Hypoxic Effects in Contraction of the Ductus Arteriosus in the Chicken Embryo
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Hypoxic Effects in Contraction of the Ductus Arteriosus in the Chicken Embryo
Adrian Cadar and Edward DzialowskiDept. of Biological Sciences
University of North TexasDenton, Texas
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The Ductus Arteriosus The ductus arteriosus (DA) is a fetal
blood vessel
Diverts blood from the right ventricle →aorta →body →fetal gas exchanger
Closure of the ductus occurs during the first few hours after birth
Inability to close results in a patent DA
Fig. 1 Fetal Heart Diagram
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The Avian DA Two distinct DA
Hatching differences Pre-pipped (Day 18) Internally pipped (IP) Externally pipped (EP)
Interested in DA changes during hatching
rpato lung
lpato lung
aortapa
RDA
rv
da
lv
LDA
Pre-pipped IP EP
Fig. 2 Chicken DA and Developmental stages
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Hypoxia A link between human development in chronic hypoxia and
the incidence of PDA in preterm infants (26 - 32 weeks gestation) was found (Rakza et al. 2007)
Incidence of PDA significantly higher in children born at higher altitudes (3847-4533m) in comparison to children born at lower altitudes (1650-2835m or 2792-4360m; Chen, et al., 2008)
60% of preterm infants with intrauterine growth restriction (IUGR) developed PDA
15% of preterm infants without IUGR developed PDA
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ExtracellularSpace
SmoothMuscleCell
Po2
DuctusContraction
Mechanism of Contraction
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ExtracellularSpace
SmoothMuscleCell
Po2
DuctusContraction
Mitochondria
[H2O2]H2O2
IIII II IVKv
Mechanism of Contraction
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ExtracellularSpace
SmoothMuscleCell
Po2
DuctusContraction
[Ca2+]
L-ty
pe
Ca2+
Mitochondria
[H2O2]H2O2
IIII II IVKv
Mechanism of Contraction
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ExtracellularSpace
SmoothMuscleCell
Po2
DuctusContraction
MLC + MLCKCa2+ +CaM
MLC - P P
[Ca2+]
L-ty
pe
Ca2+
Mitochondria
[H2O2]H2O2
IIII II IVKv
Mechanism of Contraction
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ExtracellularSpace
SmoothMuscleCell
Po2
DuctusContraction
MLC + MLCKCa2+ +CaM
MLC - P P
[Ca2+]
L-ty
pe
Ca2+
Mitochondria
[H2O2]H2O2
IIII II IVKv
MLCPROK
Mechanism of Contraction
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ExtracellularSpace
SmoothMuscleCell
Po2
DuctusContraction
MLC + MLCKCa2+ +CaM
MLC - P P
[Ca2+]
L-ty
pe
Ca2+
Mitochondria
[H2O2]H2O2
IIII II IVKv
MLCPROK
Mechanism of Contraction
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HypothesisHypoxia delays maturation in the Rho-kinase
pathway
Hyperoxia accelerates the maturation of the Rho-kinase pathway
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Materials and Methods White leghorn chicken eggs were
incubated at 37.5ºC and relative humidity of 70%
Eggs were incubated in three groups: Normoxia Hypoxia (15% O2) Hyperoxia (30% O2)
Excised Day 18, IP, EP vessels
Blocked Rho-kinase pathway with Y-27632
Fig. 3 Myograph Diagram Adapted from (Mulvany and Aalkjaer, 1990)
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Experimental Protocol
5 min
0.2
N m
-1
O2
-8 -7.5 -7 -6.5 -6 -5.5 -5 -4.5 -4[Y-27632] (Log M)
Fig. 4 Exposed to a step-wise increase in the Rho-kinase inhibitor Y-27632 (10-8 to 10-4 M)
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Relaxation Response in Day 18 Embryos
[Y-27632] (Log M)-8 -7 -6 -5 -4
Rel
axat
ion
(%)
0
200
400
600
800
1000
Day 18 Hypoxia
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Relaxation Response in Day 18 Embryos
[Y-27632] (Log M)-8 -7 -6 -5 -4
Rel
axat
ion
(%)
0
200
400
600
800
1000
Day 18 Hypoxia Day 18 Normoxia
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Relaxation Response in Day 18 Embryos
[Y-27632] (Log M)-8 -7 -6 -5 -4
Rel
axat
ion
(%)
0
200
400
600
800
1000
Day 18 Hypoxia Day 18 Normoxia Day 18 Hyperoxia
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Relaxation Response in IP Embryos
[Y-27632] Log M-8 -7 -6 -5 -4
Rel
axat
ion
(%)
0
50
100
150
200
IP Hypoxia
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Relaxation Response in IP Embryos
[Y-27632] Log M-8 -7 -6 -5 -4
Rel
axat
ion
(%)
0
50
100
150
200
IP HypoxiaIP Normoxia
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Relaxation Response in IP Embryos
[Y-27632] Log M-8 -7 -6 -5 -4
Rel
axat
ion
(%)
0
50
100
150
200
IP HypoxiaIP NormoxiaIP Hyperoxia
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Relaxation Response in EP Embryos
[Y-27632] Log M-8 -7 -6 -5 -4
Rel
axat
ion
(%)
-100
0
100
200
300
400
500
EP Hypoxia
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Relaxation Response in EP Embryos
[Y-27632] Log M-8 -7 -6 -5 -4
Rel
axat
ion
(%)
-100
0
100
200
300
400
500
EP HypoxiaEP Normoxia
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Conclusions
Y-27632 inhibited the Rho-kinase pathway, allowing significant relaxation of the DA in all stages and all incubation environments.
Day 18 hyperoxic DA showed a greater relaxation than day 18 hypoxic and normoxic DA, suggesting a higher Rho-kinase activity in eggs incubated in hyperoxia.
Incubation under chronic hypoxia or hyperoxia did not affect the Y-27632 induced relaxation when compared to normoxia during IP.
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Conclusions Cont.
Because the onset of IP and EP in embryos incubated under chronic hypoxia and hyperoxia is delayed or accelerated, further experiments will characterize the response to Y-27632 in relation to the length of development.
Given the differences observed during Day 18 and EP, we are currently examining expression levels of Rho-kinase in hypoxic, normoxic, and hyperoxic incubated embryos to gain a better understanding on their role in DA contraction.
The Rho kinase pathway is important for the contraction of the chicken DA.
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AcknowledgmentsThis study was supported by an American Heart
Association South Central Grant to Dr. Dzialowski
The Dzialowski LabThe Ronald E. McNair Post-baccularette
Achivement Program
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References1. Belanger, C., Copeland, J., Muirhead, D., Hienz, D., Dzialowski, E.M. 2008. Morphological changes in the
chicken ductus asteriosi during closure at hatching. Anat. rec. 291: 1007-1015.
2. Cogolludo, A., Moral-Sanz, J., Sterren, S., Frazziano, G., Cleef, A., Menedez, C., et al (2009). Maturation of O2 sensing and signaling in the chicken ductus arterious. American Journal of Physiology- Lung Cellular and Molecular Physiology, 297, 619-630
3. Greyner, H., Dzialowski, E.M. 2008. Mechanisms mediating the oxygen-induced vasoreactivity of the ductus arteriosus in the chicken embryo. Am. J. Physiol. 295: R1647-R1649.
4. Kajimoto, H., Hashimoto, K., Archer, S., Bonnet, S., Haromy, A., Harry, G., et al. (2007). Oxygen Activates the Rho/Rho-Kinase Pathway and Induces RhoB and Rock-1 Expression in Humnan and Rabbit Ductus Arteriosus by Increasing Mitochondria-Derived Reactive Oxygen Species: ANewly Recognized Mechanism for Sustaining Ductal Constriction. Circulation Journal of the American Heart Association, 115, 1777-1788. Retrieved February 20, 2010, from http://circ.ahajournals.org/cgi/reprint
5. Malgorzata Szkutnik, Ramiro Menacho-Delgadillo, Enrique Palmero-Zilveti and Jacek Bialkowski. 2007. Transcatheter Closure of Patent Ductus Arteriosus Among Native High- Altitude Habitants. Pediatr Cardiol. 29:624-627.
6. Mulvany , M., Aalkjaer, C., 1990. Structure and Function of Small Arteries. Am. J. Physiol. 70: 921-961.
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