Ounay Ishak

Elodie Breton

Karine Choquet

Anne Josset

Paolo Cabras

Jonathan Vappou

Monitoring MR-guided high intensity focused ultrasound therapy using transient supersonic shear wave MR-elastography

Keywords: MR Elastography, Supersonic shear wave, Thermal ablation, HIFU therapy, Therapy monitoring, Interventional MRI

Objective

The aim of the paper is to propose an all-in-one method based on Magnetic Resonance-Supersonic Shear wave Imaging (MR-SSI) and Proton Resonance Frequency Shift (PRFS) to monitor High Intensity Focused Ultrasound (HIFU) thermal ablations.

Approach

Mechanical properties have been shown to be related to tissue damage induced by thermal ablations. Monitoring elasticity in addition to temperature changes may help in ensuring the efficacy and the accuracy of HIFU therapies. For this purpose, an MR-SSI method has been developed where the ultrasonic transducer is used for both mechanical wave generation and thermal ablation. Transient quasi-planar shear waves are generated using the acoustic radiation force, and their propagation is monitored in motion-sensitized phase MR images. Using a singleshot gradient-echo echo-planar-imaging (EPI) sequence, MR images can be acquired at a

Introduction

High Intensity Focused Ultrasound (HIFU) is a non-invasive, non-ionizing thermal ablation method that has shown great potential for cancer treatment, when combined with appropriate and accurate monitoring methods [START_REF] Geoghegan | Methods of monitoring thermal ablation of soft tissue tumors -A comprehensive review Medical Physics[END_REF]. To date, Magnetic Resonance Thermometry (MRT) is considered as the gold standard for non-invasive monitoring of temperature changes during thermal ablations. Proton Resonance Frequency Shift (PRFS) MRT is a relatively tissueindependent temperature estimation method, with the exception of adipose tissue [START_REF] Ishihara | A precise and fast temperature mapping using water proton chemical shift[END_REF][START_REF] Quesson | Magnetic resonance temperature imaging for guidance of thermotherapy[END_REF][START_REF] Rieke | MR thermometry[END_REF]. In practice, temperature is used as an instantaneous biomarker of the ablation, but this information does not directly inform the physician about the structural integrity of the tissue [START_REF] Odéen | Magnetic resonance thermometry and its biological applications -Physical principles and practical considerations Progress in[END_REF]. In theory, thermal dose can provide such information [START_REF] Sapareto | Thermal dose determination in cancer therapy[END_REF], but it has the drawback of being highly sensitive to noise since it relies on the integration of temperature over time. Therefore, systematic errors on temperature will also be integrated in time and thus, may lead to amplified errors on thermal dose estimates. Therefore, complementary biomarkers are needed to ensure permanent feedback on the state of the tissue during and after ablation.

It has been shown that mechanical properties are sensitive to tissue damage caused by heating and thus, can be used as a complementary biomarker for HIFU monitoring [START_REF] Arnal | Monitoring of thermal therapy based on shear modulus changes: II. Shear wave imaging of thermal lesions[END_REF][START_REF] Brosses | Temperature dependence of the shear modulus of soft tissues assessed by ultrasound[END_REF], Corbin et al 2016[START_REF] Chen | Assessment of in vivo laser ablation using MR elastography with an inertial driver: Laser Ablation MRE with Inertial Driver[END_REF][START_REF] Mariani | Real time shear waves elastography monitoring of thermal ablation: in vivo evaluation in pig livers[END_REF][START_REF] Konofagou | Harmonic Motion Imaging (HMI) for Tumor Imaging and Treatment Monitoring CMIR[END_REF]. Elastography monitoring methods were first introduced in ultrasound imaging to assess tissue damage during thermal ablations [START_REF] Arnal | Monitoring of thermal therapy based on shear modulus changes: II. Shear wave imaging of thermal lesions[END_REF][START_REF] Mariani | Real time shear waves elastography monitoring of thermal ablation: in vivo evaluation in pig livers[END_REF]. Magnetic Resonance Elastography (MRE), initially used for diagnostic purposes [START_REF] Lewa | Magnetic Resonance Imaging in the Presence of Mechanical Waves Spectroscopy[END_REF], Muthupillai et al 1996), has also been shown to be applicable for monitoring thermal ablations. MRE has been used for assessing elasticity changes before and after the procedure (Chen et al 2014, Wu et al A c c e p t e d M a n u s c r i p t 2001), as well as during the ablation in real time [START_REF] Corbin | Interventional MR elastography for MRI-guided percutaneous procedures[END_REF][START_REF] Yuan | Preliminary assessment of one-dimensional MR elastography for use in monitoring focused ultrasound therapy[END_REF]. Nonetheless, MRE requires a mechanical exciter for shear wave generation, which significantly complicates the experimental setup of the MR-guided HIFU procedure. From this perspective, Acoustic Radiation Force (ARF)-based harmonic MRE approaches have been proposed where the mechanical excitation is generated using the radiation force of the focused ultrasound itself with no need for supplementary equipment [START_REF] Wu | MR imaging of shear waves generated by focused ultrasound Magnetic Resonance in[END_REF][START_REF] Chatelin | An automatic differentiation-based gradient method for inversion of the shear wave equation in magnetic resonance elastography: specific application in fibrous soft tissues[END_REF], Vappou et al 2018b). Yet, these harmonic MRE approaches are limited because of shear wave near-field effects, making them highly inaccurate in the central zone around the ultrasound focus.

Several transient ARF-based methods have been proposed as an alternative [START_REF] Souchon | Transient MR elastography (t-MRE) using ultrasound radiation force: Theory, safety, and initial experiments in vitro[END_REF][START_REF] Nightingale | Acoustic radiation force impulse imaging: in vivo demonstration of clinical feasibility[END_REF][START_REF] Mcdannold | Magnetic resonance acoustic radiation force imaging: Magnetic resonance acoustic radiation force imaging[END_REF][START_REF] Larrat | MR-guided transcranial brain HIFU in small animal models[END_REF][START_REF] Bour | Real-time monitoring of tissue displacement and temperature changes during MR-guided high intensity focused ultrasound: Monitoring of Changes During MRgHIFU[END_REF][START_REF] De Bever | Simultaneous MR thermometry and acoustic radiation force imaging using interleaved acquisition: Simultaneous MR Thermometry and ARFI[END_REF][START_REF] Hofstetter | Efficient shear wave elastography using transient acoustic radiation force excitations and MR displacement encoding[END_REF][START_REF] Bercoff | Supersonic shear imaging: a new technique for soft tissue elasticity mapping[END_REF][START_REF] Liu | Concurrent Visualization of Acoustic Radiation Force Displacement and Shear Wave Propagation with 7T MRI ed A A Oberai[END_REF], 2017). ARF Imaging (ARFI) is a transient elastography method that was first introduced in ultrasound imaging [START_REF] Nightingale | Acoustic radiation force impulse imaging: in vivo demonstration of clinical feasibility[END_REF]. MR-ARFI was initially proposed for the targeting of MR-guided HIFU, since it allows visualizing the focal spot on the image [START_REF] Mcdannold | Magnetic resonance acoustic radiation force imaging: Magnetic resonance acoustic radiation force imaging[END_REF]Maier 2008, Larrat et al 2010). Several studies have reported the use of the MR-ARFI displacement to monitor thermal ablations, relying on the fact that this displacement is related to the underlying tissue elasticity [START_REF] Auboiroux | ARFI-prepared MRgHIFU in liver: Simultaneous mapping of ARFI-displacement and temperature elevation, using a fast GRE-EPI sequence Magnetic Resonance in[END_REF][START_REF] Bour | Real-time monitoring of tissue displacement and temperature changes during MR-guided high intensity focused ultrasound: Monitoring of Changes During MRgHIFU[END_REF][START_REF] De Bever | Simultaneous MR thermometry and acoustic radiation force imaging using interleaved acquisition: Simultaneous MR Thermometry and ARFI[END_REF]. One limitation of these methods is that MR-ARFI displacement is not only related to local tissue elasticity, but also to the surrounding mechanical environment and to potential changes of tissue acoustic absorption (and hence radiation force) due to temperature. This has led to the development of shear-wave-based methods, as shear wave velocity (c) is quantitatively related to the underlying shear modulus (µ) by:

μ = ρc 2 (1)
A c c e p t e d M a n u s c r i p t with ρ being the density (considered to be equal to 1000 kg.m -3 ) and assuming that the biological tissue is purely linear elastic, isotropic, locally homogeneous, and incompressible [START_REF] Manduca | Magnetic resonance elastography: Non-invasive mapping of tissue elasticity[END_REF]. Hofstetter et al proposed an ARF-based method that consists in applying multiple ARF pushes at several positions to allow for composite shear modulus mapping and real-time elastography monitoring during HIFU procedures [START_REF] Hofstetter | Efficient shear wave elastography using transient acoustic radiation force excitations and MR displacement encoding[END_REF]. An alternative method to map mechanical properties over an extended area is the Supersonic Shear wave Imaging (SSI)

technique proposed by Liu et al [START_REF] Liu | Concurrent Visualization of Acoustic Radiation Force Displacement and Shear Wave Propagation with 7T MRI ed A A Oberai[END_REF][START_REF] Liu | Supersonic transient magnetic resonance elastography for quantitative assessment of tissue elasticity Physics in[END_REF] based on the ultrasonic SSI method [START_REF] Bercoff | Supersonic shear imaging: a new technique for soft tissue elasticity mapping[END_REF]. It consists in generating quasi-planar shear waves in the tissue with ARF and tracking their spatial propagation over time, so as to compute the shear wave velocity map from which an elastogram can be deduced. Although the MR-SSI method was shown to be particularly accurate with high field small animal preclinical setup [START_REF] Liu | Concurrent Visualization of Acoustic Radiation Force Displacement and Shear Wave Propagation with 7T MRI ed A A Oberai[END_REF][START_REF] Liu | Supersonic transient magnetic resonance elastography for quantitative assessment of tissue elasticity Physics in[END_REF], reported acquisition times of several minutes prevent this method from being used in its current form for real-time HIFU monitoring.

The aim of this work is to develop a new HIFU ablation monitoring method capable of providing temperature and elasticity maps simultaneously and in real time, both information being derived from the same MR phase images. In the next section, we present the experimental setup and the developed algorithm for MR-SSI elastography. Then, we expose the results obtained on a calibrated phantom as well as on a gelatin phantom undergoing HIFU heating.

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Methods

Focused ultrasound sequence

As introduced by Bercoff et al, the SSI method is based on the generation of a quasi-planar mechanical wave in the medium [START_REF] Bercoff | Supersonic shear imaging: a new technique for soft tissue elasticity mapping[END_REF]. Thanks to the electronic steering abilities of the multi-element ultrasound transducer, several ARF pushes are generated successively, hence creating multiple quasi-punctual shear wave sources. When the pushes are generated at a sufficiently high speed, these waves interfere and form a quasi-planar shear wave, the propagation of which reflects the mechanical properties of the tissue. Subsequently, the HIFU heating is performed directing the focus a few millimeters away from the SSI push line (Figure 1). This set of pushes constitutes a cycle that will be repeated for each MR image acquisition (i.e., each TR)

during the whole heating procedure. Once the heating completed, SSI pushes alone will be generated to maintain elasticity monitoring during the cooling down period. A quasi-planar supersonic shear wave is generated using 4 successive SSI pushes aligned on a straight line, followed by the HIFU heating located a few millimeters on the side of the SSI push line. Note that THIFU is sensitively longer than Tpush (typically hundreds of ms vs a few ms). This time sequence is repeated in each MR image acquisition, i.e., each TR.

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As the generated mechanical wave is cylindrical, the wavefronts will have different appearances depending on the orientation of the imaging plane (Figure S1 supplementary material). When the imaging plane is parallel to the SSI push line, the shear wave appears as 2 quasi-planar wavefronts propagating in opposite directions. When the imaging plane is perpendicular to the SSI push line, a circular wavefront is observed.

MR-SSI sequence

The displacements induced by the quasi-planar shear waves are encoded with bipolar Motion-Sensitizing Gradients (MSG) added to a single-shot gradient-echo echo-planar-imaging (EPI)

sequence. The bipolar MSG is synchronized with the mechanical excitation using a variable time offset ΔtTRIG; thus, tissue displacement is encoded at different moments of the wave propagation, below designated by "phase offsets" (Figure 2). Knowing the displacement field induced by the shear wave, the wave equation can be used to estimate the shear modulus of the underlying medium. It should be noted that for each ΔtTRIG value, two acquisitions are performed with opposed MSG polarities. The average between these two phase images is used for PRFS thermometry, since they bear opposite motion encoding that null each other, whereas their difference is used for shear wave reconstruction, which doubles the displacement encoding and suppresses the background phase in the phase difference image [START_REF] Corbin | Interventional MR elastography for MRI-guided percutaneous procedures[END_REF], Le et al 2006a, 2006b).

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Post-processing and elastogram reconstruction

Elastograms are reconstructed using an edge detection approach followed by a time-of-flight-based method for the computation of the shear wave velocity [START_REF] Mccracken | Mechanical transient-based magnetic resonance elastography Magnetic Resonance in[END_REF][START_REF] Liu | Supersonic transient magnetic resonance elastography for quantitative assessment of tissue elasticity Physics in[END_REF].

The whole process is implemented using Matlab (The Mathworks Inc., MATLAB R2021a).

Inputs

The processing program is provided with phase difference images acquired for a set of ΔtTRIG values (Figure 3A). Thus, the main input of the process is a 3-dimensional matrix containing displacement images over time (2 spatial dimensions and 1 temporal dimension).

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Preparation of the data and wavefront enhancement

The spatial domain is truncated to a region of interest (ROI) containing shear wavefronts. A Hessian-based multiscale filtering approach is applied to enhance the wavefronts on the images [START_REF] Frangi | Multiscale vessel enhancement filtering Medical Image Computing and Computer-Assisted Intervention -MICCAI[END_REF].

Shear wave velocity estimation on each line of the field of view (FOV)

To estimate the shear wave velocity, the position of each wavefront is tracked across all the propagation lines. In other words, the shear wave velocity is mapped on all the lines that are perpendicular to the wavefront. For each line, a chronogram represents the line in question over the different phase offsets sampling its propagation over time (Figure 3B). An edge detection approach (Canny filter [START_REF] Canny | A Computational Approach to Edge Detection[END_REF])) is implemented to delineate both edges of the wavefront (Figure 3C). These edges are referred to as "inner" and "outer" edges with respect to the SSI push line. For both edges, the arrival time of the wavefront is represented against its distance from the SSI push line. This curve is smoothed with a local regression (Figure 3D), then the local slope is estimated using a simple differential approach (Figure 3E). Finally, the shear wave velocity is locally deduced as it is inversely proportional to the previously estimated slope (Figure 3F).

Elastogram reconstruction

Depending on the imaging slice, the process will be repeated either in Cartesian or radial lines of the image. Once the shear wave velocity is estimated on all the lines of the image, we obtain two distinct maps: one using the inner edges of the wavefront and the other one using the outer edges (Figure 3G). A compound map is then reconstructed from these complementary maps, with a mean value taken on the pixels where both maps overlap (Figure 3H). The shear modulus map is deduced A c c e p t e d M a n u s c r i p t 10 using equation ( 1). Finally, a median filter (4×4 kernel) is applied to attenuate potential outliers (Figure 3I).

Experimental protocol

MRI acquisitions

All MR experiments were performed in a 1.5 T MRI system (MAGNETOM Aera, Siemens Healthcare, Erlangen, Germany) using a combination of Spine and 18-element body matrix coils.

The single-shot gradient-echo EPI sequence was modified for real-time monitoring with MR-SSI.

Relevant parameters for the acquisition were: TR/TE 500/29 ms, FOV 256 mm × 176 mm, slice thickness 5 mm, matrix 128 × 88 (80% phase lines), MSG duration 10 ms at 30 mT/m, bandwidth 1502 Hz/px, flip angle 55°, GRAPPA factor 2.

SSI experimental setup

A 128-element spherical HIFU transducer (1 MHz central frequency, 6 cm radius of curvature, 6 cm in aperture; Imasonic, Voray sur l'Ognon, France) was used to generate SSI pushes and HIFU heating (Figure 1) driven by the MR-compatible HIFU generator (Image Guided Therapy, Pessac, France). The transducer was positioned on the upper surface of the phantom with its focal axis perpendicular to the phantom surface. The acoustic coupling between the transducer and the phantom was realized using a balloon filled with degassed water, and a thin layer of degassed water over the phantom surface.

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Proof-of-concept of MR-SSI in CIRS phantom

Phantom

The first experiment was realized on a commercial elastography phantom (Computerized Imaging Reference Systems, Inc. (CIRS), Norfolk, USA). The phantom contains 4 inclusions of 2 cm in diameter, having each a different shear modulus. The shear modulus values estimated with MR-SSI were compared to those provided by the manufacturer in the datasheet (obtained by static indentation), as well as to the results obtained using conventional MRE.

MR-SSI

The SSI wave was provoked by four pushes respectively at 50, 60, 70 and 78 mm from the top of the transducer active surface. Each push lasted 1.6 ms with an estimated acoustic power of 230 W.

MR-SSI images were acquired in a plane perpendicular to the SSI push line, corresponding to a coronal slice for a patient in supine position. The imaged slice was chosen to cross all 4 inclusions and, at the same time, to pass through the center of the SSI pushes (i.e., at approximately 64 mm from the top of the transducer active surface). At this point of the study, we could not determine yet the number of SSI steps necessary for the shear modulus reconstruction. Therefore, a total of 46 phase offsets were acquired with ΔtTRIG starting from 0 ms to 22.5 ms with increment ΔtSSI = 0.5 ms. The longer ΔtTRIG were chosen so that all 4 inclusions were covered by the propagating shear wave. Table 1 summarizes the relevant temporal parameters used in the experiments.

This experiment had three objectives: (i) to assess the ability to map the 4 inclusions of the CIRS phantom, (ii) to evaluate the repeatability of the MR-SSI process and (iii) to investigate the number of required SSI steps. For the second objective, the acquisition was repeated 8 times. As for the A c c e p t e d M a n u s c r i p t last objective, we decimated the acquired dataset, and identified the minimal number of SSI steps providing similar values to those obtained with the entire dataset.

MRE

The commercial MR Elastography system (Resoundant, Rochester, MN) was set at a mechanical frequency of 150 Hz. Relevant parameters of the MRE gradient-echo sequence were: TR/TE 50/12.4 ms, FOV 300 mm × 300 mm, slice thickness 5.5 mm, matrix 128 × 128, MSG duration 6.7 ms at 28 mT/m, bandwidth 1000 Hz/px, flip angle 20°. The shear modulus was reconstructed using a Local Frequency Estimation algorithm [START_REF] Knutsson | Local multiscale frequency and bandwidth estimation[END_REF][START_REF] Manduca | Local wavelength estimation for magnetic resonance elastography[END_REF] under

the assumption of a purely linear elastic material.

Definition of ROIs

For each inclusion, the shear modulus was averaged within an ROI defined on the magnitude image. The same process was applied to the 8 datasets acquired for MR-SSI as well as to MRE images.

Monitoring of HIFU heating in a gel phantom

Phantom

The second experiment consisted in monitoring a HIFU heating in a gelatin phantom using MR-SSI elastography and PRFS thermometry. The phantom was made of 2 kg of water, 400 g of condensed milk, and 100 g of gelatin. Before refrigeration, the mixture was degassed using a vacuum chamber.

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MR-SSI

Images were acquired in a plane parallel to the SSI push line, corresponding to a transverse slice for a patient in supine position, centered at the ultrasound focus. SSI pushes were generated at depths of 42, 54, 66 and 78 mm from the top of the transducer active surface with a duration of 1.6 ms for each push and an estimated acoustic power of 230 W. The HIFU heating focus was located at a depth of 60 mm from transducer active surface (coinciding with the midpoint of the segment containing the SSI pushes) and 16 mm laterally from the SSI push line. The SSI push line and the HIFU heating focus are both contained in the chosen transverse monitoring plane. The HIFU heating shot was performed during 250 ms (duty cycle 50% compared to 1.3% for the SSI push sequence) at an estimated acoustic power of 92 W.

A pre-heating acquisition was performed to determine the phase offsets needed in order to cover the desired ROI with the spatial extent of the wave propagation. Based on the results of the first experiment and the pre-heating acquisition, a total of 10 SSI steps, ranging from ΔtTRIG = 1.5 ms to 15 ms with increment ΔtSSI = 1.5 ms (Table 1), were acquired for each SSI cycle, allowing the reconstruction of one elastogram. A temporal interpolation of factor 3 was used in the MR-SSI process.

Knowing that 2 images with opposite MSG polarities are acquired for each of the 10 SSI steps, and with a TR set to 500 ms, the elasticity map is updated every 10 s, corresponding to a refresh rate of 0.1 Hz. The refresh rate for thermometry was 1 Hz, because a thermometry map is obtained for each SSI step from averaging the 2 phase images acquired with opposed MSGs.

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The HIFU heating phase lasted 120 s, followed by a cooling down phase of 120 s, in which the HIFU heating shot was not performed while SSI pushes continued to be applied so that both tissue temperature and elasticity were still measured.

Experimental data processing

Phase images were spatially unwrapped prior to SSI computations and temporally unwrapped for thermometry. B0 field drift effects in PRFS were corrected by applying phase subtraction with respect to a non-heated ROI chosen close to the HIFU heating focus. 2). Recall that N: number of SSI steps, Tpush: duration of each of the 4 successive SSI pushes, TMSG: duration of the MSG, ΔtTRIG_1: temporal offset between the SSI pushes and the MSG at the first SSI step, ΔtTRIG_N: : temporal offset between the SSI pushes and the MSG at the last SSI step, ΔtSSI: temporal increment between 2 successive SSI steps, THIFU: duration of the HIFU heating push.

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Results

Validation on CIRS phantom

Circular wavefronts acquired with increasing phase offsets can be seen from left to right in Figure 4 with the corresponding detected edges represented in red (all images are provided in Video 1supplementary material). In the two phase difference images corresponding to longer phase offsets (Figure 4), one can notice that the wave propagates faster on the right side (green arrow), and slower on the left side (blue arrow). These observations are consistent with the location of the rigid and soft inclusions across the imaged slice, to the right and left side, respectively. By applying the SSI reconstruction process, we obtained the shear modulus map presented in Figure 5A (obtained with one of the 8 acquired datasets). Mean shear moduli in each ROI are given in Table 2 in kPa with standard deviations as percentages. Note that the values shown for MR-SSI represent the average of the 8 datasets (at each cell, are given the average of the 8 obtained shear moduli and the average of the 8 standard deviations).

The MR-SSI computed shear modulus of the background was 6.1 kPa (compared to 5.9 kPa from the datasheet). For inclusions I-IV, the shear moduli were measured to 4.5, 4.8, 7.3 and 11.5 kPa respectively. In comparison with the CIRS datasheet, MR-SSI provided higher values for soft inclusions (types I and II) and lower values for rigid ones (types III and IV). A better consistency was found between SSI and MRE results, with a maximum difference of 1.3 kPa between these two methods. In terms of homogeneity, the shear moduli estimated with MR-SSI have standard deviations of 15%, 12%, 22%, 22% and 17% for inclusions I-IV and the background respectively. Figure 6 represents the boxplots obtained from the 8 shear modulus maps acquired on the CIRS phantom (using the full datasets i.e., without decimation). The standard deviations between the 8 results are of 4%, 9%, 6%, 11% and 2% for inclusions I-IV and the background respectively which shows the high repeatability of our process.

In order to increase the temporal resolution of MR-SSI for real-time monitoring, we tested whether the number of SSI steps could be reduced while maintaining sufficient information. It was found that with a decimation of factor 3 (i.e. keeping 1 out of each 3 SSI steps) followed by an interpolation of the same factor (to compensate the missing phase offsets), the elasticity map (Figure 5B) and the mean shear moduli (Table 2, last line) remained close to those obtained with the full dataset. The maximum difference between the two datasets is equal to 0.1 kPa. ΔtSSI was therefore set to 1.5 ms (3×0.5 ms) for the monitoring experiment.

Monitoring of HIFU heating in a gel phantom

Figure 7 presents the SSI wavefronts before and at the end of the HIFU heating. It can be noticed that at the end of the heating, the wavefront on the right side did not go as far as it had gone before Page 18 of 30 AUTHOR SUBMITTED MANUSCRIPT -PMB-113856.R2

A c c e p t e d M a n u s c r i p t the heating (red vertical line in Figure 7) suggesting that planar shear waves slow down over time (Video 2supplementary material). This decrease of the shear wave velocity was expected because the gel softens during heating. This is consistent with the elasticity maps obtained with MR-SSI where the shear modulus is found to decrease on the right side, at the same position where the temperature increased during the HIFU heating (Video 3supplementary material). Figure 8 presents elasticity and temperature changes averaged within a 5×3-pixel ROI selected around the heating focus on the thermometry map. As expected, temperature and shear modulus remained nearly constant during the whole experiment on the left side (symmetrical ROI with regards to the SSI push line). As for the right side, the temperature increase ΔT reached 9 °C while the shear modulus µ decreased from 2.9 kPa ± 5% to 1.8 kPa ± 17% between the beginning and

Discussion

In this paper, an MRI protocol and a reconstruction process have been proposed for the MR-SSI elastography method for the monitoring of elasticity and temperature changes during HIFU ablation. The proposed reconstruction process relies on two main steps: (i) shear wavefront tracking using an edge detection approach and (ii) shear wave velocity mapping based on a timeof-flight method. This elastography technique was first validated on a calibrated phantom where inclusions with different shear moduli could be mapped with high repeatability. Second, the HIFU heating experiment emphasized the ability to map in real time the irreversible elasticity changes caused by HIFU in a gel phantom. The shear modulus decrease at the heating focus was monitored on elasticity maps refreshed every 10 s. The shear modulus remained steady during the whole cooling down phase, which emphasizes the relevance of mechanical properties as biomarkers of thermal damage.

As presented previously, our MR-SSI elastogram reconstruction process is based on a time-offlight computation, a principle also proposed by [START_REF] Mccracken | Mechanical transient-based magnetic resonance elastography Magnetic Resonance in[END_REF]Liu et al (McCracken et al 2005, Liu et al 2017) for shear wave velocity estimation. However, instead of extracting the maximum peak on the wave profiles, we chose to detect both the inner and outer edges of the wavefront. By doing so, the information is duplicated and 2 distinct elastograms can be obtained which allows averaging the shear modulus on the area where both maps overlap and so, reduce the number of potential outliers in this region.

Several limitations of the proposed method need to be highlighted. First, elasticity estimation may be subject to significant averaging effects. This is mainly explained by the fact that spatial resolution of the SSI elastograms is entirely dependent on the temporal resolution of SSI images.

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In other words, the more SSI steps are acquired, the better the resolution of the computed elasticity maps is. However, the number of SSI steps should be maintained reasonably low to ensure a high temporal update rate of elasticity. Thus, it is necessary to find an acceptable compromise between temporal and spatial resolutions of the estimated elastograms. In addition, operations such as interpolation and smoothing have supplementary averaging effects on the final elasticity values. Second, several phenomena may limit the spatial coverage and the accuracy of the obtained elastograms. As for spatial coverage, elasticity cannot be mapped neither near the SSI push line because of shear wave near-field effects, nor too far from this same line because of wave attenuation, which makes the wavefront detection more difficult. As for accuracy, guided wave phenomena can lead to inaccuracy of shear wave velocity estimates at interfaces between media with different mechanical properties. These limitations could justify the differences between the MR-SSI results and the values provided in the CIRS datasheet. Indeed, the previously mentioned averaging effects may partially explain why the shear moduli obtained for the inclusions softer than the background were higher than expected, and inversely for the rigid inclusions. It is also important to note that elasticity values reported by the manufacturer have been obtained by mechanical indentation, a static mechanical testing method that could also account for some of the observed differences. All these reasons may explain the better consistency between MR-SSI and conventional MRE as they are both dynamic elastography approaches, based on shear wave propagation. This was further highlighted by the repeatability study performed on the CIRS which shows that MR-SSI and MRE results are in good agreement. Although its accuracy needs to be further evaluated, particularly in heterogeneous media for the aforementioned reasons, MR-SSI was shown to be a repeatable method providing a quantitative biomarker of structural damage induced by heating.

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A key parameter of the SSI method is the temporal step ΔtSSI that must be determined with attention. Indeed, the higher the rigidity of the tissue, the smaller the ΔtSSI should be to ensure a sufficient temporal resolution within an SSI cycle. Therefore, preliminary acquisitions are required to obtain an estimation of the expected tissue elasticity, since this will influence the value of the SSI step ΔtSSI that will be chosen. In practice, a pre-ablation MR-SSI acquisition may be performed so the user can estimate the range and number of SSI phase offsets that will provide adequate spatial coverage of the propagating wave for the following monitoring acquisitions. The results presented in this paper are limited since they do not include in vitro or in vivo tissues. Therefore, further investigation is necessary to find the optimal range of SSI phase offsets as well as the ΔtSSI step as they will depend on the elasticity of the tissue as well as its heterogeneities.

In vitro or in vivo tissue experiments would allow reaching much higher temperatures such as those used clinically during HIFU ablations. Gelatin is not a suitable model for high temperature experiments as it melts, thus preventing the generation and propagation of shear waves. In practice, higher temperatures can be achieved by increasing the acoustic power of the HIFU heating push (which was set at only 40% of the maximal power of the transducer for the heating experiment on gelatin) and/or its duty cycle (set to 50% here). It should be noted that higher duty cycles can be achieved for the HIFU heating push. This duty cycle could be further increased through the use of a real-time industrial version of the communication software between the HIFU generator and the MRI console, or through the use of longer TR, though at the expense of the SSI temporal resolution.

Several approaches have already been proposed for MR-guided HIFU monitoring using the mechanical properties of the tissue. As opposed to MRE, MR-SSI does not require an additional mechanical exciter since the ARF is used to generate the pushes. Nonetheless, a potential risk of A c c e p t e d M a n u s c r i p t this method could be to induce some thermal effects along the SSI push line. This is mainly avoided by the short duration as well as the low duty cycle of the pushes. The mild heating observed along the SSI push line in the second experiment (+6 °C maximum) does not entail any change in the phantom structure, and it is deemed to be lower in a perfused organ. Furthermore, this heating along the SSI push line was mainly observed in experiments where a HIFU heating was performed simultaneously with the MR-SSI elastography measurements. No significant heating was observed in MR-SSI experiments without HIFU heating phase. This could be explained by the fact that the HIFU wave delivers a certain amount of energy along the whole ultrasound cone. Although this energy is mostly absorbed at the focus, the summation of energy on the areas where several ultrasound cones are superimposed may induce a mild temperature increase (especially in the presence of a long HIFU heating push, and in the absence of perfusion). This is visible in Video 3, as mild heating occurs along the SSI push line only during the HIFU heating phase, followed by a plateau during the cooling down phase despite the fact that the SSI pushes were still generated.

When comparing to other ARF-based approaches, MR-SSI is distinguished by its ability to provide an extended spatial mapping of elasticity whereas other methods such as MR-ARFI estimate a single value at the focus (Vappou et al 2018). The method proposed by Hofstetter et al [START_REF] Hofstetter | Efficient shear wave elastography using transient acoustic radiation force excitations and MR displacement encoding[END_REF] also allows for spatial elasticity mapping. It relies on the generation of multiple pushes in a plane perpendicular to the ultrasound propagation axis, allowing reconstructing elasticity maps every 12 seconds. Our method instead relies on the generation of quasi-cylindrical supersonic shear waves, which, in theory, are more contrasted and less attenuated than spherical waves. This may explain the larger spatial extent of our MR-SSI method in a coronal slice (a circular field of view of about 13 cm in diameter on the CIRS phantom) compared to the method of Hofstetter et al where elasticity has been measured on a circular field of view of 5 cm in diameter. Another A c c e p t e d M a n u s c r i p t relevant point of comparison is the applicability of our MR-SSI method in different imaging planes as the composite shear wave used by Hofstetter et al makes the method only applicable in coronal slices. Nonetheless, this latter was also demonstrated in 3D providing elasticity measurements over several slices with an acquisition time of 144 seconds. Finally, both approaches allow mapping elasticity in 2D with similar refresh rates compatible with real-time monitoring.

Being equivalently applicable in different imaging planes adds a supplementary degree of freedom to MR-SSI. A transverse imaging slice has been chosen for the HIFU heating experiment as the HIFU focus has an ellipsoidal shape of about 1 cm in length and 1-2 mm in diameter. Thus, mapping the elasticity in a transverse plane is much more relevant in order to detect the variations along the length of the ellipsoidal lesion. Therefore, the possibility to choose the imaging plane according to the shape and size of the expected tissue heterogeneities is an advantage of the proposed method.

Although theoretically, the MR-SSI method can be used with non-EPI sequences, the need for high temporal resolution virtually imposes the use of single-shot EPI acquisitions, with all related artifacts and limitations. Single-shot EPI with longer TR also offers optimal duty cycle balance between the expected low duty cycle at the SSI push line (limited heating), and the high duty cycle necessary at the HIFU ablation spot.

A c c e p t e d M a n u s c r i p t Conclusion

The proposed MR-SSI method allows simultaneous temperature and elasticity mapping. This method was first evaluated on a heterogeneous phantom and was then used for the monitoring of HIFU ablations, with MR-SSI elastograms updated every 10 seconds and relative temperature maps updated every second. In the case of a gelatin phantom, the shear modulus was found to decrease during the HIFU heating, thus reflecting that MR-SSI can be a promising technique to assess the structural changes of tissue during a HIFU treatment.

A c c e p t e d M a n u s c r i p t

Figure 1 -

 1 Figure 1 -Operating sequence of the focused ultrasound transducer. A quasi-planar supersonic shear wave is

Figure 2 -

 2 Figure 2 -Illustration of the acquired images at different SSI steps. By varying the offset ΔtTRIG between the SSI pushes and the encoding gradient (MSG), different phases of the propagating shear wave can be captured. Note that for simplicity, only one of the two MSG polarities has been represented. In practice, one wavefront image is obtained from the difference between two phase images acquired with the same ΔtTRIG but with opposed MSG polarities.

Figure 3 -

 3 Figure 3 -MR-SSI reconstruction process. Illustration in the case of a circularly propagating SSI wavefront, i.e. the imaging plane is parallel to the SSI push line. (A) For each radial line (or Cartesian line in the case of linearly propagating SSI wavefronts, i.e., the imaging plane is perpendicular to the SSI push line), (B) a chronogram is built

Figure 4 -

 4 Figure 4 -Wavefronts observed at different SSI steps on the CIRS phantom. Phase difference images for increasing phase offsets (ΔtTRIG) between SSI push and MSG phase encoding. Red circles represent the detected inner and outer edges of the wavefronts. Arrows on the last frame show the regions where the wave propagated faster (green) and slower (blue) due to the presence of inclusions with different shear moduli.

Figure 5 -

 5 Figure 5 -Shear modulus maps obtained on the CIRS phantom. (A) using the entire dataset and (B) the decimated dataset (decimation 1/3 then interpolation ×3). Black circles represent the selected ROIs in the inclusions. Inclusions are referred to as type I, II, III and IV (from left to right) by the manufacturer.

  moduli for the 4 inclusions and the background of the CIRS phantom, obtained from the datasheet, MRE 150 Hz, MR-SSI with the entire datasets (phase offset 0 to 22.5 ms with 0.5 ms step) and MR-SSI with the decimated datasets (1/3 followed by interpolation ×3). Note that the values shown for MR-SSI correspond to the average of the 8 datasets ± the average of the 8 standard deviations in the ROI.

Figure 6 -

 6 Figure 6 -Boxplot of the shear moduli obtained with the MR-SSI process in the CIRS phantom (8 datasets)

Figure 7 -

 7 Figure 7 -Shear wavefront, thermometry and elastography maps before and after the HIFU heating. (A) The red dotted line indicates the inner edge of the right-side wavefront at the beginning of the heating. At t = 120 s, the wavefront on the right side is slowed down, suggesting local gel softening as a result of the HIFU heating. Temperature increase and shear modulus decrease occurring on the heating side can be seen on ΔT (B) and Δµ (C) maps.

  the heating. While the temperature started decreasing once the HIFU heating stopped, the shear modulus remained steady at 1.8 kPa during the following 120 s.

Figure 8 -

 8 Figure 8 -Changes in temperature and shear modulus averaged within a 5×3-pixel ROI on the left side (control) and on the right side (HIFU heating) of the SSI push line. Error bars represent the standard deviation of temperature over the selected ROI. On the right-side, heating the gelatin results in significant softening, whereas no significant changes are observed on the left side (control).

Table 1 -Relevant temporal parameters of ultrasound and MRI sequences (as defined in Figure

 1

Table 2 -

 2 Shear

Acknowledgements

This work has benefitted from funding of the FUI (Fonds Unique Interministériel, BPI France) for the UFOGUIDE project, and the ANR (Agence Nationale de la Recherche) French national program "Investissements d'Avenir" for the LABEX-CAMI (ANR-11-LABX-004) and the TechnoFUS joint laboratory project (ANR-21-LCV3-0007-01), and the IHU Strasbourg (Institute of Image Guided Surgery, ANR-10-IAHU-02).


Svenja Specovius

Hanna G Zimmermann

Frederike Cosima Oertel

Claudia Chien

Charlotte Bereuter

Lawrence J Cook

Marco Lana Peixoto

Mariana Andrade Fontenelle

Ho Jin Kim

Jae-Won Hyun

Marco Aurélio

Lana Peixoto

Su-Kyung Jung

Jacqueline Palace

Adriana Roca-Fernandez

Alejandro Rubio Diaz

Maria Isabel Leite

Srilakshmi M Sharma

Fereshte Ashtari

Rahele Kafieh

Alireza Dehghani

Mohsen Pourazizi

Lekha Pandit

Anitha Dcunha

Orhan Aktas

Marius Ringelstein

Philipp Albrecht

Eugene May

Caryl Tongco

Letizia Leocani

Marco Pisa

Marta Radaelli

Elena H Martinez-Lapiscina

Hadas Stiebel-Kalish

Mark Hellmann

Itay Lotan

Sasitorn Siritho

Jérôme De Seze

Thomas Senger

Joachim Havla

Romain Marignier

Caroline Tilikete

Alvaro Cobo Calvo

Denis Bernardi Bichuetti

Ivan Maynart Tavares
email: tavares@imaynart

Nasrin Asgari

Kerstin Soelberg

Ayse Altintas

Rengin Yildirim

Uygur Tanriverdi

Anu Jacob

Saif Huda

Zoe Rimler

Allyson Reid

Yang Mao-Draayer

Ibis Soto De Castillo

Michael R Yeaman

Terry J Smith

Alexander U Brandt

Friedemann Paul
email: friedemann.paul@charite.de

Cohort profile: a collaborative multicentre study of retinal optical coherence tomography in 539 patients with neuromyelitis optica spectrum disorders (CROCTINO)

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

Future plans We are pursuing several scientific projects based on the repository, such as analysing retinal layer thickness measurements, in this cohort in an attempt to identify differences between distinct disease phenotypes, demographics and ethnicities. The dataset will be available for further projects to interested, qualified parties, such as those using specialised image analysis or artificial intelligence applications.

Strengths and limitations of this study

► The Collaborative OCT in neuromyelitis optical spectrum disorders repository cohort comprises the largest number of retinal optical coherence tomography (OCT) images from patients with a neuromyelitis spectrum disorder, a rare autoimmune disease of the central nervous system. ► Besides imaging data, information on clinical and functional scores as well as laboratory parameters were assessed. ► We collect OCT images as original files, allowing for a detailed quality reading and standardised, deviceindependent OCT analysis. ► Despite standardised image analysis, the image heterogeneity due to different OCT machines and scan protocols remains a challenge.

INTRODUCTION

Neuromyelitis optical spectrum disorders (NMOSD) are rare autoimmune neuroinflammatory diseases spanning a broad age range. They are clinically characterised by recurrent attacks of optic neuritis (ON), myelitis and less frequently by the brainstem and cerebral attacks, and profoundly impact patients' quality of life. [START_REF] Jarius | Neuromyelitis optica: clinical features, immunopathogenesis and treatment[END_REF][START_REF] Sepulveda | Late-onset neuromyelitis optica spectrum disorder: the importance of autoantibody serostatus[END_REF][START_REF] Beekman | Neuromyelitis optica spectrum disorder: patient experience and quality of life[END_REF][START_REF] Souza | Diagnostic procedures in suspected attacks in patients with neuromyelitis optica spectrum disorders: results of an international survey[END_REF] The current concepts of NMOSD are rapidly changing. An international panel of experts published the latest NMOSD diagnostic criteria in 2015. [START_REF] Wingerchuk | International consensus diagnostic criteria for neuromyelitis optica spectrum disorders[END_REF] Pathogenic serum autoantibodies against aquaporin-4 (AQP4-IgG), an astrocytic water channel protein, can be detected in 60%-80% of patients with NMOSD. 6 7 The diagnostic criteria differentiate patients with positive or negative/unknown AQP4-IgG status. In the latter case, strict rules apply to the clinical presentation and paraclinical findings, in particular those from MRI for diagnosing NMOSD. 5 8 In some AQP4-IgG seronegative patients within the NMO disease spectrum, for example with isolated recurrent ON or myelitis, serum antibodies against myelin oligodendrocyte glycoprotein (MOG) can be detected. [START_REF] Kim | Antibodies to MOG in adults with inflammatory demyelinating disease of the CNS[END_REF][START_REF] Chalmoukou | Anti-MOG antibodies are frequently associated with steroid-sensitive recurrent optic neuritis[END_REF][START_REF] Narayan | Mog antibody disease: a review of MOG antibody seropositive neuromyelitis optica spectrum disorder[END_REF][START_REF] Waters | Mog cell-based assay detects non-MS patients with inflammatory neurologic disease[END_REF] As a different cellular target is involved, most experts consider MOG-IgG autoimmunity, or MOG-IgG-associated encephalomyelitis, as a separate disease entity, pathogenetically distinct from classic AQP4-IgG-associated NMOSD. [START_REF] Zamvil | Does MOG Ig-positive AQP4-seronegative opticospinal inflammatory disease justify a diagnosis of NMO spectrum disorder?[END_REF][START_REF] Jarius | MOG encephalomyelitis: international recommendations on diagnosis and antibody testing[END_REF][START_REF] Kwon | Peripherally derived macrophages as major phagocytes in MOG encephalomyelitis[END_REF] The term myelin oligodendrocyte glycoprotein antibody disease has recently been proposed for this disorder. [START_REF] Wynford-Thomas | Neurological update: MOG antibody disease[END_REF] ON is one of the most common clinical manifestation of NMOSD. It frequently results in severe structural optic nerve damage and visual impairment, often occurs bilaterally, with common relapses. 3 17 18 Patients often develop a visual disability as a result of decreased high-contrast visual acuity (HCVA) and low-contrast visual acuity, as well as colour and visual field defects. These functional limitations result in impaired vision-related quality of life and a high incidence of legal blindness. [START_REF] Schneider | Optical coherence tomography reveals distinct patterns of retinal damage in neuromyelitis optica and multiple sclerosis[END_REF][START_REF] Outteryck | A comparative optical coherence tomography study in neuromyelitis optica spectrum disorder and multiple sclerosis[END_REF][START_REF] Schmidt | Severe structural and functional visual system damage leads to profound loss of vision-related quality of life in patients with neuromyelitis optica spectrum disorders[END_REF] Optical coherence tomography (OCT) acquires highresolution retinal images and plays an important role in assessing ON-associated damage in NMOSD [START_REF] Oertel | Optical coherence tomography in neuromyelitis optica spectrum disorders: potential advantages for individualized monitoring of progression and therapy[END_REF] and other neuroinflammatory disorders associated with ON. 23 24 After transection of optic nerve axons following acute ON, retrograde neurodegeneration leads to neuroaxonal damage in the retina. [START_REF] Brandt | Frequent retinal ganglion cell damage after acute optic neuritis[END_REF] Retinal post-inflammatory neuroaxonal degeneration typically progresses for about 6 months after the onset of acute idiopathic ON. It can be assessed by OCT as peripapillary retinal nerve fibre layer thickness (pRNFL) or ganglion and inner plexiform layer thickness (GCIP). [START_REF] Costello | The temporal evolution of structural and functional measures after acute optic neuritis[END_REF] Due to the rarity of NMOSD, adequate disease-specific studies on temporal ON dynamics are scant. [START_REF] Stiebel-Kalish | Does time equal vision in the acute treatment of a cohort of AQP4 and MOG optic neuritis?[END_REF] Thus, it remains uncertain which time frame accurately reflects the disease. For example, in a recent population-based study of all acute ON in Southern Denmark, there was not a single AQP4-IgG seropositive case and only two with MOG-IgG in the 50 patients presenting with de novo acute ON during study duration. [START_REF] Soelberg | Optical coherence tomography in acute optic neuritis: a population-based study[END_REF] In MOG-IgG seropositive patients, ON appears to be milder than in patients with detectable AQP4-IgG [START_REF] Akaishi | MRI and retinal abnormalities in isolated optic neuritis with myelin oligodendrocyte glycoprotein and aquaporin-4 antibodies: a comparative study[END_REF] and leads to a better outcome despite equally severe retinal thinning. [START_REF] Sotirchos | Aquaporin-4 IgG seropositivity is associated with worse visual outcomes after optic neuritis than MOG-IgG seropositivity and multiple sclerosis, independent of macular ganglion cell layer thinning[END_REF] However, MOG-IgG seropositive patients display a higher relapse frequency, potentially leading to comparable cumulative retinal damage and loss of vision in the two subgroups. 31 32 Furthermore, MOG-IgG seropositive patients have a more pronounced pRNFL thinning in the temporal quadrant, while temporal and nasal quadrants are equally affected in AQP4-IgG positive disease. 19 33 It is currently unclear, to what extent the retina and vision are affected by NMOSD independently of ON. [START_REF] Oertel | Optical coherence tomography in neuromyelitis optica spectrum disorders: potential advantages for individualized monitoring of progression and therapy[END_REF] In multiple sclerosis (MS), diseaseassociated and progressive neurodegeneration can occur in eyes unaffected by ON. 34 35 Studies of NMOSD have led to conflicting results. ON in NMOSD tends to be more posterior and closer to the chiasm than that occurring in MS, and chiasmal affection could lead to carry-over effects in the less affected companion eye. 36 37 However, recently described microstructural changes in the retina also suggest a primary retinopathy in NMOSD. [START_REF] Oertel | Microstructural visual system changes in AQP4-antibody-seropositive NMOSD[END_REF][START_REF] Jeong | Subclinical primary retinal pathology in neuromyelitis optica spectrum disorder[END_REF][START_REF] Yamamura | Foveal thinning in neuromyelitis optica: a sign of retinal astrocytopathy?[END_REF][START_REF] Motamedi | Altered fovea in AQP4-IgG-seropositive neuromyelitis optica spectrum disorders[END_REF] This is potentially mediated by AQP4-expressing retinal Müller cells and could be independent of ON. Longitudinal data demonstrating progressive GCIP loss in AQP4-IgG positive patients with NMOSD independent of ON further supports this notion. 42 43 These observations remain to be independently confirmed. In contrast, a recent exploratory longitudinal study in MOG-IgG seropositive patients suggested only progressive RNFL loss but not longitudinal GCIP reduction. [START_REF] Oertel | Optical coherence tomography in myelin-oligodendrocyte-glycoprotein antibody-seropositive patients: a longitudinal study[END_REF] Besides neuroaxonal damage, macroscopic retinal findings have been reported in NMOSD. Macular microcysts occur in the inner nuclear layer and are associated with severe ON in approximately 20% of patients with NMOSD. [START_REF] Kaufhold | Optic neuritis is associated with inner nuclear layer thickening and microcystic macular edema independently of multiple sclerosis[END_REF][START_REF] Sotirchos | In vivo identification of morphologic retinal abnormalities in neuromyelitis optica[END_REF][START_REF] Gelfand | Microcystic inner nuclear layer abnormalities and neuromyelitis optica[END_REF] Pathology and clinical significance of macular microcysts, sometimes called microcystic macular oedema, remain unclear; however, macular processes appear to occur more frequently than other processes such as vitreous traction, which was found in one MOG-IgG positive patient with microcysts. [START_REF] Brandt | Dynamic formation of macular microcysts independent of vitreous traction changes[END_REF] Investigating OCT for clinically and pathologically meaningful information is hampered by the rareness and heterogeneity of NMOSD. [START_REF] Mori | Worldwide prevalence of neuromyelitis optica spectrum disorders[END_REF] In 2015, the Guthy Jackson Charitable Foundation International Clinical Consortium (GJCF-ICC) agreed to establish an image repository platform (neuromyelitis optical imaging repository (NOIR)), the purpose of which is to facilitate multinational and multicentre neuroimaging studies in NMOSD. NOIR is intended to help identify imaging pitfalls in NMOSD, develop and clinically validate imaging biomarkers of the disease, clinical disability and to define imaging endpoints for clinical trials. Here we report the outcome of the Collaborative OCT in NMOSD repository study (CROCTINO) as the initial effort in establishing a platform for investigating retinal abnormalities using OCT in NMOSD.

COHORT DESCRIPTION Study design

The study was designed as a multinational and multicentre repository study collecting longitudinal OCT data as well Open access as relevant clinical data from patients with NMOSD and healthy controls (HCs). Participating centres were asked to contribute both retrospective and prospective data that was collected over a defined period extending from 2000 to 2018. Scientific coordination and OCT reading were performed at the Charité-Universitätsmedizin Berlin Translational Neuroimaging Group. Participating centres were mainly recruited from the GJCF-ICC, which includes international researchers and clinicians focusing on NMOSD. Additional experts who had previously published studies using OCT in NMOSD but who were not members of the GJCF-ICC were contacted and invited to participate. For this purpose, a questionnaire screening each centre for the number of eligible patients and the type of OCT instruments used at their site was sent to the entire GJCF-ICC and to other identified experts. Centres giving a positive response to the recruitment questionnaire received further instructions on how to contribute OCT images and the associated demographical and clinical data of their patients and HCs. The overall study design-including the workflowis illustrated in figure 1.

Inclusion and exclusion criteria

Subjects were included with (a) a diagnosis of NMOSD as specified by the 2015 International Panel criteria 5 or (b) longitudinal extensive transverse myelitis and/or (c) recurrent ON or (d) HCs (without matching requirements). OCT source data, demographic and clinical metadata from patients and HCs were required, including age, sex, disease subtype, autoantibody status and clinical history including details of ON.

Data collection and workflow

All demographic and clinical data were assessed in an electronic repository based on Research Electronic Data Capture (REDCap), [START_REF] Harris | Research electronic data capture (REDCap)-a metadata-driven methodology and workflow process for providing translational research informatics support[END_REF] located at the University of Utah Data Coordination Center. OCT images were transferred by participating centres via TeamBeam (Skalio GmbH, Hamburg, Germany), a commercial web-based medical data exchange service certified for secure data transfer. OCT images were then stored and analysed using a secure server at Charité-Universitätsmedizin Berlin, Berlin, Germany.

Study preparation began in January 2016 by identifying potential participating centres, setting up the information technology (IT) infrastructure and developing the electronic case report forms (eCRFs) in REDCap. eCRFs were sent to all participants for review and were revised accordingly. The data collection was launched on 1 September 2016, when the final repository was released, and centres received REDCap accounts for data entry. Data collection officially closed on 30 September 2018. After a detailed plausibility check, identified missing data could be submitted until 31 December 2018. A detailed timeline is illustrated in figure 2.

Collected dataset

The data elements that were collected are listed in online supplemental file 1 (including indications of which data elements were mandatory or optional) and are summarised as follows:

Demographic and clinical data

In addition to age and sex, ethnicity was assessed based on published NIH categories (https:// grants. nih. gov/ grants/ guide/ notice-files/ not-od-15-089. html). Possible selections comprised 'American Indian or Alaska Native', 'Asian', 'Black or African American', 'Hispanic or Latino', 'Native Hawaiian or Other Pacific Islander', 'White' and 'Other'; up to two selections were possible. Height and weight were collected. Information concerning the presence of ophthalmological comorbidities other than those related to NMOSD was requested. The possible answers were: 'Unknown', 'No (excluded by examination)', 'No (excluded by history taking)' and 'Yes'. In case of 'Yes', the condition had to be specified. Furthermore,

Optical coherence tomography

Scans from all common OCT machines were accepted, resulting in scans from three different types of machines: (a) Spectralis SD-OCT (Heidelberg Engineering, Heidelberg, Germany), (b) Cirrus HD-OCT (Carl Zeiss Meditec, Dublin, California, USA) and (c) Topcon 3D OCT-1 (Topcon, Tokyo, Japan). We requested that centres upload two scan types per eye from each visit:

► A peripapillary ring scan with 12° or approximately 3.4 mm diameter around the optic disc. For those using Cirrus and Topcon, an optic disc volume scan was uploaded since peripapillary data are extracted from this scan. ► A macular volume scan, centred on the fovea, with a minimum size of 20°×20° (approximately 6 mm×6 mm). There were no restrictions regarding OCT image averaging or quality. At least one scan per visit was required to be included in the repository. After images were transferred, their quality was assessed based on modified OSCAR-IB criteria. 53 54 Modifications were made for better applicability to macular scans (see online supplemental file 2). pRNFL segmentation was performed with device-internal algorithms, intraretinal layer segmentation with a device-independent segmentation pipeline. [START_REF] Motamedi | Normative data and minimally detectable change for inner retinal layer thicknesses using a semi-automated OCT image segmentation pipeline[END_REF] Visual function HCVA was requested with a selection menu ranging from 20/10 (-0.3 logMAR) to 'no light perception'. Information on the method of testing was not requested, but details of whether testing was performed best corrected, habitually corrected or uncorrected were recorded. Monocular HCVA was mandatory while binocular HCVA was optional. Additionally, low contrast letter acuity (LCLA) from 2.5% contrast Sloan charts, normal/ abnormal classifications of visual evoked potentials (VEP) P100 latencies and mean deficit and pattern standard deviation from visual fields could be entered as optional information.

Statistical analysis

Cohort and statistical description for this cohort profile were performed with R V.3.4.4 [START_REF] Core | R: A language and environment for statistical computing[END_REF] in R Markdown and RStudio (RStudio, Boston, Massachusetts, USA).

Open access

Cohort overview

Centres from North and South America, Asia and Europe participated in CROCTINO. In total, data from 539 patients from 22 centres and 114 HCs from 5 centres were collected. Centres and the size of their patient and HC cohorts are listed alphabetically in table 1.

In total, we collected 1868 peripapillary ring scans/ optic nerve volume scans and 1672 macular volume scans fulfilling defined specifications. Only scans with corresponding clinical data were considered. Device and scan specifications are depicted in table 2.

A cohort characterisation is depicted in table 3. The mean patient age (mean±SD) was 43.1±14.8 years with 444 (82%) women. The age of HCs (mean±SD) was 32.1±9.8 years with 72 (63%) women. The majority of patients were White or Middle Eastern (n=315), followed by Asian (n=128) and Black or African American (n=27).

Among all patients, 515 (95.5%) fulfilled the 2015 diagnostic criteria for NMOSD. [START_REF] Wingerchuk | International consensus diagnostic criteria for neuromyelitis optica spectrum disorders[END_REF] Of those not meeting the 2015 criteria, 21 (3.9%) carried the diagnosis of LETM, and 3 (0.6%) recurrent ON. Among those fulfilling NMOSD diagnostic criteria, 369 (72%) were AQP4-IgG seropositive, leaving 146 (28%) with negative or unknown AQP4-IgG status. Among the AQP4-IgG seronegative patients, 54 (37%) were MOG-IgG seropositive, 34 (23%) were double-negative for AQP4-IgG and MOG-IgG and 58 (40%) had an unknown antibody status. In the entire cohort, 369 (68%) were AQP4-IgG seropositive, 54 (10%) MOG-IgG seropositive, 52 (10%) had double negative and 64 (12%) had an unknown antibody status. There were 400 (74%) patients with at least one episode of ON, and 410 (76%) patients with a history of myelitis. The mean time since disease onset (mean±SD) was 5.47±19.47 years and mean age at initial symptom onset (mean±SD) was 36.18±15.05 years. Information about current N,N-Dimethyltryptamine (DMT) was available in 398 (74%) patients. Ninety-seven patients (18.0%) received a combination of multiple DMTs. Most frequent DMT options comprised rituximab (n=128; 24%), azathioprine (n=121; 22%), oral prednisolone (n=98; 18%), mycophenolate mofetil (n=77; 14%) and methotrexate (n=11; 2%).

Open access

The majority of the centres provided OCT source data from a Spectralis machine (17 centres), followed by Cirrus (6 centres) and one centre contributed Topcon OCT images (table 1).

HCVA was available for 529 (98%) patients and 83 (73%) HCs. Most of the patients received best-corrected HCVA (47%), 28% were habitually corrected and 25% not corrected. LCLA was obtained from 99 (18%) patients and 40 (35%) HCs. VEP were available for 177 (33%), and visual fields for 90 (17%) patients applying devices at the discretion of each centre.

Longitudinal data were available for 157 (29%) patients from 11 centres. Mean follow-up time (mean±SD) was 27±17 months.

Patient and public involvement

Patients and the Public were not explicitly involved in the design or conduct of this study. However, the results of this study will be presented on a Guthy-Jackson Charitable Foundation's International NMO Patient Day, giving patients, relatives and researchers the opportunity to share ideas for projects based on this cohort.

Findings to date

To our knowledge, the CROCTINO repository comprises the largest dataset on retinal OCT in NMOSD. Cohort demographics are in line with previous epidemiological NMOSD studies: gender ratio, age at symptom onset and prevalence of AQP4-IgG and MOG-IgG antibodies are similar to previously published studies and suggest a cohort that is representative of the disease spectrum. [START_REF] Pandit | Demographic and clinical features of neuromyelitis optica: a review[END_REF][START_REF] Jarius | Contrasting disease patterns in seropositive and seronegative neuromyelitis optica: a multicentre study of 175 patients[END_REF][START_REF] Borisow | Influence of female sex and fertile age on neuromyelitis optica spectrum disorders[END_REF] OCT quality and quantitative analyses are currently being investigated as first scientific derivatives of the work and will be published in the near future. During the study, many challenges in conducting an investigator-driven academic repository study were experienced. First, this study was implemented without centre reimbursement. While we are unaware of specific guidelines in this regard, we felt that data quality would potentially be higher when centres receive no reimbursement. Although without reimbursement we expected a lower motivation for interested centres to participate at all, we reckoned that data quality might be higher from participating centres contributing out of sole scientific interest. Despite the absence of financial compensation, centre motivation was excellent, and we were able to include substantially more datasets (n=539) than initially anticipated (n=200). Lack of reimbursement did preclude the participation of some centres, particularly in North America. Sharing of data requires legal compliance, including the conclusion of data transfer agreements (DTA). Although we provided an initial DTA template for this study, almost all centres required modifications, the consequence of different legal systems in this multinational setting. This process resulted in a substantially delayed data collection. On the other hand, the DTA templates for each of the involved centres in combination with concluded DTA for this repository now provide a powerful platform and network for future studies in NMOSD.

During the conduct of this study, we developed several technical solutions, which will be made available beyond the scope of this repository. First, the technical infrastructure and newly established workflows will be instrumental in conducting future neuroimaging studies in NMOSD. We further developed a device-independent intraretinal layer segmentation pipeline based on an established segmentation algorithm. [START_REF] Lang | Retinal layer segmentation of macular OCT images using boundary classification[END_REF] This pipeline is capable of importing and segmenting OCT scans from all devices included in the study and will presumably lead to better comparability of OCT data obtained from different devices. [START_REF] Motamedi | Normative data and minimally detectable change for inner retinal layer thicknesses using a semi-automated OCT image segmentation pipeline[END_REF] Finally, we modified the widely used OSCAR-IB criteria [START_REF] Tewarie | The OSCAR-IB consensus criteria for retinal OCT quality assessment[END_REF] for assessing OCT image quality to be applicable to macular volume scans and all OCT devices. The modified criteria will be validated using data from our repository. The study has several limitations, many of which are inherent to multicentre settings. First, the inclusion of retrospective data precluded application of a standardised, homogeneous imaging protocol. Heterogeneity in the dataset is further caused by (a) use of different OCT machines, (b) different scan protocols and (c) no homogeneous visual function measures. Several geographic regions are not represented in the repository (eg, Australia, Africa), and HC scans are not matched demographically and are only available for some ethnicities and from certain regions. Although great care was taken in collecting, completing and verifying the data, we could not ensure complete individual data set validity. For example, different methods for antibody testing [START_REF] Waters | Multicentre comparison of a diagnostic assay: aquaporin-4 antibodies in neuromyelitis optica[END_REF] with often uncertain sensitivity and specificity and a non-standardised documentation of patient-reported information could lead to some noise in respective analyses.

Collaboration and future directions

The study coordinating centre is pursuing several scientific projects based on the repository in collaboration with the participating centres, such as the analysis of pRNFL and intramacular layer thickness measurements in this cohort in an attempt to identify differences between distinct disease phenotypes, demographics and ethnicities. We encourage collaboration with interested researchers in additional scientific projects. The CROCTINO dataset will be made available to all participating centres and to qualified investigators on request (details see below). The study coordinators are also interested in further expanding the repository by including (a) additional data from currently underrepresented geographic regions, (b) additional HC data to provide expanded ethnicity comparators and (c) more prospective, longitudinal data, also extending already existing follow-up periods of the included patients. The established NOIR platform and the CROCTINO cohort will serve to investigate retinal imaging biomarkers in NMOSD in the future.

Collaboration and future directions The study coordinating centre is pursuing several scientific projects based on the repository in collaboration with the participating centres, such as the analysis of pRNFL and intramacular layer thickness measurements in this cohort in an attempt to identify differences between distinct disease phenotypes, demographics and ethnicities. We encourage collaboration with interested researchers in additional scientific projects. The CROCTINO dataset will be made available to all participating centres and to qualified investigators upon request (details see below). The study coordinators are also interested in further expanding the repository by including (a) additional data from currently underrepresented geographic regions, (b) additional healthy control data to provide expanded ethnicity comparators and (c) more prospective, longitudinal data, also extending already existing follow-up periods of the included patients. The established NOIR platform and the CROCTINO cohort will serve to investigate retinal imaging biomarkers in NMOSD in the future.

  Protected by copyright. on January 10, 2023 at Universite Louis Pasteur Strasbourg. http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2019-035397 on 29 October 2020. Downloaded from

  Open access the presence of other comorbidities was assessed. Information about NMOSD diagnosis was mandatory, with possible selections of 'NMO (2006 criteria)', 'AQP4-IgG seropositive NMOSD (2015 criteria)', 'AQP4-IgG seronegative NMOSD (2015 criteria)', 'MOG-IgG-associated encephalomyelitis/NMOSD', 'RON/CRION (Recurrent Optic Neuritis)', 'LETM (Longitudinal Extensive Transverse Myelitis)'. We inquired about the AQP4 and MOG antibody statuses separately (both mandatory) with possible answers 'Seropositive', 'Currently seronegative, but at least one previous test was positive', 'Seronegative', 'Not known/Never assessed'. Patients were considered to be seropositive for AQP4-IgG or MOG-IgG if antibodies were detected in at least one assay. Centres were also asked which assay they used for antibody testing. Provision of the Expanded Disability Status Scale (EDSS) score was optional.[START_REF] Kurtzke | Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS)[END_REF] History of clinical attacks was requested with a focus on ON. Information concerning each eye was recorded separately as was the number of ON attacks and date of last episode. History of other NMOSD defining attacks, such as transverse myelitis, area postrema syndrome, brainstem syndromes, symptomatic narcolepsy or acute diencephalic clinical syndrome with NMOSDtypical diencephalic MRI lesions, and symptomatic cerebral syndrome with NMOSD-typical brain lesions was requested without dates and with the option 'unknown'. Immunotherapy was assessed with a selection of common NMOSD treatments[START_REF] Trebst | Update on the diagnosis and treatment of neuromyelitis optica: recommendations of the neuromyelitis optica Study Group (NEMOS)[END_REF] and possible selections 'Current' and 'Previous use'.

Figure 2

 2 Figure2Detailed study timeline including all study phases with start and end dates, as well as a brief description of each phase. eCRFs, electronic case report forms; OCT, optical coherence tomography.

Table 1

 1 Participating centres with number of patients, number of HCs and used OCT device

	Centre	Patients HC OCT device
	Bangkok, Thailand	25	0 Cirrus
	Barcelona, Spain	13	13 Spectralis or Cirrus
	Belo Horizonte, Brazil 57	0 Spectralis
	Berlin, Germany	76	39 Spectralis
	Duesseldorf, Germany 11	28 Spectralis
	Goyang-si, Korea	50	0 Topcon OCT
	Isfahan, Iran	40	18 Spectralis
	Istanbul, Turkey	8	0 Cirrus
	Liverpool, UK	8	0 Spectralis
	Lyon, France	10	0 Spectralis
	Mangalore, India	40	16 Spectralis
	Maracaibo, Venezuela 3	0 Spectralis
	Michigan, USA	5	0 Spectralis
	Milan, Italy	30	0 Spectralis
	Munich, Germany	11	0 Spectralis
	New York, USA	6	0 Spectralis and Cirrus
	Odense, Denmark	9	0 Spectralis
	Oxford, UK	48	0 Spectralis
	Petah-Tikva, Israel	25	0 Cirrus
	Sao Paulo, Brazil	9	0 Spectralis
	Seattle, USA	30	0 Cirrus
	Strasbourg, France	25	0 Spectralis

HC, healthy control; OCT, optical coherence tomography.

Table 2

 2 Technical and scan specifications

	Protected by copyright.	http://bmjopen.bmj.com/
	Protocol* Spectralis Mac-1 3.9 62 Axial resolution (µm) Acquisition (A-scans/s) n 40 000 62 651 61 B-scans A-scans per B-scan Size (mm) † 768 6×6 Spectralis Mac-2 240 25 512 6×6 Spectralis Mac-3 225 25 1024 7.5×9 Spectralis Ring-1 1143 1 1536 3.4 Spectralis Ring-2 226 1 768 3.5 Cirrus Mac 5.0 63 27 000 63 293 128 512 6×6 Cirrus Ring 388 200 200 6×6/3.4 ‡ Topcon Mac 6.0 64 50 000 64 263 128 512 6×6 Topcon Ring 111 128 512 6×6/3.4 ‡	on January 10, 2023 at Universite Louis Pasteur Strasbourg.

*Only protocols making up more than 5% of the total macular scan rates were considered. †Scan size can vary as it depends on the eye length. ‡Peripapillary ring scan extracted from optic nerve head volume scan. Mac, macular volume.

Table 3

 3 Cohort description

			Healthy
			controls Patients
	Subjects (n)		114	539
	Centres (n)		5	22
	Age (years; mean±SD)	32.1±9.8 43.1±14.8
	Sex (woman; n (%))	72 (63.2) 444 (82.4)
	Time since disease onset (years;	-	5.5±19.5
	mean±SD)
	Age at initial symptom onset	-	36.2±15.1
	(years; mean±SD)
	Ethnicity	White or Middle	96 (84.2) 315 (58.4)
	(n (%))	Eastern
		Asian	17 (14.9) 128 (23.7)
		Black or African	0 (0)	27 (5.0)
		American
		Hispanic or Latino 1 (0.9)	11 (2.0)
		Other	0 (0)	23 (4.3)
		Not reported	0 (0)	35 (6.5)
	Patients fulfilling the 2015	-	515 (95.5)
	diagnostic criteria for NMOSD
	(fulfilled; n (%))
	AQP4-IgG seropositive NMOSD	-	369 (68.5)
	(n (%))
	MOG-IgG seropositive NMOSD	-	54 (10.0)
	(n (%))
	Double-negative NMOSD (n (%)) -	34 (6.3)
	NMOSD with unknown antibody-	-	58 (11.0)
	status (n (%))
	Patients with a history of optic	-	400 (74.2)
	neuritis (n (%))
	Patients with a history of myelitis	-	410 (76.1)
	(n (%))
	AQP4-IgG, aquaporin-4 IgG antibodies; MOG-IgG, myelin
	oligodendrocyte glycoprotein IgG antibodies; NMOSD,
	neuromyelitis optica spectrum disorders.

Specovius S, et al. BMJ Open 2020;10:e035397. doi:10.1136/bmjopen-2019-035397

Protected by copyright. on January 10, 2023 at Universite Louis Pasteur Strasbourg. http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2019-035397 on 29 October 2020. Downloaded from

Funding The creation of this repository was supported by a grant from the Guthy Jackson Charitable Foundation.

Patient consent for publication Not required.

Provenance and peer review Not commissioned; externally peer reviewed.

Data availability statement All data collected will be electronically stored in a repository and can be accessed by all participants for research purposes upon request. GJCF-ICC and the principal investigators of the repository will coordinate projects and access. Investigators not involved in CROCTINO data collection who desire access to the data should approach the corresponding author or the GJCF-ICC for access. Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.


Chloé Cerutti

Sophie Leroux

Paul Terzian

Joanna Lledo

David Gourichon

Rémy-Félix Serre

Céline Vandecasteele

Christophe C Klopp

Carole Iampietro

Christine Gaspin

Transgenerational epigenetics in quail

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

-Methods

The influence of the prenatal environment on the adult phenotype development is partially mediated by epigenetic phenomena. Recently, an increasing number of studies highlighted the transmission of epigenetic marks between generations following an environmental exposure. However, there is much debate about their acquired transmission beyond the exposed individuals. Recent studies revealed that non-genetic inheritance was probably present in avian species. In one of them, fertilized eggs were injected with an endocrine disruptor (genistein) and after three generations without any other injection, several traits were impacted by the ancestor treatment such as the reproduction and the behavior.

Here we analyse the DNA methylation between control and treated lines of the third generation.

-Results

Thousands

1

  GenPhySE, Université de Toulouse, INRAE, ENVT, 31320 Castanet-Tolosan, France 2 MIAT, PF Bioinfo GenoToul, Université de Toulouse, INRAE, Chemin de Borde Rouge, 31320 Castanet-Tolosan, France 3 GeT-PlaGe -Génome et Transcriptome -Plateforme Génomique, GET -INRAE, 31320 Castanet-Tolosan, France 4 INRAE, PEAT, 37380 Nouzilly, France Contact : chloe.cerutti@inrae.fr Chloé Cerutti¹, Sophie Leroux¹, Paul Terzian², Joanna Lledo³, David Gourichon⁴, Rémy-Félix Serre³, Céline Vandecasteele³,

  of DMC detected between epilines Identification of the biological mechanisms involved Exclude the genetic causes for the observed differences between epilines Transgenerational effects of genistein on the DNA methylation Optimising SNP filtering and minimising false positives: SNP calling with GemBS for WGBS data Gene Ontology (GO) analysis to be finalised: found in ONT (from 8,974,565 CpG in quail assembly) Carole Iampietro³, Christine Gaspin², Denis Milan¹, Cécile Donnadieu³, Frédérique Pitel¹, Guillaume Devailly¹