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1 From the Department of Anatomy and Cell Biology; and the 2 Kresge Eye Institute, Wayne State University, Detroit, Michigan.
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Abstract |
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METHODS. Newborn rats were first raised in either room air (controls) or
variable oxygen (50%/10%) for 14 days. The experimental rats were
recovered during the next 6 days (until day 20) in either room air
(21% O2) or supplemental oxygen (28%). All
groups were then exposed to room air for an additional 6 days (until
day 26). On day 20, magnetic resonance imaging (MRI) was used to
determine the panretinal oxygenation response
(
PO2, mm Hg) to a carbogen (95%
O2/5% CO2) inhalation
challenge. On days 20 and 26, the retinas from a different
subset of control, room air–recovered, or SOR-recovered animals were
analyzed using ADPase stained or fluorescein-labeled dextran infused
retinal flatmounts.
RESULTS. On day 20, the panretinal PO2 of
the room air–recovered group (125 ± 5 mm Hg, mean ± SEM,
n = 12) was significantly (P < 0.05)
lower than that of the control group (179 ± 6 mm Hg,
n = 11). The panretinal
PO2 value for the SOR
group (87 ± 5 mm Hg, n = 7) was significantly
(P < 0.05) lower than both the room air–recovered
group and the control group. The NV incidence and severity were
significantly reduced (P < 0.05) in the SOR animals
compared with the room air–recovered animals. In contrast, on day 26
(after 6 days in room air), the NV incidence was statistically
(P < 0.05) greater in the animals that had been
exposed to SOR compared with room air–recovered animals.
CONCLUSIONS. After 28% SOR, the expected decrease in NV incidence and severity occurred but with an unexpected decrease in panretinal oxygenation ability. The present data strongly support an association between subnormal panretinal oxygenation ability and increased NV risk in the newborn rat ROP model. MRI appears to be a powerful new approach for quantitatively and noninvasively measuring retinal oxygenation and may be applicable to study other ischemic or ischemia-related retinopathies in addition to ROP, such as diabetic retinopathy, sickle cell retinopathy, macular degeneration, and glaucoma.
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Introduction |
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The majority of experimental efforts to study the effect of supplemental oxygen on the NV outcome involved the kitten ROP model.1 2 In this model, kittens are placed in a relatively high oxygen environment (>70%) for 4 to 5 days. This process produces extensive panretinal vessel obliteration. The animals were then allowed to recover in either room air (21% O2) or supplemental oxygen. In contrast, present-day premature infants are not exposed to constantly high oxygen levels. Instead, they frequently experience relatively smaller fluctuations above and below systemic normoxia.5 Recently, a newborn rat model of ROP has been developed with similarities to the clinical conditions.6 7 8 In this model, newborn rats are exposed to variable oxygen for the first 14 days and allowed to recover in room air for the next 6 days.6 7 8 This procedure produces NV in 100% of the eyes by day 20.6 7 8 The location of the NV at the border of the vascular and avascular retina and its morphology resembles that found in human ROP.6 7 8
Previously, we demonstrated a novel magnetic resonance imaging (MRI)
method that noninvasively investigates retinal oxygenation in newborn
rats. We found agreement between the MRI retinal oxygenation
measurement and that determined using an oxygen electrode in the normal
rat retina under similar conditions.9
The MRI method
measures the panretinal oxygenation response
(PO2, in mm Hg) produced during a
carbogen (95% O2/5% CO2)
inhalation challenge.9
10
11
12
Normally, carbogen breathing
induces a relatively larger oxygenation response from the retinal
circulation, compared with that produced during 100% oxygen
breathing.9
It is thought that this larger oxygenation
response is produced by minimizing the hyperoxia-induced
vasoconstriction/autoregulation.9
However, in the vascular
retina, if perfusion or perfusion reserve is low and/or retinal
autoregulation dysfunctional, then a smaller than normal
PO2 will be produced during the
carbogen challenge (see below). Previously, we found a subnormal
panretinal oxygenation response to carbogen breathing before the
development of NV in the newborn rat model of ROP.12
In
the present study, we used the newborn ROP model to examine whether or
not the panretinal oxygenation response is also subnormal during the
appearance of NV in rats recovered from the variable oxygen procedure
in either room air or 28% supplemental oxygen. The long-term objective
of this research is to better understand the role of retinal
oxygenation in the development of intraretinal and preretinal NV so
that more effective diagnostic, treatment, and prevention strategies
may be developed.
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Methods |
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Animal Model
The newborn rat model of ROP has been described in detail
elsewhere.6
7
8
Briefly, Sprague–Dawley mothers and
litters (12–15 pups/litter) were housed in a modified pediatric
incubator where the oxygen levels were varied every 24 hours between
50% and 10% oxygen for the first 14 days after birth. Note that the
dams received were not always "proven breeders" and did not always
produce enough milk for the pups; litters less than 12 pups were not
used in this study. Rats were then allowed to recover in either room
air (21% O2) or supplemental oxygen (28%)
during the next 6 days (days 14–20). An additional subset of animals
from each group was exposed to room air for the next 6 days. The
different subsets of animals were examined either histologically on
days 20 and 26 or by MRI on day 20 (during the appearance of
NV.6
7
8
Animals studied by MRI were not studied
histologically.
MRI Examination
The MRI procedure has been described in detail
elsewhere.12
Briefly, on the day of the examination,
urethane-anesthetized animals (0.083 ml of a 36% solution of
urethane/20 g animal weight, intraperitoneally, freshly made daily;
Aldrich, Milwaukee, WI) were gently positioned on a homemade
MRI-compatible holder with their noses placed in a plastic nose cone
and allowed to breathe spontaneously. Core temperature and pulse and
hemoglobin oxygen saturation (data not shown) were continuously
monitored and maintained. MRI data were acquired on a 4.7-T system
using a two-turn surface coil (1.5-cm diameter) placed over the eye and
a spin-echo imaging sequence (repetition time, TR, 1 second; echo time,
TE, 22.7 msec; number of acquisitions NA 1; matrix size 128 x
256; slice thickness 1 mm; field of view 28 x 28 mm; sweep width
25,000 Hz; 2 min/image). The 2-minute image acquisition time provides a
compromise condition between the conflicting demands of achieving high
signal-to-noise and adequate resolution to observe oxygen filling of
approximately half the vitreous in the newborn rat eye.12
A capillary tube (1.5-mm inner diameter) filled with distilled water
was used as the external standard. Seven sequential 2-minute images
were acquired as follows: six control images while the animal breathed
room air and one image during carbogen breathing. The animals were
switched over to carbogen at the same phase encode step near the end of
the acquisition of the sixth image. This procedure was standardized for
every animal. If some accident prevented this timing, the experiment
was aborted and restarted again after a 10-minute "reset" time.
Animals were returned to room air for 15 minutes (to allow recovery
from the inhalation challenge) and removed from the magnet. While
maintaining the core body temperature, blood from the abdominal aorta
was collected immediately after a second 2-minute carbogen challenge
and analyzed for PaO2,
PaCO2, pH, and glucose concentration. After the
blood collection, the animal was euthanatized with an intracardiac
potassium chloride injection.
Attempts to measure preretinal vitreous PO2 by subtracting images obtained during room air breathing and death (or hypoxemia) were not successful (data not shown). The major problem was that ocular perfusion pressure and eye shape changed from normal during death or hypoxemia. These changes significantly confounded quantitative pixel-by-pixel interpretation of the data. Furthermore, producing death or hypoxemia in humans is clearly difficult. Thus, we chose carbogen breathing because this method induces a maximum oxygenation response from the retinal circulation (compared with oxygen alone,9 makes detection of subtle differences between groups more robust and precise, and has clinical potential.
Data Analysis
To be included in the MRI part of this study, the animal must have
demonstrated minimal movement (eye and/or head) during the MRI
examination, nongasping respiratory pattern before the MRI examination
(i.e., no repeated and visible difficulties in breathing enough to move
the head), core temperatures in the range of 36.5°C to 38.5°C
during the MRI examination, and PaO2 > 350 mm Hg
and PaCO2 ranging from 45 to 65 mm Hg during the
carbogen challenge. The number of animals examined by MRI on day 20
that satisfied the inclusion criterion for the room air control, room
air–recovered, and SOR groups were 11, 12, and 7, respectively. Forty
percent of the animals studied by MRI were not included in the final
analysis because, primarily, they did not satisfy condition 4 above.
This was because of poor maintenance of the animals’ core temperature
during blood collection. Improvements in core temperature maintenance
has brought the rejection rate to 5% to 10%.
The MRI data were studied by first converting, on a pixel-by-pixel basis, signal intensity changes during carbogen breathing to oxygenation response values. All pixels along a 1-pixel-thick line (200 µm), drawn at the boundary of retina/choroid and vitreous from the superior ora at top, through the optic nerve, to the inferior ora at the bottom (identified by the clear contrast differences between the preretinal vitreous, retina/choroid, and ciliary body/iris), were set to black. Next, a different 1-pixel-thick line was drawn in the preretinal vitreous space (immediately adjacent to the black pixels), and 54 pixels along this region of interest were extracted into a preretinal vitreous oxygenation response band. Each pixel (i.e., color band) is the median oxygenation response (volume averaged over a 1-mm section of preretinal vitreous in the nasotemporal direction) from across all retinas in that group at that distance from the optic nerve. Because these data were sampled from similar preretinal vitreous volumes, the potentially confounding effect of preretinal oxygen gradients on the retinal oxygenation measurement is minimized. Calculations suggest that oxygen diffusing from the hyaloidal circulation during a 2-minute carbogen challenge could confound interpretation of the regions within 0.5 mm from the optic nerve. Consequently, we did not analyze regions ±0.5 mm from the optic nerve.12 To illustrate this, these regions were blanked out in the preretinal vitreous oxygenation response bands. No statistical evidence (P > 0.05) for an asymmetrical hemiretinal (i.e., superior to the optic nerve versus inferior to the optic nerve) oxygenation response was found in any group. Therefore, the superior and inferior hemiretinal values for each pixel equidistance from the optic nerve were averaged. The average values were used as the set of observations for each animal for further comparisons.
The hemiretinal averaged oxygenation responses for a superior–inferior pixel (excluding those within 5 mm of the optic nerve) for each group were not normally distributed and were compared using a Mann–Whitney rank sum (after log transformation) test, a Kolmogorov–Smirnov two-sample test, and a Kruskal–Wallis multiple comparison test. P < 0.05 was considered significant. To illustrate variations in the oxygenation response between animals within the same group, the averaged hemiretinal data are presented as a scattergram (Fig. 1) . To illustrate the differences in the shape of the distribution of oxygenation responses between the groups, the data are also presented as a histogram (Fig. 1) . To illustrate the spatial variations of the retinal oxygenation responses for each group, a median oxygenation response band for each group was constructed on a pixel-by-pixel basis from the individual oxygenation response bands for each animal in that group (Fig. 2) . To illustrate the relationship between the MRI data (which represents a measure of retinal perfusion, see below) and retinal vessel patency, the composite median oxygenation response band for a group was superimposed on a representative fluorescein-labeled dextran-infused retinal flatmount for that group (Fig. 2) .
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Results |
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Systemic Physiology
A summary of the blood parameters measured during a 2-minute
carbogen challenge is present in Table 2
. Although there were some differences between the groups, all the blood
gas values fell within the expected range for carbogen breathing.
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PO2 was approximately
30% lower in the supplemental oxygen–recovered group
(P = 0.0003, Kruskal–Wallis multiple comparison test). |
Discussion |
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PO2 is expected to reflect
primarily the change in retinal oxygen supply and be sensitive to a
variety of vascular physiological processes governing retinal oxygen
supply during the carbogen challenge, such as retinal perfusion,
perfusion reserve, and vessel autoregulation. Thus, we speculate that
the variable oxygen exposure produced damage to some combination of
retinal perfusion, perfusion reserve, or autoregulation. The SOR
procedure used in this study appears to further damage the ability of
retinal circulation to oxygenate. Experiments are ongoing in this
laboratory to further examine these possibilities. It is possible that the MRI retinal oxygenation response differences between the groups in this study were due to systemic physiological differences in the response to carbogen rather than to local retinal effects. To address this concern, various physiological parameters were measured and compared for each group. All the values measured fell within the expected range for carbogen breathing.9 Only the arterial blood oxygen tensions and glucose levels were significantly higher (P < 0.05) between the SOR group and each of the other 2 groups. Could the higher arterial oxygen tensions in the SOR group account for its relatively lower oxygenation response? This is considered unlikely because during carbogen breathing the hyperoxia occurs with hypercapnia. Because the arterial carbon dioxide levels are elevated to the same degree in all groups, and because hypercapnia-related vasodilation occurs even during hyperoxia,9 we do not expect that differences in arterial oxygenation of this magnitude between groups to account for the differences in retinal oxygenation response. Similarly, the higher blood glucose level seen in the SOR group versus those in the control and room air–recovered groups might be expected, if it has any effect, to decrease the retina’s ability to autoregulate in response to oxygen.16 However, this would tend to increase retinal oxygenation and, presumably, the response to carbogen breathing. Differences in arterial glucose levels of this magnitude do not appear to account for the observed differences in retinal oxygenation. Thus, the retinal response differences between the groups of rats appear to be a local retinal phenomenon and not due to systemic physiological differences between groups.
Could the relatively lower panretinal oxygenation response of the SOR be due to its relatively lower NV tuft density? This is considered unlikely because the NV makes up a small fraction of the retinal vasculature and so is not likely to contribute substantially to the panretinal oxygenation response. Furthermore, because the NV typically consists of multicellular tufts or sheets without lumina, this would not be expected to significantly contribute to retinal perfusion or oxygenation.14
In this study, immediately after SOR, the NV incidence and severity decreased on day 20 compared with room air-recovered animals in this model. Even though there are species, dose, timing, and insult differences between the newborn rat and kitten ROP models, the decrease in NV severity observed in the present study supports the results of previous supplemental oxygen studies in the kitten.1 2 In addition, our finding of a similar NV severity at day 26 for animals exposed to either supplemental oxygen or room air (1 clock-hour) also appears to agree with the work of Chan–Ling et al.2 These authors report no differences in "vascular pathology" in kittens exposed to either room air or 50% oxygen for 8 days after stopping supplemental oxygen treatment. Unfortunately, there are no reports in the kitten literature on the effect of supplemental oxygen on NV incidence either immediately after supplemental oxygen or after some additional period in room air. Thus, our finding of a greater NV incidence 6 days after stopping supplemental oxygen, compared with the room air–recovered animals, is novel.
The exact mechanism underlying the reduction in NV incidence and severity on day 20 after SOR is not known. SOR is expected to elevate the retinal PO2, and this is thought to relieve the presumed retinal hypoxia that is hypothesized to play a key role in the development of NV in ROP. The reduction in NV incidence and severity in the SOR group on day 20, relative to the room air–recovered group, is consistent with the concept that elevated tissue oxygen levels relieve, to some extent, the presumed hypoxia. The relative increase in NV incidence in the SOR group on day 26, compared with room air–recovered animals may be due to the following. The retinal demand for oxygen is likely increasing due to continuing maturation between days 20 and 26 in both the SOR and room air–recovered groups. However, because the oxygenation ability of the retinal circulation in the SOR group appears more impaired than in the room air–recovered group, a mismatch in oxygen supply and demand is likely to be relatively more severe in the SOR animals. This might lead to the relatively longer continuation of NV in a greater number of animals in the SOR group. The low oxygenation response observed in the present study is also consistent with the presence of hypoxia, but it cannot yet be unambiguously interpreted as a measure of hypoxia. Thus, the data in this work only indirectly provide evidence that the retina is hypoxic. Experiments in this laboratory are ongoing to directly measure retinal oxygen levels after SOR in experimental ROP.
In the present study, both room air–recovered and SOR groups had subnormal oxygenation responses during the appearance of NV. This result complements and extends our previous findings of a subnormal panretinal oxygenation response before the appearance of NV in this model.12 One weakness of the present study is that only 89% of the SOR animals developed NV on day 20. It is possible that some animals without NV were studied by MRI and may have skewed that group’s medians. However, retinas from animals without NV are expected to have a relatively greater panretinal oxygenation response than retinas with NV. This would decrease, not increase, the differences in retinal oxygenation between groups. In addition, the relatively smaller panretinal oxygenation response in the SOR groups was associated with a relatively greater NV incidence on day 26, compared with the room air–recovered animals. Taken together, these data underscore our previous hypothesis that a subnormal panretinal oxygenation response is strongly associated with an increased risk of retinopathy in experimental ROP.
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Acknowledgements |
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Footnotes |
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Submitted for publication March 1, 1999; revised August 6, October 15, and December 15, 1999; accepted January 18, 2000.
Commercial relationships policy: N.
Corresponding author: Bruce A. Berkowitz, Department of Anatomy and Cell Biology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201. baberko{at}med.wayne.edu
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