Neuronal ceroid lipofuscinosis: clinical and morphologic findings in nine affected Polish Owczarek Nizinny (PON) dogs.

OBJECTIVE: The aim of this study was to characterize the clinical and morphologic features of neuronal ceroid lipofuscinosis (NCL) in the Polish Owczarek Nizinny (PON) breed of dog. ANIMALS: Nine Swedish PON dogs of both sexes were included in the study. PROCEDURE: All dogs underwent a detailed clinical evaluation, with emphasis on ophthalmic exams. Histopathology and electron microscopy were performed on the eyes, brain and various internal organs. Immunohistochemical staining for detection of sphingolipid activator proteins (SAPs) and mitochondrial ATP synthase (SCMAS) was performed on the eyes and brain. RESULTS: The dogs showed behavioral abnormalities, motor disturbances and visual impairment or blindness. Pupillary responses were abnormal while fundus changes varied from normal to severe retinal atrophy. Electroretinography (ERG) showed variable changes, from slight alterations in the process of dark adaptation to severely reduced or nonrecordable ERG a- and b-wave amplitudes. Histopathology revealed intracytoplasmic storage bodies within neurons of the brain and in retinal cells, especially the retinal pigment epithelium (RPE). Round to oval granular type of inclusion bodies, known as granular osmiophilic dense deposits (GRODS), were found in neuronal cells in the brain and in the retina. Immunohistochemistry identified the storage material in the brain and retina as consisting of SAPs. CONCLUSION: The presently described NCL disease in PON dogs shows similarities to previously recorded cases in the Miniature Schnauzer. The closest human equivalent to this disease is infantile NCL (CLN1), in which the major stored proteins are SAPs and the ultrastructure of the inclusion bodies of neuronal cells is granular.

Clinical, electrophysiological and morphological changes in a case of hereditary retinal degeneration in the Papillon dog.

An autosomal recessive retinal disease with a late onset in Swedish Papillon dogs has recently been described. A 7-year-old Papillon dog showed no obvious signs of visual impairment and only minor ophthalmoscopic changes. Cone ERG b-wave amplitudes were within normal limits, while rod responses were nonrecordable or severely abnormal. Ultrastructural examination showed a generalized retinal degenerative disease, most prominent in the peripheral areas. The inferior retina was more severely affected than the superior areas. Both rods and cones showed morphological changes. The Papillon dog is another dog breed affected by progressive rod-cone degeneration, with similarities to the canine retinal disease given the gene symbol prcd.

Central retinal vein occlusion combined with occlusion of a cilioretinal artery. A case report.

An otherwise healthy 39-year-old man with a dark spot in the visual field of his left eye showed retinal whitening, indicating a cilioretinal arterial obstruction and minor signs of venous stasis at the initial examination. The affected cilioretinal artery filled normally during fluorescein angiography. The visual acuity was 1.0 bilaterally. One week later, the retinal whitening had decreased and signs of central retinal venous occlusion (venous dilatation, retinal haemorrhages and papillary oedema) predominated in the fundus picture. The patient was treated with oral betamethasone and acetylsalicylic acid. The patient was free of symptoms and the fundus normalized within 10 months. The pathogenesis of cilioretinal arterial obstruction combined with central retinal venous occlusion is not established. The clinical course in this case seems to favour a hypothesis of a primary arterial affection.

Increased accuracy in MR diagnosis of uveal melanoma by carbohydrate loading. A simple method that reflects rapid metabolic change.

PURPOSE: To evaluate patients with clinically diagnosed uveal melanoma. MATERIAL AND METHODS: Forty-eight consecutive patients were examined with spin-echo (SE) and fast spin-echo (FSE) MR sequences that utilized glucose-fructose enhancement together with a subtraction technique on a 1.5 T unit. RESULTS: Twenty-seven patients were enucleated and the eyes histologically examined for tumours (spindle cell, mixed cell, and epitheloid cell). The remaining patients were referred for other treatment. There were no significant differences in T2 although T2 was longer in the amelanotic lesions. Carbohydrate loading in combination with a subtraction technique gave: an increased signal intensity; a prolongation of T2; and an increased tumour size. The FSE sequences were as good as the SE sequences in the visualization of uveal malignant melanoma. CONCLUSION: MR imaging performed with carbohydrate loading registers metabolic changes induced in the tumour. This gives the method great validity in the diagnosis of uveal melanoma. The method is especially useful in amelanotic tumours that have longer relaxation times than melanotic tumours. The SE technique can be replaced with the FSE technique.

Lipofuscin accumulation in cultured retinal pigment epithelial cells causes enhanced sensitivity to blue light irradiation.

Lipofuscin accumulates with age within secondary lysosomes of retinal pigment epithelial (RPE) cells of humans and many animals. The autofluorescent lipofuscin pigment has an excitation maximum within the range of visible blue light, while it is emitting in the yellow-orange area. This physico-chemical property of the pigment indicates that it may have a photo-oxidative capacity and, consequently, then should destabilize lysosomal membranes of blue-light exposed RPE. To test this hypothesis, being of relevance to the understanding of age-related macular degeneration, cultures of heavily lipofuscin-loaded RPE cells were blue-light-irradiated and compared with respect to lysosomal stability and cell viability to relevant controls. To rapidly convert primary cultures of RPE, obtained from neonatal rabbits, into aged, lipofuscin-loaded cells, they were allowed to phagocytize artificial lipofuscin that was prepared from outer segments of bovine rods and cones. Following blue-light irradiation, lysosomal membrane stability was measured by vital staining with the lysosomotropic weak base, and metachromatic fluorochrome, acridine orange (AO). Quantifying red (high AO concentration within intact lysosomes with preserved proton gradient over their membranes) and green fluorescence (low AO concentration in nuclei, damaged lysosomes with decreased or lost proton gradients, and in the cytosol) allowed an estimation of the lysosomal membrane stability after blue-light irradiation. Cellular viability was estimated with the delayed trypan blue dye exclusion test. Lipofuscin-loaded blue-light-exposed RPE cells showed a considerably enhanced loss of both lysosomal stability and viability when compared to control cells. It is concluded that the accumulation of lipofuscin within secondary lysosomes of RPE sensitizes these cells to blue light by inducing photo-oxidative alterations of their lysosomal membranes resulting in a presumed leakage of lysosomal contents to the cytosol with ensuing cellular degeneration of apoptotic type. The suggested mechanism may have bearings on the development of age-related macular degeneration.

Electrophysiology in some animal and human hereditary diseases involving the retinal pigment epithelium.

The present paper surveys slow electrophysiological responses recorded by a d.c. technique in some hereditary eye diseases involving the retinal pigment epithelium (RPE) in animals (English setter dogs and Polish Owczarec Nizinny (PON) dogs with ceroid lipofuscinosis and Briard dogs with a slowly progressive rod-cone dystrophy associated with RPE inclusions) and in humans (Best's disease). The electroretinogram c-wave was typically either decreased in amplitude, lacking or replaced by a negative wave. These c-wave changes could be seen at fairly early stages of disease, when the a- and b-waves of the electroretinogram were still within normal limits.

Lipofuscin formation in cultured retinal pigment epithelial cells exposed to photoreceptor outer segment material under different oxygen concentrations.

Lipofuscin accumulates in the course of time in the acidic vacuolar apparatus of retinal pigment epithelial (RPE) cells and may influence their metabolic functions. In order to study the effect of oxidative stress on lipofuscin accumulation, rabbit RPE cell cultures were kept at an ambient oxygen concentration of either 8% or 40%. To simulate the normal phagocytic function of RPE cells, bovine photoreceptor outer segments (POS) were added daily. The lipofuscin-specific autofluorescence was measured after 1, 2 and 3 weeks. RPE cells cultured under normobaric hyperoxic conditions (40% oxygen) showed significantly higher levels of lipofuscin-like autofluorescence than those kept under normobaric and probably normoxic conditions (8% oxygen) after 1 (p = 0.0050), 2 (p = 0.0001) as well as 3 (p = 0.0077) weeks. At both oxygen concentrations, the lipofuscin accumulation level was increased after 2 weeks of POS exposure (40% p = 0.0001; 8% p = 0.0037) and even further after 3 weeks (40% p = 0.0541; 8% p = 0.0377). The results suggest an involvement of oxidative mechanisms in the formation of lipofuscin from phagocytized POS by RPE cells. The autofluorescence of control cells, not exposed to POS, was significantly (40%: 1 week p = 0.0011, 2 weeks p = < 0.0001, 3 weeks p = 0.0001; 8%: 1 week p = 0.0036, 2 weeks p = 0.0063, 3 weeks p = 0.0066) lower than that of the POS-fed cells. The autofluorescence increased significantly (40% p = 0.0059; 8% p = 0.0034) between week 1 and week 3 in the control cells. This finding may reflect a contribution to lipofuscin formation by autophagocytized intracellular material. The present model seems to be useful for further studies on the mechanisms behind lipofuscinogenesis of RPE cells as well as the possible effects of lipofuscin accumulation on cell functions and viability.

Formation of lipofuscin in cultured retinal pigment epithelial cells exposed to pre-oxidized photoreceptor outer segments.

Accumulation of lipofuscin in the retinal pigment epithelium (RPE) with increasing age may affect essential supportive functions for the photoreceptors. Earlier, we described a model system for the study of lipofuscinogenesis in RPE cell cultures and showed that mild oxidative stress enhances lipofuscin formation from phagocytized photoreceptor outer segments (POS). In the present study, bovine POS were photo-oxidized, and turned into a lipofuscin-like material, by irradiation with UV light. Transmission electron microscopy of irradiated POS showed loss of the normal stacks of the disk membranes with conversion into an amorphous osmiophilic electron-dense mass. The formation of thiobarbituric acid reactive substances (TBARS), estimated during the irradiation process, indicated lipid peroxidation. Irradiated POS also showed a strong granular yellow autofluorescence. RPE cell cultures, kept at 21% ambient oxygen, were fed daily for 3, 5 or 7 days with either (i) UV-peroxidized POS, (ii) native POS or (iii) culture medium only. RPE cells fed irradiated POS showed significantly higher levels of lipofuscin-specific autofluorescence compared to cells exposed to native POS after 3 days (p = 0.0056), 5 days (p = 0.0037) and 7 days (p = 0.0020), and to the non-exposed control cells (3 days: p = 0.005, 5 days: p = 0.0037, 7 days: p = 0.0094). The lipofuscin content of cells exposed to irradiated POS increased significantly between days 3 and 7 (p = 0.0335). Ultrastructural studies showed much more numerous and larger lipofuscin-like inclusions in RPE cells fed irradiated POS compared to cells exposed to native POS. In the control cells, lipofuscin-like granules were small and sparse. It appears that exposing RPE cells to previously peroxidized POS, thus artificially converted to lipofuscin and obviously not digestible by the lysosomal enzymes, accelerates the formation of severely lipofuscin-loaded cells. The results will be useful for further studies of possible harmful effects of lipofuscin in heavily loaded RPE cells.

Accumulation of lipofuscin within retinal pigment epithelial cells results in enhanced sensitivity to photo-oxidation.

Retinal pigment epithelial (RPE) cells are largely postmitotic. They continuously phagocytose the outer tips of the photoreceptor outer segments (POS). Over the life span of an individual, this activity results, although surprisingly slowly, in the intralysomal accumulation of lipofuscin, or age-pigment. Native lipofuscin shows orange-red autofluorescence when exposed to blue light. The loss of energy resulting from the conversion of excitatory blue light into emitted orange-red light may induce photo-oxidative reactions. We exposed neonatal rabbit RPE cells in culture to purified POS from cow eyes. The material were either native or peroxidized by irradiation with UV-light before being added to the RPE cultures. Lipofuscin accumulation was studied by transmission electron microscopy and measured by microfluorometric registration of its autofluorescence. Cells exposed to peroxidized POS accumulated much more lipofuscin than those exposed to native POS, indicating that peroxidized outer segments are not digestable by lysosomal enzymes. Furthermore, lipofuscin-loaded RPE cells were considerable more sensitive to visible blue light than unloaded control cells. The former ones showed lysosomal membrane destabilization with ensuing leakage of lytic enzymes and eventually cell death. We suggest that photo-oxidation of lysosomal membranes surrounding accumulated lipofuscin may be of importance for the development of age-related macular degeneration.

Neuronal ceroid lipofuscinosis in the Polish Owczarek Nizinny (PON) dog. A retinal study.

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.

Slowly progressive changes of the retina and retinal pigment epithelium in Briard dogs with hereditary retinal dystrophy. A morphological study.

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.

Ultrastructural changes of the retina and the retinal pigment epithelium in Briard dogs with hereditary congenital night blindness and partial day blindness.

The offspring of two Briard dogs (brother and sister) with congenital, clinically stationary night blindness showed an aggravation of the disease with severe impairment of day vision in addition to night blindness. This ultrastructural study was performed on four such second generation puppies at the age of 4 months. The neuroretina and retinal pigment epithelium (RPE) from four locations were studied: the central area (immediately temporal to the optic disc); the centre of the tapetal area; the upper periphery (border of tapetal area); and the lower periphery (non-tapetal area). The RPE showed large inclusions, seemingly lipid in nature, mainly in the central and tapetal areas of the retina. Small, membrane bound, electron-dense inclusions were scattered in the RPE cytoplasm in all areas examined. The small inclusions were found to be less numerous in normal than in affected dogs and may be lysosomal in nature. Forty to fifty percent of the rod outer segments in the tapetal area showed disorientation of the disc membranes, whereas the corresponding figures were 20-40% in the central and lower peripheral areas and 6-25% in the upper peripheral area. No structural abnormalities were found in the rod inner segments or synaptic bodies. The cones were better preserved. The inner retina appeared normal. These electron microscopic findings seem to correspond to a previously published electrophysiologic evaluation, indicating a defective and delayed rod function (virtually no scotopic a- and b-waves), a better preserved cone function (photopic flicker responses present, although reduced) and impaired RPE activity (a prominent, slow negative potential of long latency at the site of the c-wave). It appears that these Briard dogs, showing structural changes of the rod outer segments in addition to pigment epithelial inclusions, mainly located in the posterior pole, comprise a pigment epitheliopathy and retinopathy morphologically different from other hereditary canine retinopathies that have been described earlier in the literature and different from animal models of congenital night blindness.

Changes in the DC electroretinogram in Briard dogs with hereditary congenital night blindness and partial day blindness.

Five Briard dogs, 7-12 months old, with congenital night blindness and severely reduced day vision (offspring of a sister and brother with congenital and supposedly stationary night blindness but with normal or nearly normal day vision) and three normal control dogs were studied by means of direct current (DC) electroretinography in order to analyse fast and slow retinal and pigment epithelial (RPE) potentials. No definite a- and b-waves were seen in the affected dogs in the dark-adapted state, which indicates severely impaired rod function. All affected dogs responded to 30 Hz flickering light in the light-adapted state, although with an amplitude reduced by 50-70%. Thus, cone function was better preserved than rod function. The control dogs showed a small c-wave and a deep negative trough between the b- and c-waves, indicating that slow PIII from the Müller cells, as well as the photoreceptor potential, are very prominent. In the affected dogs, there was no c-wave, but from a stimulus intensity of 3 log U above the normal b-wave threshold, a slow negative potential appeared, the latency and peak time of which were very long, 5-7 and 11-15 sec, respectively. With increasing stimulus intensities, both parameters decreased substantially, whereas the amplitude increased to a maximum of 2400 microV. In the light-adapted state, the dog with the best day vision showed a negative potential of short duration (peak time about 0.2 sec), followed by a positive potential (peak time about 1.2 sec).(ABSTRACT TRUNCATED AT 250 WORDS)

The Briard dog: a new animal model of congenital stationary night blindness.

Congenital stationary night blindness (CSNB), apparently inherited in an autosomal recessive manner, was observed in a litter of Briard dogs in Sweden. Of nine litter mates five had nyctalopia. The results of different clinical tests, including electroretinography (ERG), were compared with the results found in four human cases of CSNB, three of which were most probably associated with autosomal recessive inheritance and one with X-linked inheritance. The congenital and stationary nature of the disease, ophthalmoscopically normal appearing fundi, and recordable but reduced photopic flicker responses were some of the similarities found between canine and human cases. The single-flash ERG response was abnormal in the humans as well as in the affected Briards. However, the human cases showed a "negative' ERG, whereas in the Briards both the a and b waves were extremely reduced and present only at a photopic level. Cases similar to these Briards have been described also in man, where rhodopsin concentration and regeneration were found to be normal, suggesting a disturbed transduction mechanism. It thus appears that the Briard dog may become a valuable model of human CSNB.

Early morphological changes of intracellular bacteria in Salmonella typhimurium infection of the guinea pig conjunctival epithelium.

Conjunctival epithelium of the guinea pig was incubated with a virulent strain of Salmonella typhimurium 395 MS for 1 h. Intracellular bacteria were observed in superficial and intermediate cell layers, but not in basal cells. The majority of the bacteria were located within primary or secondary phagosomes; a few were seen free in the cytoplasm. A number of intraphagosomal bacteria showed morphological signs of degradation. Ultrastructurally, the initial phases of Salmonella typhimurium infection of the guinea pig conjunctival epithelium appear to be consistent with endocytic uptake of bacteria by the epithelial cells, followed by their degradation in secondary phagosomes. The conjunctival epithelial cells seem able to inactivate a certain number of virulent bacteria and thus, to a degree, to control the infection in its early phase with defence mechanisms pertaining to the cells themselves, without support from the professional phagocytic cells.

Pneumatic retinopexy in 50 patients.

Pneumatic retinopexy was introduced in our clinic in 1986. This paper reports the results from the first 50 cases. Detachments with multiple breaks within 3 clock hours and proliferative vitreoretinopathy up to grade C1 were included. This technique compares favorably to buckling procedures in selected cases. The primary success rate was 83%, and the overall success rate was 90% (pneumatic retinopexy +/- scleral buckling +/- vitrectomy).

Corneal thickness in daily contact lens wear. A comparison of a low water content lens and a high water content lens.

The corneal responses to daily wear of Scanlens 75 high water content lenses, average centre thickness 0.23 mm, and of Bausch & Lomb Soflens U3 low water content lenses, centre thickness 0.07 mm, were compared. Calculations of oxygen transmissibility indicated that corneal swelling might be less for the former lenses than for the latter lenses. This assumption was proved to be correct. The average increase in corneal centre thickness over the day (14 h) during a 4 week period was 1.6% for Scanlens 75 and 2.8% for Soflens U3. This difference was statistically highly significant (P less than 0.001). The thickness increase was greatest during the first week. Tests with thicker lenses showed that the thicker the lenses are, the more important it is to choose high water content lenses. The closed eye period increased corneal swelling in extended lens wear. The results favour a program based on certain high water content lenses and a daily wear schedule.