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Over 200 hereditary diseases have been identified and reported in the cat, several of which affect the eye, with homology to human hereditary disease. Compared with traditional murine models, the cat demonstrates more features in common with humans, including many anatomic and physiologic similarities, longer life span, increased size, and a genetically more heterogeneous background. The development of genomic resources in the cat has facilitated mapping and further characterization of feline models. During recent years, the wealth of knowledge in feline ophthalmology and neurophysiology has been extended to include new diseases of significant interest for comparative ophthalmology. This makes the cat an extremely valuable animal species to utilize for further research into disease processes affecting both cats and humans. This is especially true in the advancement and study of new treatment regimens and for extended therapeutic trials. Groups of feline eye diseases reviewed in the following are lysosomal storage disorders, congenital glaucoma, and neuroretinal degenerations. Each has important implications for human ophthalmic research.

Aim: To see if any a-wave measure segregated normal cats from those affected by a recessively inherited Abyssinian rod/cone dystrophy more efficiently than amax to scotopic Imax.Method:A-waves to electroretinograms (ERGs) evoked by a 4 cd·s/m2 scotopic flash were extracted from 241 ERG sessions using 108 cats. They were either wild type or from an affected Abyssinian stock. Fourty four were bred by back-crossing to have a 50% probability of being affected. Most were diagnosed by retinal appearance or by the pattern of loss in a long protocol ERG. Eight were still unclassified. The diagnostic efficiency of amplitudes at 7, 8, 9, and 10 ms and amax, of amax peak time, age at testing, and the main components of principal components factor analysis were compared by scaling their ability to segregate affected and normal cats.Results: Variance and overlap between the groups both decreased as time along the a-wave increased. The loading of each animal on the largest factor also gave considerable overlap. There was a small absolute separation between groups when amax itself was used. Age and peak time were uncorrelated with disease. The light intensity used could be calculated to be equivalent to one sufficient for about 75% of full saturation in man.Conclusion:Amax is a simple measure that is already in routine clinical use. When the flash is very bright and the animal fully dark adapted, this single measure is the most efficient sign of this rod/cone degeneration and possibly of all degenerations involving rods.

Aim: To assess retinal structure and function over a 3-year period in a group of five RPE65−/− dogs treated by unilateral rAAV- mediated subretinal gene transfer. Methods: Post-operative functional follow-ups were performed using simultaneous, bilateral, full-field ERGs. Structure was evaluated by SLO using FL and ICG angiography and by EM. Results: Significant improvement of retinal function was observed through ERGs approximately 4 weeks following surgery. Scotopic b-wave amplitudes peaked 3 months after surgery. Then there was a successive reduction, although greater amplitudes than base-line values were observed at all post-operative time points. A-wave amplitudes increased at a later time than b-wave amplitudes and were sustained throughout the follow-up period. The increased cone function was preserved longer than the rod function. Angiography showed structural changes at the site of injection, corroborated by photoreceptor destruction observed ultrastructurally. Immediately adjacent to the subretinal injection area photoreceptor outer segments appeared normal. Conclusion: Despite local structural alterations at the subretinal injection site, subretinal gene transfer in the RPE65 null mutation dog effectively increases retinal function for at least 3 years after surgery.

In vivogene transfer in a large group of RPE65 null mutation dogs have been recently performed. The present study was aimed at determining, through visual behavioral and electroretinographic (ERG) testing, if there is a volume effect of the gene construct administered. Eleven Beagle-Briard dogs homozygous for the RPE65 null mutation and two unaffected control dogs were included. Affected animals were unilaterally treated with either a high (70–100 μl; N = 6) or a low volume (30–60 μl; N = 5) of subretinally injected rAAV.RPE65 construct, at the age of 4 months to 2.5 years. Fellow eyes were treated with a subretinal injection of rAAV.GFP or sham operated and used as internal controls. Retinal function was measured pre- and 10–12 weeks post-surgically, using simultaneous bilateral full-field flash ERGs. A significant improvement in all ERG responses studied was identified for the high volume treated group compared to pre-surgical parameters. A significant improvement for the high intensity scotopic a-wave response for the low volume rAAV.RPE65 treated group was also found. Objective and subjective dim and day light visual maze testing, in eight of the affected treated animals, and the two control dogs, revealed better vision in daylight than in dim light for all animals. Vision in dogs treated with the high volume of gene construct was significantly better in day light than in dim light. No significant difference was noted between day and dim light testing for the control group or those animals treated with a low volume of the gene construct. Significantly better vision was noted in the control group when compared with the low volume group under dim light conditions, and the high volume group under day light conditions. No significant difference in functional vision could be identified between the high volume treated animals and control animals in day light conditions. These findings support the hypothesis that functional vision is improved by subretinal rAAV.RPE65 injection in a volume-dependent manner.

These procedures described for the dog ERG were approved at the 1st European Conference on Veterinary Visual Electrophysiology in Vienna, Austria, May 30, 2000. Dr. Narfström was Chair of the Committee for a Harmonized ERG Protocol, appointed by the European College of Veterinary Ophthalmology (ECVO), and Dr. Ofri was secretary. The other coauthors are committee members. Guidelines for ERG procedures in other animal species for clinical and laboratory studies are planned for in the future and the present guidelines are planned to be revised on a biannual basis. A brief report of the recommended procedures is available in the Conference Proceedings book.

This study evaluates the feline retina following surgical placement of a semiconductor-based microphotodiode array (MPA) into the subretinal space. Post-operative evaluations of implant durability and clinical biocompatibility have been carried out in these animals. Here, we examine the integrity of the implanted retina using anatomical techniques and immunocytochemical metabolic indicators. After appropriate fixation, the retina was divided into strips to compare areas directly over the implant versus those adjacent to the implant or in the opposite, unimplanted eye. In addition to histological analyses, the distribution of glial fibrillary acidic protein (GFAP), Na, K-ATPase, and the neurotransmitters (glutamate, glycine, and GABA) was examined using immunohistochemistry. Directly above the implant there was a near-complete loss of photoreceptor outer and inner segments and the outer nuclear layer. In comparison, the retina immediately adjacent to the implant appeared normal. In the inner nuclear layer overlying the implant, some cellular disorganization was present, however, the content was not significantly reduced. Also GFAP was up-regulated in the Müller cells directly overlying the MPA, but the retina adjacent to the implant showed a normal distribution of GFAP in the astrocytes located in the ganglion cell layer. The distributions of Na, K-ATPase adjacent to and overlying the implant were not different. Glutamate showed a decrease in overall labeling, but no change in the inner retinal layers. Glycine was found to be up-regulated in the inner nuclear layer immediately overlying the implant, while GABA showed decreased labeling over the MPA. Since photoreceptors overlying the implant degenerate, we compared the changes observed in the implanted retina to those in the Abyssinian cat model of photoreceptor degeneration. Generally, the retinal changes observed over the implant were similar to those seen in the Abyssinian cat, indicating that they may be associated with photoreceptor degeneration. Future studies will concentrate on MPAs designed to improve circulation to the outer retina which may decrease cell loss.

Introduction:Multifocal electroretinography (MF-ERG) is widely used in the detection of local retinal dysfunction. However, the position of the stimulus on the retina and the stability of fixation are usually not directly accessible. Thus, devices have been designed for a continuous fundus visualization during recording. Methods:MF-ERGs were recorded with a RetiScanTMsystem connected to two different Scanning-laser ophthalmoscopes (SLOs) that use either a red (633 nm) or green (415 nm) laser for stimulation, and a VERISTM4 system connected to a piggyback stimulator prototype that added the stimulus to the optical pathway of the SLO by means of a wavelength-sensitive mirror. Fundus visualization was achieved with the infrared lasers of the SLOs (780 and 835 nm). Results:The most extensive study so far with a green laser stimulus in a cat model of retinal degeneration demonstrated the capability of the device to detect retinal landmarks and the different stages of degeneration. Also, the advantages of exactly reproducible stimulus positioning for averaging within and comparison between disease groups became apparent. The results with the same setup in transgenic mice suggest a pure cone origin of the responses. In humans, recordings show that fixation is sufficiently good in most subjects. It is not clear yet whether red or green laser stimulation (or both) is preferable. The results with the prototype were very similar to the MF-ERGs obtained with a standard CRT screen. Conclusions:All three devices allowed us to record MF-ERGs with continuous fundus monitoring. Although further refinements are necessary, it is obvious that fundus controlled methods will improve the reliability of MF-ERG in future research on glaucoma, transplantation studies, and evaluation of gene therapy.

TheRPE65gene encodes a 65-kDa microsomal protein expressed exclusively in retinal pigment epithelium (RPE). Mutations in the humanRPE65gene have recently been identified in patients with autosomal recessive, severe, childhood-onset retinal dystrophy. Here we report the characterization of a 2.4-kb canineRpe65cDNA. The longest open reading frame predicts a 533-amino-acid protein with a calculated molecular mass of about 61 kDa prior to protein modification. Sequence comparison shows thatRPE65is highly conserved throughout mammalian evolution. We have identified a homozygous 4-bp deletion (485delAAGA) in putative exon 5 of the canineRpe65gene in affected animals of a highly inbred kinship of Swedish briard/briard–beagle dogs, in which an autosomal recessive, early-onset, and progressive retinal dystrophy segregates. The deletion results in a frameshift and leads to a premature stop codon after inclusion of 52 canine RPE65-unrelated amino acids from residue 153 onward. More than two-thirds of the wildtype polypeptide chain will be missing, and the mutant protein is most likely nonfunctional (null allele). Clinical features of the canine disease are quite similar to those described in human. Therefore this form of canine retinal dystrophy provides an attractive animal model of the corresponding human disorder with immediate significance for various therapeutic approaches, including RPE transplantation.

Light-adapted cone ERG in response to white stimuli with long duration (200 ms) was studied in seven normal cats and six cats with early to moderate, inherited retinal dystrophy. The stimuli typically elicited an ERG consisting of an a- and b-wave in response to light onset, whereas light-offset was followed by a cornea-positive d-wave and subsequent negative dip and occasionally a second positive peak in both normal and dystrophic cats, although b- and d- waves had less distinct peaks in cats with moderately advanced retinal dystrophy. Linear regression models indicated a positive correlation between d-wave amplitude and stimulus luminance, whereas a negative correlation was found between amplitude and background light luminance in normal cats. The d-wave implicit time was independent of both stimulus and background light luminance in normal cats. The d-wave amplitude was not significantly different in dystrophic cats, whereas the implicit time was increased when affected cats were compared to normal cats. The significant increase in implicit time in dystrophic cats could not be explained with a reduced sensitivity of the off-pathway to background or stimulus light. This is supported by the finding that d-wave amplitude was not significantly altered in early to moderately advanced dystrophic cats.

Purpose. Mutations of genes encoding various retina-specific proteins are known to cause a wide spectrum of inherited retinal dystrophies in different species. In the canine, several types of genetic retinal dystrophies have been described affecting primarily the photoreceptors and/or the retinal pigment epithelium. We are performing a systematic analysis of canine candidate genes for such diseases to identify the one mutated in the retinal dystrophy in Swedish Briard dogs. Methods. We isolated and characterised the full length cDNA of canine retinal arrestin by the method of rapid amplification of cDNA ends (RACE). Results. The full length cDNA isolated by us is 1,575 base pairs (bp) long and contains a 1,218 bp-long open reading frame. Conclusions. The homology of the canine arrestin protein is highest with the human analogue (88.9%) and lowest with mouse arrestin (85.3%). The most obvious sequence differences among the different arrestins are in the extreme carboxyl terminus. PCR-SSCP (single strand conformation polymorphism) analysis and direct sequencing of retinal cDNA didn't provide any evidence that mutations in the canine arrestin gene are responsible for the retinal dystrophy seen in the Swedish strain of Briard dogs. Similar data were obtained for the genes encoding rhodopsin and the ß-subunit of photoreceptor-specific phosphodiesterase by segregation analysis.

Levels of interphotoreceptor retinoid-binding protein (IRBP) protein and message in retinas of Abyssinian cats homozygous for progressive rod-cone degeneration were determined at early ages, well before the onset of clinical retinal degeneration. IRBP gene expression was assessed by immunochemical quantitation of IRBP protein, and by Northern blotting and slot-blotting of total RNA using a human IRBP cDNA probe. Morphology was assessed by electron microscopy and immunocytochemistry. Levels of both IRBP protein and message in affected Abyssinian cat retinas were significantly reduced below normal as early as 4 weeks of age at the earliest stage of retinal disorientation. Opsin mRNA was more abundant in affected Abyssininian cat retinas than in control retinas. This was at least 1 year before the onset of clinical symptoms. The reduction in IRBP gene expression to levels significantly below normal well before the onset of retinal degeneration in affected Abyssinian cat retinas indicates that this represents a primary defect or at least an early problem that could itself cause adverse effects.

Seven eyes from 2 generations of Briard dogs (5 weeks — 7 years old) with congenital night blindness and (in the second generation) impairment of day vision to varying degrees, were examined by light and electron microscopy. Specimens from 4 locations were studied: the central area, the midperiphery of the tapetal area, the upper periphery and the lower periphery. Disorientation of rod outer segment disc membranes was seen in the 5-week-old dog. Large electron-lucent inclusions were found in the RPE at 3.5 months of age. These inclusions occurred most frequently in the central and midperipheral-tapetal areas and seemed to increase in numbers and spread towards the periphery with increasing age. The content of these inclusions is not elucidated. Rod photoreceptor degeneration was apparent from 7 months of age and was most prominent in the peripheral areas. The cones were better preserved. The 7-year-old dog showed reduction of photoreceptors in the central and midperipheral-tapetal areas and almost complete photoreceptor degeneration in the periphery. This dog also showed severe changes of the inner retina in the peripheral fundus. It appears that these Briard dogs suffer from a very slowly progressive retinal degeneration, in which the photoreceptor degenerative changes do not correlate anatomically to the changes in the RPE cells. The disease seems to be different from the retinopathy described in the English Briards. It is not clear yet whether the lipid type of retinopathy found in American Briards is identical to the present disease.

Ophthalmoscopic and electroretinographic (ERG) findings were correlated in a group of Abyssinian cats affected by a slowly progressive and hereditary retinal degenerative disease. According to ophthalmoscopic findings the disease was divided into stages. At stage 1 and 2 retinal changes were minor; showing a gray discoloration most often in the peripheral and midperipheral tapetal fundus. At stage 3 discoloration was generalized and there was marked vascular attenuation. A generalized retinal atrophy was found at stage 4. ERG recordings showed an abnormally depressed stimulus response curve for the b-wave at stage 1 of disease when 30-Hz cone flicker responses were indistinguishable from normal. With progression of disease there was a successive decrease of a- and b-wave amplitudes before there was a significant reduction also of the c-wave amplitude (first seen at stage 3). The ERG was nonrecordable at stage 4. These findings suggest that the photoreceptors are affected primarily by the disease, before there is a functional involvement also of the pigment epithelium. The rod system seems to be affected early in the disease as compared with the cone system. A staging of the disease by ophthalmoscopy correlated more to the function of the rods than to that of the cones.

Reduced blood levels of long chain polyunsaturated fatty acids have been reported in humans and animals with inherited retinal degenerations. The lipid and fatty acid compositions of plasma, retina, and retinal pigment epithelium of the Swedish Briard dog, which has a very slowly progressive retinal dystrophy that is inherited in an autosomal recessive manner were analysed. The lipid class composition of the pigment epithelium was not different between affected and normal dogs; however, significant differences were found between the retinas of the two groups. Affected dogs had relatively more phosphatidylethanolamine and phosphatidylinositol and less phosphatidylcholine than normal dogs. There was no difference in the fatty acid compositions of plasma and retinal pigment epithelium between affected and normal dogs. However, the retinas of affected dogs had significantly lower levels of 22:5n-3 and 22:6n-3 and higher levels of 18:2n-6, 20:4n-6, and 22:5n-6. The total n-3 fatty acid content was significantly lower in affected retinas (P<0.001), whereas the content of n-6 fatty acids was significantly higher in affected retinas (P<0.001). These studies provide evidence for yet another animal model of inherited retinal degeneration with a defect in retinal polyunsaturated fatty acid metabolism. The fatty acid pattern in affected dogs resembles that seen in the retina in n-3 fatty acid deficiency.

Visual dysfunction and neurological symptoms were found in Polish Owczarek Nizinny (PON) dogs. Two dogs were examined, one at 2 years of age and the other one at 4 years. The oldest dog was totally blind. The 2-year-old dog developed mental disturbances and the 4-year-old dog became severely ataxic. Ophthalmoscopical findings were retinal hyper-reflectivity, attenuation of the retinal vessels and the presence of greyish to brown spots in the fundus. Electrophysiological and ultrastructural studies were performed in the 2-year-old dog. Scotopic ERG responses were absent, whereas 30 Hz cone flicker responses were recordable, although with an amplitude reduced to about 30% of the normal level. A slow negative potential replaced the c-wave, indicating a dysfunction of the RPE. Intracellular inclusions with a granular appearance or containing membranous fingerprint-like or curvilinear profiles, resembling ceroid, were found in different retinal cells. The RPE cells in the central areas were charged with autofluorescent material having similar structure, Photoreceptor degeneration was most severe in the central areas, corresponding to the RPE changes. It appears than the PON dog may provide a new animal model for neuronal ceroid lipofuscinosis.

We used a simple and reproducible technique with full-field electroretinography and a special-purpose computer system to test and evaluate outer retinal function in dogs. The standardized protocol included the following five basic responses: (1) a stable initial light-adapted, mainly cone derived response, (2) a dark-adapted rod response, (3) chromatically separated rod and cone responses, (4) a maximal rod and cone response and (5) an isolated cone flicker response. For evaluating the electroretinographic responses, a graphic-presentation was used that included data from the tested animal as well as normative data from dogs of the specific breed and age group, presented as the percentage of the median in which limits of normality were depicted in percentiles.

Electroretinography was performed in 10 Abyssinian cats, homozygous for a hereditary retinal degenerative disease but still with an ophthalmoscopically normal retina, and in 11 mixed-breed controls, all between the ages of 8 and 104 weeks. A significant reduction of maximum dark-adapted b-wave amplitude was found in affected kittens as young as 8–16 weeks when compared with controls, although there was no major difference in dark-adapted b-wave threshold or implicit time for the b-wave between affected and controls. For cats 33–104 weeks, similar results were obtained except for the b-wave threshold, which was elevated 2.5 log units in one of the affected cats. No significant difference in 30-Hz cone flicker responses were found between affected and controls at any age studied. In the time period 17–32 weeks affected Abyssinian kittens could not be differentiated from controls by means of the electroretinogram. The significant reduction in scotopic b-wave maximum amplitudes in young affected kittens (8–16 weeks) in conjunction with normal thresholds suggests an early drop-out of rods. It is clear that affected kittens can be differentiated electrophysiologically from controls long before there are ophthalmoscopic signs of retinal disease.

Retinae of Abyssinian cats homozygous for a retinal degeneration gene, and normal controls, have been investigated using antibodies directed against opsin, transducin a (TD-a), S-antigen (48K protein), interphotoreceptor retinoid-binding protein (IRBP), and cone outer segments. IRBP-immunoreactivity (IR) is much reduced at stage 2 of the disease in affected retinae; later massive photoreceptor cell death occurs. In cats, at a late stage of the disease, the retina exhibits few S-antigen-IR cells in the peripheral part of the retina whereas, in the central part, some patches of cells exhibiting opsin-IR, TD-a-IR, and S-antigen-IR are present in remnants of the outer nuclear layer (ONL). No IRBP-IR is detectable at this stage. The form and size of the majority of these remaining cells, however, does not resemble that of normal photoreceptors. No, or only rudimentary, inner and outer segments are present; long bifurcating basal protrusions often occur. These cells, which could be remains of cone elements, are S-antigen immunoreactive. Double labelling for different retina-specific proteins reveals a co-localization of opsin, TD-a and S-antigen in some, but not all, remaining photoreceptor elements. Cells exhibiting opsin-IR also show TD-a-IR and S-antigen-IR located within the entire cell and its protrusions. In control retinae and retinae at early stages of the disease, immunoreactions are comparable with all antibodies used. However, TD-a-IR is less intensive in the photoreceptor terminals. S-antigen-IR cones are most frequently present in the peripheral retina. Reduction of IRBP at an early stage of the disease could be one of the factors leading to photoreceptor cell death at later stages.