Irnin Journl of Veterinry Reserch, Shirz University 119 Clinicl studies on progressive retinl trophy in 31 dogs Kelwl, D. N. 1 ; Ptil, D. B. 2* ; Prikh, P. V. 2 ; Sheth, M. J. 3 ; Joshi, C. G. 4 nd Reddy, B. 4 1 Ph.D. Scholr in Veterinry Surgery nd Rdiology, Deprtment of Veterinry Surgery nd Rdiology, College of Veterinry Science nd Animl Husndry, Annd Agriculturl University, Annd, Gujrt-388001, Indi; 2 Deprtment of Veterinry Surgery nd Rdiology, College of Veterinry Science nd Animl Husndry, Annd Agriculturl University, Annd, Gujrt-388001, Indi; 3 Consultnt Vitreoretinl Surgeon, Govind Eye Clinic, Vdodr, Indi; 4 Deprtment of Animl Biotechnology, College of Veterinry Science nd Animl Husndry, Annd Agriculturl University, Annd, Gujrt-388001, Indi * Correspondence: D. B. Ptil, Directorte of Reserch, Kmdhenu University, Krmyogi Bhvn, Block-1, Wing B-1, 3rd Floor, Sector 10-A, Gndhingr, Gujrt-382010, Indi. E-mil: d1608@gmil.com Summry (Received 8 Jun 2016; revised version 22 Nov 2016; ccepted 10 Dec 2016) During 2-yer period, 31 cses of hereditry retinl degenertion in dogs red in Indi were found minly suspected for progressive retinl trophy (PRA) with typicl history of initil nyctlopi followed y hemerlopi. Out of 31 PRA suspected dogs, 8 dogs (26%) were from the ge group of 1-5 yers, 15 (48%) 6-10 yers nd the rest (26%) 11-15 yers. The most predominnt reed ws Spitz (18 dogs, 58%). Detiled ophthlmologic exmintions included Schirmer s ter test, fluorescein stin, pplntion tonometry, slit lmp iomicroscopy nd oculr ultrsound in pproprite cses. Ophthlmoscopic nd fundoscopic chnges included hyperreflectivity nd discolortion of the tpetl re, mrked ttenution of retinl vessels, depigmenttion in non-tpetl re nd optic disc trophy with sclloped orders. Electroretinogrms (ERG) recorded in 13 PRA-ffected cses reveled non-recordle extinguished (fltline) ERG responses. A reduction minly of - nd -wve mplitudes in the ERG indicted generlized photoreceptor disese. Key words: Dog, Electroretinogrphy, Funduscopy, Progressive retinl trophy Introduction Progressive retinl trophy (PRA) is the umrell term used to descrie numer of inherited neuroretinl degenertions nd shows genetic heterogeneity (Bedford, 2006), cused y severl muttions nd ffects more thn 100 dog reeds (Him et l., 1992; Petersen-Jones, 1998). Progressive retinl trophy is comprehensive term for severl different types of hereditry defects primrily ffecting the photoreceptors. In the mjority of forms the rod photoreceptors re initilly ffected, leding to loss of night-vision. As the disese progresses cone vision deteriortes s well, resulting in impirment of vision in dylight nd eventully complete lindness (Nrfstrom nd Petersen-Jones, 2013). The disese cuses gret del of discomfort in the reeding industry s well s to the owners s the disese is incurle (Millichmp, 1990). In some instnces, more thn one form of PRA my e segregting within the sme reed (Downs et l., 2011). Mny of the cnine PRAs re utosoml recessively inherited, ut X-linked forms nd one utosoml dominnt form hve lso een reported. At lest 24 muttions in 18 genes hve een ssocited with cnine retinl disese (Miyder et l., 2012). Typiclly, the ssocition etween the gene muttion nd the development of PRA is strightforwrd, with most eing fully penetrnt, lthough occsionlly the sitution my pper to e more complex (Miyder et l., 2012). The dignosis is sed on clinicl history, ophthlmologic nd complementry exmintion such s funduscopy, electroretinogrphy (ERGy) nd genetic testing (Gomes et l., 2013). The clinicl chrcteriztion of specific forms of PRA is importnt to otin relile models for trnsltionl reserch. This report descries the fetures of retinl trophy in 31 dogs red in Indi. Mterils nd Methods The study ws performed with the pprovl of Institutionl Animl Ethics Committee (permission letter: AAU/GVC/CPCSEA-IAEC/108/2013). In spn of 2 yers, 340 clinicl cses of dogs underwent detiled ophthlmic exmintion for vrious oculr normlities which included complete history pertining to visul normlity nd clinicl ssessment of visul functions y mence response nd reflexes like, pupillry light, 4th purkinje nd tpetl, different tests like fluorescein stining, Schirmer s ter test, tonometry, complete ophthlmic exmintion with hnd-held slitlmp iomicroscope nd inoculr indirect ophthlmoscope nd B-mode ultrsonogrphic exmintion in pproprite cses to rule out posterior segment normlity. Additionlly, visul performnce ws ssessed y distriution of short questionnire mong owners of PRA-ffected dogs regrding owner s perception of the onset nd severity of visul deficits oserved in the dogs,
120 Irnin Journl of Veterinry Reserch, Shirz University such s the ility to see sttionry or moving ojects under different light conditions nd negotiting stircse in drk conditions. All oculr normlities were documented nd in mny cses retinl photogrphs were tken with hndheld fundus cmer (Smrtscope VET2: Optomed, Finlnd). Prior to fundoscopy recording, the dog s pupils were dilted with short-cting mydritic 1% tropicmide, 30 min prior to exmintion nd fter proper focusing of the cmer, imge of the posterior segment ws tken using ophthlmoscopic fundus imging cmer in drk room. For ech dog, tpetl nd non-tpetl colour, tpetl reflectivity, shpe of tpetl nd non-tpetl re, homogenicity of tpetum nd non-tpetl, the junction of the tpetl nd nontpetl order nd loction, colour, shpe, order nd degree of myelintion of optic disc were evluted. Electroretinogrms (ERG) were recorded in 13 PRAffected cses under generl nesthesi y conventionl tle top ERG unit (RETIport ERG nd VEP: S&V Technologies AG, Acrivet Veterinry Division, Germny) with n utomted protocol. Before performing ERG dogs were fsted for t lest 12 h. The pupils were dilted using 1% tropicmide. The electrodignosis of retin for confirmtion of PRA ws performed under generl nesthesi using mixture of Ketmine HCl (50 mg/ml) nd Midzolm (10 mg/ml) in the rtio 2:1 IV for induction long with Atropine Sulphte @ 0.03 mg/kg SC s premedicnt. Positioning of ptient nd electrodes for ERG redings ws dopted from Nrfström et l. (2002) (Figs. 1, ). Phot.Scot. LED progrmme ws selected in the RETIport ERG system for quick ERG recording nd the photopic nd scotopic retinl function ws tested with stndrd flsh (3 cds/m 2 ). ERG were recorded utomticlly nd the - nd - wve mplitudes (µv), nd implicit times (ms) were uto generted using the dog dignostic protocol pre-progrmmed for the equipment which cme from the mnufcturer tht wy. After the preliminry preprtions, ll the dogs were sujected to drk dption for minimum of 20 min. Lter the rod function ws tested (scotopic ERG) initilly for the right eye followed y left eye. Following scotopic ERG of oth eyes the dogs were sujected to light dpttion for minimum of 10 min. Cone function ws tested (photopic ERG) in similr pttern. The -wve mplitude ws mesured from the seline to the -wve through, nd the -wve mplitude ws mesured from the -wve trough to the -wve pek. Then / rtios of mplitude were clculted. The - nd - wve implicit times were mesured from the stimulus onset to the - wve trough nd -wve pek, respectively. Results During the study period, out of 31 cses of PRA in dogs red in Indi, 18 dogs (58%) were mle nd 13 (42%) femle, with ge vrying from 2 to 13 yers, of which eight (26%) were from the ge group 1-5 yers, 15 (48%) etween 6-10 yers nd the rest (26%) 11-15 yers. The most predominnt reed ws Spitz (18, 58%) followed y Lrdor Retriever (8, 26%), Cocker Spniel (2, 7%), Cndin Pointer, Lhs Aphso nd Germn Shepherd (1 ech, 3%). Fig. 1: Plcement of electrode in dogs for ERG ( nd ) Most of the dogs were presented with initil signs of nyctlopi, followed y progressive hemerlopi until the ffected niml is totlly lind. The ge t onset of nyctlopi nd the rte of progression of vision loss vried widely. When exmined lmost ll the dog (n=27, 87%) hd difficulty in voiding ojects in dim light, dilted pupils with slow pupillry light responses nd chrcteristic shine from the eye (tpetl hyper reflectivity). In norml dogs, the ophthlmoscopic nd fundoscopic exmintions showed norml reflectivity of the tpetl region, norml vsculr size nd norml colortion of the tpetl fundus (Figs. 2, ). Ophthlmoscopy in dogs with PRA showed ilterl nd generlized chnges. Only sutle color chnges were noted in the centrl prts of the oculr fundi, ut in the peripherl prts there were signs of choroidl spoking nd slight hyper-reflectivity. Lesions of the fundus were vrile nd t erly stges (n=13), focl or multifocl hyperreflective lesions of the tpetum ssocited with thinning of the retin, with n inconsistent symmetry
Irnin Journl of Veterinry Reserch, Shirz University 121 were frequently oserved ut the hyperreflective nd surrounding the tpetl non-tpetl junction ws rre nd lood vessels were not completely ttenuted (Figs. 3, ) ut nrrowed wy from the disc; while t dvnced stges (n=15), generlized hyperreflexion coexisted either with hyperreflective colescent foci in the tpetum, nd only ghost vessels re visile with pllor of disc nd hypopigmenttion of nontpetum re (Figs. 4, ). Fundoscopy reveled shrinking of the lood vessels, decresed pigmenttion of the non-tpetl fundus, incresed reflection from the tpetum nd trophied optic disc with sclloped orders. There ws generlized ut mild vsculr ttenution with decrese in numer nd size of retinl vessels nd pigmentry chnges long with circulr nd trophied optic disc with sclloped or indistinct orders nd erly loss of myelin over the optic nerve hed. Fundus chnges were not detected in ny of the exmined dogs elow 4 yers of ge (n=3) though the owners hd complints regrding their dog showing signs of fer in drk. Electroretinogrphy ws crried out s complementry exmintion in 13 dogs. Initil light-dpted ERG resulted in photopic -wve with somewht reduced mplitude compred to ERGs during drk dpttion which did not show ny recordle responses. However, the mplitude of the -wve ws significntly decresed t ll light intensities in the PRA cses. The - wve mplitude ws lso significntly lower t ll light intensities for PRA-ffected dogs. At the dvnced stge of disese, there were non-recordle extinguished (fltline) ERG responses for the scotopic nd photopic ERGs (Fig. 5). Dogs showing pprent fer in drk (n=4) hd reduced ERG mplitudes ut norml fundus ppernce. The ERG recorded from the PRA-ffected dogs ws chrcterized y - nd -wves of very low mplitudes. The men±se of / rtio for PRA dogs ws 3.92 ± 0.77. Exon cpture nd sequence nlysis of PRA-ffected nd norml dogs reveled 25657 single nucleotide polymorphisms (SNPs) in totl. The cse control nlysis reveled muttions ssocited with PRA-ffected nd norml dogs. Five muttions [chr 7: 19509452 (T/A), chr 7: 19509598 (A/G), chr 7: 19510446 (C/T), chr 7: 19510503 (G/A), nd chr 7: 19509281 (G/T)] in Phosducin (PDC) showed significnt ssocition with PRA in Spitz reed. Pthogeneticlly relevnt muttions were not found in Rhodopsin (RHO) for PRA in cnine reeds studied. Muttions in PRCD, XLPRA1 nd 2, CNBG nd GALK1 were not linked to PRA-ffected Spitzs. Muttion in PRCD ws linked to PRA-ffected Lrdor Retriever (n=8) nd Cocker Spniel (n=2). Fig. 2: Norml fundus imges for Spitz dog, 6 yers ( nd ). OD: Optic disc, T: Tpetum, nd NT: Non tepetum Fig. 3: Erly progressive retinl trophy ( nd ). Here lood vessels were not completely ttenuted, ut nrrowed wy from the disc. Hyperreflectivity of tpetum
122 Irnin Journl of Veterinry Reserch, Shirz University Fig. 4: An dvnced cse of retinl degenertion ( nd ). The tpetl fundus is hyperreflective, nd only ghost vessels re visile Fig. 5: Fltline electroretinogrm of PRA dog. The mplitude of the -wve ws significntly decresed t ll light intensities in the PRA cses. The -wve mplitude ws lso significntly lower t ll light intensities for PRA-ffected dogs. Nonrecordle extinguished (fltline) ERG responses for the scotopic nd photopic ERGs Discussion Inherited retinl diseses re mong the leding cuses for incurle vision loss in the humn nd cnine popultions. In dogs, most of these conditions re clssified s PRA, nd, similr to mny forms of retinitis pigmentos (RP) in humn ptients, primrily ffect rod photoreceptors leding to initil night-vision loss followed y loss of cone photoreceptors nd susequent dy-lindness, culminting in complete lindness (Clements et l., 1996; Miyder et l., 2012). All the 31 ffected dogs descried in the present report showed no ovious visul impirment except nyctlopi t initil stge which grdully progresses to dy-lindness in few months to yers resulting in complete lindness nd there were only minor ophthlmoscopic signs of generlized retinl disese. Dogs of oth genders, 18 mles nd 13 femles were ffected y PRA, which mkes it unlikely tht the condition is sex-linked. The ophthlmoscopic signs re similr for ech type of PRA, ut the etiologies vry considerly (Bedford, 2006). Although ophthlmoscopic findings t vrious stges of PRA were rther similr, the vrition in ge of onset of disese in few dogs ws remrkle. Through the genelogy of the ffected dogs, it ws found tht certin fmilies were ffected erly with more rpid progression of disese, compred to other dogs tht developed PRA t lter time. Progressive retinl trophy in Spitz is lte onset form nd is dignosed in middle-ged dogs. A possile explntion of this devition my e genetic vrition due to inreeding. Furthermore, ge of disese onset nd rte of disese progression cn vry considerly, even in closely relted nimls nd the novelty of this disese is not only sed on the retinl disese phenotype, ut lso the vrition in ge of onset nd rte of disese progression (Cooper et l., 2014). The disese is chrcterized y progressive ttenution nd thinning of retinl lood vessels. As the disese progresses the rteries decrese in numer nd the lrge veins ecome noticely thinner. The optic disc ecomes ple to gry rown owing to loss of cpillries on its surfce nd demyelintion nd trophy of the nerve fiers cused y extensive degenertion of the retin (Mggs, 2013). The most ovious nd consistent finding in ffected dogs is greyish discolortion of the peripherl tpetl fundus tht slowly spreds towrd the centrl prt of the tpetl re. Funduscopy reveled typicl signs of PRA. It included chnge in tpetl reflectivity, i.e. tpetum showed focl or multifocl res of hyper reflectivity in erly stges nd progressed to diffused hyperreflectivity in dvnced stge. The estlished / rtio t intensity of 3 cds.s/m 2 in helthy Spitz dog is 2.33 ± 0.73 (Kelwl, 2014). Electroretinogrms indicted severe functionl deficits in ll the 13 dogs. Electroretinogrms responses in the PRA-ffected dogs tested were non-recordle extinguished (fltline) for ll levels of light stimuli used in the drk nd light dpted sttes. The fltline responses documented in dogs with PRA confirmed loss of retinl function of the eyes, while n incresed / rtio (3.92 ± 0.77) could point towrds n ffection of the outer retinl lyers. Men pek to pek mplitudes seemed to e smller for ech dog. The severely reduced
Irnin Journl of Veterinry Reserch, Shirz University 123 single flsh nd flicker responses of the PRA-ffected English springer spniel dog indicte photoreceptor disorder with severe effects on cones nd inner retin (Nrfstrom et l., 2006). Electroretinogrms recordings of PRA-ffected Miniture Schnuzer tested, ll ERG responses were non-recordle in ll of the PRA-ffected dogs for ll light intensities used (Jeong et l., 2013). The ERG exmintion reveled reduced rod responses during drk dpttion, nd the scotopic mixed rod-cone responses nd cone only responses were slightly reduced. Reduced rod function, detected y ERG, is present efore fundus chnges develop (Svensson et l., 2015). In the present study dogs elow 4 yers of ge (n=3) hd reduced ERG mplitudes ut norml fundus ppernce. Consistent reduction of rod function is the first detected ERG normlity. This suggests tht visul testing procedures routinely used in dogs re crude nd not lwys relile, nd indictes the necessity of more ojective testing methods, such s ERG. Moleculr, iologicl nd genetic dt which llow the gene responsile to e identified gretly fcilitte erly detection of ffected nd crrier dogs. In the present study, five muttions involving PDC responsile for PRA in Spitz is reported (Reddy et l., 2015). Thus, PRA represents n expnding clinicl spectrum nd it is importnt to recognise nd descrie new forms since much my e lerned regrding retinl iology from the study of its disese. Acknowledgements We re grteful to the owners of the dogs for their willingness nd permission for conducting funduscopy nd electroretinogrphy nd even for the lood smples. This work ws supported y grnts from the Deprtment of Biotechnology (DBT), Govt. of Indi. Conflict of interest The uthors declre tht they hve no conflicts of interest. References Bedford, P (2006). Hereditry retinl diseses. 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