Desaturations, Intermittent Hypoxia, Hyperoxia: New ... · Why is the Choroid Sensitive to IH? It...

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


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.


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

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

** ** *

*####

##

# #§§

§§

§

§


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

*

**

**** **

#

##

###

§§

§§

§§ §§


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

§

§

§§

§§


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§§§

##


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

##

##

####

§§§

§§§

§§§§§§

§§


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

#### ##

§§§§


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

## ##

####

##

§§


Ch

oro

idal H

2O

2 L

evels

( M

/mg

pro

tein

)

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


Saline eye drops/Saline IP


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


Tube Formation at 72 hours


Nx

50% O2

50/10% O2

Oxidative Stress at 72 hours of IH


Superoxide

anion

(MitoSOX)

Lipid

Peroxidation

(Image-iT)

HIF-1α

Antioxidants at 72 hours of IH


SOD-1

(CuZn)

SOD-2

(mt)

SOD-3

(extracellular)

Catalase

VEGF Signaling at 72 hours of IH


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??

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