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3 Materials and methods

By Alex Martin,2014-05-07 10:28
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3 Materials and methods

Preface

    The thesis of Martin Hansen is framed within a larger research project to understand the effects of genetic selection for growth on the cardiovascular physiology of broiler chickens. The project is funded by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) Dnr. 221-2004-1331.

Content

    1 Abstract…………………………….………………........................ 1 2 List of abbreviations ……………………………………………… 2

    3 Introduction………………………………..……………………… 2 4 Material and Methods………………….……….………………… 3

     4.1 Incubation conditions……………………………………… 3

     4.2 Handling of embryos……………………….……………… 3

     4.3 Histological sampling…………............................................ 4

     4.3.1 Dissection and fixation……………………………... 4

     4.3.2 Dehydration, paraffin inclusion and sectioning…... 4

     4.4 Pressure-diameter loops………………………………….. 5

     4.5 Data analysis……………………………………………….. 6

     4.6 Statistics……………………………………………………. 6 5 Results……………………………………………………………... 6

     5.1 Egg morphology…………………………………………… 6

     5.2 Effects of incubation………………………………………. 7

     5.3 Effects of fixation………………………………………….. 7

     5.4 Effects of hypoxia on aortic morphology………………… 8

     5.5 Effects of alterations in relative humidity……………….. 9

     5.6 Pressure-diameter loops…………………………………... 10 6 Discussion…………………………………………………………. 10

     6.1 Effects of fixation ………………………………………….. 10

     6.2 Aortic morphology………………………………………… 11

     6.3 Conclusion…………………………………………………. 13 7 Acknowledgements……………………………………………….. 13 8 References…………………………………………………………. 13

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1 Abstract

    Stress during prenatal development leads to effects on the cardiovascular system. Rouwet et al.

    (2002) stated that White Leghorn chicken embryos incubated under hypoxic conditions (15 %

    O) developed aortic hypertrophy, seen as a decrease in lumen diameter and an increase in 2

    wall/lumen ratio. In this study four different chicken strains were used: two broiler strains

    (Swedish, Linköping and Dutch, Maastricht) and White Leghorn and the wild close relative of

    the domestic chicken, the red jungle fowl. The chicken embryos were incubated under

    normoxic (21 % O) and hypoxic (14 % O) conditions, sampled at day 19 and processed by 22

    use of regular histological techniques to estimate the dimensions of the aorta. Wall/lumen ratio was not significantly different between hypoxia and normoxia in any of the four strains: 0.71?0.23 vs. 0.67?0.11 in normoxic and hypoxic broilers (Linköping) respectively, 0.93?0.22 versus 0.91?0.16 in broilers (Maastricht) respectively, 0.90?0.20 vs. 0.73?0.27 in jungle fowl and 0.62?0.15 vs. 0.70?0.12 in White Leghorns. However, wall thickness was significantly different in jungle fowl (0.33?0.04 vs. 0.27?0.03, normoxic and hypoxic respectively, P<0.05), and lumen diameter in White Leghorns (0.53?0.06 vs. 0.46?0.05, P<0.05) embryos. The effect of hypoxia on wall elasticity in broiler embryos was tested. No difference was found. In conclusion, no evidence of aortic hypertrophy was found, but this cannot exclude alterations in the composition of the aortic wall which will be the subject of further studies.

Keywords:

    Hypertrophy, hypoxia, elasticity, pressure-diameter loops, aorta.

2 List of abbreviations

DPX DPX mountant for histology LD lumen diameter

    ep externally pipped TD total vessel diameter

    ip internally pipped WT wall thickness

    KRB Krebbs Ringer Bicarbonate buffer

3 Introduction

    Chronic stress during embryonic development leading to a low birth weight is associated with an increased risk to develop coronary heart disease, hypertension, and non-insulin dependent diabetes later in life, a phenomenon widely known as the fetal origins hypothesis (Barker’s hypothesis, Barker 1993). The most common cause of embryonic growth retardation is placental insufficiency, which is a combination of both malnutrition and chronic hypoxia (Ruijtenbeek et al. 2003b). The effect of these pathologies is thought to be linked to alterations caused by hypertrophy in the heart and arteries (Rouwet et al. 2002). It has previously been illustrated by Rouwet et al. (2002) that White Leghorn chicken embryos incubated under hypoxic (15 % O

    ) conditions had smaller embryonic mass and 2

    expressed alterations in aortic morphology by a decrease in lumen diameter, leading to a high

    wall/lumen ratio.

    The aim of this study was to investigate the effect of hypoxia on the mechanical properties of

    the aortic wall in the 19 days old broiler chicken embryo. The broiler chicken has been breed

    for fast growth and a high body mass, which in turn means a lot of strain to the cardiovascular

    system during development. The hypothesis therefore is that not only will the broiler chicken

    embryo show aortic hypertrophy but also effects on wall elasticity.

    4 Materials and methods

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4.1 Incubation conditions

    Four different chicken strains: Broiler Linköping (fast-growing strain Ross 308), broiler

    Maastricht, White Leghorn and the wild close relative of the domestic chicken, the red jungle

    fowl were used in this study. The broiler eggs were obtained from Kläckeribolaget (Väderstad,

    Sweden), the jungle fowls eggs were obtained from Götala Research Station (SLU, Skara),

    the White Leghorns eggs were obtained from (`t Anker, Ochten, The Netherlands), broiler

    Maastricht were obtained from a commercial supplier in the Netherlands. All eggs were

    stored at 18 ?C for up to seven days and turned twice a day before incubation. -1, Apoteket AB) or by decapitation. The eggs were set in the refrigerator after euthanasia for 4.2 Handling of embryos half an hour up to a maximum of 4 h previous to dissection. All eggs were weighed before Embryos were euthanized by an injection of 0.5 mL of sodium pentobarbital (60 mg mLand after incubation to the nearest tenth of a gram and the total length and width were

    measured with Vernier callipers to the nearest millimetre.

    All procedures were approved by the local ethical committee (diary number 45-03).

    4.3 Histological sampling

    4.3.1 Dissection and fixation

    The aortic arch from the 19 days old broiler Linköping embryos and 19 days old embryos

    from broiler Maastricht, White Leghorn and jungle fowl was dissected and the

    brachiocephalic arteries and left right and ductus arteriosus were tied off. Pictures of the

    aortic arch were taken with a digital camera (Nikon coolpix 990). A sapphire ball (3.15 mm in

    diameter) was placed next to the aortic arch as a reference.

    A needle (BD MicrolanceTM 3) with a diameter of 0.9 mm was connected to a polyethylene

    tube (Intramedic? I.D 0.86 mm). The needle was inserted into the proximal end of the aortic

    arch and tied of between the right and left brachiocephalic arteries. The aortic arch was cut off

    right after the right ductus arteriosus joins with the thoracic aorta leading into the abdominal

    aorta.

4.3.2 Dehydration, paraffin inclusion and sectioning

    Following the fixation procedure all aortas were washed in 70 % ethanol several times and

    then dehydrated in 95 % ethanol for two hours and 99.5 % ethanol for three hours. After this

    the aortas were cut right after the left brachiocephalic artery to simplify later sectioning.

    Following this the aortas was further dehydrated in 1:1 99.5 % ethanol/xylene for one hour

    and then in xylene for one hour and finally impregnated with paraffin overnight before

    embedding.

4.4 Pressure-diameter loops

    The aorta was then stretched under physiological pressure corresponding to the specific stage

    of development (between 2.7 3.0 kPa) as measured by Altimiras and Crossley (2000) for

    half an hour. After this the pressure was relieved and certain volume of ringer was perfused

    (0.5 µL 1.0 µL or 2.0 µL depending on the vessel) and the increase in aortic diameter was

    photographed by a video camera (CCD C4200, Hamatsu Photosonic K.K.) connected to a

    stereoscopic zoom microscope (Olympus SZH-ILLD, Olympus Optical CO. LTD.), and the

    corresponding pressure was recorded.

4.5 Data analysis

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    For morphometric analysis of the digital pictures taken from the sections a custom made program using the LabView programming environment (LabView version 6.1, IMAQ vision v.5.1, National Instruments) was used, which allowed the quantification of total area, lumen area of the vessel and wall thickness.

4.6 Statistics

    All data, except for pressure-diameter loops, were analyzed with independent sample t-test and Levene's Test for Equality of Variances using SPSS for windows version 6.2. Results are presented as mean ? SD throughout the thesis unless otherwise stated (Curran-Everett and Benos 2004). The fiduciary level of significance was set p=0.05.

5 Results

5.1 Egg morphology

    Outer dimensions and total egg mass for the eggs of the different strains used are given in Table 1. The two broiler strains used had the largest eggs with an egg mass of 68.3 ? 4.8 g and 68.3 ? 3.3 g for broiler Linköping and broiler Maastricht respectively and outer dimensions egg length of 6.0 ? 0.2 cm and egg width of 4.53 ? 0.11 cm and egg length 6.0 ? 0.3 cm and width 4.6 ? 0.3 cm for broiler Linköping and broiler Maastricht respectively. The jungle fowl embryos had the smallest eggs 36.5 ? 3.8 g and an egg length of 4.83 ? 0.20 and an egg width of 3.70 ? 0.13.

5.2 Effects of incubation

    Hypoxic treated embryos were significantly smaller then the control in three out of four cases as shown in Table 2. The significant ones were Broiler Linköping with 35.4 ? 3.6 g and 21.9 ? 2.3 g (t= 7.76; p<0.05) for the control and hypoxic treatments respectively, White (10)Leghorn with 27.2 ? 1.1 g and 23.5 ? 1.2 g (t=6.37; p<0.05) respectively, and the jungle (14)

    fowl with 21.7 ? 3.5 g and 17.2 ? 2.5 g (t=2.91; p<0.05) respectively. (13)

    Table 2. Embryonic mass of 19 days old chicken embryos and amount of water lost during incubation. Values shown as mean ? SD. The normoxic treatment for each strain is considered as control. Significant values set as p=0.05

Strain Treatment Fetal Mass t-test % of control waterloss (%) t-test n

    Broiler Normoxic 35.4 ? 3.6 100 12.6 ? 1.6 6

    (Linköping) Hypoxic 21.9 ? 2.3 t=7.76 62 11.0 ? 1.0 ns 6 (10)

     25 % relative 32.9 ? 4.0 ns 93 14.0 ? 2.3 ns 10

     humidity

     70 % relative 34.5 ? 3.7 ns 97 6.2 ? 0.8 t=10.69 10 (14)

     humidity

Broiler Normoxic 31.9 ? 4.7 100 11.4 ? 2.3 8

    (Maastricht) Hypoxic 28.0 ? 2.7 ns 88 11.7 ? 2.1 ns 8

White Normoxic 27.2 ? 1.1 100 13.0 ? 1.4 8

    Leghorn Hypoxic 23.5 ? 1.2 t=6.37 87 10.6 ? 1.2 t=3.73 8 (14)(14)

    Jungle Normoxic 21.7 ? 3.5 100 14.8 ? 4.7 7

    fowl Hypoxic 17.2 ? 2.5 t=2.91 79 14.7 ? 4.2 ns 8 (13)

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    = 3.58; p<0.05) for those aortas that were in fixation for a total time of 24 hours (72)5.3 Effects of fixation (Figure 1). The shrinkage effect were significantly larger for those aortas that were in formalin fixative for one to two weeks which had a shrinkage of 20.4 ? 12.7 % in comparison with 11.8 ?

    7.8 % (t

    35

    30

    25*

    20

    15Shrinkage (%)10

    5

    0

    Figure 1.Effects of long versus short fixation. The white

    column is aortas fixed under a longer period (1-2 weeks)

    n= 28, and the black column is aortas fixed for a short

    period of time (24 hours) n= 46.

5.4 Effects of hypoxia on aortic morphology

    There were no significant changes in total diameter for fresh or fixed aortas between the

    normoxic and hypoxic treatments in any of the four strains as shown in Table 3. There was a

    significant difference between the normoxic and hypoxic White Leghorn in the lumen

    diameter 0.53 ? 0.06 mm and 0.46 ? 0.05 mm (t

    = 2.25; p<0.05); respectively as shown in (14)Figure 2. There was also a significant difference in wall thickness in the jungle fowl between

    the normoxic and the hypoxic treatment 0.33 ? 0.04 and 0.27 ? 0.03 (t=3.19 ;p<0.05) (13)

    respectively. No significance could be found on wall/Lumen ratio.

Table 3. Effect of hypoxia on total diameter fresh as well as fixed. The normoxic treatment for

    each strain is considered as control.

Strain Treatment Fresh diameter Fixed diameter n

    Broiler Linköping Normoxic 1.58 ? 0.12 1.32 ? 0.21 6

     Hypoxic 1.45 ? 0.05 1.26 ? 0.12 6

    Broiler Maastricht Normoxic 1.40 ? 0.07 1.30 ? 0.06 8

     Hypoxic 1.31 ? 0.10 1.21 ? 0.12 8

    White Leghorn Normoxic 1.30 ? 0.13 1.20 ? 0.08 8

     Hypoxic 1.34 ? 0.10 1.14 ? 0.12 8

    Jungle fowl Normoxic 1.19 ? 0.07 1.08 ? 0.07 7

     Hypoxic 1.13 ? 0.09 1.00 ? 0.08 8

5.5 Effects of alterations in relative humidity

    The effects of alterations in relative humidity are shown in Table 4. There were no significant

    differences seen when the broiler Linköping were treated with different

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5.6 Pressure-diameter loops

    The results of the pressure-diameter loops performed on the normoxic broiler Linköping

    aortas for two different ages, 19 days old and 20 days externally pipped embryos, are shown

    in Figure 3. The two lines are fairly similar. A

     0,7*0,6

    0,5

    0,4

    0,3

    0,2

    0,1Lumen diameter (mm)

    0

    Broiler LinköpingBrolerWhite LegghornJungle fowlMaastricht

    Strain

    B

    0,50,450,4*0,350,30,250,20,150,1Wall thickness (mm)0,050

    Broiler LinköpingBrolerWhite LegghornJungle fowlMaastricht

    Strain

    C1,4

    1,2

    1

    0,8

    0,6

    0,4Wall/lumen ratio0,2

    0

    Broiler LinköpingBrolerWhite LegghornJungle fowl

    Maastricht

    strain

    Figure 2. The effect of hypoxia on vessel morphology. The

    white columns are aortas are control and the black

    columns are aortas treated with hypoxia. Panel A is the

    lumen diameter for the different Panel B shows the wall

    thickness and Panel C shows the wall/lumen ratio. Values

    are mean ? SD; *p<0.05.

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    2,40

    1,80

    Diameter (mm)

    1,20

    02468

    Pressure (kPa)

    Figure 3. Pressure-diameter loops. Open circles are 19

    days old normoxic broiler Linköping embryos (n=6) and

    the closed circles are 19 days old hypoxic broiler

    Linköping embryos (n=5). Values are mean ? SD; *p<0.05.

6 Discussion

6.1 Effects of fixation

    Controls and hypoxic embryos within each strain were treated the same fixation time so an

    equal shrinkage effect can therefore be assumed.

6.2 Aortic morphology

    Ruijtenbeek et al. (2003a) also noted that the embryos incubated in hypoxia had an altered

    arterial function seen as a reduction in endothelial-dependent relaxation. Alterations to the

    arterial system caused by hypoxia has also been seen by Rouwet et al. (2002), who saw that

    hypoxia induced hypertrophic growth in the aortic wall in the White leghorn chicken embryo.

    This hypertrophy was seen as an increase in aortic wall thickness and a decrease in lumen

    diameter leading to an increased wall/lumen ratio.

6.3 Conclusion

    In conclusion, no evidence of aortic hypertrophy was found, but differences in responses to

    hypoxia could be seen in the different strains used. The differences between the broilers and

    the White Leghorn found in this study might be because of the difference in selection pressure

    for different treats done by breeding (Currie 1999, Dewil et al. 1996). Therefore Even though

    no aortic hypertrophy was found in this study, it cannot exclude alterations in the composition

    of the aortic wall. Looking at the specific layers in the aortic wall would be the good subject

    of further studies and investigations in the alterations in the wall composition would be of

    interest.

7 Acknowledgements

    Many thanks to my supervisor Dr Jordi Altimiras for his enthusiasm and devotion to the

    project ,Thomas Östholm for histological consultation and Henrik Gustavsson from Svenska

    Kläckeribolaget AB in Väderstad for the generous provision of eggs. Tord Jonsson for

    staining assistance, Marta Diaz for scanning assistance and Pia Ågren for incubating my eggs

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    down in Maastricht. Dr Eduardo Villamor for his hospitality. Martin Andersson, Henrik Bergman, Dr Dane Crossley and Isa Lindgren for there great support.

8 References (Journal of experimental Biology format)

    Altimiras, J. and Crossley, D. A. II, (2000). Control of blood pressure mediated by baroreflex changes of heart rate in the chicken embryo (Gallus gallus). American Journal of Physiology

    278, R980-R986.

    Curran-Everett, D. and Benos, D. J. (2004). Guidelines for reporting statistics in journals published by the Americal Physiological Society. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 287, R247-R249.

    Enell, L. (2004). Histological study of chicken embryo aorta during last week of development. Graduate thesis in biology, Department of Physics and Measurement Technology, Linköping University, LiU-IFM-Biol-Ex-1282.

    Jonsson, T. (2005). Structural changes in the aortic wall of chicken foetuses under chronic hypoxic incubation. Graduate thesis in biology, Department of Physics and Measurement Technology, Linköping University, LiU-IFM-Biol-Ex-05/1457-SE.

    Speckmann, E. W. and Ringer, R. K. (1966). Volume-Pressure Relations of the Turkey Aorta. Canadian Journal of Physiology and Pharmacology. 44, 901-907.

    Wells, S. M., Langile, B.L., Lee, J. M. and Adamson, S. L. (1999).Determinants of mechanical properties in the developing ovine thoracic aorta. American Journal of Physiology Heart and Circulatory Physiology 277, H1385-H1391.

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