Desaturations, Intermittent Hypoxia, Hyperoxia: New Concepts and Impact on
Retinopathy of Prematurity
Kay D. Beharry
SUNY Downstate Medical Center
Brooklyn, NY USA
Rio de Janeiro – August 2014
Goals and Scope:
1. Impact of Intermittent hypoxia on the development of
organ damage (i.e. Retinopathy of Prematurity).
2. The Critical Threshold of Intermittent Hypoxia (Apnea) at
organ damage becomes irreversible.
3. The importance of the pattern of Intermittent Hypoxia
(clustered vs. dispersed) on the severity of damage.
4. The damge to the eye in response to Intermittent
Hypoxia is probably more severe than the
ophthalmologist’s findings.
5. Drugs (Caffeine, NSAIDs) may correct some damage.
Intermittent Hypoxia Intermittent Hypoxia is almost universal in preterm
infants <1000 grams at birth.
Defined as breathing pauses that last >20 seconds; or
for >10 seconds if associated with bradycardia (<80
bpm); or arterial oxygen desaturation <80-85%.
There is a controversy regarding the duration of apnea
that is considered pathologic.
There is no agreement regarding the degree of change
in O2 saturation or severity of bradycardia that
constitutes an important apnea event.
The definition, diagnosis and treatment of apnea have
not been standardized. Finer et al. Pediatrics 2006
No. arterial oxygen desaturations in preterm infants (24-28 weeks GA) over
first 8 weeks of life.
Martin RJ et al. Neonatology 2011
Martin RJ et al. Neonatology 2011
Intermittent Hypoxia & ROS
• Combined adequate pulse oximetry monitoring and strict O2 administration and management in infants with BW 500-1500 g.
• The incidence of stage 3 & 4 ROP decreased consistently in a 5-year period from 12.5% in 1997 to 2.5% in 2001.
• The need for ROP laser treatment decreased from 4.5% in 1997 to 0%.
Restriction of O2 Variability
Chow et al. Pediatrics 2003
“Physiologic Hypoxia”
Lutty et al. Mol Vis. 2006
TERM RETINA FULLY DEVELOPED
>38 weeks
PRETERM UNDEVELOPED RETINA
<28 weeks
Assoc. for ROP & Related Diseases
IH & VEGF
40%
FiO2
VEGF
VEGF
20%
FiO2
50%
FiO2
VEGF
VEGF
VEGF
VEGF
Angiogenesis
VEGF Template
Assoc. for ROP
Stage 1:
vasoobliteration line
of demarcation.
Stage 2: line becomes
elevated ridge of
tissue.
Stage 3: Abnormal
blood vessels grow
toward the center of
the eye.
Plus Disease: Vessels
dilate & tortuous.
Stage 3 and plus
disease: treatment is
indicated (Laser or
cryo-therapy).
Treatment can
decrease abnormal
vessels with
potentially good
vision.
ROP can continue to
progress and the retina
detaches. Partial
detachment is Stage
4A. If center involved,
it is 4B.
Stage 5: Total retinal
detachment -
blindness.
No. preterm infants needing laser therapy.
Martin RJ et al. Neonatology 2011
Mean duration of intermittent hypoxemia in infants with and without severe
ROP.
Di Fiore et al. Pediatr Res 2012
Retinal Layers
21% O2 in Retinal Layers
Yu et al, 1996
Two Phases of ROP
Phase 1 – Vasoobliteration • premature infants have incompletely vascularized
retinas
• exposed to hyperoxia
• normal vessel growth ceases
Phase 2 – Vasoproliferation • nonvascularized retina becomes metabolically active
and increasingly hypoxic (32-34 weeks PMA)
• Vascular proliferation or neovascularization
Beharry IH Model
RA A B
Coleman et al. Pediatr Res 2008
Question???
How many desturations or apneas can a baby
tolerate?
What is the critical number of intermittent
hypoxia episodes beyond which an organ like
the retina will not recover and will be damaged
permanently???
Experimental Design
Pooled Rats
(n=18/litter)
Normoxia
(Room Air)
(21% O2)
Hyperoxia (50%
O2)/ Hypoxia
(12% O2) H/H
Cycling
P7 (RA)
(1 litter)
P21 (RA)
(1 litter)
P14 (RA)
(1 litter)
P0-P7 (H/H) P0-P14 (H/H) P0-P7 (H/H)
P7-P21 (RA)
P0-P14 (H/H)
P14-P21 (RA)
0, 2, 4, 6,
8, 10, 12
hypoxic
episodes
0, 2, 4, 6,
8, 10, 12
hypoxic
episodes
0, 2, 4, 6,
8, 10, 12
hypoxic
episodes
0, 2, 4, 6,
8, 10, 12
hypoxic
episodes
50% O2 only
OIR Model (50%O2/12%O2)
0 30 52 74
12
50
1 2 3
Time (minutes)
Oxyg
en
%
2: every 12 hr.
4: every 6 hr.
6: every 4 hr.
8: every 3 hr.
10: every 2.4
hr.
12: every 2 hr.
RA Groups
P0 P7
P14 P21
Plasma Cortisol Levels
R A 5 0 % 2 4 6 8 1 0 1 2
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
2 0 0 0 0
Pla
sm
a C
ortis
ol
Le
ve
ls
(p
g/m
L)
7 D -O 2
P 2 1 -7 D -O 2
N o . H y p e ro x ic /h y p o x ic c y c le s /d a y
*p < 0 .0 5 ; **p < 0 .0 1 v s 7 D -R A
#p < 0 .0 5
# # #p < 0 .0 0 1 v s 2 1 D -R A
§ §p < 0 .0 1 ;
§ § §p < 0 .0 0 1 v s 7 D -O 2
#
# # #
# # #
# # # # # # # # ## # #
* ****
***
§ § §§ § §
§ § §§ § §
§ § §
§ § §
§ § §
§ §
R A 5 0 % 2 4 6 8 1 0 1 2
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
2 0 0 0 0
N o . H y p e ro x ic /h y p o x ic c y c le s /d a y
Pla
sm
a C
ortis
ol
Le
ve
ls
(p
g/m
L)
1 4 D -O 2
P 2 1 -1 4 D -O 2
*
#
# #
#
# #
# #
# #
*p < 0 .0 5 v s 7 D -R A
#p < 0 .0 5
# #p < 0 .0 1 v s 2 1 D -R A
§ §p < 0 .0 1 ;
§ § §p < 0 .0 0 1 v s 7 D -O 2
§ §
§ § §
§ § §
§ § §
§ § §
§ § §
§ § §
50%O2 7DO2 14DO2
P21-7DO2 P21-14DO2
21D-14DO2 cycling – 8/day
21D-7DO2 cycling – 8/day
RA 50% 2 4 6 8 10 120
5000
10000
15000
20000
250007DO2
P21-7DO2
*p<0.05, **p<0.01 vs 7D-RA##p<0.01 vs 21D-RA
§p<0.05; §§p<0.01 vs 7D
**
* *
**
** ** **
##
##
## ## ##
§§
§§
§§
§
No. Hyperoxic/hypoxic cycles/day
Reti
nal 8-i
so
PG
F2 L
evels
(pg
/mg
pro
tein
)
RA 50% 2 4 6 8 10 120
5000
10000
15000
20000
250007D-O2
P21-7D-O2
*p<0.05, **p<0.01 vs 7D-RA#p<0.05; ##p<0.01 vs 21D-RA§p<0.05; §§p<0.01 vs 7D
** ** *
*####
##
# #§§
§§
§
§
No. Hyperoxic/hypoxic cycles/day
Ch
oro
idal 8-i
so
PG
F2
Levels
(p
g/m
g p
rote
in)
RA 50% 2 4 6 8 10 120
10000
20000
3000014DO2
P21-14DO2
*p<0.05, **p<0.01 vs 14D-RA#p<0.05; ##p<0.01 vs 21D-RA§§p<0.01 vs 14D
*
**
**** **
#
##
###
§§
§§
§§ §§
No. Hyperoxic/hypoxic cycles/day
Reti
nal 8-i
so
PG
F2 L
evels
(pg
/mg
pro
tein
)
RA 50% 2 4 6 8 10 120
10000
20000
3000014DO2
P21-14DO2
###
* * *
*p<0.05 vs 14D-RA##p<0.01 vs 21D-RA§p<0.05; §§p<0.01 vs 14D
§
§
§§
§§
No. Hyperoxic/hypoxic cycles/day
Ch
oro
idal 8-i
so
PG
F2
Levels
(p
g/m
g p
rote
in)
Beharry KD et al. IOVS 2013
Beharry KD et al. IOVS 2013
RA 50% 2 4 6 8 10 120.0
0.1
0.2
0.37DO2
P21-7DO2
**
**p<0.01 vs 7D-RA##
p<0.01 vs 21D-RA§§§
p<0.001 vs 7DO2§§§
##
No. Hyperoxic/hypoxic cycles/day
Reti
nal H
2O
2 L
evels
( M
/mg
pro
tein
)
RA 50% 2 4 6 8 10 120
1
2
3
47DO2
P21-7DO2
**
* ** **
**p<0.01 vs 7D-RA##
p<0.01 vs 21D-RA§§
p<0.01;§§§
p<0.001 vs 7DO2
##
##
####
§§§
§§§
§§§§§§
§§
No. Hyperoxic/hypoxic cycles/day
Ch
oro
idal H
2O
2 L
evels
( M
/mg
pro
tein
)
RA 50% 2 4 6 8 10 120.0
0.1
0.2
0.3
0.414DO2
P21-14DO2
*
*p<0.05 vs 14D-RA##
p<0.01 vs P21-RA§§
p<0.001 vs 14DO2
#### ##
§§§§
No. Hyperoxic/hypoxic cycles/day
Reti
nal H
2O
2 L
evels
( M
/mg
pro
tein
)
RA 50% 2 4 6 8 10 120
2
4
614DO2
P21-14DO2
**
**
**
**
**p<0.01 vs 14D-RA##
p<0.01 vs P21-RA§§
p<0.01 vs 14DO2
## ##
####
##
§§
No. Hyperoxic/hypoxic cycles/day
Ch
oro
idal H
2O
2 L
evels
( M
/mg
pro
tein
)
IH & the Choroid
IH & the Choroid
IH & the Choroid
Why is the Choroid Sensitive to IH?
It is the major blood supply to the retina.
90% of oxygen delivered to the retina is
consumed by the photoreceptors and 90% of
oxygen comes from the choroidal circulation.
This requires a steep gradient of oxygen tension
which is maintained by a high choroidal blood
flow
Choroidal blood flow is the highest of any tissue
in the body per unit tissue weight – 10-fold higher
than the brain!
Nickla DL & Wallman J, Prog Ret Eye Res 2010
Why is the Choroid Sensitive to IH?
Oxygen tension in the choroid remains high with
arterial/venous difference of 3% versus 38% in
the retina.
The retinal capillaries have tight junctions with no
fenestrations. The choroid is highly permeable
with large fenestrations.
The choroid does not autoregulate in response to
changes in O2 or blood pressure, but responds to
increases in PCO2 with increased blood flow
(1.5% per mmHg PCO2).
Nickla DL & Wallman J, Prog Ret Eye Res 2010
Increasing O2 in the Rat Retina
Yu et al, 1999
Inner Retina Outer Retina
Oxygen Consumption
Cringle et al. IOVS 2002
Question???
Can drugs (i.e. Caffeine, NSAIDs) protect
against damage caused by Intermittent
Hypoxia???
Experimental Design
Pooled rat pups at P0
(n=18/litter)
Normoxia
50%/12% O2 cycles
(8/day) P0-P14
RA P14-P21
Saline
(P0-P14)
Caffeine
(P0-P14)
Ketorolac eye drops
(0.25 mg) P5-P7
Sac at P14
Ketorolac eye drops
(0.25 mg) P5-P7
Sac at P21
Saline
(P0-14)
Caffeine
(P0-P14)
Ketorolac eye drops
(0.25 mg) P5-P7
Sac at P14
Ketorolac eye drops
(0.25 mg) P5-P7
Sac at P21
Ketorolac Administration
(P5-P7) – is it safe? Ketorolac administration
Immediately post
1 week post-P14 2 week post-P21
Eye Opening at P14
Keto/ Sal RA
n=36
Keto/
Sal IH
n=36
Keto/
Caff RA
n=36
Keto/
Caff IH
n=36
Sal/
Caff RA
n=36
Sal/
Caff IH
n=36
Sal/
Sal RA
n=36
Sal/
Sal IH
n=36
Left Eye
21 (58%)
33 (92%)
32
(89%) 33
(92%) 27
(75%) 28
(78%) 21
(58%)
10
(28%)
***
Right Eye
20
(56%) 33
(92%) 30
(83%) 27
(75%) 27
(75%) 26
(72%) 22
(61%)
8
(22%)
***
***p<0.0001 vs Treated groups
21D-14DO2 cycling – 8/day
Saline eye drops/Saline IP
21D-14DO2 cycling – 8/day
Ketorolac eye drops/Caffeine IP
Phalanx Cells: MMPs
(quiescent, form
tubes)
Tip Cells: VEGF,
VEGFR-2, NP-1,
sVEGFR-1
(migrate, navigate) Angiogenesis
The Angiogenesis Trio
Stalk Cells: Notch,
Dll4, Jagged1
(proliferate)
Endothelial Cells
Siemerink MJ et al. J Histochem Cytochem 2012
Tip cells migrate,
navigate; don not
proliferate.
Stalk cells trail
behind tip cells,
proliferate.
Phalanx cells are
quiescent, line
vessel walls.
In ROP, the number of tip cells and their
filopodia are highly increased.
They penetrate the vitreous and form
disorganized vascular tufts
They extend numerous filopodia which are
shorter, grow in all directions, and form
tufts.
Tip Cells & ROP
Effects HRECs 72 hours post IH
Saline Caffeine Ketorolac Caff+Keto
Nx
50% O2
50/10% O2
Optimization-Block-IT
Nx
IH
24 48 72
Membrane Permeability at
72 hours (IH)
Control
Ketorolac
Caffeine
Caffeine & Ketorolac
Tight Junction Proteins at 72 hours of IH
ZO-1
Claudin-5
XO
Saline Caffeine Ketorolac Caff+Keto
Tube Formation at 72 hours
Saline Caffeine Ketorolac Caff+Keto
Nx
50% O2
50/10% O2
Oxidative Stress at 72 hours of IH
Saline Caffeine Ketorolac Caff+Keto
Superoxide
anion
(MitoSOX)
Lipid
Peroxidation
(Image-iT)
HIF-1α
Antioxidants at 72 hours of IH
Saline Caffeine Ketorolac Caff+Keto
SOD-1
(CuZn)
SOD-2
(mt)
SOD-3
(extracellular)
Catalase
VEGF Signaling at 72 hours of IH
Saline Caffeine Ketorolac Caff+Keto
VEGF-A
VEGFR-1
VEGFR-2
VEGFR-3
Take Home Messages
1. Intermittent hypoxia plays a key role in the development
of severe Retinopathy of Prematurity.
2. The Critical Threshold of Intermittent Hypoxia (Apnea)
beyond which there is irreversible damage is 8 in
rats….unknown in humans.
3. Clustering Intermittent Hypoxic episodes is more
dangerous than the occasional dispersed apneas.
4. The damage to the eye in response to Intermittent
Hypoxia is probably more severe than what the
ophthalmologists’ find.
5. Drugs (e.g. Caffeine, NSAIDs) are beneficial.
Thank You
Bench Bedside
Questions??