Hyperoxia Therapy of Pre-Proliferative Ischemic
Retinopathy in a Mouse Model
Wenbo Zhang,2,3,4,5Harumasa Yokota,2,3,5,6Zhimin Xu,2,3Subhadra P. Narayanan,2,3
Lauren Yancey,2,3Akitoshi Yoshida,6Dennis M. Marcus,7Robert W. Caldwell,4
Ruth B. Caldwell,*,1,2,3,8,9and Steven E. Brooks*,3,9
PURPOSE. To investigate the therapeutic use and mechanisms of
action of normobaric hyperoxia to promote revascularization
and to prevent neovascularization in a mouse model of oxygen-
induced ischemic retinopathy.
METHODS. Hyperoxia treatment (HT, 40%–75% oxygen) was
initiated on postnatal day (P) 14 during the pre-proliferative
phase of ischemic retinopathy. Immunohistochemistry, ELISA,
and quantitative PCR were used to assess effects on retinal
vascular repair and pathologic angiogenesis in relation to glial
cell injury, VEGF protein, and mRNA levels of key mediators of
pathologic angiogenesis. Effects of intravitreal injections of
VEGF and the VEGF inhibitor VEGFR1/Fc fusion protein were
RESULTS. Administration of HT during the ischemic pre-prolif-
erative phase of retinopathy effectively accelerated the process
of revascularization while preventing the development of vit-
reous neovascularization. HT enhanced the formation of spe-
cialized endothelial tip cells at the edges of the repairing
capillary networks and blocked the overexpression of several
molecular mediators of angiogenesis, inflammation, and extra-
cellular proteolysis. HT markedly reduced the reactive expres-
sion of GFAP in Mu ¨ller cells and improved the morphology of
astrocytes in the avascular region of the retina. Exogenous
VEGF administered into the vitreous on P14 was not sufficient
to cause vitreous neovascularization in the HT mice. Injection
of the VEGF antagonist VEGFR1/Fc blocked both pathologic
and physiological angiogenesis and did not rescue astrocytes.
CONCLUSIONS. HT may be clinically useful to facilitate vascular
repair while blocking neovascularization in the pre-prolifera-
tive stage of ischemic retinopathy by correcting a broad range
of biochemical and cellular abnormalities. (Invest Ophthalmol
Vis Sci. 2011;52:6384–6395) DOI:10.1167/iovs.11-7666
cation of diabetes, retinal vein occlusion, and retinopathy of
prematurity, with loss of vision occurring as a result of retinal
hypoxia, increased vascular permeability, and pathologic neo-
vascularization (NV). Considerable scientific and clinical inves-
tigation have focused on identifying the molecular mediators
involved in initiating pathologic angiogenesis in the retina and
developing effective inhibitors. Anti-VEGF agents are now in
widespread use in the treatment of patients with subretinal
neovascularization associated with age-related macular degen-
eration and are being investigated in clinical trials in diabetic
retinopathy.1Several antiangiogenic agents are in clinical use
to manage a wide variety of conditions involving pathologic
angiogenesis and vascular permeability in the eye.1Several
issues, however, detract from the potential clinical value of
Whereas angiogenic inhibitors are effective in reversing
vascular permeability and eliminating NV, this therapy does
not address the issue of retinal ischemia or promote the normal
vascular repair that is essential to ending tissue hypoxia and
maintaining normal function. In fact, an inhibitor’s lack of
antiangiogenic selectivity might adversely impact the process
of physiological vascular repair.2,3Similarly, suppression of
VEGF-mediated cell survival pathways in hypoxic neurons
might inhibit neuronal survival and function.4In addition,
antiangiogenic therapy is not indicated at the pre-proliferative
stage of ischemic retinopathy, a time when metabolic support
of the retina and repair of the damaged capillary beds are most
critical. Delivery of antiangiogenic agents by intravitreal injec-
tion also carries a risk for vision-threatening endophthalmitis
The therapeutic use of oxygen has been extensively studied
in wound healing,7,8especially in clinical situations in which
tissue perfusion is compromised by arterial insufficiency,9di-
abetes,10,11or previous radiation treatment for neoplasia.12
Numerous studies support the use of hyperbaric oxygen as
adjunctive treatment for nonhealing lower extremity wounds
in patients with diabetes,10,11compromised tissue flaps and
grafts,13and radiation-induced ischemic osteonecrosis.14Ex-
periments in various animal models of ischemia have suggested
that supplemental oxygen can improve the rate of wound
healing and reduce apoptosis in the affected tissue.8,15In
addition to supporting oxidative phosphorylation, oxygen
plays a critical role in redox signaling for cytokines, including
VEGF and PDGF, and in cell motility, integrin function, and
schemic retinopathy is the leading cause of blindness in
persons younger than 60 in the United States. It is a compli-
From the2Vascular Biology Center,3Vision Discovery Institute,
and the Departments of4Pharmacology and Toxicology,8Cellular Bi-
ology and Anatomy, and
University, Augusta, Georgia; the6Department of Ophthalmology, Asa-
hikawa Medical College, Asahikawa, Japan; the7Southeast Retina Cen-
ter, Augusta, Georgia; and the1VA Medical Center, Augusta, Georgia.
5These authors contributed equally to the work presented here
and should therefore be regarded as equivalent authors.
Supported by the Georgia Health Sciences University Vision Discov-
ery Institute (SEB, RBC); National Eye Institute Grants R01 EY04618 (RBC)
and R01 EY11766 (RBC, RWC); Veterans Administration MRA
(RBC); National Heart, Lung, and Blood Institute Grant R01 HL70215
(RWC); American Heart Association Grant 11SDG4960005; and Juve-
nile Diabetes Research Foundation Grant JDRF 10-2009-575 (WZ).
Submitted for publication April 1, 2011; revised May 29, 2011;
accepted June 14, 2011.
Disclosure: W. Zhang, None; H. Yokota, None; Z. Xu, None; S.P.
Narayanan, None; L. Yancey, None; A. Yoshida, None; D.M. Mar-
cus, None; R.W. Caldwell, None; R.B. Caldwell, None; S.E. Brooks,
*Each of the following is a corresponding author: Steven E.
Brooks, Vision Discovery Institute, Georgia Health Sciences University,
Augusta, GA 30912-2500; email@example.com.
Ruth B. Caldwell, Vascular Biology Center, Georgia Health Sciences
University, Augusta, GA 30912-2500; firstname.lastname@example.org.
9Ophthalmology, Georgia Health Sciences
Retinal Cell Biology
Investigative Ophthalmology & Visual Science, August 2011, Vol. 52, No. 9
Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc.
The therapeutic effects of oxygen supplementation in isch-
emic retinopathy have not been well characterized. However,
it has been shown that hyperbaric oxygen treatment reduces
breakdown of the blood-retinal barrier in streptozotocin-in-
duced diabetic rats.17In humans, small case series have sug-
gested that normobaric supplemental oxygen can reduce vas-
cular permeability and retinal thickness in diabetic macular
edema18and central retinal vein occlusion.19,20Oxygen sup-
plementation immediately after vaso-obliteration but before
the onset of retinal hypoxia was also shown to reduce the
severity of vitreous NV and to attenuate the degeneration of
astrocytes in oxygen-induced retinopathy (OIR) models.21–23
These clinical and scientific studies support the potential value
of oxygen therapy as a primary or adjunctive treatment for
vision-threatening ischemic retinopathies.24
In the present study, we investigated the therapeutic ben-
efit of oxygen supplement during the ischemic pre-prolifera-
tive phase of ischemic retinopathy in an OIR mouse model.
Unlike other studies that start treatment of ischemic retinopa-
thy before the onset of hypoxia, we initiated hyperoxia treat-
ment (HT) after a period of retinal ischemia, when the multiple
signs of ischemic retinopathy—such as no perfusion in the
central retina, tortuous and dilated vessels,25and upregulation
of many angiogenic and inflammatory genes in the retina—are
evident. This delay was chosen to determine the potential
clinical use of HT in the pre-proliferative stage of ischemic
retinopathy because most patients with ischemic retinopathy
seek evaluation before NV has developed. We show that nor-
mobaric HT after a period of retinal ischemia selectively blocks
the development of pathologic NV while it simultaneously
accelerates the process of physiologic revascularization. Mech-
anistically, we further demonstrate the beneficial effect of HT
may be attributed to its pleiotropic effect by correcting a broad
range of biochemical and cellular abnormalities. Our data
strongly support the potential of hyperoxia as an effective
primary or adjunctive therapy for pre-proliferative ischemic
Treatment of Animals
All procedures with animals were performed in accordance with the
ARVO Statement for the Use of Animals in Ophthalmic and Vision
Research and were approved by the institutional animal care and use
committee (Animal Welfare Assurance no. A3307-01). OIR was in-
duced by exposing C57BL/6J mice to 75% oxygen from postnatal day
(P) 7 to P12, followed by a return to room air until P17 or P20 (OIR
groups). To investigate the effect of HT, OIR mice were maintained in
room air from P12 to P14 and were then returned to 75% or 40%
oxygen for various times (HT groups). Age-matched mice kept in room
air served as room air control. Nutritional status is known to alter
retinal neovascularization during OIR, and hyperoxia and return to
room air can stress mice, which affects feeding. Thus, handling of the
pups was kept to a minimum in experiments. The dams and pups were
monitored closely during experiments, and surrogate dams were in-
troduced if signs of stress were observed. On P14, pups were weighed;
the weight range was 4.5 to 4.8 g. At the end of the experiment, the
mice were killed, and one eye from each mouse was fixed in 4%
paraformaldehyde for morphology studies. The contralateral retina was
dissected, frozen in liquid nitrogen, and used for the measurement of
mRNA by quantitative RT- PCR. All mice were weighed before kill, and
there were no significant differences in body weight between the OIR
and HT groups (Table 1).
Mice were anesthetized by intraperitoneal injection of anesthetic (tri-
bromoethanol; Avertin; 625 mg/kg). Intravitreal injections were per-
formed by delivering 1 ?L PBS containing 2 ?g VEGFR1/Fc fusion
protein or mouse VEGF (R&D Systems, Minneapolis, MN) or vehicle
only with a 36-gauge needle mounted to a 10-?L Hamilton syringe. The
tip of the needle was inserted under the guidance of a dissecting
microscope (Wild M650; Leica, Bannockburn, IL) through the dorsal
limbus of the eye. Injections were performed slowly throughout a
period of 2 minutes.
Immunostaining on Wholemount Retinas
Retinas were carefully dissected, blocked, and permeabilized in PBS
containing 10% goat serum and 1% Triton-X-100 for 30 minutes. Then
retinas were incubated with Alex 594–labeled isolectin B4 (Griffonia
simplicifolia; Invitrogen, Carlsbad, CA) and polyclonal rabbit anti-
mouse glial fibrillary acidic protein (GFAP) (1:200; Dako, Carpinteria,
CA) overnight at 4°C with gentle rocking. After washing in PBS, retinas
were incubated with Alex 488–conjugated goat anti–rabbit antibody at
1:400 (Invitrogen) for 4 hours at 4°C. Retinas were washed with PBS,
mounted on microscope slides in mounting medium (Vectashield;
Vector Laboratories, Burlingame, CA), and examined by fluorescence
microscopy (Axiophot; Carl Zeiss, Thornwood, NY) and by confocal
microscopy (Zeiss 510; Carl Zeiss). Areas of vaso-obliteration and
vitreoretinal neovascular tufts were quantified by using ImageJ soft-
ware (developed by Wayne Rasband, National Institutes of Health,
Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html) as re-
Immunostaining on Retinal Sections
After eyes were fixed in 4% paraformaldehyde, retinas were carefully
dissected, equilibrated in 30% sucrose, embedded in optimal cutting
temperature compound frozen in liquid nitrogen, and cut into 10-?m
sections. Retinal sections were permeabilized with PBS containing 1%
Triton X-100 for 30 minutes at room temperature and blocked with 3%
normal goat serum for 30 minutes. Sections were incubated overnight
at 4°C with rabbit polyclonal anti–GFAP antibody. After washing in
PBS, sections were incubated with Alexa 488–conjugated goat anti–
rabbit antibody at 1:500 (Invitrogen) for 1 hour at room temperature,
washed with PBS, covered in mounting medium containing DAPI
(Vector Laboratories) under a coverslip, and examined by fluorescence
microscopy (Carl Zeiss).
Total RNA was isolated using a kit (RNA 4PCR kit; Applied Biosystems,
Austin, TX) according to the manufacturer’s instructions. Total RNA
was reverse transcribed with reverse transcriptase (M-MLV; Invitrogen)
TABLE 1. Mouse Body Weights by Postnatal Day and Group
(n ? 17)
HT (75% O2)
(n ? 17)
(n ? 9)
HT (75% O2)
(n ? 9)
HT (40% O2)
(n ? 6)
Body weight, g5.1 ? 0.15.3 ? 0.25.9 ? 0.15.8 ? 0.1 6.3 ? 0.3
n, number of mice.
IOVS, August 2011, Vol. 52, No. 9
Hyperoxia Therapy in Ischemic Retinopathy6385
to generate cDNA. Quantitative PCR was performed (StepOne PCR
system; Applied Biosystems) with Power SYBR Green.27The fold
difference in various transcripts was calculated by the CT method
using 18S as the internal control. After PCR, a melting curve was
constructed in the range of 60°C to 95°C to evaluate the specificity of
the amplification products. Primer sequences for mouse transcripts are
shown in Table 2.
Mouse eyeballs (or retinas) were lysed by sonication with RIPA lysis
buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid,
1% NP-40, 1 mM EDTA) supplemented with protease inhibitors. The
lysate was cleared of debris by centrifugation, and the supernatant was
used for ELISA with a mouse VEGF ELISA kit (R&D Systems). Protein
concentration in the lysates was determined by a BCA assay (Pierce
Biotechnology, Rockford, IL). VEGF concentration was normalized to
total protein in the lysate and then calculated as fold increase relative
to the control.
The results are expressed as mean ? SEM. Group differences were
evaluated by using one-way ANOVA followed by post hoc Student’s
t-test. Results were considered significant at P ? 0.05.
HT Accelerates Retinal Revascularization in
Pre-Proliferative Ischemic Retinopathy and
Studies were performed in the mouse model of ischemic reti-
nopathy (OIR) in which obliteration of the immature retinal
vessels is induced by exposure to 75% oxygen from P7 through
P12. Immediately on return to room air, the avascular central
retina becomes hypoxic, leading to the upregulation of many
angiogenic and inflammatory genes.28However, vitreous NV is
not initiated until P15, which is 3 days after the onset of retinal
hypoxia.25,29This model has been widely used for studies of
mechanisms and strategies for the blockade of pathologic
NV,2,25,30–36but very little is known about the mechanisms of
vascular repair. Given that most patients with ischemic reti-
nopathy present for evaluation before the development of NV,
we initiated HT after a period of retinal ischemia to determine
its potential clinical use in the pre-proliferative stage of isch-
emic retinopathy. Our data (Fig. 1) show that the HT prevented
vitreous NV and accelerated revascularization of the central
retina compared with room air ischemic controls. These ef-
fects were highly significant by P20, at which time retinal NV
was reduced by 96% and the avascular area was reduced by
35%. Confocal microscopy of lectin-labeled capillaries (Fig. 2)
showed that vascular recovery in hyperoxia was enhanced in
both the deep and the superficial plexuses. Supplemental ox-
ygen was effective in promoting revascularization and blocking
NV at concentrations as low as 40%. There was no statistical
difference between 75% and 40% oxygen supplements (Fig. 1).
Retinas analyzed at P17, after 3 days of rescue hyperoxia,
showed a reduction of NV by 98%, though improved revas-
cularization was not yet evident (Supplementary Fig. S1,
HT Increases the Formation of Endothelial
To examine potential cellular mechanisms underlying the ben-
eficial effects of HT, the formation of endothelial tip cells was
analyzed at P17 after 3 days of HT. Endothelial tip cells are
critical for the development of new capillaries. They are char-
acterized by the presence of specialized apical filopodia that
mediate directional migration. Their migration is thought to be
guided in part by receptor-mediated responses to cytokine
gradients and by interactions with astrocytes, extracellular
matrix, and cell surface adhesion molecules. In OIR, these cells
were notably sparse at the junction of the vascular and avas-
cular retina on P17 (Fig. 3). In areas lacking endothelial tip
cells, the capillaries were poorly developed, and pathologic
extension of vessels into the vitreous was present. On the
other hand, in mice treated with hyperoxia beginning on P14,
the density and distribution of endothelial tips cells was in-
creased 3.4-fold compared with untreated controls (Fig. 3E),
indicating that cellular and molecular factors critical to a nor-
mal angiogenic response were rescued.
HT Rescues Astrocytes
Astrocytes form the vascular template that guides physiological
angiogenesis during retinal development.37,38Aberrant astro-
cyte function, such as overactive CK2 or hypoxia-induced
astrocyte degeneration, is involved in ischemia-induced vitre-
ous neovascularization.22,39,40OIR retinas analyzed on P17
showed extensive alterations in astrocyte morphology and dis-
tribution. The density of astrocytes in the central retina was
increased, and many showed a spindle-shaped morphology
with extensive overlapping (Fig. 4A). In mice treated with HT,
astrocytes in the central retina formed a better network than
those with OIR and retained their normal stellate/dendritic
morphology (Fig. 4A). Their distribution was also more uni-
form and lacked the irregular overlap and variations in density
seen with OIR. The transition zone between the vascular and
avascular retina was also a transition zone for astrocyte mor-
phology and distribution (Fig. 4B) in both OIR and hyperoxia-
TABLE 2. Primers Used for q-PCR
Gene NameForward PrimerReverse Primer
6386Zhang et al.
IOVS, August 2011, Vol. 52, No. 9
treated eyes. In areas with a capillary plexus, astrocytes were
evenly distributed and were arranged in a reticular configura-
tion, mirroring the capillaries. In HT-treated eyes, the density
of capillaries was reduced compared with OIR eyes, though
the capillaries that were present conformed to the template of
underlying astrocytes. The formation of an astrocyte plexus
slightly preceded the leading edge of developing capillaries,
similar to the pattern described for developmental angiogene-
sis in the neonatal mouse retina.37
HT Reduces GFAP Expression in Mu ¨ller Cells
Mu ¨ller cells are activated and involved in the development of
pathologic angiogenesis.41,42The reaction of Mu ¨ller cells was
determined by immunolabeling GFAP, a marker for Mu ¨ller cell
activation.39,43Under normoxic conditions, GFAP-containing
intermediate filaments are robustly expressed in processes
associated with the superficial and deep vessels (Fig. 5A). OIR
induces the activation of Mu ¨ller cells in the central retina
during the ischemic phase, as indicated by their robust expres-
sion of GFAP-containing filaments throughout their processes.
Activation is minimal in peripheral areas of the retina, where
capillary perfusion is preserved. Although GFAP expression in
the nerve fiber layer is not changed, its expression in the
Mu ¨ller cell body in the central retina is suppressed by HT (Fig.
5A) compared with OIR. Quantitative PCR analysis of whole
retina on P17 demonstrates that ischemia-induced expression
of GFAP mRNA (11.6 ? 1.2-fold of control) is completely
blocked by HT (1.2 ? 0.1-fold of control) (Fig. 5B). The ex-
pected lag between decreased mRNA expression and turnover
of intermediate filament proteins would explain the residual
GFAP immunolabeling seen on P17 in the HT retinas in spite of
mRNA levels having already returned to normal.
HT Normalizes Molecular Mediators of
Angiogenesis and Inflammation
To investigate the molecular mechanisms underlying the ben-
eficial effect of HT, the expression of known mediators of NV,
mation and accelerates retina revas-
cularization in OIR. OIR mice were
treated with hyperoxia (75% or 40%
oxygen; HT) or were maintained in
room air (OIR) from P14 to P20. (A)
Retinas from OIR mice treated with
75% oxygen were whole flatmounted
at P20. Representative images are
shown. Original magnification, ?5.
(B) NV areas (red) and (C) avascular
areas (yellow) were quantified (n ?
6–9 retinas from 6–9 mice). *P ?
0.05 compared with OIR.
HT reduces NV tufts for-
IOVS, August 2011, Vol. 52, No. 9
Hyperoxia Therapy in Ischemic Retinopathy 6387
including angiogenic factors (VEGF, FGF-2, PDGF-A, angiopoi-
etin 2, erythropoietin, IGFBP-3), inflammatory molecules
(TNF-?, ICAM-1, MCP-1, iNOS), and extracellular proteolytic
molecules (urokinase plasminogen activator receptor [uPAR],
PAI-1), were determined by quantitative PCR. As expected, the
expression of these molecules is significantly increased by 0.4-
to 54-fold after 2 or 5 days of OIR (Fig. 6). However, HT
completely normalized the expression of each of these genes
to the levels seen in the controls (non-OIR, room air). The
observed downregulation of hypoxia-responsive genes, includ-
ing VEGF and erythropoietin (EPO, suggests that HT decreases
HIF-1? expression to normoxic levels. The downregulation of
iNOS, ICAM-1, and MCP-1 suggests that supplemental oxygen
also exerts a potent anti-inflammatory effect. Interestingly,
PDGF-A, which is normally expressed by retinal ganglion cells
and is known to play a prominent role in astrocyte recruitment
and vascular patterning during retinal development,37was not
affected by either OIR or HT.
Intravitreal Injection of a VEGF Antagonist Blocks
Pathologic and Physiological Angiogenesis
In addition to the increase in VEGF mRNA shown by quantitative
PCR, analysis of VEGF protein in the retina also revealed close to
a 3.2-fold increase that was decreased to the level of room air
control by HT (1.2 ? 0.1-fold of control; Supplementary Fig. S2,
DCSupplemental). To compare the relative efficacy of HT with
anti-VEGF therapy, VEGFR-1/Fc (VEGF trap) was administered by
intravitreal injection on P14 in OIR mice. VEGF blockade potently
suppressed vitreous NV by 79% but also blocked retinal revascu-
larization and increased the avascular area by 82% (Fig. 7A). It also
larization of deep layers. OIR mice
were treated with hyperoxia (75%
oxygen; HT) or were maintained in
room air (OIR) from P14 to P20. Ret-
inas were whole flatmounted at P20.
Retinal vasculature in nerve fiber layer
(NFL), inner plexiform layer (INL), and
outer plexiform layer (OPL) was visu-
alized by confocal microscopy. Rep-
resentative images are shown (n ? 9
retinas from 9 mice). Original magni-
fication, ?20. (A) Vessels in the pe-
ripheral retina. (B) Vessels in the cen-
HT accelerates revascu-
6388 Zhang et al.
IOVS, August 2011, Vol. 52, No. 9
induced further regression of the primary vascular plexus and
thus reduced vascular density in the peripheral retina. These
findings confirm the essential role of VEGF in normal retinal
angiogenesis and in the formation of pathologic vessels in the
vitreous. In contrast with the improved astrocyte morphology
observed with HT (Fig. 4), alterations in astrocyte morphology
and distribution found in VEGF antagonist-treated eyes were sim-
ilar to those in vehicle-treated OIR eyes (Fig. 7B).
Intravitreal VEGF Does Not Cause Pathologic NV
in Hyperoxia-Treated Eyes
HT effectively blocks the upregulation of VEGF in eyes made
ischemic by OIR (Supplementary Fig. S2, http://www.iovs.org/
test whether blockade of VEGF expression is the critical mecha-
nism by which hyperoxia prevents vitreous NV, we administered
exogenous VEGF intravitreally on P14 in mice receiving HT. Our
results showed that HT continued to prevent NV in spite of a
5.3-fold elevated level of VEGF protein (Fig. 8), suggesting that
VEGF is necessary but not sufficient for the development of
extraretinal NV. Exogenous VEGF did not alter the rate of revas-
cularization compared with hyperoxia alone.
Ischemic retinopathy is characterized by a period of retinal
ischemia (pre-proliferative stage) caused by vessel regression
or occlusion, followed by pathologic retinal NV (proliferative
OIR. OIR mice were treated with hy-
peroxia (75% oxygen; HT) or were
maintained in room air (OIR) from
P14 to P17. Retinas were whole flat-
mounted at P17. Representative im-
ages of retinal tip cells (white aster-
isk) and filopodia (yellow arrow) are
shown. (A, B) OIR and HT. Original
magnification, ?20. (C, D) High-mag-
nification image from top of A and B.
Quantification of numbers of tip cells
(n ? 3 retinas from 3 mice). *P ?
0.05 compared with OIR.
HT increases tip cells in
IOVS, August 2011, Vol. 52, No. 9
Hyperoxia Therapy in Ischemic Retinopathy6389
stage). It is at the pre-proliferative stage of ischemic retinopa-
thy that many patients first present to the ophthalmologist.
Few therapeutic options exist at that point, and clinical obser-
vation for progression to NV is typically recommended. In this
study, we determined the effects of supplying oxygen during
the pre-proliferative stage of ischemic retinopathy. We demon-
strate that HT with a range of oxygen concentrations after a
period of retinal ischemia prevents the development of NV as
effectively as supplying oxygen before the onset of retinal hyp-
oxia21,23in pre-proliferative ischemic retinopathy. Moreover, HT
promoted the recovery and repair of damaged capillary beds
within the retina, thus offering the possibility of significantly
improving visual outcomes compared with current antiangio-
genic or retinal ablative interventions. This study, together with
previous studies showing similar beneficial effects when oxygen
is supplied immediately after vessel regression but before retinal
ischemia,21,23indicates a broad therapeutic time window for HT
in the pre-proliferative stage of ischemic retinopathy. The nonin-
vasive and nondestructive nature of this intervention makes it
particularly appealing for clinical use.
astrocyte network and morphology.
OIR mice were treated with hyper-
oxia (75% oxygen; HT) or were main-
tained in room air (OIR) from P14 to
P17. Mice maintained in room air (RA)
from P1 to P17 are control. Retinal
flatmounts were stained with isolectin
B4 (red) and anti-GFAP (green). Repre-
sentative confocal images are shown
(n ? 9 retinas from 9 mice). (ar-
rows) Examples of astrocytes with
different morphology in OIR (spin-
dle-shaped) and HT (stellate). The
punctate staining for GFAP in the flat-
mounts represents cross-sections of
Mu ¨ller cells expressing GFAP. Origi-
nal magnification, ?20. (A) Central
retina. (B) Revascularization area.
HT improves the status of
6390 Zhang et al.
IOVS, August 2011, Vol. 52, No. 9
Thus far, only limited investigation of oxygen therapy in
human ischemic retinopathy has been reported. Its use in
human retinopathy of prematurity is limited by the clinical
challenges of tightly controlling blood oxygenation in neonates
whose conditions are unstable and by the potential for induc-
ing oxygen-related pulmonary complications in the premature
infant.44–46In spite of these limitations, a randomized trial of
supplemental oxygen did demonstrate a notable trend toward
producing better retinal outcomes in infants with pre-thresh-
old ROP.44Oxygen therapy has also been evaluated in a small
case series of patients with chronic diabetic macular edema18
and was found to cause significant and prolonged reduction in
central macular thickness and improved visual function. We
are not aware of any trials evaluating oxygen therapy in pa-
tients with diabetic retinopathy with significant ischemia (pre-
proliferative or proliferative); however, case reports have been
published19,20in which hyperbaric oxygen therapy has been
successfully used in central retinal vein occlusion with good
Because hypoxia is thought to be a driving force behind
angiogenesis, it is surprising that hyperoxia promotes retinal
vascular recovery. This observation suggests that key phys-
iological differences exist between the control of reparative
angiogenesis and developmental angiogenesis in the retina.
Although we did not directly measure tissue oxygen levels in
the retina, the fact that VEGF and EPO mRNA expression
were reduced to normoxic control levels by hyperoxia in-
dicates that relief of cellular hypoxia was biologically signif-
icant. These data suggest that the expansion and survival of
capillary networks during retinal revascularization is neither
sensitive to high levels of inspired oxygen nor critically
related to hypoxia-driven expression of VEGF or EPO, as has
been shown for capillaries involved in developmental angio-
genesis.33,47Although it has been reported that oxygen-
induced vaso-obliteration is significantly reduced in the ret-
inas of transgenic mice that overexpress PDGF-? or IGFBP-3
and is enhanced in mice lacking IGFBP-3,48–50our analysis
showed that PDGF-? expression in hyperoxia-treated eyes
was not significantly different from PDGF-? expression in
ischemic controls and that IGFBP-3 levels were reduced by
hyperoxia to those of room air control mice, suggesting
protection from hyperoxia during vascular repair is medi-
ated by other factors. Clinically, these data indicate that the
use of therapeutic oxygen is not likely to adversely affect
retinal revascularization in pre-proliferative ischemic reti-
nopathy used outside the early developmental window seen
in premature infants.
One cellular mechanism that may explain the improved
vascular recovery in HT-treated eyes is the emergence of
increased numbers of endothelial tip cells. It has been
shown that these cells, which are critical to angiogenesis,
depend on a physiological gradient of VEGF acting through
the KDR/flk-1 receptor.51Such gradients may be disrupted
in severely ischemic tissue. The presence of appropriately
directed endothelial tip cells in HT retinas indicates active,
directional endothelial cell migration,51,52implying physio-
logical rather than pathologic regulation of angiogenesis.
Conversely, the relative paucity of tip cells in non–HT-
treated eyes suggests a disruption of normal signaling and
cell-cell interactions involved in revascularization.
HT-induced rescue of astrocytes may also be crucial be-
cause the migration of endothelial tip cells is thought to be
mediated in part by their interaction with cell adhesion
molecules such as R-cadherin, expressed on the surfaces of
astrocytes.38Our immunohistochemical analysis of GFAP
expression indicates that HT decreases the activation of
Mu ¨ller glia and improves the status of the astrocyte network
in the avascular areas, facilitating the formation of a tem-
were maintained in room air (OIR) from P14 to P17. Mice maintained in room air (RA) from P1 to P17 are
control. (A) Retinal frozen sections were stained with anti-GFAP (green) and DAPI (blue). Arrows: Mu ¨ller
cell processes expressing GFAP. Representative fluorescence microscopy images of central and peripheral
retinas (n ? 3 retinas from 3 mice). Original magnification, ?20. (B) GFAP mRNA in retinas was quantified
by q-PCR and normalized to RA control (n ? 6–9 retinas from 6–9 mice). *P ? 0.05 compared with RA.
#P ? 0.05 compared with OIR.
HT reduces GFAP expression. OIR mice were treated with hyperoxia (75% oxygen; HT) or
IOVS, August 2011, Vol. 52, No. 9
Hyperoxia Therapy in Ischemic Retinopathy6391
plate over which proliferating intraretinal capillaries can
form. Appropriate astrocyte-endothelial cell interactions
would not only promote repair of the superficial vascular
plexus but would also serve to limit pathologic extension of
the vessels into the vitreous,53–55thereby maintaining
proper vascular compartmentalization.
To identify critical molecular mediators involved in HT,
we performed RT-PCR analysis of several genes known to
play key roles in pathologic NV. These included angiogenic
cytokines, inflammatory mediators, and extracellular proteo-
lytic molecules involved in cell migration. Interestingly, HT
normalized the expression of each of these genes, demon-
(75% oxygen; HT) or were maintained in room air (OIR) from P14 to P17. Mice maintained in room air (RA) from P1 to P17 are control. mRNA
levels of specified molecules in retinas were quantified by q-PCR and normalized to RA control (n ? 6–9 retinas from 6–9 mice). *P ? 0.05
compared with RA. #P ? 0.05 compared with OIR.
HT normalizes the expression of angiogenic, inflammatory, and proteolytic molecules in OIR. OIR mice were treated with hyperoxia
6392Zhang et al.
IOVS, August 2011, Vol. 52, No. 9
strating its pleiotropic effect in ischemic retinopathy. Al-
though it is not possible to determine from this analysis
which of these many factors might have been most critical,
it is reasonable to assume that all might have had important
and potentially interdependent contributions. Supporting
this notion, whereas administration of a VEGF antagonist
was sufficient to block pathologic NV in normoxic eyes
subjected to OIR, exogenously administered intravitreal
VEGF did not promote pathologic NV in eyes treated with
hyperoxia. Selective suppression of pathologic NV in the
presence of elevated VEGF expression has also been
achieved consistently in the rodent model of OIR by the
pharmacologic inhibition of retinal iNOS,35,56TNF-?,34or
the angiotensin type 1 receptor57or by the activation of
peroxisome proliferator-activated receptor gamma.58In this
regard, the pleiotropic effect of HT makes it an appealing
therapeutic approach for ischemic retinopathy, particularly
because it does not aversely affect vascular repair, as seen
with VEGF blockade. However, it is also expected that the
beneficial role of HT will be transient unless such treatment
is maintained until physiological vascular repair is complete.
Further investigation is needed to establish the relevant
the astrocyte network. OIR mice were intravitreally injected with PBS (Veh) or VEGF blocker (VEGFR1/Fc,
2 ?g/eye) at P14. Retinal vasculature (isolectin B4, red) and astrocyte network (GFAP, green) were
analyzed at P17 in flatmount. (A) Representative images of retinal flatmount and quantification of NV and
avascular areas (n ? 4 retinas from 4 mice). *P ? 0.05 compared with Veh. Original magnification, ?5.
(B) Representative confocal images of astrocyte in the central retina are shown (n ? 4 retinas from 4
mice). Original magnification, ?20.
Blocking VEGF inhibits NV and revascularization in OIR but does not prevent the disruption of
IOVS, August 2011, Vol. 52, No. 9
Hyperoxia Therapy in Ischemic Retinopathy6393
dose-response relationship of HT and to better define the
key molecular and cellular mechanisms involved in tip cell
formation, glial rescue, and vascular repair during such
1. Tolentino MJ. Current molecular understanding and future treat-
ment strategies for pathologic ocular neovascularization. Curr Mol
2. Ishida S, Usui T, Yamashiro K, et al. VEGF164-mediated inflamma-
tion is required for pathological, but not physiological, ischemia-
induced retinal neovascularization. J Exp Med. 2003;198:483–489.
3. McLeod DS, Taomoto M, Cao J, Zhu Z, Witte L, Lutty GA. Local-
ization of VEGF receptor-2 (KDR/Flk-1) and effects of blocking it in
oxygen-induced retinopathy. Invest Ophthalmol Vis Sci. 2002;43:
4. Nishijima K, Ng YS, Zhong L, et al. Vascular endothelial growth
factor-A is a survival factor for retinal neurons and a critical neu-
roprotectant during the adaptive response to ischemic injury. Am J
5. Ness T, Feltgen N, Agostini H, Bohringer D, Lubrich B. Toxic
vitreitis outbreak after intravitreal injection. Retina. 30:332–338.
6. Pilli S, Kotsolis A, Spaide RF, et al. Endophthalmitis associated with
intravitreal anti-vascular endothelial growth factor therapy injec-
tions in an office setting. Am J Ophthalmol. 2008;145:879–882.
7. Sen CK. Wound healing essentials: let there be oxygen. Wound
Repair Regen. 2009;17:1–18.
8. Zhang Q, Chang Q, Cox RA, Gong X, Gould LJ. Hyperbaric oxygen
attenuates apoptosis and decreases inflammation in an ischemic
wound model. J Invest Dermatol. 2008;128:2102–2112.
9. Knighton DR, Silver IA, Hunt TK. Regulation of wound-healing
angiogenesis-effect of oxygen gradients and inspired oxygen con-
centration. Surgery. 1981;90:262–270.
10. Abidia A, Laden G, Kuhan G, et al. The role of hyperbaric oxygen
therapy in ischaemic diabetic lower extremity ulcers: a double-
blind randomised-controlled trial. Eur J Vasc Endovasc Surg.
11. Faglia E, Favales F, Aldeghi A, et al. Adjunctive systemic hyperbaric
oxygen therapy in treatment of severe prevalently ischemic dia-
betic foot ulcer: a randomized study. Diabetes Care. 1996;19:
12. Chong KT, Hampson NB, Corman JM. Early hyperbaric oxygen
therapy improves outcome for radiation-induced hemorrhagic cys-
titis. Urology. 2005;65:649–653.
13. Monies-Chass I, Hashmonai M, Hoere D, Kaufman T, Steiner E,
Schramek A. Hyperbaric oxygen treatment as an adjuvant to re-
constructive vascular surgery in trauma. Injury. 1977;8:274–277.
14. Feldmeier JJ, Hampson NB. A systematic review of the literature
reporting the application of hyperbaric oxygen prevention and
treatment of delayed radiation injuries: an evidence based ap-
proach. Undersea Hyperb Med. 2002;29:4–30.
not induce NV in hyperoxia-treated
OIR mice. OIR mice were intravitre-
ally injected with PBS (Veh) or VEGF
(2 ?g/eye) at P14 and then were
treated with hyperoxia (75% oxygen)
from P14 to P17. (A) VEGF levels in
the ocular of P17 mice were deter-
mined by ELISA and normalized to
vehicle control (n ? 3 eyes from 3
mice). (B) Retinal vasculature (isolec-
tin B4, red) at P17 in flatmount and
quantification of NV and avascular ar-
eas were shown (n ? 6 retinas from 6
mice). Original magnification, ?5.
VEGF supplement does
6394Zhang et al.
IOVS, August 2011, Vol. 52, No. 9
15. Gordillo GM, Sen CK. Revisiting the essential role of oxygen in Download full-text
wound healing. Am J Surg. 2003;186:259–263.
16. Sen CK, Roy S. Redox signals in wound healing. Biochim Biophys
17. Chang YH, Chen PL, Tai MC, Chen CH, Lu DW, Chen JT. Hyper-
baric oxygen therapy ameliorates the blood-retinal barrier break-
down in diabetic retinopathy. Clin Exp Ophthalmol. 2006;34:
18. Nguyen QD, Shah SM, Van Anden E, Sung JU, Vitale S, Campo-
chiaro PA. Supplemental oxygen improves diabetic macular
edema: a pilot study. Invest Ophthalmol Vis Sci. 2004;45:617–
19. Roy M, Bartow W, Ambrus J, Fauci A, Collier B, Titus J. Retinal
leakage in retinal vein occlusion: reduction after hyperbaric oxy-
gen. Ophthalmologica. 1989;198:78–83.
20. Wright JK, Franklin B, Zant E. Clinical case report: treatment of a
central retinal vein occlusion with hyperbaric oxygen. Undersea
Hyperb Med. 2007;34:315–319.
21. Chan-Ling T, Gock B, Stone J. Supplemental oxygen therapy: basis
for noninvasive treatment of retinopathy of prematurity. Invest
Ophthalmol Vis Sci. 1995;36:1215–1230.
22. Chan-Ling T, Stone J. Degeneration of astrocytes in feline retinop-
athy of prematurity causes failure of the blood-retinal barrier.
Invest Ophthalmol Vis Sci. 1992;33:2148–2159.
23. Gu X, Samuel S, El-Shabrawey M, et al. Effects of sustained hyper-
oxia on revascularization in experimental retinopathy of prematu-
rity. Invest Ophthalmol Vis Sci. 2002;43:496–502.
24. Oguz H, Sobaci G. The use of hyperbaric oxygen therapy in
ophthalmology. Surv Ophthalmol. 2008;53:112–120.
25. Smith LE, Wesolowski E, McLellan A, et al. Oxygen-induced reti-
nopathy in the mouse. Invest Ophthalmol Vis Sci. 1994;35:101–
26. Stahl A, Connor KM, Sapieha P, et al. Computer-aided quantifica-
tion of retinal neovascularization. Angiogenesis. 2009;12:297–301.
27. Zhang W, Rojas M, Lilly B, et al. NAD(P)H oxidase-dependent
regulation of CCL2 production during retinal inflammation. Invest
Ophthalmol Vis Sci. 2009;50:3033–3040.
28. Ishikawa K, Yoshida S, Kadota K, et al. Gene expression profile of
hyperoxic and hypoxic retinas in a mouse model of oxygen-
induced retinopathy. Invest Ophthalmol Vis Sci. 51:4307–4319.
29. Skoura A, Sanchez T, Claffey K, Mandala SM, Proia RL, Hla T.
Essential role of sphingosine 1-phosphate receptor 2 in patholog-
ical angiogenesis of the mouse retina. J Clin Invest. 2007;117:
30. Ritter MR, Banin E, Moreno SK, Aguilar E, Dorrell MI, Friedlander
M. Myeloid progenitors differentiate into microglia and promote
vascular repair in a model of ischemic retinopathy. J Clin Invest.
31. Connor KM, SanGiovanni JP, Lofqvist C, et al. Increased dietary
intake of omega-3-polyunsaturated fatty acids reduces pathological
retinal angiogenesis. Nat Med. 2007;13:868–873.
32. Kubota Y, Hirashima M, Kishi K, Stewart CL, Suda T. Leukemia
inhibitory factor regulates microvessel density by modulating ox-
ygen-dependent VEGF expression in mice. J Clin Invest. 2008;118:
33. Chen J, Connor KM, Aderman CM, Smith LE. Erythropoietin defi-
ciency decreases vascular stability in mice. J Clin Invest. 2008;118:
34. Gardiner TA, Gibson DS, de Gooyer TE, de la Cruz VF, McDonald
DM, Stitt AW. Inhibition of tumor necrosis factor-alpha improves
physiological angiogenesis and reduces pathological neovascular-
ization in ischemic retinopathy. Am J Pathol. 2005;166:637–644.
35. Sennlaub F, Courtois Y, Goureau O. Inducible nitric oxide syn-
thase mediates the change from retinal to vitreal neovasculariza-
tion in ischemic retinopathy. J Clin Invest. 2001;107:717–725.
36. Zamora DO, Davies MH, Planck SR, Rosenbaum JT, Powers MR.
Soluble forms of EphrinB2 and EphB4 reduce retinal neovascular-
ization in a model of proliferative retinopathy. Invest Ophthalmol
Vis Sci. 2005;46:2175–2182.
37. Fruttiger M, Calver AR, Kruger WH, et al. PDGF mediates a neuron-
astrocyte interaction in the developing retina. Neuron. 1996;17:
38. Dorrell MI, Aguilar E, Friedlander M. Retinal vascular development
is mediated by endothelial filopodia, a preexisting astrocytic tem-
plate and specific R-cadherin adhesion. Invest Ophthalmol Vis Sci.
39. Kramerov AA, Saghizadeh M, Pan H, et al. Expression of protein
kinase CK2 in astroglial cells of normal and neovascularized retina.
Am J Pathol. 2006;168:1722–1736.
40. Ljubimov AV, Caballero S, Aoki AM, Pinna LA, Grant MB, Castellon
R. Involvement of protein kinase CK2 in angiogenesis and retinal
neovascularization. Invest Ophthalmol Vis Sci. 2004;45:4583–
41. Bai Y, Ma JX, Guo J, et al. Mu ¨ller cell-derived VEGF is a significant
contributor to retinal neovascularization. J Pathol. 2009;219:446–
42. Pierce EA, Avery RL, Foley ED, Aiello LP, Smith LE. Vascular
endothelial growth factor/vascular permeability factor expression
in a mouse model of retinal neovascularization. Proc Natl Acad Sci
U S A. 1995;92:905–909.
43. Kirsch M, Trautmann N, Ernst M, Hofmann HD. Involvement of
gp130-associated cytokine signaling in Mu ¨ller cell activation fol-
lowing optic nerve lesion. Glia. 58:768–779.
44. Supplemental Therapeutic Oxygen for Prethreshold Retinopathy
Of Prematurity (STOP-ROP), a randomized, controlled trial. I: pri-
mary outcomes. Pediatrics. 2000;105:295–310.
45. McGregor ML, Bremer DL, Cole C, et al. Retinopathy of prematu-
rity outcome in infants with prethreshold retinopathy of prematu-
rity and oxygen saturation ?94% in room air: the high oxygen
percentage in retinopathy of prematurity study. Pediatrics. 2002;
46. Lloyd J, Askie L, Smith J, Tarnow-Mordi W. Supplemental oxygen
for the treatment of prethreshold retinopathy of prematurity. Co-
chrane Database Syst Rev. 2003;CD003482.
47. Alon T, Hemo I, Itin A, Pe’er J, Stone J, Keshet E. Vascular endo-
thelial growth factor acts as a survival factor for newly formed
retinal vessels and has implications for retinopathy of prematurity.
Nat Med. 1995;1:1024–1028.
48. Yamada H, Yamada E, Ando A, et al. Platelet-derived growth factor-
A-induced retinal gliosis protects against ischemic retinopathy.
Am J Pathol. 2000;156:477–487.
49. Lofqvist C, Chen J, Connor KM, et al. IGFBP3 suppresses retinop-
athy through suppression of oxygen-induced vessel loss and pro-
motion of vascular regrowth. Proc Natl Acad Sci U S A. 2007;104:
50. Kielczewski JL, Jarajapu YP, McFarland EL, et al. Insulin-like
growth factor binding protein-3 mediates vascular repair by en-
hancing nitric oxide generation. Circ Res. 2009;105:897–905.
51. Gerhardt H, Golding M, Fruttiger M, et al. VEGF guides angiogenic
sprouting utilizing endothelial tip cell filopodia. J Cell Biol. 2003;
52. Gerhardt H. VEGF and endothelial guidance in angiogenic sprout-
ing. Organogenesis. 2008;4:241–246.
53. Dorrell MI, Aguilar E, Jacobson R, et al. Maintaining retinal astro-
cytes normalizes revascularization and prevents vascular pathol-
ogy associated with oxygen-induced retinopathy. Glia. 58:43–54.
54. Stone J, Chan-Ling T, Pe’er J, Itin A, Gnessin H, Keshet E. Roles of
vascular endothelial growth factor and astrocyte degeneration in
the genesis of retinopathy of prematurity. Invest Ophthalmol Vis
55. Zhang Y, Stone J. Role of astrocytes in the control of developing
retinal vessels. Invest Ophthalmol Vis Sci. 1997;38:1653–1666.
56. Banin E, Dorrell MI, Aguilar E, et al. T2-TrpRS inhibits preretinal
neovascularization and enhances physiological vascular regrowth
in OIR as assessed by a new method of quantification. Invest
Ophthalmol Vis Sci. 2006;47:2125–2134.
57. Downie LE, Pianta MJ, Vingrys AJ, Wilkinson-Berka JL, Fletcher EL.
AT1 receptor inhibition prevents astrocyte degeneration and re-
stores vascular growth in oxygen-induced retinopathy. Glia. 2008;
58. Murata T, Hata Y, Ishibashi T, et al. Response of experimental retinal
neovascularization to thiazolidinediones. Arch Ophthalmol. 2001;
IOVS, August 2011, Vol. 52, No. 9
Hyperoxia Therapy in Ischemic Retinopathy6395