control sections of PBS (J) or PEG (K) treated eyes.

proliferation of RPE cells and significant photoreceptor loss were observed. We reported before that 1.0 mg of PEG-400 induces CNV in mice (Lyzogubov et al., 2011). In the present study we propose a novel mouse model for dry AMD by subretinal injection of lower dose (0.5 mg) of PEG. This model is fast as well as cost effective. Two

hallmarks of dry AMD pathology are retinal changes and degeneration of RPE cells (Zarbin, 2004). Degeneration of RPE and photoreceptors lead to impairment of the blooderetinal barrier (Sarks et al., 1999; Zarbin, 2004). Progression of dry AMD is also associated with proliferation of RPE (Christenbury et al., 2013; Venza et al., 2012). Our results clearly demonstrated degenerative changes in

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Table 1 Functional characteristics of genes whose expression changed in RPE-choroid and retina of PEG treated animals. All genes presented in this table were up-regulated in the PEG treated group compared to PBS control. Function

AMD or RPE pathology Apoptosis and proliferation

Immunoregulation and inflammation Autophagy and phagocytosis

Localization RPE-choroid

Retina

C3, Htra1, Mmp9 Cdkn2b, Htr2b, Fbln2, Timp1, Mmp9, Fabp5, Clec7a, Serpina3n, Prc1, Plk1, Gadd45a, Fblim1, Casp1, Nckap1l, Card11, Gtse1, Lgals3, Vav1, Samsn1, C3, Aurka, Rrm2 Casp1, Selplg, Lgals3, Il10ra, Vav1, Ncf4, Slc11a1, Samsn1, Csf2rb2, Btk, Mefv, Ifi205, Scgb1a1, Alox5ap, Grn, Tlr13, C3, Slamf7, Pira3, Chi3l3 Trem2, Gadd45a, Lyz1, Lyz2, Laptm5, Slamf7, Casp1, Aurka

C3, Cfi, Serping1, lpl Timp1, Lcn2, Bcl3, Socs3, Edn2, Nupr1, Ifi27, Tubb6, Casp1, C3, S100a6, S100a11, Gal, Cd44, Bst2, Trf, Pcolce, Cebpb, Mvp, Ifitm3, Ifit3, Parp14, Scotin, Eif2ak2, Cdkn1a, Junb, Egr1, Gadd45b, Chi3l4 Lyzs, Bcl3, Serpina3n, Fcgr4, Serping1, Rsad2, Cd52, B2m, Fyb, Osmr, Bst2, Lgals3bp, Igtp, Psmb9, Usp18, Cfi, Ccl4, Oasl2, H2-T23, Ifitm3, Irf9, Gbp2, Clec2d, H2-Q7, Vcam1, ifi47Efemp2, H2-Q8 Cebpb, Gadd45b, Lyz2, Casp1

RPE (reduction of RPE thickness, depigmentation, and deposition of “drusen”-like structures), and proliferation of RPE (expression of PCNA and increase of the density of RPE cell nuclei) in PEG treated mouse eyes compared to PBS controls. Results from our present study provide evidence that PEG-induced pathological changes in mouse eyes were similar to the pathological changes seen in human dry AMD. In the present study we used a dose (0.5 mg) of PEG and time point (day 5 after subretinal injection) to create numerous signs mimicking key events in dry AMD. In our previous work (Lyzogubov et al., 2011) we used 1 mg of PEG to induce CNV however, in the present study we used 0.5 mg of PEG injection to focus on degenerative changes in RPE and retina after subretinal injection of PEG, with this concentration (0.5 mg) we see tiny amount of CNV or no CNV. However, the changes we observed after 0.5 mg PEG injection closely resemble to dry AMD like changes. In the present work changes in neuronal retina and RPE were investigated. Our results demonstrate that PEG affects photoreceptors in the ONL. Photoreceptors die by caspase dependent apoptosis, which leads to reductions of ONL thickness, density of cells in the ONL, and shortening of PIS&POS length. These changes in the retina are similar to photoreceptor loss in human dry AMD (Coleman et al., 2008; Sarks et al., 1999; Zarbin, 2004). In our PEGinduced model pathological changes in retina occur in parallel to changes in the RPE. We found both degeneration (depigmentation, reduction of size, accumulation of phagosomes with membranous material, and “drusen”-like structures) and proliferation of RPE cells after injection of PEG. We suggest that PEG primarily affects function of RPE cells by activating the complement system as shown in our previous publication (Lyzogubov et al., 2011). Accumulation of C3, MAC and action of others components of the complement system may lead to RPE cell dysfunction (Gehrs et al., 2010; Klein et al., 2005; Nozaki et al., 2006). Depigmentation of the RPE in our study was correlated with two processes: 1) autophagy, and 2) proliferation of RPE. During autophagy portions of cytoplasm including pigment granules become digested, which contributes to loss of pigmentation of RPE. Proliferation and dedifferentiation of RPE cells may also lead to reduction of pigmentation. Depigmentation of the RPE reduces the protective functions of these cells and reduces support and nutrition of the neuronal retina (Loskutova et al., 2013). Death of photoreceptors and resultant formation of debris is another factor affecting RPE, which becomes overloaded with phagosomes containing POS and cellular fragments. Increased phagocytosis and autophagy may lead to accumulation of lipofuscin e one of the important players in pathogenesis of dry AMD (Coleman et al., 2008). We found in the present work a correlation between “drusen”-like structures seen on the light microscopic level and macroautophagosomes observed by electron microscopy. We believe that it should be possible to investigate the pathogenesis of drusen formation using this PEGinduced model.

Autophagy was previously described in human and mouse RPE (Frost et al., 2014; Lee et al., 2014; Yao et al., 2014). Autophagy or phagocytosiseautophagy pathways have a role in clearance of photoreceptors outer segment, maintenance of retinoid levels (Kim et al., 2013; Yao et al., 2014), cell survival (Frost et al., 2014; Lee et al., 2014). Macrophagosomes were observed in wild type C57BL/6 mice under normal cycling light conditions (Yao et al., 2014). We observed PEG-induced formation of double membrane autophagosomes in RPE and also observed the increase of rhodopsin positive material in RPE after PEG treatment. Thus, we and other investigators, mentioned above, believe that classic autophagy occurs in RPE. We found several genes up-regulated which are involved in regulation of response to injury; regulation of the immune system, cell death and proliferation. It is noteworthy that some of the genes up regulated in PEG injected eyes were similar to human AMD or RPE pathology (Chen et al., 2010; Cipriani et al., 2012; DeWan et al., 2007; Gehrs et al., 2010; Holliday et al., 2013; Hussain et al., 2011; Lee et al., 2010; Merle et al., 2013; Seddon et al., 2013; Tian et al., 2012; Wang, 2014). Complement components C3 and CFI belong to the complement system, well known to be involved in AMD (Cipriani et al., 2012; Chen et al., 2010; Gehrs et al., 2010; Klein et al., 2005; Nozaki et al., 2006; Seddon et al., 2013; Tian et al., 2012). Component of the classic complement activation Serping1 is supposed to have a role in AMD (Lee et al., 2010). Our data show up-regulation of Serping1 in PEG injected retinas but not RPE-choroid. Matrix metalloproteinase 9 (MMP9) is a proteolytic enzyme participates in regulation of Bruch's membrane homeostasis and plays a role in pathogenesis of AMD (Hussain et al., 2011). Increase of mmp9 gene expression in PEG-induced retinal degeneration may lead to increased expression of MMP9 protein and predispose to increased degradation or remodeling of Bruch's membrane. We found up-regulation of htra1 in mouse retinas of eyes injected with PEG. High-temperature requirement A serine peptidase 1 (HTRA1) is involved in degradation of the extracellular matrix. HTRA1 gene and protein is expressed in human retina and RPE. HTRA1 is elevated in AMD and may be detected in drusen (Gehrs et al., 2010; Tian et al., 2012). HTRA1 gene polymorphism is strongly linked to AMD (Cipriani et al., 2012; DeWan et al., 2007; Gehrs et al., 2010; Lee et al., 2010; Tian et al., 2012; Wang, 2014). Fibroblast growth factor 2 is considered as a trophic and protective factor for photoreceptors in different pathologic conditions (Gao and Hollyfield, 1996; Yamada et al., 2001). Up-regulation of Serping1, C3, Casp1 and Cfi was detected in mouse retina in the optic nerve crush (ONC) mouse model e the model exploring the role of innate immunity in diseases like glaucoma and AMD (Templeton et al., 2013). We also found Osmr and Serpina3n increase in the retina and Edn2 and Cebpb increase in the RPE-choroid of PEG treated eyes. Osmr, Serpina3n and Cebpb up-regulation probably reflects a non-specific response of damage to the retina. Two mouse models of retinal detachment and light-damage (Rattner et al.,

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2008) showed increase of Osmr and Serpina3n transcripts in the RPE and inner nuclear layer and increase of Edn2 and Cebpb in retina in the detachment model e a model of retinal detachment. We found up-regulation of several genes involved in phagocytosis and autophagy in RPE-choroid and retina of PEG-injected eyes (Table 1). Trem2 is a phagocytic receptor and has a significant neurodegeneration role in Alzheimer's disease (Lucin et al., 2013). Gadd45a is involved in cell cycle arrest and induction of autophagy (Ebert et al., 2012; Galluzzi et al., 2012) and atrophy (Ebert et al., 2012). Slamf7 up-regulation was detected in RPE-choroid and retina of PEG injected eyes. Slamf7 belongs to the SLAM (CD150) family, which are possible partners of Beclin 1 e a key regulator of autophagy (Kang et al., 2011). Casp1 is involved in regulation of autophagy and has a protective role against hepatocyte cell death (Sun et al., 2013). Activation of autophagy in RPE exerts a protective effect against cell death (Repnik et al., 2012). These gene expression findings further support the idea that this dry AMD model should be very valuable to study dry AMD. We conclude that changes in morphology and gene expression caused by PEG in RPE and retina are consistent with human dry AMD. PEG is well known activator of complement system and very useful tool to induce proliferation and death of RPE cells. This simple and fast model may be useful to investigate the pathogenesis of retinal degeneration and dry AMD like changes. Acknowledgments This work was supported in part by Dr. Phil Palade and Department of Ophthalmology Research foundation, Pat & Willard Walker Eye Research Center, Jones Eye Institute, Little Rock, AR. We thank Dr. Philip Palade for the critical review and comments on the manuscript. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.exer.2014.07.021. References Ambati, J., Anand, A., Fernandez, S., Sakurai, E., Lynn, B.C., Kuziel, W.A., Rollins, B.J., Ambati, B.K., 2003. An animal model of age-related macular degeneration in senescent Ccl-2- or Ccr-2-deficient mice. Nat. Med. 9, 1390e1397. Chang, B., Hawes, N.L., Hurd, R.E., Wang, J., Howell, D., Davisson, M.T., Roderick, T.H., Nusinowitz, S., Heckenlively, J.R., 2005. Mouse models of ocular diseases. Vis. Neurosci. 22, 587e593. Chen, W., Stambolian, D., Edwards, A.O., Branham, K.E., Othman, M., Jakobsdottir, J., Tosakulwong, N., Pericak-Vance, M.A., Campochiaro, P.A., Klein, M.L., Tan, P.L., Conley, Y.P., Kanda, A., Kopplin, L., Li, Y., Augustaitis, K.J., Karoukis, A.J., Scott, W.K., Agarwal, A., Kovach, J.L., Schwartz, S.G., Postel, E.A., Brooks, M., Baratz, K.H., Brown, W.L., , Complications of Age-Related Macular Degeneration Prevention Trial Research Group, Brucker, A.J., Orlin, A., Brown, G., Ho, A., Regillo, C., Donoso, L., Tian, L., Kaderli, B., Hadley, D., Hagstrom, S.A., Peachey, N.S., Klein, R., Klein, B.E., Gotoh, N., Yamashiro, K., Ferris 3rd, F.,
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