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1 From the Institute of Ophthalmology, University College London, United Kingdom; and the 2 Laboratorio de Oftalmología, Facultad de Medicina, Universidad de Murcia, Spain.
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Abstract |
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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.
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Introduction |
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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).
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Materials and Methods |
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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
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Results |
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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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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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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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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) .
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Discussion |
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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.
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Acknowledgements |
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Footnotes |
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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
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