SPIOenhanced magnetic resonance imaging ... - Wiley Online Library

Basic science

DOI: 10.1111/j.1471-0528.2011.03251.x www.bjog.org

SPIO-enhanced magnetic resonance imaging study of placental perfusion in a rat model of intrauterine growth restriction B Deloison,a,b N Siauve,a,c S Aimot,a D Balvay,a R Thiam,a CA Cuenod,a,c Y Ville,b O Clement,a,c LJ Salomona,b a INSERM, U970, Paris Cardiovascular Research Center – PARCC, Paris, France b Universite´ Paris Descartes, Sorbonne Paris Cite, Assistance Publique-Hoˆpitaux de Paris, Hoˆpital Necker Enfants Malades, Paris, France c Universite´ Paris Descartes, Sorbonne Paris Cite, Assistance Publique-Hoˆpitaux de Paris, Hoˆpital Europe´en Georges Pompidou, Paris, France Correspondence: Dr B Deloison, INSERM U970, Paris Cardiovascular Research Center – PARCC, 56 rue Leblanc, 75015 Paris, France. Email [email protected]

Accepted 13 November 2011. Published Online 20 January 2012.

Objective To assess placental perfusion with magnetic resonance

recommendations for animal care.

Results Fifty-four kinetic curves of placental perfusion were obtained in 11 rats. The mean placental blood flow was significantly lower in the ligated horns than in the normal horns (108.1 versus 159.4 ml/minute/100 ml, p = 0.0004). The mean fractional volume of the maternal vascular placental compartment did not differ significantly between the pathological (42.8%) and normal placentas (39.2%).

Population Thirty-two rats at day 16 of gestation underwent

Conclusions Placental perfusion, including changes during

surgical ligation of the left uterine vessel to induce IUGR.

experimental IUGR, can be measured in rats by using MRI with SPIO. These findings could have implications for human studies of placental microcirculation and for the management of disorders related to placental dysfunction.

imaging (MRI) and superparamagnetic iron oxide (SPIO) in a rat model of intrauterine growth restriction (IUGR). Design Experimental animal study. Setting The study complied with US National Institutes of Health

Methods Eighteen rats were examined by MRI 3 days later, after

bolus injection of ferucarbotran. Main outcome measure Signal intensities were measured in the

maternal left ventricle and in the placentas of the two horns. Quantitative microcirculation parameters were calculated and compared between the placentas of the two horns.

Keywords Functional MRI, intrauterine growth restriction, micro-

circulation, placenta, SPIO.

Please cite this paper as: Deloison B, Siauve N, Aimot S, Balvay D, Thiam R, Cuenod C, Ville Y, Clement O, Salomon L. SPIO-enhanced magnetic resonance imaging study of placental perfusion in a rat model of intrauterine growth restriction. BJOG 2012;119:626–633.

Introduction Adequate uteroplacental blood flow is vital for fetal growth and development. Indeed, increased uterine vascular resistance and/or decreased uterine blood flow may reduce placental perfusion and cause fetal growth restriction, as well as maternal vascular complications.1 However, the etiologies and pathogenesis of uteroplacental disorders are poorly documented, and placental function remains difficult to assess and quantify in routine clinical practice.1–3 Fetal and placental development are currently assessed mainly by means of standard and Doppler ultrasonography

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(US), but this imaging modality has limited predictive value for pre-eclampsia, fetal growth restriction, and perinatal morbidity and mortality.1,2 Moreover, Doppler ultrasound cannot be routinely used to quantify placental perfusion, a parameter that could in theory be measured in millilitres of blood per gram of placenta per minute, even though several studies have measured uterine artery blood flow volume during pregnancy.4–7 Several experimental approaches have been developed to assess placental function, including the use of radioactive microspheres and angiography, but their routine clinical use is severely restricted by concerns for both fetal and maternal safety.8

ª 2012 The Authors BJOG An International Journal of Obstetrics and Gynaecology ª 2012 RCOG

Placental perfusion in MRI

Magnetic resonance imaging (MRI) is being increasingly used as a diagnostic tool in human pregnancy, and recent reports suggest that it may provide important information on placental function.9–13 Perfusion can be measured in several ways with MRI, including arterial spin labelling (ASL), echo planar imaging (EPI), and intravoxel incoherent motion (IVIM).14–16 We have previously described a novel MRI-based approach to study placental function in a living mouse model, based on the use of gadolinium contrast agents and dynamic contrast enhancement (DCE) MRI. This has the capacity to measure placental perfusion and permeability in both physiological and pathological settings;10,12–16 however, gadolinium is not recommended for use during human pregnancy. The objective of this study was therefore to develop a new MRI-based approach to measure placental function, using superparamagnetic iron oxide (SPIO), an iron oxide-based contrast agent commonly used to detect liver metastases, and which is considered safe for clinical use in MRI.17,18

Methods Animal model All the experiments conformed to both French law and US National Institutes of Health recommendations for animal care. Janvier Laboratories (Le Genest St Isle, France) provided pregnant Sprague–Dawley rats. The rats were mated at the supplier’s laboratory, and all MRI imaging studies were performed on day 19 of the 22-day gestation period. A previously validated model of fetal growth restriction was developed based on the ligation of one of the two uterine vessels on day 16 of pregnancy, as first described by Wigglesworth in 1964.19,20 This ligation alters fetal growth in the horn with restricted perfusion, leading to a 20% reduction in the birthweight of live fetuses in the ligated horn at 22 days of gestation.19–23 This model also induces brain injuries related to intrauterine growth restriction (IUGR) in pups, and also fetal deaths.21–23 It is still used today, especially to study these brain injuries.21–24 For this study, 32 female rats were anaesthetised on day 16 of pregnancy by intraperitoneal injection of 2% xylazine (Rompum; Bayer, Puteaux, France) and ketamine (Imalge`ne; Rhoˆne Me´rieux, Lyon, France). The abdomen was shaved and a midline laparotomy incision was made under aseptic conditions. The number of fetoplacental units (FPUs) in each uterine horn was determined, and a ligature was placed round the left uterine artery near the lower end of the horn (Figure 1). This reduces blood supply to the horn, which is then only fed by the ovarian artery. The non-ligated side serves as a control, thus minimising interrat variability.12 The abdominal incision was sutured in two layers, using a standard surgical technique.

Figure 1. Presentation of the animal model of intrauterine growth restriction, inspired by Wiggleworth.19,20 During laparotomy, the two horns of the uterus are exposed. The left uterine vessels are ligated near the lower end of the left horn at day 16 of gestation (cross). This induces intrauterine growth restriction of fetuses in the ligated horns.

On day 19 of gestation, the rats were anaesthetised with the same protocol as that used for ligation. A 23-gauge catheter was placed in a tail vein to inject the contrast agent prior to MRI. On day 21 of gestation, the fetuses were delivered by caesarean section under general anaesthesia. Fetal weights were measured separately in each horn.

Contrast agent The SPIO used here was ferucarbotran (Resovist, Schering, Berlin, Germany), a microparticle consisting of magnetite (Fe3O4) and maghemite (Fe2O3) coated with carboxydextran. Its characteristics are summarised in Table S1. It has an overall hydrodynamic diameter of approximately 62 nm, as measured by electron microscopy. In humans, Ferucarbotran has a rapid initial intravascular phase (half-life 3.9– 5.8 minutes), and a second distribution phase of 3 hours, which corresponds to ferucarbotran capture by the reticuloendothelial system.18 Ferucarbotran is usually studied during the late capture phase, with T2 (transverse relaxation time)-weighted sequences. Its magnetic properties in T2 are characterised by high relaxivity: r2 = 190 l/mmol/second (Table S1).18,25,26 Biodistribution studies in humans and rats have shown that 80% of the intravenous dose is captured by the liver.18,25 Ferucarbotran can also be used with T1-weighted sequences, like gadolinium chelates, during its initial intravascular distribution phase.27 Its magnetic properties in T1 are characterised by relaxivity r1 = 20 l/mmol/second


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(Table S1).18,25,26 It is the only SPIO that can be injected intravenously as a bolus. It has been developed for contrast-enhanced MRI of human liver metastases,17,18 and was used in this study for the potential angiographic positive T1 enhancement it provides during the intravascular distribution phase.27–30 We used a clinical dose of 10 lmol Fe/kg. Ferucarbotran is safe and well tolerated. Safety data obtained during clinical phase I–III trials showed that 7.1% of patients had drug-related adverse outcomes. The majority of events occurred within the first 3 h and were mild (headache, nausea, tiredness, etc.).17,18 There is no toxicity study of this iron oxide during pregnancy, but an iron supplement of 60 mg/day is usually used during pregnancy to prevent iron-deficiency anaemia.31

Imaging procedure and evaluation All MRI studies were performed with the same 1.5-T unit (Signa; GE Medical Systems, Milwaukee, WI, USA), with the anaesthetised animals being placed in a knee high-definition coil. The following two anatomic MRI sequences were first used to locate the maternal left ventricle, the placentas, and the fetal rats. • A coronal fast spin-echo T1-weighted sequence: TR (Repetition Time) 340 ms and TE (Echo Time) 24 ms; FA (Flip Angle) 90; matrix 256 · 192; field of view 20 · 8 cm; section thickness 3.7 mm; bandwidth 10.4 kHz. • A fast imaging employing steady-state acquisition (FIESTA) 2D sequence: TR 8.2 ms and TE 2.2 ms; FA 60; matrix 256 · 256; field of view 20 · 8 cm; section thickness 3.5 mm; bandwidth 125 kHz. A two-dimensional fast spoiled gradient-echo multisection (FSPGR) sequence was then used for perfusion imaging. • FSPGR sequence: TR 4 ms and TE 1.5 ms; FA 40; matrix 256 · 140; field of view 22 · 8.8 cm; section thickness 3 mm; bandwidth 62.5 kHz. The number of images was eight. The temporal resolution was 1.5 seconds. After about 10 baseline images, the SPIO was injected as a bolus and the enhancement of the vascular input function (maternal left ventricle), placentas, and fetuses was monitored for 3 minutes 45 seconds (150 phases of 1.5 seconds).

image and then propagated automatically to all other images using PhysioD3D image software, developed in our laboratory.32 The appropriate positioning of the ROIs was then checked manually by one of the authors, and adjustments were made when necessary. For each explored organ, the signal intensity was represented as a function of time, to obtain a kinetic curve. Two kinetic curves were then obtained: the arterial kinetic curve, with the maternal left ventricle, and the placental kinetic curve (Figure S1).

Compartmental analysis Compartmental analysis is the translation in differential equations of the variation in the number of molecules of a contrast agent, after intravenous injection, over time within the placenta. We used a single-compartment model (Figure S2) that is based on physiological data,33–36 and on previous reports.10,12,13 In this model, the placenta is considered to be supplied by an arterial input (uterine arteries) and drained solely by venous output. The contrast agent is considered as a tracer. The model also assumes that the contrast agent is not taken-up by cells. The differential equation for the compartmental model is: dq2 =dt ¼ kð2;1Þ : q1 kð0;2Þ : q2 ; where q2 is the quantity of contrast agent in the maternal vascular compartment of the placenta, q1 is the quantity of contrast agent in the maternal left ventricle, k(2,1) is the transfer constant related to the arterial input, and k(0,2) is the transfer constant related to the venous output (also described in Figure S2). The transfer constants k(2,1) and k(0,2) were adjusted using the numerical modelling program PhysioD3D with the kinetic curves obtained by MRI in the maternal left ventricle and placental regions. Goodness-of-fit was examined qualitatively on the PhysioD3D-computed curves. Two quantitative microcirculation parameters were calculated as described in previous studies.10,12,13,32 Mean placental blood flow, designated F (perfusion in 100 ml/ second/100 ml), was calculated from the transfer constant k(2,1). The fractional volume of the maternal vascular placental compartment (Vb in %) was defined as the ratio between the placental blood volume and the entire placental volume.

Kinetic enhancement curves Signal intensities (SIs) were measured on all the perfusion images by placing regions of interest (ROIs) over the maternal left ventricle (to measure the vascular input function) as well as the placentas. The ROIs were as large as possible to cover the entire studied structure, to ensure reproducibility, and to avoid a selection bias. ROIs were placed manually within the first

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Statistical analysis Based on previous reports, we postulated that normal placental perfusion, reported to be around 150 ml/minute/ 100 ml in rats, would be reduced by about 30% after uterine vessel ligation. We also postulated that, because of fetal deaths, 50% of placentas in the ligated left horn would not be included in the analysis. We therefore aimed to



include a total of at least 44 placentas (18 in the ligated group and 26 in the non-ligated group, i.e. from the right horn), in order to achieve 80% power to detect a difference between the two groups of placentas, with an alpha risk of 5%. Rats were therefore included in the experiment until the target number of assessable placentas was reached. We used the Wilcoxon two-sample test to compare placental blood flow in the two groups. All tests were twotailed, and statistical significance was assumed at P < 0.05.

Results IUGR model A total of 32 rats were used in the study (Figure 2). Five rats died of surgical complications and three rats were excluded because of unicornuate uterus, peritonitis, or evisceration. The validation of the IUGR model was obtained with the remaining 24 rats (Table 1). At the time of artery ligation (day 16 of pregnancy), there were 133 fetuses in the left horns and 131 fetuses in the right horns. At caesarean delivery, on day 21 of pregnancy, 79 (59%) and 4 (3%) dead fetuses were observed in the left and right horns (p < 10)4), respectively. The mean weight (SD) of live pups was 3.6 ± 0.6 and 4.3 ± 0.7 g in the left and right horns, respectively, with a statistically significant difference of 16% (Table 1). The model was thus considered validated with respect to the difference of 20% reported in the literature for birthweight at 22 days of gestation.

Six rats were excluded from MRI analysis because of failed catheter installation or failed contrast medium injection. Eighteen rats were therefore studied by MRI (Figure 2). Anatomic MRI sequences were studied first. Figure 3 shows an example of an MRI anatomic T1-weighted sequence. The maternal heart was identified first, together with the uterus and its two horns. Observable FPUs were then counted. In each FPU, the fetus was in the middle, surrounded by amniotic fluid, and the placenta was located laterally. The signal intensity of the placenta before injection was similar to that of muscle on T1-weighted sequences. The fetal signal intensity was similar to that of water. Figure 4 shows a set of FSPGR images for perfusion imaging. Perfusion images showed enhancement of the placenta in 11 of the 18 rats studied by MRI. The other seven rats were subsequently excluded from the perfusion analysis (Figure 2). The spatial and temporal patterns of placental enhancement were observed qualitatively. The fetuses were not clearly distinguishable from amniotic fluid on perfusion sequences. However, no enhancement was observed when ROIs were placed over the fetal regions. Among the 11 rats included in the perfusion analysis, the mean number of observable placentas on FSPGR sequences was 2.1 in the left horn and 2.8 in the right horn. We were therefore able to analyse 54 sets of placental kinetic data, 23 for the left (ligated) horn and 31 for the right horn (Table 1).

Kinetic curves The FSPGR dynamic sequences showed enhancement of the maternal left ventricle and the placentas: the left ventricle exhibited rapid enhancement, with a peak during the first pass, whereas the placentas showed slower enhancement. Figure S1 shows an example of enhancement in the left maternal ventricle and in a placenta located in a right horn.

32 pregnants rats with maternal left horn ligation

8 complications of surgery and anesthesia

Imaging procedure and image evaluation

24 rats without complications

Compartmental analysis and physiological parameters

6 cases of failed catheter instalation or failed contrast agent injection

18 rats with complete protocole

7 rats without visualization of placental enhancement

11 rats with placental enhancement

31 placentas in the right non ligated horns

Figure 2. Flowchart of experiments.

23 placentas in the left ligated horns

Data fitting was successful in all 11 rats with detectable enhancement in the maternal left ventricle. The quality-offit of the PhysiD3D curves was examined qualitatively and was considered satisfactory in every case. As shown in Table 1, the mean placental blood flow (F) was significantly lower in the ligated group (left horn placentas) than in the non-ligated group (108.1 ± 41 versus 159.4 ± 54.6 ml/minute/100 ml). In contrast, the mean fractional volume in the maternal placental vascular compartment (Vb) was not significantly different between the two groups (42.8 ± 16.7% in the ligated horns and 39.2 ± 11.9% in the non-ligated horn; Table 1).


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Table 1. Validation of the experimental model of intrauterine growth restriction Horn

Right Left

Fetuses

Placental perfusion

Total number of fetuses

Number of dead fetuses

Mean birthweight of live fetuses (g)

Number of placentas

Mean blood flow (ml/minute/100 ml)

Mean fractional volume (%)

131 133

4* 79*

4.3 (±0.7)* 3.6 (±0.6)*

31 23

159.4 (±54.6)** 108.1 (±41.0)**

39.2 (±11.9)*** 42.8 (±16.7)***

The validation was obtained first on the fetal data: weight of fetuses, number of alive and dead fetuses at caesarean delivery. Then, the functional MRI validation was obtained by comparing perfusion and fractional volume of the maternal placental vascular compartment in the right horns (not ligated) and in left horns (ligated). Numbers in parenthesis represent the standard deviation; *P < 0.0001; **P = 0.0004; ***P = 0.24.

Figure 4. Example of coronal images obtained during the FSPGR perfusion sequence. On the left, the image was acquired before injection; the left ventricle, placental, and fetal areas cannot be identified. On the right, the image was acquired after ferucarbotran injection: note the enhancement of the heart (red) and placentas (purple). Figure 3. Anatomical T1-weighted magnetic resonance imaging sequence. This coronal view of a rat shows the heart contoured in yellow, the liver in green, and the right horn of the uterus containing four feto-placental units. Placentas are contoured in red and fetuses in blue.

Discussion This study shows that placental perfusion can be evaluated in rats by means of dynamic MRI with SPIO, an iron oxide-based contrast agent, using its T1 effect. To our knowledge, this is the first time that SPIO has been used to evaluate placental function. The ability to measure placental perfusion during human pregnancy would represent an advance in the understanding and management of pregnancies compromised by

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placental dysfunction. Gowland et al.14 used high-speed echoplanar imaging (EPI) to measure human placental perfusion. Arterial spin labelling (ASL) and intravoxel incoherent motion (IVIM) studies have also been used to measure placental perfusion with MRI.15,16 We have previously used a gadolinium-based contrast agent to study placental function in mice,10–13,15,16 and we were able to detect the acute reduction in placental blood flow induced by noradrenalin injection. Although no effect on fetal outcome or postnatal development has been reported in human pregnancies exposed to gadolinium chelate,37–39 the use of this agent is still not recommended during pregnancy. With SPIO, placental perfusion measured in the right (normal) horn of the rat uterus was 159 ml/minute/100 ml



of placenta in this study, a value very similar to that obtained in our previous studies (128–180 ml/minute/ 100 ml of placenta),10,12,13,15,16 and to that calculated by other teams using techniques such as EPI (176 ml/minute/ 100 ml).14 Our MRI-based compartmental model showed a significant reduction in placental blood flow (108 ml/minute/ 100 ml) after uterine artery ligation, which is associated with an increased risk of intrauterine death and growth restriction, as confirmed here. The fractional volume of the maternal placental vascular compartment, which represents the relative volume of blood in the placenta, was not significantly different between the two horns (42.8 versus 39.2% in the ligated and non-ligated horns, respectively; p = 0.24). These results compare favourably with the 35.3% obtained in a morphometric study of placentas sampled during caesarean section.40 They also suggest that the proportion of blood within the placenta is not reduced by chronic hypoperfusion or fetal growth restriction: only placental perfusion appears to be reduced. This result could be explained by the absence of a significant change in the anatomy of the placenta during IUGR, but only by a modification of the perfusion of blood within the placenta. Although the signal enhancement obtained with SPIO was lower than in our previous study using a gadolinium chelate, we were nonetheless able to visualise the reduction in placental blood flow following uterine artery ligation. However, we were not able to detect placental enhancement in all the animals. One explanation could be the loss of signal-to-noise ratio caused by partial-volume effects, related to the small size of the placentas: when the whole placenta is not included in the slice, the modification of the signal intensity resulting from placental enhancement is lower. A second possible explanation is an excessive decrease in blood flow in the placentas. This lack of detection probably constitutes a selection bias, leading to underestimated placental blood flow in the IUGR fetuses. This inability to detect placental enhancement was particularly noteworthy for the fetuses that died soon after ligation, leading to far smaller fetuses and placentas at the time of MRI. We tried to measure placental blood flow in several placentas of dead fetuses but were never able to visualise enhancement in these evanescent placentas. This lack of sensitivity would probably not represent a problem in humans, because of the large size of the placenta. Dynamic MRI could offer a new way of exploring the pathophysiology of fetal growth restriction, as altered perfusion observed on uterine Doppler US fails to explain a large proportion of cases in routine practice. MRI is becoming increasingly available, and our new approach using ferucarbotran could make it feasible during human pregnancy. The safety of SPIO during human pregnancy

has not yet been established, but iron oxides are known to be well tolerated.18 However, possible fetal toxicity must be precisely evaluated. SPIO is commonly used to detect liver metastases, but has never been used in pregnant women or in children. One limitation of our study is the use of a surgical model of IUGR may not fully reproduce human vascular IUGR, but it is used and validated as an IUGR model in rats in literature. However, it mimics a chronic reduction in placental blood flow. A period of 6 days of IUGR during a 22-day period of gestation may induce chronic lesions. The criteria of validation of IUGR in this model are less accurate than in humans, being based on brain injuries in pups (very similar to those observed in IUGR babies) and a 20% reduction in birthweight. The proportion of dead fetuses in the left horns was high, because of the anatomy of the horn. The quality of the arterial supply from the ovarian vascular pedicle is highly variable. This model, in which one horn is ligated and the other serves as a control, represents one way of overcoming the problems of inter-animal variability that we encountered in a previous study.12 It also provides greater statistical power for a similar sample size, by enabling the use of paired tests.

Conclusion This study demonstrates that dynamic MRI with SPIO contrast enhancement can be used to quantify placental blood flow in rats, and that it can detect reduced flow resulting from uterine artery ligation causing intrauterine growth restriction. This approach could be of value in experimental studies of placental vascular abnormalities, and, if proven to be safe in humans, might become a useful research tool for studying intrauterine growth restriction.

Disclosure of interests There is no conflict of interest in this article.

Contribution to authorship BD was the principal investigator and performed all the experiments, analysed the data, and wrote and corrected the article. NS conceived and designed the experiments, analysed the data, and corrected the article. SA-M performed the experiments. DB analysed the data. RT analysed the data. C-AC helped with the data analysis. YV is the head of the obstetrics department: he helped with the data analysis and corrected the article. OC is the head of the laboratory: he helped with the data analysis and corrected the article. LJS conceived and designed the experiments, analysed the data, and corrected the article.


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Details of ethics approval All the experiments conformed to both French law and US National Institutes of Health recommendations for animal care. We also received the approval of the INSERM ethics committee of Paris University.

Funding This study was funded by INSERM.

Acknowledgements We want to thank Dr Catherine Verney from INSERM U676 at Robert Debre Hospital who taught us the technique of ligature of the uterine vessels.

Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Example of the two kinetic curves. Figure S2. The one-compartment model. Table S1. Main characteristics of ferucarbotran in humans and rats.18,25 Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author. j

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