Vascular changes and their mechanisms in the feline model of retinopathy of prematurity.

20 Pages·2012·3.13 MB·English

Investigative Ophthalmology & Visual Science, Vol. 33, No. 7, June 1992
Copyright © Association for Research in Vision and Ophthalmology
Vascular Changes and Their Mechanisms in the Feline
Model of Retinopafhy of Prematurity
Tailoi Chan-Ling,* Simon Tour,* Horstmar Hollander,! and Jonathan Stone*
This study documents changes to retinal vasculature during the feline form of retinopathy of prematur-
ity (ROP). The authors describe the closure and obliteration of retinal vessels during exposure to high
oxygen, the pattern and tempo of growth of proliferative vasculature, which, after the return of the
animal to room air, extends from the optic disc in a spectacular "rosette" pattern, the formation of
preretinal vascular growths, and an initial lack of barrier properties in the new vessels. Finally, the
response of the vasculature to the relief of hypoxia is reported, including the gradual establishment of
barrier properties in the intraretinal vessels, the partial normalization of the proliferative vessels, and
the abnormalities that persist. It is suggested that the vascular changes occur in successive stages:
closure and obliteration during hyperoxia, vasoproliferation induced by hypoxia, and normalization
after the relief of hypoxia with distinct cellular mechanisms and stimuli. It is argued that the same
stages can be seen in the human form of ROP; two possible stimuli for the fibroplasia that damages the
retina in human ROP are discussed. Invest Ophthalmol Vis Sci 33:2128-2147,1992
In the cat,l>2 as in humans,13 the formation of the the retinal and choroidal circulations.15 The mecha-
retinal vasculature begins during gestation and ex- nism of that interaction can be understood, we be-
tends through early postnatal life. The pattern and lieve, in the different regulatory properties of choroi-
timing of its development suggest12'4 that the forma- dal and retinal vessels. Retinal vessels regulate their
tion of retinal vasculature is driven by a rise in the flow in response to changes in arterial pressure and
metabolic requirements of the retina related to the tissue oxygen tension.16 When the tissue partial oxy-
onset of retinal function. Conversely, angiogenesis in gen pressure (pO) rises, these vessels narrow, limiting
2
the retina ceases when oxygen tension in the retina is the flow of blood through them. The drop in flow
raised, eg, by raising the level of oxygen in the inspired limits the rise in tissue PO and, as a consequence, the
2
air.56 The control of angiogenesis by tissue oxygen closure of the vessel. The choroidal vessels do not regu-
tension7 may be exerted through an angiogenic fac- late their flow in a comparable way. During hyper-
tor. ' Recently, an angiogenic factor was isolated from oxia, their high blood flow is maintained16 or only
the retina and identified as one of the family of fibro- slightly reduced,1718 raising PO in the inner layers of
2
blast growth factors.7"12 retina to levels higher than normally established by
The control of angiogenesis by oxygen tension has the retinal circulation.19"21 In the developing retina,
been shown in various other tissues, such as the chick the high PO in the inner retina stops the growth of
2
chorioallantoic membrane.13 It is probably found in new vessels and, if continued for a sufficient time,
all tissues, but the retina is uniquely vulnerable to a drives the constriction of existing vessels to complete
breakdown of this control.14 It has been suggested that and irreversible closure.56
this vulnerability results from an interaction between When animals with retinas thus made avascular are
returned to room air, the inner layers of the retina
become hypoxic, and a massive growth of new vessels
From the Department of Anatomy, University of Sydney, Syd- results.5 This new growth of vessels presumably re-
ney, Australia, and the |Neuroanatomy Laboratory, Department lieves the hypoxia that induced it, and the new vascu-
of Neuromorphology, Max Planck Institute for Psychiatry, lature undergoes a period of normalization. In the cat
Miinich, Munich, Germany.
form of the disease, the retina survives these events,
Supported by a grant from the National Health and Medical Re-
although several vascular abnormalities persist. In hu-
search Council of Australia, Canberra, Australia, and The Clive and
Vera Ramaciotti Foundation, Sydney. TC-L is the recipient of a R. mans, the retina often survives, but it may be de-
Douglas Wright Award from the National Health and Medical Re- stroyed by a proliferation of fibrous tissue not seen in
search Council. the cat. The fibrous stage of the disease was the first
Submitted for publication: May 3, 1991; accepted January 6,
stage detected in humans and led to the condition
1992.
being called retrolental fibroplasia.22 Subsequently,
Reprint requests: Dr. Tailoi Chan-Ling, Department of Anat-
omy, University of Sydney, Sydney, NSW 2006, Australia. the importance of the early stages of the disease was
2128
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No. 7 FELINE RETINOPATHY OF PREMATURITY / Chan-Ling er ol 2129
recognized, and the term "retinopathy of prematur- vasculature. Second, the vascular tree was kept largely
ity" (ROP) introduced.23 The early stages of this dis- intact, reducing the leakage of marker substances
ease have been studied most intensively in the feline from vessels, which is inevitable when they are sec-
model of ROP. tioned. Although the relative depth of structures in
We examined the topography, cellular detail, and the retina was not observed as readily as in cross-sec-
barrier properties of the retinal vasculature during tions, the careful use of focal depth with higher power
ROP in the cat, using both older (eg, ink injection) (>40x) objectives allowed a clear distinction between
and newer techniques (eg, lectin histochemistry, intra- preretinal and intraretinal structures and between the
vascular perfusion with horseradish peroxidase principal layers of the retina (see, for example, Fig. 10
[HRP], light microscopy of whole-mount prepara- in Chan-Ling et al2).
tions of the retina, and electron microscopy of HRP-
perfused retinas). We draw two principal conclusions. Indian Ink Perfusion
First, we build on earlier descriptions56'24'25 to identify
One cat aged P4/6 was given an overdose of sodium
successive stages in the vascular changes of ROP, with
pentobarbital and perfused transcardially with phos-
distinct pathologic findings and causative mecha-
phate-buffered saline (PBS), followed by India ink
nisms. Second, we suggest that the blinding fibropla-
(Rotring drawing ink; Rotring, Hamburg, Germany).
sia seen in human ROP is not caused directly by the Following the earlier procedure,2 the eyes were post-
pathologic state of the vasculature, but by a distinct
fixed (minimum, 2 hr) in paraformaldehyde 4% in 0.1
still-unidentified mechanism. Two possibilities for
mol/1 phosphate buffer (PB) at pH 7.4. The retina was
this mechanism are discussed: (1) the fibroplasia is
dissected under PBS and spread on a gelatinized slide.
induced by an angiogenic factor and (2) it is induced
The radial incisions needed to flatten the retina were
by proteins leaked from the proliferative vasculature
placed, where possible, to avoid cutting the vascula-
that forms during the hypoxic stage of the condition.
ture. The retina was allowed to dry onto the slide and
then was mounted, without dehydration, using Aqua-
mount (BDH Limited, Poole, England).
Materials and Methods
Exposure to Hyperoxia and Return to Air Labeling With Fluorescence-Conjugated Lectin
Four litters of kittens of various sizes were placed To demonstrate retinal vessels and vascular precur-
within hours after birth in a large incubator with an sor cells,2 retinas were labeled with the Griffonia sim-
atmosphere consisting of 70-80% oxygen. Oxygen lev- plicifolia (Bandeirae) isolectin B (conjugated to fluo-
4
els inside the incubator were monitored using a Datex rescein isothiocyanate; Sigma, St. Louis, MO). Using
oxygen and temperature monitor (OT 102; Datex In- earlier protocols,26 the retinas were fixed by perfusion
struments Corp., Helsinki, Finland) with a hand-held with paraformaldehyde 4% in 0.1 mol/1 phosphate
polarographic oxygen sensor. The litters remained buffer at pH 7.4. After dissection, the retinas were
under these conditions with their mothers for 96 hr. incubated in the G. simplicifolia lectin (0.2 /xg in 5 ml
The mother was exercised regularly and given periods of PBS) for 24-48 hr at 4°C. The tissue was washed in
in room air. After this period in high oxygen, the lit- three changes (each, 10 min) of PBS with Triton
ters were returned to air and killed 0, 3, 6, 7, 10, 11, X-100 1% and mounted in PBS glycerol. The isolectin
13,18,19, 20, 27, 28, 42, and 88 d later. Their retinas has a major affinity for terminal a-D-galactosyl resi-
were processed for lectin histochemistry, perfused in- dues.
travascularly with ink, or perfused intravascularly
with HRP for light and electron microscopy. To indi- Labeling With HRP-Conjugated Lectin and
cate both age and oxygen exposure, we adopted a no- Toluidine Blue Counterstain
tation such that "P4/11," for example, denotes an an-
Retinal vessels and vascular precursor cells were vi-
imal raised in high oxygen for 4 d from birth and
sualized using the G. simplicifolia lectin conjugated
returned to room air for 11 d before they were killed.
with peroxidase (GSA 1-B-HRP; Sigma). The isolec-
All procedures conformed to the ARVO Resolution 4
tin was prepared using 0.2 mg/ml in Tris-bufFered sa-
on the Use of Animals in Research.
line (TBS). The retinas were washed in PBS and Tri-
ton 1% for 30 min before incubation in lectin over-
Use of Whole Mounts
night at 4°C, washed in TBS for 20 min, followed by
Our observations were made using whole-mount another wash in nickel TBS (0.04%) at pH 7.4. The
preparations of the retina. This provided two distinct peroxidase was visualized by applying 3,3' diamino-
advantages. First, they allowed a much more powerful benzidine tetrahydrochloride (DAB) 0.05% and hy-
description of the topography of changes in retinal drogen peroxide 0.02% in nickel TBS for 3-5 min.
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2130 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / June 1992 Vol. 33
After a final wash in TBS, the retinas were spread onto in the intervascular space. Where the level of extra vas-
gelatinized slides with the nerve fiber layer upper- cular HRP was close to the background levels, a rating
most. Some retinas were counterstained,26 using tolu- of 0.5 was recorded.
idine blue 0.1% in sodium tetraborate 1% for 1 min,
rinsed with distilled water, and fixed using the mor-
Mapping of Vascular Spread
dant, ammonium molybdate 5%, for 30-60 min, be-
fore dehydration, clearing, and mounting in D.P.X. From a series of whole mounts perfused with ink or
(BDH Chemicals, Fairy, Australia). HRP or reacted with the G. simplicifolia lectin, maps
were made of the vascularization of the retina and the
leakiness of the vasculature at various times. In each
Intravascular Perfusion of HRP and
retina, retinal boundaries, the outer limit of the
Toluidine Blue Counterstaining
spread of spindle cells, and the outer limit of patent
The barrier properties of retinal vessels were tested vessels in both the inner and outer layers of vascula-
by intravascular injection of HRP (200 mg/kg of body ture were mapped using a microscope equipped with
weight, type 2; Boehringer-Mannheim) 10% solution a measuring eyepiece.27
in sterile saline. The HRP solution was injected into
the common carotid artery of the eye to be examined
Electron Microscopy
using a 25-gauge winged infusion set (Terumo Surflo;
Melbourne, Australia). The animal was killed by ad- Fixation and HRP perfusion: Kittens exposed to
ministration of a sodium pentobarbital overdose after oxygen as described were anesthetized with a mixture
30 min had elapsed. The globe was enucleated and of ketamine and xylazine (0.3 ml and 0.07 ml, respec-
immersion fixed in paraformaldehyde 4% in 0.1 mol/1 tively, per 250-g body weight injected intramuscu-
PB. The retina was dissected in TBS followed by an- larly). Anesthesia was maintained with supplemen-
other wash in nickel TBS (0.04%) at pH 7.4. The per- tary doses of this solution. After deep anesthesia was
oxidase was visualized by applying DAB 0.05% and obtained, a solution of promethazine (0.1 mg/100 g
hydrogen peroxide 0.02% in nickel TBS for 3-5 min- body weight) was injected intramuscularly. Ten min-
utes. After a final wash in TBS, the retinas were spread utes later, HRP was injected intravascularly (10 mg/
onto gelatinized slides with the axon layer uppermost, 100 g body weight in 30 mg/ml solution of Grade II
dehydrated, and cleared before mounting in Per- HRP, dissolved in Hartmann's solution; Boehringer-
mount. Some retinas were counterstained with tolu- Mannheim).
idine blue 0.1% as described previously. After the HRP had circulated for 10-15 min, the
The barrier properties of the retinal vasculature animal was perfused intracardially for 30 sec with
were rated using an arbitrary scale of 0-4. Where Hartmann's solution at 37°C, then for 20 min with
leaked HRP masked the outlines of the vasculature fixative (glutaraldehyde 5% in 0.1 mol/1 cacodylate
and hemorrhage was evident, a rating of 4 was re- buffer, pH 7.3, containing 2 mmol/1 calcium chloride
corded. Where the leaked HRP was copious in the and sucrose 3.4%) at 37°C. Simultaneously, cold (0-
intervascular spaces, but the outlines of the vessels 4°C) fixative was dripped directly onto the exposed
could be discerned, a rating of 3 was assigned. A grad- retinal surface. Perfusion pressure was kept low to
ing of 1.5-2 was used when the outlines of the vessels minimize the possibility of rupturing vessels. At the
were delineated clearly by intravascular HRP accu- end of the perfusion, the eyes were removed and post-
mulation, but only small quantities of HRP were seen fixed for 40 min in ice-cold fixative.
Fig. 1. Effect of hyperoxia on vessel growth.
Left: In a normal P4 retina, vessels have ex-
tended from the optic disc, concentrating in
three lobes, over approximately half the ret-
ina. Large vessels have developed, some of
which are sketched. The vessels have skirted
above and below the area centralis, marked by
an X. Right: In the retina of an animal raised
in high levels of oxygen for 4 days from birth,
patent vessels extend only 1-2 mm from the
optic disc. In the area marked by the dotted
line, scattered remnants of vessels remain.
The X marks the area centralis.
P4 CONTROL P4 80% OXYGEN
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No. 7 FELINE RETINOPATHY OF PREMATURITY / Chan-Ling er ol 2131
HRP histochemistry: The fixed retinas were sepa- the closure of the vessels irreversible; this irreversible
rated intact from the eye, rinsed in cacodylate buffer closure is the vasoobliteration described by others.24
for 2 hr, and cleaned of adhering fragments of vitreous When exposure to high oxygen was extended to 6 d,
humor. The retinas were incubated for 30 min in caco- no retinal vessels remained patent, and only small
dylate buffer containing 1 mg/ml DAB and then remnants of vessels could be detected (Fig. 2C).
transferred to fresh solution containing 0.1 ml of hy- The vessels shown extending from the optic disc in
drogen peroxide 3% and 10 ml of DAB solution to Figure 1 (right) were considered patent because they
produce a colored HRP reaction product. After ap- appeared continuous with vessels in the optic nerve
proximately 5 min, the retinas were rinsed in cacodyl- head and contained no blood cells (removed by the
ate buffer and photographed with a dissecting micro- perfusion of the fixative). Surrounding the patent ves-
scope. Small regions of interest were removed for pro- sels, in the region indicated by the dotted line in Fig-
cessing for electron microscopy. ure 1 (right), remnants of vessels were present. Like
Electron microscopy: The tissue blocks were fixed the patent vessels, they were labeled by the G. simpli-
in osmium tetroxide 1% in 0.1 mol/1 cacodylate cifolia lectin, but they were separate from the patent
buffer for 2 hr at 4°C and then rinsed overnight in vessels and often contained trapped blood cells, which
buffer at 4°C. The blocks were dehydrated in ascend- label with the lectin2 (Figs. 2E-F). The region in
ing grades of alcohol and embedded in Spurr's resin which remnants were found corresponded approxi-
(Polysciences, Warrington, PA). In some cases, ul- mately to the extent of retinal vasculature at birth
trathin sections were taken directly from the blocks. when the exposure to high oxygen began. When the
In other instances, 10-/xm sections were cut from the period of exposure to high oxygen was extended to 9
blocks with glass knives, mounted in glycerol, photo- d, lectin-positive remnants almost were eliminated.
graphed, and examined for regions of interest. Such During normal development, the spread of vessels
regions were remounted onto resin stubs, and ul- over the cat retina is preceded by a movement of spin-
trathin sections were taken. These were mounted dle-shaped precursor cells.2 When the spread of vascu-
onto Butvar(Electron Microscope Sciences, Washing- lature is stopped, the spindle cells persist, and weak
ton, D.C.)-coated slot grids and stained with uranyl labeling with the G. simplicifolia lectin occurs (Figs.
acetate and lead citrate with an LKB [Bromma, Swe- 2E, 2G).
den] Ultrostainer. Stained sections were examined
Vascular Changes Induced by Hypoxia
under the electron microscope at 60 kV.
When neonatal animals kept in high oxygen for 4 d
Results are returned to room air, the inner retina becomes
hypoxic and remains so until proliferative vessels
Vascular changes in ROP in the cat can be divided
spread from the optic disc. During this period, the
into three stages: closure and obliteration during hy-
following vascular changes were observed.
peroxia, vasoproliferation during hypoxia, and nor-
Vascular remnants reopened: One prompt response
malization after the relief of hypoxia.
to the return to room air was a reopening of remnants
of vessels persisting in the retina. Cells in the rem-
Closure and Obliteration of the Retinal
nants extended multiple filopodia (Fig. 2G) and con-
Circulation Induced by Hyperoxia
nected together to reform capillary beds (Fig. 2H).
During normal development, vessels extend across This process of revascularization was limited to the
the inner layers of the retina from the optic disc.1'21424 remnant-containing region indicated in Figure 1
At birth, vessels cover a three-lobed region 60 mm2 in (right).
area; thereafter, they spread across the retina, extend- New vessels spread from the disc in an abnormal
ing from the optic disc at approximately 0.20 mm/d.2 pattern: A more prominent response to hypoxia was
The map in Figure 1 (left) shows the substantial area the growth of new vessels from the optic disc, in a
over which the vessels normally have spread by the distinctive circular "rosette" pattern. Figures 3A and
fourth postnatal day (P4). In a cat kept in oxygen 80% 3B show this pattern, visualized using G. simplicifolia
for the first 4 postnatal days, by contrast, patent ves- lectin and India-ink perfusion, 3 and 6 d after return
sels were restricted to a region of a few square milli- to room air. In both cases, some reopening remnants
meters near the optic disc (Fig. 1, right). Thus, the of vessels can be seen beyond the edge of the rosette
normal growth of the vasculature (Fig. 2 A) during this and joining with it (Fig. 3C). Within 11 d of the return
period had been stopped, and most existing vascula- to room air, the reopening and newly growing vessels
ture had closed (Figs. 2B, 2D). Because the lectin merged and could no longer be differentiated.
binds to endothelial cells, the pattern of labeling sug- The rosette formed by newly growing vessels ini-
gested that the endothelium had degenerated, making tially was radially symmetric, forming a circle (Figs.
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2132 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / June 1992 Vol. 33
•SHE
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No. 7 FELINE RETINOPATHY OF PREMATURITY / Chan-Ling er ol 2133
Fig. 2. Regrowth of vascular remnants, shown by labeling with the G. simplicifolia lectin conjugated to FITC. (A) Normally developing
vessels in the midperipheral region of a control P4 retina .(B, D) Vessels undergoing closure and obliteration after 4 days exposure to 70-80%
oxygen. (C) In a retina exposed to 70-80% oxygen for 6 days, the vessels have closed, and degenerated almost completely. (E, F) Vessel
remnants a P4/0 retina. As vessels close, they retain blood cells, which can be seen brightly labeled, as described previously2 .In (E), the
spindle-cell precursors of vasculature are lightly labeled. (G, H) Revascularization in a P4/3 retina. When animals are placed in room air after 4
days in high oxygen, endothelial cells in the vessel remnants grow filopodia, and reconnect with surrounding remnants to form a capillary
plexus. The lightly labeled cells in (G) are spindle cells.
3A-B). These new vessels did not form in the three- Newly growing vessels were initially abnormal in
lobed pattern seen during normal vascularization morphology: Three abnormalities were seen. First, as
(Fig. 1). As the rosette grew, however, its neat circular- the rosette formed, the vessels at its edge were rela-
ity broke down. Its edge became scalloped, and shal- tively unbranched and finger-like (Figs. 6A-B), a mor-
low lobes appeared, with "corners" between them phology suggesting30 that the vessels were formed by a
(Fig. 4A). Except at the area centralis, however, these process of budding from existing vessels. In normal
lobes were not consistent in position and did not development, by contrast, the vessels extending from
match the lobes of normal vascularization. In addi- the optic disc form a network of capillaries, consid-
tion, the newly growing vessels, like the original vascu- ered to be generated by transformation of precursor
lature (Fig. 1, left), did not spread over the area cen- spindle cells.2 As the rosette of new vessels expanded,
tralis but skirted around it. By P4/11, two consistent the vessels at its edge became exuberant, being more
"lobes" of vasculature had formed temporal to the numerous and larger in caliber than the capillaries of
optic disc, one above and one below the area centralis the adult retina. A comparable exuberance was seen
(Fig. 4B). By P4/18, the newly growing vessels had in vessels forming during normal development,2 but
spread across the raphe region temporal to the area the vessels at the edge of the rosette (Fig. 6C) were
centralis. coarser, more numerous, and more chaotic. Third,
The number of artery-vein pairs emerging from the the vessels of the rosette commonly formed more
optic disc was higher in the experimental retinas (Fig. than a single layer of capillaries. In addition to the
4C) than in the normal. Instead of the three major layer of capillaries found normally in the ganglion cell
pairs of the normal retina,28'29 extending into inferon- and axon layers, a more superficial layer was formed
asal, inferotemporal, and superonasal retina, numer- in the inner part of the axon layer. This innermost
ous artery-vein pairs formed, and their orientation layer was seen most clearly where it crossed larger
was not consistent. vessels (Fig. 6D).
The growth of new vessels over the hypoxic retina The new vessels initially lacked barrier properties:
also was abnormal in its tempo and eventual extent. We used intravascular perfusion of HRP as a test of
The abnormality of its tempo was related largely to the barrier properties of retinal vessels. Normally de-
the delay caused by the period of hypoxia. The spread veloping retinal vessels were tight as soon as they were
of new vessels from the optic disc began when the patent; we could not detect leakage of HRP from even
retina was made hypoxic, at P4 (19 d after normal the most peripheral (and therefore most immature)
vascularization begins at E502), and thereafter pro- vessels of normally developing vasculature at PI2
ceeded steadily (Fig. 5). To compare the rate of spread (Fig. 7A). Intervascular regions close to the optic disc
of these new vessels with that of normally developing in the same PI2 retina also appeared clear and free of
vessels, we measured the area vascularized during suc- HRP diffusing from the vessels (Fig. 7B). In addition,
cessive ages. For each age, we calculated the radius of in vessels formed by the reopening of remnants in
a circle of area equivalent to the area vascularized, experimental retinas, there was little evidence of leak-
and plotted its radius as a function of age. The slope of age of HRP. In proliferative vasculature, by contrast,
the line of best fit for the radius as a function of age leakage of HRP was observed consistently. Brown
was taken as a measure of the rate of vascular spread. HRP reaction product colored the intervascular re-
The rate of growth estimated in this way for the new gions and blurred the outlines of vessels. At P4/10, the
vasculature (0.26 mm/d) was somewhat greater than vessels of the rosette appeared to be leaky both near
that of normal vasculature (0.20 mm/d). the optic disc (Fig. 7C) and in the midperipheral re-
In the hypoxic retinas, as in the normal, the periph- gions of the rosette (Fig. 7D). In addition, focal hemor-
eral margin of the retina was not vascularized. The rhages were evident at the edge of the rosette (Fig. 7E).
margin remaining unvascularized was considerably At P4/20, the vessels near the optic disc had retained
wider in the experimental retinas (up to 2 mm, Fig. 5 HRP, but the vessels at the edge of the rosette were
[P4/42, P4/88]) than in the normal ones (< 1 mm, very leaky (Fig. 7F).
Fig. 8 in Chan-Ling et al2). Figure 8 shows the leakage of HRP observed in the
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2134 INVESTIGATIVE OPHTHALMOLOGY b VISUAL 5CIENCE / June 1992 Vol. 33
into the surrounding extracellular space. Figure 8B
shows, at higher power, a site of HRP leakage through
a distinct gap between endothelial cells (Figs. 8A-B,
arrows). Glial processes were present around the ves-
sel; the large process labeled "m" was the inner foot of
Miiller cell as assessed by its shape (compare with Fig.
2 in Hollander et al31). Our procedures did not allow
us to exclude the presence of astrocyte processes. How-
ever, the nuclei of astrocytes were less common in this
material than in normal material, and the vessel in
Figure 8 A lacks the close glia limitans of vessels in
normal retinal tissue (eg, Fig. 5 of Hollander et al31).
Preretinal vasculature formed and lacked barrier
properties: Beginning as early as P4/7, preretinal ves-
sels appeared. They budded (Figs. 9A-B) from the
vessels of the inner layer and broke through the inner
glia limitans of the retina. They were highly abnormal
in appearance, consisting of swirls of capillaries folded
closely on each other. They typically formed in
clumps, which varied in position, number, and size
(Fig. 9C). Such vessels were common close to the op-
tic disc (Fig. 9D) and were not observed at the area
centralis. In some retinas, many small (25 nm) scat-
tered clumps had formed; in other retinas, only a few
relatively large (approximately, 100 fim) clumps of
preretinal vessels were observed. In several animals,
the preretinal vessels were so extensive (Fig. 9E) that
they formed preretinal membranes.
In favorable preparations, preretinal vessels were
seen in the light microscope to leak HRP. Confirma-
tion of their leakiness is shown in Figure 10. This is an
electron micrograph of a preretinal vessel from a P4/
42 retina. The vessels lack a glia limitans, and there
are, as a consequence, no narrow extracellular chan-
nels in which leaked HRP might accumulate. The
HRP contained in the lumen was, however, seen leak-
ing through distinct gaps between endothelial cells
(Figs. 10A-B, arrows). Figure 10B shows one of these
gaps at higher magnification.
Events After the Relief of Hypoxia
Fig. 3. Proliferative vascularisation—early stages. (A) The optic
disc region of a P4/3 retina labelled with the G. simpticifolia lectin. After new vessels spread over a patch of retina, it
Vessels growing out from the disc form a circular rosette whose edge presumably receives its normal supply of oxygen and
fuses with vessels reforming from vascular remnants. Superior is at
other nutrients. Several changes to the structure of the
top, temporal at left. (B) At P4//6, the rosette, here shown by ink
vessels followed.
injection, has expanded, and larger vessels have begun to differen-
tiate near the optic disc. Again, at its edges the "rosette" fuses with Exuberant vessels matured in morphology: The
vessels reforming from remnants. Superior is at top, temporal at highly exuberant vessels formed during vasoprolifera-
right. (C) The border between the exuberant vessels at the edge of
tion undergo a process of maturation comparable to
the rosette (bottom) and the vessels formed from vascular rem-
that seen in normal development. By P4/11, rela-
nants, atP4/IO.
tively large vessels began to form, apparently (as in
normal development2) by selection among capillaries
electron microscope. Figure 8A shows a vessel near (Figs. 9A-B). Capillary-free zones became apparent
the inner surface of a P4/42 retina, near the edge of along the flanks of some large vessels, presumably ar-
the rosette of vessels. HRP has leaked from the vessels teries (Fig. 4C, arrow). By P4/42, the larger vessels
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No. 7 FELINE RETINOPATHY OF PREMATURITY / Chan-Ling er ol 2135
were increasingly smooth in appearance, and the capil- flanking them. As in the normal retina, large vessels
laries were reduced in caliber toward the normal adult formed only in the inner layer and did not cross the
form. Even at this age, however, the capillaries form- area centralis. However, some unusual branching pat-
ing at the edge of the rosette were somewhat' exuber- terns persisted at least until P4/42, including the addi-
ant (Fig. 11C). By P4/88, the vessels at the inner sur- tional layer of capillaries at the inner surface of the
face of the retina appeared normal in many respects. retina (Fig. 1 ID).
Veins and arteries commonly were found in pairs, The blood-retinal barrier was established in intra-
and within each pair, arteries could be distinguished retinal vessels: The gradual establishment of barrier
by their smaller caliber and the capillary-free zones properties in the new vessels of ROP was apparent in
Fig. 4. Proliferative vascularisation—later stages. (A) The edge of the rosette a tP4/11, showing an irregularity in the previously smooth
circumference of the rosette. (B) At P4/t 1, the vasculature at the edge of the rosette skirts the area centralis, which lies to the right side of this
figure. The capillaries bordering the area centralis are less exuberant than those in more peripheral retina (A). (C) The optic disc region of a
retina from a P4/42 animal. Large vessels have differentiated, and capillary-free zones have formed along the flanks of the arteries (arrows at
top left). The number of large vessels entering and leaving the disc is higher than normal, and the three large artery/vein pairs seen in normal
development cannot be identified. Superior is at top, temporal at left.
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2136 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / June 1992 Vol. 33
P4/3 P4/7 P4/11
Fig. 5. Maps of the regrowth of ves-
sels in retinae after 4 days hyperoxia.
The area of "revascularisation" is the
area over which vasculature formed by
the reopening of vascular remnants.
The shaded areas show the regions in
which the inner and outer layers of vas-
culature have formed. The X repre-
sents the area centralis, and the open
circle represents the optic disc.
• Revascularisation
Inner
• Vasculature
!| Outer
Vasculature
two ways. First, leakiness was most marked in the outer layer formed first in the vicinity of the area cen-
most recently formed vessels, those at the edge of the tralis (not at the optic disc) were fine in caliber and
rosette (Figs. 7E-F, 8, 12). As the rosette expanded, less exuberant than the vessels of the inner layer (Fig.
for example between P4/10 and P4/42, the newest 13B). Capillary-sized buds grew outward from the in-
vessels, at the edge of the rosette, remained leaky, but ner vessels and extended through the inner plexiform
the earlier-formed vessels near the optic disc became layer to form a plexus of capillaries (Fig. 13B) located
tight (Figs. IOC, 12). Second, the leakiness of even the (Fig. 13C) at the junction of the inner nuclear and
newest vessels gradually decreased, and by P4/88, all outer plexiform layers. At P4/18, the outer layer was
the vasculature of the inner layer appeared tight, both formed only over a small horizontally elongated re-
at the edge of the rosette (Fig. 11E) and nearer the gion at the area centralis (Fig. 5). The layer was ini-
optic disc (Fig. 1 IF). tially patchy (Fig. 13D) but became continuous (Fig.
The area centralis became vascularized, and the 13E) and extended over most of the retina (Fig. 5) by
outer layer of vasculature formed: Beginning at be- P4/88.
tween P4/11 and P4/18, a growth of fine capillaries Abnormalities were noticed in the tempo and topog-
over the area centralis occurred. The first such vessels raphy of the formation of the outer layer of vessels.
extended across the area centralis in the ganglion cell The formation of the outer vessels was initially more
layer. Figures 4A and 4B show the capillaries at the patchy (Fig. 13D) than in normal development, and
edge of the rosette at the area centralis (B) and, by the outline of the area of vessel formation was harder
contrast, in more peripheral retina (A) at P4/11. The to define. As in normal development, the outer layer
capillaries at the area centralis are finer and more did not reach the edge of the retina. The margin lack-
adult-like. By P4/20, relatively fine capillaries have ing the outer layer was considerably wider in the exper-
extended over the area centralis at its inner surface imental retinas (up to 4 mm, Fig. 5, P4/42 and P4/88)
(Fig. 13A). As in the normal retina, large vessels do than in the normal ones (< 1 mm, Fig. 82). The for-
not form at the area centralis. mation of the outer layer of vasculature began late,
As in normal vascularization, the vessels of the probably at P4/15-18 compared with P7-10 in nor-
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No. 7 FELINE RETINOPATHY OF PREMATURITY / Chan-Ling er al 2107
Fig. 6. Proliferative vascularisation—details. (A, B) The vessels that extend from the optic disc at P4/3 initially show few interconnections.
They are straight and finger-like in morphology, typical of vessels formed by the budding of endothelial cells. (C) By P4/11, the new vessels
formed by budding from the rosette and revascularisation have merged. The vessels at the edge of the rosette are coarse and exuberantly
interconnected. (D) By P4/11, large vessels have begun to differentiate. This plate is focused on a superficial plexus of capillaries, distinct from
a slightly deeper layer, here out of focus. This superficial plexus is not seen in normal vasculature.
mally developing retinas.2 Thereafter, its formation the closure and obliteration of the retinal circulation.
continued to lag behind the normal development of These changes involve two steps. First, the normal
the outer layer. formation of retinal vessels, still underway at birth,
ceases. Second, vessels already formed close. The stim-
Discussion
ulus for both reactions is a rise in tissue oxygen ten-
Our study of the feline model of ROP leads us to sion in the retina after inspiration of high levels of
make two suggestions concerning the vascular oxygen.20-21'24'32 The normal formation of vessels
changes that occur in this condition. First, we suggest ceases, it is believed,33 because the rise in oxygen ten-
that successive stages can be distinguished during sion reduces levels of an angiogenic factor that nor-
these vascular changes, with distinct mechanisms and mally drives vessel formation. Retinal vessels auto-
pathologic findings. Second, we suggest that questions regulate,16 and high Po maintained in the inner ret-
2
about the genesis, prevention, and treatment of the ina by the choroid circulation may drive the
retinopathy can be formulated best in terms of these autoregulatory mechanism until the retinal vessels
stages. close completely. The degeneration of endothelial
cells, which makes the closure irreversible (oblitera-
Vascular Changes in Feline ROP:
tive), occurs only in the immature retina, and its
Stages and Their Mechanisms
mechanism is unknown. It may be caused by an accu-
mulation of toxic radicals during hyperoxia, which
In successive stages of ROP, the vascular changes
may destroy the endothelial cells directly.34 Vision is
are distinct and are driven by distinct mechanisms.
not affected by the closure and obliteration of the reti-
In hyperoxia, vascular closure and obliteration: The
nal circulation.35
first vascular changes in the genesis of feline ROP are
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