www.elsevier.com/locate/yexnrExperimental Neurology 189 (2004) 150–161
Transient exposure of rat pups to hyperoxia at normobaric and hyperbaric
pressures does not cause retinopathy of prematurity
John W. Calvert,a,b Changman Zhou,a and John H. Zhanga,b,*
aDepartment of Neurosurgery, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932, USAbDepartment of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, LA, USA
Received 5 March 2004; revised 23 April 2004; accepted 18 May 2004
Available online 3 July 2004
Abstract
We have shown that hyperoxia reduces brain damage in a rat model of hypoxia– ischemia. The purpose of this study was to examine the
possibility of hyperoxia in inducing vision-threatening retinopathy. Two different experiments were conducted in this study. PART 1: seven-
day-old rat pups were subjected to unilateral carotid artery ligation followed by 2 h of hypoxia (8% O2 at 37jC). Pups were treated with
100% oxygen at 1 ATA, 1.5 ATA, and 3.0 ATA for a duration of 1 h. PART 2: Newborn rat pups were exposed to 100% oxygen at 1, 1.5, or
3.0 ATA for 1 h, the same treatment protocol used for brain protection after hypoxia– ischemia. Retinopathy was evaluated by the degree of
neovascularization (measuring retinal vascular density), by the structural abnormalities (histology) in the retina, and by the expression of
hypoxia–hyperoxia sensitive proteins including hypoxia-inducible factor-1a (HIF-1a) and vascular endothelial growth factor (VEGF) at 24
h, 1, 2, and 10 weeks after hyperoxia exposure. Hyperoxic treatment at all pressures administered significantly reduced the hypoxia–
ischemic-induced reduction in brain weight. Retinal vascular density measurements revealed no signs of neovascularization after hyperoxia
exposure. There were also no abnormalities in the structure of the retina and no changes in the protein expression of HIF-1a and VEGF
following hyperoxia exposure. Exposure to hyperoxia for 1 h at normobaric or hyperbaric pressures did not result in the structural changes
or abnormal vascularization that is associated with retinopathy of prematurity, suggesting that hyperoxia is a safe treatment for hypoxic
newborn infants.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Retinopathy; VEGF; HIF-1a; HBO; Neonates
Introduction
The emergence of retinopathy of prematurity as a leading
cause of blindness in infants has occurred over the past 60
years, seemingly as a result of the advances in neonatal
intensive care practices that have allowed for the survival of
premature infants who have significant immature retinal
vasculature (Brooks et al., 2001; Miyamoto et al., 2002;
Smith, 2002; Stout and Stout, 2003). The incidence for
threshold (disease progression to the point of necessitating
peripheral retinal ablation therapy) retinopathy of prematu-
rity for premature infants weighing <1.25 kg is roughly 5%,
with about 20–30% of these infants becoming blind despite
treatment (Palmer, 2003; Reynolds, 2001).
0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.expneurol.2004.05.030
* Corresponding author. Department of Neurosurgery, Louisiana State
University Health Sciences Center, 1501 Kings Highway, Shreveport, LA
71130-3932. Fax: +1-318-675-8805.
E-mail address: [email protected] (J.H. Zhang).
Oxidant stress appears to play a role in the retinal vaso-
obliteration associated with retinopathy of prematurity
(Beauchamp et al., 2002; Weinberger et al., 2002). Exposure
to hyperoxia affects developing retinas by leading to micro-
vascular degeneration which produces inner retinal hypoxia,
which in turn leads to structural and functional changes.
These changes can lead to abnormal vascularization resulting
in the development of vision-threatening retinopathy (Beau-
champ et al., 2002; Brooks et al., 2001). Hypoxia–ischemia
is a common cause of brain injury in the perinatal period
leading to mental impairment, seizures, and permanent motor
deficits, such as cerebral palsy (Ferriero, 2001; Johnston,
2001). The role of supplemental oxygen therapy in the
development of retinopathy of prematurity was suggested
in the 1950s (Stout and Stout, 2003) and is a main reason for
not giving oxygen to hypoxic or premature infants (Neuba-
uer, 2002). But, recent data indicate that it is the withdrawal
from the oxygen environment that causes retinopathy of
prematurity and that subsequent oxygen exposure can cure
J.W. Calvert et al. / Experimental Neurology 189 (2004) 150–161 151
the situation (The STOP-ROP Multicenter Study Group,
2000; Neubauer, 2002). We have shown that a single treat-
ment of hyperbaric oxygen (HBO) (100% oxygen) for 1 h at 3
atmospheres absolute (ATA) attenuates brain damage caused
by a hypoxia–ischemia insult on the neonatal rat brain by
reducing the progression of apoptotic neuronal injury and
increasing sensorimotor function (Calvert et al., 2002, 2003).
Consideration of HBO treatment for hypoxia–ischemia is
derived from the belief that oxygen under increased pressure
might salvage the still viable, though nonfunctioning, tissue
surrounding the area of insult by increasing the amount of
oxygen dissolved in blood plasma, thereby increasing oxygen
delivery to the brain (Nighoghossian and Trouillas, 1997).
Also, by raising the amount of oxygen available to the brain
tissue, hyperbaric oxygen may trigger a mechanism control-
ling cellular and vascular repair, presumably allowing time
for collateral circulation to develop (Mink and Dutka, 1995).
But, questions still remain about the safety of elevated levels
of oxygen for the treatment of newborns, because experi-
mentally, it has been shown that newborn rat pups exposed to
oxygen at 1.8 ATA for 10 days did not show any signs of
retinopathy of prematurity (Ricci and Calogero, 1988),
whereas when newborn rat pups were exposed to a higher
pressure of 5 ATA for 5 h, retinopathy of prematurity was
evident (Torbati et al., 1995). Therefore, the purpose of this
study was to test the hypothesis that a single exposure to
100% oxygen for 1 h at various pressures (1, 1.5, and 3.0
ATA) would not produce retinopathy of prematurity in
newborn rat pups.
Materials and methods
Groups and oxygen exposure
The Animal and Ethics Review Committee at the Loui-
siana State University Health Sciences Center-Shreveport
evaluated and approved the protocol used in this study.
Timed pregnant female Sprague–Dawley rats were obtained
from Harlan Labs (USA). After birth, pups were housed
with the dam under a 12:12 h light/dark cycle, with food and
water available ad libitum throughout the study. This study
was divided into two parts.
Part 1: the model used in this study is based on the Rice–
Vannucci (Rice et al., 1981) model previously described
(Calvert et al., 2002, 2003). Unsexed 7-day-old (day 0 =
day of birth), Sprague–Dawley (Harlan) rats were anesthe-
tized by inhalation with isoflurane (0.1%) in oxygen. The
pups were kept at a temperature of 37jC as the right common
carotid artery of each pup was exposed and ligated with 5–0
surgical sutures. The duration of the anesthesia did not exceed
20 min, and the pups were allowed to recover with their dams
for 2 h. They were then placed in a jar perfused with a
humidified gas mixture (8% oxygen balanced nitrogen) for 2
h. Both the jar and the gas mixture were kept at 37jC. Thepups were returned to their dams after the hypoxic exposure.
The pups that underwent hyperoxia treatment were allowed
to recover from the hypoxic exposure for 1 h before being
placed in the HBO chamber (Sechrist Industries, Inc., Ana-
heim, CA). The hyperoxic treatment of 100% oxygen was
administered at pressures of 3 atmospheres absolute (ATA),
1.5 ATA, and 1.0 ATA for 1 h and the pups were then returned
to their cages and dam after the treatment. Only one hyper-
oxic exposure was conducted for each pup. These pups were
sacrificed 2 weeks after the hypoxic–ischemic insult. The
pups in this part of the study were divided into the following
groups: (1) control group (n = 5), (2) hypoxia–ischemia (HI)
group (n = 15), (3). HI + 3.0 ATA group (n = 15), HI + 1.5
ATA group (n = 15), (4). HI + 1.0 ATA group (n = 15).
Part 2: unsexed 7-day-old (day 0 = day of birth) pups
were divided into the following groups: (1) control group
(n = 28), (2) normobaric group, 1.0 ATA group (n = 34), (3)
1.5 ATA group (n = 30), (4) 3.0 ATA group (n = 32), and (5)
retinopathy of prematurity group (n = 12). Pups were placed
in a hyperbaric oxygen chamber (Sechrist Industries, Inc) to
expose them to oxygen. For 1, 1.5, and 3 ATA groups,
oxygen (100%) was administered for duration of 1 h at the
following pressures: 1 (normobaric), 1.5, and 3.0 ATA. Each
pup only received one exposure. Control pups were not
exposed to oxygen. The pups were sacrificed at various time
points after hyperoxia exposure: 24 h, 1, 2, and 10 weeks.
For the retinopathy of prematurity group, postnatal day 5
(P5) pups were exposed to 100% oxygen at a pressure of 3
ATA for 10 h. These pups were sacrificed at 24 h and 2
weeks to obtain positive controls for retinopathy.
Brain weight
The pups from Part 1 were sacrificed under deep anes-
thesia. After removal of the brain, the cerebellum and brain
stem were removed from the forebrain. The hemispheres
were separated by a midline incision and then weighed on a
high precision balance (sensitivity F 0.001 g). The cerebel-
lum was also weighed. Brain damage was expressed as the
percent reduction of the ipsilateral (right) hemisphere com-
pared to the contralateral (left) hemisphere.
Retinal vascular density
Retinal vascular density was determined as previously
described (Torbati et al., 1995). The thorax was opened and
the left ventricle was perfused with 4 ml of India ink (Design
Higgins, Sanford, USA) for pups in the 1 and 2 weeks groups
and 12 ml for the rats in the 10-week group. After infusion of
ink, eyes were enucleated and placed in 2% formalin. Under a
dissecting microscope, the retinas were separated placed on
glass microscope slides and mounted with coverslips. Images
were captured using an imaging system that included an
Olympus BX51 microscope with MagnaFire capturing soft-
ware (SP2.1B) at magnifications of 1.25� and 10� and
examined with Scion Image for windows (Scion Corp.).
The retinal vascular density within an image was defined as
Fig. 1. Brain weight 2 weeks after hypoxia– ischemia and subsequent
hyperoxia exposure. #P<0.05 compared with control, **P < 0.05 compared
with HI, and yP < 0.05 compared with both control and HI (ANOVA).
J.W. Calvert et al. / Experimental Neurology 189 (2004) 150–161152
the ratio of the total number of pixels representing the retinal
vessels divided by the total number of pixels representing
both the retinal tissue and retinal vessels.
Histology and cell counts
At the appropriate time (24 h, 2 and 10 weeks) after
hyperoxia exposure, rats (n = 3 per group) were anesthetized
with a-chloralose (40 mg/kg, i.p.)/urethane (400 mg/kg, i.p.).
The thorax was opened and the left ventricle was perfused
with 60 ml of 0.1M PBS (pH 7.4) followed by 60 ml of 2%
glutaraldehyde and 2% formalin in 0.1M PBS (pH 7.4).
Following perfusion, the eyes were enucleated and post-fixed
in the same fixative and stored at 4jC. The eyes were then
placed in 3% sucrose just before being sectioned. The eyes
were cut across the conjunctiva and the cornea and crystalline
lens were removed. The eye was then cut in half and placed in
tissue freezing medium (Triangle Biomedical Sciences,
USA). Four-micron sections were cut using a cryostat (Leica
LM 3050S). Sections prepared from samples taken at 24
h after hyperoxia exposure were used for immunohistoche-
mistry and sections prepared from samples taken at 2 and 10
weeks were stained with hematoxylin and eosin.
To analyze the retinal sections stained with hematoxylin
and eosin, images of intact sections were captured at a
magnification of 40� and analyzed using Image-Pro Plus
4.5.1 software. For each eye, two slides containing four
sections were analyzed (representing whole eye). The num-
ber of neurons in the inner nuclear layer (INL), the number
of neurons in the outer nuclear layer (ONL), the thickness of
the outer plexiform layers (OPL), and the areas of both the
INL and ONL were determined. For each section, these
measurements were made in five distinct areas of the retina
(see schematic in Fig. 4). The counts from each of these
areas were then averaged to represent the counts for each
section. The counts for each of these sections were then
averaged to represent the counts for each eye.
Immunohistochemistry
Immunohistochemistry was done as previously described
(Zhou et al., 2003) using an ABC Staining System (Santa
Cruz Biotechnology, USA). Briefly, sections prepared from
the samples taken at 24 h after hyperoxia exposure were
used for immunohistochemistry. These sections were
allowed to air dry before being washed in 0.01M PBS
followed by incubation in 3% hydrogen peroxide (H2O2)
to prevent reactions with endogenous peroxidases. This was
followed by washing in PBS and then incubation with 3%
normal serum in PBS (blocking solution). Sections were
then incubated with either rabbit anti-VEGF antibodies
(147) or rabbit anti-HIF-1a antibodies (H206) (Santa Cruz
Biotechnology) diluted in blocking solution (1:100) over-
night at 4jC followed by washing in PBS and incubation
with anti-rabbit secondary antibodies. After washing again
in PBS, sections were incubated with avidin–peroxidase
complex solution containing avidin–peroxidase conjugate.
The sections were then washed in PBS and peroxidase
activity was revealed by dipping the sections in a mixture
containing 3-3V diaminobenzidine (DAB) and H2O2 fol-
lowed by washing in distilled water. All the procedures were
conducted at room temperature. Sections were allowed to air
dry before being dehydrated and mounted with coverslips.
Images of the slides were captured using the imaging system
described above. Application of a control serum instead of
the primary antibody on sections provided negative controls.
Statistical analysis
The retinal vascular density values as well as the values
from the histological analysis are expressed as the means
F SEM. The values were analyzed by one-way analysis of
variances (ANOVA), followed by Tukey test. A P value less
than 5% was considered significant.
Results
Brain weight
To demonstrate that elevated levels of oxygen at normo-
baric and hyperbaric pressures can be neuroprotective, we
subjected neonatal rats to a hypoxic–ischemic insult fol-
lowed by 1 h of 100% oxygen at 1 ATA, 1.5 ATA, and 3
ATA. Two weeks after the insult and subsequent oxygen
treatment, the pups were sacrificed and the brains of those
pups were divided into two regions, contralateral hemi-
sphere and ipsilateral hemisphere. Brain damage was then
assessed by dividing the ipsilateral hemispheric weight by
the contralateral hemispheric weight and expressing this as a
percentage (Fig. 1). This rat model of hypoxia–ischemia
yields a reproducible pattern of hemispheric injury ipsila-
teral, but not contralateral to the ligated carotid artery (Han
et al., 2000). Since animals that are subjected to a hypoxic–
J.W. Calvert et al. / Experimental Neurology 189 (2004) 150–161 153
ischemic insult show retardation in brain growth, the percent
reduction in ipsilateral brain weight to contralateral brain
weight allows for the evaluation and testing of neuropro-
tective agents and strategies. The ipsilateral hemisphere was
Fig. 2. Flat mounts of retinas of newborn rats exposed to hyperoxia at 1 atmosphe
week after hyperoxia exposure. (B) Inversion of retinal images in A to provide a b
Inversion of images in C. (E) Ten weeks after hyperoxia exposure. (F) Inversion
found to be 54.3% of the contralateral hemisphere after the
hypoxic– ischemic insult and hyperoxic treatment at all
pressures administered significantly reduced this damage,
as brain weights were found to be 78.168%, 81.230%, and
re absolute (ATA), 1.5 ATA, or 3.0 ATA and age-matched controls. (A) One
etter look at the vasculature. (C) Two weeks after hyperoxia exposure. (D)
of images in E. Scale bar = 1 mm.
Fig. 3. Typical sections of the retinas taken from newborn rat pups 1, 2, and 10 weeks after exposure to hyperoxia. These are higher magnification (10�)
images of those found in Fig. 1 and provide an image from which the retinal vascular density may be calculated. (A–C) Age-matched controls. (D) 1 ATA
group, 1 week. (E) 1 ATA, 2 weeks. (F) 1 ATA group, 10 weeks. (G) 1.5 ATA group 1 week. (H) 1.5 ATA group, 2 weeks. (I) 1.5 ATA group, 10 weeks. (J) 3.0
ATA group, 1 week. (K) 3.0 ATA group, 2 weeks. (L) 3.0 ATA group, 10 weeks. Scale bar = 50 Am.
J.W. Calvert et al. / Experimental Neurology 189 (2004) 150–161154
70.497% after administration of 100% oxygen at 3.0 ATA,
1.5 ATA, and 1.0 ATA, respectively.
Retinal vascular density
We next tested if the hyperoxia that produced neuro-
protection would result in retinopathy of prematurity. Ret-
inal vasculature of newborn rat pups exposed to hyperoxia
Table 1
Structural assessment of retina after hyperoxia exposure
Groups INL
(no. of cells)
INL no./area
(no./Am2)
OPL thickne
(Am)
Control 2 weeks 118 F 7.077 0.0127 F 0.000583 16.527 F 0.
1 ATA 2 weeks 112 F 3.228 0.0136 F 0.000322 16.882 F 0.
1.5 ATA 2 weeks 123 F 1.350 0.0144 F 0.000512 16.387 F 0.
3.0 ATA 2 weeks 117 F 1.802 0.0133 F 0.000577 16.759 F 0.
ROP 2 weeks 106 F 1.951 0.0166 F 0.00147 9.150 F 0.
Control 10 weeks 116 F 4.655 0.0130 F 0.000429 16.068 0.381
1 ATA 10 weeks 121 F 1.562 0.0131 F 0.000715 16.718 0.232
1.5 ATA 10 weeks 121 F 1.280 0.0134 F 0.000121 16.195 0.345
3.0 ATA 10 weeks 122 F 1.822 0.0124 F 0.000269 16.777 F 0.
Data are represented as mean F SEM. INL indicates inner nuclear layer; OPL, out
*P < 0.001 (ANOVA) compared to all other groups in 2-week time point.
at various pressures (1, 1.5, and 3 ATA) is shown in Fig. 2.
Higher magnification (10�) sections of the retinas (Fig. 3)
were used in the calculation of retinal vascular density for
each group (Table 1). No differences were observed in the
retinal flat mounts at low magnification or high magnifica-
tion and retinal vascular density calculations showed no
difference among the control, 1.0, 1.5, and 3.0 ATA groups
at 1 (data not shown), 2, and 10 weeks after hyperoxia
ss ONL
(no. of cells)
ONL no./area
(no./Am2)
RVD
115 317 F 0.391 0.0229 F 0.000930 0.0421 F 0.00596
286 326 F 9.426 0.0258 F 0.000521 0.0463 F 0.00252
187 331 F 8.161 0.0259 F 0.000589 0.0383 F 0.00374
168 325 F 6.423 0.0238 F 0.000146 0.0416 F 0.00410
453* 334 F 6.001 0.0281 F 0.00119 0.110 F 0.00667*
336 F 9.346 0.0241 F 0.000103 0.0457 F 0.00304
335 F 6.624 0.0242 F 0.000147 0.0444 F 0.0163
336 F 4.809 0.0246 F 0.000568 0.0442 F 0.00460
410 332 F 2.517 0.0242 F 0.000328 0.0436 F 0.00524
er plexiform layer; ONL, outer nuclear layer; RVD, retinal vascular density.
Fig. 4. Retinal vasculature of newborn rat pups 2 weeks after exposure to 100% oxygen at 3 ATA for 10 h. (A) Retinal flat-mount of age-matched control. (B)
Retinal flat-mount of pup from retinopathy of prematurity group. (C) Inversion of image in A. (D) Inversion of image in B. (E) Typical section of retina from an
age-matched control pup at higher magnification (10�). (F) Typical section of retinal from a pup from retinopathy of prematurity group at higher magnification
(10�). Note the increase in vasculature seen in the retina from the retinopathy of prematurity pup (B, D, and F). Scale bar = 1 mm in A–D. Scale bar = 50 Amin E–F.
J.W. Calvert et al. / Experimental Neurology 189 (2004) 150–161 155
exposure. We did expose some pups to 100% oxygen at 3
ATA for 10 h to induce retinopathy. Newborn rat pups in
this retinopathy of prematurity group did show signs of
increased vascular proliferation in their retinas 2 weeks after
hyperoxia exposure when compared to age-matched con-
trols (Fig. 4). There was also a significant increase (P <
0.001, ANOVA) in retinal vascular density values for the
retinopathy of prematurity group at 2 weeks compared to the
other groups at the same time point (Table 1).
Histological analysis
The effects of hyperoxia on the structure of the retina
were further assessed on serial sections of the retina (Fig.
5). The following parameters were measured: the number
of cells in the inner nuclear layer (INL) and outer nuclear
layer (ONL), the thickness of the outer plexiform layer
(OPL) (Am), and the area of both the INL and ONL (Am2)
(Table 1). The number of cells per unit area was also
determined. No difference was found in the number of cells
in the INL, the thickness of the OPL, and the number of
cells in the ONL among the control, 1, 1.5 and 3.0 ATA
groups at both 2 and 10 weeks. Retinas of newborn pups in
the retinopathy of prematurity group (Fig. 4) had an OPL
that had a thickness of 9.150 F 0.453, which was signif-
icantly (P < 0.001, ANOVA) thinner (arrow Fig. 4) than
the OPL of the age-matched control group as well as the 1,
1.5, and 3.0 ATA groups at 2 weeks after hyperoxia
exposure. There were no differences in any of the other
parameters measured.
J.W. Calvert et al. / Experimental Neurology 189 (2004) 150–161156
Fig. 6. HIF-1a protein expression 24 h after hyperoxia exposure. (A) Age-matched control, arrow denotes positive HIF-1a staining. (B) 1 ATA group.
(C) 1.5 ATA group. (D) 3.0 ATA group. (E) retinopathy of prematurity group, arrow denotes HIF-1a expression in the inner limiting membrane. 100�,
Scale bar = 10 Am.
J.W. Calvert et al. / Experimental Neurology 189 (2004) 150–161 157
HIF-1a and VEGF protein expression
HIF-1a and VEGF protein expressions were assessed
in the retinas of newborn rat pups 24 h after hyperoxia
exposure. HIF-1a protein expression (Fig. 6) was evident
in retinas of pups in all groups examined. Labeling was
observed at the level of the inner retinal surface near the
nerve fiber layer and within the INL. No differences in
HIF-1a protein expression were noted between the con-
trol, 1, 1.5, and 3.0 ATA groups. However, HIF-1a
protein expression pattern was different in the retinas of
retinopathy of prematurity pups. The majority of HIF-1a
Fig. 5. Hematoxylin and eosin staining of retinas from newborn rat pups 2 and 10 w
group, 2 weeks. (D) 1 ATA group, 10 weeks. (E) 1.5 ATA group, 2 weeks. (F) 1.5 A
(I) retinopathy of prematurity group, 2 weeks. Note the thinning of the OPL in the
panel A: INL, inner nuclear layer; ONL, outer nuclear layer; OPL, outer plexifor
section. Scale bar = 20 Am.
expression was found to be in the inner limiting mem-
brane (Fig. 6E, arrow), which is the layer closest to the
retinal vessels and there was also less expression evident
in the inner retinal surface near the nerve fiber layer and
within the INL. Labeling of VEGF (Fig. 7) was also
observed at the level of the inner retinal surface near the
nerve fiber layer (NFL) and within the INL. No differ-
ences in the extent of expression of VEGF were noted
between the control group, 1, 1.5, and 3.0 ATA groups.
VEGF protein expression in the retinopathy of prematurity
group, like the HIF-1a protein expression, was also found
to be in the inner limiting membrane with more intense
eeks after exposure to hyperoxia. (A–B) Age-matched controls. (C) 1 ATA
TA group, 10 weeks. (G) 3.0 ATA group, 2 weeks. (H) 3.0 ATA, 10 weeks.
retina from the retinopathy of prematurity group (I, arrow). Abbreviations in
m layer. Schematic in right lower corner shows regions analyzed for each
Fig. 7. VEGF protein expression 24 h after hyperoxia exposure. (A) Age-matched control group, arrow denotes positive VEGF staining. (B) 1 ATA group.
(C) 1.5 ATA group. (D) 3.0 ATA group. (E) Retinopathy of prematurity group, arrow denotes VEGF expression in the inner limiting membrane. 100�,
Scale bar = 10 Am.
J.W. Calvert et al. / Experimental Neurology 189 (2004) 150–161158
staining seen (Fig. 7E, arrow) and there was less expres-
sion evident in the inner retinal surface near the NFL and
within the INL.
Discussion
The retina appears to only be susceptible to oxidant stress
during developmental stages because maturational events
occurring around the 36th postconceptual week in humans
and the third postnatal week in rodents make the vessels
resistant to hyperoxia-induced obliteration (Brooks et al.,
2001). If the use of supplemental oxygen therapy (pressur-
ized or not) is to be used in the treatment of premature or
hypoxic infants, questions concerning the safety of hyper-
oxia, especially in regards to the retina, need to be
addressed. We show here, that hyperoxia at pressures
ranging from 1.0 ATA to 3 ATA is neuroprotective when
administered after a hypoxic– ischemic insult and that
hyperoxia, as used in this study, does not cause the struc-
tural changes or abnormal vascularization in the retina that
are associated with retinopathy of prematurity. We also
found that there are not any differences in the expression
of HIF-1a or VEGF in the retinas of pups exposed to
hyperoxia when compared to control pups. Thus, a single
1-h application of oxygen, either normobaric or hyperbaric,
appears to be a safe treatment protocol for neonates after a
hypoxia–ischemia insult.
Hypoxic conditions can trigger neovascular proliferation
through the induction of potent angiogenic factors such as
VEGF (Ricci et al., 2000), which is regulated by HIF-1a
under hypoxic conditions (Miyamoto et al., 2002). During a
hypoxic– ischemic insult, the HIF-1a-VEGF system is
thought to provide protective measures by reconstructing
the vasculature through the development of collateral ves-
sels, thus acting as a rescue mechanism. However, this does
not appear to be the case in the eye (Miyamoto et al., 2002).
The pathophysiology of retinopathy of prematurity can be
J.W. Calvert et al. / Experimental Neurology 189 (2004) 150–161 159
separated into two distinct phases. Phase I is termed hyper-
oxia-vasocessation and occurs when an infant or animal is
placed in a hyperoxic environment. The hyperoxia can
cause a downregulation of VEGF leading to the cessation
of normal retinal vascularization (Reynolds, 2001; Shih et
al., 2003). Phase II is termed hypoxia-vasoproliferation and
occurs when the infant or animal is moved from the hyper-
oxic environment back to room air causing a relative
hypoxia to develop in the retina. An increase in VEGF in
response to this hypoxia can lead to the proliferation of
vessels that can invade the vitreous and the retina appearing
as the pathological phenomena that cause the various
clinical and experimental stages of retinopathy of prematu-
rity (Provis et al., 1997; Stone et al., 1995). Subjecting mice
to 75% oxygen from postnatal day (P) 7-P12 leads to the
development of extraretinal neovascularization by P17
(Brooks et al., 2001) through an increase in VEGF protein
expression when the mice are returned to room air (Brooks
et al., 2001; Pierce et al., 1996). Our results showed that a
single exposure to hyperoxia did not result in an over-
expression of VEGF when the pups were returned to room
air for 24 h, nor did we see any indications of an increase in
retinal vascularization within 10 weeks after the exposure.
Torbati et al. (1995) have shown that retinopathy of prema-
turity can develop in newborn rats exposed to 100% oxygen
at 5 ATA for 5 h, whereas Ricci and Calogero (1988) have
shown that exposing newborn rats to 80% oxygen at 1.8
ATA for 10 days after birth did not cause retinopathy of
prematurity. However, Ricci and Calogero (1988) also
showed that exposing newborn rat pups to 80% oxygen at
normobaric pressure for 5 or 10 days after birth did cause
retinopathy of prematurity. The vasoconstrictive response
caused by hyperbarism can be used to explain the results of
both studies. Hyperbarism can constrict the choriocapillaries
and reduce the amount of oxygen transported from the
choroid to the inner retina during the period of hyperoxia.
The vasoconstrictive response will vary with the degree of
hyperbarism, so at high pressures the constriction can cause
a severe and prolonged reduction in choroidal and retinal
blood flow. On return to room air after exposure to low
levels of hyperbarism, the hypoxemic environment the
retina finds itself in will be reduced and the stimulus for
vasoproliferation will be decreased (Ricci and Calogero,
1988). On return to room air from exposure to higher levels
of hyperbarism, oxidative damage created by hypoxia–
ischemia can lead to the induction of retinal vasoprolifera-
tion (Torbati et al., 1995). These results and those of this
study suggest that it is the duration of the exposure that is
important when hyperoxia is given at normobaric pressure,
that the pressure is important when hyperoxia is given at
hyperbaric pressures, and that hyperoxic exposures can be
safe when administered at the appropriate pressure and
duration.
Exposure to hyperoxia during the first 14 days of
postnatal life in rat pups can prevent the normal develop-
ment of the OPL and result in long-lasting effects that can
hamper vision (Dembinska et al., 2001; Lachapelle et al.,
1999). The OPL is the retinal layer where synaptic contacts
between the photoreceptors and second order neurons occur
and its formation in rats begins around P5 and commences
on P12 (Lachapelle et al., 1999). Dembinska et al. (2001)
have shown that a progressive increase in the duration of
hyperoxia causes a gradual thinning of the OPL. Our results
show that a single exposure to hyperoxia at normobaric and
hyperbaric pressures did not result in the thinning of the
OPL (Table 1), suggesting that this short exposure does not
cause the structural anomalies in the retina that are associ-
ated with prolonged hyperoxia exposure.
Despite vast improvements that have been made in
neonatal intensive care practices, the incidence of retinop-
athy of prematurity has been stable over the last 2 decades
(Reynolds, 2001). The idea that elevated levels of oxygen
causes retinopathy of prematurity is a main reason why
physicians are hesitant about treating hypoxic or premature
infants with normobaric or hyperbaric oxygen, even though
a recent clinical trial showed that supplemental oxygen
given to infants with pre-threshold retinopathy of prematu-
rity did not result in a worsening of the disease (The STOP-
ROP Multicenter Study Group, 2000) and experimentally it
has been shown that supplemental oxygen therapy can
actually reduce the proliferative vasculopathy of retinopa-
thy of prematurity in kittens (Tailoi et al., 1995). This
stigma regarding supplemental oxygen has been around
since the early 1950s (Campbell, 1951; Crosse and Evans,
1952; Patz et al., 1952) when for lack of a better explana-
tion, it was thought that the oxygen used to treat the infants
was causing the blindness (Neubauer, 2002) even though
retinopathy of prematurity can occur in term and pre-term
infants not exposed to elevated levels of oxygen (Lucey and
Dangman, 1984). Retinopathy of prematurity shares its
pathophysiological characteristics with other common ocu-
lar diseases such as diabetic retinopathy, central vein
occlusion, and age-related macular degeneration (Mechou-
lam and Pierce, 2003), all of which share the common
feature of the development of a somewhat hypoxic state
(Frank, 2004). The use of oxygen in neonatal intensive care
units in the care of premature infants normally includes
keeping the infants on ventilators for days to weeks (Sinha
and Tin, 2003) which will certainly result in hypoxic
conditions when the infants are taken off the ventilators.
Given that long-term exposures to elevated levels of oxy-
gen can result in the onset of a hypoxic state on return to
normal room air, the incident of the hyperoxia–hypoxia
swings needs to be managed accordingly with possibly
some sort of step down strategy that will prevent the onset
of severe hypoxia. Recently, Chow et al. (2003) have
reported that through a strict management of oxygen
delivery and monitoring the incidence of retinopathy of
prematurity has decreased significantly in their neonatal
intensive care center. The authors also reported that the
strict guidelines of the program allowed for the monitoring
of oxygen levels to avoid the repeated episodes of hyper-
J.W. Calvert et al. / Experimental Neurology 189 (2004) 150–161160
oxia–hypoxia in very low birthweight infants (Chow et al.,
2003). In light of the potential toxic effects and caution
surrounding the use of elevated levels of oxygen at normo-
baric pressures, treatment with oxygen at increased pressure
may be a more suitable treatment for premature and
hypoxic infants. Mild hyperbarism, as mention above, has
been shown to be safe, in regards to the retina, when
administered to rats for 10 days (Ricci and Calogero,
1988) and HBO is being used to treat newborns and
children with radiation-induced bone and soft tissue com-
plications, cyanotic congenital heart disease, and CO poi-
soning (Ashamalla et al., 1996; Chuba et al., 1997; Rudge,
1993). As mentioned above, consideration of HBO treat-
ment for hypoxia–ischemia is derived from the belief that
HBO therapy might salvage the still viable, though non-
functioning tissue surrounding the area of insult through an
increase in the amount of oxygen available to the brain
tissue (Nighoghossian and Trouillas, 1997). Other possible
effects of HBO include the reduction of pressure within the
brain caused by swelling, the restoration of blood–brain
barrier function and cell membrane function, the scaveng-
ing of free radicals, and the reduction in the stickiness of
blood products (Neubauer, 2002). All of which aid in the
reduction of brain injury. In recent years, HBO treatment
has emerged as a treatment for cerebral palsy (CP), a
common pathology associated with a hypoxic–ischemic
insult during the perinatal period. Cases studies have shown
that administration of HBO (sometimes years after the
initial injury) to children who had experienced perinatal
hypoxia– ischemia led to the dramatic improvement of
these patients in regards to behavior and alertness (Neu-
bauer, 2002).
It needs to be pointed out that the purpose of this study
was not to induce retinopathy of prematurity in neonatal
rats (hence, we did not use the established model of
oxygen-induced retinopathy), only to test if a single
treatment of hyperoxia at various pressures would result
in the retinal changes associated with retinopathy. In
conclusion, we have shown that a single exposure to
100% oxygen for 1 h at normobaric or hyperbaric pres-
sures did not cause retinopathy in newborn rat pups,
suggesting that when used correctly hyperoxia does not
lead to the development of retinopathy of prematurity. The
results obtained in this study may help to open the door for
the use of hyperoxia therapy for the treatment of hypoxic
newborn infants since both normobaric oxygen (Singhal et
al., 2002a,b) and hyperbaric oxygen (Badr et al., 2001; Yin
et al., 2002) have been shown to be effective in neonate
and adult models of stroke.
Acknowledgments
This work was partially supported by American Heart
Association Bugher Foundation for Stroke Award and by
NIH NS45694, NS43338, and HD43120 to JHZ.
References
Ashamalla, H.L., Thom, S.R., Goldwein, J.W., 1996. Hyperbaric oxygen
therapy for the treatment of radiation-induced sequelae in children. The
University of Pennsylvania experience. Cancer 77, 2407–2412.
Badr, A.E., Yin, W., Mychaskiw, G., Zhang, J.H., 2001. Effect of hyper-
baric oxygen on striatal metabolites: a microdialysis study in awake
freely moving rats after MCA occlusion. Brain Res. 916, 85–90.
Beauchamp, M.H., Marrache, A.M., Hou, X., Gobeil Jr., F., Bernier, S.G.,
Lachapelle, P., Abran, D., Quiniou, C., Brault, S., Peri, K.G., Roberts,
J., Almazan, G., Varma, D.R., Chemtob, S., 2002. Platelet-activating
factor in vasoobliteration of oxygen-induced retinopathy. Invest. Oph-
thalmol. Vis. Sci. 43, 3327–3337.
Brooks, S.E., Gu, X., Samuel, S., Marcus, D.M., Bartoli, M., Huang,
P.L., Caldwell, R.B., 2001. Reduced severity of oxygen-induced
retinopathy in eNOS-deficient mice. Invest Ophthalmol. Vis. Sci.
42, 222–228.
Calvert, J.W., Yin, W., Patel, M., Badr, A., Mychaskiw, G., Parent, A.D.,
Zhang, J.H., 2002. Hyperbaric oxygenation prevented brain injury in-
duced by hypoxia– ischemia in a neonatal rat model. Brain Res. 951,
1–8.
Calvert, J.W., Zhou, C., Nanda, A., Zhang, J.H., 2003. Effect of hyperbaric
oxygen on apoptosis in neonatal hypoxia– ischemia rat model. J. Appl.
Physiol. 95, 2072–2080.
Campbell, K., 1951. Intensive oxygen therapy as a possible cause of retro-
lental fibroplasia; a clinical approach. Med. J. Aust. 2, 48–50.
Chow, L.C., Wright, K.W., Sola, A., 2003. Can changes in clinical practice
decrease the incidence of severe retinopathy of prematurity in very low
birth weight infants? Pediatrics 111, 339–345.
Chuba, P.J., Aronin, P., Bhambhani, K., Eichenhorn, M., Zamarano, L.,
Cianci, P., Muhlbauer, M., Porter, A.T., Fontanesi, J., 1997. Hyperbaric
oxygen therapy for radiation-induced brain injury in children. Cancer
80, 2005–2012.
Crosse, V.M., Evans, P.J., 1952. Prevention of retrolental fibroplasia. AMA
Arch. Ophthalmol. 48, 83–87.
Dembinska, O., Rojas, L.M., Varma, D.R., Chemtob, S., Lachapelle, P.,
2001. Graded contribution of retinal maturation to the development of
oxygen-induced retinopathy in rats. Invest Ophthalmol. Vis. Sci. 42,
1111–1118.
Ferriero, D.M., 2001. Oxidant mechanisms in neonatal hypoxia– ischemia.
Dev. Neurosci. 23, 198–202.
Frank, R.N., 2004. Diabetic retinopathy. N. Engl. J. Med. 350, 48–58.
Han, B.H., D’Costa, A., Back, S.A., Parsadanian, M., Patel, S., Shah, A.R.,
Gidday, J.M., Srinivasan, A., Deshmukh, M., Holtzman, D.M., 2000.
BDNF blocks caspase-3 activation in neonatal hypoxia– ischemia. Neu-
robiol. Dis. 7, 38–53.
Johnston, M.V., 2001. Excitotoxicity in neonatal hypoxia. Ment. Retard.
Dev. Disabil. Res. Rev. 7, 229–234.
Lachapelle, P., Dembinska, O., Rojas, L.M., Benoit, J., Almazan, G.,
Chemtob, S., 1999. Persistent functional and structural retinal anoma-
lies in newborn rats exposed to hyperoxia. Can. J. Physiol. Pharmacol.
77, 48–55.
Lucey, J.F., Dangman, B., 1984. A reexamination of the role of oxygen in
retrolental fibroplasia. Pediatrics 73, 82–96.
Mechoulam, H., Pierce, E.A., 2003. Retinopathy of prematurity: molecular
pathology and therapeutic strategies. Am. J. Pharmacogenomics 3,
261–277.
Mink, R.B., Dutka, A.J., 1995. Hyperbaric oxygen after global cerebral
ischemia in rabbits reduces brain vascular permeability and blood flow.
Stroke 26, 2307–2312.
Miyamoto, N., Mandai, M., Takagi, H., Suzuma, I., Suzuma, K., Koyama,
S., Otani, A., Oh, H., Honda, Y., 2002. Contrasting effect of estrogen on
VEGF induction under different oxygen status and its role in murine
ROP. Invest Ophthalmol. Vis. Sci. 43, 2007–2014.
Neubauer, R.A., 2002. New hope for the neurologically damaged child,
cerebral palsy, anoxic ischemic encephalopathy, and traumatic brain
injury. In: Joiner, J.T. (Ed.), The Proceedings of the 2nd International
J.W. Calvert et al. / Experimental Neurology 189 (2004) 150–161 161
Symposium on Hyperbaric Oxygenation for Cerebral Palsy and the
Brain-injured child. Best Publishing Company, Flagstaff, pp. 3–7.
Nighoghossian, N., Trouillas, P., 1997. Hyperbaric oxygen in the treatment
of acute ischemic stroke: an unsettled issue. J. Neurol. Sci. 150, 27–31.
Palmer, E.A., 2003. Implications of the natural course of retinopathy of
prematurity. Pediatrics 111, 885–886.
Patz, A., Hoeck, L.E., De La, C.R.U.Z., 1952. Studies on the effect of high
oxygen administration in retrolental fibroplasia: I. Nursery observa-
tions. Am. J. Ophthalmol. 35, 1248–1253.
Pierce, E.A., Foley, E.D., Smith, L.E., 1996. Regulation of vascular endo-
thelial growth factor by oxygen in a model of retinopathy of prematu-
rity. Arch. Ophthalmol. 114, 1219–1228.
Provis, J.M., Leech, J., Diaz, C.M., Penfold, P.L., Stone, J., Keshet, E.,
1997. Development of the human retinal vasculature: cellular relations
and VEGF expression. Exp. Eye Res. 65, 555–568.
Reynolds, J.D., 2001. The management of retinopathy of prematurity. Pae-
diatr. Drugs 3, 263–272.
Ricci, B., Calogero, G., 1988. Oxygen-induced retinopathy in newborn
rats: effects of prolonged normobaric and hyperbaric oxygen supple-
mentation. Pediatrics 82, 193–198.
Ricci, B., Ricci, F., Maggiano, N., 2000. Oxygen-induced retinopathy in
the newborn rat: morphological and immunohistological findings in
animals treated with topical timolol maleate. Ophthalmologica 214,
136–139.
Rice III, J.E., Vannucci, R.C., Brierley, J.B., 1981. The influence of im-
maturity on hypoxic– ischemic brain damage in the rat. Ann. Neurol. 9,
131–141.
Rudge, F.W., 1993. Carbon monoxide poisoning in infants: treatment with
hyperbaric oxygen. South. Med. J. 86, 334–337.
Shih, S.C., Ju, M., Liu, N., Smith, L.E., 2003. Selective stimulation of
VEGFR-1 prevents oxygen-induced retinal vascular degeneration in
retinopathy of prematurity. J. Clin. Invest. 112, 50–57.
Singhal, A.B., Dijkhuizen, R.M., Rosen, B.R., Lo, E.H., 2002a. Normo-
baric hyperoxia reduces MRI diffusion abnormalities and infarct size in
experimental stroke. Neurology 58, 945–952.
Singhal, A.B., Wang, X., Sumii, T., Mori, T., Lo, E.H., 2002b. Effects of
normobaric hyperoxia in a rat model of focal cerebral ischemia-reper-
fusion. J. Cereb. Blood Flow Metab. 22, 861–868.
Sinha, S.K., Tin, W., 2003. The controversies surrounding oxygen therapy
in neonatal intensive care units. Curr. Opin. Pediatr. 15, 161–165.
Smith, L.E., 2002. Pathogenesis of retinopathy of prematurity. Acta Pae-
diatr., Suppl. 91, 26–28.
Stone, J., Itin, A., Alon, T., Pe’er, J., Gnessin, H., Chan-Ling, T., Keshet,
E., 1995. Development of retinal vasculature is mediated by hypoxia-
induced vascular endothelial growth factor (VEGF) expression by neu-
roglia. J. Neurosci. 15, 4738–4747.
Supplemental Therapeutic Oxygen for Prethreshold Retinopathy Of Pre-
maturity (STOP-ROP), 2000. A randomized, controlled trial: I. Primary
outcomes. Pediatrics 105, 295–310.
Stout, A.U., Stout, J.T., 2003. Retinopathy of prematurity. Pediatr. Clin.
North Am. 50, 77–87.
Tailoi, C.L., Gock, B., Stone, J., 1995. Supplemental oxygen therapy. Basis
for noninvasive treatment of retinopathy of prematurity. Invest Ophthal-
mol. Vis. Sci. 36, 1215–1230.
Torbati, D., Peyman, G.A., Wafapoor, H., Shaibani, S.B., Khoobehi, B.,
1995. Experimental retinopathy by hyperbaric oxygenation. Undersea
Hyperb. Med. 22, 31–39.
Weinberger, B., Laskin, D.L., Heck, D.E., Laskin, J.D., 2002. Oxy-
gen toxicity in premature infants. Toxicol. Appl. Pharmacol. 181,
60–67.
Yin, W., Badr, A.E., Mychaskiw, G., Zhang, J.H., 2002. Down regulation
of COX-2 is involved in hyperbaric oxygen treatment in a rat transient
focal cerebral ischemia model. Brain Res. 926, 165–171.
Zhou, C., Li, Y., Nanda, A., Zhang, J.H., 2003. HBO suppresses Nogo-A,
Ng-R, or RhoA expression in the cerebral cortex after global ischemia.
Biochem. Biophys. Res. Commun. 309, 368–376.
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