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(Investigative Ophthalmology and Visual Science. 2000;41:537-545.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

Progressive Optic Axon Dystrophy and Vascular Changes in rd Mice

Shaomei Wang1, Maria Paz Villegas–Pérez2, Manuel Vidal–Sanz2 and Raymond D. Lund1

1 From the Institute of Ophthalmology, University College London, United Kingdom; and the 2 Laboratorio de Oftalmología, Facultad de Medicina, Universidad de Murcia, Spain.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To examine how the vascular plexuses in the rd mouse retina are affected by the loss of photoreceptors and how this compares with the Royal College of Surgeons (RCS) rat. To examine whether the profound effects of vascular pathology on retinal ganglion cells (RGCs) and their axons seen in RCS rats are also found in rd mice.

METHODS. Vascular patterns were studied in flatmounted and sectioned retinas using either nicotinamide adenine dinucleotide phosphate(NADPH)-diaphorase histochemistry or vessel filling with horseradish peroxidase. Optic axons were visualized using RT97 (an antibody against the 200-kDa neurofilament subunit), and RGCs were labeled by retrograde transport of fluorescence label, the Fluorogold, applied to the superior colliculus.

RESULTS. The present study showed that in the rd mouse, similar to the RCS rat, vascular complexes developed in association with retinal pigment epithelial cells at the outer border of the retina. The number and distribution of complexes were very different from the rat, but as in the rat, progressive axonal dystrophy was seen in the optic fiber layer. RGC loss, rather than being local was more broadly distributed, but some, at least, appeared to be secondary to axonal dystrophy caused by vessels supplying vascular formation.

CONCLUSIONS. Photoreceptor loss in the rd mouse leads to RGC axonal dystrophy and loss. The lesser degree and different distribution of RGC loss caused by abnormal vasculature associated with vascular formations in the outer retina in the rd mouse may be due to the early atrophy of the deep vascular plexus in this animal.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
There is a close relationship between neural activity and levels of vascular perfusion. In the retina, photoreceptor death is accompanied by a major reduction in the vascular plexuses. This has been documented for the Royal College of Surgeons (RCS) rat as well as for the rd mouse.1 2 3 4 5 6 7 8 In addition to the diminution of vascular plexus, there is condensation of vessels associated with the invasion of retinal pigment epithelial (RPE) cells into the retina. We have studied this process in the RCS rat and found that one consequence of the formation of these RPE–vascular complexes is that the vessels supplying them come under traction and first deform and then later ligate the bundles of optic axons that they cross. The result is loss of retinal ganglion cells (RGCs).9 10 This is a progressive process beginning at approximately 6 months of age. Although no direct homology with a known human disease has yet been defined, the RCS rat may serve as a model for assessing treatments that may be of value in age-related macular degeneration.11 12

However, the rd mouse has direct homology with a form of retinitis pigmentosa (RP).13 In rd mouse, unlike the RCS rat, the primary defect lies in the photoreceptors themselves, rather than in the RPE cell layer.5 14 15 We examined the consequences of photoreceptor loss in this rodent and found not only a thinning of the vascular plexus and development of vascular formations, but also a loss of RGCs. A more detailed examination of this animal is prompted by several observations of RGC loss over time in patients with RP.16 17 18 19 There is also evidence in RP of thinning of the vascular plexus and development of aberrant vascular formations.20 21 22 These similarities suggested that a more detailed study of the changes in the rd mouse would usefully contribute to an understanding of the changes seen in RP. For example, it has been unclear in the rd mouse whether RGC loss is due to transneuronal atrophy, to the type of vascular changes seen in the RCS rat, or to more general vascular remodeling and loss. Such work also provides a foundation for transplantation studies, because it is not known whether the altered vascular network can accommodate to the increased metabolic demands of a newly introduced photoreceptor layer.

In this study, we used the nicotinamide adenine dinucleotide phosphate(NADPH)-diaphorase reaction in parallel with horseradish peroxidase (HRP) labeling to visualize blood vessels. The NADPH-diaphorase method has the advantage of outlining the whole vascular tree (providing endothelial cells survive), and arteries and veins are differentially stained.23 24 Optic axons were visualized using RT97, and RGCs were labeled by retrograde transport of a fluorescence label (Fluorogold; Fluorochrome, Englewood, CO) applied to the superior colliculus (SC).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The present investigation was performed in mice of both sexes of the C57BL/6J strain, homozygous for the rd allele (rd/rd), with ages ranging from 3 weeks to 13 months. All animals were housed and cared for in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research guidelines and Home Office (UK) regulations for the care and use of laboratory animals, and the U. K. Animals (Scientific Procedures) Act (1986).

Wholemounted Retinas
Twenty-five mutant rd mice were studied, ranging from 3 weeks to 13 months of age. Ten nondystrophic rd mice aged between 1 and 8 months were examined as control subjects. All animals were anesthetized with a lethal dose of sodium pentabarbitone (Euthatal; Rhône Mérieux, Harlow, UK) and perfused intracardially with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde fixative in PBS. The dorsal pole of each eye was marked with a suture before enucleation, and the tissue was postfixed in the same fixative for 30 minutes before transferring to PBS for dissection. Wholemounts of the retinas were prepared as described previously.9 The retinas were postfixed for 1 hour in the same fixative, washed, and preincubated for 1 hour in a solution containing 3% Triton X-100 (Merck, Poole, UK) and 1% bovine serum albumen (Sigma, Poole, UK) in PBS. The retinas were then incubated overnight in monoclonal 200-kDa neurofilament protein antibody (RT97; 1:1000, the generous gift of Roger Morris, Guy’s Hospital, London, UK) at 4°C. After washing in PBS, retinas were incubated in a solution containing Triton X-100 and bovine serum albumin as above plus 1:50 fluorescein-isothiocyanate–conjugated goat anti-mouse IgG (Sigma) for 1 hour. Lastly, the retinas were rinsed with PBS before transferring to an incubation medium for NADPH-diaphorase staining modified from Takemura et al.25 The incubation medium contained 3% Triton X-100, 0.02% NADPH-diaphorase (Sigma), and 0.04% nitroblue tetrazolium (Sigma) in PBS and before use was mixed until it turned purple. The retinas were agitated in this solution for 90 minutes at 37°C before finally mounting in glycerol on subbed (gelatin-alum) slides.

Sectioned Material
Twelve mutant and control mice were used in this part of the study, with ages ranging from 3 weeks to 13 months. Animals were perfused as described. The eyes were removed and postfixed in the same fixative for 1 hour before transferring to PBS for 30 minutes. After dehydration through a graded series of alcohol to 95%, the eyes were processed through mixtures of 95% alcohol and polyester wax26 (Merck) to pure polyester wax. Sections were cut at 8-µm thickness and mounted onto gelatin-alum subbed slides. Sections were dewaxed in 95% alcohol and dried in a cool current of air for 1 hour before immersing in 5% defatted milk in PBS for 30 minutes. Finally, sections were processed for RT97 immunocytochemistry (as described earlier) and counterstained with thionine.

HRP Labeling
Two dystrophic 8-month-old and two nondystrophic 5-month-old mice received injections of 10% type I HRP (1 ml; Sigma) in saline in the femoral vein. Fifteen minutes later, the animals were given a lethal dose of sodium pentabarbitone. The retinas were dissected as wholemounts as before, postfixed, and processed for HRP histochemistry using a modified Hanker–Yates reaction27 to visualize the retinal vasculature (for details see Reference 9). Retinae were subsequently incubated for RT97 immunohistochemistry as before.

Fluorescence Labeling
Five dystrophic mice, 11 months (n = 2) and 13 months (n = 3) old, and three nondystrophic 8-month-old animals were used for fluorescence labeling of RGCs, using previously described methods.9 28 Briefly, the animals were anesthetized, both SCs were exposed, the pia overlying the SC was removed, and a piece of gelfoam soaked in a solution of 2% Fluorogold and 10% dimethylsulphoxide in saline was placed on top of the SC, and the skin was sutured. Seven days later, the animals were perfused first with PBS and then with 4% paraformaldehyde in 0.1 M phosphate buffer. The eyes were enucleated, and the retinas were dissected and postfixed as described before, washed, and mounted on gelatinized slides with a mounting medium for fluorescence. Retinae were observed by fluorescence microscopy and 12 standard rectangular areas of each retina were photographed. Each measured 0.565 x 0.365 mm2, and three such regions were examined in each quadrant (superotemporal, superonasal, inferotemporal, and inferonasal) at 0.5, 1.25, and 2 mm, respectively, from the optic disc. Three retinas (two from dystrophic and one from nondystrophic animals) were not photographed because of poor visualization of the labeling. Fluorogold-labeled cells in the photographs were counted and pooled to obtain the density of labeled cells per square millimeter in each retina. The mean densities of fluorescence-labeled cells in dystrophic and nondystrophic animals were compared using the Kruskal–Wallis test.29


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Nondystrophic Mouse Retina
Previous studies1 have indicated the presence of three vascular plexuses at different depths within the mouse retina. How they relate to the overall retinal circulation has not been described in detail. In the present specimens stained with NADPH-diaphorase, elucidation of the laminar distribution of plexuses in flatmounted specimens is helped by the fact that a subclass of amacrine cell is also stained. These cells have bodies lying on the deep border of the inner nuclear layer (see Fig. 2B ) and processes distributing in the inner plexiform layer. The NADPH-diaphorase method has the added advantage that arteries tend to stain somewhat darker than veins, although this distinction may be less clear than in rats.



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  		Figure 2. NADPH-diaphorase–stained flatmount of a normal retina focused on the superficial (A) and deep (B) vascular plexuses. Note that the radial artery (a) and vein (v) are differentially stained. In (B), a deep drainage venule (dv) can be seen associated with small vessels of the deep plexus. Some amacrine cells are NADPH-diaphorase positive (arrows). Scale bar, 50 µm.

 
There are usually five to six radial arteries with robust branches that supply the vascular input to the superficial plexus (Figs. 1A , 2 A) This plexus is formed of terminal branches of the arterioles. It distributes deep in the retinal layers to supply the intermediate and deep plexuses (Fig. 2B) . In addition, vessels of the superficial plexus drain directly into radial veins through very fine lateral branches. For the most part such arteriovenous connections are indirect. The radial veins are interposed between the radial arteries. In most cases when one of the dorsally disposed radial veins and one ventral vein reach the edge of the retina they continue for some distance as circumferential vessels running around the retinal border and supplying tributaries back into the retina (Fig. 1A) . Apart from the fine branches entering the radial veins from the superficial plexus, coarser lateral branches are periodically encountered that run through the depth of the retina from the deep plexus (Figs. 1A 2B) .



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  		Figure 1. Flatmount of retina from nondystrophic and dystrophic mice stained with HRP after vascular perfusion to show the overall vascular patterns. (A) Five-month normal retina; (B) 8-month dystrophic retina. In (B), the reduced vascular plexus and a large area of vascular leakage radiating from the region around the optic nerve head can be seen. Scale bar, 500 µm.

 
The intermediate plexus is a loose reticulum located at the border of the nuclear layer and inner plexiform layers at the level of the NADPH-positive amacrine cells. It is symmetrically disposed and for the most part appears to connect predominately with the two adjacent plexuses.

The deep plexus lying within the outer plexiform layer is a complex reticulum and is dominated by deep drainage venules that run through the retina to the radial veins (Fig. 2B) . These venules frequently run at right angles to the radial veins, but occasionally a radial vein will itself run through the retina to become a deep drainage vessel.

In summary, the superficial plexus is mainly an arterial-input plexus, and the deep plexus is associated with the venous output. The intermediate plexus connects both superficial and deep plexuses.

Dystrophic rd Mouse
We studied the changes seen from 3 weeks to 13 months from the time immediately after substantial loss of rods to a point at which there is advanced cone loss throughout the retina.

At 3 weeks and 1 month of age, each of the plexuses was still clearly evident (Figs. 3A 3B ), but both deep drainage venules and the more peripheral parts of the radial veins were clearly dilated along their course (Fig. 3B) . In some cases, the retina appeared little different from normal. In other instances, sometimes even in the opposite eye of the same animal, the plexuses (especially the deep plexus) were severely reduced and the venules either dilated or collapsed. Cross sections showed the loss of rod photoreceptors at 1 month (Fig. 7A) .



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  		Figure 3. Figure 3.

NADPH-diaphorase–stained flatmounts of dystrophic retinas. (A, B) Retina of a 1-month-old rd mouse at the levels of the superficial (A) and deep (B) plexuses. At this age, each plexus is well developed. (C, D) Dystrophic retina at 3 months of age at the levels of the superficial (C) and deep (D) plexuses. Note the substantial thinning of each plexus, especially the deep plexus, where the drainage venules (dv) are the most prominent feature. Scale bar, 50 µm.

 


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  		Figure 7. Cross sections of dystrophic retinas at 1 month (A) and 11 months (B), stained with cresyl violet and RT97. Note the outer nuclear layer had been reduced to a single layer of cones (arrows) at 1 month, but by 11 months there was major disruption of lamination, associated with a vascular complex (vc). An obliquely running vessel can be seen supplying the complex (vc), and axon bundles (ax) can be seen entering the retina. Scale bars, 120 µm.

 
By 3 months of age, the deep plexus showed a consistent and substantial loss of fine capillaries, and as a result, the drainage venules appeared more prominent (Fig. 3D) . Many venules were clearly dilated along their whole course. They followed a straighter course than normal, and at their entry point into the radial vein, they often appeared collapsed.

Over the succeeding months, the branches from these venules became progressively shorter, until by 7 months of age, the vessels of the deep plexus were largely lost. The intermediate plexus similarly became diminished over time, was substantially reduced by 7 months, and had largely disappeared by 13 months. The superficial plexus, by contrast, was evident throughout the time series studied (Figs. 3A 3C 4D 6A ) and was still present at 13 months, although both it and the radial vessels of the inner retina showed significant changes over this period. The most prominent change was that some of the radial veins showed evidence of collapse and apparent twisting one third to one half the distance out from the disc (Fig. 6A) . In some cases, but not invariably, this corresponded to the point at which a radial vein runs deep into the retina. With time, the more distal part of such veins was completely lost or distributes into a number of small branches (Fig. 6A) . This was also found in those veins, the terminal branches of which overlapped with branches from the circumferential vessels. No shortening of the radial arteries was seen, even at advanced ages. The superficial plexus became reduced with time; one consequence of this was that the connections between radial arteries and veins became more obvious, and direct arteriovenous connections could be seen (Fig. 6B) . At intermediate time points, vessel loops were seen that were not present in normal animals, suggesting either substantial resculpting or new vessel formation. Such vessels were most evident in older animal (Figs. 5B 5D 6A ).



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  		Figure 4. (A, B) Retina of a 7-month-old dystrophic mouse in dark field reacted with RT97 (A) and in bright field with NADPH-diaphorase (B). In (A), note several points of deviation of nerve bundles where vessels cross them (arrows). In (B), the limited vasculature of the superficial plexus is seen together with vascular–pigment complexes (arrows) associated with the vessels that distort axon bundles. (C, D) Nine-month-old dystrophic retina at the level of the superficial (C) and deep (D) vascular plexuses. In (C), reacted for RT97, axonal distortion can be seen (arrow). In (D), the vessel associated with that distortion can be seen supplying a pigment-invested vascular complex. Note that some vessels (see arrowhead) seem to be pulled toward the vascular complex. Scale bar, 50 µm.

 


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  		Figure 6. Views of two retinas aged 10 (A) and 9 (B) months, showing a shortened radial vein (arrow, A), associated with complex formations of the superficial plexus, and large numbers of direct arteriovenous anastomoses (B). Scale bars, 50 µm.

 


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  		Figure 5. Views of abnormalities in the optic fiber layer of dystrophic mice. (A, C) Nine- and 13-month-old animals, respectively, showed disordered axon disposition, increased intensity of RT97 staining of compromised ganglion cells and their axons, and abortive sprouting (C, arrow) associated with vessel crossings (arrows). (B, D) Eight- and 10-month-old mice, respectively, showed vascular complexes in HRP- (B) and NADPH-stained sections (D). In (D), the lighter vascular staining allowed visualization of pigmented cells. Scale bar, 50 µm.

 
From 6 months onward, small foci were seen within the outer retina composed of RPE cells and a local vascular complex. Sometimes the pigmented cells were difficult to visualize in these foci, because RPE cells in rd mice generally had less pigment than those in control animals. These foci were broadly distributed across the retina but were more common in an intermediate position halfway out to the periphery of the retina (Figs. 4D 5D 6A) . At 7 months there were approximately 30 (mean, 29.9 ± 4.3; n = 6) per retina; by 8 to 9 months there were approximately 50 (46 ± 8.2; n = 6). There was a tendency toward fewer vascular complexes in the superotemporal retina.

The RPE cells were usually associated with vessels arising from either the intermediate plexus or, more commonly, the superficial plexus (Figs. 4D 6A) . Some were directly associated with radial veins (Figs. 5D 6A) , and frequently at such points, the vein formed a loop running deep into the retina and back to the surface, or it ended, continuing on as a number of very small vessels (Fig. 6A) . With time these formations became more frequent, and some had increased complexity. The vessels supplying them usually ran perpendicularly through the retina. Occasionally, obliquely disposed vessels were seen (Fig. 7B ). Some of these followed a course toward an area of pigmented cells and appeared extremely thin (Figs. 4B 4D 5A 5C) , often to a point beyond resolution. These were most commonly arteriolar branches and at the points where they were thinnest, they could usually be seen disrupting optic axon bundle patterns (see following description and Figs. 4A 4C ). The parent vessels of these smaller branches sometimes deviated at the point of origin of the branch.

Up to 5 months of age, the optic axon bundles coursed across the retina in an orderly fashion. By 6 months, the first indications of axon disruption could be seen. Axon bundle alterations correlating in location with the vascular formations were found throughout the retina with no preferential distribution pattern. Several events were evident. First, the course of bundles deviated laterally (Figs. 4A 4C) . Second, axon bundles could sometimes be seen entering the retina; this was best seen in sectioned tissue (Fig. 7B) . Third, axons were disrupted, and swollen endings of blocked axons (retraction bulbs) were seen (Fig. 5A) . Some individual axons were stained extremely heavily at either side of an interruption point. Fourth, individual axons were seen coursing over the retina in a disorganized fashion suggesting abortive sprouting (Fig. 5A) . Fifth, swollen ganglion cells were also seen from 8 months onward (Figs. 5A 5C) . Sixth, some thinning of the fiber bundles was also seen with time, both fewer axons per bundle and fewer bundles. In most cases the point at which the axon bundle was disrupted corresponded to a crossing point of an arterial branch as it ran deep to supply a vascular complex in the outer retina (Figs. 7B) .

In the animals whose RGCs had been labeled with Fluorogold from the SC, fluorescence-labeled RGCs were found evenly distributed throughout the retina. We never found sectors devoid of RGCs as in RCS rats, although we periodically observed local patches devoid of RGCs (Fig. 8B ). These were situated in the midperiphery, associated with vessels that formed loops into the retina and seemed to be formed by lateral displacement of RGCs by tractional loops.



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  		Figure 8. Retrograde fluorescence labeling of RGCs in nondystrophic (A) and dystrophic (B) retinas allowed determination of RGC density. The densities were significantly higher in nondystrophic than in dystrophic retina (Kruskal–Wallis; P < 0.04). Scale bar, 500 µm.

 
Mean densities of fluorescence-labeled RGCs from the SC were 3149 ± 497 cells/mm2 in nondystrophic mice (n = 5; Fig. 8A ) and 2458 ± 314 cells/mm2 in dystrophic mice (n = 6; Fig. 8B ). Thus, the overall densities of RGCs were lower in the dystrophic animals, and the Kruskal–Wallis test showed the difference between the two groups of mice to be statistically significant (P < 0.04).

In the four retinas taken from the 8-month-old dystrophic mice injected with HRP, leakage was observed in different regions of the retina (Fig. 1B) . A large area, accounting for one third to one half of the central retina, showed leakage of HRP in the outer layers of the retina. Also HRP leakage was observed around the foci of vascular–RPE formations in the outer layers of the retina (Fig. 5B) .


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study on the rd mouse, there was as in the RCS rat evidence of progressive axonal dystrophy in the optic fiber layer and RGC loss. In both species, the axonal dystrophy is associated with vessels that supply vascular formations at the interface of the retina and RPE cell layer. There are, however, considerable differences in the way the vascular disorders develop and in the pattern of RGC loss in the two animal models.

In the pigmented RCS rat, photoreceptor loss occurs secondary to a buildup of debris in the subretinal space.14 30 31 Rods die over a period of several months in a gradient running from center to periphery and ventral to dorsal.32 33 During this period, there is also a progressive increase in threshold sensitivities across the visual field recording from the SC and responses to focal stimulation are largely lost by 6 months34 (Sauvé Yves, unpublished data, May 1999). Although it is very difficult to identify photoreceptors at advanced ages, visual reflexes can still be elicited, even at 1 year or more of age,35 and these are presumed to be attributable to the few remaining cones. The early development of vascular network of the retina is quite normal, and it is only at approximately 3 months of age that the first sign of loss of the deep vascular plexus is seen. The initial changes are the most evident around sites where RPE cells migrate among the vessels of the deep capillary plexus, creating local complex vascular formations surrounded by areas devoid of plexus.2 3 11 36 37 38 These vascular complexes are especially prevalent in the ventral retina close to the optic disc.9 The vessels from the inner retina cross nerve bundles before running deep to supply complexes, and as they run deep, they initially distort the bundles and later pull them into the retina and ligate them. This is particularly evident close to the ventral disc. As a result, axons supplying a whole wedge of retina are affected, and that leads to loss of RGCs from that wedge. Interestingly, a count made outside these wedges showed normal RGC numbers, which argues against the presence of general dystrophy.

In the rd mouse, rod photoreceptor loss is much more rapid, being largely complete by 3 weeks.39 40 Accordingly, an ordered map of the visual field on the SC is rapidly lost41 (Sauvé unpublished data). In contrast to the rat, a distinct single layer of cone cell bodies remains over much of the outer retina, although this gradually disappears with time. It is reported that vascular complexes develop at points where the cone layer is deficient.42 Unlike the rat, there is no concentration of vascular complexes immediately ventral to the optic disc, and the overall numbers of foci are considerably less. Instead they are more broadly dispersed over the retina, with the exception of the upper temporal retina, where they are less prevalent. This differs from a recent report on sectioned specimens where complexes were absent from the whole temporal retina.43 However, we have found differences in the progress of change among animals and even between the two eyes of the same mouse. It is likely also that slight differences among strains, as well as epigenetic factors, may cause variance in the development of the vascular anomalies.

Another feature recently reported in rd mouse retinas44 and seen also in this study was a loss of RPE cells from Bruch’s membrane over local areas of retina. How this may relate to the migration of RPE cells into the retina or the development of vascular complexes is not clear.

The first vascular complexes are found, as in the rat, in close relation to RPE cells, although this is generally harder to see, because in the rd mouse, these cells have much less pigment. In contrast to the rat, the first complexes are seen relatively late, at approximately 6 months. This may be because of the continued presence of a coherent cone layer, which would delay the development of a close association between RPE cells and vessels of the deep plexus. This late onset is in contrast to the overall changes in retinal vasculature. By the time vascular foci are first seen, the deep plexus has largely disappeared, and only the deep drainage venules remain, with short stubby branches. This is in contrast to the RCS rat, in which the formation of the vascular complexes precedes loss of the deep plexus and seems to precipitate its loss. This difference in timing is also likely to affect the configuration of the vessels contributing to the complexes. They more commonly originate from vessels of the intermediate and superficial plexus, running perpendicularly rather than obliquely through the retina. Deep drainage venules with clusters of pigmented cells and local dilatations (seen in the RCS rat) are much less common in the rd mouse.

Despite a somewhat different pattern of development of the vascular foci in rat and mouse, the vessels serving them are involved in the disruption of optic axons and loss of ganglion cells. The distribution of axonal disruption correlates closely with the distribution of vascular foci and is particularly prevalent close to the optic disc in rats but more widely distributed in mice. In contrast to RCS rats, which show sector loss of RGCs, but normal RGC densities outside the sectors, rd mice show only local loss of RGCs correlated with vessel traction, but reduced numbers of RGCs over the whole retina.

The possibility of ganglion cell loss occurring in rd mice was previously raised in a study showing loss of cells in the ganglion cell layer in older mice.45 In this case it was attributed to transneuronal atrophy associated with the very early developmental loss of photoreceptors in the first 3 weeks of life. The results from the present study, when compared with those in our previous studies on RCS rats, suggest again that some cell death can be correlated with axonal dystrophy and retrograde degeneration caused by vascular changes. However, because of the broader distribution of cell loss, it is not possible to exclude a transneuronal effect, although the late onset and slow progress argue against this. Another possibility is that the substantial overall reduction in the vascular plexus may be insufficient to support all the remaining cells of the retina, leading to their degeneration. Previous works in albino RCS rat has noted that there is loss of inner nuclear layer cells,46 but whether this is due to light damage effects or to the RCS mutation alone is not clear. In further studies (Shaomel Wang, unpublished data, July 1997), we have found that with time there is reduction in the number of NADPH-diaphorase positive amacrine cells in rd mice suggesting that there may indeed be generalized neuronal cell loss.

This poses the question of which mechanism may play a role in the loss of RGCs over time in patients with RP17 18 19 47 and which animal serves as a better model. Certainly, the RCS rat shows a histopathology that is very similar to RP including the loss of photoreceptors, invasion of RPE cells into the inner retina, narrowing of blood vessels,21 the presence of vascular formations,22 leaky blood vessels,20 and progressive loss of retinal ganglion cells.

The rd mouse, although homologous to a form of RP, differs in the very early loss of rods and of the deep vascular plexus. Although neither transneuronal atrophy nor generalized vascular loss appears to play a role in RGC loss in RCS rats, these cannot be completely ruled out in rd mice, although the late onset, as in humans, makes transneuronal atrophy unlikely. It appears therefore that both focal and general RGC loss secondary to vascular events should be considered to be complications occurring after the photoreceptor loss that occurs in RP.

The vascular changes have repercussions for repair strategies in the rd mouse. For example, the rapid loss of the deep vascular plexus in the rd mouse presents a potential problem for transplantation of new photoreceptors. For such transplants to be stable and functional, the deep plexus may have to be re-established. Two studies have indicated that such transplants deteriorate with time,48 49 and the loss of the plexus clearly may contribute to this. Furthermore, efficacy of transplantation at too late a stage–in the rd mouse after 7 months of age–could be further compromised by ganglion cell loss.


   Acknowledgements
 
The authors thank J. Lawrence for comments and help regarding this study; Toby Holmes for expertise in producing wholemount pictures; Anthony S. L. Kwan for help and advice; Niyi Ademuso for technical assistance; and the Department of Psychology, University of Sheffield for providing some of the animals.


   Footnotes
 
Supported by Grants MRC, EU CT 96-0976, FIS 98/0341, FIS 99/1090, and Seneca PB 18 FS97 from Foundation Fighting Blindness, Action Research.

Submitted for publication June 15, 1999; revised September 21, 1999; accepted October 5, 1999.

Commercial relationships policy: N.

Corresponding author: Raymond Lund, Department of Pathology, Institute of Ophthalmology, University College London, Bath Street, London, EC1V 9EL, UK. r.lund{at}ucl.ac.uk


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Blanks, JC, Johnson, LV (1986) Vascular atrophy in the retinal degenerative rd mouse J Comp Neurol 254,543-553[Medline][Order article via Infotrieve]
  2. Caldwell, RB, Roque, RS, Solomon, SW (1989) Increase vascular density and vitreo-retinal membranes accompany vascularization of the pigment epithelium in the dystrophic rat retina Curr Eye Res 8,923-937[Medline][Order article via Infotrieve]
  3. El-Hifnawi, E. (1987) Pathomorphology of the retina and its vasculature in hereditary retinal dystrophy in RCS rats Zrenner, E Krastel, H Goebel, HH eds. Research in Retinitis Pigmentosa Advances in the Biosciences Vol 62,417-434 Pergamon Oxford, UK.
  4. Fitzgerald, ME, Slapnick, SM, Caldwell, RB (1989) Alterations in lectin binding accompany increase permeability in the dystrophic rat model for proliferative retinopathy Prog Clin Biol Res 314,409-425[Medline][Order article via Infotrieve]
  5. Matthes, MT, Bok, D. (1984) Blood vascular abnormalities in the degenerative mouse retina (C57BL/6J-rd le) Invest Ophthalmol Vis Sci 25,364-369[Abstract/Free Full Text]
  6. Tobe, T, Okamoto, N, Vinores, MA, et al (1998) Evolution of neovascularization in mice with overexpression of vascular endothelial growth factor in photoreceptors Invest Ophthalmol Vis Sci 39,180-188[Abstract/Free Full Text]
  7. Van Driel, D, Provis, J, Billson, FA (1988) Morphology of intraretinal new vessels in the PETH rat Graefes Arch Clin Exp Ophthalmol 226,576-582[Medline][Order article via Infotrieve]
  8. Wecker, JR, Ison, JR (1986) Visual function measured by reflex modification in rats with inherited retinal dystrophy Behav Neurosci 100,679-684[Medline][Order article via Infotrieve]
  9. Villegas–Pérez, MP, Lawrence, JM, Vidal–Sanz, M, et al (1998) Ganglion cell loss in RCS rat retina: a result of compression of axons by contracting intraretinal vessels linked to the pigment epithelium J Comp Neurol 392,58-77[Medline][Order article via Infotrieve]
  10. Villegas-Pérez, MP, Vidal-Sanz, M, Lund, RD (1996) Mechanism of retinal ganglion cell loss in inherited retinal dystrophy NeuroReport 7,1995-1999[Medline][Order article via Infotrieve]
  11. Herron, JWL, Riegel, BW, Brennan, E, et al (1974) Retinal dystrophy in the pigmented rat Invest Ophthalmol Vis Sci 13,87-94[Abstract/Free Full Text]
  12. Herron, JWL, Jr (1977) The dystrophic rat as a model for clinical research Lauders, MB Wolbarsht, ML Dowling, JE Laties, AM eds. Retinitis Pigmentosa. Clinical Implications of Current Research ,137-152 Plenum Press New York.
  13. Farber, DB, Flannery, JG, Bowesrickman, C. (1994) The rd mouse story: Seventy years of research on an animal model of inherited retinal degeneration Prog Retinal Eye Res 13,31-64
  14. LaVail, MM, Sidman, RL, O’Neil, D. (1972) Photoreceptor-pigment epithelial cell relationships in rats with inherited retinal dystrophy J Cell Biol 53,185-209[Abstract/Free Full Text]
  15. LaVail, MM, Sidman, RL (1974) C57BL/6J Mice with inherited retinal degeneration Arch Ophthalmol 91,394-400[Medline][Order article via Infotrieve]
  16. Li, Z-Y, Possin, DE, Milam, AH (1995) Histopathology of bone spicule pigmentation in retinitis pigmentosa Ophthalmology 102,805-817[Medline][Order article via Infotrieve]
  17. Newman, NM, Stevens, RA, Heckenlively, JR (1987) Nerve fibre layer loss in diseases of the outer retinal layer Br J Ophthalmol 71,21-26[Abstract/Free Full Text]
  18. Santos, A, Humayun, MS, de Juan, E, Jr, et al (1997) Preservation of the inner retina in retinitis pigmentosa: a morphometric analysis Arch Ophthalmol 117,511-515
  19. Stone, JL, Barlow, WE, Humayun, MS, et al (1992) Morphometric analysis of macular photoreceptors and ganglion cells in retinas with retinitis pigmentosa Arch Ophthalmol 110,1634-1639[Abstract]
  20. Spalton, DJ, Bird, AC, Cleary, PE (1978) Retinitis pigmentosa and retinal oedema Br J Ophthalmol 62,174-182[Abstract/Free Full Text]
  21. Milam, AH, Li, ZY (1995) Retinal pathology in retinitis pigmentosa: Considerations for therapy Anderson, RE LaVail, MM Hollyfield, JG eds. Degenerative Diseases of the Retina ,275-284 Plenum Press New York.
  22. Uliss, AE, Gregor, ZJ, Bird, AC (1986) Retinitis pigmentosa and retinal neovascularization Ophthalmology 93,1599-1602[Medline][Order article via Infotrieve]
  23. Chakravarthy, U, Stitt, AW, McNally, J, et al (1995) Nitric-oxide synthase activity and expression in retinal capillary Curr Eye Res 14,285-294[Medline][Order article via Infotrieve]
  24. Provis, JM, Leech, J, Diaz, C, et al (1997) Development of the human retinal vasculature: cellular relations and VEGF expression Exp Eye Res 65,555-568[Medline][Order article via Infotrieve]
  25. Takemura, M, Wakisaka, S, Iwase, K, et al (1996) NADPH-diaphorase in the developing rat: lower brainstem and cervical spinal cord, with special reference to the trigemino-solitary complex J Comp Neurol 365,511-525[Medline][Order article via Infotrieve]
  26. Steedman, HF (1957) Polyester wax: new ribboning embedding medium for histology Nature 179,1345-1345
  27. Linden, R, Perry, VH (1983) Massive retinotectal projection in rats Brain Res 272,145-149[Medline][Order article via Infotrieve]
  28. Peinado–Ramon, P, Salvador, M, Villegas–Pérez, MP, et al (1996) Effects of axotomy and intraocular administration of NT-4, NT-3, and brain-derived neurotrophic factor on the survival of adult rat retinal ganglion cells: a quantitative in vivo study Invest Ophthalmol Vis Sci 37,489-500[Abstract/Free Full Text]
  29. Kruskal, WH, Wallis, WA (1952) Use of ranks in one-criterion variance analysis J Am Stat Assoc 47,583-621
  30. Bok, D, Hall, MO (1971) The role of the pigment epithelium in the etiology of inherited retinal dystrophy in the rat J Cell Biol 49,664-682[Abstract/Free Full Text]
  31. Mullen, RJ, LaVail, MM (1976) Inherited retinal dystrophy: primary defect in pigment epithelium determined with experimental rat chimeras Science 192,799-801[Abstract/Free Full Text]
  32. Dowling, JE, Sidman, RL (1962) Inherited retinal dystrophy in the rat J Cell Biol 14,73-107[Abstract/Free Full Text]
  33. LaVail, MM (1975) Influence of eye pigmentation and light deprivation on inherited retinal dystrophy in the rat Exp Eye Res 21,167-192[Medline][Order article via Infotrieve]
  34. Sauvé, Y, Klassen, H, Whiteley, SJO, et al (1996) Retinotopic analysis of response parameters in the superior colliculus of RCS rats following retinal pigment cell transplants Soc Neurosci Abstr 22,1978
  35. Whiteley, SJO, Young, MJ, Litchfield, TM (1994) Changes in pupillary light reflex with age in RCS rats and the effects of intraretinal transplantation of retinal pigment epithelium. (ARVO Abstract) Invest Ophthalmol Vis Sci 35(4),1524Abstract nr 1254
  36. Gerstein, DD, Dantzker, DR (1969) Retinal vascular changes in hereditary visual cell degeneration Arch Ophthalmol 81,99-105[Medline][Order article via Infotrieve]
  37. Matthes, MT, Bok, D. (1985) Blood vascular abnormalities in animals with inherited retinal degeneration LaVail, MM Hollyfield, JG Anderson, RE eds. Retinal Degeneration: Experimental and Clinical Studies ,209-237 Alan R. Liss New York.
  38. Weber, ML, Mancini, MA, Frank, RN (1989) Retinovitreal neovascularization in the Royal College of Surgeons rat Curr Eye Res 8,61-74[Medline][Order article via Infotrieve]
  39. LaVail, MM (1979) The retinal pigment epithelium in mice and rats with inherited retinal degeneration Zinn, KM Marmor, MF eds. The Retinal Pigment Epithelium ,357-389 Harvard University Press Cambridge, MA.
  40. Sidman, R, Green, MC (1965) Retinal degeneration in the mouse J Hered 56,23-29[Free Full Text]
  41. Drager, UC, Hubel, DH (1978) Studies of visual function and its decay in mice with hereditary retinal degeneration J Comp Neurol 180,85-114[Medline][Order article via Infotrieve]
  42. Oglivie, JM, Tenkova, T, Lett, JM, Speck, J, Landgraf, M, Silverman, MS (1997) Age-related distribution of cones and ON-bipolar cells in the rd mouse retina Curr Eye Res 16,244-251[Medline][Order article via Infotrieve]
  43. Nishikawa, S, LaVail, MM (1998) Neovascularization of the RPE: temporal differences in mice with rod photoreceptor gene defects Exp Eye Res 67,509-515[Medline][Order article via Infotrieve]
  44. Neuhardt, T, May, CA, Wilsch, C, et al (1999) Morphological changes of retinal pigment epithelium and choroid in rd-mice Exp Eye Res 68,75-83[Medline][Order article via Infotrieve]
  45. Grafstein, B, Murray, M, Ingoglia, N. (1972) Protein synthesis and axonal transport in retinal ganglion cells of mice lacking visual receptors Brain Res 44,37-48[Medline][Order article via Infotrieve]
  46. Kohler, K, Hartmann, J, Fischer, S, Zrenner, E. (1997) Degenerative processes in the inner retina of the RCS rat Invest Ophthalmol Vis Sci 38,173
  47. Li, ZY, Kljavin, J, Milam, AH (1995) Rod photoreceptor neurite sprouting in retinitis pigmentosa J Neurosci 15,5429-5438[Abstract]
  48. del Cerro, M, Ison, JR, Bowen, GP, et al (1991) Intraretinal grafting restores visual function in light-blinded rats Neuroreport 2,529-532[Medline][Order article via Infotrieve]
  49. Gouras, P, Du, J, Kjeldbye, H, et al (1992) Reconstruction of degenerate rd mouse retina by transplantation of transgenic photoreceptors Invest Ophthalmol Vis Sci 33,2579-2586[Abstract/Free Full Text]




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