A pictorial essay on fetal rabbit anatomy using micro-ultrasound and magnetic resonance imaging
Philip DeKoninck1,2, Masayuki Endo1,2, Inga Sandaite1,3, Jute Richter1,2, Luc De Catte1,2, Ben Van Calster1, Jaan Toelen1,2, Uwe Himmelreich3, Filip Claus3, Jan Deprest1,2*
1
Organ systems cluster, Department of Development and Regeneration, and 2Center for
Surgical Technologies, KU Leuven, Leuven, Belgium. 3
Department of Radiology, University Hospitals Leuven, Leuven, Belgium
4
Biomedical MRI cluster, Department of Imaging and Pathology, KU Leuven, Leuven,
Belgium
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/pd.4259
This article is protected by copyright. All rights reserved.
Funding sources: ME and PD are beneficiaries of a Marie Curie Industria-Academia Partnership and Pathways grant sponsored by the European Commission (251356). JDP is beneficiary of a fundamental clinical research grant of the Fonds Wetenschappelijk Onderzoek (FWO) Vlaanderen (1.8.012.07.N.02) and JR, JT and LD from the “Klinische Opleidings- en Onderzoeks- Raad” of the University Hospitals Leuven. BVC is a postdoctoral fellow of the Research Foundation Flanders (FWO). Our experimental program is supported by the Flemish Hercules foundation (large infrastructure investments AKUL/09/033) and by the KU Leuven (OT/13/115).
Running title: Pictorial essay on fetal rabbit development
* Corresponding author: Prof. J. Deprest, University Hospitals Leuven – Division Of Woman and Child, Department of Obstetrics and Gynecology, Fetal Medicine Unit - Herestraat 49, B3000 Leuven, Belgium - Tel: +32 16 344215 - Fax: +32 16 344205 - Email: [email protected]. Keywords: MRI, micro-ultrasound, Doppler ultrasound, biometry, rabbit
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Bulleted statements What’s already known about this topic? In the recent decades more advanced ultrasound platforms have been developed, providing high resolution images. Thereby allowing a more detailed structural, as well as functional evaluation of fetal development in several animal models. The rabbit model is used in numerous fields of research and therefore detailed imaging with micro-ultrasound could provide interesting new insights for many researchers.
What does this study add? We have provided gestational age specific reference ranges for various biometric variables in the rabbit, as well as an anatomical overview of the fetal thoracic development. This could be useful for researchers planning future experiments in the rabbit model.
This article is protected by copyright. All rights reserved.
Abstract: Introduction With this pictorial essay we aimed to provide gestational age specific reference ranges of relevant fetal structures using micro- ultrasound, as well as its correlation with postmortem MRI and whole body sections. Material and Methods Time-mated pregnant rabbits (n=24) were assessed once at various gestational ages in the second half of pregnancy (15, 17, 21-23, 25-28 and 30-31 days; term=31 days). We obtained biometric data, together with Doppler flow patterns in the ductus venosus (DV), umbilical artery (UA) and umbilical vein. After euthanasia, micro-ultrasound images were compared with images obtained by 9.4 Tesla MRI and whole body paraffin sections at 15,23,26 and 28 days. Results We constructed biometric normative curves, which showed a significant correlation with gestational age. The pulsatility index (PI) in the UA decreased with gestation (PI= 5.746 – 0.2969(GA) + 0.004931(GA)2; R²=0.30), whereas pulsatility index for veins (PIV) in the DV remained constant (median PIV= 0.82 (0.60-1.12)). In this report we provide an anatomical atlas of fetal thoracic development using both micro-ultrasound and MRI. Conclusion We describe anatomical fetal leporine development as can be visualized by microultrasound and MR imaging. The reported reference ranges may be useful for researchers using the fetal rabbit model.
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Introduction The development of new hardware platforms, such as micro-ultrasound or micro MRI, allows non-invasive fetal assessment of small experimental animals. The fetal rabbit is used in different research areas, including that of pulmonary development, intrauterine growth and prematurity.1-3 The relatively short duration of gestation (term = 31 days), the large litter size (average=8) and the relatively large fetal size (at term = 50g) make this animal an interesting model for fetal surgical research. Ultrasound (US) imaging has been proven safe in the evaluation of the developing fetus. Moreover as no ionizing radiation is necessary there is no additional hazard for the operator. A second advantage of ultrasound is the option of both real-time evaluation as well as offline image processing, allowing the assessment of both static and dynamic processes. With conventional US imaging resolution is limited (300 to 500 µm). Resolution scales directly with frequency, so that significant higher scanning frequencies are required to allow assessment of small animals at the expense of penetration depth.4,5 Newer generation micro-ultrasound platforms (also called ultrasound biomicroscopy scanners) provide excellent resolution with sufficient depth.4,5 Apart from structural evaluation, high frequency probes also allow detailed functional analysis with newer imaging modalities such as 3D image acquisition and tissue Doppler. The use of such novel micro-ultrasound platforms for the evaluation of fetal rabbit development has not been described yet. In 2008, Chavatte-Palmer et al. published an overview of the development of leporine large fetal structures (body length, heart length, etc) using a low frequency (7.5 MHz) ultrasound system.6 Imaging of small structures as limbs or umbilical vessels was less successful.6 With this pictorial essay we aimed to provide a detailed anatomical atlas, together with normative ranges for a number of fetal rabbit
This article is protected by copyright. All rights reserved.
anatomical structures and several physiological parameters. Micro-ultrasound was combined with postmortem MRI and further illustrated with whole body paraffin sections.
Materials and methods Animals Time-mated pregnant rabbits (hybrid of Dendermonde and New Zealand White) at various gestational ages were used for this study. Prior to the experiment, animals were housed in separate cages at normal room temperature and daylight, with free access to food and water. All animals were treated according to the current guidelines on animal well-being. The Ethics Committee for Animal Experimentation of the Faculty of Medicine of the KU Leuven approved the experiments. Premedication consisted of ketamin 35 mg/kg (Ketamin 1000®; CEVA Sante Animale, Libourne, France) and xylazine 6 mg/kg (Vexylan®; CEVA Sante Animale). General anesthesia was maintained using a face mask with isoflurane 1.5% (Isoba®Vet; Abbott Laboratories Ltd., Queenborough, Kent, UK) in oxygen at 2 l/min. The uterine horn with the fetus of interest was exteriorized after a midline laparotomy. All ultrasound scans were performed directly on the uterus, with minimal manipulation of the fetuses. During the examination the exposed uterus was continuously irrigated with warmed saline, standard ultrasound gel was applied for imaging. We evaluated up to four fetuses per doe, i.e. both ovarian-end fetuses, as well as the second next fetus from the ovarian end, as these are the preferred locations for surgical experiments. At the end of the experiment the mother was euthanized with an IV bolus (0.3 ml/kg) of a mixture of embutramide 200 mg/ml, mebezonium 50 mg/ml and tetracain hydrochloride 5 mg/ml (T61® Intervet Belgium NV, Mechelen, Belgium). The fetuses were harvested following
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hysterotomy, their weight was noted, and they were randomly assigned to either postmortem MRI evaluation or further processing for whole body sections.
Ultrasound imaging All images were obtained with a micro-ultrasound platform (Vevo2100, VisualSonics Inc, Toronto, Ontario, Canada) with either the linear 20 or 30 MHz probe (MS-250 resp. MS-400). Providing a 30 micron resolution and frame rates up to 740 fps. In each fetus we first measured the blood flow in the ductus venosus (DV), umbilical vein (UV) and umbilical artery (UA). After identification with color-flow mapping we used pulsed Doppler to assess the flow pattern. The insonation angle was kept close to 0 degrees; whenever necessary we used angle correction with a maximum of 20 degrees. We then systematically acquired images of the fetal head at level of the orbits, the thorax at level of the 4-chamber view, the abdomen at the level of the stomach, the humerus and femur, the umbilical vein, one of the umbilical arteries, amniotic fluid and the placenta at the insertion of the umbilical vessels. All images were stored digitally and were analyzed offline with the provided software (VEVO 2100 Imaging System software version 1.2.0). We measured the bi-orbital diameter (BOD), defined as the distance between the external borders of the eyeballs on a coronal section. This is contrary to what is generally used clinically. However, in our experience the biparietal diameter in rabbits is difficult to measure in a reproducible way, the eyes on the other hand are easy to visualize. We measured the abdominal circumference (AC), cardiac circumference (CC) and thoracic circumference (TC), from which the cardio-thoracic index (CTI=CC/TC) was derived. Humerus- (HL) and femur length (FL) were defined as the distance between the outer edges of the ossified bone diaphysis. Furthermore, we obtained the umbilical vein and artery diameter (UVD and UAD), deepest vertical pocket of amniotic fluid
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(DVP) and placental thickness (PLAC). The pulsatility index of the UA was calculated using the following formula PI = peak systolic velocity (PSV) – end diastolic flow velocity (EDF) / mean velocity. The flow in the ductus venosus was expressed as pulsatility index for veins (PIV = Vmax (S-wave) – Vmin (a-wave) / mean velocity). Further we measured the peak flow velocity in the umbilical vein, except for fetuses with a pulsatile flow in the UV (n=18).
Postmortem MRI imaging Rabbit fetuses were kept at 4°C until immediately before MRI was done. Whole-body MR images were acquired using a Bruker Biospec 9.4 Tesla small animal MR scanner (Bruker Biospin, Ettlingen, Germany; horizontal bore, 20 cm) equipped with actively shielded gradients (600 mT/m) and using a 3.5 cm quadrature coil (volume resonator, Rapid Biomedical, Rimpar, Germany). For planning purposes, two-dimensional 2D sagittal and coronal (T2-weighted) MR images were recorded. High-resolution three-dimensional (3D) MR images were acquired using either a gradient-echo sequence (FLASH, TR=120 ms, TE=10 ms; flip angle 60°, FOV: 90 x 30 x 30 mm, isotropic resolution of 117 µm, number of averages (NA)=2); a spin echo sequence (TurboRARE, TR=1300ms, TE=30ms, RARE factor of 16, FOV: 30 x 30 x 12 mm, resolution 176 x 117 x 117 µm, NA=2) or a 2D MDEFT protocol (TR=11ms, TE=3.5ms, N segments=8 (segment TR=3500ms, segment duration=154ms), NA=6, flip angle=20°, 50 slices of 0.5mm thickness, FOV: 90 x 30 mm, in plane resolution 234µm). The analysis of obtained images was performed by manual delineation and measurement of the respective anatomical parameters using the Bruker software ParaVision 5.1.
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Histology: The harvested fetuses were completely immersed in paraformaldehyde solution (4%) for several days and subsequently embedded in paraffin, cut in 5 µm thickness whole body sections and stained with hematoxylin and eosin for gross microscopic imaging.
Statistics Data were analyzed with Prism for Windows version 5.0 (Graphpad software, San Diego, CA, USA). Continuous data were expressed as medians and IQR. Ordinary least-square regression analysis was used to model the mean evolution of each parameter using the method described by Royston et al.7 The mean was modeled as a polynomial function of gestational age, with maximal degree of 3 (i.e. cubic polynomial: a + b*GA + c*GA^2 + d*GA^3). Using statistical significance and goodness-of-fit indexes we determined whether a cubic, quadratic (i.e. d=0), or linear (i.e. c=d=0) effect was most adequate. Then, using scaled absolute residuals of the resulting model for the mean, the standard deviation was modeled using a similar approach7, but bearing in mind that gestational age typically has a less complex relationship with SD than with the mean. Using the predicted mean and SD by gestational age, model fit was checked using scatter plots of the Z score by gestational age.7 From the predicted mean and SD equations, we calculated the 10th and 90th centile using the formula: centile = mean + K x SD, where K is ± 1.28.7 We analyzed the correlations between various biometrical measurements and the fetal weight with Spearman’s correlation coefficient.
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Results A total of 85 fetuses from 24 different does were evaluated at various gestational ages 15, 17, 21-23, 25-28 and 30-31 days. All fetuses survived until sacrifice. In the vast majority all structural measurements were feasible, except for FL and HL measurements at 15 and 17 days. This is mainly related to the limited ossification of the bony structures early in gestation. Figure 1 displays gestational age specific reference ranges for the BOD, AC, TC, CC and CTI. MRI measurements at different gestational ages are also reported, but not included in the calculation of reference ranges. Amniotic fluid quantity increased up to 22 days of gestation followed by a decline towards term (figure 2). Placental thickness also increased gradually with gestational age (figure 2). The humerus and the femur length measurements with ultrasound and MRI are presented in figure 3. Figure 4 displays the normative ranges for vessel diameters of the umbilical artery and vein. We observed reversed end diastolic flow in the umbilical artery in all fetuses at 15 days of gestation. From 17 days onwards the pulsatility index in the umbilical artery decreased gradually (PI = 5.746 – 0.2969(GA) + 0.004931(GA)2; R²=0.30). The flow in the ductus venosus remained constant throughout gestation (median PIV = 0.82 (0.60-1.12). In the majority of fetuses (75%) we observed a non-pulsatile flow pattern in the umbilical vein, with a median peak velocity of 66.7 cm/s (IQR: 47.6-85.6 cm/s). Cases with observed pulsatile flow in the umbilical vein were randomly distributed throughout gestation. Figure 5 gives an overview of flow patterns in the ductus venosus and the umbilical artery and vein. When evaluating the correlation between biometric measurements and fetal weight, we found the best correlation for femur length (r = 0.97) as shown in figure 6. Together with images acquired by post mortem MRI and whole body sections we aimed to provide a chronological and detailed structural overview of a transverse section of the
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thorax at the level of the 4-chamber view of the heart throughout the pregnancy. Detail is sufficient to localize the trachea, thymus and the fetal heart anatomy. Figure 7 and 8 show an anatomic correlation between postmortem imaging and in vivo micro-ultrasound at various gestational ages. At later gestational ages (>28 days) postmortem whole body images were of insufficient quality and therefore these were left out.
Discussion We report gestational age specific reference ranges, for both anatomical biometric structures as well as functional parameters of blood flow in a selection of fetal vessels. We also provide a chronologic series of images of the fetal chest with advancing pregnancy, showing a good anatomical correlation between micro-ultrasound, high resolution MRI images and histology sections. However at early gestational ages micro-ultrasound allows a more detailed evaluation of the fetal anatomy and at that stage this modality is clearly superior to MRI. Moreover, micro-ultrasound allows evaluation of smaller structures such as umbilical vessels, which is an improvement compared to what is reported with lower frequency ultrasound probes6. Also, functional evaluation of blood flow in fetal arteries as well as cardiac assessment is feasible with these high frequency probes. We observed a steady decrease in umbilical arterial pulsatility index, as previously documented with low frequency probes by Polisca et al.8 In contrast, the peak flow velocity in the umbilical vein and the PI in the ductus venosus remained constant throughout gestation. Yet, anesthetic products as xylazine and isoflurane potentially alter fetal cardiac function, as such influencing the measured data.9,10 Therefore alternative anesthetic methods such as
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epidural analgesia or anesthetic agents with less impact on cardiac function should be considered when assessing the fetal heart and function. Although it is possible to obtain high quality images without exteriorizing the uterine horns, hence make longitudinal studies, previous reports already indicate that identification of the total number and exact location of fetuses is not easy.6 We confirmed this in our experiment (data not shown). The use of MRI in-vivo could potentially overcome these difficulties, however in light of performing experimental procedures this is not yet applicable. In conclusion, this pictorial essay provides typical images and biometric data on leporine fetal development. Micro-ultrasound allows detailed in vivo examination of small fetal structures, including intrathoracic structures that may be of interest for interventional studies.
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References
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Eixarch E, Figueras F, Hernandez-Andrade E, Crispi F, Nadal A, Torre I, et al. An experimental model of fetal growth restriction based on selective ligature of uteroplacental vessels in the pregnant rabbit. Fetal. Diagn. Ther. 2009;26(4):203–11.
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Wu J, Yamamoto H, Gratacós E, Ge X, Verbeken E, Sueishi K, et al. Lung development following diaphragmatic hernia in the fetal rabbit. Hum. Reprod. 2000 Dec;15(12):2483–8.
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Gras-Le Guen C, Denis C, Franco-Montoya M-L, Jarry A, Delacourt C, Potel G, et al. Antenatal infection in the rabbit impairs post-natal growth and lung alveolarisation. European Respiratory Journal. 2008 Dec 1;32(6):1520–8.
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Foster FS, Zhang MY, Zhou YQ, Liu G, Mehi J, Cherin E, et al. A new ultrasound instrument for in vivo microimaging of mice. Ultrasound Med Biol. 2002 Sep;28(9):1165–72.
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Moran CM, Pye SD, Ellis W, Janeczko A, Morris KD, McNeilly AS, et al. A comparison of the imaging performance of high resolution ultrasound scanners for preclinical imaging. Ultrasound Med Biol. 2011 Mar;37(3):493–501.
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Chavatte-Palmer P, Laigre P, Simonoff E, Chesné P, Challah-Jacques M, Renard J-P. In utero characterisation of fetal growth by ultrasound scanning in the rabbit. Theriogenology. 2008 Apr 15;69(7):859–69.
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Royston P, Wright EM. How to construct “normal ranges” for fetal variables. Ultrasound Obstet Gynecol. 1998 Jan;11(1):30–8.
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Polisca A, Scotti L, Orlandi R, Brecchia G, Boiti C. Doppler evaluation of maternal and fetal vessels during normal gestation in rabbits. Theriogenology. 2010 Feb;73(3):358– 66.
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Baumgartner C, Bollerhey M, Ebner J, Laacke-Singer L, Schuster T, Erhardt W. Effects of ketamine-xylazine intravenous bolus injection on cardiovascular function in rabbits. Can. J. Vet. Res. 2010 Jul;74(3):200–8.
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Sanford TD, Colby ED. Effect of xylazine and ketamine on blood pressure, heart rate and respiratory rate in rabbits. Lab. Anim. Sci. 1980 Jun;30(3):519–23.
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Figure 1:
Polynomial functions of the mean, together with 10th and 90th centiles, for BOD (1a: BOD = 51.68 – 7.466(GA) + 0.3808(GA)2 – 0.005632(GA)3); AC (1b: AC = 242.9 – 36.47(GA) + 1.863(GA)2 – 0.02785(GA)3); CC (1c: CC = -11.8 + 1.397 (GA)); TC (1d: TC = -12.43 + 2.146(GA)) and CTI (1e: CTI = 0.7216 – 0.02054(GA) + 0.0005559(GA)²), with GA in days. The red squares represent MRI measurements at various gestational ages.
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Figure 2:
Polynomial functions of the mean, together with 10th and 90th centiles, for DVP (2a: DVP = -52.88 + 5.70(GA) - 0.1282(GA)²) and placental thickness (2b: placental thickness = 0.1947 + 0.4655(GA), with GA in days
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Figure 3:
Polynomial functions of the mean, together with 10th and 90th centiles, for HL (3a: HL = -15.1 + 0.8788(GA)) and FL (3b: FL = -18.15 + 0.9947(GA)) starting from GA day 21, with GA in days. Before 21 days only MRI measurements (red squares) were feasible.
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Figure 4:
Polynomial functions of the mean, together with 10th and 90th centiles, for UA and UV diameter (4a: UAd = 0.08336 + 0.03025(GA); 4b: UVd = -0.4570 + 0.07026(GA)), with GA in days.
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Figure 5:
a-c: Doppler flow patterns in ductus venosus, umbilical vein and umbilical artery, respectively. 5d represents polynomial function of the mean, together with 10th and 90th centiles, for pulsatility index in the umbilical artery (UA PI = 5.746 – 0.2969(GA) + 0.004931(GA)²), with GA in days
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Figure 6:
Scatterplot showing the correlation between FL and fetal weight (FW), Spearman’s r 0.97
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Figure 7:
Transverse sections of the fetal thorax at level of cardiac 4-chamber view, at various gestational ages (day 15, 23, 26 and 28). Top: micro-ultrasound images; middle: MRI images (RARE sequences); bottom: paraffin sections.
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Figure 8:
Sagittal and transverse sections of rabbit fetus at 15 gestational days. Left: micro-ultrasound; right: paraffin sections. H: heart; Li: liver; arrow: umbilical vessels.
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Philip DeKoninck1,2, Masayuki Endo1,2, Inga Sandaite1,3, Jute Richter1,2, Luc De Catte1,2, Ben Van Calster1, Jaan Toelen1,2, Uwe Himmelreich3, Filip Claus3, Jan Deprest1,2*
1
Organ systems cluster, Department of Development and Regeneration, and 2Center for
Surgical Technologies, KU Leuven, Leuven, Belgium. 3
Department of Radiology, University Hospitals Leuven, Leuven, Belgium
4
Biomedical MRI cluster, Department of Imaging and Pathology, KU Leuven, Leuven,
Belgium
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/pd.4259
This article is protected by copyright. All rights reserved.
Funding sources: ME and PD are beneficiaries of a Marie Curie Industria-Academia Partnership and Pathways grant sponsored by the European Commission (251356). JDP is beneficiary of a fundamental clinical research grant of the Fonds Wetenschappelijk Onderzoek (FWO) Vlaanderen (1.8.012.07.N.02) and JR, JT and LD from the “Klinische Opleidings- en Onderzoeks- Raad” of the University Hospitals Leuven. BVC is a postdoctoral fellow of the Research Foundation Flanders (FWO). Our experimental program is supported by the Flemish Hercules foundation (large infrastructure investments AKUL/09/033) and by the KU Leuven (OT/13/115).
Running title: Pictorial essay on fetal rabbit development
* Corresponding author: Prof. J. Deprest, University Hospitals Leuven – Division Of Woman and Child, Department of Obstetrics and Gynecology, Fetal Medicine Unit - Herestraat 49, B3000 Leuven, Belgium - Tel: +32 16 344215 - Fax: +32 16 344205 - Email: [email protected]. Keywords: MRI, micro-ultrasound, Doppler ultrasound, biometry, rabbit
This article is protected by copyright. All rights reserved.
Bulleted statements What’s already known about this topic? In the recent decades more advanced ultrasound platforms have been developed, providing high resolution images. Thereby allowing a more detailed structural, as well as functional evaluation of fetal development in several animal models. The rabbit model is used in numerous fields of research and therefore detailed imaging with micro-ultrasound could provide interesting new insights for many researchers.
What does this study add? We have provided gestational age specific reference ranges for various biometric variables in the rabbit, as well as an anatomical overview of the fetal thoracic development. This could be useful for researchers planning future experiments in the rabbit model.
This article is protected by copyright. All rights reserved.
Abstract: Introduction With this pictorial essay we aimed to provide gestational age specific reference ranges of relevant fetal structures using micro- ultrasound, as well as its correlation with postmortem MRI and whole body sections. Material and Methods Time-mated pregnant rabbits (n=24) were assessed once at various gestational ages in the second half of pregnancy (15, 17, 21-23, 25-28 and 30-31 days; term=31 days). We obtained biometric data, together with Doppler flow patterns in the ductus venosus (DV), umbilical artery (UA) and umbilical vein. After euthanasia, micro-ultrasound images were compared with images obtained by 9.4 Tesla MRI and whole body paraffin sections at 15,23,26 and 28 days. Results We constructed biometric normative curves, which showed a significant correlation with gestational age. The pulsatility index (PI) in the UA decreased with gestation (PI= 5.746 – 0.2969(GA) + 0.004931(GA)2; R²=0.30), whereas pulsatility index for veins (PIV) in the DV remained constant (median PIV= 0.82 (0.60-1.12)). In this report we provide an anatomical atlas of fetal thoracic development using both micro-ultrasound and MRI. Conclusion We describe anatomical fetal leporine development as can be visualized by microultrasound and MR imaging. The reported reference ranges may be useful for researchers using the fetal rabbit model.
This article is protected by copyright. All rights reserved.
Introduction The development of new hardware platforms, such as micro-ultrasound or micro MRI, allows non-invasive fetal assessment of small experimental animals. The fetal rabbit is used in different research areas, including that of pulmonary development, intrauterine growth and prematurity.1-3 The relatively short duration of gestation (term = 31 days), the large litter size (average=8) and the relatively large fetal size (at term = 50g) make this animal an interesting model for fetal surgical research. Ultrasound (US) imaging has been proven safe in the evaluation of the developing fetus. Moreover as no ionizing radiation is necessary there is no additional hazard for the operator. A second advantage of ultrasound is the option of both real-time evaluation as well as offline image processing, allowing the assessment of both static and dynamic processes. With conventional US imaging resolution is limited (300 to 500 µm). Resolution scales directly with frequency, so that significant higher scanning frequencies are required to allow assessment of small animals at the expense of penetration depth.4,5 Newer generation micro-ultrasound platforms (also called ultrasound biomicroscopy scanners) provide excellent resolution with sufficient depth.4,5 Apart from structural evaluation, high frequency probes also allow detailed functional analysis with newer imaging modalities such as 3D image acquisition and tissue Doppler. The use of such novel micro-ultrasound platforms for the evaluation of fetal rabbit development has not been described yet. In 2008, Chavatte-Palmer et al. published an overview of the development of leporine large fetal structures (body length, heart length, etc) using a low frequency (7.5 MHz) ultrasound system.6 Imaging of small structures as limbs or umbilical vessels was less successful.6 With this pictorial essay we aimed to provide a detailed anatomical atlas, together with normative ranges for a number of fetal rabbit
This article is protected by copyright. All rights reserved.
anatomical structures and several physiological parameters. Micro-ultrasound was combined with postmortem MRI and further illustrated with whole body paraffin sections.
Materials and methods Animals Time-mated pregnant rabbits (hybrid of Dendermonde and New Zealand White) at various gestational ages were used for this study. Prior to the experiment, animals were housed in separate cages at normal room temperature and daylight, with free access to food and water. All animals were treated according to the current guidelines on animal well-being. The Ethics Committee for Animal Experimentation of the Faculty of Medicine of the KU Leuven approved the experiments. Premedication consisted of ketamin 35 mg/kg (Ketamin 1000®; CEVA Sante Animale, Libourne, France) and xylazine 6 mg/kg (Vexylan®; CEVA Sante Animale). General anesthesia was maintained using a face mask with isoflurane 1.5% (Isoba®Vet; Abbott Laboratories Ltd., Queenborough, Kent, UK) in oxygen at 2 l/min. The uterine horn with the fetus of interest was exteriorized after a midline laparotomy. All ultrasound scans were performed directly on the uterus, with minimal manipulation of the fetuses. During the examination the exposed uterus was continuously irrigated with warmed saline, standard ultrasound gel was applied for imaging. We evaluated up to four fetuses per doe, i.e. both ovarian-end fetuses, as well as the second next fetus from the ovarian end, as these are the preferred locations for surgical experiments. At the end of the experiment the mother was euthanized with an IV bolus (0.3 ml/kg) of a mixture of embutramide 200 mg/ml, mebezonium 50 mg/ml and tetracain hydrochloride 5 mg/ml (T61® Intervet Belgium NV, Mechelen, Belgium). The fetuses were harvested following
This article is protected by copyright. All rights reserved.
hysterotomy, their weight was noted, and they were randomly assigned to either postmortem MRI evaluation or further processing for whole body sections.
Ultrasound imaging All images were obtained with a micro-ultrasound platform (Vevo2100, VisualSonics Inc, Toronto, Ontario, Canada) with either the linear 20 or 30 MHz probe (MS-250 resp. MS-400). Providing a 30 micron resolution and frame rates up to 740 fps. In each fetus we first measured the blood flow in the ductus venosus (DV), umbilical vein (UV) and umbilical artery (UA). After identification with color-flow mapping we used pulsed Doppler to assess the flow pattern. The insonation angle was kept close to 0 degrees; whenever necessary we used angle correction with a maximum of 20 degrees. We then systematically acquired images of the fetal head at level of the orbits, the thorax at level of the 4-chamber view, the abdomen at the level of the stomach, the humerus and femur, the umbilical vein, one of the umbilical arteries, amniotic fluid and the placenta at the insertion of the umbilical vessels. All images were stored digitally and were analyzed offline with the provided software (VEVO 2100 Imaging System software version 1.2.0). We measured the bi-orbital diameter (BOD), defined as the distance between the external borders of the eyeballs on a coronal section. This is contrary to what is generally used clinically. However, in our experience the biparietal diameter in rabbits is difficult to measure in a reproducible way, the eyes on the other hand are easy to visualize. We measured the abdominal circumference (AC), cardiac circumference (CC) and thoracic circumference (TC), from which the cardio-thoracic index (CTI=CC/TC) was derived. Humerus- (HL) and femur length (FL) were defined as the distance between the outer edges of the ossified bone diaphysis. Furthermore, we obtained the umbilical vein and artery diameter (UVD and UAD), deepest vertical pocket of amniotic fluid
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(DVP) and placental thickness (PLAC). The pulsatility index of the UA was calculated using the following formula PI = peak systolic velocity (PSV) – end diastolic flow velocity (EDF) / mean velocity. The flow in the ductus venosus was expressed as pulsatility index for veins (PIV = Vmax (S-wave) – Vmin (a-wave) / mean velocity). Further we measured the peak flow velocity in the umbilical vein, except for fetuses with a pulsatile flow in the UV (n=18).
Postmortem MRI imaging Rabbit fetuses were kept at 4°C until immediately before MRI was done. Whole-body MR images were acquired using a Bruker Biospec 9.4 Tesla small animal MR scanner (Bruker Biospin, Ettlingen, Germany; horizontal bore, 20 cm) equipped with actively shielded gradients (600 mT/m) and using a 3.5 cm quadrature coil (volume resonator, Rapid Biomedical, Rimpar, Germany). For planning purposes, two-dimensional 2D sagittal and coronal (T2-weighted) MR images were recorded. High-resolution three-dimensional (3D) MR images were acquired using either a gradient-echo sequence (FLASH, TR=120 ms, TE=10 ms; flip angle 60°, FOV: 90 x 30 x 30 mm, isotropic resolution of 117 µm, number of averages (NA)=2); a spin echo sequence (TurboRARE, TR=1300ms, TE=30ms, RARE factor of 16, FOV: 30 x 30 x 12 mm, resolution 176 x 117 x 117 µm, NA=2) or a 2D MDEFT protocol (TR=11ms, TE=3.5ms, N segments=8 (segment TR=3500ms, segment duration=154ms), NA=6, flip angle=20°, 50 slices of 0.5mm thickness, FOV: 90 x 30 mm, in plane resolution 234µm). The analysis of obtained images was performed by manual delineation and measurement of the respective anatomical parameters using the Bruker software ParaVision 5.1.
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Histology: The harvested fetuses were completely immersed in paraformaldehyde solution (4%) for several days and subsequently embedded in paraffin, cut in 5 µm thickness whole body sections and stained with hematoxylin and eosin for gross microscopic imaging.
Statistics Data were analyzed with Prism for Windows version 5.0 (Graphpad software, San Diego, CA, USA). Continuous data were expressed as medians and IQR. Ordinary least-square regression analysis was used to model the mean evolution of each parameter using the method described by Royston et al.7 The mean was modeled as a polynomial function of gestational age, with maximal degree of 3 (i.e. cubic polynomial: a + b*GA + c*GA^2 + d*GA^3). Using statistical significance and goodness-of-fit indexes we determined whether a cubic, quadratic (i.e. d=0), or linear (i.e. c=d=0) effect was most adequate. Then, using scaled absolute residuals of the resulting model for the mean, the standard deviation was modeled using a similar approach7, but bearing in mind that gestational age typically has a less complex relationship with SD than with the mean. Using the predicted mean and SD by gestational age, model fit was checked using scatter plots of the Z score by gestational age.7 From the predicted mean and SD equations, we calculated the 10th and 90th centile using the formula: centile = mean + K x SD, where K is ± 1.28.7 We analyzed the correlations between various biometrical measurements and the fetal weight with Spearman’s correlation coefficient.
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Results A total of 85 fetuses from 24 different does were evaluated at various gestational ages 15, 17, 21-23, 25-28 and 30-31 days. All fetuses survived until sacrifice. In the vast majority all structural measurements were feasible, except for FL and HL measurements at 15 and 17 days. This is mainly related to the limited ossification of the bony structures early in gestation. Figure 1 displays gestational age specific reference ranges for the BOD, AC, TC, CC and CTI. MRI measurements at different gestational ages are also reported, but not included in the calculation of reference ranges. Amniotic fluid quantity increased up to 22 days of gestation followed by a decline towards term (figure 2). Placental thickness also increased gradually with gestational age (figure 2). The humerus and the femur length measurements with ultrasound and MRI are presented in figure 3. Figure 4 displays the normative ranges for vessel diameters of the umbilical artery and vein. We observed reversed end diastolic flow in the umbilical artery in all fetuses at 15 days of gestation. From 17 days onwards the pulsatility index in the umbilical artery decreased gradually (PI = 5.746 – 0.2969(GA) + 0.004931(GA)2; R²=0.30). The flow in the ductus venosus remained constant throughout gestation (median PIV = 0.82 (0.60-1.12). In the majority of fetuses (75%) we observed a non-pulsatile flow pattern in the umbilical vein, with a median peak velocity of 66.7 cm/s (IQR: 47.6-85.6 cm/s). Cases with observed pulsatile flow in the umbilical vein were randomly distributed throughout gestation. Figure 5 gives an overview of flow patterns in the ductus venosus and the umbilical artery and vein. When evaluating the correlation between biometric measurements and fetal weight, we found the best correlation for femur length (r = 0.97) as shown in figure 6. Together with images acquired by post mortem MRI and whole body sections we aimed to provide a chronological and detailed structural overview of a transverse section of the
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thorax at the level of the 4-chamber view of the heart throughout the pregnancy. Detail is sufficient to localize the trachea, thymus and the fetal heart anatomy. Figure 7 and 8 show an anatomic correlation between postmortem imaging and in vivo micro-ultrasound at various gestational ages. At later gestational ages (>28 days) postmortem whole body images were of insufficient quality and therefore these were left out.
Discussion We report gestational age specific reference ranges, for both anatomical biometric structures as well as functional parameters of blood flow in a selection of fetal vessels. We also provide a chronologic series of images of the fetal chest with advancing pregnancy, showing a good anatomical correlation between micro-ultrasound, high resolution MRI images and histology sections. However at early gestational ages micro-ultrasound allows a more detailed evaluation of the fetal anatomy and at that stage this modality is clearly superior to MRI. Moreover, micro-ultrasound allows evaluation of smaller structures such as umbilical vessels, which is an improvement compared to what is reported with lower frequency ultrasound probes6. Also, functional evaluation of blood flow in fetal arteries as well as cardiac assessment is feasible with these high frequency probes. We observed a steady decrease in umbilical arterial pulsatility index, as previously documented with low frequency probes by Polisca et al.8 In contrast, the peak flow velocity in the umbilical vein and the PI in the ductus venosus remained constant throughout gestation. Yet, anesthetic products as xylazine and isoflurane potentially alter fetal cardiac function, as such influencing the measured data.9,10 Therefore alternative anesthetic methods such as
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epidural analgesia or anesthetic agents with less impact on cardiac function should be considered when assessing the fetal heart and function. Although it is possible to obtain high quality images without exteriorizing the uterine horns, hence make longitudinal studies, previous reports already indicate that identification of the total number and exact location of fetuses is not easy.6 We confirmed this in our experiment (data not shown). The use of MRI in-vivo could potentially overcome these difficulties, however in light of performing experimental procedures this is not yet applicable. In conclusion, this pictorial essay provides typical images and biometric data on leporine fetal development. Micro-ultrasound allows detailed in vivo examination of small fetal structures, including intrathoracic structures that may be of interest for interventional studies.
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Figure 1:
Polynomial functions of the mean, together with 10th and 90th centiles, for BOD (1a: BOD = 51.68 – 7.466(GA) + 0.3808(GA)2 – 0.005632(GA)3); AC (1b: AC = 242.9 – 36.47(GA) + 1.863(GA)2 – 0.02785(GA)3); CC (1c: CC = -11.8 + 1.397 (GA)); TC (1d: TC = -12.43 + 2.146(GA)) and CTI (1e: CTI = 0.7216 – 0.02054(GA) + 0.0005559(GA)²), with GA in days. The red squares represent MRI measurements at various gestational ages.
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Figure 2:
Polynomial functions of the mean, together with 10th and 90th centiles, for DVP (2a: DVP = -52.88 + 5.70(GA) - 0.1282(GA)²) and placental thickness (2b: placental thickness = 0.1947 + 0.4655(GA), with GA in days
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Figure 3:
Polynomial functions of the mean, together with 10th and 90th centiles, for HL (3a: HL = -15.1 + 0.8788(GA)) and FL (3b: FL = -18.15 + 0.9947(GA)) starting from GA day 21, with GA in days. Before 21 days only MRI measurements (red squares) were feasible.
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Figure 4:
Polynomial functions of the mean, together with 10th and 90th centiles, for UA and UV diameter (4a: UAd = 0.08336 + 0.03025(GA); 4b: UVd = -0.4570 + 0.07026(GA)), with GA in days.
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Figure 5:
a-c: Doppler flow patterns in ductus venosus, umbilical vein and umbilical artery, respectively. 5d represents polynomial function of the mean, together with 10th and 90th centiles, for pulsatility index in the umbilical artery (UA PI = 5.746 – 0.2969(GA) + 0.004931(GA)²), with GA in days
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Figure 6:
Scatterplot showing the correlation between FL and fetal weight (FW), Spearman’s r 0.97
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Figure 7:
Transverse sections of the fetal thorax at level of cardiac 4-chamber view, at various gestational ages (day 15, 23, 26 and 28). Top: micro-ultrasound images; middle: MRI images (RARE sequences); bottom: paraffin sections.
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Figure 8:
Sagittal and transverse sections of rabbit fetus at 15 gestational days. Left: micro-ultrasound; right: paraffin sections. H: heart; Li: liver; arrow: umbilical vessels.
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