PhD Guillaume Duhamel
email: guillaume.duhamel@univ-amu.fr

V H Prevost

M Cayre

A Hertanu

S Mchinda

V N Carvalho

G Varma

P Durbec

D C Alsop

O M Girard

Validating the sensitivity of inhomogeneous Magnetization Transfer (ihMT) MRI to myelin with fluorescence microscopy

Keywords: Myelin imaging, MRI, inhomogeneous magnetization transfer, ihMT, plp-GFP, fluorescence microscopy Funding

Inhomogeneous magnetization transfer (ihMT) was validated as a myelin sensitive imaging technique against fluorescence microscopy

• ihMT signal was strongly and significantly correlated with myelin-related plp-GFP (proteolipid -Green Fluorescence Protein) signal

• Short dipolar relaxation time (T1D) filtering is an efficient way to reduce non-myelin contribution in ihMT signal

• MT signal was more weakly correlated with plp-GFP signal and had a much larger nonmyelin contribution

• ihMT contrast can be varied with pulse timing, leading to different signal properties in terms of sensitivity and specificity for myelin content

Introduction

Magnetization transfer (MT) [START_REF] Wolff | Magnetization transfer contrast (MTC) and tissue water proton relaxation in vivo[END_REF] and inhomogeneous Magnetization Transfer (ihMT) (Varma et al., 2015a) are MRI techniques sensitive to the macromolecular content of biological tissues. Whereas conventional MT analysis considers the classic Zeeman order magnetization of macromolecules, ihMT highlights dipolar order (Varma et al., 2015b), an additional degree of freedom associated to dipolar-broadened lineshapes. Dipolar order corresponds to the polarization of dipolar-coupled spins within their local magnetic fields [START_REF] Korb | Pulse saturation behavior of inhomogeneously broadened lines. II. Rapid-spectral-diffusion case[END_REF][START_REF] Provotorov | Magnetic resonance saturation in crystals[END_REF] and is associated with a relaxation time constant, T1D.

Relatively long T1D values have been measured in lipid bilayer membranes [START_REF] Swanson | Molecular, dynamic, and structural origin of inhomogeneous magnetization transfer in lipid membranes[END_REF] and in brain white (WM) and grey (GM) matter [START_REF] Varma | In vivo measurement of a new source of contrast, the dipolar relaxation time, T1D , using a modified inhomogeneous magnetization transfer (ihMT) sequence[END_REF]. In contrast, much shorter T1Ds were estimated in other tissues and samples [START_REF] Manning | The physical mechanism of "inhomogeneous" magnetization transfer MRI[END_REF]Varma et al., 2015b), which most likely explains the strong selectivity of the ihMT images for myelin-rich structures. This myelin selectivity was further supported by quantitative calculation of the ihMT ratio (ihMTR) values, the ihMT signal normalized by the unsaturated reference signal (S0): ihMTR~4-10% in GM and WM [START_REF] Girard | Magnetization transfer from inhomogeneously broadened lines (ihMT): Improved imaging strategy for spinal cord applications[END_REF][START_REF] Girard | Magnetization transfer from inhomogeneously broadened lines (ihMT): Experimental optimization of saturation parameters for human brain imaging at 1.5 Tesla[END_REF] and ihMTR≾1% in muscle tissue [START_REF] Prevost | Optimization of inhomogeneous magnetization transfer (ihMT) MRI contrast for preclinical studies using dipolar relaxation time (T1D ) filtering[END_REF]. In addition to this strong contrast for myelinated tissues, the ihMT technique also benefits from trivial processing, simply based on the subtraction of a minimum of 2 MT images obtained with different radiofrequency (RF) irradiation parameters. Overall, these properties have made ihMT an attractive method to assess myelin-related information in vivo, in the clinical context of demyelinating diseases [START_REF] Rasoanandrianina | Region-specific impairment of the cervical spinal cord (SC) in amyotrophic lateral sclerosis: A preliminary study using SC templates and quantitative MRI (diffusion tensor imaging/inhomogeneous magnetization transfer)[END_REF][START_REF] Van Obberghen | Evaluation of the Sensitivity of Inhomogeneous Magnetization Transfer (ihMT) MRI for Multiple Sclerosis[END_REF] or in the context of aging studies [START_REF] Geeraert | A comparison of inhomogeneous magnetization transfer, myelin volume fraction, and diffusion tensor imaging measures in healthy children[END_REF][START_REF] Taso | Tract-specific and age-related variations of the spinal cord microstructure: a multi-parametric MRI study using diffusion tensor imaging (DTI) and inhomogeneous magnetization transfer (ihMT): Spinal Cord Microstructure Assessment Using DTI and ihMT[END_REF]. In addition, other studies have shown that the ihMT signal correlates with established myelin-sensitive MR imaging techniques such as myelin water fraction (MWF) [START_REF] Ercan | Microstructural correlates of 3D steady-state inhomogeneous magnetization transfer (ihMT) in the human brain white matter assessed by myelin water imaging and diffusion tensor imaging[END_REF][START_REF] Geeraert | A comparison of inhomogeneous magnetization transfer, myelin volume fraction, and diffusion tensor imaging measures in healthy children[END_REF]. To date, however, no validation of ihMT as a myelin imaging technique against an established histologic standard has been reported.

The existence of an ihMT signal is related to the creation of dipolar order by offresonance RF irradiation. The ihMT signal is consequently weighted by the associated relaxation time constant T1D. In practice, the sensitization of the ihMT signal for a given T1D value is driven by power and timing parameters of the RF irradiation. Higher RF power tends to increase the ihMT signal of all T1Ds [START_REF] Prevost | Optimization of inhomogeneous magnetization transfer (ihMT) MRI contrast for preclinical studies using dipolar relaxation time (T1D ) filtering[END_REF], but may reduce ihMT contrast by greatly increasing contribution to the signal from short T1D (i.e. few hundreds of µs to ~1ms) components [START_REF] Manning | The physical mechanism of "inhomogeneous" magnetization transfer MRI[END_REF] relative to longer T1Ds (i.e. few ms or longer). Fortunately, when using a train of RF pulses for irradiation, the repetition time of consecutive pulses (∆t) can be tuned to attenuate or filter out the signal contribution from such short T1D components [START_REF] Prevost | Optimization of inhomogeneous magnetization transfer (ihMT) MRI contrast for preclinical studies using dipolar relaxation time (T1D ) filtering[END_REF]. As stressed in recent studies, concentrating RF power within bursts over the long irradiation period, as opposed to RF power regularly distributed along this period, increases the ihMT MRI signal in various tissues [START_REF] Mchinda | Whole brain inhomogeneous magnetization transfer (ihMT) imaging: Sensitivity enhancement within a steady-state gradient echo sequence[END_REF][START_REF] Varma | Low duty-cycle pulsed irradiation reduces magnetization transfer and increases the inhomogeneous magnetization transfer effect[END_REF].

Overall, the changes in ihMT signal induced by the modification of the irradiation parameters are different depending on the brain structures and type of tissues. Both intensity and contrast in ihMT images depend on the parameters of the RF irradiation, raising additional questions about which acquisitions would correlate best with myelin content. Hence several variants of ihMT acquisitions should be validated against a gold standard myelin measure.

We address this need below by comparing ihMTR values derived from ihMT experiments with different irradiation parameters with myelin-content assessed with a reference technique. Several methods for ex vivo myelin visualization and quantification are available, including histochemical Luxol Fast Blue staining [START_REF] Kluver | A method for the combined staining of cells and fibers in the nervous system[END_REF], Myelin Basic Protein (MBP) immunostaining, myelin fluorescent reporter mice or transmission electron microscopy. Genetically-modified plp-GFP (proteolipid protein-Green Fluorescent Protein) mouse is the animal model in which the green fluorescent protein (GFP) is specifically expressed in myelinating oligodendrocytes [START_REF] Bras | Oligodendrocyte development in the embryonic brain: the contribution of the plp lineage[END_REF][START_REF] Spassky | The early steps of oligodendrogenesis: insights from the study of the plp lineage in the brain of chicks and rodents[END_REF]. The GFP fluorescence, which is directly observable and quantifiable with fluorescence microscopy, reflects the expression of proteolipid protein, the major myelin protein in the central nervous system. In the adult brain of these mice, GFP-expressing cells are mature oligodendrocytes but not oligodendroglial progenitors [START_REF] Ferent | Investigation of the proteolipid protein promoter activity during demyelination and repair[END_REF]. Therefore, fluorescence microscopy performed on plp-GFP mice represents a direct and very valuable tool to observe and quantify myelin content. We chose plp-GFP fluorescence as the reference technique for validating ihMT as a myelin imaging technique.

Material and Methods

Ethics statement

Animal experiments were conducted on C57Bl/6J mice and plp-GFP mice and were performed following French guidelines for animal care from the French Department of Agriculture (Animal Rights Division), the directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 and approved by our institutional committee on Ethics in animal research (Comite ́ d'Ethique de Marseille n°14, project authorization APAFIS#1747-2015062215062372v6).

IhMT technique and irradiation schemes

An ihMT image is generated by the subtraction of an MT image obtained with a single frequency-offset RF irradiation at +∆f, MT + , and an MT image obtained with a symmetric dual frequency-offset RF irradiation at ±∆f, MT +-, both performed with identical average RF power.

Single frequency-offset RF irradiation of the macromolecular pool creates dipolar order [START_REF] Manning | The physical mechanism of "inhomogeneous" magnetization transfer MRI[END_REF]Varma et al., 2015b), which reduces RF saturation effects contributing to the MT + signal. On the other hand, simultaneous symmetric dual frequency-offset RF irradiation eliminates any dipolar order contribution from the MT +-signal (Varma et al., 2015b) and leads to enhanced RF saturation effects [START_REF] Swanson | Molecular, dynamic, and structural origin of inhomogeneous magnetization transfer in lipid membranes[END_REF]. Hence, the (MT + -MT +-) image, that is ihMT, isolates the dipolar order contribution from the MT effects. To minimize sensitivity to off-resonance and asymmetry of the macromolecular line, a second ihMT image using negative frequency irradiation for the single frequency image is also acquired and added to the first [START_REF] Prevost | Minimizing the effects of magnetization transfer asymmetry on inhomogeneous magnetization transfer (ihMT) at ultra-high magnetic field (11.75 T)[END_REF]Varma et al., 2015a). It has been shown that dipolar order effects are weighted by dipolar relaxation mechanisms (characterized by the dipolar relaxation time T1D) and this additional weighting can be exploited to attenuate/filter out the signal from short T1D components. This is achieved by a modification of the dual frequency-offset RF irradiation experiment, performed sequentially using repetition of frequency alternated RF pulses, as opposed to simultaneously using multiband (e.g. cosine-modulated) RF pulses [START_REF] Varma | In vivo measurement of a new source of contrast, the dipolar relaxation time, T1D , using a modified inhomogeneous magnetization transfer (ihMT) sequence[END_REF]. By doing so, dipolar order is allowed to relax in between repeated RF pulses hence providing sensitivity to this contrast mechanism. Based on the above, it may be inferred that the ihMT signal output depends upon the value of T1D, the RF power absorption, and the implementation of the dual frequency-offset RF irradiation. The latter two can be developed to either enhance or attenuate the ihMT signal intensity of components based on their T1D values. The irradiation strategies presented in Fig. 1 were used for this study. Concentration of RF power was achieved using bursts of Np RF pulses followed by a mixing time without RF power, repeated every BTR. Dashed boxes were zoomed in to display the main features of the RF pulses: Hann-shaped pulses (normalized power integral 0.375), duration PW, pulse repetition time ∆t, peak power b1 (standard protocols) or b1 ' (boosted protocols), frequency +∆f or -∆f. MT +-was achieved either by frequency alternation (from +∆f to -∆f), allowing T1D-filtering, or with cosinemodulated pulses (CM, peak power increased by a √2 factor), producing simultaneous irradiation at +∆f and -∆f. CM pulses did not allow T1D-filtering. Identical irradiation power, b1,rms, was used for all experiments. Note that the indicated b1,rms formulas applied for Hann-shape pulses.

The first strategy, referred to as standard ihMT (Fig. 1a) in the following, corresponds to the one used in previous reports of preclinical and clinical studies [START_REF] Girard | Magnetization transfer from inhomogeneously broadened lines (ihMT): Experimental optimization of saturation parameters for human brain imaging at 1.5 Tesla[END_REF][START_REF] Prevost | Optimization of inhomogeneous magnetization transfer (ihMT) MRI contrast for preclinical studies using dipolar relaxation time (T1D ) filtering[END_REF]Varma et al., 2015a). It employed for RF irradiation, a train of uniformly distributed pulses (peak power, b1) depositing an average root-mean-square power, b1,rms, over the total irradiation time,τ. The second strategy, referred to as boosted ihMT (Fig. 1b) in the following, employed a concentrated RF power deposition scheme. Bursts of Np RF pulses followed by relatively long mixing periods (i.e. periods with RF power set off) were repeated every BTR (burst repetition time) for a total irradiation time, τ. Peak power of individual RF pulses (b1') were adjusted such that the deposited average root-mean-square power was identical to that of the standard ihMT experiment, b1,rms. The mixing periods, during which both relaxation and magnetization transfer occur, can be properly tuned to enhance the ihMT signal for a given set of irradiation parameters: In humans, 100-200% ihMT signal increase is possible with b1,rms applied in a concentrated manner rather than with uniform distribution of RF power [START_REF] Mchinda | Whole brain inhomogeneous magnetization transfer (ihMT) imaging: Sensitivity enhancement within a steady-state gradient echo sequence[END_REF][START_REF] Varma | Low duty-cycle pulsed irradiation reduces magnetization transfer and increases the inhomogeneous magnetization transfer effect[END_REF].

Dual frequency-offset irradiation of both standard and boosted ihMT experiments was achieved either sequentially by alternating the offset frequency (+∆f to -∆f) of consecutive RF pulses or, simultaneously, by using cosine-modulated (CM), i.e. dual-band, shaped pulses.

Simultaneous irradiation at +∆f and -∆f leads to actual decoupling of the dipolar order from the Zeeman order (Varma et al., 2015b) independent of the T1D value. Conversely, the frequencyalternating approach introduces an additional degree of freedom ∆t, the time between consecutive pulses, which can be adjusted to modulate the ihMT signal. Fast frequencyalternation (Δt <<T1D) averages out dipolar order effects occurring during the individual RF pulses, hence leading to a MT +-signal tending towards the one obtained using the simultaneous CM approach. On the other hand, long Δt values (Δt > T1D) allow dipolar order relaxation to occur between individual RF pulses and leads to a dual frequency-offset irradiation experiment virtually equivalent to the single frequency-offset one for infinitely long ∆t values (Varma et al., 2017). For this configuration, the ihMT signal would naturally vanish. In other words, Δt is setting a limit for efficient saturation of both frequency offsets and can be adjusted to attenuate or even filter out the ihMT signal of components associated with short T1D values. Further details of this short T1D-filtering mechanism can be found in [START_REF] Prevost | Optimization of inhomogeneous magnetization transfer (ihMT) MRI contrast for preclinical studies using dipolar relaxation time (T1D ) filtering[END_REF].

To summarize, the implementations in Fig. 1 lead to distinct ihMT protocol variants, specifically referred to as the following: standard ihMT with or without T1D-filtering (Fig 1a)

and boosted ihMT with or without T1D-filtering (Fig 1b). The value of ∆t drives the strength of the T1D-filtering.

MRI experimental setup

MR experiments were performed with a preclinical 11.75T scanner (Bruker Avance 500 MHz/89 mm wide bore vertical imager, Ettlingen, Germany) and a transmit/receive volume birdcage coil (Bruker, Ettlingen, Germany; length L=3 cm, diameter Ø=2 cm). Experiments were conducted on healthy female C57Bl/6J mice and plp-GFP mice. After isoflurane induction in an anesthetic chamber at 3%, mice were maintained under anesthesia during MR experiments by spontaneous respiration of a mixture of air and isoflurane (1.5%; constant flow, 300 mL/min; Univentor 400 anesthesia unit, Zejtun, Malta) through a dedicated nose cone. Respiration and temperature were controlled throughout the experiments with an MR-compatible monitoring and gating system (SA Instruments, Stony Brook, NY, USA). A heating blanket connected to a water bath system was placed on the back of the mice to maintain physiological temperature (T = 37.0 ± 0.5°C).

Prior to ihMT image acquisition, first and second order B0 shimming was performed using fastmap (Bruker) with a 1.15x1.15x1.15 cm 3 volume centered in the mouse brain. The MT irradiation schemes of Fig. 1 followed by a single-shot RARE (Rapid Acquisition with Relaxation Enhancement) readout were implemented in Bruker's Paravision 5.1 software. The following imaging parameters were used for all experiments: axial orientation; slice thickness, 1 mm; field of view, 20 mm; acquisition matrix, 64 × 64; minimum TE and echo spacing, 1.82 ms; TE eff = 12.74 ms; RARE factor = 38; partial Fourier acceleration = 1.7; linear phase encoding; bandwidth = 400 kHz; acquisition train length ~70 ms; TR = 3 s.

At very high magnetic field, the chemical shift of the macromolecular line induces MT asymmetry effects, which need to be accounted for to obtain reliable ihMT measurements [START_REF] Prevost | Minimizing the effects of magnetization transfer asymmetry on inhomogeneous magnetization transfer (ihMT) at ultra-high magnetic field (11.75 T)[END_REF]. Compensation was realized experimentally by i) shifting the center frequency, fc, of the irradiation offset-frequency ∆f from fc=0Hz to fc=-100Hz, and ii) by taking for the single frequency-offset MT image, the average of a MT image obtained with irradiation realized at fc+∆f, (MT + ) and one obtained with irradiation realized at fc-∆f, (MT -) [START_REF] Prevost | Minimizing the effects of magnetization transfer asymmetry on inhomogeneous magnetization transfer (ihMT) at ultra-high magnetic field (11.75 T)[END_REF]. Finally, acquisition of two dual frequency-offset MT image (MT +-and MT -+ ) allows calculation of the final ihMT image as:

ℎ = ( + ) -( + ) equation 1
An extra image acquired with the RF irradiation power set to zero, S0, served as reference signal for normalization and calculation of the semi-quantitative ihMT ratio:

ℎ = equation 2
Single-offset and dual-offset MT ratios (MTR +∆f and MTR +/-∆f ) can be derived from the same image dataset as Standard and boosted ihMT protocols with 3 different T1D-filtering strengths were applied on 3 plp-GFP mice. PW corresponds to the pulse length, ∆t to the repetition time of consecutive pulses, Np to the number of pulses per burst, BTR to the repetition time of consecutive bursts and b1 ' to the peak-power of the irradiation pulses. The * symbol indicates that the standard protocol without T1D-filtering was applied on 2 mice only. Other irradiation parameters were b1,rms=6.7µT, ∆f=10kHz and τ=900ms.

∆ = 1 - equation 3 / ∆ = 1 - equation 4

Validation of ihMT with fluorescence microscopy

After in vivo MR experiments, mice were deeply anesthetized (ketamine 150 mg/kg with xylazine 15 mg/kg) and perfused (intra-cardiac) with the fixative paraformaldehyde (PFA) 4%. Mouse brains were then extracted, post-fixed 2 hours in 4% PFA and cryopreserved overnight in 20% sucrose. Brains were frozen and carefully sectioned into 20-μm thick histological slices (Cryostat Leica CM3050S) targeting the same axial planes as imaged by MRI (rostro-caudal levels -0.7mm and -3.2mm from bregma). Sections were mounted with Dabco anti-fading agent. GFP fluorescence images were acquired using a Zeiss fluorescence microscope with a 5x objective and a fixed exposure time.

Image processing

MRI data processing was realized with Matlab (vR2012, MathWorks Inc., Natick, MA, USA). For each configuration of table 1, individual 256x256 MT images were reconstructed by Fourier transform after cosine windowing [START_REF] Harris | On the use of Windows for harmonic analysis with the discreteFourier transform[END_REF] and 4x zero-filling of the 64x64 initial k-space. Magnitude MT images were averaged over the NEX and processed using custom routines to generate the final ihMT and ihMTR images using equations 1 and 2, as well as MTR +∆f and MTR +/-∆f images using equations 3 and 4. Note that strictly-speaking the concept of T1D-filtering does apply for dual-offset frequency irradiation MT and ihMT, but not for single-frequency irradiation MT. For single frequency RF irradiation, weak and no T1D-filtering experiments were identical (single-offset RF pulses repeated every (∆t=1.3ms)). However, for consistency in presentation, we refer to the different T1D-filtering conditions for ihMTR, MTR +/- ∆f , as well as MTR +∆f .

Quantitative analyses were performed in 7 different brain structures: internal capsule (IC), corpus callosum (CC), optic nerves (ON), thalamus (Th), cortex (Cx), hippocampus (HC)

and Inter-Peduncular Nucleus (IPN). Regions-of-interest (ROIs) were manually placed in these 7 brain structures on ihMTR and GFP fluorescence images (Fig. 3d) by two different observers (V.H.P and M.C) using a mouse brain atlas as reference. The analysis was restricted to these structures, considered relatively homogeneous throughout the 1mm-thick slices, in order to limit partial volume effects in MRI measurements. Mean values of ihMTR, MTR +∆f and MTR +/- ∆f were measured in each ROI for all mice. Quantification of myelin content in each ROI was obtained by measuring the mean GFP fluorescence intensity (ImageJ software (NIH)) in 3 20µm-thick histological sections, spaced by 160µm each, hence covering a total thickness of 340µm. Background fluorescence (measured in the lateral ventricle) was subtracted from the mean GFP fluorescence intensity. The resulting signal was normalized, taking as a reference the ROI with the highest GFP signal, and was referred to as ! in the following.

Statistical analyses

Statistical analyses were performed using JMP software (v9.0.1; SAS Institute, Cary, NC).

One-way analysis of variance followed by a Tukey-Kramer HSD test corrected for multiple comparisons was used to test for differences between brain structures for ihMTR values and GFP values. Three groups of structures were considered for such comparison: white matter, obtained by averaging measurements in IC, CC and ON ROIs; mixed structures, obtained by averaging measurements in Th and IPN ROIs; and grey matter obtained by averaging measurements in Cx and HC ROIs.

Pearson's correlation coefficient (r) and linear regression analysis were used to determine associations of the relationship between individual ihMTR values (derived from the 6 variants of table 1) and myelin-content (GFP fluorescence intensity, !) across ROIs in IC, CC, ON, Th, Cx, HC and IPN and animals (n=3). Linear regression analysis was also performed for mean ihMTR values in each ROI calculated over the three animals. A correlation coefficient r>0.7

(r 2 >0.5) being considered as significantly strong with P-value<0.05. Significance of differences between Pearson's correlation coefficients for the 6 sequence variants was tested using Fisher's r-to-z transformation and asymptotic z-test. Slopes and intercepts of regression equations were compared between protocol variants using analysis of covariance (ANCOVA) where ihMTR values were used as a dependent variable, GFP fluorescence intensity ( !) was used as a covariate, and the protocol variant was used as a categorical factor. In addition, the agreement between ihMT and GFP fluorescence was assessed by a Bland-Altman analysis [START_REF] Bland | Statistical methods for assessing agreement between two methods of clinical measurement[END_REF]. IhMTR values in different anatomical structures were normalized with respect to values in IC. Differences between normalized ihMTR values ("#ℎ $) and ! values were then plotted against their averages. Significance of the bias between ihMT and GFP measurements was evaluated using the one sample t-test for the mean difference. The limits of agreement were calculated as the mean difference ± 1.96 standard deviation of the mean difference. Similar correlation analyses with GFP were performed for MTR +∆f and MTR +/-∆f .

Results

Representative standard and boosted ihMTR images obtained at -3.2mm and -0.7mm from bregma with the 6 ihMT protocols outlined in table 1 and (and for all ihMT configurations). Relative contrast values between WM and GM were higher for T1D-filtered ihMTR images (0.52-0.56) compared to non-filtered ones (0.42-0.45).

Conversely, the non-filtered ihMTR images provided the highest SNR values. As for CNR, higher values were obtained for weakly-filtered and non-filtered ihMTR images. Plain markers correspond to results from standard ihMT protocols (Fig. 1a) and open markers from boosted ihMT protocols (Fig. 1b). The different colors indicate variable T1D-filtering strengths. Linear fits ( ℎ = ' ( ! + )) to scatterplot data were obtained by least-square regression and correlation coefficients, r 2 , (assessed by a bivariate (Pearson) correlation test) were reported for all protocols. Shaded areas correspond to confidence curves for line fits with a α-level=0.1. All correlations were significant (p <0.0001).

R4

The significance of the linear relationship between ihMTRs and ! ( ℎ = ' ( ! + )) was very high for all ihMT protocol variants for both individual data (0.87 ≤ r 2 ≤ 0.97, p <0.0001 (Fig. 4a)) and averaged data (0.96 ≤ r 2 ≤ 0.99, p <0.0001 (Fig. 4b)). Moreover, no significant differences were found for correlation strength of the regression lines between ihMTR variants, thereby validating ihMT as a myelin-sensitive imaging technique, regardless of the selected irradiation parameters. Bland-Altman plots (Fig. B.1) show good agreements between ! and "#ℎ $ for all configurations with a significant bias ranging from ~12%

for T1D-filtered protocols to ~20% for non-filtered ones. A proportional bias (~ -0.5) was observed indicating closer agreement for measurements in WM. This proportional bias was reduced for T1D-filtered ihMTR configurations, consistent with the lower intercept obtained from the regression analysis.

R4

n represents the number of data points used for regression analysis. Sp, calculated as 01 = 2 2 3 corresponds to the component of ihMTR signal associated specifically to myelin and NSp, calculated as 401 = 1 -01 = 5 6 5 to the non-specific signal component; δα, δβ, δSp and δNSp correspond to the standard deviations of each metric. Coefficients of correlation between ihMTR and !, r, were assessed by a bivariate (Pearson) correlation test, with p<0.05 considered as significant.

Of interest, ihMT was much more highly correlated with myelin, as quantified by GFP, than conventional MTR measurements obtained from the data (Tables S1 andS2 in supplementary materials and corresponding plots shown in Figs. 5): 0.24 ≤ r 2 ≤ 0.64, p <0.02 for MTR +∆f individual data (Fig. 5a) and 0.47 ≤ r 2 ≤ 0.69, 0.02 < p <0.09 for MTR +∆f averaged data (Fig. 5b); 0.67 ≤ r 2 ≤ 0.78, p <0.0001 for MTR +/-∆f individual data (Fig. 5c) and 0.73 ≤ r 2 ≤ 0.82, 0.03 < p <0.07 for MTR +/-∆f averaged data (Fig. 5d). show much poorer agreement for MTR +∆f and MTR +/-∆f with ! as compared to ihMTR, with constant biases in the order of ~40-45% and proportional biases 3 times higher (~ -1.5) than those obtained with ihMTR. Despite the excellent correlations between ihMTR values and !, all ihMT protocol variants are not equivalent. Boosted configurations produced significantly higher slope (α) and intercept (β) values (p<0.01) than their counterparts acquired with standard configurations, with the exception of strong-T1D filtering sequences, for which no differences were found in the intercepts between standard and boosted protocols (p=0.16). For standard protocols, significant differences in the slopes were found between no T1D-filtering and strong T1D-filtering configurations only (p=0.01), whereas intercepts were different between all T1D-filtering conditions (p<0.03). For boosted protocols, slopes and intercepts were different between all T1D-filtering conditions (p<0.0001). The differences in linear regression α and β parameters provide key information regarding the sensitivity and specificity of ihMTR for myelin. Within the framework of brain tissue analysis, a sensitivity of ihMTR for myelin may be defined by the slope, α, and a corresponding specificity as 01 = 2 2 3 . Indeed, for !=1 (case of the most myelinated structure taken as reference for normalization), the component of the ihMTR signal associated with this GFP signal (and hence with myelin) is ' for a total detected ihMTR signal of ' + ). Hence, Sp corresponds to the fraction of the ihMTR signal that is specific to myelin. In other words, 01 = 70% means that for the most myelinated structure ( !=1), 70% of the ihMTR signal can be associated specifically with myelin. By extension, one can also define the non-specific ihMTR signal component as 401 = 1 -01 = 3 2 3 . The importance of power concentration and T1D-filtering for the sensitivity and specificity of ihMT to myelin is quantitatively highlighted in table 3. Higher slope values (α) obtained with the boosted protocols signify higher sensitivity for myelin as compared to their standard ihMT variant counterparts. In addition, stronger T1D-filtering resulted in lower sensitivity (lower α) but higher specificity (Sp) of ihMTR for myelin. Of particular interest, the myelin-specificity of ihMT is preserved for boosted protocols associated with T1D-filtering, as shown by identical values of Sp obtained for standard and boosted ihMT with strong T1D-filtering (Sp=0.69±0.06)

or weak T1D-filtering (Sp=0.65±0.04). Without T1D-filtering, a slight decrease of specificity was observed for boosted ihMT (Sp=0.53±0.03) compared to standard ihMT (Sp=0.56±0.08).

The sensitivity of boosted ihMTRs was generally higher than that of MTR +∆f , and for non-filtered conditions, similar to MTR +/-∆f . Both MTR +∆f and MTR +/-∆f were associated with lower values of Sp, with minor dependence on the T1D-filtering strengths (0.08≤ Sp ≤0.12 for MTR +∆f and 0.16≤ Sp ≤0.18 for MTR +/-∆f , Tables S1 andS2 in supplementary material) in comparison to ihMTR.

Discussion

Validation of ihMT with GFP fluorescence microscopy

Notwithstanding the dependence of signal intensity on irradiation parameters, all ihMT protocol variants produced ihMTRs highly correlated with the GFP signal (r 2 >0.9, Fig. 4). Since GFP in our model is expressed under the control of the promoter of plp, the major protein constituting myelin, it seems reasonable to assume a linear relationship between GFP signal and myelin content (Le [START_REF] Bras | Oligodendrocyte development in the embryonic brain: the contribution of the plp lineage[END_REF][START_REF] Spassky | The early steps of oligodendrogenesis: insights from the study of the plp lineage in the brain of chicks and rodents[END_REF], at least in the context of mature and intact myelin. These strong correlations generally validate ihMT as a myelinsensitive imaging technique. It is important to note that no other correction than the removal of the background noise signal (measured in the ventricle) was applied for calibrating the GFP signal. In particular, the tissue autofluorescence, which may interfere with GFP fluorescence and introduce a signal-offset, was not considered. Since this potential GFP-signal offset would in turn translate into a quantification bias of β, a variability in the absolute values of Sp and NSp would be expected. However, for a given mouse, since the same GFP-offset bias occurred for all ihMT analyses, the relative differences in α, β, Sp and NSp observed between the ihMT variants can be generalized.

The validation of ihMT as a myelin-sensitive imaging technique is an important step for the use of ihMT in preclinical and clinical research for diagnosis, therapy follow-up or neuroscience studies.

MT imaging applied in demyelinating disorders showed that MTR variations can be associated with demyelination [START_REF] Zaaraoui | Monitoring demyelination and remyelination by magnetization transfer imaging in the mouse brain at 9.4 T[END_REF], inflammation [START_REF] Gareau | Magnetization transfer and multicomponent T2 relaxation measurements with histopathologic correlation in an experimental model of MS[END_REF] and edema [START_REF] Dousset | Experimental allergic encephalomyelitis and multiple sclerosis: lesion characterization with magnetization transfer imaging[END_REF]. The sensitivity of MT to these multiple mechanisms limits the potential of the semi-quantitative MTR parameter to univocally characterize demyelination/remyelination processes in myelin diseases such as multiple sclerosis [START_REF] Moll | Multiple sclerosis normal-appearing white matter: Pathology-imaging correlations[END_REF]. On the other hand, quantitative MT (qMT) provides us with f, the macromolecular bound pool fraction [START_REF] Sled | Quantitative Interpretation of Magnetization Transfer in Spoiled Gradient Echo MRI Sequences[END_REF], a quantitative metric that is highly-correlated with myelin content in various neurological conditions [START_REF] Thiessen | Quantitative MRI and ultrastructural examination of the cuprizone mouse model of demyelination: MRI IN CUPRIZONE DEMYELINATION[END_REF][START_REF] Underhill | Fast bound pool fraction imaging of the in vivo rat brain: Association with myelin content and validation in the C6 glioma model[END_REF], albeit not specific. The main issue with this model-based technique is the rather long protocol and complex processing [START_REF] Portnoy | Modeling pulsed magnetization transfer[END_REF], which requires several parametric images for robust multi-parameter fitting, even for the most optimized ones [START_REF] Naumova | High-resolution three-dimensional macromolecular proton fraction mapping for quantitative neuroanatomical imaging of the rodent brain in ultra-high magnetic fields[END_REF][START_REF] Yarnykh | Fast macromolecular proton fraction mapping from a single offresonance magnetization transfer measurement[END_REF].

In contrast, ihMT allows probing an additional degree of freedom within the myelin resonance spectrum and can have an interesting role to play. IhMT variants generally outperformed their conventional MT counterparts for myelin detection, as indicated by much weaker correlations between MTR values and ! (Fig. 5). The specificity of ihMTR for myelin was about 4 to 6 times higher than that of MTR +∆f and MTR +/-∆f (Sp values, Tables 3, S1 andS2). The irradiation parameters explored offered limited opportunities to sensitize MT sequences to a specific T1D range for modulation of the myelin-specificity, unlike ihMT protocols. This is demonstrated by the MTR +/-∆f data having minor differences in both α and Sp for varying MR parameters (table S2, supplementary material), consistent with the dualfrequency offset irradiation being naturally insensitive to T1D because of decoupling of Zeeman order from dipolar order. For single-frequency offset irradiation, although the contribution of dipolar order is embedded in the large MT signal, it may still have an effect: When strong dipolar order is created such as for boosted protocols, the sensitivity and specificity for myelin sensitivity, f and R associated to the different structures may further contribute to the final signal and contrast.

In summary, ihMTRs are semi-quantitative parameters trivial to process, highly correlated to myelin content and whose sensitivity and specificity to myelin can be modulated by sequence parameters variation. These properties are all assets, which warrant future studies to evaluate the potential of ihMT for studies of myelin related disorders.

What is the optimal ihMT protocol for myelin imaging?

Boosted ihMT protocols should benefit all applications of the technique, but the best choice of cosine-modulated pulses or frequency-alternated pulses for dual-offset irradiation would depend on the specific target of the study. Hence, if very high sensitivity (α value) for myelin is sought, then boosted ihMT protocols without any T1D-filtering (i.e. use of cosinemodulated pulses), which additionally provide the highest value of SNR and high CNR, are best options. However, the extent to which a significant signal arising from non-myelin protons (NSp~0.5, table 3) could interfere with the unambiguous interpretation of ihMTR variations as a change in myelin with this protocol needs to be evaluated. Conversely, protocols with frequency-alternating pulses provide more opportunities to modulate the ihMT contrast with T1D-filtering, offer highest relative contrast values and higher specificity (Sp≥0.65, table 3) for myelin. As such, boosted ihMT configurations associated with weak T1D-filtering (∆t~1ms), which additionally provide high SNR values and similar CNR than that of non-filtered protocols, can be considered as optimal ihMT protocols.

Interestingly, T1D-filtered and non-filtered ihMT protocols could also be combined together (e.g. by subtraction) to filter-out signal of long-T1D components and modulate specificity for short T1D values. Such short-T1D weighted data would for instance be particularly interesting in animal models that involve loss of the myelin compact multilamellar structure (e.g. Experimental Allergic Encephalomyelitis (EAE), a relevant model of multiple sclerosis) [START_REF] Ohler | Role of lipid interactions in autoimmune demyelination[END_REF], for which a decrease in T1D values might be expected.

In any case, ihMTR values are not unique for a given tissue but depend on the irradiation scheme and associated parameters. Hence, in order to ease future comparisons, it is recommended to carefully note the type of protocols used, RF irradiation power and T1Dfiltering conditions in future reports of ihMT studies.

Limitations of the study

One of the main limitations of this study is the small number of investigated animals (n=3).

Additionally, ihMTR analyses were performed on relatively low resolution and thick slice 2D images, which limited the ability to perform a voxel-by-voxel comparison with GFP images and only permitted the ROI analysis to be performed in a few regions of the brain. Highly resolved 3D imaging and image coregistration would improve the comparison of ihMTR with GFP images. The acquisition time for the multiple NEX used to obtain ihMTR images in this study was rather long, which could prevent their use in multimodal imaging studies. However, halving the number of NEX, and therefore reducing the acquisition time for boosted ihMTR images to ~6 minutes, did not change the correlation results (data not shown) nor the conclusions regarding sensitivity and specificity of the ihMTR variants for myelin. This is rather important for human studies, for which a high number of scan repetition is usually not feasible. Finally, no absolute quantification of the measurements was achieved in this study.

Hence, even though ihMTR correlates well with GFP fluorescence it does not provide an absolute quantitative measure of myelin content. In this perspective refined quantitative ihMT metrics, including for instance the value of the dipolar order relaxation time, T1D, or the fraction of macromolecules associated to dipolar order, f, could be obtained by application of the ihMT R2.7 models [START_REF] Varma | In vivo measurement of a new source of contrast, the dipolar relaxation time, T1D , using a modified inhomogeneous magnetization transfer (ihMT) sequence[END_REF](Varma et al., , 2015b) on data acquired with several different irradiation parameters.

Conclusion

This work reports a formal validation of the ihMT sensitivity relative to a reference myelin density measurement technique, plp-GFP fluorescence microscopy.

All tested ihMT protocol variants, standard and boosted with or without T1D-filtering,

showed very high correlations with GFP detection in the plp-GFP reporter mouse line, thereby generally validating ihMT as a myelin-sensitive technique. Short-T1D filtering reduced the fraction of signal unrelated to myelin, hence making protocols with frequency-alternating pulses for dual-offset frequency irradiation more myelin-specific than those using cosinemodulated pulses.

IhMT protocols generally outperformed their conventional MT counterparts as indicated by higher correlations between ihMTR values and GFP fluorescence signal, 4-6 times higher specificity for myelin, and a broad capacity to sensitize ihMTR to specific T1D ranges.

These assets provide motivation for the use of ihMT and to evaluate its potential in future studies of myelin disorders.

Appendices

A.1 Characterization of the boosted ihMT protocols

A comprehensive characterization of the boosted ihMT protocol is presented and discussed in this appendix. It extended analyses of previous studies performed in a clinical context [START_REF] Mchinda | Whole brain inhomogeneous magnetization transfer (ihMT) imaging: Sensitivity enhancement within a steady-state gradient echo sequence[END_REF][START_REF] Varma | Low duty-cycle pulsed irradiation reduces magnetization transfer and increases the inhomogeneous magnetization transfer effect[END_REF] by characterizing the effects of power concentration on the ihMT signal for various T1D-filtering conditions and high RF-power irradiation conditions (B1,rms=6.7µT), that are not achievable within typical clinical settings at conventional field strengths (: ; ≥ 1.5 ). This characterization enabled determining a set of boosted ihMT protocols providing high signal selectivity for myelin-rich structures, which were used for the validation study (section 2.4).

A.2 Methods

Female C57Bl/6J mice (12 weeks, 23±2g) underwent a 2-hour protocol during which axial single-slice (position: -0.7mm from bregma) boosted ihMT RARE images (Fig. 1b) were acquired using the parameters in table A1. A total number of 6 mice was necessary to cover the 39 sets of parameters in order to obtain a total number of n=3 mice per parameter set. 27.8, 54.4, 87.8 24.3, 31.4, 42.2, 50.7 17.2, 22.2, 29.8, 35.8 14.0, 18.1, 24.3, 29.3 No (CM, ∆t=1.3ms) 2 6 12 18 13, 90, 150 30, 50, 90, 130 30, 50, 90, 130 30, 50, 90, 130 1 1 1 1 27.8, 73, 94.3 24.3, 31.4, 42.2, 50.7 17.2, 22.2, 29.8, 35.8 14.0, 18.1, 24.3, 29.3 Values of variables used for the characterization of the boosted ihMT protocol (Fig. 1b) at constant averaged RF irradiation power, b1,rms=6.7µT. PW corresponds to the pulse length, ∆t to the repetition time of consecutive pulses, Np to the number of pulses per burst, BTR to the repetition time of consecutive bursts and b1 ' to the peak-power of the irradiation pulses. Other irradiation parameters were derived from previous optimization of the ihMT signal in brain internal capsule using the standard ihMT experiment, ∆f=10kHz, and τ=900ms [START_REF] Prevost | Optimization of inhomogeneous magnetization transfer (ihMT) MRI contrast for preclinical studies using dipolar relaxation time (T1D ) filtering[END_REF]. CM stands for Cosine-Modulated pulses; Six mice were necessary to cover the 39 sets of parameters with a total number of n=3 mice per parameter set.

For each set of parameters, 20 repetitions of the (MT + , MT -, MT +-, MT -+ ) images were acquired and ihMT processing was realized with the methodology described in the section 2.5.

IhMTR values were evaluated in ROIs manually drawn in the internal capsule (IC), cortex (Cx) and muscle (Mu) (Fig. A1) and were further reported as mean in-ROI values ± group (n=3) standard deviation.

A.3 Results

The effect of RF pulse parameters on the ihMT technique is illustrated in Fig. A.1, which shows ihMTR images acquired using the boosted ihMT protocol with different levels of RF power concentration and strength of T1D-filtering. Variation of signal intensities and contrasts between long-T1D myelinated WM and GM and shorter-T1D muscle are evidenced depending on saturation parameters. The effect of T1D-filtering can be appreciated by comparing ihMTR images obtained for identical power settings ( Arrows indicate internal capsule (IC, red), cortex (Cx, blue) and muscle (Mu, green). Highlyconcentrated RF power (Np=2) resulted in reduced signal when combined with T1D-filtering (a) but in a very large increase without T1D-filtering (d), for which highest ihMTR values were obtained in all brain structures. This highlighted the non-equivalence of frequency-alternated pulses and cosine-modulated pulses for dual-offset irradiation. For less-concentrated RF power, T1D-filtering, (b), allowed better contrast between highly-myelinated WM (e.g. IC) and muscle at the cost of a slight signal decrease compared to that without T1D-filtering, (c). (d-f), weak T1D-filtering, ∆t=1.3ms; (g-i), no T1D-filtering, CM) and the RF power concentration (adjusted by variable values of {Np, BTR}, the number of pulses per burst and the burst repetition time). ihMTR values derived from optimized standard ihMT protocols were taken from [START_REF] Prevost | Optimization of inhomogeneous magnetization transfer (ihMT) MRI contrast for preclinical studies using dipolar relaxation time (T1D ) filtering[END_REF].

For long-T1D tissues, in comparison with the standard ihMT protocol, the boosted ihMT protocols provided a signal gain (higher ihMTR values) whose magnitude depended on the T1Dfiltering strength: whereas the gain was very modest with strong T1D-filtering (∆t=3.3ms) (Figs. (∆t=1.3ms) (Figs. A.2d,e) and quadrupled without T1D-filtering (Figs. A.2g,h), as compared to standard protocols. Very long BTR values (BTR ≥130ms) were inefficient as indicated by the onset of decrease of ihMTR {boosted} values. RF power concentration from 12 to 2 pulses was inefficient when T1D-filtering was used, leading to decreased ihMTRs.

Boosted ihMT protocols using Np=12 and BTR=90ms yielded high signal difference between myelin-rich structures and others for all T1D-filtering conditions and were hence chosen for the validation experiment (table 2).

ihMT signal enhancement in short T1D-structures

The three to eight-fold (without T1D-filtering) ihMTR signal increase obtained for muscle tissue, emphasized the stronger signal enhancement of short-T1D tissues using boosted ihMT protocols. This effect can be explained by the mechanisms underlying the creation of the ihMT signal, which relies on the influence of dipolar order on RF saturation effects. Exchange of magnetization between Zeeman and dipolar order reservoirs of the bound pool during singlefrequency offset irradiation is mediated by the competitive effects of RF power ( ? @ ∝ B C D )

and dipolar order relaxation ( CE ) (Eq. 2 of (Varma et al., 2015b), such that, if dipolar relaxation is too fast, the dipolar order magnetization will remain negligible with no net effect on the RF saturation of the Zeeman magnetization. In case, the effects of single-frequency offset and dual-frequency offset irradiations would be similar (Eq. 2 tending toward Eq. 3 in (Varma et al., 2015b)), hence resulting in no ihMT effect. A necessary condition for an observable ihMT effect was proposed and formulated by the coarse rule, ? @ CE > 0.01 [START_REF] Manning | The physical mechanism of "inhomogeneous" magnetization transfer MRI[END_REF]. Varma et al., 2015b) was implemented using a MATLAB solver (ode45) and the methodology described in [START_REF] Mchinda | Whole brain inhomogeneous magnetization transfer (ihMT) imaging: Sensitivity enhancement within a steady-state gradient echo sequence[END_REF] adapted to the ihMT-prepared boosted sequence (i.e. simulating the boosted ihMT RARE sequence used experimentally) instead of an ihMT-steady-state boosted sequence (i.e. simulating the boosted ihMT-GRE sequence). Simulations considered the following tissue parameters, adapted from literature [START_REF] Mchinda | Whole brain inhomogeneous magnetization transfer (ihMT) imaging: Sensitivity enhancement within a steady-state gradient echo sequence[END_REF]: This includes: a rapid increase to a maximum, followed by a relatively slower decrease at long BTR values; a higher absolute increase for cosine-modulated based experiments for both long and short T1Ds tissues; for short T1Ds tissues, a higher relative increase between standard ihMT and boosted ihMT; and finally, when reducing Np from 12 to 2, a different variation of ihMTR whether T1D-filtering was used (large decrease, Fig. A.2d and Fig. A.3a), or not (large increase, Figs. A.2g,Fig. A.3b). These opposed variations are important features of boosted ihMT protocols performed at high irradiation power, and confirm the major difference between the sequential frequency-alternated pulses and simultaneous cosine-modulated pulses used for the dual-frequency offset irradiation, consistent with previous theoretical predictions (See Fig. 7 of [START_REF] Mchinda | Whole brain inhomogeneous magnetization transfer (ihMT) imaging: Sensitivity enhancement within a steady-state gradient echo sequence[END_REF]). The variable efficiency of both approaches for eliminating dipolar order is responsible for this difference as demonstrated by MT simulations (Fig. A.4). Concentration of RF power was achieved using bursts of Np RF pulses followed by a mixing time without RF power, repeated every BTR. Dashed boxes were zoomed in to display the main features of the RF pulses: Hann-shaped pulses (normalized power integral 0.375), duration PW, pulse repetition time ∆t, peak power b1 (standard protocols) or b1 ' (boosted protocols), frequency +∆f or -∆f. MT +-was achieved either by frequency alternation (from +∆f to -∆f), allowing T1D-filtering, or with cosinemodulated pulses (CM, peak power increased by a √2 factor), producing simultaneous irradiation at +∆f and -∆f. CM pulses did not allow T1D-filtering. Identical irradiation power, b1,rms, was used for all experiments. Note that the indicated b1,rms formulas applied for Hann-shape pulses. Plain markers correspond to results from standard ihMT protocols (Fig. 1a) and open markers from boosted ihMT protocols (Fig. 1b). The different colors indicate variable T1D-filtering strengths. Linear fits ( ℎ = ' ( ! + )) to scatterplot data were obtained by least-square regression and correlation coefficients, r 2 , (assessed by a bivariate (Pearson) correlation test) were reported for all protocols. Shaded areas correspond to confidence curves for line fits with a α-level=0.1. All correlations were significant (p <0.0001). scatterplot data were obtained by least-square regression and correlation coefficients, r 2 , (assessed by a bivariate (Pearson) correlation test) were reported for all protocols. Shaded areas correspond to confidence curves for line fits with a α-level=0.1. All correlations were significant (P <0.0001 for MTR +/-∆f v.s. GFP, and P <0.02 for MTR +∆f v.s. GFP). Arrows indicate internal capsule (IC, red), cortex (Cx, blue) and muscle (Mu, green). Highlyconcentrated RF power (Np=2) resulted in reduced signal when combined with T1D-filtering (a) but in a very large increase without T1D-filtering (d), for which highest ihMTR values were obtained in all brain structures. This highlighted the non-equivalence of frequency-alternated pulses and cosine-modulated pulses for dual-offset irradiation. For less-concentrated RF power, T1D-filtering, (b), allowed better contrast between highly-myelinated WM (e.g. IC) and muscle at the cost of a slight signal decrease compared to that without T1D-filtering, (c). A piecewise numerical integration of the ihMT theory [START_REF] Mchinda | Whole brain inhomogeneous magnetization transfer (ihMT) imaging: Sensitivity enhancement within a steady-state gradient echo sequence[END_REF]Varma et al., 2015b) was implemented using a MATLAB solver (ode45) and the methodology described in [START_REF] Mchinda | Whole brain inhomogeneous magnetization transfer (ihMT) imaging: Sensitivity enhancement within a steady-state gradient echo sequence[END_REF] adapted to the ihMT-prepared boosted sequence (i.e. simulating the boosted ihMT RARE sequence used experimentally) instead of an ihMT-steady-state boosted sequence (i.e. simulating the boosted ihMT-GRE sequence). Simulations considered the following tissue parameters, adapted from literature [START_REF] Mchinda | Whole brain inhomogeneous magnetization transfer (ihMT) imaging: Sensitivity enhancement within a steady-state gradient echo sequence[END_REF]:

CH = 0.65J, = 65s -1 , K = 0.

B.1 Bland-Altman Analysis

Fig 1 .

 1 Fig 1. Standard and boosted ihMT protocols. a) standard and b) boosted ihMT protocols obtained with distributed RF power and concentrated RF power throughout the irradiation period, τ, respectively.

  Fig. 3d.Table 2 summarizes the statistics of GFP fluorescence and ihMTR measurements in

Fig 2 .

 2 Fig 2. standard ihMTR and boosted ihMTR (Np=12, BTR=90ms) maps obtained on the same mouse for different T1D-filtering strengths at a) -3.2mm and b) -0.7mm from bregma. Other irradiation parameters were B1,rms=6.7µT, ∆f=10kHz and τ=900ms. These maps were used for correlation analyses with GFP fluorescence by comparing ihMTR values and normalized GFP intensity measured in ROIs outlined in Fig. 3d.

Fig 4 .

 4 Fig 4. Linear regressions of ihMTR values derived from the 6 ihMT protocols described in table 1 on myelin density according to ! values in brain structures for (a) individual measurements in each animal and each ROI of Figs. 3d and (b) mean values in each ROI (error bars representing standard deviations across mice).Plain markers correspond to results from standard ihMT protocols (Fig.1a) and open markers from boosted ihMT protocols (Fig.1b). The different colors indicate variable T1D-filtering strengths. Linear fits ( ℎ = ' ( ! + )) to scatterplot data were obtained by least-square regression and correlation coefficients, r 2 , (assessed by a bivariate (Pearson) correlation test) were reported for all protocols. Shaded areas correspond to confidence curves for line fits with a α-level=0.1. All correlations were significant (p <0.0001).

R2. 5 Fig 5 :

 55 Fig 5: Linear regressions of MTR +∆f (a,b) and MTR +/-∆f (c,d) values derived from the 6 protocols described in table 1 on myelin density according to ! values in brain structures for (a, c) individual measurements in each animal and each ROI of Figs. 3d, and (b, d) mean values in each ROI (error bars representing standard deviations across mice). The different colors indicate variable T1D-filtering strengths. Linear fits ( ∆ = ' ( ! + ) (a,b) and / ∆ = ' ( ! + ) (c,d)) to scatterplot data were obtained by least-square regression and correlation coefficients, r 2 , (assessed by a bivariate (Pearson) correlation test) were reported for all protocols. Shaded areas correspond to confidence curves for line fits with a α-level=0.1. All correlations were significant (P <0.0001 for MTR +/-∆f v.s. GFP, and P <0.02 for MTR +∆f v.s. GFP).

  Fig. A.1c); Use of T1D-filtering attenuated signal in short-T1D muscle, yielding ihMTR images

Fig A. 1 .

 1 Fig A.1. Representative boosted ihMTR brain images. IhMTR values and contrasts between structures are modulated by the combined effects of T1D-filtering and the concentration of RF power.Arrows indicate internal capsule (IC, red), cortex (Cx, blue) and muscle (Mu, green). Highlyconcentrated RF power (Np=2) resulted in reduced signal when combined with T1D-filtering (a) but in a very large increase without T1D-filtering (d), for which highest ihMTR values were obtained in all brain structures. This highlighted the non-equivalence of frequency-alternated pulses and cosine-modulated pulses for dual-offset irradiation. For less-concentrated RF power, T1D-filtering, (b), allowed better contrast between highly-myelinated WM (e.g. IC) and muscle at the cost of a slight signal decrease compared to that without T1D-filtering, (c).

Figure A .

 A Figure A.2 shows boosted ihMTR values (ihMTR {boosted} ) measured in internal capsule

  Fig A.2. Dependence of ihMTR with T1D-filtering and RF power concentration. Variations of ihMTR values measured in Internal Capsule (IC, (a), (d), (g)), Cortex (Cx, (b), (e), (h)) and muscle (Mu, (c), (f), (i)) as a function of the T1D-filtering strength ((a-c), strong T1D -filtering, ∆t=3.3ms; (d-f), weak T1D-filtering, ∆t=1.3ms; (g-i), no T1D-filtering, CM) and the RF power concentration (adjusted by

A

  .2a,b), the ihMTR signal measured in IC and Cx almost doubled for weak T1D-filtering

The

  b1 values of RF pulses in standard ihMT protocols made the previous inequality barely met in short-T1D muscle, hence resulting in reduced ihMTR values. Conversely, concentrating RF pulses at constant average b1,rms such as done in boosted ihMT protocols, naturally resulted in pronounced increase of pulse power B C G , thus making the previous inequality met for a broader range of T1D values, hence revealing ihMT signal from short T1D components. This mechanism likely explains the very large increase of ihMTR in muscle observed, for instance, with boosted ihMT protocols and no T1D-filtering (Fig. A.1c). Non-equivalence of cosine-modulated pulses and frequency-alternated pulses for dual-offset irradiation The general variations of boosted ihMTR values with {Np, BTR} shown on Fig. A.2 are in agreement with previous experimental results obtained in humans with a 3D steady-state GE sequence and frequency-alternating RF pulses (Mchinda et al., 2017) and with a 2D ihMT prepared sequence with CM RF pulses for dual frequency irradiation (Varma et al., 2018), and are furthermore well supported by the theoretical ihMT model illustrated by numerical simulations (Fig. A.3).

Fig A. 3 .

 3 Fig A.3. Numerical simulations of the dependence of ihMTR with RF power concentration with and without T1D-filtering. A piecewise numerical integration of the ihMT theory[START_REF] Mchinda | Whole brain inhomogeneous magnetization transfer (ihMT) imaging: Sensitivity enhancement within a steady-state gradient echo sequence[END_REF]

  65 , D@ = 12.5 µs, 1 ( CH DH ) Simulations are shown for T1D values of 6.2ms (mimicking Internal capsule case) (a,b) and 1.0ms (mimicking muscle) (c,d) with weak T1D-filtering (a,c) and without T1D-filtering (b,d). RF power concentration was varied with changes in {Np, BTR} values.

Fig A. 4 .

 4 Fig A.4. Numerical simulations of MT + and MT +/-. Variations of MT + (grey curves) and MT +-

Fig B. 1

 1 Fig B.1 Bland-Altman plots comparing the normalized ihMTR and

Fig B. 2

 2 Fig B.2 Bland-Altman plots comparing the normalized MTR +∆f and

Figure Captions Fig 1 .

 Captions1 Figure Captions

Fig 2 .

 2 Fig 2. standard ihMTR and boosted ihMTR (Np=12, BTR=90ms) maps obtained on the same mouse for different T1D-filtering strengths at a) -3.2mm and b) -0.7mm from bregma. Other irradiation parameters were B1,rms=6.7µT, ∆f=10kHz and τ=900ms. These maps were used for correlation analyses with GFP fluorescence by comparing ihMTR values and normalized GFP intensity measured in ROIs outlined in Fig. 3d.

Fig 4 .

 4 Fig 4. Linear regressions of ihMTR values derived from the 6 ihMT protocols described in table 1 on myelin density according to ! values in brain structures for (a) individual measurements in each animal and each ROI of Figs. 3dand (b) mean values in each ROI (error bars representing standard deviations across mice). Plain markers correspond to results from standard ihMT protocols (Fig.1a) and open markers from boosted ihMT protocols (Fig.1b). The different colors indicate variable T1D-filtering strengths. Linear fits ( ℎ = ' ( ! + )) to scatterplot data were obtained by least-square regression and correlation coefficients, r 2 , (assessed by a bivariate (Pearson) correlation test) were reported for all protocols. Shaded areas correspond to confidence curves for line fits with a α-level=0.1. All correlations were significant (p <0.0001).

Fig 5 :

 5 Fig 5: Linear regressions of MTR +∆f (a,b) and MTR +/-∆f (c,d) values derived from the 6 protocols described in table 1 on myelin density according to ! values in brain structures for (a, c) individual measurements in each animal and each ROI of Figs. 3d, and (b, d) mean values in each ROI (error bars representing standard deviations across mice). The different colors indicate variable T1D-filtering strengths. Linear fits ( ∆ = ' ( ! + ) (a,b) and / ∆ = ' ( ! + ) (c,d)) to

Fig A. 1 .

 1 Fig A.1. Representative boosted ihMTR brain images. IhMTR values and contrasts between structures are modulated by the combined effects of T1D-filtering and the concentration of RF power.Arrows indicate internal capsule (IC, red), cortex (Cx, blue) and muscle (Mu, green). Highlyconcentrated RF power (Np=2) resulted in reduced signal when combined with T1D-filtering (a) but in a very large increase without T1D-filtering (d), for which highest ihMTR values were obtained in all brain structures. This highlighted the non-equivalence of frequency-alternated pulses and cosine-modulated pulses for dual-offset irradiation. For less-concentrated RF power, T1D-filtering, (b), allowed better contrast between highly-myelinated WM (e.g. IC) and muscle at the cost of a slight signal decrease compared to that without T1D-filtering, (c).

Fig A. 2 .

 2 Fig A.2. Dependence of ihMTR with T1D-filtering and RF power concentration. Variations of ihMTR values measured in Internal Capsule (IC, (a), (d), (g)), Cortex (Cx, (b), (e), (h)) and muscle (Mu, (c), (f), (i)) as a function of the T1D-filtering strength ((a-c), strong T1D -filtering, ∆t=3.3ms;(d-f), weak T1D-filtering, ∆t=1.3ms; (g-i), no T1D-filtering, CM) and the RF power concentration (adjusted by variable values of {Np, BTR}, the number of pulses per burst and the burst repetition time). ihMTR values derived from optimized standard ihMT protocols were taken from[START_REF] Prevost | Optimization of inhomogeneous magnetization transfer (ihMT) MRI contrast for preclinical studies using dipolar relaxation time (T1D ) filtering[END_REF]

  Simulations are shown for T1D values of 6.2ms (mimicking Internal capsule case) (a,b) and 1.0ms (mimicking muscle) (c,d) with weak T1D-filtering (a,c) and without T1D-filtering (b,d). RF power concentration was varied with changes in {Np, BTR} values.

Fig A. 4 .

 4 FigA.4. Numerical simulations of MT + and MT +/-. Variations of MT + (grey curves) and MT +- (achieved with frequency-alternated pulses (yellow curves) and cosine-modulated pulses (orange curves)) with BTR are shown for NP=2 (a) and NP=12 (b). T1D was set to 6.2ms for both cases. This figure explains the different behavior of frequency-alternated pulses and cosine-modulates pulses for dual irradiation at high RF power concentration, at the origin of the opposed variations of ihMTR for NP=2 (Fig.A.2d vs Fig. A.2g, and Fig. A.3a vs Fig. A.3b).

Table 1 :

 1 Parameters for the ihMT protocols used in studies for comparison with fluorescence

	This experiment evaluated to what extent ihMTR values obtained with different ihMT
	protocols correlate with myelin-content. Two axial ihMT RARE slices (position: -0.7mm and -
	3.2mm from bregma) were acquired on plp-GFP mice (n=3, 12 weeks, 23±2g) with six different
	ihMT variants consisting of both standard and boosted protocols parameterized with different
	T1D-filtering strengths: no filtering, achieved by CM; weak filtering; and, strong filtering, the
	latter two achieved by sequential frequency alternation and increasing values of ∆t. The

microscopy protocol T1D-filtering Np BTR (ms) PW (ms) b1 or b1' (µT) standard

		Strong (∆t=3.3ms)	N/A	N/A	3	11.4
		Weak (∆t=1.3ms) No (CM, ∆t=1.3ms) *	N/A N/A	N/A N/A	1 1	12.4 12.4
		Strong (∆t=3.3ms)	12	90	3	17.2
	boosted	Weak (∆t=1.3ms) No (CM, ∆t=1.3ms)	12 12	90 90	1 1	29.8 29.8

Table 2 :

 2 Quantitative ihMTR and ! values in brain structuresMean values ± standard deviation, calculated by pooling the measurements of the 3 mice in Cx and HC ROIs for GM; in Th and IPN ROIs for mixed structures and the measurements in IC, CC and ON ROIs for WM. The relative contrast between WM and GM was calculated as (ihMTR WM -ihMTR GM )/ihMTR WM for MRI and as ( ! WM -! GM )/ ! WM for fluorescence. The CNR was calculated as (ihMTR WM -ihMTR GM )/σ, and the SNR as ihMTR WM /σ, with σ, the noise of the ihMTR image. The # symbol indicates statistical difference (ANOVA, Tukey-Kramer HSD corrected for multiple comparisons) with values in both GM and mixed structures and the * symbol indicates statistical difference with values in GM.

	ihMTR (%)

Table 3 :

 3 Linear regression analysis of associations between ihMTR and

	! -Sensitivity and

Individual data (Fig. 4a)

		Standard	ihMT	Strong (∆t=3.3ms) Weak (∆t=1.3ms)	0.97 0.98	0.94 0.96	23 23	<.0001 <.0001	3.62 ± 0.20, p<.0001 4.02 ± 0.19, p<.0001	1.60 ±0.12, p<.0001 2.17 ± 0.11, p<.0001 0.65 ± 0.04 0.69 ± 0.06	0.31 ± 0.03 0.35 ± 0.03
				No (CM, ∆t=1.3ms)	0.93	0.87	15	<.0001	4.78 ± 0.51, p<.0001	3.70 ± 0.31, p<.0001 0.56 ± 0.08	0.44 ± 0.06
		ihMT		Strong (∆t=3.3ms)	0.97	0.94	23	<.0001	4.43 ± 0.25, p<.0001	1.95 ± 0.15, p<.0001 0.69 ± 0.06	0.31 ± 0.03
		Boosted		Weak (∆t=1.3ms) No (CM, ∆t=1.3ms)	0.98 0.98	0.96 0.96	23 23	<.0001 <.0001	6.57 ± 0.28, p<.0001 8.41 ± 0.38, p<.0001	3.52 ± 0.17, p<.0001 0.65 ± 0.04 7.29 ± 0.23, p<.0001 0.53 ± 0.03	0.35 ± 0.02 0.47 ± 0.02
	Structure-averaged data (Fig. 4b)	Standard ihMT ihMT Boosted		Strong (∆t=3.3ms) Weak (∆t=1.3ms) No (CM, ∆t=1.3ms) Strong (∆t=3.3ms) Weak (∆t=1.3ms) No (CM, ∆t=1.3ms)	0.99 0.99 0.98 0.99 0.99 0.99	0.98 0.98 0.96 0.97 0.98 0.99	7 7 7 7 7 7	<.0001 <.0001 <.0001 <.0001 <.0001 <.0001	3.56 ± 0.23, p=.0001 3.99 ± 0.23, p<.0001 4.84 ± 0.42, p<.0001 4.28 ± 0.31, p<.0001 6.47 ± 0.35, p<.0001 8.40 ± 0.37, p<.0001	1.65 ± 0.15, p<.0001 0.68 ± 0.07 2.20 ± 0.15, p<.0001 0.65 ± 0.05 3.74 ± 0.28, p<.0001 0.56 ± 0.07 2.06 ± 0.20, p=.0002 0.68 ± 0.07 3.61 ± 0.23, p<.0001 0.64 ± 0.05 7.33 ± 0.24, p<.0001 0.53 ± 0.03	0.32 ± 0.04 0.35 ± 0.03 0.44 ± 0.05 0.32 ± 0.04 0.36 ± 0.03 0.47 ± 0.02

Table A1 :

 A1 boosted ihMT protocol parameterization

	T1D-filtering	Np	BTR (ms)	PW (ms)	b1' (µT)
		6	60, 90, 130	3	19.9, 24.3, 29.3
	Strong (∆t=3.3ms)	12	60, 90, 130	3	14.0, 17.2, 20.7
		18	60, 90, 130	3	11.5, 14.0, 16.9
		2	13, 50, 130	1
	Weak (∆t=1.3ms)	6 12	30, 50, 90, 130 30, 50, 90, 130	1 1
		18	30, 50, 90, 130	1

This work was supported by the national research agency ANR [ANR-17-CE18-0030], VERISMO project. V. P. received funding from the French Government's 'Investissements d'Avenir' program A*MIDEX, [ANR-11-IDEX-0001-02]. S. M. received funding from ARSEP 2017 grant (Association pour la Recherche sur la Sclérose en Plaques) V. N. C. received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement N o 713750. Also, it has been carried out with the financial support of the Regional Council of Provence-Alpes-Côte d'Azur and with the financial support of the A*MIDEX (n° ANR-11-IDEX-0001-02), funded by the Investissements d'Avenir project funded by the French Government, managed by the French National Research Agency (ANR).

Bruker format file) will be shared

were reduced (see α and Sp in table S1). This is a rather paradoxical situation: the stronger the dipolar order effects are generated in myelinated tissues, the stronger the attenuation of MT + saturation effects, hence limiting sensitivity and specificity for these tissues.

Correlations of ihMTRs with myelin histology imaging are as good as those obtained with f, the bound pool fraction extracted from qMT analyses [START_REF] Khodanovich | Histological validation of fast macromolecular proton fraction mapping as a quantitative myelin imaging method in the cuprizone demyelination model[END_REF][START_REF] Underhill | Fast bound pool fraction imaging of the in vivo rat brain: Association with myelin content and validation in the C6 glioma model[END_REF], which was found to be the best predictor of pathological myelin content among metrics derived from other advanced MR techniques (DTI, T1, T2, MWF) [START_REF] Thiessen | Quantitative MRI and ultrastructural examination of the cuprizone mouse model of demyelination: MRI IN CUPRIZONE DEMYELINATION[END_REF]. The intercepts of the linear equations describing the relationship between myelin content and ihMTRs (Fig. 4) and between myelin content and f [START_REF] Thiessen | Quantitative MRI and ultrastructural examination of the cuprizone mouse model of demyelination: MRI IN CUPRIZONE DEMYELINATION[END_REF][START_REF] Underhill | Fast bound pool fraction imaging of the in vivo rat brain: Association with myelin content and validation in the C6 glioma model[END_REF] were both non-zero, thereby indicating that structures other than myelin contribute to these measured parameters. Fortunately, with ihMT, T1D-filtering can be used to tailor protocols with variable contributions of non-myelin protons. Although the myelin nonspecific signal component could represent almost half of the total ihMT signal without T1D-filtering, it reduces to a third of the total when modest short-T1D filtering was used (NSp values, table 3). This indicates that, on average, ihMT-responding non-myelin protons are associated with T1D values shorter than myelin ones. Though further investigation of the precise nature of those protons is merited, the faster dipolar relaxation of these protons could result from faster water exchange processes [START_REF] Manning | The physical mechanism of "inhomogeneous" magnetization transfer MRI[END_REF]. Hence, membrane protons of less motionallyrestricted glial cells, which represent nearly 35% of mouse brain cells [START_REF] Herculano-Houzel | Cellular scaling rules for rodent brains[END_REF] are reasonable candidates. In principle, even larger ∆t values might entirely remove the contribution from short-T1D non-myelin structures, leading to 100% myelin-specific ihMTR images, albeit with lower sensitivity. Note also that further investigation of the contribution of other ihMT parameters (f, the fraction of dipolar order associated to T1D and R, the exchange rate with the free pool) is merited. Indeed, if the strength of T1D-filtering scales the initial


MD Emeline Maisonneuve
email: emelinem@yahoo.com

MD Laure Debain

MD Catherine Garel

MD Erik Hervieux

Bertrand Lafon

MD, PhD Ferdinand Dhombres

MD, PhD Gilles Kayem

J.-M Jouannic

MD, PhD Jean-Marie Jouannic

Diagnostic prénatal et issue néonatale des calcifications intra-abdominales isolées : étude rétrospective sur 10 ans

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Toute découverte de calcifications intra-abdominales (iA) lors d'une échographie obstétricale nécessite une consultation au sein d'un Centre Pluridisciplinaire de Diagnostic Prénatal (CPDPN) afin de confirmer le diagnostic, préciser le caractère unique ou multiple ainsi que la localisation (intra-hépatique, vésiculaire, splénique, péri-hépatique ou péritonéale), rechercher une malformation associée et discuter d'éventuelles explorations prénatales. Les calcifications intra-hépatiques diffuses ou évolutives peuvent être observées dans les foetopathies infectieuses, alors que les calcifications intra-hépatiques stables sont le plus souvent en rapport avec une cause vasculaire. Les calcifications péri-hépatiques, localisées à la surface du foie, et les calcifications intra-péritonéales sont plutôt évocatrices de péritonite méconiale [START_REF] Carroll | The significance of echogenic areas in the fetal abdomen[END_REF][START_REF] Zerhouni | Can we select fetuses with intra-abdominal calcification for delivery in neonatal surgical centres?[END_REF][START_REF] Le | Isolated echogenic foci in the left upper quadrant of the fetal abdomen: are they significant?[END_REF]. Cependant, il existe peu de données dans la littérature étudiant, en anténatal, à la fois les cas de calcifications iA isolées intra et extra-hépatiques. Notre objectif était d'étudier l'étiologie et le pronostic des calcifications iA isolées.

MATERIEL ET METHODES

Nous avons réalisé une étude descriptive rétrospective monocentrique portant sur les enfants ayant eu un diagnostic anténatal de calcification iA isolée confirmé par une échographie de référence à l'hôpital Armand Trousseau de 2008 à 2018.

Les critères d'inclusion de notre étude étaient : les nouveau-nés ayant eu un diagnostic de calcification iA isolée en échographie de référence anténatale ainsi qu'un avis au CPDPN de l'hôpital Armand Trousseau et nés entre le 1 er janvier 2008 et le 30 juin 2018. Le diagnostic de calcification iA était posé devant une image au moins iso-échogène par rapport à la côte adjacente, retrouvée sur au moins deux plans de coupe et après avoir supprimé les harmoniques et réduit le gain au maximum, sans vascularisation en mode Doppler couleur, ayant dans la majorité des cas un cône d'ombre postérieur, celui-ci étant dépendant de la taille de la calcification et de sa teneur en calcium [START_REF] Garel | Imagerie: du foetus au nouveau-né[END_REF]. Les calcifications iA comprenaient : les calcifications intra-hépatiques situées au sein du parenchyme hépatique foetal, les calcifications péri-hépatiques situées autour du foie, et les calcifications péritonéales situées le plus souvent au contact des anses intestinales.

Les critères d'exclusion étaient : les calcifications situées à l'intérieur des anses digestives (entérolithiases), les calcifications rétro-péritonéales et les calcifications iA associées à d'autres anomalies digestives (dilatation digestive, ascite) ou autres malformations congénitales, les petits poids pour l'âge gestationnel (PAG) < 5ème percentile à l'échographie, les hyperclartés nucales ≥

RESULTATS

Parmi les 70 cas de calcifications iA retrouvés sur les 10 dernières années, 32 cas étaient isolés (46%) et 38 cas (54%) associés à d'autres anomalies échographiques ont été exclus (cf. diagramme de flux, figure 1). Les données néonatales étaient manquantes pour neuf dossiers, que nous avons également exclus. Au total, 23 cas de calcifications iA isolées ont été retenus : 15 concernaient des calcifications intra-hépatiques, 5 des péri-hépatiques, 2 des péritonéales et un foetus était porteur à la fois de calcifications intra-et péri-hépatiques. Les aspects échographiques de ces différentes localisations sont présentés en figure 2. Il n'y avait pas de calcifications spléniques, ni dans la vésicule biliaire dans notre série.

Concernant les 23 cas de calcifications iA isolées, l'âge moyen des patientes était de 32 ans et s'étendait de 22 à 43 ans. Le diagnostic échographique était réalisé entre 21 et 31 SA, et dans 21 cas sur 23 au cours de l'échographie de dépistage du 2ème trimestre. Toutes localisations confondues, les calcifications iA étaient dans 71% des cas au nombre d'une, dans 5% des cas au nombre de deux et 24% des cas, de trois ou plus. Une PLA a été réalisée pour 4 cas, retrouvant un caryotype foetal normal, les recherches de virus négatives et des enzymes digestives normales. Les sérologies maternelles ont permis de diagnostiquer un seul cas de séroconversion maternelle pour le CMV estimée à un mois avant la date de conception. Pour ce cas, la recherche de CMV urinaire était négative à naissance. La recherche des mutations les plus fréquentes de la mucoviscidose chez les parents a été systématiquement proposée en cas de localisation péri-hépatique ou intra-péritonéale.

Elle a été réalisée dans 16 cas sur 23, et a permis d'exclure le risque d'homozygotie ou d'hétérozygotie composite pour l'enfant à naitre pour les mutations les plus fréquentes. Le sexe ratio était de 16 garçons (70%) pour 7 filles (30%). L'âge gestationnel de naissance s'étendait de 31SA à 41SA+6j, avec une moyenne de 38SA+5j. Trois naissances sur 23 ont eu lieu prématurément avec une césarienne programmée à 36SA+5j pour allo-immunisation plaquettaire, une césarienne en urgence à 36SA+2j pour métrorragies sur placenta praevia et un accouchement voie basse à 31SA dans un contexte de chorioamniotite. Le poids de naissance moyen des nouveau-nés était voisin du 50 ème percentile et dans 4 cas sur 23 (17%) le poids de naissance était inférieur au 10ème percentile (selon les courbes Audipog à la naissance). L'examen clinique réalisé par le pédiatre dans les 24 premières heures de vie était normal dans tous les cas. La recherche de CMV salivaire ou urinaire a concerné la moitié des enfants et était toujours négative. Sur les 18 échographies abdominales réalisées, 3 (17%) ne retrouvaient plus la ou les calcifications décrites en anténatal et toutes les autres retrouvaient un aspect comparable à celui décrit en prénatal et ne décrivaient aucune autre anomalie associée. Enfin, le dépistage de la mucoviscidose par le test de Guthrie (dosage de la trypsine immuno-réactive) a été réalisé systématiquement et était négatif dans tous les cas.

Parmi les 38 cas de calcifications iA associées, 11 cas de péritonite méconiale et/ou atrésie digestive, une trisomie 13, une trisomie 21 et 2 foetopathies à CMV ont été diagnostiqués.

DISCUSSION

Dans notre étude, en cas de calcification iA isolée après échographie de diagnostic, le bilan étiologique anténatal a toujours été normal et les issues de grossesses étaient toutes favorables.

A notre connaissance, cette série est la seule portant sur les calcifications iA isolées regroupant les calcifications hépatiques, péri-hépatiques et péritonéales. Les forces de notre étude sont le recueil exhaustif des cas de calcifications, suivis en diagnostic prénatal, présentés en réunion pluridisciplinaire et ayant accouché dans notre centre, ainsi que la pratique systématique d'échographies de diagnostic, en particulier par des radiopédiatres qui revoient les enfants également en postnatal. Cependant, notre étude présente plusieurs limites. D'une part, la proportion de calcifications associées parmi toutes les calcifications est probablement surestimée du fait d'un biais de recrutement lié à un effet centre associé à un service de chirurgie pédiatrique. D'autre part, la taille de l'échantillon de calcifications isolées est trop réduite pour conclure définitivement sur le pronostic.

A l'étude des différentes échographies, nous avons observé qu'il existait une variabilité inter et parfois même intra-observateur pour décrire et préciser la localisation des calcifications. De même, le terme d'"hyperéchogénicité" a parfois été retrouvé dans des dossiers pour parler d'une calcification déjà mentionnée dans une précédente échographie, sans que l'aspect de celle-ci ait changé. Une calcification est reconnaissable par ses contours nets, sa forte hyperéchogénicité et son cône d'ombre postérieur, dépendant de l'absorption du faisceau ultrasonore par le calcium. Ainsi, les fines calcifications ne donnent pas toujours de cône d'ombre postérieur à l'échographie. Les calcifications sont également visibles à l'examen foetopathologique, aussi bien en macroscopie qu'en coupe histologique. [START_REF] Sahlin | Fetal calcifications are associated with chromosomal abnormalities[END_REF] Les calcifications intra-hépatiques sont le plus souvent en rapport avec une cause vasculaire si elles sont isolées et stables (défaut de perfusion hépatique, thrombose veineuse portale ou hépatique, hématome sous-capsulaire) [START_REF] Garel | Imagerie: du foetus au nouveau-né[END_REF][START_REF] Simchen | Fetal hepatic calcifications: prenatal diagnosis and outcome[END_REF]. Lorsqu'elles sont diffuses et évolutives, on évoque souvent l'hypothèse d'un granulome calcifié secondaire à une infection.

Dans la littérature, nous avons retenu six études qui ont analysé des séries de calcifications iA, présentées dans le tableau 1. Nos données concordent avec les issues favorables retrouvées sur les calcifications hépatiques isolées [START_REF] Le | Isolated echogenic foci in the left upper quadrant of the fetal abdomen: are they significant?[END_REF][START_REF] Simchen | Fetal hepatic calcifications: prenatal diagnosis and outcome[END_REF][START_REF] Koopman | Fetal intrahepatic hyperechogenic foci: prenatal ultrasound diagnosis and outcome[END_REF][START_REF] Achiron | Prenatal ultrasonographic diagnosis of fetal hepatic hyperechogenicities: clinical significance and implications for management[END_REF]. Leur prévalence était de 1/1037 à 1/1500 [START_REF] Koopman | Fetal intrahepatic hyperechogenic foci: prenatal ultrasound diagnosis and outcome[END_REF][START_REF] Achiron | Prenatal ultrasonographic diagnosis of fetal hepatic hyperechogenicities: clinical significance and implications for management[END_REF][START_REF] Bronshtein | Prenatal diagnosis of liver calcifications[END_REF]. Dans l'étude d'Achiron et al, sur cinq cas de calcifications, seuls 3 foetus présentaient des calcifications intra-hépatiques réellement isolées avec une évolution favorable [START_REF] Achiron | Prenatal ultrasonographic diagnosis of fetal hepatic hyperechogenicities: clinical significance and implications for management[END_REF]. La plus grande étude est celle de Chez les 10 foetus présentant des calcifications intra-hépatiques isolées (71%), tous sont nés à terme, eutrophes avec un examen néonatal normal. Dans l'étude rétrospective de Koopman et al, 7 cas de foci hyperéchogènes hépatiques ont été retenus, dont 5 isolés (71%) [START_REF] Koopman | Fetal intrahepatic hyperechogenic foci: prenatal ultrasound diagnosis and outcome[END_REF]. Les auteurs n'employaient pas le mot "calcification" et ne précisaient pas si il existait un cône d'ombre postérieur. Chez les 5 foetus avec calcifications isolées, l'issue néonatale était favorable avec une naissance normale à terme. L'échographie postnatale retrouvait le ou les foyers hépatiques hyperéchogènes dans 2 cas sur 5. Dans l'étude de Stein et al, 33 cas de calcifications intra-hépatiques incluant 25 cas de calcifications intra-hépatiques isolées ont été étudiés [START_REF] Stein | Fetal liver calcifications: sonographic appearance and postnatal outcome[END_REF] ; parmi ceux-ci, un cas de foetopathie à CMV a été retrouvé. Le point d'appel initial était une calcification initialement isolée à 21 SA, mais sont apparues secondairement de multiples calcifications, ainsi qu'une microcéphalie. L'issue a été défavorable avec un décès néonatal. De notre point de vue, ce cas ne peut pas être considéré comme un cas de calcification isolée. D'ailleurs, les auteurs soulignent l'intérêt de réaliser des contrôles échographiques mensuels pour s'assurer du caractère isolé et non évolutif de la ou des calcifications.

Les 24 autres foetus présentaient réellement un diagnostic de calcifications intra-hépatiques isolées et tous sont nés à terme avec un devenir postnatal normal. Ces études concernaient seulement les calcifications hépatiques et sont relativement anciennes [START_REF] Simchen | Fetal hepatic calcifications: prenatal diagnosis and outcome[END_REF][START_REF] Koopman | Fetal intrahepatic hyperechogenic foci: prenatal ultrasound diagnosis and outcome[END_REF][START_REF] Achiron | Prenatal ultrasonographic diagnosis of fetal hepatic hyperechogenicities: clinical significance and implications for management[END_REF][START_REF] Bronshtein | Prenatal diagnosis of liver calcifications[END_REF]. D'autres auteurs se sont intéressés à évaluer le pronostic de calcifications diagnostiquées dans le quadrant supérieur gauche de l'abdomen sans préciser l'organe concerné [START_REF] Le | Isolated echogenic foci in the left upper quadrant of the fetal abdomen: are they significant?[END_REF]. Les issues périnatales ont pu être analysées pour 20 des 26 foetus avec calcifications iA isolées : ils étaient tous nés à terme, eutrophes, avec un examen clinique normal.

Les progrès obtenus depuis quinze ans dans la qualité de l'image échographique laissent penser que nous sommes plus performants dans la recherche de malformations associées et dans l'information aux couples sur le pronostic foetal. L'échographie de diagnostic est donc primordiale pour rechercher d'autres anomalies associées, en particulier en lien avec des foetopathies infectieuses ou d'autres signes digestifs évocateurs de péritonite méconiale, afin d'anticiper le lieu de naissance et une éventuelle prise en charge chirurgicale [START_REF] Zerhouni | Can we select fetuses with intra-abdominal calcification for delivery in neonatal surgical centres?[END_REF]. Une série récente montre bien que parmi les enfants ayant présenté une péritonite méconiale, les calcifications iA n'étaient pas isolées, les signes les plus souvent associés étant l'ascite, l'intestin hyperéchogène, la dilatation digestive, l'hydramnios et le pseudokyste méconial [START_REF] Ping | Meconium Peritonitis: Correlation of Antenatal Diagnosis and Postnatal Outcome -An Institutional Experience over 10 Years[END_REF].

Concernant les examens complémentaires postnataux, la recherche du CMV urinaire/salivaire a été réalisée dans la moitié des cas. En reprenant les examens pré et postnataux (sérologies CMV de la grossesse et recherche de CMV à la naissance), il n'existait que 2 cas sur 23 pour lesquels on ne pouvait pas exclure formellement une foetopathie à CMV. Néanmoins, tous les enfants avaient un examen clinique normal. Les échographies abdominales ont été réalisées dans les trois quarts des cas et les pratiques récentes dans notre centre sont plutôt de ne plus en réaliser si l'examen clinique du nouveau-né en suites de couches est normal (absence de vomissement ou de ballonnement et transit normal). Par ailleurs, la gêne occasionnée par l'air du tube digestif après la naissance peut faire méconnaitre des calcifications intra-abdominales extra-hépatiques qui étaient évidentes en échographie prénatale.

Aucun cas de mucoviscidose n'a été retrouvé ni dans notre série, ni dans la littérature en cas de calcification iA isolée [START_REF] Simchen | Fetal hepatic calcifications: prenatal diagnosis and outcome[END_REF][START_REF] Stein | Fetal liver calcifications: sonographic appearance and postnatal outcome[END_REF]. Concernant la mucoviscidose, elle ne devrait être recherchée qu'en cas de calcifications péri-hépatiques ou péritonéales, c'est-à-dire en lien avec une possible péritonite méconiale, mais pas en cas de calcifications intra-hépatiques (Figure 3). En cas de calcifications péri-hépatiques ou péritonéales, il faut rechercher la visualisation de la vésicule biliaire, car le seul critère de non-visualisation de la vésicule biliaire a un rapport de vraisemblance de 2,7 pour la mucoviscidose [START_REF] Bergougnoux | Isolated Nonvisualization of the Fetal Gallbladder Should Be Considered for the Prenatal Diagnosis of Cystic Fibrosis[END_REF]. Toutefois, notre effectif est une fois encore trop réduit et une étude prospective avec un effectif plus important devrait être réalisée avant de pouvoir se passer de la recherche non invasive des mutations de la mucoviscidose chez les parents.

En tenant compte des cas rapportés dans la littérature et de ceux de notre étude, sur 100 enfants porteurs de calcification iA isolée en anténatal, un cas de trisomie 21 a été rapporté (cf. tableau 1).

Une étude réalisée sur des examens foetopathologiques provenant d'interruptions médicales de grossesses ou de morts foetales in utero, a retrouvé que l'association d'une anomalie congénitale et d'une calcification foetale (localisée dans le foie principalement) était associée à l'existence d'une aneuploïdie. En revanche, les foetus présentant des calcifications isolées n'étaient pas plus porteurs d'aneuploïdies que les foetus ne présentant pas de calcifications. Les auteurs recommandent ainsi fortement la réalisation d'un caryotype lorsqu'une malformation est associée à une calcification foetale [START_REF] Sahlin | Fetal calcifications are associated with chromosomal abnormalities[END_REF]. Par ailleurs, aucune étude n'a retrouvé de lien entre le nombre ou la localisation des calcifications et l'association à une infection, une aneuploïdie ou d'autres anomalies.

CONCLUSION

La moitié des cas de calcifications intra-abdominales foetales sont échographiquement isolées. Dans notre étude, parmi les 23 cas de calcifications iA isolées, la majorité ont été stables tout au long de la grossesse. Aucun cas d'aneuploïdie, de foetopathie infectieuse, ni de mucoviscidose n'a été retrouvé.

Le pronostic de ces enfants était favorable avec un examen clinique normal dans tous les cas, aucune chirurgie nécessaire et une échographie retrouvant la ou les calcifications dans 83% des cas. Il apparaît donc qu'une échographique de référence, suivie d'une surveillance échographique mensuelle, ainsi qu'une recherche de foetopathie à CMV et des mutations les plus fréquentes de la mucoviscidose paraissent suffisants pour rassurer les couples devant des calcifications iA isolées échographiquement chez leur foetus.

Légende

3 .

 3 5mm au premier trimestre et les dossiers avec données manquantes sur l'issue de grossesse et/ou l'examen néonatal. Le recueil des informations a été effectué par recherche de mots-clés ("calcification" et "hyperéchogénicité isolée") sur le logiciel Viewpoint® pour les données d'échographie prénatale et de consultation de médecine foetale et sur le logiciel PACS pour les échographies faites en radiologie (anté et postnatales). Les données collectées sur les calcifications iA étaient : leur nombre, leur localisation, leur forme, leur taille, leur évolution au cours de la grossesse, l'âge gestationnel au moment du diagnostic. Au cours de l'étude, le bilan étiologique de ces calcifications reposait de manière systématique sur la réalisation d'une échographie de référence, d'une sérologie maternelle pour le Cytomégalovirus (CMV) et de la recherche des mutations les plus fréquentes de la mucoviscidose chez les parents. Les autres sérologies infectieuses maternelles (Toxoplasmose, Rubéole, Herpès, Syphilis) n'étaient réalisées qu'en cas de calcification intra-hépatique. Pour chaque cas, la réalisation d'une ponction de liquide amniotique (PLA) avec caryotype était discutée en tenant compte notamment du calcul de risque combiné pour la trisomie 21. En cas de calcification iA isolée sur une échographie de référence, le bilan était ainsi la plupart du temps non invasif. En cas de calcification iA associée, une amniocentèse était systématiquement proposée pour les analyses suivantes : caryotype standard, recherche de CMV et de parvovirus B19 par Polymerase Chain Reaction (PCR), dosage de l'interféron et analyse des enzymes digestives du liquide amniotique. L'Analyse Chromosomique par Puce à ADN (ACPA) était également proposée aux couples à partir de 2013 dans notre centre. Les issues de grossesse et le devenir des enfants ont été collectés à partir des dossiers obstétricaux et pédiatriques pour les données néonatales (examen clinique néonatal, imagerie postnatale, bilan postnatal : CMV urinaire et/ou salivaire, recherche de mucoviscidose sur le test de Guthrie et éventuelle indication chirurgicale). Il n'y avait pas de suivi postnatal spécifique en cas de calcification iA isolée. Cette étude a reçu l'avis favorable du Comité d'Ethique de la Recherche en Obstétrique et Gynécologie (CEROG 2019-OBS-0101).

  Simchen et al qui ont analysé de manière prospective tous les cas de calcifications intra et périhépatiques au CPDPN de Toronto de 1992 à 2001 : sur 61 foetus, seulement 21 (34%) présentaient des calcifications isolées [6]. Le bilan était systématique pour chaque cas et comportait toutes les sérologies maternelles de potentielles foetopathies infectieuses, la recherche des mutations les plus fréquentes de la mucoviscidose chez les parents et une amniocentèse était systématiquement proposée pour la réalisation d'un caryotype et d'une recherche virale par PCR. Concernant leurs 21 cas de calcifications isolées, il est intéressant de noter qu'ils retrouvé un cas d'infection à Parvovirus B19 et un cas de trisomie 21 diagnostiqué en postnatal. Il faut souligner que dans ce cas précis de trisomie 21, l'amniocentèse n'avait pas été souhaitée par les parents et les auteurs n'ont pas précisé le risque combiné pour la trisomie 21. Ce foetus avait pour seul signe échographique une calcification hépatique isolée, diagnostiquée à 17 SA. Au demeurant, aucun cas de mucoviscidose n'a été rapporté et aucune corrélation n'a été mise en évidence entre le nombre de calcifications ou leur localisation et l'association à une infection, une aneuploïdie ou d'autres anomalies. Dans l'étude de Bronshstein et al, des calcifications intra-hépatiques ont été diagnostiquées chez 14 foetus [9].

Figure 2 :

 2 Figure 2 : Aspect échographique des calcifications intra-abdominales isolées (2a-2f) et associées (2g-2i)

Figure 1 :

 1 Figure 1 : Diagramme de flux de l'étude

TORCH : sérologies maternelles pour Toxoplasmose, syphilis, varicelle, PB19, Rubéole, CMV et HSV; QSG : quadrant supérieur gauche; PLA : ponction de liquide amniotique; Toxo : toxoplasmose ; HSV : herpès simplex virus ; CMV : Cytomégalovirus ; PB19 : parvovirus B19 ; 45X0 : monosomie X ; T18 : trisomie 18 ; T21 : trisomie 21 ; T13 : trisomie 13 ; 4p-: syndrome de Wolf-Hirschhorn ; 22q+ : duplication bras long chromosome 22 ; 8p+ : duplication bras court chromosome 8 ; Dvpt Nl : développement normal; neuro: neurologique ; IMG: interruption médicale de grossesse ; MFIU : mort foetale in utero.