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1 From the Child Life and Health, Reproductive and Developmental Sciences, University of Edinburgh; and 2 Princess Alexandra Eye Pavilion, Edinburgh, Scotland, United Kingdom.
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Abstract |
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METHODS. Arterial oxygen data from a preterm infant who developed severe retinopathy of prematurity (ROP) was translated to equivalent values for the rat. Newborn rat pups were raised for 14 days in a cage in which a computer controlled the atmosphere to mimic the fluctuating oxygen profile (group V). Positive controls (P) of 12-hour cycles of 80% and 21% were run concurrently, as were room air controls (C). All were killed at day 14.
RESULTS. Groups V and P had significantly larger avascular retinal areas than C [median, interquartile range (IQR): 1.7%, 0–7.9%; 10%, 8.1–13%; 0%, 0–0%, respectively; each group n = 30]. Group P had a higher capillary branch count than C (median, IQR: 310/mm2; 253–311 mm2; versus 277/mm2, 272–364/mm2, respectively), but this was not significant using a multilevel analysis. Group V had significantly reduced capillary counts compared with C (median, 261/mm2; IQR, 215–290/mm2; P < 0.05 multilevel analysis). No neovascularization was seen in any group, though abnormal terminal vessels were seen at the avascular/vascular retina interface in 73% of rats in group P and 21% of rats in group V. In situ hybridization on serial sections demonstrated VEGF in the inner nuclear layer of the retina in P and V, whereas C showed trace levels only.
CONCLUSIONS. The vaso-obliterative stage of ROP can be induced in rats using clinically relevant oxygen levels.
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Introduction |
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Although ROP is now considered by clinicians to be multifactorial, animal work has continued to identify oxygen as a factor in development. Animal work on oxygen-induced retinopathy (OIR) that represents ROP has predominantly been based on the extreme oxygen injury model that was responsible for retinopathy in relatively mature preterm infants in the 1940s.4 5 More recently, Penn6 and others7 have varied the inspired oxygen delivered to models over 6- to 48-hour periods in an active endeavor to more closely resemble the arterial oxygen of preterm infants, though they recognize that this only partially mimics what is experienced by an extremely preterm infant. Relative hypoxia appears to be an important contributor to retinopathy,8 and our understanding of the control of vascular development has increased with the identification of vascular endothelial growth factor (VEGF) and its hypoxic induction via hypoxia inducible factors (HIFs).9 Recent preliminary work by Madan et al.10 has suggested that at least one of the HIF proteins may be involved in the development of ROP.
Practically, preterm infants have significant swings in their arterial oxygen though they stay predominantly within clinically acceptable "safe limits." Our previous work with a group of preterm infants identified that severe ROP was associated with greater variability in transcutaneous (arterialized) oxygen in the first 2 weeks of life.11 We hypothesize that these frequent small changes in oxygen may cause retinopathy by a frequent interruption of the process of ordered retinal vascularization and stabilization, which is controlled by relative hypoxia and hyperoxia in response to blood supply and metabolic demands. We have, therefore, developed an oxygen delivery system that enables us to mimic the transcutaneous arterialized oxygen values recorded from an infant who developed severe ROP.12 This is a more representative model of newborn arterial oxygen than has previously been possible.
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Methods |
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Oxygen Profile Development and Delivery
A computerized physiological monitoring system has gathered data
from infants admitted to the Neonatal Intensive Care in Edinburgh since
1990.13
Arterial oxygen is continuously monitored
transcutaneously and a mean of data points is stored each minute. The
computerized transcutaneous oxygen profile of one infant who developed
severe (threshold) ROP was selected, and the first 14 days were used. A
stream of transcutaneous oxygen values was obtained, representing
partial pressure of arterial oxygen, one value per minute. Published
data were used to translate arterial oxygen in preterm
humans14
to arterial oxygen in the rat. In brief, a
preterm infant in our unit is maintained between 6 and 10 kPa (45–75
mm Hg), with a mean of 8 kPa (60 mm Hg). A newborn rat breathing 21%
oxygen has an arterial value of 12.9 kPa (96.8 mm Hg). Therefore to
each arterial value gained from the preterm infant we added 4.9 kPa
(36.8 mm Hg) to give an equivalent value in the rat. From this set of
values we derived the inspired oxygen in the rat that would produce the
equivalent arterial oxygen,15
one value per minute (Fig. 1)
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Animal Groups
Three groups of animals were studied, each for 14 days. A control
group (C) consisted of animals raised in room air. A 12-hour-variable
model (P) consisted of a group raised in oxygen fluctuating between
80% and 21% (room air) on 12-hour cycles. Our experimental model, the
minute-variable group (V), were animals exposed to our oxygen
profile, with changes in inspired oxygen concentration each minute.
Pregnant animals were acclimatized to isolators at least 24 hours before delivery. A minimum of 12 pups was required per litter, which all rat mothers produced in these experiments. Experiments were begun immediately after the delivery of the final pup in each litter. Bedding was changed every 7 days, at which point the profile was paused and restarted a few minutes later. No other interruption to the profile was required.
At 14 days the rat pups were weighed and anesthetized by intraperitoneal injection of ketamine (2.5 mg/kg) and xylazine (1 mg/kg). Paraformaldehyde (PFA) was then directly perfused (0.4 ml 0.5%) into the left ventricle, and then pups were euthanatized by intracardiac injection of pentobarbitone (80 mg/kg). Both eyes were enucleated.
Preparation of Tissues
Retinal Wholemounts.
The retinas were dissected using a modification of the method of
Chan-Ling.16
Enucleated eyeballs were fixed whole in 2%
PFA for 2 hours before being washed in 1 M phosphate-buffered saline
(PBS), pH 7.4. Under a dissecting microscope an incision was made
between the cornea and sclera. Scissors were then used to cut around
the junction between the cornea and sclera until the cornea could be
removed. The lens was gently removed, taking care not to remove the
retina. The eyecup was transferred to 1 M PBS for further dissection.
The retina was gently eased from the sclera using fine forceps, taking
care to leave the ora serrata intact because it defines the edge of the
retina. The retina was then placed onto a TESPA
(3'-aminopropyltriethoxysilane)-coated slide and flattened by making
four or five incisions perpendicular to its outer edge. At this stage
as much vitreous as possible was removed using cellulose sponges and
scissors.
The flattened, wholemounted retinas were permeabilized in 70% vol/vol ethanol (kept at -20°C) for 20 minutes and then in 1 M PBS/1% Triton X-100 for 30 minutes. The retinas were then incubated with biotinylated Griffonia simplicifolia (Bandeiraea) isolectin B4 (ICN, Hampshire, UK) at 5 µg/ml in 1 M PBS overnight at 4°C. They were rinsed in 1 M PBS/1% Triton X-100 for 10 minutes and then twice in 1 M PBS for 10 minutes. Streptavidin-conjugated fluorescein isothiocyanate (FITC; Sigma, St. Louis, MO) at 25 µg/ml in 1 M PBS was added for 4 hours at room temperature, and the slides were rinsed twice in 1 M PBS for 10 minutes each. The retinas were mounted in PBS:glycerol (2:1), and the coverslip was sealed with nail varnish.
The stained, flatmounted retinas were viewed using an argon krypton laser confocal microscope (Leica Microsystems GmBH, Heidelberg, Germany), which allowed low- and high-powered images to be taken and digitally stored for later analysis.
Capillary Density.
Capillary bed sample areas were chosen in the central retina with no
major vessels present in the fields analyzed. Five areas of capillary
vasculature in each retina were imaged at x100 magnification and
stored for later analysis. All stored files were assigned a random
number to mask the observers, and counts of the number of branches were
made. One observer counted all files, and a second observer counted a
subset to compare results. There was a statistically significant
correlation (P < 0.05) between the two observers using
a Pearson correlation and no evidence of bias assessed using a
Bland-Altmann plot.
Avascular Areas
Digitized images of the total retinal area and peripheral
avascular area were measured using Scion Image Software (Scion
Corporation, Frederick, MD), and the avascular area was expressed as a
percentage of the total retinal area.
Immunohistochemistry
The whole enucleated eyeball was pierced by a needle and fixed in
4% PFA for 2 hours. It was washed twice in 1 M PBS (pH 7.4) before
being embedded in agarose (1.5% in 1 M PBS, pH 7.4, supplemented with
5% sucrose). The solid agarose blocks were trimmed and left in 30%
sucrose overnight at 4°C or until the agarose block sank. The blocks
were then frozen slowly and stored at -70°C until sectioning. Serial
10-µm-thick cryosections were made from whole frozen eyes and
incubated for 1 hour using biotinylated G. simplicifolia
(Bandeiraea) isolectin B4 (ICN) at 12.5 µg/ml in Tris-buffered saline
(TBS). After successive washes in TBS, the slides were then incubated
with peroxidase-labeled streptavidin (Dako, High Wycombe, UK) at 8.75
µg/ml for 1 hour. After washing, DAB was applied for 5 minutes, and
the slides were rinsed in tap water and counterstained in hematoxylin
before being mounted in Depex (BDH Chemicals, Poole, UK) and viewed
under a light microscope. Slides were analyzed for preretinal vessels
that grow out from the surface of the retina.
Cryosections
Sections of 10 µm were cut from frozen eyes (as above) using a
Leica CM 1300 cryostat and transferred to TESPA-coated slides. Slides
were air-dried for at least 30 minutes before storage at -20°C until
required.
VEGF Probes.
DNA from exons 1 to 4 (392 bp) were cloned into a pBluescript II KS
(Stratagene, La Jolla, CA) at the SacII site (gift
from Steve Charnock-Jones, University of Cambridge, UK). The plasmid
was digested with SacI restriction endonuclease and
end-filled using T4 DNA polymerase. A DIG-labeled sense probe was
generated using T3 RNA polymerase (Roche Molecular
Biochemicals, East Sussex, UK). The antisense probe was
generated by digesting the plasmid with BamHI restriction
endonuclease. After purification using Elutip columns (Schleicher &
Schuell, Keene, NH), the DNA was transcribed in vitro with T7
polymerase in the presence of DIG-UTP (Roche Molecular Biochemicals).
The size of both probes was confirmed using agarose gel
electrophoresis.
Methods.
Frozen sections were allowed to defrost at room temperature for at
least 1 hour and hybridized with probe overnight at 65°C. Slides were
washed at 65°C in SSC buffer (3 M sodium chloride, 0.3 M sodium
citrate, pH 7) with 50% formamide and 0.1% Triton X-100 and then at
room temperature in TBST (0.14 M NaCl, 2.7 mM KCl, 0.025 M Tris-HCl, pH
7.5, 1% Triton X-100). Slides were then blocked in 10%
heat-inactivated sheep serum (in TBST) for >l hour at room
temperature. Anti-DIG AP-Fab fragment (in 10% heat-inactivated sheep
serum in TBST) was then added to each section and incubated overnight
in a humidified chamber at 4°C. Slides were washed in TBST at room
temperature and then in NTMT (100 mM NaCl, 100 mM Tris-HCl, pH 9.5, 50
mM MgCl2, 0.1% Triton X-100). Staining was
performed in the dark with nitroblue tetrazolium (NBT + 3.5%
5-bromo-4-chloro-3-indolyl phosphate [BCIP] in NTMT. After a few
hours, staining reaction was checked, though color development
could take up to 24 hours at 4°C. The reaction was stopped using
distilled water, and the sections fixed in 4% PFA/0.1% glutaraldehyde
for 20 minutes. The sections were dehydrated through a series of
alcohols and then counterstained with filtered 0.1% eosin in 95%
ethanol for 20 to 30 seconds. Rinses were made in 95% and then in
100% ethanol before transferral to histoclear and mounting in Vecta
mount (Vector Laboratories, Peterborough, UK).
VEGF Concentration in Retinal Sections.
The presence or absence of VEGF mRNA assessed by in situ hybridization
in retinal sections was qualitatively scored by two masked, independent
observers.
Statistics
Summary statistics are presented as means ± SD or median and
interquartile range. Correlation between groups was by Pearson
correlation and correlation of between-group differences was made by
Mann–Whitney U. The capillary branching was
compared between groups by multilevel analysis using the software
package MLWin (Institute of Education, University of London).
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Results |
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Retinal Wholemounts
Peripheral Avascularity.
Thirty animals were assessed in each of the three groups at 14 days
postnatal age. Control animals had fully vascularized retinas; those
exposed to 12-hour-variable oxygen injury had a median 10% peripheral
avascular area, and the minute-variable oxygen had median 1.7%
peripheral avascularity (Table 1)
. The differences between groups were statistically significant
in all cases (P < 0.001; Mann–Whitney U).
The degree of peripheral avascularity in group P is consistent with
that seen with other extreme oxygen injury models in the rat.
Penn17
produced 8% peripheral avascularity at 14 days
after a 24-hour-variable 80/40% oxygen regimen but without
neovascularization.6
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Abnormal Vessels
Observers noted no extraretinal neovascularization on the flatmounts,
though these are often difficult to distinguish in these preparations.
However, two masked observers noted abnormal terminal dilatations
present at the vascular/avascular interface of 73% of retinas from
group P and in 21% of retinas from group V. Figure 2J
shows a typical
example of one of these terminal dilatations from the group P. They
were stained with endothelial cell–specific lectin and may represent
endothelial cell proliferations.
Assessment of VEGF by In Situ Hybridization
VEGF mRNA was demonstrated in retinas using in situ hybridization
and scored semiquantitatively. Sense controls were included and showed
no staining (results not shown). VEGF was found in all three groups in
both the anterior retina and the retina as a whole, though staining was
more intense in the anterior retina in most specimens. There was an
increasing strength of staining present in group V compared with group
C, and in group P when compared with group V. Figures 2G
2H
2I
demonstrate a typical cryosection with VEGF mRNA stained in black from
a retina in each of the three groups. VEGF is evident in the inner
nuclear layer as previously reported.18
Immunohistochemistry
Immunohistochemistry was performed on serial cryosections from
each group, but no evidence of extraretinal neovascularization was
seen.
Postexperimental Weights
Mean body weights at 14 days were as follows: group C, 29.2 g
(95% confidence interval [CI], 28.3–30.2 g); group P, 28.6 g
(CI, 27.8–29.5 g); and group V, 23.7 g (CI, 23.0–24.4 g). The
difference between the variable group and other two was significant
(P < 0.001; t-test), but there was no
difference between group P and group C.
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Discussion |
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The development of models for identifying the pathogenesis of ROP has predominantly concentrated on extreme oxygen injury and are based on early and pivotal work by Ashton19 that first established that oxygen was important in disrupting retinal blood vessel development. Although these models are able to induce retinopathy with neovascularization,4 5 they have not closely represented the arterial oxygen of the preterm infant who might in current times develop the disease, and this has been considered a potential weakness in understanding the precise pathogenesis of the disease in extremely preterm infants. Work by Penn6 15 clearly delineated that the relative degree of hyperoxia and hypoxia were important in inducing OIR,17 and his most successful model, which produced severe OIR with neovascularization in all retinas, used fluctuations between 10% and 50% inspired oxygen. Penn’s levels of oxygen mimic the extremes that a preterm neonate could experience and support the concept that our understanding of retinopathy may be enhanced by models more closely representing preterm arterial oxygen levels. We have extended that concept and modeled our fluctuations directly on those that were experienced by a preterm infant that developed severe ROP. The extremes of our oxygen fluctuations were similar to Penn’s (9.2–41.5%); however, each step change was relatively small but frequent. Although both our model and the extreme injury models are able to induce retinopathy, only the extreme injury models are successful in inducing the later, proliferative stages of the disease.
The ability of small frequent fluctuations in oxygen to create retinopathy may be linked to the intrinsic nature of VEGF induction and suppression. VEGF has a relatively short half-life in normoxia (40 minutes) when compared with other mammalian mRNA but is stabilized during hypoxia.20 Although this enables a rapid response rate to stimulus, in the preterm infant, persistently fluctuating oxygen may make this system too responsive and inefficient in producing stable new blood vessels. In our model periods of relative hyperoxia alternate with periods of relative hypoxia (Fig. 1) , and presumably, stimulation would alternate rapidly with suppression. Pierce et al.21 have demonstrated that hypoxic stimulation of VEGF can be reversed by a 24-hour exposure to hyperoxia, which reduced VEGF mRNA to undetectable levels and reduced VEGF protein to 70% of its hypoxic level. Their study did not assess the effects of shorter periods of hypoxia/hyperoxic stimulation and suppression of VEGF. We assessed VEGF mRNA in our model using in situ hybridization, which does not quantify the amount of VEGF mRNA present. However, in group V, VEGF mRNA was clearly present centrally within the inner nuclear layer and both peripherally and centrally throughout the retina. In contrast, room air controls had little or no VEGF mRNA present.
Neovascularization was not produced in our model. However, in both our minute-variable and 12-hour-variable pups we did see abnormal terminal dilatations present at the vascular/avascular interface of the retina. These could either represent precursors of pathologic neovascularization or simply endothelial proliferation at the leading edge of retinal vasculogenesis. A period in room air has induced neovascularization in the 12-hour-variable model6 and might have done so in our model. However, we chose not to have a room air exposure period at the end of the experiment in any of our experimental groups because preterm infants do not experience an acute switch from high to relatively low oxygen during which they develop neovascularization. They progress to threshold ROP while still experiencing variable arterial oxygen, probably as a result of more chronic lung disease.
Although the lack of neovascularization in our minute-variable model
may be seen as indicating a poor model of OIR, we believe that the
pathophysiological process induced by our model may be more
representative of the earlier stages of ROP than the extreme oxygen
injury models. We ran an extreme oxygen injury model of 12-hour
variations between 80% and 21% for 14 days as a comparison for our
minute-variable model. In the 12-hour-variable group we demonstrated a
large avascular peripheral retina and VEGF staining in the inner
nuclear layer but no central vaso-obliteration. The larger vessels had
capillary free zones (Fig. 2E)
, but the capillaries between these areas
were dilated compared with controls, and there were more capillary
branches, which suggested that the vasculature may be immature. Other
investigators have demonstrated occlusion and obliteration of
capillaries both centrally and peripherally15
17
22
23
using an extreme oxygen injury models in rats, and our inability to
produce central vaso-obliteration is difficult to interpret. The
experimental protocol we used was not identical with others, in that we
used a combination of Reynauds23
60%
Fio2 and Penn’s17
12-hour cycles.
Further work is needed with our model to assess the precise pathophysiology induced by frequent small fluctuations and to assess the degree of capillary obliteration and astrocyte injury. In addition, we need to further assess the influence of frequent oxygen fluctuation on vascular stabilization in our minute-variable group.
Our novel animal model, based on the arterial oxygen data derived from an infant with ROP, is able to more closely represent the retinal oxygenation of a preterm infant developing ROP than has previously been possible. Though the retinopathy produced was not as severe as with other extreme injury models, perhaps this will enable us in the future to delineate a pathophysiological process in the disease that has not been represented by previous animal models.
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Acknowledgements |
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Footnotes |
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Supported by Royal Blind School, Edinburgh, United Kingdom; Mason Medical Foundation; Ross Foundation for the Prevention of Blindness; Research into Eye Disease Trust; and the Royal Society.
Submitted for publication June 6, 2000; revised August 3, 2000; accepted August 17, 2000.
Commercial relationships policy: N.
Corresponding author: Janet R. McColm, Child Life and Health, Reproductive and Developmental Sciences, University of Edinburgh, 20 Sylvan Place, Edinburgh EH9 1UW, UK. jan.mccolm{at}ed.ac.uk
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