Anat Embryol (2005) 209: 263–267 DOI 10.1007/s00429-004-0451-9
O R I GI N A L A R T IC L E
C. Chapon Æ F. Franconi Æ J. Roux J. J. Le Jeune Æ L. Lemaire
Prenatal evaluation of kidney function in mice using dynamic contrast-enhanced magnetic resonance imaging
Accepted: 6 December 2004 / Published online: 15 March 2005 Springer-Verlag 2005
Abstract Glomerular differentiation starts as soon as embryonic stage 12 in mice and suggests that kidneys may be functional at this stage. Dynamic contrast-enhanced magnetic resonance microscopy, a noninvasive imaging technique, was used to assess renal function establishment in utero. Indeed, in adults (n=3), an intravenous injection of gadolinium-DOTA induced in a first step a massive and rapid drop in kidney signal intensity followed, in a second step, by a drop in bladder signal intensity. The delay in signal changes between kidney and bladder reflected glomerular filtration. Pregnant mice underwent anatomical and dynamic contrast-enhanced magnetic resonance microscopy on postcoital days 12–13 (n=2), 13–14 (n=1), 14–15 (n=3), 15–16 (n=2), 16–17 (n=3), 17–18 (n=3), and 18–19 (n=1). Kidneys and bladder were unambiguously depicted prior to contrast agent injection on stage 15–16 embryos. Contrast agent injection allowed kidney, detection as early as stage 12–13 but not bladder. Kinetics of signal changes demonstrated that glomerular filtration is established at embryonic stage 15–16 in mice. Thus, anatomical and dynamic contrast-enhanced magnetic resonance microscopy may be a powerful noninvasive method for in vivo prenatal developmental and functional studies. Keywords In utero functional imaging Æ MRI Æ Contrast agent Æ Gadolinium Æ Embryo Æ Kidney C. Chapon Æ J. J. Le Jeune Æ L. Lemaire (&) INSERM U646 ‘‘Inge´nierie de la Vectorisation’’, Universite´ d’Angers, 10 rue Boquel, 49100 Angers, France E-mail:
[email protected] Tel.: +33-0241-735006 Fax: +33-0241-735007 F. Franconi Service Commun d’Analyses Spectroscopiques, Universite´ d’Angers, France J. Roux Service Commun Animalerie Hospitalo-Universitaire, Universite´ d’Angers, France
Introduction In the fast-developing era of transgenic mouse models, any tool providing information on the effect of gene manipulation is of great interest to the biologist. The mouse (genetically engineered or wild) is the major model system for studying the genetic basis of mammalian development. Systematic reference description of the phenotype of the mouse embryo is still under establishment using invasive techniques such as histology (Kaufman 1992; Brune et al. 1999) and magnetic resonance (MR) microscopy (Dhenain et al. 2001; Schneider et al. 2003). In parallel, noninvasive approaches for studying in utero anatomy have been developed to follow up embryos’ development using either ultrasound biomicroscopy (Turnbull et al. 1995) or MR microscopy (Smith et al. 1998; Chapon et al. 2002; Weintraub et al. 2004). Complementary to these noninvasive anatomical descriptions of mouse embryo genesis, functional imaging of the set-up of the biological processes has been used, mainly in addressing fluxes in the embryonic cardiovascular system (Aristiza´bal et al. 1998; Linask and Huhta 2000; Hogers et al. 2000). Addressing renal function is equally of major interest because malformations of the urological tracts account for about 9% of embryonic malformations in humans (Congenital Malformations Australia 1981–97), among which almost 70% correspond to cystic kidney disease, obstructive defects of the renal pelvis/ureter, or stenosis of the urethra. Theoretically, kidney function could also be assessed in utero using techniques currently available in the clinic, such as ultrasound Doppler, scintigraphy, or dynamic contrast-enhanced magnetic resonance imaging (MRI) (Grattan-Smith et al. 2003). Studies of kidney function in embryonic mice are challenging and require high-resolution imaging because the length of the kidney is about 2 mm by the end of the gestational period. Contrast agents are required to depict the structure of interest, and rapid imaging is needed in order to evaluate
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signal evolution post-contrast agent. We have previously set up the technical requirements for such a study in which particular contrast agents were used to attempt to evaluate glomerular filtration, assuming that the agent would penetrate the nonmature kidney but not the mature kidney (Chapon et al. 2002). Unfortunately, the particles tested appeared to be stopped by the placenta, and we were unable to test our hypothesis. To bypass the tight placental microcirculation that limits crossing of the fetoplacental barrier (Firth and Leach 1996), we used diffusive gadolinium-DOTA. Gadolinium-DOTA can cross the placenta and be recovered in the embryonic bladder (Tanaka et al 2001). It was used to assess mouse kidney function establishment in utero.
Materials and methods Animals and anesthesia Virgin female OF1 mice 9-11 weeks old were mated for 24 h (n=35) starting at 8:00 a.m. with male of the same genetic constitution. Because of the long mating period, it was impossible to accurately stage the embryos. Therefore, embryonic stages presented in this paper took into account this 24-h confidence interval and will always be labeled as such. A fecundation rate of 43% was obtained. The 15 pregnant mice received a bolus of 37.5 lmol of gadolinium-DOTA (Dotarem, Guerbet, France) via the catheterized tail vein using an 80-cmlong prefilled catheter (Folioplast, France) equipped with a 29-gauge needle (Terumo Europe, Belgium). During MRI examination, mice were anesthetized by spontaneous inhalation of a mixture of isoflurane/oxygen (1.5–4%)/(1–1.5 l/min), and body temperature was controlled at 36–37C. Animal care was administered in strict accordance to the French Ministry of Agriculture regulations.
frequency-encoding direction to avoid aliasing. Five to nine 0.8–1-mm contiguous slices were acquired to cover the entire embryo. T2-weighted images were acquired using an imaging method known as the multislice RARE method (Henning et al. 1986). This method employs a single excitation step followed by the collection of multiple phaseencoded echoes. The repetition time (TR) was 2,000 ms and the echo time (TE) was 7.5 ms. A train of eight echoes was used to fill the k-space, and the effective TE was 31.7 ms. Four averages for each phase encoding were performed, resulting in a total acquisition time of 2 min 11 s. Bruker ParaVision software was used for data acquisition and processing on a Silicon Graphics O2 workstation with the IRIX 6.5 operating system. Regions of interest were manually drawn over kidneys and bladder, and signal changes were expressed as a percentage of initial signal intensity of the corresponding region of interest prior to contrast agent.
Results Figure 1 shows a qualitative and quantitative evaluation of signal changes after gadolinium-DOTA injection in E14/15 pregnant mice. A preliminary experiment showed that a minimal dose of 35 lmol of gadolinium– DOTA is required to observe signal changes within the
Magnetic resonance imaging Experiments were performed on a Bruker Avance DRX 300 equipped with a vertical superwide-bore magnet operating at 7T and a shielded gradient system (maximum gradient strength 144 mT/m, rising time
