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From the 1 Departments of Anatomy and Histology and 2 Clinical Ophthalmology, University of Sydney, New South Wales, Australia; and the 3 Departments of Biological Structure and 4 Ophthalmology, University of Washington, Seattle.
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Abstract |
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METHODS. Retinal sections and flatmounts from monkeys aged between fetal day (Fd)80 and 2 years of age were double labeled using antisera to CD31 or von Willebrand factor to detect vascular endothelial cells and antiserum to glial fibrillary acidic protein to detect astrocytes. Sections were studied by fluorescence or confocal microscopy.
RESULTS. From Fd88 to 115, vessels on the horizontal meridian were found only at the level of the ganglion cell layer (GCL)–inner plexiform layer (IPL) border where they form the ganglion cell layer plexus (GCP). Stellate astrocytes accompany GCP vessels and extend closer to the fovea than vessels. The foveal avascular zone was present within the GCP at Fd101, and at Fd105 a shallow foveal depression encircled by the GCP was present. The GCP foveal margin had the same dimensions as the adult foveal pit. Both blood vessels and astrocytes were excluded from the emerging fovea throughout development. After Fd140, capillary plexuses in the outer retina anastomosed with the GCP on the foveal slope to form a perifoveal plexus, but this plexus did not mature until a month or more after birth. After Fd142, astrocytes rapidly disappeared from the GCP and most of central retina.
CONCLUSIONS. An avascular area is outlined by the GCP before the foveal pit begins to form, suggesting that molecular factors in this region exclude both vessels and astrocytes. These factors may also guide neuronal migration to form the pit. Because the perifoveal plexus is formed during late gestation, both capillary growth and foveal development may be affected adversely by prematurity.
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Introduction |
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The location of the future fovea is evident at 11 weeks’ gestation (WG) in the human,24 or fetal day (Fd) 55 in the macaque,25 when a region of the outer nuclear layer containing only cones is discernible at the posterior pole. Beginning at midgestation and continuing well after birth, cones are displaced centrally2 to establish adult foveal cone densities of 200,000 to 300,000/mm2, which provide the anatomic substrate for the high-resolving power of the fovea.2 26 27 28 The foveal depression is not detectable until approximately 25 WG in the human and Fd110 in the macaque.1 3 4 25 28 29 30 Studies in primates have found that the foveal region is never vascularized during normal development,19 21 but suggest that there is a close temporal relationship between formation of the perifoveal vasculature and the early stages of formation of the foveal depression.4 18 21 It is not known whether the fovea forms before a foveal avascular zone is established or within an already prescribed avascular region. Clarification of this temporal relationship may help unravel the mechanisms directing and controlling foveal development.
In mammals, retinal blood vessels are first evident in the inner retina surrounding the optic disc and later grow toward the peripheral retinal margin.31 32 This disc-to-periphery maturation of blood vessels contrasts with the fovea-to-periphery maturation sequence of retinal neurons.2 4 29 33 34 35 The primary retinal blood vessels are also associated with astrocytes that accompany and lead the developing blood vessels.18 21 23 36 37 Astrocytes are thought to sense relative hypoxia in the more peripheral avascular regions and, in response, to stimulate endothelial proliferation through the expression of vascular endothelial growth factor.18 38 39 Formation of capillary beds in the outer retina as far as the outer border of the inner nuclear layer (INL) occurs by budding from the primary blood vessels16 31 32 and is thought to be stimulated by vascular endothelial growth factor released from the Müller cells.38 40
In primates, the first blood vessels are in the nerve fiber layer plexus that forms at the nerve fiber layer–ganglion cell layer (GCL) interface and initially is distributed in four lobes, one for each retinal quadrant. Vessel growth in the two temporal lobes mimics the arcuate fiber pattern created by ganglion cell axons that pass around rather than through the future foveal region.16 18 20 41 Other retinal plexuses are formed by budding of the nerve fiber layer plexus. In the macaque at Fd120 the inner capillary plexus (ICP) is present at the inner plexiform layer (IPL)–INL border near the optic disc. By Fd130 the outer capillary plexus (OCP) at the INL–outer plexiform layer (OPL) border has also formed near the disc.16 However, there are some indications that perifoveal blood vessel development may be different. Growth of human blood vessels toward the foveal region along the horizontal meridian has been described as "retarded,"19 and a recent study of the nerve fiber layer plexus development in 14- to 23-WG fetal human retina found very low levels of cell proliferation along the horizontal meridian.23
In the present study we investigated growth of the retinal vasculature around the fovea in fetal and newborn macaque monkeys to establish the temporal relationship between definition of the foveal avascular zone and formation of the foveal depression. Our studies found a distinctive pattern of blood vessel development in this region. The nerve fiber layer plexus was absent, and instead a ganglion cell layer plexus (GCP) was established at the GCL-IPL boundary, from which the ICP and OCP developed by budding. A perifoveal ring formed by the GCP defined the future depression before cell migrations begin within the inner retina. Near birth, the GCP, ICP, and OCP anastomosed to form a perifoveal capillary plexus surrounding the foveal depression.
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Methods |
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Immunohistochemistry
Adjacent sections through the foveal region and optic disc were
immunolabeled in single or double-label combinations. Astrocytes were
labeled using anti-glial fibrillary acidic protein (GFAP; rabbit
anti-bovine at 1:500; Dako, Carpinteria, CA). Müller cells were
labeled using antibody to cellular retinaldehyde-binding protein
(CRALBP; rabbit anti-bovine at 1:20,000, gift of John Saari)
and endothelial cells were labeled using mouse anti-human antibodies to
the vascular differentiation markers CD31 (at 1:50 to 1:100; Dako) or
von Willebrand factor (at 1:50) .
Sections were incubated for 1 hour in Tris-buffered saline containing 0.2% Triton (TBS-Triton) and 10% goat serum, then incubated overnight in a primary antiserum for single labeling or a mixture of a polyclonal and a monoclonal primary antiserum for double labeling. For single labeling, sections were sequentially incubated for 45 minutes at 37o in species-specific biotinylated IgG (1:100) followed by avidin-Texas red (Molecular Probes, Eugene, OR; 1:1000). For double labeling, sections were incubated sequentially in anti-mouse biotinylated IgG followed by a mixture of avidin-Texas red (1:1000) and anti-rabbit IgG tagged with fluorescein isothiocyanate (FITC; 1:100). Sections were then washed and coverslipped in 80% glycerol in phosphate buffer.
Flatmounts of the retina were double labeled to show the distribution of immunoreactive (IR) GFAP astrocytes and CD31-IR blood vessels. Blood vessels in wholemounts at Fd155 or older were labeled with a mixture of CD31 and von Willebrand factor antisera because of a decrease in CD31-IR with age. Whole retinas were rinsed in filtered TBS (pH 7.6) containing 0.002% sodium azide overnight at 4°C, blocked in TBS containing 0.4% saponin and 10% donkey serum for 3 to 4 days, and then incubated in GFAP antiserum in TBS–2% donkey serum at 4°C for 3 to 5 days, thoroughly rinsed in TBS, and incubated in anti-rabbit IgG conjugated to Cy2 (1:100; Amersham, Arlington Heights, IL) for 24 hours at 4°C. Retinas were washed in TBS containing 0.2% saponin and 10% donkey serum for 24 hours, incubated at 4°C in one or a mixture of both blood vessel antisera in TBS–2% donkey serum for 3 to 4 days, washed for 3 hours in TBS, then incubated sequentially in biotinylated anti-mouse IgG (1:50) for 24 to 36 hours and streptavidin conjugated Cy3 (1:100, Amersham) for 30 minutes. Retinas were finally rinsed in TBS and mounted in glycerol with the inner retina uppermost.
Microscopy
Controls for section staining consisted of the elimination of
either one primary or one secondary IgG. There was no bleed-through
using the Texas red–FITC filter set used for conventional microscopy
and photography. All material presented in this study was judged to be
specific, compared with control sections. Double labeling by
conventional microscopy was imaged by single-exposure photography for
each label, digital scanning of the images, and combination into a
single image using the red and green channels of a document generated
by computer (Photoshop; Adobe, San Jose, CA).
Some sections and all flatmounts were imaged in a confocal microscope (Leica, Deerfield, IL) using an argon krypton laser and associated software (TCSNT; Leica). To prepare montages, flatmounts were optically sectioned using a x16 objective lens in 12 to 19 planes parallel to the retinal surface at 5- to 10-µm intervals beginning at the nerve fiber layer and ending at the outermost capillary plexus. Optical sections were viewed in separate layers using a see-through mode in the image analysis software (Photoshop; Adobe). For analysis and figures, optical sections through the GCP and through the ICP and OCP were merged into single layers.
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Results |
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A flatmount of the fellow Fd105 retina shows a well-developed GCP 375 to 425 µm wide encircling the avascular depression (Fig. 2B) . This ring was formed by the anastomoses of four to five large radial vessels feeding into the GCP and had a denser and tighter capillary meshwork than vessels in adjacent retina (Fig. 2C) . Many large, stellate astrocytes were in contact with the perifoveal capillaries, and the larger blood vessels (Figs. 2B , arrowhead, and 2D small arrows), but their overall distribution did not mimic the pattern of the smaller blood vessels. Fd105 appeared to be the peak of astrocyte density in the perifoveal GCP. Astrocytes formed an almost complete circle just central to the perifoveal capillary ring, extending their processes approximately 50 µm onto the slope of the developing fovea. Short, blind capillary branches reached into the fovea as far as this circle, and astrocyte cell bodies seemed to cluster at these branches (Figs. 2B 2D , arrowhead).
Formation of the Outer Retinal Vascular Plexuses: Fd115 through 145
The outer retina was avascular in sections or a flatmount up to
Fd110. In sections at Fd115 near the optic disc, branches of the GCP
had grown across the IPL to form the ICP (Fig. 3D
, small arrows). A few ICP branches crossed the INL (Fig. 3D
arrowheads), indicating that formation of the OCP also had begun.
In contrast, near the fovea, blood vessels only were starting
to penetrate the IPL (Fig. 3E
, small arrows). All GCP blood vessel
sprouts were free of GFAP-IR astrocytes. By Fd140, near the optic disc,
the four capillary layers were established (Fig. 3F)
, but as late as
Fd132 only a single layer of GCP astrocytes and blood vessels
surrounded the emerging fovea (Figs. 3G
;
4A
4B
).
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A flatmount at Fd142 showed that a month before birth, the GCP was well established (Fig. 5A ), but the OCP was only partially formed near the fovea (Fig. 5B) . Anastomoses between the two capillary layers (Fig. 5B , small arrows) were present 150 to 250 µm from the perifoveal ring, but not on the foveal rim. At Fd142 the GCP perifoveal capillary bed was more dense than at Fd105 with many short, blind-ending vascular sprouts reaching toward the foveal depression (Fig. 5C , thick arrows). The number of GCP astrocytes was much reduced (Figs. 4C 5C) , but Müller cells were more heavily labeled. The overall dimensions of the avascular area were 500 x 300 µm, similar to Fd105, suggesting that there had been no further growth of GCP capillaries into the developing fovea.
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By P3 week, both the perifoveal plexus and the foveal pit appeared mature and anastomoses were complete (Fig. 4G 4H) . The mature perifoveal plexus was formed by three strata of GCP, ICP, and OCP capillaries with the final step occurring when the GCP and OCP anastomosed with the ICP to form a single layer of vessels at the level of the ICP. The completed perifoveal plexus was seen clearly in a P-2-year flatmount (Fig. 5E 5F) where a single layer of capillaries encircled the avascular fovea. Within 100 µm of the avascular area, branch points could be identified that connected the capillaries of the GCP-ICP (Fig. 5E , white arrows) and OCP (Fig. 5F , white arrows).
Changing Relationship of Astrocytes to the Perifoveal Capillaries
In all specimens Fd130 or younger, astrocytes lay closer to the
fovea than blood vessels in both the GCP and the perifoveal ring (Figs. 1
and 2)
, as seen in other parts of the retina.18
21
However, by Fd132 to 140 astrocytes lagged behind blood vessels in the
central retina (Figs. 4A
4B
4C
4D)
, and there were many fewer
astrocytes associated with the perifoveal GCP (compare Fig. 2B
with
Fig. 5C ). In the Fd142 flatmount, astrocytes no longer clustered around
the perifoveal capillaries (Fig. 5C
, large arrows). By birth, the
immediate vicinity of the fovea was virtually astrocyte free (Figs. 4E
4G
, arrowheads), and at P2 year, only eight stellate astrocytes were
present in an 800 x 800-µm field (Fig. 5E
, green cells). During
late gestation and shortly after birth, foveal Müller cells
labeled heavily for GFAP, but by P2 year GFAP-IR had disappeared (Fig. 5E
5F)
.
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Discussion |
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Growth and Guidance of Retinal Vessels
An early vasculogenesis hypothesis proposed that vascular
precursor cells become aligned and then differentiate to form nerve
fiber layer capillaries.41
Recent studies using
endothelial cell–specific markers have failed to label these cells in
primate retina.20
Rather, elongated cells in front of the
growing vasculature have been found to be GFAP-IR, indicating that many
are astrocytes. Double-label experiments have found that all cell
proliferation associated with human inner retinal vascular
development can be accounted for within astrocyte and endothelial cell
populations,23
suggesting that if present, vascular
precursor cells do not proliferate. Gariano et al.20
conclude that almost all the vessel growth in the monkey nerve fiber
layer occurs by budding of existing endothelial cells and that this may
be under the control of factors released from the astrocytes. This is
supported by findings in human retina showing that astrocytes at the
actively growing capillary front express vascular endothelial growth
factor mRNA.18
Almost all researchers agree that the extension of vessels into the outer layers of the retina involves budding of the existing vasculature.16 18 20 32 Müller cells are suggested to be the main source of vascular endothelial growth factor during growth of vessels toward the outer retina in the rat,38 but to date this has not been demonstrated in the primate. In the cat it has been reported that the outer retinal plexus forms first in the region of the area centralis,32 the homologue of the primate fovea. However, in agreement with previous findings in the primate, the present study clearly demonstrates that first the ICP and later the OCP formed first near the optic disc, and then gradually spread toward the fovea, where the full complement of perifoveal capillaries was not established until between Fd160 and P day 1. We also found that in the foveal region, the OCP formed earlier than the ICP, which may be a response to increased metabolic demand due to elevating foveal cone density around birth.27 It also has been suggested that in the cat the early development of the OCP may be driven by the metabolic needs of central photoreceptors that are born earlier and may become active ahead of those in more peripheral locations.32
Although the migration pathways of astrocytes can clearly influence the growth patterns of retinal vessels, the factors that induce astrocytes into the retina and direct them to migrate toward the periphery are less clear. In general, retinal astrocytes grow in a pattern that reflects the arrangement of ganglion cell axons,18 20 40 44 suggesting that axon bundles provide some mechanical guidance or growth factor stimulation. In primate nerve fiber layer, bipolar astrocytes and vessels migrate in a plane different from that of axons,20 suggesting that direct contact with axons is not required for either astrocyte or blood vessel growth. In the present study, astrocytes preceded vessels as they grew first across the GCL to establish the GCP and later along the GCL-IPL border. There is no known axonal pathway that might guide this migration, and there is no evidence of involvement of any other type of neuronal or nonneuronal cell. The present findings indicate, therefore, that at least some astrocytes do not require an underlying axon guidance pathway and that factors other than simple cell–cell interactions underlie establishment of the GCP.
Defining the Foveal Avascular Region
Much recent work in retinal angiogenesis has emphasized the
dynamic role astrocytes play in normal and abnormal retinal vascular
development, including the secretion of growth factors in response to
relative hypoxia.18
38
39
45
Despite the high density of
neurons in central retina, which presumably creates a high metabolic
demand and in turn should stimulate vessel growth, our present results
show that both vessels and astrocytes were inhibited from growing into
the incipient fovea. Although human fetal astrocytes were shown to
express vascular endothelial growth factor in the nerve fiber
layer,18
because that study could not be extended after
midgestation to allow examination of fovea development, the role of
stimulating factors in the development of the ICP, OCP, and perifoveal
capillaries remains to be explored.
The role of inhibitory vascular factors in the retina is poorly understood,22 46 47 but such factors are likely to have a critical role in modulating the action of vasoproliferative factors to control vessel growth. We have shown recently that cell proliferation in the retinal vasculature is significantly reduced along the horizontal meridian of the human fetal retina, including the vicinity of the developing fovea.23 This occurs despite the increased number of neurons in the foveal region4 and the probable poor oxygenation of the inner retina by the immature fetal choriocapillaris16 that could be expected to enhance endothelial cell proliferation. Both endothelial cells and astrocytes proliferate in fetal retina,23 and studies are in progress to determine whether proliferation rates are equally affected. Those findings imply that a factor that inhibits cell proliferation is expressed along the horizontal meridian. In addition, the present finding of blind-ending capillaries that are directed toward but never grow into the foveal region is another indication of the presence of a negative angiogenic factor creating a "no-go" region at the incipient fovea. Viewed together, these findings suggest that a focal concentration of an antiproliferative factor at the incipient fovea defines the avascular region.
An unexpected finding of this study was the observation that by Fd105 both the stellate astrocytes and blood vessels of the GCP formed a ring that was spatially coincident with the rim of the early foveal depression. Inner retinal neurons were displaced peripherally toward the foveal rim as the foveal depression was formed, but the mechanisms underpinning these displacements remain enigmatic.1 2 4 48 The spatial coincidence of the astrocyte–vascular ring and the foveal rim raised the possibility that whatever factor(s) defined the GCP no-go zone may also act to exclude or repel neurons from within the incipient fovea. If such a factor were activated around the time that the avascular area is defined, it could trigger the commencement of the peripheral displacements forming the foveal depression. Alternatively, we have suggested previously that inner retinal neurons within the avascular area may be metabolically stressed, because they are very numerous but must rely on diffusion of oxygen from the relatively distant choriocapillaris.4 Inner retinal neurons may therefore be triggered to migrate toward the vascular ring to resolve their metabolic needs.
Our observation that from approximately Fd105 GCP astrocytes became less numerous in central monkey retina is consistent with a previous report on postnatal retina,49 although in the present study, this decline in astrocyte populations began prenatally. The density of astrocytes in postnatal monkey central retina declined from 400/mm2 at P day 1 to 23/mm2 in the adult, associated with loss of GFAP-IR around the fovea.49 This observation strongly suggests that stellate astrocytes play an important but transient role in establishing the perifoveal plexus. What remains to be determined is whether the disappearance of astrocytes occurs by programmed cell death, withdrawal to the periphery, and/or downregulation of GFAP to undetectable levels. This could resolve whether astrocytes are not necessary to sustain the perifoveal plexus or are still present but undetectable and could respond if needed.
Implications for Foveal Development in Premature Infants
In the present results, definition of the avascular area,
deepening of the foveal depression by neuronal and Müller cell
migration, and maturation of the perifoveal plexus took place between
Fd105 and P3 week in the Macaca monkey. In the human a
comparable period is 25 WG to P3 month.25
Human infants
are at high risk of development of retinopathy of prematurity if
delivered before 28 WG.50
Long-term follow-up studies
indicate that even when retinopathy is not present in the neonatal
period, premature infants of 31WG or less are at increased risk of
development of some form of ocular disorder, including myopia,
hypermetropia, strabismus, astigmatism, or poor visual
acuity.50
51
52
53
54
The results of the present study suggest
that the astrocyte–vascular interactions that establish the human
perifoveal plexus occur during this vulnerable period, as do the
neuronal migrations that form the foveal depression and elevate cone
density.4
28
Because perifoveal plexus development and
formation of the foveal depression appear interdependent, it seems
likely that the changes in retinal
PO2 experienced by
premature infants could affect these normal developmental interactions,
retarding growth of the perifoveal plexus at a time when the foveal
depression is beginning to form. This in turn may indirectly affect
neuronal displacements, resulting in subtle abnormalities that may
underlie the visual difficulties experienced by premature infants.
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Acknowledgements |
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Footnotes |
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Submitted for publication May 27, 1999; revised March 7, 2000; accepted March 17, 2000.
Commercial relationships policy: N.
Corresponding author: Anita E. Hendrickson, Biological Structure, Box 357420, University of Washington, Seattle, WA 98195. anitah{at}u.washington.edu
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