Phosphodiesterase Inhibition Induces Retinal Degeneration, Oxidative

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Experimental Eye Research xxx (2013) 1e12

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Experimental Eye Research

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1 56 2 Phosphodiesterase inhibition induces retinal degeneration, oxidative stress and 57 3 58 4 inflammation in cone-enriched cultures of porcine retina 59 5 60 6 a a a b 61 Q4 Cristina Martínez-Fernández de la Cámara ,M Dolores Sequedo , Ulises Gómez-Pinedo , 7 Teresa Jaijo a,c, Elena Aller a,c, Patricia García-Tárraga d, José Manuel García-Verdugo d, 62 8 63 a,c,e a,* 9 José María Millán , Regina Rodrigo 64 10 Q1 a Laboratory of Sensorineural Disorders, Instituto de Investigación Sanitaria-La Fe, Avda. Campanar, 21, 46009 Valencia, Spain 65 11 b Laboratory of Regenerative Medicine/Neurology and Neurosurgery, Instituto de Neurociencias, IdISSC, Hospital Clínico San Carlos, C/Profesor Martín Lagos, S/N. 28040 Madrid, Spain 66 12 c Centre for Biomedical Network Research on Rare Diseases, Valencia, Spain 67 d 13 Laboratory of Comparative Neurobiology, Instituto Cavanilles de Biodiversidad y Biología Evolutiva, Universidad de Valencia, C/Catedrático José Beltrán, 2, Ciberned, 46980 Paterna, 68 València, Spain 14 e Genetics Unit, Hospital Universitario La Fe, Avda. Campanar, 21, 46009 Valencia, Spain 69 15 70 16 71 17 article info abstract 72 18 73 19 Article history: Inherited retinal degenerations affecting both rod and cone photoreceptors constitute one of the causes 74 20 Received 19 September 2012 of incurable blindness in the developed world. Cyclic guanosine monophosphate (cGMP) is crucial in the 75 21 Accepted in revised form 19 March 2013 phototransduction and, mutations in genes related to its metabolism are responsible for different retinal 76 Available online xxx 22 dystrophies. cGMP-degrading phosphodiesterase 6 (PDE6) mutations cause around 4e5% of the retinitis 77 23 pigmentosa, a rare form of retinal degeneration. The aim of this study was to evaluate whether phar- 78 Keywords: 24 macological PDE6 inhibition induced retinal degeneration in cone-enriched cultures of porcine retina 79 retinal degeneration similar to that found in murine models. PDE6 inhibition was induced in cone-enriched retinal explants 25 cGMP 80 26 Zaprinast from pigs by Zaprinast. PDE6 inhibition induced cGMP accumulation and triggered retinal degeneration, 81 27 oxidative stress as determined by TUNEL assay. Western blot analysis and immunostaining indicated that degeneration 82 was accompanied by caspase-3, calpain-2 activation and poly (ADP-ribose) accumulation. Oxidative stress 28 TNF-alpha 83 poly (ADP-ribose) markers, total antioxidant capacity, thiobarbituric acid reactive substances (TBARS) and nitric oxide 29 84 caspase measurements revealed the presence of oxidative damage. Elevated TNF-alpha and IL-6, as determined 30 retinitis pigmentosa by enzyme immunoassay, were also found in cone-enriched retinal explants treated with Zaprinast. Our 85 31 photoreceptor death study suggests that this ex vivo model of retinal degeneration in porcine retina could be an alternative 86 32 model for therapeutic research into the mechanisms of photoreceptor death in cone-related diseases, 87 33 thus replacing or reducing animal experiments. 88 34 Ó 2013 Published by Elsevier Ltd. 89 35 90 36 91 37 92 38 93 39 1. Introduction photosensitive ganglion cells of the retina. The phototransduction 94 40 cascade occurs via a type of G-protein coupled receptors (GPCR) 95 41 Inherited retinal degeneration affecting both rod and cone called opsins, which contain the chromophore 11-cis retinal. When 96 42 photoreceptors constitutes one of the causes of incurable blindness struck by a photon, 11-cis retinal undergoes photoisomerization to 97 43 in the developed world. Several genes causing these genetic dis- all-trans retinal, which changes the conformation of the opsin GPCR, 98 fi 0 44 eases have been identi ed including those involved in visual leading to transducing activation. Active transducing increases 3 , 99 0 45 phototransduction. 5 ,-cyclic guanosine monophosphate (cGMP) phosphodiesterase 100 46 Visual phototransduction is the process by which light is con- activity (PDE6), thereby lowering the concentration of cGMP. 101 47 verted into electrical signals in the rod cells, cone cells and Decrease in cGMP concentration leads to the closure of cGMP- 102 þ 2þ fl þ 48 regulated Na and Ca ion channels, decreased in ux of Na and 103 2þ 49 Ca , hyperpolarization of membrane potential and decreased 104 Abbreviations: RP, retinitis pigmentosa; cGMP, cyclic guanosine mono- 50 glutamate release. Following isomerization and release from the 105 phosphate; PDE6, phosphodiesterase 6; PAR, poly (ADP-ribose); PARP, poly (ADP- opsin protein, all-trans retinal generated during phototransduction 51 ribose) polymerase. 106 52 * Corresponding author. Tel.: þ34 961973324. is reduced to all-trans retinol in the retinal pigmented epithelium 107 53 E-mail address: [email protected] (R. Rodrigo). (RPE) and travels back to the rod outer segment where it can be 108 54 109 e Ó 55 0014-4835/$ see front matter 2013 Published by Elsevier Ltd. 110 http://dx.doi.org/10.1016/j.exer.2013.03.015

Please cite this article in press as: Martínez-Fernández de la Cámara, C., et al., Phosphodiesterase inhibition induces retinal degeneration, oxidative stress and inﬂammation in cone-enriched cultures of porcine retina, Experimental Eye Research (2013), http://dx.doi.org/10.1016/ j.exer.2013.03.015 YEXER6134_proof ■ 12 April 2013 ■ 2/12

2 C. Martínez-Fernández de la Cámara et al. / Experimental Eye Research xxx (2013) 1e12

111 conjugated again to opsin to form a new, functional visual pigment. retina similar to the degeneration found in murine models of RP 176 112 Rods and cones recover from excitation and recover their light- with non-functional PDE6. Secondly, we assessed whether the 177 113 sensitivity by both inactivating the PDE6 cascade and synthetizing damage was accompanied with oxidative stress and induction of 178 þ 114 new cGMP by Ca2 -sensitive membrane guanylyl cyclases (RetGCs). inflammatory mediators. 179 þ 115 RetGCs are controlled by a calcium negative feedback through Ca2 / We found that PDE6 inhibition triggered retinal degeneration 180 þ 116 Mg2 binding proteins, GCAPs (guanylyl cyclase activating proteins) with caspase-3, calpain-2 activation and PAR accumulation (indi- 181 117 (Burns et al., 2002; Koch and Stryer, 1988). Alterations in any step of cator of PARP activity) and induced oxidative stress and cytokine 182 118 the phototransduction cascade could lead to photoreceptor cell induction in cone-enriched cultures of porcine retina. These results 183 119 death. suggest that the cone-enriched organotypic culture of porcine 184 120 Cyclic GMP is essential for the ability of rods and cones to retina exposed to PDE inhibitor could be a complementary model 185 121 respond to the light stimuli and the control of its level is critical for for therapeutic research into the mechanisms of retinal degenera- 186 122 proper functioning of photoreceptors. Mutations in genes involved tion, thus replacing or reducing animal experiments. 187 123 Q2 in cGMP synthesis (RetGCs and GCAPs) or degradation (PDE6) 188 124 (Chang et al., 2009; Dizhoor, 2000; Grau et al., 2011; Piri et al., 2. Materials and methods 189 125 2005; Hunt et al., 2010) can lead to various forms of retinal dys- 190 126 trophies such as some types of retinitis pigmentosa (Bowes et al., 2.1. Porcine retinal explant cultures 191 127 1990; McLaughlin et al., 1993), progressive cone dystrophy 192 128 (Thiadens et al., 2009), dominant cone degeneration (Behnen et al., Sixty eyes (both left and right eye) from small miniature pigs 3e 193 129 2010; Jiang and Baehr, 2010), cone-rod dystrophy (Buch et al., 2011; 7 months old were obtained from the local slaughterhouse. Neu- 194 130 Sokal et al., 2005; Tucker et al., 1999) and Leber congenital amau- roretina explants enriched in cones were carried out as previously 195 131 rosis (Perrault et al., 2000, 1996). described (Fernandez-Bueno et al., 2008) with some modifications. 196 132 Retinitis Pigmentosa (RP) is a common form of rod-cone dys- Briefly, each eyeball was immersed in 70% ethanol and washed in 197 133 trophy, constituting the largest Mendelian genetic cause of blind- Dulbecco’s Modified Eagle Medium (DMEM). All extraocular tissues 198 134 ness in the developed world. Patients with RP typically loose night were removed and the sclera was punctured with a 22 gauge 199 135 vision in adolescence, peripheral vision in young adulthood, and needle at the ora serrata and bisected, dividing the ocular globe into 200 136 central vision later in life due to progressive loss of rod and cone anterior and posterior eyecups. The vitreous was removed, and the 201 137 photoreceptor cells. Photoreceptor cell death starts with rod posterior eyecup was placed into a dish with phosphate buffer 202 138 photoreceptor degeneration and eventually cone cell death that is saline (PBS). A paintbrush was used to mechanically detach the 203 139 the major problem affecting RP patients, because it leads to loss of neuroretina from the RPE, and the optic nerve was cut with 204 140 central vision (Kalloniatis and Fletcher, 2004). Westcott scissors. The visual streak with a high cone density 205 141 Mutations in genes encoding a and b-subunit of PDE6 have been (Hendrickson and Hicks, 2002) was cut into 5 5 mm explants. 206 Ò 142 reported to cause recessive RP in humans (Corton et al., 2010; Dryja Explants were transferred to Transwell culture dishes (Corning 207 143 et al., 1999; Huang et al., 1995; McLaughlin et al., 1995). Inc., Corning, NY) with photoreceptor side down, containing 1.5 mL 208 144 PDE6Anmf363/nmf363, PDE6Anmf282/nmf2823 (Sakamoto et al., 2009), culture medium composed of Neurobasal A medium supplemented 209 rd1 rd10 145 PDE6B and PDE6B mice are models of human autosomal with 2% B-27 (Invitrogen, Life Technologies, Madrid, Spain), 2 mM L- 210 146 recessive RP that carry mutations on the a or the b-subunit of PDE6 glutamine (Invitrogen, Life Technologies, Madrid, Spain), 100 U/mL 211 147 (Bowes et al., 1990; Chang et al., 2002), that triggers photoreceptor penicillin, and 100 ng/mL streptomycin (Invitrogen, Life Technolo- 212 148 degeneration (Farber and Lolley, 1974; Paquet-Durand et al., 2009). gies, Madrid, Spain). Explants were cultured at 37 C with 5% CO2 in 213 149 Others than the available murine models have been reported to a humidified atmosphere. The culture medium level was main- 214 150 carry mutations in PDE6 subunits such as the canine models rcd1 tained in contact with the support membrane beneath the explant. 215 151 and rcd2 (Petersen-Jones et al., 1999; Sargan et al., 1994; Suber Treatments were added the day of the culture and maintained them 216 152 et al., 1993; Tuntivanich et al., 2008; Wang et al., 1999). for 24 h or 48 h. 217 153 Although murine models provide invaluable information about To evaluate the effect of PDE6 inhibition we used Zaprinast 218 154 photoreceptor cell death, they present different eye size and (Zhang et al., 2005) (100, 200 and 500 nmol/L). Zaprinast (Sigmae 219 155 anatomic differences in retinal structures compared to humans; for Aldrich, Madrid, Spain) was prepared in dimethyl sulfoxide (DMSO) 220 156 example, they lack macula and fovea. This is why the use of large (AppliChem, Darmstadt, Germany). For controls, the same amount 221 157 animal models seems to be critical for the development of retinal of DMSO was added to the culture medium. Some retinal explants 222 158 rescue strategies (Stieger et al., 2009). Anatomically, the pig eye is were also exposed to 2 mmol/L A231187, a calcium ionophore, as 223 159 remarkably similar to the human eye and it is well-endowed with apoptotic inducer (SigmaeAldrich, Madrid, Spain). 224 160 cones (Gerke et al., 1995) especially, in a large horizontal band Freshly detached neuroretinas were also obtained for normal 225 161 extending across the retina covering the optic disc and horizontal morphologic and biochemical evaluation. 226 162 meridian (Hendrickson and Hicks, 2002). This relative abundance 227 163 of cones makes the porcine eye a good model for therapeutic 2.2. Tissue processing and histology 228 164 research into the mechanisms of photoreceptor degeneration in 229 165 cone-related diseases. For morphological characterization retinal explants were 230 166 The mechanisms responsible for photoreceptor cell death still fixed in 4% filtered paraformaldehyde (SigmaeAldrich, Madrid, 231 167 remain unclear however, increasing evidence suggests that Spain) and 2.5% glutaraldehyde (Electron Microscopy Sciences, 232 168 oxidative stress (Komeima et al., 2006; Shen et al., 2005) and Hatfield, UK) in 0.1 M PBS (pH 7.4) for 2 h. Afterwards explants 233 169 inflammation (de Kozak et al., 1997; Yang et al., 2007; Yoshida were post-fixed with 2% osmium tetroxide, rinsed, dehydrated and 234 170 et al., 2013a,b) contribute to the pathogenesis of RP. Recently, embedded in Durcupan resin (Fluka, SigmaeAldrich, Madrid, 235 171 Yoshida et al. (2013a,b) have been found elevated inflammatory Spain). Semi-thin sections were cut at 1.5 mm, mounted on gelatin- 236 172 mediators in the eye of rd10 mice and of patients with RP coated slides and stained with 1% toluidine blue. These sections 237 173 including TNF-alpha. were examined under an Eclipse 80i microscope (NIKON In- 238 174 This study investigated whether PDE6 inhibition produced struments, Badhoevedorp, The Netherlands) and images were 239 175 ex vivo retinal degeneration in cone-enriched cultures of porcine captured with a DS-Qi1 digital camera (NIKON Instruments, 240

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241 Badhoevedorp, The Netherlands). ImageJ software was used to of selected area mean fluorescence of background) (Burgess et al., 306 242 quantify the thickness of the retinal explants. After calibration, 2010). Data are expressed as mean SEM. 307 243 total area of at least three visual fields per retinal explant was For co-localization with cleaved caspase-3 or PAR, staining was 308 244 calculated. The total area was expressed as mm2. Data are followed by TUNEL staining. 309 245 expressed as mean SEM. 310 246 To evaluate apoptosis with the terminal deoxynucleotidil 311 2.5. Cyclic GMP determination 247 transferase dUTP nick and labeling (TUNEL) assay, retinal explants 312 248 were fixed in 4% filtered paraformaldehyde (SigmaeAldrich, 313 cGMP was measured by using the BIOTRAK cGMP enzyme 249 Madrid, Spain) in 0.1 M PBS (pH 7.4) and cryoprotected in a sac- 314 immunoassay kit (GE Healthcare Europe GmbH, Barcelona, Spain). 250 charose gradient (15e20e30%) (Panreac Química, Barcelona, 315 Ò Retinal explants were homogenised in 5% trichloroacetic acid and 251 Spain). Samples were frozen embedded in Tissue-Tek O.C.T.Ô 316 neutralized with 2 M potassium bicarbonate. Neutralized super- 252 Compound (Sakura Finetek Europe B.V., Zoeterwoude, The 317 natant was used for cGMP determination. Protein content was 253 Netherlands). Next, 10 mm sections were cut with a cryostat (Leica 318 measured by the bicinchoninic acid (BCA) protein assay (BCA Kit; 254 CM1900, Nussloch, Germany) and placed on Super Frost Ultra Plus 319 Pierce Scientific, CA). The tissue cGMP levels were expressed as 255 treated slides (Thermo Scientific, Barcelona, Spain). 320 pmol/mg protein. 256 321 257 322 2.3. TUNEL assay 258 2.6. Caspase-3 activity assay 323 259 324 The TUNEL assay was performed on 10 mm cryosections by 260 Caspase-3 activity was measured with a colorimetric tetrapep- 325 means of an in situ cell death detection kit conjugated with rhoda- 261 tide (DEVD-pNA) cleavage assay kit following the manufacturer’s 326 mine fluorochrome (ApopTag Red In Situ) (Millipore, Schwalbach, 262 instructions (Bio-Vision, Mountain View, CA). Total retinal protein 327 Germany) according to the manufacturer’s instructions. The speci- 263 was extracted from retinal explants and measured by the BCA 328 mens were examined under a Eclipse 80i microscope (NIKON In- 264 protein assay. Caspase-3 activity was expressed as arbitrary units 329 struments, Badhoevedorp, The Netherlands) and images were 265 (au)/mg of protein. 330 captured with a DS-Qi1 digital camera (NIKON Instruments, Bad- 266 331 hoevedorp, The Netherlands). The apoptotic (TUNEL-positive) 267 332 nuclei per visual field were counted in at least three visual fields per 2.7. Nitrites and nitrates (NOX) determination 268 333 each retinal explant using NIS-Elements imaging software (NIKON 269 334 Instruments, Badhoevedorp, The Netherlands). The data were ana- Intracellular and extracellular nitrites (stable end-product of 270 335 lysed quantitatively and, only cells with red intensity were consid- NO) and nitrates (NOX) were measured in retinal explants by 271 336 ered TUNEL-positive. The number of apoptotic nuclei was spectrophotometric GRIESS reaction (El-Mlili et al., 2008) using 272 337 normalised to the SYTOX Green-labelled cell nuclei. Results are nitrate reductase. The tissue NOX levels were expressed as nmol/ 273 338 given as percentage of apoptotic nuclei/total nuclei. Data are mg protein (intracellular) or nmol/mL (extracellular). 274 339 expressed as mean SEM. 275 340 276 2.8. Oxidative stress evaluation 341 277 2.4. Immunofluorescence of Caspase-3 and PAR 342 278 Retinal explants were assayed for total antioxidant capacity 343 (TAC) and thiobarbituric acid reactive substances (TBARS) forma- 279 Immunofluorescence was carried out on 10 mm cryosections. 344 tion as indicator of malonyldialdehyde (MDA) formation. 280 Sections were post-fixed for 15 min at room temperature in 4% 345 Retinal explants were homogenized in 5 mM phosphate buffer 281 filtered paraformaldehyde (SigmaeAldrich, Madrid, Spain) in 346 pH 7, 0.9% NaCl, 0.1% glucose, centrifuged at 10,000 g for 15 min at 282 0.1 M PBS (pH 7.4). Sections were incubated for 1 h in blocking 347 4 C, and then the supernatants were used to determine TAC and 283 solution containing 5% normal goat serum, 1% bovine serum al- 348 TBARS. Protein concentrations were measured by the BCA protein 284 bumin and 0.25% Triton X-100. They were then incubated with 349 assay. 285 primary antibody against cleaved Caspase-3 (1:200, Cell Signaling 350 TAC was measured using a commercial kit (Cayman Chemical, 286 Technology, Barcelona, Spain) or PAR (poly-(ADP-ribose) (1:200, 351 Ann Arbor, MI) (Kowluru et al., 2006). The tissue TAC levels were 287 Enzo Life Sicence, Madrid, Spain) overnight at 4 C in blocking 352 expressed as nmol/mg protein. 288 solution. After washing with PBS three times, samples were 353 MDA levels were detected by a colorimetric method involving 289 incubated for 1 h at room temperature with the fluorescence- 354 thiobarbituric acid (TBA) adduct formation (Cayman Chemical, Ann 290 conjugated secondary antibody Alexa Fluor 647 (Invitrogen, Life 355 Arbor, MI). Tissue TBARS levels were expressed as nmol/mg protein. 291 Technologies, Madrid, Spain). After washing with PBS three times, 356 292 sections observed under a confocal microscope (Leica TCS SP5 357 293 Confocal microscope, Leica Microsistemas S.L.U, Barcelona, Spain). 2.9. TNF-alpha and IL-6 measurement 358 294 SYTOX Green (Molecular Probes, Paisley, UK) were used as a 359 295 specific nuclear counterstain. Cells were counted 40 magnifica- For detection of cytokine levels, retinal explants were homog- 360 296 tion, and the number of caspase-3 positive cells was counted enized in 20 mM TriseHCl pH 7.4, 10 mM EDTA containing protease 361 297 manually in four visual fields per each retinal explant. The number inhibitor cocktail (Complete Protease Inhibitor Cocktail; Roche, 362 298 of cells positive for the cleaved caspase-3 immunolabelling was Basel, Switzerland) and 200 mM phenylmethylsufloxifluoride 363 299 normalised to the SYTOX Green-labelled cell nuclei. Results are (PMSF). The TNF-a and IL-6 protein levels were estimated with the 364 300 given as percentage of caspase-3 positive cell/total nuclei. Data are corresponding ELISA kit (Diaclone, Besancon, France), according to 365 301 expressed as mean SEM. the manufacturers’ instructions. Tissue cytokine levels were 366 302 PAR positive cells were difficult to count in outer nuclear layer expressed as pg/mg protein. 367 303 (ONL). For the quantification of PAR immunostaining we used the Values for cGMP, caspase-3 activity, NOX, oxidative markers and 368 304 following formula to calculate the corrected fluorescence (CF) for cytokines are given as the mean SEM of at least ten different 369 305 each cell layer: CF ¼ Integrated density of the selected area (area cultures. For each experiment samples were measured in duplicate. 370

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371 2.10. Western blot of these results, 100 nmol/L seemed to be the best concentration of 436 372 Zaprinast for further studies. 437 373 Retinal explants were homogenized in lysis buffer (50 mM Trise 438 374 HCl buffer pH 7.4 containing 50 mM NaCl, 5 mM EDTA, 1% SDS, 3.2. Evaluation of organotypic retina cultures 439 375 protease inhibitor cocktail (Complete Protease Inhibitor Cocktail; 440 376 Roche, Basel, Switzerland) and 1 mM PMSF). Samples (30 mg) were Toluidine blue staining of semi-thin sections demonstrated that 441 377 subjected to electrophoresis and immunoblotting as described in the overall architecture of the cultured retina was maintained 442 378 Corbalán et al. (2002). The following primary antibodies were used: throughout the culture period (Fig. 2A). However, morphometric 443 379 cleaved caspase-3 (Asp175) rabbit polyclonal antibody (1:1000, Cell analysis revealed that explants treated with 100 nmol/L Zaprinast 444 380 Signaling Technology, Danvers, MA, USA), calpain-2 rabbit poly- were significantly thinner (47,915 2938 mm2, ManneWhitney 445 381 clonal antibody (1:1000; SigmaeAldrich, Madrid, Spain); and test, p < 0.01 and 33,129 1365 mm2, ManneWhitney test, 446 382 b-actin mouse monoclonal antibody (1:2000; SigmaeAldrich, p < 0.05), compared with controls (58,940 861 mm2 and 447 383 Madrid, Spain). The images were captured using an EPSON SCAN 42,000 2220 mm2) at 24 h and 48 h respectively (Fig. 2B). Zap- 448 384 from EPSON Corporation (EPSON IBERICA, Barcelona, Spain) and rinast also decreased photoreceptor outer segments (OS) compared 449 385 quantified using the Alpha Imager 2200 (version 3.1.2) software to control explants (Fig. 2A). 450 386 (AlphaInnotech Corporation, San Francisco, CA, USA). 451 387 3.3. PDE inhibition induces apoptosis in cultured porcine retina 452 388 2.11. Statistical analyses 453 389 The TUNEL assay was performed to measure the apoptotic cell 454 390 For parametric data, ANOVA followed by NewmaneKeul’s post- death in fresh isolated retinas, control explants and explants 455 391 hoc test was used. When only two samples were compared the treated with different Zaprinast concentrations (Fig. 3). While fresh 456 392 Student’s t-test was used. isolated retina did not present any apoptotic nuclei, control ex- 457 393 For non-parametric data, ManneWhitney test was used. Sig- plants showed a few apoptotic nuclei (2.4 0.7% of TUNEL-positive 458 394 nificance levels were set at a ¼ 0.05. cells) after 24 h in culture. 459 395 TUNEL staining demonstrated that inhibition of PDE induced 460 396 3. Results photoreceptor degeneration overall after 48 h of Zaprinast treat- 461 397 ment. As shown in Fig. 3A and Table 1, apoptotic nuclei were found 462 398 3.1. PDE inhibition triggers cGMP accumulation in cultured porcine in outer nuclear layer (ONL), in inner nuclear layer (INL) and also in 463 399 retina the ganglionar layer (GCL) at the lower dose of Zaprinast (100 nmol/ 464 400 L). The major effect was observed in ONL (4.6 1.3% of apoptotic 465 e < 401 Retinal explants were cultured for 48 h in the presence or cells, Mann Whitney test, p 0.0001). Moreover, Zaprinast caused 466 fi 402 absence of Zaprinast, a selective PDE5/6 inhibitor which raises signi cant increase of TUNEL-positive cells (ANOVA test, post-hoc 467 e < 403 intracellular cGMP levels in a concentration-dependent manner Newman Keuls, p 0.05) after 24 h in a concentration- 468 404 and causes cGMP-dependent photoreceptor degeneration in small dependent manner (Fig. 3B). 469 405 animals closely resembling the rd1 degeneration (Sahaboglu et al., As shown in Fig. 3 control explants showed low numbers of 470 406 2010). TUNEL-positive cells after 24 h in culture (2.4 0.7% of apoptotic 471 407 Successful PDE6 inhibition was confirmed by a significant cGMP cells) but, this number increased across the whole retinal explant 472 408 increase at all time points evaluated (Fig. 1). While untreated retina (ONL, INL and GCL) after 48 h (6.7 1.0% of apoptotic cells). This cell 473 409 essentially maintained the same level of cGMP, the Zaprinast- death was consequence of retinal detachment and culturing time. 474 410 treated retina showed a strong increase of cGMP accumulation at To further investigate the impact of PDE inhibition on down- 475 411 24 h in a dose-dependent manner (1.72 0.3; 2.04 0.2, stream effectors of cell death, we analysed the activity of caspase-3, 476 412 2.78 0.27 pmol/mg protein at 100, 200 and 500 nmol/L respec- and activation of calpain-2. 477 413 tively, One-way ANOVA post-hoc NewmaneKeuls p < 0.05). Under After 24 h, caspase-3 activity (Fig. 4A) and protein content of 478 414 our experimental conditions, the effect of Zaprinast concentration cleaved caspase-3 (Fig. 4B) were further up regulated in retina 479 415 on cGMP accumulation disappeared at 48 h. treated with 100 nmol/L Zaprinast (2.4 0.1 au/mg protein and 480 416 100 nmol/L Zaprinast was the lowest concentration that 2.1 0.4 fold over control retina respectively, student t-test, 481 < 417 induced a significant cGMP accumulation at 24 h and 48 h. In view p 0.05). This up regulation was maintained at 48 h (2.4 0.2 au/ 482 418 mg protein and 2.7 0.8 fold over control retina, student t test, 483 419 p < 0.05). Immunofluorescence of cleaved caspase-3 revealed 484 420 that PDE inhibition significantly up regulated caspase-3 in INL 485 421 (2.6 0.3% of caspase-3 positive cells, p < 0.0005) and GCL 486 422 (1.7 0.2% of caspase-3 positive cells, p < 0.01) but not in ONL 487 423 (0.3 0.1% of caspase-3 positive cells), after 24 h in culture (Table 1 488 424 and Fig. 4C). Control explants also showed up regulation of caspase- 489 425 3 mainly in GCL after 48 h. 490 426 Western blot analysis showed that PDE6 inhibition also induced 491 þ 427 calpain-2 activation (Fig. 4D). A Ca2 ionophore, A231187, was used 492 428 to confirm calpain-2 activation. 493 429 Strong activation of poly(ADP-ribose) polymerase (PARP) has 494 430 been found in animal models of retinal degeneration with subse- 495 431 Fig. 1. PDE6 inhibition induces cGMP accumulation in cone-enriched cultures of quent accumulation of poly(ADP-ribose) (PAR) polymers (Paquet- 496 432 porcine neuroretina. Control retinal explants (C) or explants treated with 100 (Z100), Durand et al., 2007; Kaur et al., 2011). Excessive PARP activation 497 200 (Z200) or 500 (Z500) nmol/L Zaprinast were prepared as described in Materials 433 may contribute to caspase-independent photoreceptor death 498 and methods. The levels of cGMP at different times in culture are shown. Values are 434 the mean SEM of twelve cultures. Values significantly different from own control are (Paquet-Durand et al., 2007; Kaur et al., 2011). To investigate 499 435 indicated by asterisks *p < 0.05, **p < 0.01 (ANOVA NewmaneKeuls post-hoc). indirectly PARP activity in Zaprinast-treated explants, we 500

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501 566 502 567 503 568 504 569 505 570 506 571 507 572 508 573 509 574 510 575 511 576 512 577 513 578 514 579 515 580 516 581 517 582 518 583 519 584 520 585 521 586 522 587 523 588 524 589 525 590 526 591 527 592 528 593 529 594 530 595 531 596 532 597 533 598 534 599 535 600 536 601 537 602 538 603 539 604 540 605 541 606 542 607 543 608 544 609 545 610 546 611 547 612 548 613 549 614 550 615 551 616 552 617 553 Fig. 2. PDE6 inhibition reduces retinal thickness of cone-enriched cultures of porcine neuroretina. Control retinal explants or Zaprinast-treated retinal explants were prepared as 618 described in Materials and methods. (A) Light micrographs from toluidine blue-stained semi-thin sections of control and 100 nmol/L Zaprinast-treated retinal explants. Abbre- 554 viations: OS (outer segment); outer nuclear layer (ONL); inner nuclear layer (INL); ganglion cell layer (GCL). (B) Quantitative analysis of retinal thickness. Control (C); 100 nmol/L 619 555 Zaprinast (Z100). Values are the mean SEM of six different cultures. Values that are significantly different from own control are indicated by asterisks *p < 0.05 (ANOVA Newmane 620 556 Keuls post-hoc). 621 557 622 558 performed PAR immunostaining. PDE6 inhibition significantly large extent with TUNEL-positive cells in GCL and ONL. A few 623 559 increased accumulation of poly(ADP-ribosyl)ated proteins in ONL number of cells in INL also co-localized with PAR. However, caspase- 624 560 (23,744 5304 corrected fluorescence, ManneWhitney test, 3 positive cells did not co-localize with TUNEL-positive cells except 625 561 p < 0.05) compared to control (7497 722 corrected fluorescence) for a subset of cells in INL. Moreover, double-immunostaining of 626 562 after 24 h in culture (Table 1). caspase-3 and PAR reflected co-localization in a subset of cells in GCL 627 563 To determine whether cleaved caspase-3 or PAR co-localize with and INL. Caspase-3 activation occurs mainly in INL and it may 628 564 TUNEL-positive cells, we performed double labelling (Fig. 5). In partially contribute to cell death in this cell layer. Moreover, caspase- 629 565 Zaprinast-treated explants PAR immunostaining co-localized to a 3 activation also occurs in GCL and could contribute to the future cell 630

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631 696 632 697 633 698 634 699 635 700 636 701 637 702 638 703 639 704 640 705 641 706 642 707 643 708 644 709 645 710 646 711 647 712 648 713 649 714 650 715 651 716 652 717 653 718 654 719 655 720 656 721 657 722 658 723 659 724 660 725 661 726 662 727 663 728 664 729 665 730 666 731 Fig. 3. PDE6 inhibition induces apoptosis in cone-enriched cultures of porcine neuroretina. Control retinal explants or Zaprinast-treated retinal explants were prepared as described 667 in Materials and methods. (A) Photomicrographs of TUNEL-stained sections visualizing apoptotic photoreceptors (red) and total cell nuclei (green) in control and 100 nmol/L 732 668 Zaprinast-treated explants. Scale bar: 50 mm. (B) Zaprinast induced apoptosis in a dose-dependent manner (100, 200, 500 nmol/L) after 24 h in culture. Values are the mean SEM of 733 669 eight different cultures. Values significantly different (p < 0.05) from Z100 are indicated by ‘a’. Values that are significantly different from own control are indicated by asterisks 734 670 *p < 0.05 (ANOVA NewmaneKeuls post-hoc). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 735 671 736 672 death. Taking together, these results suggest that PDE6 inhibition measurement of nitric oxide, TBARS formation as indicator of lipid 737 673 probably induces cell death by caspase-independent mechanisms peroxidation and total antioxidant capacity (Fig. 6). 738 674 (PARP activity) in ONL and GCL and by caspase-dependent and At 24 h, PDE6 inhibition enhanced iNOX levels compared with 739 675 caspase-independent mechanisms in INL. control (48 4 nmol/mg protein and 30 2 nmol/mg protein, 740 676 respectively, student t-test, p < 0.05). As culturing time moved for- 741 677 3.4. PDE inhibition induces oxidative stress and cytokine production ward eNOX level also increased compared to control (47 3 nmol/ 742 678 in cultured porcine retina mL and 36 2 nmol/mL, respectively, student t-test, p < 0.05) 743 679 (Fig. 6A). 744 680 It has been described that cGMP accumulation induces oxidative MDA concentration was determined by the thiobarbituric acid 745 681 stress in murine models of retinal degeneration (Sharma and (TBA) assay, which measures the amount of TBA reactivity with 746 682 Rohrer, 2007). To explore whether PDE inhibition also induced MDA formed during the acid hydrolysis of lipid peroxide com- 747 683 oxidative damage in cultured porcine retina we measured: intra- pound. Addition of 100 nmol/L Zaprinast caused a rapid increase of 748 684 cellular and extracellular nitrites formation (iNOX and eNOX) as lipid peroxide TBA value compared with control at 24 h 749 685 750 686 751 687 Table 1 752 688 Effect of Zaprinast on cell death markers in porcine retinal explants. 753 689 Layer TUNEL-positive cells (%) Caspase-3 positive cells (%) PAR content (CF) 754 690 C Z100 C Z100 C Z100 755 691 ONL 0.43 0.13 4.55 1.3*** 0.08 0.03 0.28 0.06 7497 722 23,744 5304* 756 692 INL 0.92 0.20 2.40 0.40** 0.29 0.09 2.64 0.34*** 7019 1163 9640 2380 757 693 GCL 0.71 0.15 2.66 0.44** 0.82 0.27 1.72 0.24** 9891 2011 10,413 2301 758 694 Note: ManneWhitney test was used. Values different from control are shown by *p < 0.05, **p < 0.01, ***p < 0.0005. ONL: outer nuclear layer; INL: inner nuclear layer; GCL: 759 695 ganglionar nuclear layer; C: control; Z100: 100 nM Zaprinast; CF: corrected fluorescence. 760

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761 826 762 827 763 828 764 829 765 830 766 831 767 832 768 833 769 834 770 835 771 836 772 837 773 838 774 839 775 840 776 841 777 842 778 843 779 844 780 845 781 846 782 847 783 848 784 849 785 850 786 851 787 852 788 853 789 854 790 855 791 Fig. 4. PDE6 inhibition activates caspase-3 and calpain-2 in cone-enriched cultures of porcine neuroretina. Control retinal explants or Zaprinast-treated retinal explants were 856 792 prepared as described in Materials and methods. (A) Caspase-3 activity using the substrate DEVD-pNA in homogenates of control retinal explants or explants treated with Zaprinast 857 m 793 (100 nmol/mL). Values are the mean SEM of twelve cultures. (B and D) Retinal homogenates (30 g of protein) were subjected to electrophoresis and cleaved caspase-3 (B) and 858 calpain-2 (D) activation were analysed by immunoblotting. The intensities of the bands were quantified, normalized respect to internal control (b-actin) and the values are rep- 794 resented as fold over control. Values significantly different from own control are indicated by asterisks *p < 0.05 (paired student t-test). (C) Evaluation of caspase-3 activation with 859 795 cleaved caspase-3 staining in frozen sections; scale bar, 50 mm. Q5 860 796 861 797 862 798 (3.9 0.4 nmol/mg protein and 2.0 0.2 nmol/mg protein, student retinitis pigmentosa. Between 4 and 5% of patients with retinitis 863 799 t-test, p < 0.05), remaining elevated at 48 h (4.8 0.5 nmol/mg pigmentosa present PDE6 mutations leading to non-functional 864 800 protein, student t-test, p < 0.05 (Fig. 6B). enzyme. 865 801 The overall antioxidant capacity of retinal explants treated with There is a lot of interest in using the pig as a model of retinal 866 802 100 nmol/L Zaprinast, decreased at 24 h and 48 h (174 12 mmol/mg disease and stem cell transplantation therapy due to its resem- 867 803 protein and 156 9 mmol/mg protein respectively, student t-test, blance to human retina. However, transgenic pigs are expensive 868 804 p < 0.05), compared to control retinal explants (225 21 mmol/mg and difficult to manage. Porcine organotypic culture of the retina is 869 805 protein and 192 14 mmol/mg protein, respectively) (Fig. 6B). a choice that could allow us to evaluate the effect of new thera- 870 806 TNFa and IL-6 are upregulated in several inflammatory ocular peutic drugs on some retinal changes faster and cheaper than 871 807 diseases, including AdamantiadeseBehcet disease (Durrani et al., in vivo and has the additional advantage of reducing animal ex- 872 808 2007), retinal vascular tumours (Japiassú et al., 2008), neo- periments. These organotypic cultures were originally developed to 873 809 vascular age-related macular degeneration (Seddon et al., 2005), follow cellular and cytoskeletal changes during the culture period 874 810 uveitis (Murray et al.,1990), and retinitis pigmentosa (Yoshida et al., (Allamby et al., 1997; Winkler et al., 2002) and more recently to 875 811 2013a,b). We assessed whether 100 nmol/L Zaprinast induced these study retinal detachment (Fernandez-Bueno, 2008; Fisher and 876 812 pro-inflammatory mediators in porcine retinal explants (Fig. 6C). Lewis, 2003). Here we evaluated whether PDE6 inhibition pro- 877 813 PDE inhibition induced a significant long-lasting upregulation of duced retinal degeneration in enriched-cone cultures of porcine 878 814 TNF-alpha at 24 h and 48 h (49.6 10.2 pg/mg protein and retina. 879 815 50.8 5.6 pg/mg protein, respectively, student t-test, p < 0.05) Firstly, we assessed whether this porcine model reproduces the 880 816 compared to control (26.6 3.6 pg/mg protein and 24.5 2.9 pg/ retinal degeneration observed in small animals after PDE inhibition. 881 817 mg protein, respectively). Our results showed that PDE inhibition induced cGMP accumula- 882 818 At 24 h PDE inhibition also produced IL-6 up regulation tion accompanied by retinal degeneration. These results corrobo- 883 819 (6.5 1.8 pg/mg protein, student t-test, p < 0.05) compared to rated those found in small animals both in vivo and in vitro 884 820 control (3.1 0.4 pg/mg protein). (Sahaboglu et al., 2010; Vallazza-Deschamps et al., 2005). 885 821 Sahaboglu et al. (2010) demonstrated that Zaprinast caused cGMP- 886 822 4. Discussion dependent photoreceptor degeneration closely resembling the rd1 887 823 degeneration observed in murine retinal explants. In the rd1 mouse 888 824 Mutations in genes related to cGMP metabolism as RetGC, the high levels of cGMP increase the number of the cGMP-gated 889 825 GCAPs or PDE6 are involved in several retinal dystrophies including channels in the open state, thus allowing intracellular calcium 890

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891 956 892 957 893 958 894 959 895 960 896 961 897 962 898 963 899 964 900 965 901 966 902 967 903 968 904 969 905 970 906 971 907 972 908 973 909 974 910 975 911 976 912 977 913 978 914 979 915 980 916 981 917 982 918 983 919 984 920 985 921 986 922 987 923 988 924 989 925 990 926 991 927 992 Fig. 5. PDE6 inhibition induced different cell markers in cone-enriched cultures of porcine neuroretina. Double-immunostaining of control retinal explants or explants treated with 928 Zaprinast (100 nmol/mL) were prepared as described in Materials and methods. (A) Double staining of TUNEL assay and PAR. (B) Double staining of TUNEL assay and cleaved 993 929 caspase-3. (C) Double staining of PAR and cleaved caspase-3; scale bar: 50 mm. Images A3, B3 and C3 were obtained after zooming (factor ¼ 3) with Leica TCS SP5 Confocal 994 930 microscope. 995 931 996 932 997 þ 933 (Ca2 ) to rise to toxic levels and leading to rapid photoreceptor retinal degeneration (Kaur et al., 2011; Paquet-Durand et al., 2010; 998 934 degeneration (Fox et al., 1999). Several studies have pursued Sanges et al., 2006). In our model caspase-3 activation seems to be 999 935 to reverse the effects of PDE6 loss function by blocking cGMP- involved in part of cell death in INL but neither in GCL nor ONL. 1000 þ 936 gate channel (Vallazza-Deschamps et al., 2005), Ca2 channel However, co-localization of PAR accumulation with TUNEL-positive 1001 937 (Nakazawa, 2011) or PKG activity (Paquet-Durand et al., 2009). All cells suggests that caspase-independent mechanisms are involved 1002 938 these treatments reduced photoreceptor cell death. in cell death in ONL and GCL. It is important to highlight that 1003 939 We reported that sustained elevation of intracellular cGMP in although TUNEL is used as an apoptotic marker, it also can detect 1004 940 porcine retinal explants triggered different downstream effectors of non-apoptotic DNA fragmentation, e.g. in necrosis (Grasl-Kraupp 1005 941 cell death related to caspase-dependent mechanisms (caspase-3) et al., 1995). Taken together, these findings suggest the involve- 1006 942 and caspase-independent mechanisms (calpain-2 and probably ment of multiple death signalling mechanisms (caspase-dependent 1007 943 PARP activity, measured as PAR accumulation). In 661W cells in- and caspase-independent mechanisms) in retinal cell death after 1008 þ 944 hibition of PDE, increases the intracellular Ca2 that in turns acti- PDE6 inhibition in porcine retina. 1009 945 vates the cysteine protease calpain, which executes apoptosis via We have described that PDE inhibition induced the inflamma- 1010 946 modulation of caspase-3 activity (Sharma and Rohrer, 2004). Cas- tory mediators TNF-alpha and IL-6 in porcine retina. Elevated levels 1011 947 pase activation occurs mainly through the extrinsic and intrinsic of these two inflammatory mediators have been recently described 1012 948 pathways (Bredesen et al., 2006). Although caspase-3 inhibitors in the eye of patients with RP and rd10 mice (Yoshida et al., 2013a,b). 1013 949 have been transiently effective in delaying retinal degeneration TNF-alpha or IL-6 have been also observed in other eye diseases, 1014 950 through inhibition of the apoptosis of photoreceptor cells in rd including uveitis, proliferative vitreoretinopathy, retinal detach- 1015 951 gene-carrying mice (Yoshizawa et al., 2002), the role of caspases in ment and age-related macular degeneration (El-Ghrably et al.,1999; 1016 952 inherited retinal degeneration is controversial (Doonan et al., 2003; Klein et al., 2008; Nakazawa et al., 2011). TNF-a is likely secreted 1017 953 Sanges et al., 2006; Zeiss et al., 2004). Recent studies have shown from activated macrophages, astrocytes, microglial cells and retinal 1018 954 that several caspase-independent inducers of cell death such as AIF Müller glial cells. It has been described that TNF-alpha has cytotoxic 1019 955 (apoptosis-inducing factor), calpains, PARP are activated during effects on photoreceptors (Nakazawa et al., 2006, 2011). TNF-alpha 1020

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1021 1086 1022 1087 1023 1088 1024 1089 1025 1090 1026 1091 1027 1092 1028 1093 1029 1094 1030 1095 1031 1096 1032 1097 1033 1098 1034 1099 1035 1100 1036 1101 1037 1102 1038 1103 1039 1104 1040 1105 1041 1106 1042 1107 1043 1108 1044 1109 1045 1110 1046 1111 1047 1112 1048 1113 1049 1114 1050 1115 1051 1116 1052 1117 1053 1118 1054 1119 1055 1120 1056 1121 1057 1122 1058 1123 1059 1124 1060 1125 1061 1126 1062 1127 1063 1128 1064 1129 1065 1130 Fig. 6. PDE inhibition triggers oxidative stress and cytokine production cone-enriched cultures of porcine neuroretina. Control retinal explants or explants treated with Zaprinast 1066 (100 nmol/mL) were prepared as described in Materials and methods. (A) Effect of Zaprinast on nitric oxide formation (measurement of nitrites and nitrates (NOX)). Intracellular 1131 1067 (iNOX) and extracellular nitrites and nitrates (eNOX) were measured by Griess reaction. (B) Effect of Zaprinast on the total antioxidant capacity and malonyldialdehyde formation 1132 1068 (TBARS formation). (C) Effect of Zaprinast on TNF-alpha and IL-6 content. Each sample was measured in duplicate, and the values are the mean SEM of twelve cultures. Values 1133 1069 significantly different from own control are indicated by asterisks *p < 0.05, **p < 0.01 (paired student t-test). 1134 1070 1135 1071 1136 1072 can trigger several well-characterized death-promoting (caspase- potential resulting in release of mitochondrial factors such as cy- 1137 1073 dependent and caspase-independent cell death) and survival- tochrome c or AIF. AIF rapidly translocates from mitochondria to the 1138 1074 promoting pathways, depending upon the predominating signal- nucleus and induces nuclear fragmentation and cell death by 1139 1075 ling pathway in the particular cell type (Maianski et al., 2003). TNF- autophagic degeneration (Suo et al., 2010; Munemasa et al., 2010; 1140 1076 alpha binding to cell surface receptors such as TNF receptor 1 me- Tezel and Yang, 2004; Lorenzo et al., 1999; Daugas et al., 2000). The 1141 1077 diates activation of initiator caspases (caspase-8, caspase-10), and translocation of AIF has been implicated in several types of 1142 1078 finally triggers cleavage of effector caspases (extrinsic pathway of neuronal death, including photoreceptor and ganglion cell death 1143 1079 cell death) (Nagata, 1997). TNF-alpha binding to cell surface re- (Hisatomi et al., 2001; Mizukoshi et al., 2010; Thapa et al., 2012; 1144 1080 ceptors may also elicit anti-apoptotic responses mediated by the Sanges et al., 2006). Inhibition of nuclear AIF translocation delays 1145 1081 activation of the NF-kB pathway. retinal degeneration of RCS rats, a model of retinitis pigmentosa 1146 1082 Several evidence shows that TNF-alpha is also involved in the (Murakami et al., 2008). 1147 1083 intrinsic pathway of cell death that is initiated by cellular and DNA PARP-1 activation in response to excessive DNA damage triggers 1148 1084 damage and particularly employs mitochondria. TNF-alpha and the release of AIF from mitochondria and promotes PARP-1- 1149 1085 other stimuli can reduce the mitochondrial transmembrane dependent cell death or parthanatos. AIF is a high-affinity PAR- 1150

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1151 binding protein. PAR-binding by AIF is required for its release from Uncited reference Q3 1216 1152 the mitochondria, translocation to the nucleus, and cell death 1217 1153 (Wang et al., 2011). PARP activation and PAR accumulation have Tsai et al., 2009. 1218 1154 been linked to mitochondrial death and AIF translocation to the 1219 1155 nucleus in photoreceptor cell death in rd1 mice (Paquet-Durand 1220 Acknowledgements 1156 et al., 2007). 1221 1157 The other inflammatory mediator upregulated in these cultures, 1222 We thank Iván Fernández-Bueno (University of Valladolid, 1158 IL-6 is a pleiotropic cytokine with a role in inflammation, angio- 1223 Instituto Universitario de Oftalmobiologia Aplicada (IOBA), Spain) 1159 genesis, cell differentiation and neuronal survival. In the retina, IL-6 1224 for his help with the development of the retinal organotypic cul- 1160 is synthesized by Müller cells and the RPE (Benson et al., 1992; 1225 ture, Fany Belenchon and Carlos Vila (Unidad Veterinaria-Estabu- 1161 Yoshida et al., 2001). A neuroprotective role for IL-6 has been 1226 lación Centro de Investigación, Hospital La Fe, Valencia, Spain), Ana 1162 suggested in different animal models of ocular injury, in vitro 1227 Díaz (Unidad Central de Investigación Facultad de Medicina y 1163 studies, retinal vein occlusion, diabetic macular oedema and 1228 Odontología, Universidad de Valencia, Valencia, Spain), Juan Martín 1164 experimental glaucoma (Chong et al., 2008; Funatsu et al., 2009; 1229 (Local Slaughterhouse MercaValencia, Valencia, Spain) for 1165 Noma et al., 2009) suggesting that IL-6 upregulation after injury 1230 providing pig eyes and, Rafael Vázquez-Manrique for helping us 1166 may serve to control photoreceptor apoptosis (Chong et al., 2008). 1231 with the confocal microscope (Sensory Organ Disorders, IIS-La Fe, 1167 Retinal degeneration induced by PDE inhibition was accompa- 1232 Valencia). This work was supported by the European Regional 1168 nied by oxidative stress in porcine retina. Retina is normally pro- 1233 Development Fund, Instituto de Salud Carlos III, PI08/90311 from 1169 tected from oxidative damage by the presence of enzymes such as 1234 the Ministerio de Ciencia e Innovación (MICINN) and AP-183/10 1170 superoxide dismutase and catalase (De La Paz et al., 1996). Photo- 1235 from the Conselleria de Sanitat de la Comunitat Valenciana. 1171 receptors, which are the predominant cell type in the retina, are 1236 Regina Rodrigo has a Contrato-Investigador SNS Miguel Servet 1172 particularly susceptible to free radical damage or lipid peroxidation 1237 (CP09/118) from Instituto de Salud Carlos III (MICINN). 1173 (Osborne and Wood, 2004), because retinal photoreceptor mem- 1238 1174 branes have an unusually high concentration of docosahexaenoic 1239 1175 acid. Oxidative damage is a major factor contributing to cone cell References 1240 1176 death after the death of rods has occurred (Komeima et al., 2006; 1241 1177 Shen et al., 2005). The increased levels of oxygen (hyperoxia), after Allamby, D., Foreman, D., Carrington, L., McLeod, D., Boulton, M., 1997. Cell 1242 1178 death of rods result in progressive oxidative damage to cones in a attachment to, and contraction of, the retina in vitro. Invest. Ophthalmol. Vis. 1243 Sci. 38 (10), 2064e2072. 1179 transgenic pig model of RP and in multiple mouse models, Behnen, P., Dell’Orco, D., Koch, K.W., 2010. Involvement of the calcium sensor 1244 1180 including models of recessive and dominant RP. Antioxidant GCAP1 in hereditary cone dystrophies. Biol. Chem. 391 (6), 631e637. 1245 1181 treatments can scavenger reactive oxygen species and promote Benson, M.T., Shepherd, L., Rees, R.C., Rennie, I.G., 1992. Production of interleukin-6 1246 by human retinal pigment epithelium in vitro and its regulation by other cy- 1182 cone survival and function (Komeima et al., 2006). In our study, we tokines. Curr. Eye Res. 11 (Suppl.), 173e179. 1247 1183 demonstrated the elevation of NO (NOX), which may increase Bowes, C., Li, T., Danciger, M., Baxter, L.C., Applebury, M.L., Farber, D.B., 1990. Retinal 1248 1184 peroxynitrite via the reaction the superoxide radical (Pryor and degeneration in the rd mouse is caused by a defect in the beta subunit of rod 1249 cGMP-phosphodiesterase. Nature 347 (6294), 677e680. 1185 Squadrito, 1995). NO is an important regulator of homeostatic Bredesen, D.E., Rao, R.V., Mehlen, P., 2006. Cell death in the nervous system. Nature 1250 1186 processes in the eye and its over-expression could contribute to 443, 796e802. 1251 1187 pathological conditions in RP (Komeima et al., 2008). Buch, P.K., Mihelec, M., Cottrill, P., Wilkie, S.E., Pearson, R.A., Duran, Y., West, E.L., 1252 1188 Michaelides, M., Ali, R.R., Hunt, D.M., 2011. Dominant cone-rod dystrophy: a 1253 We hypothesize that cGMP accumulation induces oxidative mouse model generated by gene targeting of the GCAP1/Guca1a gene. PLoS One 1189 stress that probably induces microglial activation, as described in rd 28 (6(3)), e18089. 1254 1190 mice, that in turns upregulates TNF-alpha contributing to the cell Burgess, A., Vigneron, S., Brioudes, E., Labbé, J.-C., Lorca, T., Castro, A., 2010. Loss of 1255 1191 death. TNF-alpha can activate different cell death pathways human Greatwall results in G2 arrest and multiple mitotic defects due to 1256 deregulation of the cyclin B-Cdc2/PP2A balance. Proc. Natl. Acad. Sci. U.S.A. 107, 1192 including caspase-3, and PARP. On the other hand, cGMP accumu- 12564e12569. 1257 1193 lation leads to calcium influx and calpain activation. We believe Burns, M.E., Mendez, A., Chen, J., Baylor, D.A., 2002. Dynamics of cyclic GMP syn- 1258 e 1194 that PDE6 inhibition activates more than one apoptotic pathway thesis in retinal rods. Neuron 36 (1), 81 91. 1259 Chang, B., Hawes, N.L., Hurd, R.E., Davisson, M.T., Nusinowitz, S., Heckenlively, J.R., 1195 (caspase-dependent and caspase-independent) as occurs in other 2002. Retinal degeneration mutants in the mouse. Vision Res. 42 (4), 517e 1260 1196 experimental models of retinal degeneration (Gómez-Vicente et al., 525. 1261 1197 2005; Kaur et al., 2011). Moreover, our findings suggest that Chang, B., Grau, T., Dangel, S., Hurd, R., Jurklies, B., Sener, E.C., Andreasson, S., 1262 Dollfus, H., Baumann, B., Bolz, S., Artemyev, N., Kohl, S., Heckenlively, J., 1198 different retinal cell types follow different apoptotic pathways. Wissinger, B., 2009. A homologous genetic basis of the murine cpfl1 mutant and 1263 1199 In summary, PDE6 mutations induce retinal degeneration in human achromatopsia linked to mutations in the PDE6C gene. Proc. Natl. Acad. 1264 1200 small animal models (rd1, rd and rd10 mice), but eye size and Sci. U.S.A. 17 (106(46)), 19581e19586. 1265 Chong, D.Y., Boehlke, C.S., Zheng, Q.D., Zhang, L., Han, Y., Zacks, D.N., 2008. Inter- 1201 anatomic differences suggest that should be useful to have alter- leukin-6 as a photoreceptor neuroprotectant in an experimental model of 1266 1202 native models for studying retinal rescue strategies or design new retinal detachment. Invest. Ophthalmol. Vis. Sci. 49 (7), 3193e3200. 1267 1203 drugs intended for humans. Porcine eyes are closer in size to human Corbalán, R., Chatauret, N., Behrends, S., Butterworth, R.F., Felipo, V., 2002. Region 1268 1204 selective alterations of soluble guanylate cyclase content and modulation in 1269 eyes and have a rich supply of rod and cones. Our organotypic brain of cirrhotic patients. Hepatology 36, 1155e1162. 1205 culture of porcine cone-enriched retina exposed to Zaprinast may Corton, M., Blanco, M.J., Torres, M., Sanchez-Salorio, M., Carracedo, A., Brion, M., 1270 1206 provide a helpful model to design and assay some treatments thus 2010. Identification of a novel mutation in the human PDE6A gene in autosomal 1271 1207 replacing or reducing animal experiments. recessive retinitis pigmentosa: homology with the nmf28/nmf28 mice model. 1272 (Letter) Clin. Genet. 78, 495e498. 1208 However, this kind of culture has its own limitations. Organo- Daugas, E., Nochy, D., Ravagnan, L., et al., 2000. Apoptosis-inducing factor (AIF): a 1273 1209 typic cell culturing involves transaction of the optic nerve and ubiquitous mitochondrial oxidoreductase involved in apoptosis. FEBS Lett. 476, 1274 e 1210 mechanical retinal detachment causing photoreceptor loss and 118 123. 1275 de Kozak, Y., Cotinet, A., Goureau, O., Hicks, D., Thillaye-Goldenberg, B., 1997. Tumor 1211 retrograde retinal ganglion cell degeneration. To minimize this necrosis factor and nitric oxide production by resident retinal glial cells from 1276 1212 problem, use of detached samples as controls is necessary. In the rats presenting hereditary retinal degeneration. Ocul. Immunol. Inflamm. 5 (2), 1277 1213 future, we aim at improving the viability of organotypic cell cul- 85e94. 1278 fi De La Paz, M.A., Zhang, J., Fridovich, I., 1996. Antioxidant enzymes of the human 1214 tures, although it is dif cult to culture retinal neurons for long retina: effect of age on enzyme activity of macula and periphery. Curr. Eye Res. 1279 1215 periods perhaps due to the high energy requirements of the retina. 15 (3), 273e278. 1280

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1281 Dizhoor, A.M., 2000. Regulation of cGMP synthesis in photoreceptors: role in Kowluru, R.A., Kowluru, V., Xiong, Y., Ho, Y.S., 2006. Overexpression of mitochon- 1346 e 1282 signal transduction and congenital diseases of the retina. Cell. Signal. 12 (11 drial superoxide dismutase in mice protects the retina from diabetes-induced 1347 12), 711e719. oxidative stress. Free Radic. Biol. Med. 41 (8), 1191e1196. 1283 Doonan, F., Donovan, M., Cotter, T.G., 2003. Caspase-independent photoreceptor Lorenzo, H.K., Susin, S.A., Penninger, J., Kroemer, G., 1999. Apoptosis inducing factor 1348 1284 apoptosis in mouse models of retinal degeneration. J. Neurosci. 23, 5723e5731. (AIF): a phylogenetically old, caspase- independent effector of cell death. Cell. 1349 1285 Dryja, T.P., Rucinski, D.E., Chen, S.H., Berson, E.L., 1999. Frequency of mutations in the Death Differ. 6, 516e524. 1350 gene encoding the alpha subunit of rod cGMP-phosphodiesterase in autosomal Maianski, N.A., Roos, D., Kuijpers, T.W., 2003. Tumor necrosis factor alpha induces a 1286 recessive retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 40 (8), 1859e1865. caspase-independent death pathway in human neutrophils. Blood 101 (5), 1351 1287 Durrani, K., Ahmed, M., Foster, C.S., 2007. AdamantiadeseBehcet disease: diagnosis 1987e1995. 1352 1288 and current concepts in management of ocular manifestations. Comp. Oph- McLaughlin, M.E., Sandberg, M.A., Berson, E.L., Dryja, T.P., 1993. Recessive mutations 1353 thalmol. Update 8 (4), 225e233. in the gene encoding the beta-subunit of rod phosphodiesterase in patients 1289 El-Ghrably, I.A., Dua, H.S., Orr, G.M., Fischer, D., Tighe, P.J., 1999. Detection of cyto- with retinitis pigmentosa. Nat. Genet. 4 (2), 130e134. 1354 1290 kine mRNA production in infiltrating cells in proliferative vitreoretinopathy McLaughlin, M.E., Ehrhart, T.L., Berson, E.L., Dryja, T.P., 1995. Mutation spectrum of 1355 1291 using reverse transcription polymerase chain reaction. Br. J. Ophthalmol. 83 the gene encoding the beta subunit of rod phosphodiesterase among patients 1356 e 1292 (11), 1296 1299. with autosomal recessive retinitis pigmentosa. Proc. Natl. Acad. Sci. U.S.A. 92 1357 El-Mlili, N., Rodrigo, R., Naghizadeh, B., Cauli, O., Felipo, V., 2008. Chronic hyper- (8), 3249e3253. 1293 ammonemia reduces the activity of neuronal nitric oxide synthase in cere- Mizukoshi, S., Nakazawa, M., Sato, K., Ozaki, T., Metoki, T., Ishiguro, S., 2010. Acti- 1358 1294 bellum by altering its localization and increasing its phosphorylation by vation of mitochondrial calpain and release of apoptosis-inducing factor from 1359 e e e 1295 calcium calmodulin kinase II. J. Neurochem. 106 (3), 1440 1449. mitochondria in RCS rat retinal degeneration. Exp. Eye Res. 91 (3), 353 361. 1360 Farber, D.B., Lolley, R.N., 1974. Cyclic guanosine monophosphate: elevation in Munemasa, Y., Kitaoka, Y., Kuribayashi, J., Ueno, S., 2010. Modulation of mito- 1296 degenerating photoreceptor cells of the C3H mouse retina. Science 186 (4162), chondria in the axon and soma of retinal ganglion cells in a rat glaucoma model. 1361 1297 449e451. J. Neurochem. 115 (6), 1508e1519. 1362 1298 Fernandez-Bueno, I., Pastor, J.C., Gayoso, M.J., Alcalde, I., Garcia, M.T., 2008. Müller Murakami, Y., Ikeda, Y., Yonemitsu, Y., Onimaru, M., Nakagawa, K., Kohno, R., 1363 and macrophage-like cell interactions in an organotypic culture of porcine Miyazaki, M., Hisatomi, T., Nakamura, M., Yabe, T., Hasegawa, M., Ishibashi, T., 1299 neuroretina. Mol. Vis. 14, 2148e2156. Sueishi, K., 2008. Inhibition of nuclear translocation of apoptosis-inducing 1364 1300 Fisher, S.K., Lewis, G.P., 2003. Müller cell and neuronal remodeling in retinal factor is an essential mechanism of the neuroprotective activity of pigment 1365 1301 detachment and reattachment and their potential consequences for visual re- epithelium-derived factor in a rat model of retinal degeneration. Am. J. Pathol. 1366 covery: a review and reconsideration of recent data. Vision Res. 43 (8), 887e 173 (5), 1326e1338. 1302 897. Murray, P.I., Hoekzema, R., van Haren, M.A., de Hon, F.D., Kijlstra, A., 1990. 1367 1303 Fox, D.A., Poblenz, A.T., He, L., 1999. Calcium overload triggers rod photoreceptor Aqueous humor interleukin-6 levels in uveitis. Invest. Ophthalmol. Vis. Sci. 1368 1304 apoptotic cell death in chemical-induced and inherited retinal degenerations. 31 (5), 917e920. 1369 Ann. N.Y. Acad. Sci. 893, 282e285. Nagata, S., 1997. Apoptosis by death factor. Cell 88, 355e365. 1305 Funatsu, H., Noma, H., Mimura, T., Eguchi, S., 2009. Hori. Association of vitreous Nakazawa, T., Matsubara, A., Noda, K., Hisatomi, T., She, H., Skondra, D., Miyahara, S., 1370 1306 inflammatory factors with diabetic macular edema. Ophthalmology 116 (1), Sobrin, L., Thomas, K.L., Chen, D.F., Grosskreutz, C.L., Hafezi-Moghadam, A., 1371 1307 73e79. Miller, J.W., 2006. Characterization of cytokine responses to retinal detachment 1372 e 1308 Gerke, C.G., Hao, Y., Wong, F., 1995. Topography of rods and cones in the retina of in rats. Mol. Vis. 12, 867 878. 1373 the domestic pig. HKMJ 1, 302e308. Nakazawa, M., 2011. Effects of calcium ion, calpains, and calcium channel blockers 1309 Gómez-Vicente, V., Donovan, M., Cotter, T.G., 2005. Multiple death pathways in on retinitis pigmentosa. J. Ophthalmol. 2011, 292040. 1374 1310 retina-derived 661W cells following growth factor deprivation: crosstalk be- Nakazawa, T., Kayama, M., Ryu, M., Kunikata, H., Watanabe, R., Yasuda, M., 1375 e 1311 tween caspases and calpains. Cell. Death Differ. 12, 796 804. Kinugawa, J., Vavvas, D., Miller, J.W., 2011. Tumor necrosis factor-alpha mediates 1376 Grasl-Kraupp, B., Ruttkay-Nedecky, B., Koudelka, H., Bukowska, K., Bursch, W., photoreceptor death in a rodent model of retinal detachment. Invest. Oph- 1312 Schulte-Hermann, R., 1995. In situ detection of fragmented DNA (TUNEL assay) thalmol. Vis. Sci. 52 (3), 1384e1391. 1377 1313 fails to discriminate among apoptosis, necrosis, and autolytic cell death: a Noma, H., Funatsu, H., Mimura, T., Harino, S., Hori, S., 2009. Vitreous levels of 1378 e 1314 cautionary note. Hepatology 21 (5), 1465 1468. interleukin-6 and vascular endothelial growth factor in macular edema with 1379 Grau, T., Artemyev, N.O., Rosenberg, T., Dollfus, H., Haugen, O.H., Cumhur Sener, E., central retinal vein occlusion. Ophthalmology 116 (1), 73e79. 1315 Jurklies, B., Andreasson, S., Kernstock, C., Larsen, M., Zrenner, E., Wissinger, B., Osborne, N.N., Wood, J.P., 2004. Metipranolol blunts nitric oxide-induced lipid 1380 1316 Kohl, S., 2011. Decreased catalytic activity and altered activation properties of peroxidation and death of retinal photoreceptors: a comparison with other 1381 1317 PDE6C mutants associated with autosomal recessive achromatopsia. Hum. Mol. anti-glaucoma drugs. Invest. Ophthalmol. Vis. Sci. 45 (10), 3787e3795. 1382 Genet. 20 (4), 719e730. Paquet-Durand, F., Silva, J., Talukdar, T., Johnson, L.E., Azadi, S., van Veen, T., 1318 Hendrickson, A., Hicks, D., 2002. Distribution and density of medium- and short- Ueffing, M., Hauck, S.M., Ekström, P.A., 2007. Excessive activation of poly(ADP- 1383 1319 wavelength selective cones in the domestic pig retina. Exp. Eye Res. 74, 435e ribose) polymerase contributes to inherited photoreceptor degeneration in the 1384 1320 444. retinal degeneration 1 mouse. J. Neurosci. 27 (38), 10311e10319. 1385 Hisatomi, T., Sakamoto, T., Murata, T., Yamanaka, I., Oshima, Y., Hata, Y., Ishibashi, T., Paquet-Durand, F., Hauck, S.M., van Veen, T., Ueffing, M., Ekström, P., 2009. PKG 1321 Inomata, H., Susin, S.A., Kroemer, G., 2001. Relocalization of apoptosis-inducing activity causes photoreceptor cell death in two retinitis pigmentosa models. 1386 1322 factor in photoreceptor apoptosis induced by retinal detachment in vivo. Am. J. J. Neurochem. 108 (3), 796e810. 1387 1323 Pathol. 158, 1271e1278. Paquet-Durand, F., Sanges, D., McCall, J., Silva, J., van Veen, T., Marigo, V., Ekström, P., 1388 1324 Huang, S.H., Pittler, S.J., Huang, X., Oliveira, L., Berson, E.L., Dryja, T.P., 1995. Auto- 2010. Photoreceptor rescue and toxicity induced by different calpain inhibitors. 1389 somal recessive retinitis pigmentosa caused by mutations in the alpha subunit J. Neurochem. 115 (4), 930e940. 1325 of rod cGMP phosphodiesterase. Nat. Genet. 11 (4), 468e471. Perrault, I., Rozet, J.M., Calvas, P., Gerber, S., Camuzat, A., Dollfus, H., Châtelin, S., 1390 1326 Hunt, D.M., Buch, P., Michaelides, M., 2010. Guanylate cyclases and associated Souied, E., Ghazi, I., Leowski, C., Bonnemaison, M., Le Paslier, D., Frézal, J., Dufier, J.L., 1391 e e fi 1327 activator proteins in retinal disease. Mol. Cell. Biochem. 334 (1 2), 157 168. Pittler, S., Munnich, A., Kaplan, J., 1996. Retinal-speci c guanylate cyclase gene 1392 Japiassú, R.M., Brasil, O.F., Cunha, A.L., de Souza, E.C., 2008. Regression of vaso- mutations in Leber’s congenital amaurosis. Nat. Genet. 14 (4), 461e464. 1328 proliferative tumor with systemic infliximab. Ophthal. Surg. Lasers Imaging 39 Perrault, I., Rozet, J.M., Gerber, S., Ghazi, I., Ducroq, D., Souied, E., Leowski, C., 1393 1329 (4), 348e349. Bonnemaison, M., Dufier, J.L., Munnich, A., Kaplan, J., 2000. Spectrum of retGC1 1394 ’ e 1330 Jiang, L., Baehr, W., 2010. GCAP1 mutations associated with autosomal dominant mutations in Leber s congenital amaurosis. Eur. J. Hum. Genet. 8 (8), 578 582. 1395 cone dystrophy. Adv. Exp. Med. Biol. 664, 273e282. Petersen-Jones, S.M., Entz, D.D., Sargan, D.R., 1999. cGMP phosphodiesterase-alpha 1331 Kalloniatis, M., Fletcher, E.L., 2004. Retinitis pigmentosa: understanding the mutation causes progressive retinal atrophy in the Cardigan Welsh corgi dog. 1396 1332 clinical presentation, mechanisms and treatment options. Clin. Exp. Optom. Invest. Phthalmol. Vis. Sci. 40 (8), 1637e1644. 1397 1333 87 (2), 65e80. Piri, N., Gao, Y.Q., Danciger, M., Mendoza, E., Fishman, G.A., Farber, D.B., 2005. 1398 Kaur, J., Mencl, S., Sahaboglu, A., Farinelli, P., van Veen, T., Zrenner, E., Ekström, P., A substitution of G to C in the cone cGMP-phosphodiesterase gamma subunit 1334 Paquet-Durand, F., Arango-Gonzalez, B., 2011. Calpain and PARP activation gene found in a distinctive form of cone dystrophy. Ophthalmology 112 (1), 1399 1335 during photoreceptor cell death in P23H and S334ter rhodopsin mutant rats. 159e166. 1400 1336 PLoS One 6 (7), e22181. Pryor, W.A., Squadrito, G.L., 1995. The chemistry of peroxynitrite: a product from the 1401 Klein, R., Knudtson, M.D., Klein, B.E., Wong, T.Y., Cotch, M.F., Liu, K., Cheng, C.Y., reaction of nitric oxide with superoxide. Am. J. Physiol. 268 (5 Pt 1), L699eL722. 1337 Burke, G.L., Saad, M.F., Jacobs Jr., D.R., Sharrett, A.R., 2008. Inflammation, com- Sahaboglu, A., Tanimoto, N., Kaur, J., Sancho-Pelluz, J., Huber, G., Fahl, E., Arango- 1402 1338 plement factor h, and age-related macular degeneration: the Multi-ethnic Gonzalez, B., Zrenner, E., Ekström, P., Löwenheim, H., Seeliger, M., Paquet- 1403 1339 Study of Atherosclerosis. Ophthalmology 115 (10), 1742e1749. Durand, F., 2010. PARP1 gene knock-out increases resistance to retinal degen- 1404 1340 Koch, K.W., Stryer, L., 1988. Highly cooperative feedback control of retinal rod eration without affecting retinal function. PLoS One 5 (11), e15495. 1405 guanylate cyclase by calcium ions. Nature 334 (6177), 64e66. Sakamoto, K., McCluskey, M., Wensel, T.G., Naggert, J.K., Nishina, P.M., 2009. New 1341 Komeima, K., Rogers, B.S., Lu, L., Campochiaro, P.A., 2006. Antioxidants reduce cone mouse models for recessive retinitis pigmentosa caused by mutations in the 1406 1342 cell death in a model of retinitis pigmentosa. Proc. Natl. Acad. Sci. U.S.A. 103 Pde6a gene. Hum. Mol. Genet. 18 (1), 178e192. 1407 e 1343 (30), 11300 11305. Sanges, D., Comitato, A., Tammaro, R., Marigo, V., 2006. Apoptosis in retinal 1408 Komeima, K., Usui, S., Shen, J., Rogers, B.S., Campochiaro, P.A., 2008. Blockade of degeneration involves cross-talk between apoptosisinducing factor (AIF) and 1344 neuronal nitric oxide synthase reduces cone cell death in a model of retinitis caspase-12 and is blocked by calpain inhibitors. Proc. Natl. Acad. Sci. U.S.A. 103, 1409 1345 pigmentosa. Free Radic. Biol. Med. 45 (6), 905e912. 17366e17371. 1410

12 C. Martínez-Fernández de la Cámara et al. / Experimental Eye Research xxx (2013) 1e12

1411 Sargan, D.R., Clements, P.J., Sohal, A., Gregory, C.Y., Bhattacharya, S.S., Petersen- Tucker, C.L., Woodcock, S.C., Kelsell, R.E., Ramamurthy, V., Hunt, D.M., Hurley, J.B., 1445 1412 Jones, S.M., 1994. Progressive retinal atrophy: a model for retinitis pigmentosa 1999. Biochemical analysis of a dimerization domain mutation in RetGC-1 1446 in companion animals. Gene Ther. 1 (Suppl. 1), S89. associated with dominant cone-rod dystrophy. Proc. Natl. Acad. Sci. U.S.A. 96 1413 Seddon, J.M., George, S., Rosner, B., Rifai, N., 2005. Progression of age-related mac- (16), 9039e9044. 1447 1414 ular degeneration: prospective assessment of C-reactive protein, interleukin 6, Tuntivanich, N., Pittler, S.J., Fischer, A.J., Omar, G., Kiupel, M., Weber, A., Yao, S., 1448 1415 and other cardiovascular biomarkers. Arch. Ophthalmol. 123 (6), 774e782. Steibel, J.P., Khan, N.W., Petersen-Jones, S.M., 2008. Characterization of a canine 1449 Sharma, A.K., Rohrer, B., 2004. Calcium-induced calpain mediates apoptosis via model of autosomal recessive retinitis pigmentosa due to a PDE6A mutation. 1416 caspase-3 in a mouse photoreceptor cell line. J. Biol. Chem. 279 (34), 35564e Invest. Ophthalmol. Vis. Sci. 50 (2), 801e813, 2009. 1450 1417 35572. Vallazza-Deschamps, G., Cia, D., Gong, J., Jellali, A., Duboc, A., Forster, V., Sahel, J.A., 1451 1418 Sharma, A.K., Rohrer, B., 2007. Sustained elevation of intracellular cGMP causes Tessier, L.H., Picaud, S., 2005. Excessive activation of cyclic nucleotide-gated 1452 oxidative stress triggering calpain-mediated apoptosis in photoreceptor channels contributes to neuronal degeneration of photoreceptors. Eur. J. Neu- 1419 degeneration. Curr. Eye Res. 32 (3), 259e269. rosci. 22 (5), 1013e1022. 1453 1420 Shen, J., Yang, X., Dong, A., Petters, R.M., Peng, Y.W., Wong, F., Campochiaro, P.A., Wang, W., Acland, G.M., Ray, K., 1999. Aguirre GD Evaluation of cGMP- 1454 1421 2005. Oxidative damage is a potential cause of cone cell death in retinitis phosphodiesterase (PDE) subunits for causal association with rod-cone 1455 e 1422 pigmentosa. J. Cell. Physiol. 203 (3), 457 464. dysplasia 2 (rcd2), a canine model of abnormal retinal cGMP metabolism. 1456 Sokal, I., Dupps, W.J., Grassi, M.A., Brown Jr., J., Affatigato, L.M., Roychowdhury, N., Exp. Eye Res. 69 (4), 445e453. 1423 Yang, L., Filipek, S., Palczewski, K., Stone, E.M., Baehr, W., 2005. A novel GCAP1 Wang, Y., Kim, N.S., Haince, J.F., Kang, H.C., David, K.K., Andrabi, S.A., Poirier, G.G., 1457 1424 missense mutation (L151F) in a large family with autosomal dominant cone-rod Dawson, V.L., Dawson, T.M., 2011. Poly(ADP-ribose) (PAR) binding to apoptosis- 1458 e 1425 dystrophy (adCORD). Invest. Ophthalmol. Vis. Sci. 46 (4), 1124 1132. inducing factor is critical for PAR polymerase-1-dependent cell death (partha- 1459 Stieger, K., Lhériteau, E., Moullier, P., Rolling, F., 2009. AAV-mediated gene therapy natos). Sci. Signal. 5 (4(167)), ra20. 1426 for retinal disorders in large animal models. ILAR J. 50 (2), 206e224. Winkler, J., Hagelstein, S., Rohde, M., Laqua, H., 2002. Cellular and cytoskeletal 1460 1427 Suber, M.L., Pittler, S.J., Qin, N., Wright, G.C., Holcombe, V., Lee, R.H., Craft, C.M., dynamics within organ cultures of porcine neuroretina. Exp. Eye Res. 74 (6), 1461 e 1428 Lolley, R.N., Baehr, W., Hurwitz, R.L., 1993. Irish setter dogs affected with rod/ 777 788. 1462 cone dysplasia contain a nonsense mutation in the rod cGMP phosphodies- Yang, L.P., Zhu, X.A., Tso, M.O., 2007. A possible mechanism of microglia- 1429 terase beta-subunit gene. Proc. Natl. Acad. Sci. U.S.A. 90 (9), 3968e3972. photoreceptor crosstalk. Mol. Vis. 13, 2048e2057. 1463 1430 Suo, G.J., Qin, J., Zhong, C.P., Zhao, Z.X., 2010. Suppressor of cytokine signaling 1 Yoshida, S., Sotozono, C., Ikeda, T., Kinoshita, S., 2001. Interleukin-6 (IL-6) pro- 1464 1431 inhibits apoptosis of islet grafts through caspase 3 and apoptosis-inducing duction by cytokine-stimulated human Muller cells. Curr. Eye Res. 22 (5), 1465 factor pathways in rats. Transplant. Proc. 42 (7), 2658e2661. 341e347. 1432 Tezel, G., Yang, X., 2004. Caspase-independent component of retinal ganglion cell Yoshida, N., Ikeda, Y., Notomi, S., Ishikawa, K., Murakami, Y., Hisatomi, T., Enaida, H., 1466 1433 death, in vitro. Invest. Ophthalmol. Vis. Sci. 45 (11), 4049e4059. Ishibashi, T., 2013a. Clinical evidence of sustained chronic inflammatory reac- 1467 1434 Thapa, A., Morris, L., Xu, J., Ma, H., Michalakis, S., Biel, M., Ding, X.Q., 2012. Endo- tion in retinitis pigmentosa. Ophthalmology 120 (1), 100e105. 1468 plasmic reticulum stress-associated cone photoreceptor degeneration in cyclic Yoshida, N., Ikeda, Y., Notomi, S., Ishikawa, K., Murakami, Y., Hisatomi, T., Enaida, H., 1435 nucleotide-gated channel deficiency. J. Biol. Chem. 287 (22), 18018e18029. Ishibashi, T., 2013b. Laboratory evidence of sustained chronic inflammatory 1469 1436 Thiadens, A.A., den Hollander, A.I., Roosing, S., Nabuurs, S.B., Zekveld-Vroon, R.C., reaction in retinitis pigmentosa. Ophthalmology 120 (1), e5ee12. 1470 1437 Collin, R.W., De Baere, E., Koenekoop, R.K., van Schooneveld, M.J., Strom, T.M., Yoshizawa, K., Kiuchi, K., Nambu, H., et al., 2002. Caspase-3 inhibitor transiently 1471 1438 van Lith-Verhoeven, J.J., Lotery, A.J., van Moll-Ramirez, N., Leroy, B.P., van den delays inherited retinal degeneration in C3H mice carrying the rd gene. Graefes 1472 Born, L.I., Hoyng, C.B., Cremers, F.P., Klaver, C.C., 2009. Homozygosity mapping Arch. Clin. Exp. Ophthalmol. 240 (3), 214e219. 1439 reveals PDE6C mutations in patients with early-onset cone photoreceptor dis- Zeiss, C.J., Neal, J., Johnson, E.A., 2004. Caspase-3 in postnatal retinal development 1473 1440 orders. Am. J. Hum. Genet. 85 (2), 240e247. and degeneration. Invest. Ophthalmol. Vis. Sci. 45, 964e970. 1474 fi 1441 Tsai, G.Y., Cui, J.Z., Syed, H., Xia, Z., Ozerdem, U., McNeill, J.H., Matsubara, J.A., 2009. Zhang, X., Feng, Q., Cote, R.H., 2005. Ef cacy and selectivity of phosphodiesterase- 1475 Effect of N-acetylcysteine on the early expression of inflammatory markers in targeted drugs in inhibiting photoreceptor phosphodiesterase (PDE6) in 1442 the retina and plasma of diabetic rats. Clin. Exp. Ophthalmol. 37, 223e231. retinal photoreceptors. Invest. Ophthalmol. Vis. Sci. 46 (9), 3060e3066. 1476 1443 1477 1444 1478