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Hereditary retinal degeneration in the Rhode Island Red chicken: Ultrastructural analysis
Erp.
Eye Res. (1985)
Hereditary
40, 865877
Retinal Degeneration in the Rhode Chicken: Ultrastructural Analysis ROBERT
Department
J. ULSHAFERAND
CLARK
Red
B. ALLEN
of Ophthalmology, College of Medicine, University Gainesville, FL 32610, 1J.S.A.
(Received 26’ November
Island
1984 and accepted 7 February
of Florida,
1985, New
York)
An electron microscopic analysis of photoreceptor degeneration in a congenitally blind strain of chickens is presented. The mutation was named rd, meaning ‘retinal degeneration’. Although the chicks were behaviorally and electrophysiologically blind at the time of hatching, their retinas appeared morphologically comparable to normal chicks at this stage. Both groups had welldeveloped photoreceptor cells, although outer segments were typically disoriented or misaligned. In the normal, and to some degree in the rd, retina, outer segments became organized within the first week posthatching. In the rd retina at that time, however, more outer segments were disorganized and disoriented. Disc-like membranes were also seen in some inner segments. Many photoreceptors had distended inner segment tips containing a granular cytoplasm. Membraneous debris was present in the subretinal space. Over the next 2-3 weeks there was a reduction in number of inner segments, outer segments and photoreceptor nuclei of both rods and cones. Photoreceptor cell bodies in the outer nuclear layer were replaced by Mueller cell processes. By the end of the second month, a larger cone:& ratio was apparent, and a large proportion of the remaining cones were double cones. Intact outer segments were rarely seen at that time. Few and sporadic cone cells, identified by a pale-staining oil droplet, were the predominant surviving photoreceptors by 6 months of age. At the later stages examined, the pigment epithelium (PE) appeared to be undergoing degenerative changes. A generai thinning of cells and hypopigmentation of PE cells was apparent, although hyperpigmented, hypertrophied PE cells were also present which bulged into the subretinal space. Pigmented cells of unknown origin were also noted in the subretinal space at the later time points. Key words: retina; photoreceptor; chicken; hereditary retinal degeneration: animal model.
1. Introduction Hereditary congenital blindness in a strain of Rhode Island Red chickens was reported by Cheng et al. (1980) to have arisen in response to a translocation occurring between one arm of the sex chromosome and the long q arm of chromosome 3. The mutation was expressed as single gene inheritance having complete penetrance and a recessive mode of transmission; behaviorally, the chickens appeared blind at the time of hatching. In a previous study it was reported that the offspring of a mating between two chickens homozygous for the disorder hatched with no measurable electroretinographic (ERG) response, while the ERG of a normal chick had a- and b-wave amplitudes near the adult level at the time of hatching. At the same time, however, the affected retina appeared morphologically normal when compared to a retina from a normal chick at the light microscopic level (L%hafer, Allen, Dawson and Wolf, 1984). That study also showed that sometime between 1 and 2 weeks after hatching retinal photoreceptors
began
degenerating.
By
4-6
months
of age
the
retinas
had
only
a few
photoreceptors left, and those remaining appeared to be predominantly cones. Based on these findings the mutation was named rd, meaning retinal degeneration. The purpose of the present study was to describe the process of photoreceptor cell Please address reprint requests and correspondence mology. Box J-284 JHMHC. University of Florida, 001~U35/85/060865
+ 13 $03.00/O
to Dr Robert J. Ulshafer, Department Gainesville. FL 32610. I‘.S.A. @ 1Q8R Academic
Press Inc.. (London)
of Ophtha].
Limited
866
Fra. 1. Normal (PE) interdigitate (E). Cone inner
R. J. IJLSHAFER
chicken among segments
retina rod have
ASI)
(‘. 13. AI,I,EN
on the day of hatching. Apical processes ofthe and cone outer segments (OS). Inner segments an oil droplet (On). x 4000.
retinal pigment (TS) contain
rpithelium an ellipsoid
death in the rd chicken at the ultrastructural level. Observations in the present report are limited to findings in the central retina since that region is the first to undergo degeneration, and degeneration in peripheral retina follows the same pattern, albeit at later times (Ulshafer and Allen, 1984; Ulshafer et al., 1984).
2. Materials
and Methods
Eggs from normal and rd chickens were incubated in a commercial, automatic rotating, forced-draft incubator until the day before hatching when they were transferred to a brooder. Chicks were raised in a light-controlled environment (12 I-12 D; 1.6 log FtL). Three to six specimens were killed during the light cycle by decapitation at the following posthatching times: 1 (day of hatching), 3, 5, 7, 9, 11, 21 days and 1, 2, 3, 4, 5. 6 and 8 months. Eyes were enucleated immediately following death and the globe anterior to the ora serrata was removed while the posterior globe was immersed into 2 O/’ glutaraldehyde in 91 N phosphate buffer overnight. The following morning the vitreous was removed and the retinas were cut
ELECTRON
MICROSCOPY
OF
rd CHICK
RETINA
867
FIG. 2. Retina from a rd chick on the day of hatching. Morphology is similar to the normal chick retina at this stage of development. CC, Choriocapillaris; OS. outer segment; IS. inner segment: PE, pigment epithelium; OD. oil droplet. x 4000.
into strips whose locations were noted. Tissues were then washed and postfixed in 1 o/0 osmium tetroxide, dehydrated through an ethyl alcohol series, cleared in propylene oxide and embedded in Epon. Sections were cut at 1 pm in a Sorval MT2 microtome, mounted on glass slides and stained with 1 o/0 toluidine blue in 1 o/0 sodium borate for light microscopy. Thin sections (70-80 nm) were cut on an LKB Ultratome III microtome, stained with a saturated solution (2 %, aqueous) of uranyl acetate followed by Reynold’s lead citrate, and photographed in a Zeiss 9s electron microscope.
3. Results An electronmicrograph of a normal, l-day-old chick retina is presented in Fig. 1. The retina was relatively well-developed at the time of hatching. Both rod and cone photoreceptors had differentiated prehatching, and both outer and inner segments resembled the adult form, albeit somewhat smaller and more disoriented. Most cones contained an oil droplet whose contents had frequently leached out during the preparatory procedures. Five types of cones (three single cones and principal and
FIG. 3. Outer synaptic zone of a l-day-old rd retina. The chick retina possesses the typical synaptic ribbons (SR) and triad arrangement of most vertebrate retinas. (+cnerally rods have H shorter synaptic expanse and fewer synaptic ribbons than cows. C’S, (‘onp nuc~lt~us: ICY’. r~,tl r~uclrus: (‘I’. ,YUIP ~~dicle; OPL, outer plexiform layer. x 10200.
accessory members of the double cone) were identified and classified, based on position and density of the oil droplet (Morris, 1970 ; Meyer and May, 1973 ; Morris and Shorey, 1967). Not all cone types are evident in Fig. 1. The chicken rod inner segment did not contain an oil droplet but had a paraboloid body. The accessory member of the double cone contained a similar or identical organelle (Meyer and May, 1973 : Morris and Shorey, 1967). The retina of an rd chick on the day of hatching is shown in Fig. 2. This chick was one of those shown t#olack an.electroret’inogram (ERG)signal in response to light under both light and dark adapted states (Ulshafer et al., 1984). Despite the chick’s apparent lack of vision, retinal morphology was comparable to a normal chick of the same age (Fig. 1). Photoreceptor cells were present and no significant pathological changes were evident. It should be noted that both rd and normal retinas contained photoreceptors with disorganized, disoriented or partially developed outer segments at this time. Apical tips of some inner segments in rd retinas, however, appear to be somewhat more
ELECTRON
MICROSCOPY
OF
rd CHICK
RETISA
ns9
Fro. 4. Photoreceptors of l-week posthatch rd retina. Some outer segments appear disorganized (arrows). Outer segment discs are seen within the inner segment of at least one photoreceptor (*). Note granular. distended tips of many inner segments (arrowheads). x 4200.
distended than normals, although not apparent in Fig. 2. The retinal pigment epithelium (PE) and choriocapillaris were also virtually identical to that from a normal chick. Synapses between photoreceptors and interneurons were present in the outer plexiform layer (OPL) in rd chicks (Fig. 3) that resembled those of normal chicks. Synaptic ribbons, 50 nm diameter vesicles and typical triad postsynaptic elements were present in dimensions and numbers similar to normals. During the first week posthatching, however, some photoreceptors appeared to lose their morphological integrity (Fig. 4). Many outer segments were disorganized, disoriented and vesiculated. Some contained granular cytoplasm similar to that’ normally found in inner segments while, in others, stacks of OS discs were identified within the inner segment. Inner segments were frequently swollen, particularly at the apical tip proximal to the outer segment. These degenerative changes were more evident during the next week, and by the end of week 2 few normal appearing outer segments were present in the central retina
870
871
FIG.
6. For
legend see p 87’7
a72
R. .J. IrLSHAFEII
AND
(‘. 13. ALLEN
(Fig. 5). In addition to those changes described in the previous paragraphs, significant alterations in morphology of the synaptic elements of many photoreceptors had occurred. Whereas, in the normal g-week-old chick, synaptic pedicles and spherules were displaced somewhat from the photoreceptor nucleus by the inner connecting fiber (Fig. 6), the structures in the rd retina were frequently seen within the soma not far from the nucleus (Fig. 5). Other organelles, such as the oil droplet and paraboloid. were also sometimes in abnormal locations (Fig. 5). By 34 weeks posthatching, a distinct lesion was present in the central retina of all rd chicks. At the light and scanning microscopic levels, this was characterized by a retinal detachment, some debris in the subretinal space, and a reduction in the number of intact photoreceptor cells (Ulshafer and Allen, 1984; Ulshafer et al., 1984). These phenomena were also observed at the ultrastructural level (Fig. 7). The debris in the subretinal space appeared to be composed of large membraneous sacs, frequently emanating from, or associated with, inner segments. Tips of inner segments were grossly distended. Inner segments contained swollen mitochondria. Also observed were what appeared to be unattached outer segments and large whorls of outer segment-like material in the subretinal space. Intact photoreceptors, rods and cones, were also seen. By 2 months after hatching the lesion had spread to mid-periphery, although peripheral retina still appeared relatively normal (Ulshafer et al., 1984). Under the electron microscope, it was observed that few photoreceptors remained in the central retina. A high proportion of those remaining were double cones (Fig. 8), but very few outer segments were intact at this stage. The subretinal space was relatively free of debris at this time. In the ONL the majority of degenerated cells had been replaced by processes of Mueller cells. The terminal bar network of these cells, which comprises the outer limiting membrane of the neural retina, appeared very pronounced. Very few synaptic ribbons were noted at this time. By G8 months after hatching only a few sporadic photoreceptors remained (Fig. 9). Their inner segments were diminutive, compared to those from a normal chicken retina at this stage, but contained recognizable organelles : ellipsoid, rough and smooth endoplasmic reticulum, and frequently a lightly staining oil droplet. Many photoreceptor nuclei had become pleomorphic and lobulated. Synaptic ribbons were still occasionally seen associated with one or more processes of retinal interneurons. At this late stage of photoreceptor degeneration, changes were also noted in the PE : a general thinning of this cell layer occurred, with fewer numbers of melanosomes in many cells or in parts of a given cell. Some PE cells appeared to be hypertrophied, bulging into the subretinal space and containing many melanosomes. Cells of unknown origin were frequently seen among the inner segments and PE processes in the
FIG. 5. Low-power electron micrograph montage of outer retina of Z-week-old rd chick. Outer segments are few and largely disorganized. Oil droplets are not as regularly alligned as in normal retina and some (straight arrow) appear at abnormal locations as do some parabaloids (curved arrows). Inner connecting fibers (arrowheads) are very short. P = Principal and A = accessory member of double cones. x 2000. FIG. 6. Low-power electron micrograph montage of outer retina of %week-old normal chick. Xote well-developed and aligned outer segments and stratification of oil droplets (OD) in the various cone types. The inner segments of double cones (P = principal member, A = accessory member) are not separated by Mueller cell microvillae. Note the long inner connecting fiber (axon-like process, large arrow) of cones ending in a pedicle and the shorter connecting fiber of a rod (small arrow) which ends in a spherule. ONL, Outer nuclear layer: INL. inner nuclear layer; PE. pigment epithelium. x 1980.
ELECTRON
MICROSCOPY
OF
rd CHICK
RETINA
873
FIG. 7. Receptor layer of a 21-day-old rd chick. Note swollen granular inner segments (IS) of most photoreceptor cells. Although one outer segment (OS) appears relatively normal, a massive accumulation of disorganized outer segment membranes (*) is notable in the subretinal space. Many ellipsoids (E) have swollen mitochondria. In this specimen apical processes of PE appear retracted and swollen. x 5.580.
874
H. J. LTLSHAFEK
AKD
(‘. R. ALI,E?;
Fra. 8. Outer retina of a 2-month-old rd chick. Further reduction in number of photoreceptor cells is evidc mced by increase in Mueller cell processes (MC), fewer nuclei in the ON, and the warty (wntinuous outer 11imiting membrane (OLM). A few synaptic ribbons are present (arrows). Remaining photoreceptors member. A = accessory member. The suhrrtinal space appear to be mostly double cones: P = principal x ~(KMI. is relat ively dehris-free.
ELECTRON
MICROSCOPY
OF
rd CHICK
RETINA
876
FIQ. 9. Outer retina of a B-month-old rd chick. A few remaining photoreceptor inner segments (IS) are seen in this section. The entire expanse of one cone can be identified from its pale staining oil droplet (OD) to its truncated connecting fiber. The nucleus (N) of this cone has become pleomorphic; a synaptic ribbon (curved arrow) is present in the pedicle which abuts on an interneuron (IN). Mueller cell (MC) processes largely fill up the outer nuclear layer; the terminal bar network of these cells forms the outer limiting membrane (OLM) of the neutral retina. The pigment epithelium (PE) has thinned considerably and reduction in number of melanin granules is especially evident in the cell to the right. Processes of cells (straight arrows) of unknown origin are present in the subretinal space. x 4666.
subretinal space. These cells usually contained debris and melanosome-like granules ; they resembled detached PE cells or macrophages originating in the vascular system. 4. Discussion As has been reported in other animal models of hereditary retinal degeneration, such as the rd mouse (Sanyal and Bal, 1973) Irish setter (Buyukmihci, Aguirre and Marshall, 1980) and collie (Santos-Anderson, Tso and Wolf, 1980), degenerative changes in photoreceptor cells in the rd chicken are first seen in the outer segments. However, an important difference between those models and the rd chick is that in
876
Il. .J. C71,SH.4PE
R ASJ)
(‘. 13. A I,I,ES
the former group photoreceptors degenerate during the initial phases of different,iation. while in the latter the cells degenerate after differentiation, although before c~omplet~c maturity. In the case of late onset photoreceptor degeneration seen in the rniniat)urr poodle, the earliest morphological changes are also seen in t’he out’rr segments (Aguirre. Alligood, O’Brien and Buyukmihci, 1982). In the pcd mouse, another model of late onset photoreceptor degeneration, the first rhanges are seen in and around the inner segments (Blanks, Mullen and LaVail, 1983). In two ot’her animal models. the Ft(% rat and the DAM chicken. pathological rhanges in photoreceptors result from a degenerative event in overlying retinal PE (Fite, Montgomery, Whitney. Boissey and Smyth, 1983; Herron, 1977). In many of the animal models of retinal degeneration, as well as in cases of retinitis pigmentosa in man, cone photoreceptors tend to persist longer than rods. The ability to easily distinguish different photoreceptor types in the chicken has allowed identification of the surviving cells in the rd chicken as cones. At around 2 months posthatching, many of the remaining photoreceptor cells can be identified as double cones. Although no double cones, per se, could be distinguished at’ the later time points (G-8 months of age) in the rd chick, a distinctly pale staining oil droplet near t’he tip of the inner segment indicates that the residual cells are probably the principal members of the double cones (Meyer and May, 1973; Morris, 1970). Whether some cones partially or completely lack the genetic abnormality, are ‘immune’ to a toxic agent, or are generally more hardy than rods has yet to be discovered in this or other cases of hereditary retinal degeneration. It seems paradoxical that the newly hatched rd chick retina should appear to be similar to that of normal chicks at the ultrastructural level yet be unable to generate a measurable ERG. Indeed, it appears that the rd chick never had the ability to see, since at the time of hatching they neither find their food easily, peck at random targets (black dots), nor avoid threatening visual stimulae as do normal chicks. Degeneration of the photoreceptor cells therefore occurs well after (l-2 weeks) the chicks appear to be behaviorally blind. It is possible that they partially or completely lack the visual pigments, enzymes or carrier proteins of the vitamin A cycle necessary for normal vision. Alternatively, other enzymes or systems crucial in maintaining normal ionic environment, membrane permeability or synaptic transmission may be involved. Although significant’ amounts of membraneous debris are seen in the subretinal spare at the early stages of the disorder, the lack of such debris at lat’er stages implies that phagocytosis by PE is not abnormal in this model, unlike the RCS rat model where reduced phagocytosis of shed discs leads to a build-up of debris in the subretinal space and is somehow toxic to the photoreceptors (Herron, 1977). The presence of large amounts of granular cytoplasm in the distended apical tips of inner segments is unique to the rd chick. Possibly this represents an accumulation of phospholipid and/or protein molecules, normally destined to be incorporated into membraneous discs of the outer segment. Their membranes may stop being generated due to a defect in protein synthesis or in t’he membrane assembly mechanism. Autoradiographic st’udies on the fate of labeled protein precursors in rd and normal chicks are presently underway which should determine if this is the case. ACKNOWI,EDGMENTG This study was supported in part by a non-restricted to Prevent Blindness, Inc. and by NIH R01 EY04590
Departmental Grant from Research (R.J.U.). This work was also aided
ELECTRON
MICROSCOPY
by Basil O’Connor Starter Research Grant Foundation. We thank MS Julie Driggers
OF No. S-438 for typing
rd CHICK
RETINA
from the March the manuscript.
of Dimes
Hi5 Births
Defects
REFERENCES Aguirre, G., Alligood, P., O’Brien, P. J. and Buyukmihci, N. (1982). Pathogenesis of progressive rod-cone degeneration in miniature poodles. InvecTt. Ophthalmol. Vis. I’%%. 23. 61&30. Blanks, J: C., Mullen, R. J. and LaVail, M. M. (1982). Retinal degeneration in the pcd cerebellar mutant mouse. II. Electron microscopic analysis. J. Camp. Nrurol. 212. 231-46. Buyukmihci, N.. Aguirre, G. and Marshall, J. (1980). Retinal degeneration in the dog. II. Development of the retina in rod-cone dysplasia. Exp. Eye Res. 30, 575-91. Cheng, K. M., Shoffer, R. N., Gelatt, K. N., Gum, G. G., Otis, J. S. and Bitgood, J. J. (1980). An autosomal recessive blind mutant in the chicken. Poultry Sci. 59, 2179-82. Fite. K. V., Montgomery, N., Whitney, T., Boissey, R. and Smyth, J. R., Jr (1983). Inherited retinal degeneration and ocular amelanosis in the domestic chicken (QaZZus domesticus). Curr. Eye Res. 2, 109-15. Herron, W. L., Jr (1977). The dystrophic rat as a model for clinical research. Bdv. Exp. Med. Biol. 77: 137-52. Meyer. D. B. and May, H. C., Jr (1973). The topographical distribution of rods and cones in the adult chicken retina. Exp. Eye Res. 17, 347-55. Morris, V. B. (1970). Symmetry in a receptor mosaic demonstrated in the chick from the frequencies, spacing and arrangement of the types of retinal receptor. J. Pomp. Newel.
140, 35S98. Morris,
V. B. and Shorey, C. B. (1967). An electron microscope study of types of receptor in the chick retina. J. Comp. Neurol. 129, 313-40. Santos-Anderson, R. M., Tso, M. 0. M. and Wolf, E. D. (1980). An inherited retinopathy in collies: a light and electron microscopic study. Invest. Ophthalmol. Vis. Sci. 19. 1281-94. Sanyal, S. and Bal, A. K. (1973). Comparative light and electron microscopic study of retinal histogenesis in normal and rd mutant mice. S. nat. Entwicklungsgesch. 142, 21S38. Ulshafer, R. J. and Allen, C. B. (1984). Scanning electron microscopy of the retina in an animal model of hereditary blindness. Scanning Electron Microscopy 1984/11, 841-8. Ulshafer. R. J., Allen. C. B., Dawson, W. W. and Wolf, E. D. (1984). Hereditary retinal degeneration in the Rhode Island Red chicaken. I. Histology and ERG. Exp. Eye RPS 39, 125-35.
Eye Res. (1985)
Hereditary
40, 865877
Retinal Degeneration in the Rhode Chicken: Ultrastructural Analysis ROBERT
Department
J. ULSHAFERAND
CLARK
Red
B. ALLEN
of Ophthalmology, College of Medicine, University Gainesville, FL 32610, 1J.S.A.
(Received 26’ November
Island
1984 and accepted 7 February
of Florida,
1985, New
York)
An electron microscopic analysis of photoreceptor degeneration in a congenitally blind strain of chickens is presented. The mutation was named rd, meaning ‘retinal degeneration’. Although the chicks were behaviorally and electrophysiologically blind at the time of hatching, their retinas appeared morphologically comparable to normal chicks at this stage. Both groups had welldeveloped photoreceptor cells, although outer segments were typically disoriented or misaligned. In the normal, and to some degree in the rd, retina, outer segments became organized within the first week posthatching. In the rd retina at that time, however, more outer segments were disorganized and disoriented. Disc-like membranes were also seen in some inner segments. Many photoreceptors had distended inner segment tips containing a granular cytoplasm. Membraneous debris was present in the subretinal space. Over the next 2-3 weeks there was a reduction in number of inner segments, outer segments and photoreceptor nuclei of both rods and cones. Photoreceptor cell bodies in the outer nuclear layer were replaced by Mueller cell processes. By the end of the second month, a larger cone:& ratio was apparent, and a large proportion of the remaining cones were double cones. Intact outer segments were rarely seen at that time. Few and sporadic cone cells, identified by a pale-staining oil droplet, were the predominant surviving photoreceptors by 6 months of age. At the later stages examined, the pigment epithelium (PE) appeared to be undergoing degenerative changes. A generai thinning of cells and hypopigmentation of PE cells was apparent, although hyperpigmented, hypertrophied PE cells were also present which bulged into the subretinal space. Pigmented cells of unknown origin were also noted in the subretinal space at the later time points. Key words: retina; photoreceptor; chicken; hereditary retinal degeneration: animal model.
1. Introduction Hereditary congenital blindness in a strain of Rhode Island Red chickens was reported by Cheng et al. (1980) to have arisen in response to a translocation occurring between one arm of the sex chromosome and the long q arm of chromosome 3. The mutation was expressed as single gene inheritance having complete penetrance and a recessive mode of transmission; behaviorally, the chickens appeared blind at the time of hatching. In a previous study it was reported that the offspring of a mating between two chickens homozygous for the disorder hatched with no measurable electroretinographic (ERG) response, while the ERG of a normal chick had a- and b-wave amplitudes near the adult level at the time of hatching. At the same time, however, the affected retina appeared morphologically normal when compared to a retina from a normal chick at the light microscopic level (L%hafer, Allen, Dawson and Wolf, 1984). That study also showed that sometime between 1 and 2 weeks after hatching retinal photoreceptors
began
degenerating.
By
4-6
months
of age
the
retinas
had
only
a few
photoreceptors left, and those remaining appeared to be predominantly cones. Based on these findings the mutation was named rd, meaning retinal degeneration. The purpose of the present study was to describe the process of photoreceptor cell Please address reprint requests and correspondence mology. Box J-284 JHMHC. University of Florida, 001~U35/85/060865
+ 13 $03.00/O
to Dr Robert J. Ulshafer, Department Gainesville. FL 32610. I‘.S.A. @ 1Q8R Academic
Press Inc.. (London)
of Ophtha].
Limited
866
Fra. 1. Normal (PE) interdigitate (E). Cone inner
R. J. IJLSHAFER
chicken among segments
retina rod have
ASI)
(‘. 13. AI,I,EN
on the day of hatching. Apical processes ofthe and cone outer segments (OS). Inner segments an oil droplet (On). x 4000.
retinal pigment (TS) contain
rpithelium an ellipsoid
death in the rd chicken at the ultrastructural level. Observations in the present report are limited to findings in the central retina since that region is the first to undergo degeneration, and degeneration in peripheral retina follows the same pattern, albeit at later times (Ulshafer and Allen, 1984; Ulshafer et al., 1984).
2. Materials
and Methods
Eggs from normal and rd chickens were incubated in a commercial, automatic rotating, forced-draft incubator until the day before hatching when they were transferred to a brooder. Chicks were raised in a light-controlled environment (12 I-12 D; 1.6 log FtL). Three to six specimens were killed during the light cycle by decapitation at the following posthatching times: 1 (day of hatching), 3, 5, 7, 9, 11, 21 days and 1, 2, 3, 4, 5. 6 and 8 months. Eyes were enucleated immediately following death and the globe anterior to the ora serrata was removed while the posterior globe was immersed into 2 O/’ glutaraldehyde in 91 N phosphate buffer overnight. The following morning the vitreous was removed and the retinas were cut
ELECTRON
MICROSCOPY
OF
rd CHICK
RETINA
867
FIG. 2. Retina from a rd chick on the day of hatching. Morphology is similar to the normal chick retina at this stage of development. CC, Choriocapillaris; OS. outer segment; IS. inner segment: PE, pigment epithelium; OD. oil droplet. x 4000.
into strips whose locations were noted. Tissues were then washed and postfixed in 1 o/0 osmium tetroxide, dehydrated through an ethyl alcohol series, cleared in propylene oxide and embedded in Epon. Sections were cut at 1 pm in a Sorval MT2 microtome, mounted on glass slides and stained with 1 o/0 toluidine blue in 1 o/0 sodium borate for light microscopy. Thin sections (70-80 nm) were cut on an LKB Ultratome III microtome, stained with a saturated solution (2 %, aqueous) of uranyl acetate followed by Reynold’s lead citrate, and photographed in a Zeiss 9s electron microscope.
3. Results An electronmicrograph of a normal, l-day-old chick retina is presented in Fig. 1. The retina was relatively well-developed at the time of hatching. Both rod and cone photoreceptors had differentiated prehatching, and both outer and inner segments resembled the adult form, albeit somewhat smaller and more disoriented. Most cones contained an oil droplet whose contents had frequently leached out during the preparatory procedures. Five types of cones (three single cones and principal and
FIG. 3. Outer synaptic zone of a l-day-old rd retina. The chick retina possesses the typical synaptic ribbons (SR) and triad arrangement of most vertebrate retinas. (+cnerally rods have H shorter synaptic expanse and fewer synaptic ribbons than cows. C’S, (‘onp nuc~lt~us: ICY’. r~,tl r~uclrus: (‘I’. ,YUIP ~~dicle; OPL, outer plexiform layer. x 10200.
accessory members of the double cone) were identified and classified, based on position and density of the oil droplet (Morris, 1970 ; Meyer and May, 1973 ; Morris and Shorey, 1967). Not all cone types are evident in Fig. 1. The chicken rod inner segment did not contain an oil droplet but had a paraboloid body. The accessory member of the double cone contained a similar or identical organelle (Meyer and May, 1973 : Morris and Shorey, 1967). The retina of an rd chick on the day of hatching is shown in Fig. 2. This chick was one of those shown t#olack an.electroret’inogram (ERG)signal in response to light under both light and dark adapted states (Ulshafer et al., 1984). Despite the chick’s apparent lack of vision, retinal morphology was comparable to a normal chick of the same age (Fig. 1). Photoreceptor cells were present and no significant pathological changes were evident. It should be noted that both rd and normal retinas contained photoreceptors with disorganized, disoriented or partially developed outer segments at this time. Apical tips of some inner segments in rd retinas, however, appear to be somewhat more
ELECTRON
MICROSCOPY
OF
rd CHICK
RETISA
ns9
Fro. 4. Photoreceptors of l-week posthatch rd retina. Some outer segments appear disorganized (arrows). Outer segment discs are seen within the inner segment of at least one photoreceptor (*). Note granular. distended tips of many inner segments (arrowheads). x 4200.
distended than normals, although not apparent in Fig. 2. The retinal pigment epithelium (PE) and choriocapillaris were also virtually identical to that from a normal chick. Synapses between photoreceptors and interneurons were present in the outer plexiform layer (OPL) in rd chicks (Fig. 3) that resembled those of normal chicks. Synaptic ribbons, 50 nm diameter vesicles and typical triad postsynaptic elements were present in dimensions and numbers similar to normals. During the first week posthatching, however, some photoreceptors appeared to lose their morphological integrity (Fig. 4). Many outer segments were disorganized, disoriented and vesiculated. Some contained granular cytoplasm similar to that’ normally found in inner segments while, in others, stacks of OS discs were identified within the inner segment. Inner segments were frequently swollen, particularly at the apical tip proximal to the outer segment. These degenerative changes were more evident during the next week, and by the end of week 2 few normal appearing outer segments were present in the central retina
870
871
FIG.
6. For
legend see p 87’7
a72
R. .J. IrLSHAFEII
AND
(‘. 13. ALLEN
(Fig. 5). In addition to those changes described in the previous paragraphs, significant alterations in morphology of the synaptic elements of many photoreceptors had occurred. Whereas, in the normal g-week-old chick, synaptic pedicles and spherules were displaced somewhat from the photoreceptor nucleus by the inner connecting fiber (Fig. 6), the structures in the rd retina were frequently seen within the soma not far from the nucleus (Fig. 5). Other organelles, such as the oil droplet and paraboloid. were also sometimes in abnormal locations (Fig. 5). By 34 weeks posthatching, a distinct lesion was present in the central retina of all rd chicks. At the light and scanning microscopic levels, this was characterized by a retinal detachment, some debris in the subretinal space, and a reduction in the number of intact photoreceptor cells (Ulshafer and Allen, 1984; Ulshafer et al., 1984). These phenomena were also observed at the ultrastructural level (Fig. 7). The debris in the subretinal space appeared to be composed of large membraneous sacs, frequently emanating from, or associated with, inner segments. Tips of inner segments were grossly distended. Inner segments contained swollen mitochondria. Also observed were what appeared to be unattached outer segments and large whorls of outer segment-like material in the subretinal space. Intact photoreceptors, rods and cones, were also seen. By 2 months after hatching the lesion had spread to mid-periphery, although peripheral retina still appeared relatively normal (Ulshafer et al., 1984). Under the electron microscope, it was observed that few photoreceptors remained in the central retina. A high proportion of those remaining were double cones (Fig. 8), but very few outer segments were intact at this stage. The subretinal space was relatively free of debris at this time. In the ONL the majority of degenerated cells had been replaced by processes of Mueller cells. The terminal bar network of these cells, which comprises the outer limiting membrane of the neural retina, appeared very pronounced. Very few synaptic ribbons were noted at this time. By G8 months after hatching only a few sporadic photoreceptors remained (Fig. 9). Their inner segments were diminutive, compared to those from a normal chicken retina at this stage, but contained recognizable organelles : ellipsoid, rough and smooth endoplasmic reticulum, and frequently a lightly staining oil droplet. Many photoreceptor nuclei had become pleomorphic and lobulated. Synaptic ribbons were still occasionally seen associated with one or more processes of retinal interneurons. At this late stage of photoreceptor degeneration, changes were also noted in the PE : a general thinning of this cell layer occurred, with fewer numbers of melanosomes in many cells or in parts of a given cell. Some PE cells appeared to be hypertrophied, bulging into the subretinal space and containing many melanosomes. Cells of unknown origin were frequently seen among the inner segments and PE processes in the
FIG. 5. Low-power electron micrograph montage of outer retina of Z-week-old rd chick. Outer segments are few and largely disorganized. Oil droplets are not as regularly alligned as in normal retina and some (straight arrow) appear at abnormal locations as do some parabaloids (curved arrows). Inner connecting fibers (arrowheads) are very short. P = Principal and A = accessory member of double cones. x 2000. FIG. 6. Low-power electron micrograph montage of outer retina of %week-old normal chick. Xote well-developed and aligned outer segments and stratification of oil droplets (OD) in the various cone types. The inner segments of double cones (P = principal member, A = accessory member) are not separated by Mueller cell microvillae. Note the long inner connecting fiber (axon-like process, large arrow) of cones ending in a pedicle and the shorter connecting fiber of a rod (small arrow) which ends in a spherule. ONL, Outer nuclear layer: INL. inner nuclear layer; PE. pigment epithelium. x 1980.
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FIG. 7. Receptor layer of a 21-day-old rd chick. Note swollen granular inner segments (IS) of most photoreceptor cells. Although one outer segment (OS) appears relatively normal, a massive accumulation of disorganized outer segment membranes (*) is notable in the subretinal space. Many ellipsoids (E) have swollen mitochondria. In this specimen apical processes of PE appear retracted and swollen. x 5.580.
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Fra. 8. Outer retina of a 2-month-old rd chick. Further reduction in number of photoreceptor cells is evidc mced by increase in Mueller cell processes (MC), fewer nuclei in the ON, and the warty (wntinuous outer 11imiting membrane (OLM). A few synaptic ribbons are present (arrows). Remaining photoreceptors member. A = accessory member. The suhrrtinal space appear to be mostly double cones: P = principal x ~(KMI. is relat ively dehris-free.
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FIQ. 9. Outer retina of a B-month-old rd chick. A few remaining photoreceptor inner segments (IS) are seen in this section. The entire expanse of one cone can be identified from its pale staining oil droplet (OD) to its truncated connecting fiber. The nucleus (N) of this cone has become pleomorphic; a synaptic ribbon (curved arrow) is present in the pedicle which abuts on an interneuron (IN). Mueller cell (MC) processes largely fill up the outer nuclear layer; the terminal bar network of these cells forms the outer limiting membrane (OLM) of the neutral retina. The pigment epithelium (PE) has thinned considerably and reduction in number of melanin granules is especially evident in the cell to the right. Processes of cells (straight arrows) of unknown origin are present in the subretinal space. x 4666.
subretinal space. These cells usually contained debris and melanosome-like granules ; they resembled detached PE cells or macrophages originating in the vascular system. 4. Discussion As has been reported in other animal models of hereditary retinal degeneration, such as the rd mouse (Sanyal and Bal, 1973) Irish setter (Buyukmihci, Aguirre and Marshall, 1980) and collie (Santos-Anderson, Tso and Wolf, 1980), degenerative changes in photoreceptor cells in the rd chicken are first seen in the outer segments. However, an important difference between those models and the rd chick is that in
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the former group photoreceptors degenerate during the initial phases of different,iation. while in the latter the cells degenerate after differentiation, although before c~omplet~c maturity. In the case of late onset photoreceptor degeneration seen in the rniniat)urr poodle, the earliest morphological changes are also seen in t’he out’rr segments (Aguirre. Alligood, O’Brien and Buyukmihci, 1982). In the pcd mouse, another model of late onset photoreceptor degeneration, the first rhanges are seen in and around the inner segments (Blanks, Mullen and LaVail, 1983). In two ot’her animal models. the Ft(% rat and the DAM chicken. pathological rhanges in photoreceptors result from a degenerative event in overlying retinal PE (Fite, Montgomery, Whitney. Boissey and Smyth, 1983; Herron, 1977). In many of the animal models of retinal degeneration, as well as in cases of retinitis pigmentosa in man, cone photoreceptors tend to persist longer than rods. The ability to easily distinguish different photoreceptor types in the chicken has allowed identification of the surviving cells in the rd chicken as cones. At around 2 months posthatching, many of the remaining photoreceptor cells can be identified as double cones. Although no double cones, per se, could be distinguished at’ the later time points (G-8 months of age) in the rd chick, a distinctly pale staining oil droplet near t’he tip of the inner segment indicates that the residual cells are probably the principal members of the double cones (Meyer and May, 1973; Morris, 1970). Whether some cones partially or completely lack the genetic abnormality, are ‘immune’ to a toxic agent, or are generally more hardy than rods has yet to be discovered in this or other cases of hereditary retinal degeneration. It seems paradoxical that the newly hatched rd chick retina should appear to be similar to that of normal chicks at the ultrastructural level yet be unable to generate a measurable ERG. Indeed, it appears that the rd chick never had the ability to see, since at the time of hatching they neither find their food easily, peck at random targets (black dots), nor avoid threatening visual stimulae as do normal chicks. Degeneration of the photoreceptor cells therefore occurs well after (l-2 weeks) the chicks appear to be behaviorally blind. It is possible that they partially or completely lack the visual pigments, enzymes or carrier proteins of the vitamin A cycle necessary for normal vision. Alternatively, other enzymes or systems crucial in maintaining normal ionic environment, membrane permeability or synaptic transmission may be involved. Although significant’ amounts of membraneous debris are seen in the subretinal spare at the early stages of the disorder, the lack of such debris at lat’er stages implies that phagocytosis by PE is not abnormal in this model, unlike the RCS rat model where reduced phagocytosis of shed discs leads to a build-up of debris in the subretinal space and is somehow toxic to the photoreceptors (Herron, 1977). The presence of large amounts of granular cytoplasm in the distended apical tips of inner segments is unique to the rd chick. Possibly this represents an accumulation of phospholipid and/or protein molecules, normally destined to be incorporated into membraneous discs of the outer segment. Their membranes may stop being generated due to a defect in protein synthesis or in t’he membrane assembly mechanism. Autoradiographic st’udies on the fate of labeled protein precursors in rd and normal chicks are presently underway which should determine if this is the case. ACKNOWI,EDGMENTG This study was supported in part by a non-restricted to Prevent Blindness, Inc. and by NIH R01 EY04590
Departmental Grant from Research (R.J.U.). This work was also aided
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by Basil O’Connor Starter Research Grant Foundation. We thank MS Julie Driggers
OF No. S-438 for typing
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from the March the manuscript.
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REFERENCES Aguirre, G., Alligood, P., O’Brien, P. J. and Buyukmihci, N. (1982). Pathogenesis of progressive rod-cone degeneration in miniature poodles. InvecTt. Ophthalmol. Vis. I’%%. 23. 61&30. Blanks, J: C., Mullen, R. J. and LaVail, M. M. (1982). Retinal degeneration in the pcd cerebellar mutant mouse. II. Electron microscopic analysis. J. Camp. Nrurol. 212. 231-46. Buyukmihci, N.. Aguirre, G. and Marshall, J. (1980). Retinal degeneration in the dog. II. Development of the retina in rod-cone dysplasia. Exp. Eye Res. 30, 575-91. Cheng, K. M., Shoffer, R. N., Gelatt, K. N., Gum, G. G., Otis, J. S. and Bitgood, J. J. (1980). An autosomal recessive blind mutant in the chicken. Poultry Sci. 59, 2179-82. Fite. K. V., Montgomery, N., Whitney, T., Boissey, R. and Smyth, J. R., Jr (1983). Inherited retinal degeneration and ocular amelanosis in the domestic chicken (QaZZus domesticus). Curr. Eye Res. 2, 109-15. Herron, W. L., Jr (1977). The dystrophic rat as a model for clinical research. Bdv. Exp. Med. Biol. 77: 137-52. Meyer. D. B. and May, H. C., Jr (1973). The topographical distribution of rods and cones in the adult chicken retina. Exp. Eye Res. 17, 347-55. Morris, V. B. (1970). Symmetry in a receptor mosaic demonstrated in the chick from the frequencies, spacing and arrangement of the types of retinal receptor. J. Pomp. Newel.
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V. B. and Shorey, C. B. (1967). An electron microscope study of types of receptor in the chick retina. J. Comp. Neurol. 129, 313-40. Santos-Anderson, R. M., Tso, M. 0. M. and Wolf, E. D. (1980). An inherited retinopathy in collies: a light and electron microscopic study. Invest. Ophthalmol. Vis. Sci. 19. 1281-94. Sanyal, S. and Bal, A. K. (1973). Comparative light and electron microscopic study of retinal histogenesis in normal and rd mutant mice. S. nat. Entwicklungsgesch. 142, 21S38. Ulshafer, R. J. and Allen, C. B. (1984). Scanning electron microscopy of the retina in an animal model of hereditary blindness. Scanning Electron Microscopy 1984/11, 841-8. Ulshafer. R. J., Allen. C. B., Dawson, W. W. and Wolf, E. D. (1984). Hereditary retinal degeneration in the Rhode Island Red chicaken. I. Histology and ERG. Exp. Eye RPS 39, 125-35.