American Journal of Medical Genetics 42:605-608 (1992)
Retina in Various Animal Models of Neuronal Ceroid-Lipofuscinosis Hans H. Goebel Division of Neuropathology, University of Mainz, Mainz The childhood forms of human neuronal ceroid-lipofuscinosis(NCL)are invariably associated with a severe progressive retinopathy which commences at the photoreceptor level morphologically and proceeds to a final loss of neuronal cells accompanied by severe gliosis. In respective spontaneousanimal conditions of NCL, in English setters, Dalmatian dogs, and New Zealand sheep,retinal involvement is not commensurate although the retina does not seem to be completely unaffected. In canine NCL, there might be functional and electro-physiological impairment of retinal cells, but retinal atrophy is not obvious. In ovine NCL, the retina, apart from accumulating NCL-specific lipopigments within neuronal perikarya, also shows loss of photoreceptors. Conversely, retinal pigment epithelial cells (RPE)in canine and ovine NCL contain peculiar lamellar inclusions,not seen in unaffected animals, possibly an indication of a disturbed phagocytic process of photoreceptor outer segments. Similar such lamellar inclusions have not been observed in RPE cells in human childhood NCL.
tion of the neuronal elements, apparently commencing a t the level of the photoreceptors. While the former process of lipopigment accretion refers to the fine structural details among the human childhood forms, the retinopathy resulting in severe atrophy of the entire retina seems to be similar, if not identical during the course of the disease resulting in a burnt-out stage of both the condition NCL and the retinal degeneration when judged by eyes obtained after death from NCL-afflicted children [Goebel et al., 1974, 1977, 19881. These light and electron microscopic studies have confirmed and augmented scant earlier findings on the retinal pathology in human childhood NCL [Tarkkanen et al., 19721. In regard to adult human NCL, to be distinguished from protracted juvenile human NCL, which may entail only blindness [Goebel et al., 19761 and pigmentary retinal degeneration, functional impairment and pathology of the retina have not been reliably elucidated [Berkovicet al., 19881. Accumulation of disease-specificlipopigments in retinal ganglion cells has been documented in adult human NCL, but retinal atrophy has not been confirmed [Martin et al., 19871 while the nosological classification has been questioned where retinal atrophy had been encountered [Ikeda et al., 1984; Martin et al., 19871. It is still controversial, whether the 2 retinal processes, i.e., lipopigment accretion and retinal atrophy, in KEY WORDS: lipopigments, English Setters, childhood NCL are related or even interdependent. Our Dalmatian dogs, South Hampstudies on retinal specimens of NCL-affected animal shire sheep, retinal degeneraspecies [Goebel et al., 1979; Goebel and Dahme, 1985, tion 1986; Goebel and Dopfmer, 19901 cast doubt on such a pathogenetic interdependence between disease-specific lipopigments and atrophy in retinae of NCL-affected INTRODUCTION English Setters, Dalmatian dogs, and South Hampshire In childhood forms of human neuronal ceroid-lipo- sheep. fuscinosis (NCL), the retina is affected by 2 different MATERIALS AND METHODS pathological processes, accumulation of disease-specific lipopigments in neuronal perikarya and retinal pigEight eyes from NCL-affected English Setters, 2 eyes ment epithelial (RPE) cells and progressive degenera- from NCL-affected Dalmatian dogs, and 11 eyes from NCL-affected South Hampshire sheep, including control eyes of unaffected animals, have been studied by Received for publication April 5,1991; revision received August light and electron microscopy-after appropriate fixa5, 1991. Address reprint requests to Hans H. Goebel, M.D., Division of tion in glutaraldehyde and embedding in resins-in 1 Neuropathology, University of Mainz, Langenbeckstr. 1, 6500 pm thick toluidine blue stained sections and subsequently ultrathin sections, respectively. Mainz/FRG.
0 1992 Wiley-Liss, Inc.
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Goebel
RESULTS NCL-Affected Dogs Morphologically, ubiquitous formation of lipopigments, characteristic for homozygosity of NCL, is present in ganglion cell and bipolar and photoreceptor cell layers (Fig. la), both in English Setters and Dalmatians. The ultrastructural spectrum of the encountered lipopigments is quite variable, comprising lamellar bodies, fingerprint profiles, curvilinear profiles, and composite bodies (Fig. lb), which usually lack the elec-
tron lucent opaque component so obvious in regular lipofuscin. RPE cells contain melanin granules, melanolipopigments, the lipopigment component, however without NCL-specific details, and phagocytosed outer segments of photoreceptors. In addition, there were densely packed lamellae, with or without associated melanin, often surrounded by a common unit membrane (Fig. lc). By light microscopy, the individual layers of the retina appeared intact (Fig. Id) including the inner and
Fig. 1. Retinal specimens of NCL-affected dogs. a: Lipopigments (arrows) in preserved photoreceptors. Dalmatian, x 3,040.b A composite and two small lipopigment bodies within a retinal process. Dalmatian x 32,300.c: Lamellar (L) inclusions and remnant of ingested photoreceptor outer segments (arrow) in retinal pigment epithelial cell. English Setter, x 33,600.d The layers of the retina are well preserved. English Setter, x 280.
Retina in Neuronal Ceroid-Lipofuscinosis
outer segments of the photoreceptors, which densely filled the subretinal space bordering on a single layer of RPE cells. They harbored melanin pigment outside of the tapetum lucidum. There was no evidence of retinal atrophy, retinal gliosis, or displacement of melanized cells between the inner and outer limiting membranes.
NCL-Affected Sheep The electron microscope disclosed disease-specific lipopigments in ganglion cells, bipolar cells, and photo-
607
receptors (Fig. 2a) again comprising lamellar and fingerprint bodies as well as curvilinear and complex inclusions. In RPE cells (Fig. 2b), densely packed lamellae, running straight or in circles, were often present, in addition to melanin granules and phagocytosed outer segments of photoreceptors. However, histological examination showed loss of photoreceptors later in the course of the disease (Fig. 2121, while early on photoreceptors were well preserved (Fig. 2d).
Fig. 2. NCL-affected South Hampshire sheep. a: Lipopigments (L)are present in the photoreceptor cells. x 14,300.b: Within RPE cells lamellar inclusions (arrows) are seen among melanin granules. x 24,700.c: At 2 years of age (and disease), the retina shows total loss of photoreceptors. Toluidine blue stained 1 p,m thick section. x 360.d At 3.5months of age (and disease), the retina shows well-preserved layers including that of photoreceptors. Toluidine blue stained 1 km thick section, X 300.
608
Goebel
DISCUSSION While the formation of lipopigments is similar in the 2 NCL-affected canine breeds, English Setters, Dalmatian dogs, and in the South Hampshire sheep, the degree of retinal atrophy differs considerably. In sheep, however, atrophy does not seem as advanced as seen in childhood forms of human NCL. Conversely, peculiar densely packed lamellae within RPE cells were present in NCL affected dogs [Goebel et al., 1983; Goebel and Dahme, 1986; Neville et al., 19801 and sheep [Goebel and Dopfmer, 19901, but were unlike human NCL RPE cells which lacked these lamellar inclusions. As the pathogenesis of these inclusions remains to be elucidated, a relation between these lamellar inclusions and physiologically ingested outer segments of photoreceptors is currently a matter of speculation rather than evidence. RPE cells in childhood NCL never harbored such lamellar inclusions, but possibly because photoreceptors in human childhood NCL have disappeared by the time of RPE examination, i.e., at the end stage of the NCL and of the degenerative process of the retina. Only additional studies on human NCL eyes, obtained when photoreceptors are still present, may answer this latter question. Our studies on the retina in NCL-affected species, man, dogs, and sheep, lead us to conclude, that the retina is not a good model to study NCL, but rather a model to examine retinal degeneration. Thus, although NCL affected dogs and sheep may represent excellent spontaneous models for human NCL, the retinal component seems to be lacking. Furthermore, retinal degeneration is uniformly profound in each childhood form of human NCL, but the responsible genes for different forms of human childhood NCL may be located on different chromosomes or at different loci. ACKNOWLEDGMENTS I am grateful to Drs. Dahme (Munich), Jolly (Palmerston-North), and Koppang (Oslo) for kindly provid-
ing respective animal retinal tissues. Mrs. I. Warlo contributed to the electron microscopic studies, Mr. W. Meffert produced photographs, and Mrs. M. Messerschmidt edited the manuscript.
REFERENCES Berkovic SF, Carpenter S, Andermann F, Andermann E, Wolfe LS (1988): Kufs’ disease: A critical reappraisal. Brain 111:27-62. Goebel HH, Dahme E (1985):Retinal ultrastructure of neuronal ceroidlipofuscinosis in the Dalmatian dog. Acta Neuropathol (Berl) 68:224-229. Goebel HH, Dahme (1986):Ultrastructure ofretinal pigment epithelial and neural cells in the neuronal ceroid-lipofuscinosisaffected Dalmatian dog. Retina 6179-187. Goebel HH, Dopfmer I (1990): An ultrastructural study on retinal neural and pigment epithelial cells in ovine neuronal ceroid-lipofuscinosis. Ophthalmic Paediatr Genet 1151-69. Goebel HH, Fix JD, Zeman W (1974):The fine structure of the retina in neuronal ceroid-lipofuscinosis.Am J Ophthalmol 77:25-39. Goebel HH, Pilz H, Gullotta F (1976):The protracted form of juvenile neuronal ceroid-lipofuscinosis. Acta Neuropathol (Berl) 36393396. Goebel HH, Zeman W, Damaske E (1977):An ultrastructural study of the retina in the Jansky-Bielschowsky type of neuronal ceroidlipofuscinosis. Am J Ophthalmol 83:70-79. Goebel HH, Koppang N, Zeman W (1979):Ultrastructure of the retina in canine neuronal ceroid-lipofuscinosis.Ophthalmic Res 1155-72. Goebel HH, Kohneke B, Koppang N, Armstrong D (1983):Ultrastructural studies on the retinal pigment epithelium in the neuronal ceroid-lipofuscinoses. Ophthalmic Paediatr Genet 3:29-37. Goebel HH, Klein H, Santavuori P, Sainio K (1988): Ultrastructural studies of the retina in infantile neuronal ceroid-lipofuscinosis. Retina 8:59-66. Ikeda K, Kosaka K, Oyanagi S, Yamada K (1984)Adult type of neuronal ceroid-lipofuscinosis with retinal involvement. Clin Neuropathol 3:237-239. Martin J J , Libert J , Ceuterick C (1987): Ultrastructure of brain and retina in Kufs’ disease (adult type of ceroid-Lipofuscinosis).Clin Neuropathol6 381-394. Neville H, Armstrong D, Wilson B, Koppang N, Wehling C (1980): Studies on the retina and the pigment epithelium in hereditary canine ceroid lipofuscinosis.111. Morphologic abnormalities in retinal neurons and retinal pigmented epithelial cells. Invest Ophthalmol Vis Sci 19:75-86. Tarkkanen A, Haltia M, Merenmies L (1972): Ocular pathology in infantile tvue of neuronal ceroid-lipofuscinosis.J. Pediatr Ophthalmol Strabismus 14:235-241.
Retina in Various Animal Models of Neuronal Ceroid-Lipofuscinosis Hans H. Goebel Division of Neuropathology, University of Mainz, Mainz The childhood forms of human neuronal ceroid-lipofuscinosis(NCL)are invariably associated with a severe progressive retinopathy which commences at the photoreceptor level morphologically and proceeds to a final loss of neuronal cells accompanied by severe gliosis. In respective spontaneousanimal conditions of NCL, in English setters, Dalmatian dogs, and New Zealand sheep,retinal involvement is not commensurate although the retina does not seem to be completely unaffected. In canine NCL, there might be functional and electro-physiological impairment of retinal cells, but retinal atrophy is not obvious. In ovine NCL, the retina, apart from accumulating NCL-specific lipopigments within neuronal perikarya, also shows loss of photoreceptors. Conversely, retinal pigment epithelial cells (RPE)in canine and ovine NCL contain peculiar lamellar inclusions,not seen in unaffected animals, possibly an indication of a disturbed phagocytic process of photoreceptor outer segments. Similar such lamellar inclusions have not been observed in RPE cells in human childhood NCL.
tion of the neuronal elements, apparently commencing a t the level of the photoreceptors. While the former process of lipopigment accretion refers to the fine structural details among the human childhood forms, the retinopathy resulting in severe atrophy of the entire retina seems to be similar, if not identical during the course of the disease resulting in a burnt-out stage of both the condition NCL and the retinal degeneration when judged by eyes obtained after death from NCL-afflicted children [Goebel et al., 1974, 1977, 19881. These light and electron microscopic studies have confirmed and augmented scant earlier findings on the retinal pathology in human childhood NCL [Tarkkanen et al., 19721. In regard to adult human NCL, to be distinguished from protracted juvenile human NCL, which may entail only blindness [Goebel et al., 19761 and pigmentary retinal degeneration, functional impairment and pathology of the retina have not been reliably elucidated [Berkovicet al., 19881. Accumulation of disease-specificlipopigments in retinal ganglion cells has been documented in adult human NCL, but retinal atrophy has not been confirmed [Martin et al., 19871 while the nosological classification has been questioned where retinal atrophy had been encountered [Ikeda et al., 1984; Martin et al., 19871. It is still controversial, whether the 2 retinal processes, i.e., lipopigment accretion and retinal atrophy, in KEY WORDS: lipopigments, English Setters, childhood NCL are related or even interdependent. Our Dalmatian dogs, South Hampstudies on retinal specimens of NCL-affected animal shire sheep, retinal degeneraspecies [Goebel et al., 1979; Goebel and Dahme, 1985, tion 1986; Goebel and Dopfmer, 19901 cast doubt on such a pathogenetic interdependence between disease-specific lipopigments and atrophy in retinae of NCL-affected INTRODUCTION English Setters, Dalmatian dogs, and South Hampshire In childhood forms of human neuronal ceroid-lipo- sheep. fuscinosis (NCL), the retina is affected by 2 different MATERIALS AND METHODS pathological processes, accumulation of disease-specific lipopigments in neuronal perikarya and retinal pigEight eyes from NCL-affected English Setters, 2 eyes ment epithelial (RPE) cells and progressive degenera- from NCL-affected Dalmatian dogs, and 11 eyes from NCL-affected South Hampshire sheep, including control eyes of unaffected animals, have been studied by Received for publication April 5,1991; revision received August light and electron microscopy-after appropriate fixa5, 1991. Address reprint requests to Hans H. Goebel, M.D., Division of tion in glutaraldehyde and embedding in resins-in 1 Neuropathology, University of Mainz, Langenbeckstr. 1, 6500 pm thick toluidine blue stained sections and subsequently ultrathin sections, respectively. Mainz/FRG.
0 1992 Wiley-Liss, Inc.
606
Goebel
RESULTS NCL-Affected Dogs Morphologically, ubiquitous formation of lipopigments, characteristic for homozygosity of NCL, is present in ganglion cell and bipolar and photoreceptor cell layers (Fig. la), both in English Setters and Dalmatians. The ultrastructural spectrum of the encountered lipopigments is quite variable, comprising lamellar bodies, fingerprint profiles, curvilinear profiles, and composite bodies (Fig. lb), which usually lack the elec-
tron lucent opaque component so obvious in regular lipofuscin. RPE cells contain melanin granules, melanolipopigments, the lipopigment component, however without NCL-specific details, and phagocytosed outer segments of photoreceptors. In addition, there were densely packed lamellae, with or without associated melanin, often surrounded by a common unit membrane (Fig. lc). By light microscopy, the individual layers of the retina appeared intact (Fig. Id) including the inner and
Fig. 1. Retinal specimens of NCL-affected dogs. a: Lipopigments (arrows) in preserved photoreceptors. Dalmatian, x 3,040.b A composite and two small lipopigment bodies within a retinal process. Dalmatian x 32,300.c: Lamellar (L) inclusions and remnant of ingested photoreceptor outer segments (arrow) in retinal pigment epithelial cell. English Setter, x 33,600.d The layers of the retina are well preserved. English Setter, x 280.
Retina in Neuronal Ceroid-Lipofuscinosis
outer segments of the photoreceptors, which densely filled the subretinal space bordering on a single layer of RPE cells. They harbored melanin pigment outside of the tapetum lucidum. There was no evidence of retinal atrophy, retinal gliosis, or displacement of melanized cells between the inner and outer limiting membranes.
NCL-Affected Sheep The electron microscope disclosed disease-specific lipopigments in ganglion cells, bipolar cells, and photo-
607
receptors (Fig. 2a) again comprising lamellar and fingerprint bodies as well as curvilinear and complex inclusions. In RPE cells (Fig. 2b), densely packed lamellae, running straight or in circles, were often present, in addition to melanin granules and phagocytosed outer segments of photoreceptors. However, histological examination showed loss of photoreceptors later in the course of the disease (Fig. 2121, while early on photoreceptors were well preserved (Fig. 2d).
Fig. 2. NCL-affected South Hampshire sheep. a: Lipopigments (L)are present in the photoreceptor cells. x 14,300.b: Within RPE cells lamellar inclusions (arrows) are seen among melanin granules. x 24,700.c: At 2 years of age (and disease), the retina shows total loss of photoreceptors. Toluidine blue stained 1 p,m thick section. x 360.d At 3.5months of age (and disease), the retina shows well-preserved layers including that of photoreceptors. Toluidine blue stained 1 km thick section, X 300.
608
Goebel
DISCUSSION While the formation of lipopigments is similar in the 2 NCL-affected canine breeds, English Setters, Dalmatian dogs, and in the South Hampshire sheep, the degree of retinal atrophy differs considerably. In sheep, however, atrophy does not seem as advanced as seen in childhood forms of human NCL. Conversely, peculiar densely packed lamellae within RPE cells were present in NCL affected dogs [Goebel et al., 1983; Goebel and Dahme, 1986; Neville et al., 19801 and sheep [Goebel and Dopfmer, 19901, but were unlike human NCL RPE cells which lacked these lamellar inclusions. As the pathogenesis of these inclusions remains to be elucidated, a relation between these lamellar inclusions and physiologically ingested outer segments of photoreceptors is currently a matter of speculation rather than evidence. RPE cells in childhood NCL never harbored such lamellar inclusions, but possibly because photoreceptors in human childhood NCL have disappeared by the time of RPE examination, i.e., at the end stage of the NCL and of the degenerative process of the retina. Only additional studies on human NCL eyes, obtained when photoreceptors are still present, may answer this latter question. Our studies on the retina in NCL-affected species, man, dogs, and sheep, lead us to conclude, that the retina is not a good model to study NCL, but rather a model to examine retinal degeneration. Thus, although NCL affected dogs and sheep may represent excellent spontaneous models for human NCL, the retinal component seems to be lacking. Furthermore, retinal degeneration is uniformly profound in each childhood form of human NCL, but the responsible genes for different forms of human childhood NCL may be located on different chromosomes or at different loci. ACKNOWLEDGMENTS I am grateful to Drs. Dahme (Munich), Jolly (Palmerston-North), and Koppang (Oslo) for kindly provid-
ing respective animal retinal tissues. Mrs. I. Warlo contributed to the electron microscopic studies, Mr. W. Meffert produced photographs, and Mrs. M. Messerschmidt edited the manuscript.
REFERENCES Berkovic SF, Carpenter S, Andermann F, Andermann E, Wolfe LS (1988): Kufs’ disease: A critical reappraisal. Brain 111:27-62. Goebel HH, Dahme E (1985):Retinal ultrastructure of neuronal ceroidlipofuscinosis in the Dalmatian dog. Acta Neuropathol (Berl) 68:224-229. Goebel HH, Dahme (1986):Ultrastructure ofretinal pigment epithelial and neural cells in the neuronal ceroid-lipofuscinosisaffected Dalmatian dog. Retina 6179-187. Goebel HH, Dopfmer I (1990): An ultrastructural study on retinal neural and pigment epithelial cells in ovine neuronal ceroid-lipofuscinosis. Ophthalmic Paediatr Genet 1151-69. Goebel HH, Fix JD, Zeman W (1974):The fine structure of the retina in neuronal ceroid-lipofuscinosis.Am J Ophthalmol 77:25-39. Goebel HH, Pilz H, Gullotta F (1976):The protracted form of juvenile neuronal ceroid-lipofuscinosis. Acta Neuropathol (Berl) 36393396. Goebel HH, Zeman W, Damaske E (1977):An ultrastructural study of the retina in the Jansky-Bielschowsky type of neuronal ceroidlipofuscinosis. Am J Ophthalmol 83:70-79. Goebel HH, Koppang N, Zeman W (1979):Ultrastructure of the retina in canine neuronal ceroid-lipofuscinosis.Ophthalmic Res 1155-72. Goebel HH, Kohneke B, Koppang N, Armstrong D (1983):Ultrastructural studies on the retinal pigment epithelium in the neuronal ceroid-lipofuscinoses. Ophthalmic Paediatr Genet 3:29-37. Goebel HH, Klein H, Santavuori P, Sainio K (1988): Ultrastructural studies of the retina in infantile neuronal ceroid-lipofuscinosis. Retina 8:59-66. Ikeda K, Kosaka K, Oyanagi S, Yamada K (1984)Adult type of neuronal ceroid-lipofuscinosis with retinal involvement. Clin Neuropathol 3:237-239. Martin J J , Libert J , Ceuterick C (1987): Ultrastructure of brain and retina in Kufs’ disease (adult type of ceroid-Lipofuscinosis).Clin Neuropathol6 381-394. Neville H, Armstrong D, Wilson B, Koppang N, Wehling C (1980): Studies on the retina and the pigment epithelium in hereditary canine ceroid lipofuscinosis.111. Morphologic abnormalities in retinal neurons and retinal pigmented epithelial cells. Invest Ophthalmol Vis Sci 19:75-86. Tarkkanen A, Haltia M, Merenmies L (1972): Ocular pathology in infantile tvue of neuronal ceroid-lipofuscinosis.J. Pediatr Ophthalmol Strabismus 14:235-241.
