Novel organotypic culture model of adult mammalian neurosensory retina in co-culture with retinal pigment epithelium

Journal of Neuroscience Methods 173 (2008) 47–58

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Novel organotypic culture model of adult mammalian neurosensory retina in co-culture with retinal pigment epithelium Stefanie Kaempf ∗ , Peter Walter 1 , Anna Katharina Salz 2 , Gabriele Thumann 3 Department of Ophthalmology and IZKF “BIOMAT.”, RWTH Aachen University, Pauwelsstr. 30, 52074 Aachen, Germany

a r t i c l e

i n f o

Article history: Received 13 August 2007 Received in revised form 13 May 2008 Accepted 16 May 2008 Keywords: Retina Tissue culture RPE GFAP Vimentin GS S100

a b s t r a c t Purpose: The purpose of this study was to assess survival of adult mammalian neurosensory retina cultured in contact with the layer of a choroid–retinal pigment epithelium (RPE) explant. Methods: The entire adult porcine neurosensory retina and RPE–choroid layer were placed in tissue culture by juxtaposing both tissues in their original orientation. Culture of the neurosensory retina alone and freshly prepared retina were used as control. After 3 days in culture retinal explants were fixed and processed for immunohistochemistry and TUNEL technique. Results: We observed limited nuclei loss and significant reduction in apoptotic cells in nuclear cell layers (GCL, INL, and ONL) and decreased Muller cell hypertrophy in retina–RPE cultures compared to retinal cultures alone. In addition, cultures were characterized by reduced upregulation of GFAP, vimentin as well as S100 and increased glutamine synthetase expression. Conclusions: As any tissue culture model, retinal tissue culture is a short-term system and since degenerative processes begin quite early it may be a good model to investigate degenerative processes in the retina. However, our model of culture of retina adjacent to the RPE–choroid layer improves the maintenance of neural retina as evidenced by reduced apoptosis in nuclear cell layers (GCL, INL, and ONL) and reduced gliosis as indicated by the diminished expression of glial-specific proteins and increased glutamine synthetase compared to cultures of retina alone. Thus the retina–RPE–choroid culture system can enable the evaluation of interactions between RPE and neural retina, the role of signaling molecules as well the effect of pharmaceuticals on retinal biology. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Cell culture experiments can provide valuable information toward the understanding of physiological and biological mechanisms. The controlled environment in cell and tissue culture provides conditions for the analysis of individual biological mechanisms such as the action of neurotrophic factors or the function of synapses (Goureau et al., 2004; Doonan et al., 2005; Lagreze et al., 2005). However, the main difficulty in studies of adult neural tissues is the limited survival in culture (Germer et al., 1998; Winkler et al., 2000; Garcia et al., 2002; Ito et al., 2004; Rzeczinski et al., 2006).

∗ Corresponding author. Tel.: +49 241 80 35 944; fax: +49 241 80 82 490. E-mail addresses: [email protected] (S. Kaempf), [email protected] (P. Walter), [email protected] (A.K. Salz), [email protected] (G. Thumann). 1 Tel.: +49 241 80 88 191. 2 Tel.: +49 241 80 35 943. 3 Tel.: +49 241 80 35 728. 0165-0270/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2008.05.018

The first organ culture of eye tissue was performed by placing whole embryonic chick eyes in plasma clots (Strangeways and Fell, 1926). Tansley (1933) advanced the technique by extending the method to mammalian tissue. Retinal explant cultures in the first half of the 20th century were primarily grown in plasma clots or in a collagen matrix using a roller tube method, also known as the flying coverslip method and variations on this method are still used today (Hild and Callas, 1967; LaVail and Hild, 1971; Feigenspan et al., 1993; Rothermel et al., 2005; Rzeczinski et al., 2006). In the 1950s Trowell (1954) developed the membrane culture in which the tissue is placed on a porous membrane on top of a wire grid and maintained at the air–medium interface. This technique with the photoreceptor cell layer uppermost has been used by a number of investigators to study retinal explants (Lucas, 1958; Lucas and Trowell, 1958; Sidman, 1963; Tamai et al., 1978). Caffe et al. (1989) have developed a method in which the neural retina is placed with the vitreal surface facing upwards on rafts made of nitrocellulose filters and polyamide gauze grids. Tissue culture models of isolated mammalian embryonic or postnatal neurosensory retinas have been used to evaluate differentiation of developing retina under variable conditions (Caffe et

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al., 1993; Germer et al., 1997a, 1998; Hoff et al., 1999; Feigenspan et al., 1993; Rowe-Rendleman et al., 1996; Engelsberg et al., 2004; Katyal and Godbout, 2004; Pinzon-Duarte et al., 2004; Ouchi et al., 2005; Rothermel et al., 2005; Zelina et al., 2005; Beier et al., 2006). In addition, mammalian retinal organ culture has provided valuable insights in patho-physiological processes of neurodegenerative diseases (Ogilvie et al., 1999; Katsuki et al., 2004; Cossenza et al., 2006; Hartani et al., 2006), has proved useful to test therapeutic substances (Katsuki et al., 2004; Cossenza et al., 2006; Syed et al., 2006) and to examine the role of growth factors (Delyfer et al., 2005; Lagreze et al., 2005; Franke et al., 2006). However, for the analysis of whole retina in vitro, tissue culture models are limited by a variety of problems, such as tissue edema (Ogilvie et al., 1999), proliferation, hypertrophy and migration of Muller cells (Caffe et al., 1993), rapid degeneration of outer segments (Caffe et al., 1993; Kuhrt et al., 2004) and thinning of the retinal layers (Caffe et al., 1989; Ogilvie et al., 1999; Kuhrt et al., 2004). Even though these shortcomings restrict the information and interpretation of the data obtained, especially in studies of microglia activation, neural and Muller cell remodeling (Mertsch et al., 2001; Johansson and Ehinger, 2005) the similarity between the characteristics of retinal detachment in vivo and models of retinal degeneration in vitro allows for investigations of pharmacological and bioengineering treatment modalities (Azadi et al., 2007; Liljekvist-Larsson and Johansson, 2007). The first neural retina–RPE culture was reported by Tamai et al. (1978) and a number of other investigators have published studies of neural retina–RPE cultures using embryonic or postnatal tissue from rodents (Caffe et al., 1989; Pinzon-Duarte et al., 2004; Soderpalm et al., 1999), chicken (Strangeways and Fell, 1926; Rizzolo et al., 1994; Katyal and Godbout, 2004), frog (Jablonski et al., 2001; Wang et al., 2005), and pig (Saikia et al., 2006). The purpose of this study was to establish cultures of adult porcine neural retina–RPE explants and to define its survival in culture. 2. Materials and methods 2.1. Tissue isolation Porcine eyes were obtained from a local slaughterhouse within 1 h of sacrifice and transported to the laboratory on ice. The eyes were washed 3 min in 70% ethanol followed by a wash with ice-cold, sterile 150 mM phosphate buffer saline (PBS), pH 7.4, containing 100 U/ml penicillin and 0.1 mg/ml streptomycin. All preparation steps were performed at room temperature. The anterior segment was then cut approximately 3.5 mm posterior to the limbus. The posterior segment was flattened by a radial incision of the sclera. The globe was turned over exposing the vitreous, neurosensory retina and RPE on the outer side. The vitreous was carefully removed using a sterilized cotton swab. The neurosensory retina was detached, leaving it attached only at the optic nerve head to guarantee correct orientation. The neurosensory retina was washed with PBS with the inner limiting membrane (ILM) facing downwards on a sterile ceramic tile. Subsequently the connection to the optic nerve head was cut using scissors. Using a cell scraper the RPE–choroid complex was separated from the underlying sclera and placed on a nitrocellulose membrane (Schleicher and Schuell, pore size 0.45 ␮m) with the choroid facing downwards. For each retina culture a separate eye were used to get tissue pieces of the central retina. Six eyes, respectively explants were used for each experimental group. Unless otherwise noted, all chemicals, tissue culture media and reagents were purchased from Sigma–Aldrich (Deisenhofen, Germany).

2.2. Tissue culture Immediately following isolation, the neural retina and the RPE–choroid explants were recombined by placing the neurosensory retina on top of the RPE cell layer with the ILM facing upwards restoring the original in vivo orientation of the tissue (retina–RPE). Then pieces of 10 mm × 10 mm close to the area centralis were cut using a surgical blade. The tissue was placed on a sterile, perforated, high-grade stainless steel grid support bench placed in a six-well culture plate and the wells were then filled with minimum essential medium (MEM) culture medium up to a level just below the upper, vitreous surface of the explant. The culture medium consisted of MEM (GIBCO BRL, Eggenstein), supplemented with 2.8 mM l-glutamine (GIBCO), 10% newborn calf serum (NCS) (GIBCO), 673 U/ml penicillin and 673 ␮g/ml streptomycin (SIGMA). Retinas were cultured for 3 days at 37 ◦ C in an atmosphere containing 5% CO2 . The culture medium was changed every 24 h. Culture of neurosensory retina alone and freshly isolated retina served as controls. Each culture was repeated 10 times and the 6 best morphological specimens without preparation damage, as visualized by H&E staining, were used for further immunohistological examinations. 2.3. Immunohistochemistry The tissue was removed from the culture medium, fixed for 24 h in 4% paraformaldehyde at room temperature, dehydrated and paraffin embedded following standard protocols. Sections were stained with hematoxilin–eosin and evaluated by light microscopy (Zeiss Axioscop, Oberkochen, Germany) for morphology. For immunohistochemistry, serial sections were mounted on polylysine-coated slides, transferred into citrate buffer (0.1 M, pH 6) and irradiated by microwave for 15 min at 500 W. After cooling, sections were washed with 50 mM PBS and blocked with 20% normal goat serum (NGS) and 2% BSA for 1 h at room temperature. Sections were exposed for 3 h to the primary antibody diluted in 10% NGS and 2% BSA in PBS at room temperature. The cultures were examined for the expression of a number of antigens: Ki67, a nuclear protein expressed in proliferating cells (antiKi67; mouse monoclonal clone MIB-1, 1:25, DAKO M-7240, Hamburg). Glial fibrillary acid protein (GFAP), an intracytoplasmic, intermediate filament, highly specific for glial cells which is upregulated following CNS injury. Functionally GFAP is thought to be important for astrocyte motility and shape maintenance by providing structural stability to astrocytic processes (Eng et al., 2000). In order to exclude false positives due to cross-reactivity to other intermediate filaments caused by structural homologies two distinct clones of antibodies were used: anti-GFAP1 (mouse monoclonal clone G-A-5, 1:100, SIGMA G-3893, Munich) and anti-GFAP2 (mouse monoclonal clone 6F2, 1:100, DAKO M0761, Hamburg), respectively. S100, a Ca2+ -binding protein abundant in glial cells; within the retina S100 identifies Muller cells (anti-S100; rabbit polyclonal, 1:300, DAKO Z-0311, Hamburg). Glutamine synthetase (GS), a key enzyme in glutamate recycling. Glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, is known to be neurotoxic, when present in excess at synaptic junctions. Metabolism and recycling of glutamate by synaptic astrocytes via GS is one of the major mechanisms for protecting neurons from glutamate-induced toxicity. GS immunoreactivity in the retina localizes in Muller cells and has been used as a specific marker for these cells (Riepe and Norenburg, 1977). GS activity is a useful marker for astrocytes and an important differentiation feature in the retina (Norenberg, 1979; Kentroti et al., 1991) (anti-GS, rabbit polyclonal, 1:300, SIGMA G 2781, Munich).

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Fig. 1. H&E staining of freshly prepared retina (A), retina culture alone (B), and retina–RPE–culture. Both retina cultures show a reduction of cell number in the ganglion cell layer (*), number of nuclear bodies in both nuclear layers and photoreceptor outer segments (***). The inner plexiform layer (**) seems to be reduced in both retina cultures compared to freshly prepared retina. Layer 1 (L1) included the inner limiting membrane (ILM), the nerve fiber layer (NFL), and the ganglion cell layer (GCL); layer 2 (L2) represent the inner plexiform layer (IPL); layer 3 (L3) comprised the inner nuclear layer (INL); layer 4 consisted of the outer plexiform layer (OPL), the outer nuclear layer (ONL) and the external limiting membrane (ELM).

Vimentin is an intermediate filament responsible for maintaining cell shape, integrity of the cytoplasm, and stabilizing cytoskeletal interactions. In order to exclude false positives due to cross-reactivity to other intermediate filaments caused by structural homologies two distinct clones of antibodies were used: anti-vimentin1 (mouse monoclonal clone VIM 13.2, 1:100, SIGMA V-5255, Munich) and anti-vimentin2 (mouse monoclonal clone V9, 1:100, SIGMA V-6630, Munich). Clone V9 was used and is of specific interest because pig eye lens was used as the immunogen in antibody production. RPE65 is a major and specific protein of the RPE and plays a critical role in the visual cycle (mouse monoclonal, 1:100, NOVUS Biologicals, NB100-355, Littleton, USA). RPE65 is expressed by RPE cells in vivo and in vitro. Downregulation of RPE65 gene expression in vitro indicates dedifferentiation, proliferation and migration (Alge et al., 2003). After exposing the sections for 3 h to a primary antibody diluted in 10% NGS and 2% BSA in PBS, the slides were washed three times with PBS and exposed for 3 h in the dark to the secondary antibody Alexa Fluor® (1:500, monoclonal Alexa Fluor® 488 goat anti-mouse IgG or monoclonal Alexa Fluor® 488 goat anti-rabbit IgG). In control experiments the primary antibody was omitted and the sections were incubated with PBS alone. Nuclear staining was accomplished by exposing the sections for 5 min to DAPI (Merck, Darmstadt, Germany, 350 ␮g DAPI/ml 50% ethanol in PBS). Immunofluorescence microscopy was performed with a Polyvar microscope (LEICA, Bensheim). All tissues were analysed for histological damage by HE staining and only histomorphologically intact retinas were processed for immunohistochemistry.

2.4. Quantification of immunohistochemistry Digital image analysis of micrographs was performed as a semiquantitative confirmation of the changes in fluorescence density, which correlates with changes in protein concentration, intracellular shift or protein expression. For each retina the six best morphological specimens that did not show any preparation damage, as visualized by H&E staining, were used. To avoid immunohistological variant results caused by regional differences of more peripheral or central retina sections within every single explant, central segments of similar retinal thickness were used for quantification of immunohistochemistry. Microscopic images were transferred to Adobe Photoshop® software (ADOBE, San Jose). Brightness and contrast were adjusted until green fluorescence was discriminated from non-stained tissue. The Software Jasc® Paint Shop Pro (ADOBE, San Jose) was utilised to overlay fluorescent images (Alexa Fluor® 488) on the DAPI-stained images. The analysis included calculation of fluores-

cent areas in defined retinal layers. Layer 1 (L1) included the ILM, the nerve fiber layer (NFL), and the ganglion cell layer (GCL); layer 2 (L2) comprised the inner plexiform layer (IPL); layer 3 (L3) consisted of the inner nuclear layer (INL); layer 4 comprised the outer plexiform layer (OPL), the outer nuclear layer (ONL) and the external limiting membrane (ELM). After visual identification of the borders of the four retinal layers, the microscopic image was divided manually into four individual micrographs corresponding to the four layers. The software Adobe Photoshop® determined the total number of pixels in the micrographs. Subsequently the software was used to separate the green fluorescent area of Alexa Fluor® 488 binding from the unstained background. The number of green fluorescent stained pixels in every defined retinal layer (L1 to L4) was compared to the total number of pixels of retinal layer to calculate the percent stained area for every retinal layer. In order to evaluate the influence of the visual determination of the borders of the retinal layers, the determination was repeated for an identical section at three time intervals without any significant differences (data not shown). Results are presented as the means ± standard deviation for six measurements from two sections of different retina cultures (n = 12). For statistical comparison between control and cultured retinas a Student’s t-test was performed and cultures were compared by one factor-ANOVA-testing. A p value of <0.05 was considered statistically significant. 2.5. Quantification of nuclei in INL and ONL To quantify the number of nuclei in the INL and ONL of retinas, 190 ␮m × 280 ␮m microscopic images from DAPI-stained retinas were used. For each retina culture alone (n = 6), retina–RPE–choroid culture (n = 6) and freshly isolated retinas, nuclei in the INL and ONL were counted in two images per single retina culture (n = 12). Results are presented as the means ± standard deviation (S.D.) for 2 measurements from 6 sections for each condition (n = 12). Statistical comparison of freshly isolated retinas and cultured retinas was performed using a Student’s t-test and comparison between conditions was performed using a one factor-ANOVA-testing. A p value of <0.05 was considered statistically significant. 2.6. In situ detection of apoptosis by TUNEL staining Detection of apoptotic cells was performed using the TUNEL test according to the manufacturer’s protocol (In situ Cell Death Detection kit, Fluorescein; ROCHE, Penzberg, Germany). Deparaffinised sections of cultured retinas were microwaved for 3 min at 360 W in citrate buffer (0.1 M, pH 6). After the stepwise exchange of hot citrate buffer with PBS (room temperature), sections were incubated in Proteinase K (20 ␮g/ml in 10 mM Tris–HCl pH 7.5,

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Fig. 2. Ki67 labeling. (A) Paraffin section of non-cultured, freshly prepared porcine neural retina without cultivation stained with anti-Ki67. The marked cells in the inner and outer plexiform layer (arrows) represent proliferating microglial cells (arrows). Inner limiting membrane (asterisk). (B) Retina alone reacted with anti-Ki67 after 3 days in culture. Note the reduced staining of the OPL with anti-Ki67 as well as several proliferating and migrating microglial or Muller cells (arrows) in the vitreal retinal layers close to the ILM (asterisk). (C) Retina–RPE reacted with anti-Ki67 after 3 days in culture. As in retina tissue culture without RPE, proliferating glial cells (arrows) are evident in the inner retinal layers. Beside these Ki67-labeled cells, proliferating RPE and endothelial cells can be detected underlying the RPE (arrowheads) and in the vitreal retinal layers close to the ILM (asterisk) (blue stain indicate nuclei stained with DAPI).

SIGMA, Germany) for 30 min and washed three times with PBS. For TUNEL labeling sections were incubated in TUNEL reaction mixture for 1 h at 37 ◦ C in the dark. The TUNEL reaction mixture contains terminal deoxynucleotidyl transferase (TdT), which catalyses the polymerisation of fluorescein labeled nucleotides to the 3 -OH DNA ends in a template-independent manner (TUNEL-reaction). Negative control sections were incubated without TdT. Sections were counterstained with DAPI, and analysed using fluorescence microscopy. For the quantification of TUNEL labeling, 190 ␮m × 280 ␮m microscopic images from TUNEL/DAPI-stained retina were used. For each retina culture alone (n = 6), retina RPE–choroid culture (n = 6) and freshly isolated retinas (n = 6) nuclei of the GCL, INL and ONL were analysed for two images per retina culture (n = 12). Number of nuclei were recorded in the blue fluorescent DAPIstained image of each nuclear layer (GCL, INL, ONL), whereas apoptotic nuclei were recorded in the green fluorescent stained TUNEL-labeled images of GCL, INL and ONL. The number of apoptotic cells is expressed as the ratio of DAPI–TUNEL double-labeled nuclei to the total number of nuclei stained with DAPI. All values are presented as means ± S.D. (n = 12, 2 microscopic micrographs per culture). Data were analysed for statistical significance by the Mann–Whitney rank sum test. A p < 0.05 was considered statistically significant.

3. Results Culture of retina–RPE resulted in fewer necrotic zones (Figs. 1C, 2C, 4C–9C). The observation of numerous microscopic images suggests that photoreceptors are better preserved in retina–RPE–choroid cultures than in cultures of retinas alone (Figs. 1, 2, 4–9). Despite progressive photoreceptor cell degeneration a continuous outer limiting membrane and no rosettes or folds could be observed in retina–RPE culture. 3.1. Ki67 In freshly prepared retinas anti-Ki67 lightly stained activated microglia cells in the OPL and IPL (Fig. 2A), as well as cells in the lumen of blood vessels. After 3 days in culture retina alone reacted with anti-Ki67 revealed several proliferating microglial cells in the IPL and NFL (Fig. 2B). In retina–RPE culture the pattern of staining by Ki67 was similar to retina alone, except for the staining of sporadic cells close to the RPE and choroid (Fig. 2C). 3.2. RPE65 The RPE is arranged as a pigmented cell monolayer close to the choroid (Fig. 3C) and shows positive staining for RPE65 whereas the choroid does not show any staining (Fig. 3D).

Fig. 3. RPE65 labeling. Light micrograph of DAPI stain (A and C) and RPE65 fluorescence (B and D) of the retina–RPE/choroid culture. Adjacent to the retina are evident dark stained cells (DAPI stained) (arrows) (A) that do not show any RPE65 staining (B). The RPE65-stained cells (RPE cells) are arranged as a monolayer adjacent to the choroid (C). Close by the RPE yellow coloured photoreceptor outer segments, bound or phagocytised by the RPE, are visible (arrows) (D).

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The intense RPE65 labeling and the intact RPE cell monolayer indicates the absence of RPE proliferation or migration. Ki67 staining showed only a few positive cells close to the RPE and choroid. If single RPE cells had migrated into the retinal layers pigmented cells should have been visible by light microscopy, in addition no RPE65 positive cells were observed (Fig. 3A). Pigmented particles observed in the section outside the retina represent cellular debris from the choroid and RPE were also RPE65 negative. The absence of RPE65-positive structures within retina excludes the possible migration of RPE cells into retinal layer within the culture time of 3 days. 3.3. GFAP As expected, in freshly prepared retinas, GFAP expression was abundant in the astrocytes of the nerve fibre layer (NFL) as well as those close to the blood vessels and was detected by both clones of antibodies used (Figs. 4A, D, and 5A, D). Differences were seen in the reaction of the antibody clones with Muller cells. Clone 6F2 (anti-GFAP2 ) was more specific for astrocytes (Fig. 5A), compared to clone G-A-5 (anti-GFAP1 ) which also lightly stained the outer radial processes of Muller cells and the Muller cell somata and microvilli (Fig. 4A). Only when Muller cells became more prominent as seen in retinal culture alone (Fig. 5B and D) a slight cross-reactivity of clone 6F2 (anti-GFAP2 ) was observed. In cultured retina alone, staining with anti-GFAP1 (clone G-5A) showed increased expression in Muller cells in all layers of the retina from the IPL to the ELM (Fig. 4B and D), which was absent in retina–RPE culture (Fig. 4C and D). The pattern of GFAP expression in retina–RPE cultures was similar to that observed in freshly prepared retina for both antibodies (Figs. 4A, C, D and 5A, C, D).

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3.4. Vimentin In freshly prepared retina vimentin was visualized in the innermost parts of Muller cells from the ILM to the NFL but was barely detectable in Muller cell somata or microvilli or in astrocytes using clone VIM13.2 antibody (anti-vimentin1 ) (Fig. 6A and D). In contrast, clone V9 (anti-vimentin2 ) showed vimentin reactivity in astrocytes as well as Muller cells throughout all layers with the highest intensity observed in the inner retina (Fig. 7A and D). In cultures of retina alone the expression of vimentin was significantly increased in Muller cells (Figs. 6B and 7B) at the level of the ILM and NFL (16 ± 6%), the IPL (30 ± 13%) and the INL (49 ± 18%), respectively (Figs. 6D and 7D). In cultures of retina–RPE the expression of vimentin more closely approached the expression observed in uncultured retina (Figs. 6A, C and 7A, C). Compared to retina cultures alone the expression of vimentin1 was reduced in the INL by 30 ± 13% (Fig. 6D), whereas expression in the ILM and NFL remained very high, 48% higher than in retina cultures alone.

3.5. S100 In freshly prepared retina S100 was present in Muller cells but not astrocytes or ganglion cells throughout all retinal layers (Fig. 8A). The highest levels of S100 staining was observed in the inner retina including ILM, NFL and GCL (48 ± 7%) and the INL (35 ± 8%) (Fig. 8D). In cultures of retina alone microscopic and quantitative analysis of the immunohistochemical images showed that S100 was increased in Muller cells in all retinal layers (Fig. 8B and D). A significant increase in S100 density was evident in the apical processes

Fig. 4. GFAP1 labeling. (A) In freshly isolated retina GFAP1 can be detected in the astrocytes of the NFL, in the astrocytes close to blood vessels (asterisk) and outer radial processes of Muller cells until the OPL (arrows). (B) After 3 days in retina culture without RPE there is increased GFAP1 expression in Muller cells from the ILM to the ELM (arrows) and reduced expression in astrocytes. (C) After the same culture period retina–RPE cultures exhibited less of an increase of GFAP expression in Muller cells (arrows) compared to non-cultured retina, and no decline in astrocytes (asterisk) and more nuclei in the ONL (DAPI staining). (D) Semiquantitative analysis of labeling with anti-GFAP1 (*p < 0.05 Student’s t-test; *p < 0.05 one factor-ANOVA; mean ± S.D.). The high GFAP1 content in apical Muller cell processes and astrocytes results in a fluorescence intensity of 44 ± 4% in the ILM and NFL (L1). The retina tissue culture without RPE shows no changes in total GFAP1 content in L1 because the increased fluorescence in Muller cells is balanced by a decline in GFAP content in astrocytes. Muller cells show a significantly higher GFAP fluorescence: 10 ± 10% in L2, 16 ± 5% in L3 (INL) and 9 ± 4% in L4. Culture of the retina in the presence of RPE compared to retina culture alone results in a reduced GFAP1 increase in Muller cells: 16 ± 8% in the IPL (L2), 21 ± 8% in the INL (L3) and 13 ± 4% in L4.

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Fig. 5. GFAP2 labeling. (A) The stratification of the freshly isolated tissue is well preserved including the photoreceptor cells. Freshly isolated retinas show no staining of Muller cells. Only astrocytes are stained by GFAP2 -antibody (asterisks). (B) Retina cultures without RPE show a slight staining increase in the NFL (asterisk) and vitreal Muller cell processes and somata (arrows). The ONL appears more irregular then in freshly prepared retinas. (C) Retina–RPE cultures show increased GFAP content only in the NFL (asterisk). (D) Semiquantitative analysis of the GFAP2 labeling (*p < 0.05 Student’s t-test; *p < 0.05 one factor-ANOVA; mean ± S.D.). In freshly isolated retinas the highest GFAP concentration was detected in the NFL (L1) representing GFAP rich astrocytes. In contrast to the apical region other retinal layers show slight staining, because of the non-reactive Muller cells. Retina cultures without RPE show a significant increase of GFAP2 content in the NFL (L1 = 20 ± 10%), in the IPL by 14 ± 10%, in INL by 13 ± 6% and in the basal Muller cell endplates (L4) by 7 ± 4%. In retina–RPE cultures compared to retina culture alone exhibited significantly lower anti-GFAP2 expression in INL and basal Muller cell processes, whereas in NFL a reduced GFAP2 content were observed.

Fig. 6. Vimentin1 labeling. (A) Freshly isolated retina. The light labeling in the ILM and NFL represents the apical Muller cell endplates and processes (arrows). The basal somata and microvilli show only very light labeling. Astrocytes, located in the nerve fiber layer also show only very light labeling. (B) Retina cultures without RPE show increased vimentin1 labeling for Muller cells (arrows) and astrocytes (asterisk) throughout the retina. (C) Compared to culture without RPE neural retina–RPE cultures show a more intense fluorescence in the NFL (asterisk) but reduced labeling in Muller cells from the IPL to the ELM. (D) Semiquantitative analysis of vimentin1 labeling (*p < 0.05 Student’s t-test; *p < 0.05 one factor-ANOVA; mean ± S.D.). Freshly prepared retina show the highest vimentin1 concentration in the apical Muller cell endplates (27 ± 4%). The other retinal layers show only very slight fluorescence. After 3 days in retina cultures alone, Muller cells and astrocytes exhibit a significant increase of vimentin1 expression throughout all retinal layers compared to freshly isolated retina with the largest increase in the apical Muller cell processes, in the IPL by 30 ± 13% and in the INL by 49 ± 8%, which contain the Muller cell somata. Retina cultured with RPE exhibited a reduced increase of vimentin1 expression in the INL and a significant increase of vimentin1 in L1, L2 and L3 compared to retina culture alone.

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Fig. 7. Vimentin2 labeling. (A) Muller cells (arrows) and astrocytes (asterisk) in freshly isolated retina exhibit only slight staining throughout all retinal layers. (B) After 3 days in culture neural retinas alone show a dramatic increase in Muller cells in the IPL (arrows) and of somata from the INL (asterisk) to the ELM. (C) Neural retina–RPE cultures show a slightly reduction of vimentin2 content, especially in the INL (asterisk) compared to cultures of neural retinas alone. The outer limiting membrane formed by the basal Muller cell endplates appears closed (arrows). (D) Semiquantitative analysis of the vimentin2 labeling (*p < 0.05 Student’s t-test; *p < 0.05 one factor-ANOVA; mean ± S.D.) Astrocytes and apical Muller cell endplates (L1) exhibit the highest vimentin2 concentration: 31 ± 8% in freshly prepared retina tissue. The L2 fluorescence represented by the thin Muller cell processes contains 15 ± 6% of the total retinal vimentin2 . The content of vimentin2 in Muller cell somata and basal microvilli is 25 ± 8% in L3 and 24 ± 10% in L4. After 3 days in culture neural retina alone shows a significant increase in vimentin2 content in the IPL (17 ± 12%), in the INL (L3) (29 ± 11%) and the ONL (L4) (17 ± 14%) Neural retina–RPE cultures show a reduced vimentin2 increase in the retinal layers L1, L3 and L4; whereas only Muller cell somata in the INL show a significant reduced increase of vimentin2 (19 ± 9%) compared to culture of retina alone.

of Muller cell (32 ± 14%), in the somata (38 ± 10%) and the microvilli of Muller cell (28 ± 17%). In cultures of retina–RPE S100 expression was reduced in the OPL/ONL and the INL, but only in the OPL/ONL expression was similar to that observed in freshly prepared retina (Fig. 8A and C). S100 expression increased significantly in the NFL in the retina–RPE culture compared to freshly prepared retina as well as compared to retina culture alone (Fig. 8D). S100 expression was upregulated in both culture techniques to a similar extent in the IPL (47/50% compared to 15% in freshly isolated retina). 3.6. GS GS expression was observed only in Muller cells; anti-GS antibodies stained the entire cytoplasm of Muller cells from the ELM to the ILM with the most intense staining localized in the end plates and somata; astrocytes were not labeled (Fig. 9A and D). Compared to freshly isolated retina, GS expression in retina cultures alone increased slightly in the IPL (8 ± 6%), but was significantly higher (33 ± 9%) in the Muller cell microvilli of the ONL (Fig. 9B and D). In retina–RPE cultures GS expression was higher in the apical Muller cell processes (NFL, IPL and the INL) while it showed a decrease in basal Muller cell microvilli (OPL/ONL) (Fig. 9C and D). 3.7. Nuclei number in INL and ONL After 3 days in culture retinal explants without RPE–choroid showed a significant decrease in thickness that is reflected by a decrease in the numbers of nuclei in the inner and ONL (Student’s t-test, p < 0.05), whereas cell nuclei number in INL and ONL of retina RPE–choroid culture were not reduced significantly (Fig. 10).

The significant difference in nuclei in the INL (39.7 ± 20.9) and the ONL (29.8 ± 18.4) in retinas cultured in the absence of the RPE–choroid suggests that glial and/or neuronal degeneration occurs within the 3 days of culture in retinas cultured alone (one factor-ANOVA; p < 0.05). 3.8. TUNEL In freshly isolated retinas no apoptotic labeled cells were observed, which represent the normative data of 0% apoptosis at the start of culture (Fig. 11A). In cultures of retina alone without RPE apoptotic cells were found in the GCL (37%), INL (42%) and in the ONL (46%) (Fig. 11B and C). In retina culture with RPE/choroid the number of apoptotic cells were significantly fewer than in retinas cultured alone, 21% in the GCL, 14% in the INL and 9% in the ONL (Fig. 11D). 4. Discussion Analysis of neuronal synapses and other physiological aspects of cell–cell interactions necessitate either in vivo models or organ culture models. Pre- and early postnatal retina organ cultures are described in numerous studies (Caffe et al., 1989; Feigenspan et al., 1993; Germer et al., 1997b, 1998; Hoff et al., 1999; Katyal and Godbout, 2004) whereas reports of adult retina tissue culture are limited (Winkler et al., 2000; Ouchi et al., 2005). There have been a number of reports on the culture of retinas alone (Winkler et al., 2000; Rzeczinski et al., 2006; Saikia et al., 2006) but very few reports on the culture of retinas attached to the underlying RPE and choroid (Ogilvie et al., 1999; Katyal and Godbout, 2004; Reidel et al., 2006). Studies of isolated retina in culture without

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Fig. 8. S100 labeling. (A) In freshly isolated retina only Muller cells are stained by anti-S100 throughout all retinal layers with the highest intensity in the apical endplates (asterisks), whereas astrocytes and ganglion cells show no fluorescence. (B) After 3 days in culture we observed higher S100 immunoreactivity in Muller cells (arrows). (C) In comparison to freshly isolated retina, neural retina–RPE cultures shows increase of S100 staining throughout all retinal layers but in contrast to neural retina cultures alone we observed higher S100 staining density in NFL (asterisk) and IPL. Furthermore we observed reduced S100 immunoreactivity in OPL (arrow) and ONL. (D) Semiquantitative analysis of the S100 labeling (*p < 0.05 Student’s t-test; *p < 0.05 one factor-ANOVA; mean ± S.D.) In freshly prepared retinas the highest S100 concentration was detected in the basal Muller cell endplates (L1) with 48 ± 7% and in the INL comprising the Muller cell somata (L3) with 35 ± 8%. The microvilli of Muller cells show a S100 density of 21 ± 7% whereas the IPL (L2) comprising the fine, radial Muller cell processes showed the lowest S100 density (15 ± 7%). After 3 days the neural retinas cultured alone show significantly increased S100 density in IPL (L2) by 32 ± 14%, in Muller cell somata (L3) by 38 ± 10% and in L4 by 28 ± 17%. In neural retina–RPE cultures we observed a significantly higher S100 density in L1 by 26 ± 13% but reduced S100 density by 12 ± 6% in L3 and by 22 ± 11% in L4.

Fig. 9. GS labeling. (A) In freshly isolated retina GS staining was only observed in macroglial cells, the label extending from the endplates of the ILM (asterisk) to the outer limiting membrane. The somata and endplates of Muller cells are very distinctly labeled. (B) In neural retina cultures alone increased labeling with anti-GS was observed throughout all retinal layers, particularly in IPL (asterisk) and Muller cell microvilli (ONL) (arrows). (C) The neural retina–RPE cultures also show an increase of GS expression throughout all retinal layers compared to uncultured retina, however labeling in the ONL appears reduced compared to neural retina culture without RPE (arrows). (D) Semiquantitative analysis of the glutamine synthetase labeling (*p < 0.05 Student’s t-test; *p < 0.05 one factor-ANOVA; mean ± S.D.). Non-cultured retinal tissue shows labeling throughout all retinal layers, with a density in the inner nuclear layer of 51 ± 3% and 43 ± 5% in the ONL (L4) and the lowest density in the IPL (L2; 31 ± 3%). Retina cultures alone showed an increase in GS concentration in L2 (8 ± 6%) and L4 (33 ± 9%) compared to freshly prepared retina. Retina–RPE culture showed a significant GS increase in L1 (19 ± 12%) and L2 (25 ± 8%) but a reduced density (17 ± 7%) in the ONL (L4).

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Fig. 10. Nuclei number in INL and ONL. In the INL and the ONL of freshly prepared retinas 178.1 ± 12.3 (INL) and 325.4 ± 12.7 (ONL) nuclei were counted per 190 ␮m × 280 ␮m. Whereas in retina cultures without RPE 128.9 ± 11.7 nuclei in INL and 292.3 ± 14.0 nuclei in ONL were counted, in retina–RPE–choroid cultures 168.6 ± 9.2 nuclei in the INL and 322.2 ± 9.2 nuclei in the ONL were counted per 190 ␮m × 280 ␮m. The differences in nuclei in freshly prepared retina and retina RPE–choroid culture are not significant, however the differences between freshly isolated retinas and retinas cultured alone are statistically significant (Student’s ttest; p < 0.05). The differences in nuclei between retina–RPE–choroid cultures and retina cultures alone are also statistically significant (one factor-ANOVA-testing; p < 0.05). Results are presented as the means ± standard deviation (S.D.; n = 12).

RPE have shown early ganglion cell apoptosis followed by photoreceptor degeneration (Caffe et al., 1989; Jablonski et al., 1999, 2001; Engelsberg et al., 2004; Katsuki et al., 2004; Kuhrt et al., 2004). The culture of isolated adult porcine neural retina cultured in conjunction with the RPE–choroid complex, described in this report, provides a novel tissue culture model that shows reduced apoptosis in the GCL, INL and ONL (Jeon et al., 1998). Although

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we could not differentiate between the cell types in the GCL by DAPI staining we assume that the apoptotic nuclei in GCL represent mostly ganglion cell death (Ogilvie et al., 1999; Engelsberg et al., 2004; Katsuki et al., 2004; Cossenza et al., 2006). DAPI and TUNEL labeling in the INL, which includes Muller cells and bipolar cells, is not sufficient to differentiate whether Muller cells or bipolar cells are apoptotic. However, since Pinzon-Duarte et al. (2004) showed that in cultures of retinas mainly bipolar cells but not Muller cells were affected by apoptosis, we assume that the TUNEL labeled nuclei of the INL in the retina cultures in our study represent mainly bipolar cells. Furthermore the lack of Ki67 labeling of Muller cells suggests that there was no glial cell proliferation in the INL and that the loss of nuclei and apoptosis in the INL is probably the result of bipolar cell death. In addition, significant Muller cell death can be excluded since GFAP-stained Muller cells are comparable in freshly prepared retinas, in cultures of retinas alone and retinas plus RPE/choroid culture. Although there is a significant reduction of bipolar cells in the explants, the majority of TUNEL positive cells are localized to the ONL indicating that the apoptotic cells represent photoreceptor cells. Despite the fact that the culture lasted only 3 days, our results show that co-cultivation of neural retina abutted to the RPE/choroid resulted in better preservation of neural retinal cells than in cultures of neural retina alone, as evidenced by the significantly lower loss of nuclei in the INL and ONL of RPE/choroid cultures. During the 3 days of culture we did not observe subretinal gliosis nor formation of rosettes in retina–RPE culture, which are usually observed when the neural retina is detached from RPE cells or in cultures of the neural retina that does not adhere to the substratum (Tansley, 1933; Ogilvie et al., 1999; Winkler et al., 2000; PinzonDuarte et al., 2004). Since no RPE65 positive cells could be detected

Fig. 11. TUNEL test. Fluorescence photomicrographs of freshly isolated porcine retina (A), retina culture alone (B) and retina RPE–choroid culture (C) processed with TUNEL technique (green) and counterstained with DAPI (blue). (A) In freshly isolated retina no apoptotic cells were detected, whereas in retina cultured alone (B) large number of apoptotic cells were observed in the GCL (*), the INL (**) and the ONL (***). (C) In retina RPE/choroid cultures the number of apoptotic cells was significantly reduced compared to cultures of neural retina alone (B). (D) The number of apoptotic, TUNEL positive cells is expressed as the ratio of DAPI–TUNEL double-labeled nuclei to the total number of DAPI-stained nuclei. In cultures of neural retina alone the number of apoptotic cells was 37% in the GCL, 42% in the INL and 46% in the ONL layer. In retina–RPE–choroid cultures apoptotic cells were diminished significantly and were 21% in the GCL, 14% in the INL and 9% in the ONL (9%) (mean ± S.D., n = 12, Mann–Whitney rank sum test, *p < 0.05). In freshly isolated retinas apoptotic cells were not detected and are set at 0%.

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in the retina, but RPE65 staining was clearly associated only with the RPE cell monolayer adjacent to the choroid, proliferation and migration of RPE cells into the retina did not occur. The 3-day follow-up was chosen because reports in the literature have described that in a detached retina the destructive glial response, photoreceptor death (Cook et al., 1995; Lewis et al., 2002a,b) and alterations in a number of cytokines reach a maximum at day 3 in retinal tissue (Nakazawa et al., 2006). Activated and migrating microglial cells modulate the release of trophic factors by the microglia Muller cell network during retinal degeneration (Harada et al., 2002) and can therefore cause secretion of factors that accelerate photoreceptor degeneration (Thanos and Mey, 1995; Roque et al., 1999). The increase of proliferating cells stained by anti-Ki67 in the cultures of retina alone could represent microglial cells similar to that observed in retinal injury (Lewis et al., 2005). A reduction of Ki67 labeled cells, likely microglia, in the inner retinal layers of retina–RPE cultures could account for the diminished neuronal degeneration and better neuronal survival. Not only microglial cells but also Muller cell and astrocytic gliotic response play an important role in retinal degeneration. In the retina stress factors such as mechanical injury lead to upregulation of intermediate filaments, specifically GFAP and vimentin (Fuchs and Cleveland, 1998; Lam et al., 2003; Lewis and Fisher, 2003; Lundkvist et al., 2004). In our retina–RPE culture model, levels of GFAP, and vimentin expression correspond closely to the levels observed in freshly isolated retina whereas the expression of these proteins were increased significantly in retinal culture alone. These results indicate that the retina–RPE culture system reduces neuronal degeneration and glial hypertrophy. Phosphorylation and assembly of the intermediate filaments GFAP and vimentin is modulated by S100B protein (Ziegler et al., 1998; Garbuglia et al., 1999) and exerts regulatory effects on neurons, astrocytes and microglia in terms of enzyme activity, dynamics of cytoskeleton, cell growth and differentiation (for review see Donato, 2003). S100B displays a concentrationdependent effect on neuronal cells, at nanomolar concentrations S100 acts as neurotrophic agent, whereas at micromolar concentrations S100 induces neuronal apoptosis (Reali et al., 2005; Bianchi et al., 2007). In addition, it has been suggested that S100B acts as an astrocytic endokine participating in the response to CNS injury (Bianchi et al., 2007). Increased S100 expression is observed in vivo in gliosis of the retina as well as following retinal injury (Marshak, 1990; Selinfreund et al., 1990; Kim et al., 2003; Lam et al., 2003). Therefore it is not surprising that the pattern of S100 expression in our culture model follows that of GFAP and vimentin. The reduced S100 concentration in the ONL of retina–RPE cultures suggests a protective influence of RPE cells on photoreceptor survival. Another important factor involved in retinal degeneration in vivo and in vitro are excessive glutamate levels (Dreyer et al., 1996; Rauen and Wiessner, 2000; Delyfer et al., 2005; Ristoff et al., 2007). Elevated levels of glutamate induce an increase in glutamate synthetase (GS) in retinal Muller cells, which is responsible for glutamate removal (Germer et al., 1997b; Shen et al., 2004). We observed significantly increased GS concentration in all parts of Muller cells in retina–RPE culture, suggesting that GS is involved in a rescue mechanism of the retina. This upregulation of GS may be associated with the increased endogeneous glutamate that is induced during tissue preparation (Heidinger et al., 1999; Hoff et al., 1999) or may be induced by photoreceptor cell degeneration (Linser and Moscona, 1979). It has been reported that GS expression regresses when a major source of glutamate is lost, such as photoreceptor death due to degeneration or retinal detachment (LaVail and Hild, 1971; Lewis et al., 1989). Our observations support these reports, in fact GS upregulation in isolated retinal culture was limited to Muller cell microvilli and GS expression was signifi-

cantly lower than in retina–RPE culture where more photoreceptors survived. Reduced GS expression has also been reported in whole mount preparations of postnatal rabbit retina compared to the in vivo retina (Germer et al., 1997b). Intact RPE and Muller cells as well as secretory factors are necessary for photoreceptor survival. A number of investigators have reported that RPE cells secrete growth factors that increase photoreceptor survival such as PEDF (Jablonski and Ervin, 2000; Jablonski et al., 2000, 2001). In an embryonic model using xenopus tadpoles it has also been shown that Muller cell and photoreceptor dismorphogenesis caused by the loss of adherens junctions is prevented by the presence of RPE cells (Jablonski and Ervin, 2000; Jablonski et al., 2000). Here we have shown that in porcine neural retinas cultured adjacent to RPE–choroid, cellular maintenance is better preserved than in cultures of neural retinas alone as indicated by the reduced apoptosis in the GCL, the INL and the ONL, the diminished expression of glial-specific proteins and the increased GS. Even though the culture model described here is only a 3-day culture, it mimics the expression of various proteins that occur in retinal detachments in vivo and thus this culture system will allow the investigation of various physiological parameters, such as cell interactions, synthesis of various growth and maintenance factors, as well as regulation of degenerative processes in the retina. The fact that the RPE–choroid is able to prevent some of the degenerative changes that are observed in cultures of retina alone, suggests that the RPE in vivo has a profound influence on the maintenance not only of photoreceptors but of cells spatially removed from the RPE cells. Disclosure None. Acknowledgements This work was supported by a grant from the Interdisciplinary Centre for Clinical Research “BIOMAT.” within the faculty of Medicine at the RWTH Aachen University. The authors thank Liliane Palm for excellent technical assistance. References Alge CS, Suppmann S, Priglinger SG, Neubauer AS, May CA, Hauck S, et al. Comparative proteome analysis of native differentiated and cultured dedifferentiated human RPE cells. Invest Ophthalmol Vis Sci 2003;44:3629–41. Azadi S, Johnson LE, Paquet-Durand F, Perez MT, Zhang Y, Ekström PA, et al. CNTF+BDNF treatment and neuroprotective pathways in the rd1 mouse retina. Brain Res 2007;1129:116–29. Beier M, Franke A, Paunel-Gorgulu AN, Scheerer N, Dunker N. Transforming growth factor beta mediates apoptosis in the ganglion cell layer during all programmed cell death periods of the developing murine retina. Neurosci Res 2006;56:193–203. Bianchi R, Adami C, Giambanco I, Donato R. S100B binding to RAGE in microglia stimulates COX-2 expression. J Leukoc Biol 2007;81:108–18. Caffe AR, Visser H, Jansen HG, Sanyal S. Histotypic differentiation of neonatal mouse retina in organ-culture. Curr Eye Res 1989;8:1083–92. Caffe AR, Soderpalm A, van Veen T. Photoreceptor-specific protein expression of mouse retina in organ culture and retardation of rd degeneration in vitro by a combination of basic fibroblast and nerve growth factors. Curr Eye Res 1993;12:719–26. Cook B, Lewis GP, Fisher SK, Adler R. Apoptotic photoreceptor degeneration in experimental retinal detachment. Invest Ophthalmol Vis Sci 1995;36:990–6. Cossenza M, Cadilhe DV, Coutinho RN, Paes-de-Carvalho R. Inhibition of protein synthesis by activation of NMDA receptors in cultured retinal cells: a new mechanism for the regulation of nitric oxide production. J Neurochem 2006;97:1481–93. Delyfer MN, Simonutti M, Neveux N, Leveillard T, Sahel JA. Does GDNF exert its neuroprotective effects on photoreceptors in the rd1 retina through the glial glutamate transporter GLAST? Mol Vis 2005;11:677–87. Donato R. Intracellular and extracellular roles of S100 proteins. Microsc Res Tech 2003;60:540–51.

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