V H Prevost

O M Girard

S Mchinda

G Varma

D C Alsop

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

Optimization of inhomogeneous Magnetization Transfer (ihMT) MRI contrast for preclinical studies using dipolar relaxation time (T 1D ) filtering

Keywords: Inhomogeneous magnetization transfer, ihMT, dipolar order, dipolar relaxation time, inhomogeneous Magnetization Transfer / inhomogeneous Magnetization Transfer ratio

Background and Introduction: A pulsed ihMT-prepared fast imaging sequence was implemented at 11.75T for preclinical studies on mouse central nervous system. A strategy based on filtering the ihMT signal originating from short dipolar relaxation time (T 1D ) components is proposed. It consists in increasing the repetition time of consecutive RF pulses of the dual saturation and allowed improving signal specificity for long T 1D myelinated structures. Furthermore, frequency offset, power and timing saturation parameters were adjusted to optimize the ihMT sensitivity.

Results: Optimization of the ihMT sensitivity while preserving strong specificity for the long T 1D component of myelinated tissues allowed measurements of ihMT ratios on the order of 4-5% in white matter (WM), 2.5% in grey matter (GM) and 1-1.3% in muscle. This led to high relative ihMT contrasts between myelinated tissues and others (~3-4 between WM and Muscle, and 2 between GM and Muscle). Conversely, higher ihMT ratios (~6-7% in WM) could be obtained using minimal T 1D filtering achieved with short saturation pulse repetition time or cosine-modulated pulses for the dual frequency saturation.

Conclusion:

This study represents a first stage in the process of validating ihMT as a myelin biomarker by providing optimized ihMT preclinical sequences, directly transposable and applicable to other preclinical magnetic fields and scanners. Finally, ihMT ratios measured in various central nervous system areas are provided for future reference.

Introduction

Inhomogeneous Magnetization Transfer (ihMT) is a new endogenous contrast mechanism that has shown great promise for Central Nervous System (CNS) imaging. This technique is based on magnetization transfer (MT), and reveals an interesting characteristic of dipolar-broadened macromolecular lines [START_REF] Varma | Magnetization transfer from inhomogeneously broadened lines: A potential marker for myelin[END_REF]. Human experiments have demonstrated strong capacities of ihMT in discriminating myelinated tissues from others [START_REF] Varma | Magnetization transfer from inhomogeneously broadened lines: A potential marker for myelin[END_REF][START_REF] Girard | Magnetization transfer from inhomogeneously broadened lines (ihMT): Improved imaging strategy for spinal cord applications: ihMT 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], with the hypothesis that the lipid membranes, highly present in myelin (4), and which have an inhomogeneously broadened spectrum [START_REF] Davis | High resolution 1H nuclear magnetic resonance of a transmembrane peptide[END_REF][START_REF] Chen | High-resolution MAS NMR spectroscopy detection of the spin magnetization exchange by cross-relaxation and chemical exchange in intact cell lines and human tissue specimens[END_REF], strongly contribute to the ihMT effect. However, the hypothesis of ihMT being a myelin biomarker needs to be further explored and validated. With this perspective, preclinical ihMT MRI applied on well-characterized and established animal models of myelin disorders (demyelination or dysmyelination) will have an important role to play. Potentially being similar to what qMT experiments offer with the macromolecular proton fraction: a truly quantitative approach for myelin-sensitive imaging [START_REF] Dula | Multiexponential T2, magnetization transfer, and quantitative histology in white matter tracts of rat spinal cord[END_REF][START_REF] Ou | The MT pool size ratio and the DTI radial diffusivity may reflect the myelination in shiverer and control mice[END_REF][START_REF] Ou | Quantitative magnetization transfer measured pool-size ratio reflects optic nerve myelin content in ex vivo mice[END_REF][START_REF] Rausch | Characterization of white matter damage in animal models of multiple sclerosis by magnetization transfer ratio and quantitative mapping of the apparent bound proton fraction f[END_REF][START_REF] Samsonov | Quantitative MR imaging of two-pool magnetization transfer model parameters in myelin mutant shaking pup[END_REF][START_REF] Turati | In vivo quantitative magnetization transfer imaging correlates with histology during de-and remyelination in cuprizone-treated mice: QUANTITATIVE MAGNETIZATION TRANSFER IMAGING IN CUPRIZONE-TREATED MICE[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]. A prerequisite to this validation stage is to derive a robust, contrast-optimized and sensitive technique for preclinical ihMT investigations; this constitutes the core of the current work.

The ihMT signal corresponds to the difference in saturation transfer between single frequency-offset and dual frequency-offset saturation performed with the same RF power. A theoretical model for ihMT has been proposed with the inclusion of a dipolar reservoir in the existing two-pool model for MT, which allowed interpreting the inhomogeneous MT signal as a dipolar order effect within motion restricted molecules [START_REF] Varma | Interpretation of magnetization transfer from inhomogeneously broadened lines (ihMT) in tissues as a dipolar order effect within motion restricted molecules[END_REF]. The name inhomogeneous MT was intended to reflect the non-uniform saturation of the spectrum of a line when single frequency off-resonance power is applied. This terminology has been questioned [START_REF] Manning | The physical mechanism of "inhomogeneous" magnetization transfer MRI[END_REF] broadening of dipolar systems induced by external factors [START_REF] Portis | Electronic structure of F centers: Saturation of the electron spin resonance[END_REF], particularly those permitting hole burning in the spectrum, and separately certain quantum properties of the system under magic angle spinning [START_REF] Maricq | NMR in rotating solids[END_REF]. In contrast, the basic underlying physical model of the ihMT effect, which relies on dipolar order theory [START_REF] Varma | Interpretation of magnetization transfer from inhomogeneously broadened lines (ihMT) in tissues as a dipolar order effect within motion restricted molecules[END_REF], is generally accepted [START_REF] Manning | The physical mechanism of "inhomogeneous" magnetization transfer MRI[END_REF][START_REF] Swanson | Molecular, dynamic, and structural origin of inhomogeneous magnetization transfer in lipid membranes: Origin of ihMT Contrast[END_REF]. For lack of an obviously more specific name and for consistency with the prior literature on ihMT, we retain the name inhomogeneous MT and use the word inhomogeneous to reflect lines whose spectrum can be non-uniformly saturated with single-offset RF.

Dipolar order may in principle be encountered in various tissue structures, providing that averaging is slowed down by motional restriction. Dipolar order is characterized by a dipolar relaxation time, T 1D, and its associated fraction, f within the bound pool. In the framework of ihMT, the finite lifetime of the dipolar order, i.e. T 1D , indicates the duration of the homogenization process within the line. Long T 1D values associated with a large fraction of the semi-solid pool are responsible for the highest ihMT ratio (ihMTR) obtained in myelinated WM structures. It is however possible to reveal ihMT signals in very short T 1D tissues when using high saturation power levels. This property has not been fully appreciated in our previous human studies [START_REF] Varma | Magnetization transfer from inhomogeneously broadened lines: A potential marker for myelin[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] because these were focused on areas of interest located in the brain and relatively low power intensities were used, as imposed by regulatory energy deposition limitations. Conversely, preliminary studies performed on mice using high power levels have evidenced a non-negligible ihMT signal in muscle [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] and in vitro studies also revealed non-zero ihMT signal in short T 1D components such as wood or hair [START_REF] Manning | The physical mechanism of "inhomogeneous" magnetization transfer MRI[END_REF]. In this context it is appropriate to envision ihMT as a T 1D -weighted imaging modality, for which various contrast regimes and tissue sensitivity could apply.

In this study, we introduced the concept of T 1D -filtered ihMT imaging and used it to optimize the contrast of preclinical ihMT images, obtained on mice at 11.75T using a 2D pulsed ihMT fast imaging sequence [START_REF] Varma | Magnetization transfer from inhomogeneously broadened lines: A potential marker for myelin[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]. The dual saturation scheme was adjusted to vary the T 1D weighting in order to remove the ihMT signal contribution from a certain range of low T 1D s while maintaining signal from the longer T 1D s components. This T 1D -filtering concept was first illustrated in vitro on phantoms and further used in vivo to attenuate the ihMT signal of the shorter T 1D muscle components, while preserving high signal from long-T 1D myelinated structures, overall enhancing the contrast of ihMT for these particular structures. Optimization of the ihMT sensitivity was achieved by adjustment of the saturation frequency offset and the other RF timing and power parameters. Finally, the resulting optimized T 1D -filtered sequence was used to establish a reference database of ihMTR values in normal mouse CNS along with relative ihMT contrast values between various structures.

Material & Methods

Animal experiments

Animal studies were conducted in agreement with the European Council Directive 2010/63/EU, the French guidelines for animal care from the French Department of Agriculture (Animal Rights Division), and approved by our institutional committee on Ethics in animal research. After isoflurane induction in an anesthetic chamber at 3%, healthy female C57Bl/6J mice (10-20 weeks, 23±2g) were maintained under anaesthesia by spontaneous respiration of a mixture of air and isoflurane (1.5%, constant flow 300mL/min, Univentor 400 anesthesia unit, Zejtun, Malta), through a dedicated nose cone. Respiration and temperature were controlled throughout the experiment with a MR compatible monitoring and gating system (SA Instruments, Stony Brook, NY). A heating blanket connected to a water bath system was placed on the mice's back to maintain physiological temperature throughout the whole experiments (T=37.0 0.5°C).

P

MR setup

In vivo experiments were performed on a preclinical 11.75T scanner (Bruker Avance 500 MHz/89 mm wide bore vertical imager, Ettlingen, Germany) with a transmit/receive volume birdcage coil (Bruker, Ettlingen, Germany; length L=30 mm, diameter Ø=20 mm). Prior to ihMT data acquisition, first and second order shimming was achieved using the Bruker Fastmap procedure in a 11.5x11.5x11.5 mm 3 3D volume covering the entire mouse brain. B 0 maps were then acquired using the Bruker FieldMap procedure, based on the acquisition of a 3D double gradient echo dataset (TE 1 /TE 2 =1.5/5.5ms, matrix 96x64x64, FOV 30x20x20 mm 3 ).

Pulsed ihMT sequence

The ihMT signal generation relies on the comparison of two different MT weighted images (1): one using a single frequency-offset (+∆f), leading to the saturated image MT + , and the other using a dual frequency-offset (+∆f and -∆f) using identical total energy, leading to the saturated image MT +-. In order to compensate for MT asymmetry effects, two additional single (MT -) and dual (MT -+ ) frequency-offset acquisitions are acquired so that the final ihMT image is generated by the following combination:

!ℎ#$ = #$ & + #$ ( -#$ ± -#$ ∓ Equation 1
At ultra-high magnetic field strength, efficient correction of MT-asymmetry signal contributions also requires the frequency offsets ∆f to be applied relative to a frequency center, fc, slightly shifted to fc=-100Hz from the free water resonance frequency [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]. The ihMT ratio is defined by ihMTR = ihMT/S 0 , where S 0 is the signal measured with RF saturation power set to zero.

MR imaging

A 2D single-slice single-shot fast imaging readout (RARE -rapid acquisition with relaxation enhancement) was used following the pulsed ihMT preparation with the imaging parameters: were also evaluated for specificity considerations.

Principle of T 1D -filtered ihMT imaging

By comparing a single frequency-offset MT image, whose expression contains a term related to T 1D (Eq. 10 of ( 14)) with a dual frequency-offset MT image, whose expression is free of any T 1D contribution (Eq. 6 of ( 14)), the dipolar contribution is isolated from traditional MT and is revealed in the ihMT signal combination. In other words, dipolar order effects associated to non-trivial T 1D components (i.e. T 1D ≠ 0) are created with single offset RF saturation, whereas they are ideally constrained to zero with a perfect dual offset RF saturation. Sensitivity of the ihMT signal to T 1D s can hence be adjusted by modulating the efficiency of the dual frequency-offset saturation. This latter may be performed in two different ways (1): 1) using a cosine-modulated envelope for the shaped MT pulses, which corresponds to a simultaneous saturation at both frequency bands (Fig 1b ); and 2) using a frequency alternated pulsed approach for which each frequency offset is excited sequentially (Fig 1c

In vivo experiments

Optimizing the ihMT contrast with T 1D -filtering imaging

Values of T 1D in mouse tissues were estimated with the technique outlined in [START_REF] Varma | In vivo measurement of a new source of contrast, the dipolar relaxation time, T 1 D , using a modified inhomogeneous magnetization transfer (ihMT) sequence: In Vivo Measurement of T 1D Using ihMT[END_REF] to

T 1D =6.1±0.

Reference ihMTR measurements in mouse CNS

Results

Principle of T 1D -filtered ihMT imaging

In vivo optimization of the ihMT contrast with T 1D -filtering imaging

In vivo T 1D -filtered ihMT imaging is highlighted in Figure 3, which shows a mouse brain T 1D

Optimizing the ihMT sensitivity

The sensitivity of ihMT as a function of ∆f, PW, ∆t, B 1,RMS and τ is reported in Figs. 4-5. Variation of ihMTR with frequency offset in myelinated tissue provided bell-shaped curves with maximum values obtained at different ∆f values depending on the brain tissue (Fig 4a-c).

For a saturation power of B

Discussion

This study aimed at optimizing the contrast and the sensitivity of ihMT in the context of preclinical investigations of the mouse CNS using T 1D -filtering strategy in combination with adapted saturation parameters. (axons lying mainly parallel to B 0 ) compared to that in CC resulted in similar ihMTR values for ∆f=20kHz, further suggesting that above this frequency offset, inversion will occur and ihMTR values will be higher in CC. Previous MT experiments also reported a dependence of qMT derived parameters (e.g. T 2B -T 2 of bound protons) with white matter anisotropy [START_REF] Pampel | Orientation dependence of magnetization transfer parameters in human white matter[END_REF][START_REF] Yarnykh | Fast macromolecular proton fraction mapping from a single off-resonance magnetization transfer measurement[END_REF].

Hence, the results obtained here warrant further investigations in order to interpret the white matter anisotropy effect on ihMT and evaluate to what extent it is a potential confound for ihMT quantification.

Absolute ihMTR values in mouse brain and spinal cord WM measured in this study were ~1.5-2 times smaller than those reported in humans at 1.5T in similar structures [START_REF] Girard | Magnetization transfer from inhomogeneously broadened lines (ihMT): Improved imaging strategy for spinal cord applications: ihMT 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].

The low resolution used in this study, and consequential partial volume effects, might have led to underestimated values, thus explaining, at least in part, these differences. Given that identical ihMT MR sequences were used for humans and animal studies and that, slightly higher ihMTR values would be expected at higher magnetic field strength due to longer T1s (Fig. S1), the reasons for lower ihMTR values could be related to physiological differences between species. The mouse WM dipolar relaxation time T 1D is only slightly shorter than that of human (T 1D ~6.1ms vs T 1D ~6.3ms) whilst the measured fraction f of the bound pool with dipolar order is almost halved [START_REF] Varma | In vivo measurement of a new source of contrast, the dipolar relaxation time, T 1 D , using a modified inhomogeneous magnetization transfer (ihMT) sequence: In Vivo Measurement of T 1D Using ihMT[END_REF]. This suggests differences in the composition and/or the structure of the WM compounds to which ihMT is sensitive. The rich-lipid composition (70% lipids, 30% proteins) [START_REF] O'brien | Lipid composition of the normal human brain: gray matter, white matter, and myelin[END_REF] and unique structure (molecular lipid-bilayer membrane tightly wrapped around axons) of myelin, which constitutes 40-50% of white matter, is thought to favor the ihMT effect. Similarities in the composition of myelin main lipids were found between mammalian species with a few exceptions (4). Thus, whereas both human myelin and mouse myelin have similar cholesterol content (~27% of total myelin lipids) and comparable galactolipids (cerebroside and sulfatide, ~27% of total lipids), differences in phospholipid content (~45% of total lipids) were noticed. In particular, mouse myelin has 2-3 times less sphingomyelin but more phosphoglycerides than human myelin [START_REF] Baumann | Lipid composition of quaking mouse myelin: comparison with normal mouse myelin in the adult and during development[END_REF][START_REF] Hogan | Composition of cerebral lipids in murine leucodystrophy: the quaking mutant[END_REF][START_REF] Horrocks | Composition of mouse brain myelin during development[END_REF].

Sphingomyelin and other sphingolipids (cerebrosides, sulfatides) mainly contain long-chain (>19 carbon atoms) saturated fatty acids [START_REF] O'brien | Fatty acid and fatty aldehyde composition of the major brain lipids in normal human gray matter, white matter, and myelin[END_REF], which contribute to a closely packed, highly stable membrane structure. In contrast, numerous mid-chain (<19 carbon atoms) unsaturated fatty acids present in phosphoglycerides contribute to a more loosely packed, less stable structure due to weaker binding interactions between neighboring molecules [START_REF] Lodish | Section 5.3, Biomembranes: Structural Organization and Basic Functions[END_REF]. Less sphingomyelin and more phosphoglycerides may lead to higher fluidity properties of the mouse myelin structure, which could favor the mechanisms of spectral homogenization of the corresponding broadened lines and thus reduce ihMT effects. Another major difference between human and mouse experiments are the use of anaesthetics, which may induce changes in myelin structure [START_REF] Mateu | The action of local anesthetics on myelin structure and nerve conduction in toad sciatic nerve[END_REF], and hence affect the ihMT signal. Performing ihMT phantom experiments on lipid components of myelin [START_REF] Kucharczyk | Relaxivity and magnetization transfer of white matter lipids at MR imaging: importance of cerebrosides and pH[END_REF] and in vivo on animal models with deficiency of myelin lipids [START_REF] Baumann | Lipid composition of quaking mouse myelin: comparison with normal mouse myelin in the adult and during development[END_REF][START_REF] Hogan | Composition of cerebral lipids in murine leucodystrophy: the quaking mutant[END_REF] would help in validating/invalidating these hypotheses.

Although this study did not address the source of ihMT contrast, it confirms that most components with non-trivial T 1D can be revealed by the ihMT technique [START_REF] Varma | Interpretation of magnetization transfer from inhomogeneously broadened lines (ihMT) in tissues as a dipolar order effect within motion restricted molecules[END_REF][START_REF] Manning | The physical mechanism of "inhomogeneous" magnetization transfer MRI[END_REF], provided that the saturation parameters are appropriately chosen. In the original ihMT paper (1), with an experimental design slightly different than that of this study, we concluded that dual saturation achieved by cosine-modulated pulses or alternated pulses approach yield similar metrics. However the present study demonstrated that both approaches are essentially different, addressing different tissue components, and measuring potentially different metrics.

Hence, simultaneous dual frequency-offset saturation approach by means of cosine-modulated pulses provided significant ihMT signal in short T 1D components such as muscle (T 1D ~1.6ms), suggesting that this strategy could enable in vivo studies of ihMT in tissues other than those found in CNS. Conversely, alternating pulses for dual saturation enables T 1Dfiltered ihMT imaging, which can be exploited to enhance the contrast between tissues with quantitative pair of parameters T 1D , f (which requires acquisitions at different frequency offsets and power levels ( 14) and potentially dual frequency switching times) may not be required for practical characterization of CNS tissues with ihMT. For that purpose, the use of short ∆t values or in the limiting case of the cosine modulated approach, would have the advantage of providing higher ihMTR and CNR values (Figs. 3b,d). However, one should notice that this would sensitize the ihMT images to shorter T 1D value components, thereby potentially modifying the source of contrast as compared to T 1D -filtered conditions. Signs of this effect can be seen with the use of CM for dual saturation. The lower relative WM/GM contrast value (~1.8 v.s 2 for alternated pulses) seems to indicate that short T 1D components participate more to the GM ihMT signal compared to that of WM. Overall this opens the questions of the specificity of ihMT signal and its relationship with myelin. Further studies will address this question by looking for correlations between relative ihMTR contrasts values of brain structures [START_REF] Kuo | In vivo measurements of T1 relaxation times in mouse brain associated with different modes of systemic administration of manganese chloride[END_REF]. Contrast-enhanced strategies by injection of gadolinium or manganese have been proposed for improving the mapping of myelin-rich WM structures using MT MRI at high magnetic field [START_REF] Watanabe | Myelin mapping in the central nervous system of living mice using contrast-enhanced magnetization transfer MRI[END_REF][START_REF] Watanabe | Myelin mapping in the living mouse brain using manganeseenhanced magnetization transfer MRI[END_REF]. Quantitative maps of the bound pool fraction extracted from multi-parametric qMT methods provide similar brain tissue contrast to that of ihMT, but at the cost of time-consuming experiments and/or more sophisticated postprocessing [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][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]. From a practical point of view and compared to qMT experiments, the implementation of ihMT described in this study suffers from low time efficiency, low spatial resolution and limited volume coverage. However strategies for 3D ihMT imaging have already been developed for humans [START_REF] Varma | 3D Acquisition of the Inhomogeneous Magnetization Transfer Effect for Greater White Matter Contrast[END_REF][START_REF] Girard | Whole Brain inhomogeneous MT using an ihMT prepared 3D GRE sequence at 1.5T[END_REF] and can be duplicated for preclinical investigations. The ihMT contrast is preserved at high magnetic field as its underlying mechanisms are related to dipolar order effects within bound molecules, which do not scale directly with B 0. Hence, the relationship between ihMTR and the saturation parameters is mostly independent of field strength, even though a slight increase of absolute ihMTR values can be observed when magnetic field increases due to T 1 increase of free water protons (Fig. S1). Since preclinical scanners typically operate in a closer range of magnetic field strengths (7T<B 0 <11.75T), differences in absolute ihMTR related to magnetic field strengths should be reduced, overall making the contrast/sensitivity optimization results of the current study highly reproducible, and directly transposable and applicable to other preclinical magnetic fields and scanners. Finally, a practical advantage of ihMT and its pronounced contrast features could be its potential for automatic brain structure extraction. For example, with the parameter set corresponding to Fig. 6, a 3.5-threshold of ihMTR values would mask all structures except WM ones. Such potential may further facilitate automatic whole-brain measurements and eliminate time-consuming manual image analysis in serial high-volume small animal studies.

Conclusions

A 2D pulsed ihMT prepared fast imaging sequence was implemented at 11.75T for preclinical studies on mouse CNS. A strategy based on filtering the signal of short T 1D components and achieved by proper tuning of the repetition time between consecutive RF pulses of the dual saturation was used to optimize the ihMT contrast for long T 1D myelinated structures. Furthermore frequency offset, power and timing saturation parameters were adjusted to optimize the ihMT sensitivity, providing measurements of ihMTR in mouse CNS for future reference. Typical ihMTR values on the order of 4-5% in WM, 2.5% in GM and 1-1.3% in muscle were obtained, thus leading to high positive relative ihMTR contrasts between myelinated tissues and other tissues (~3-4 between WM and Mu, and 2 between GM and Mu). Of particular importance, the relative ihMTR contrast between WM and GM (~ 2)

appeared to be relatively independent of the saturation parameters, supporting similar T 1D values in both brain structures. Hence, for WM/GM analyses minimizing T 1D -filtering by using short ∆t value (e.g. ∆t=1.3ms) may be a good option, offering higher CNR and higher ihMTR values. These results are of great significance and represent a first milestone in the process of validating ihMT as a biomarker of myelin. A strategy based on filtering the signal of short T 1D components was used to optimize the ihMT contrast for long T 1D myelinated structures in mouse. Typical ihMTR values on the order of 4-5% in WM, 2.5% in GM and 1-1.3% in muscle were obtained, thus leading to high positive relative ihMTR contrasts between myelinated tissues and other tissues. Of particular importance, the relative ihMTR contrast between WM and GM (~ 2) appeared to be relatively independent of the saturation parameters.

P

Optimization of inhomogeneous Magnetization Transfer (ihMT) MRI contrast for preclinical studies using dipolar relaxation time (T 1D ) filtering

  , since inhomogeneous has been used in the solid-state magnetic resonance literature to indicate

A 7 × 9 (

 79 pulsed ihMT preparation module similar to that previously described for human 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] was implemented under Bruker Paravision 5.1 software (Fig1). The saturation was achieved with a pulse train consisting of several frequency-shifted Hann-shaped pulses (duration PW) repeated every ∆t during a total saturation duration τ (total number of pulses given by τ/∆t), and which deposited a total RF energy proportional to E TR , with , -. = / 0,.23(in µT 2 •s). B 1,RMS(p) is the root mean square B 1 calculated over a single pulse and B 1,RMS is that calculated over the whole saturation period. Crusher gradients were inserted between saturation pulses. Single frequency-offset saturation MT + or MT -images were acquired with the frequency of all the saturation pulses applied at +∆f or -∆f respectively (Fig 1a). Dual frequency-offset saturation MT +/-(MT -/+ ) images were obtained either by using cosine-modulated (CM) Hannshaped pulses (Fig 1b) or by alternation of the frequency of consecutive pulses (Fig 1c).

  8ms in internal capsule, T 1D =5.6±1.2ms in cortical gray matter, and T 1D =2.2±0.6ms in muscle. The proposed T 1D -filtering strategy was thus used to attenuate the ihMT signal of the shorter T 1D components, represented by muscle here, while preserving high amount of signal of long-T 1D WM and GM, overall optimizing the contrast of ihMT for these myelinated structures. In vivo T 1D filtering was performed on 3 mice with the same parameters as those used for phantom experiments. Thirty averages (NEX=30) per tested parameter value (acquisition time ~ 8 minutes) were acquired in order to increase the signalto-noise.

Following the previous sensitivity

  and specificity characterization, reference 2D ihMT RARE images, with alternating pulses for dual frequency-offset saturation were sequentially acquired in coronal, axial (2 slices) and sagittal directions on 4 mice with ∆f=±10kHz, PW=3ms, ∆t=3.3ms, B 1,RMS =6.7µT, τ=0.9s and E TR =40 µT 2 .s. IhMT images were averaged 180 times (NEX=180, total acquisition time ~45 minutes) to improve SNR. This experiment allowed the establishment of a reference database of ihMTR/MTR values in trigeminal nerves (TN), brain white matter (internal capsule -IC, fornix -Fo, corpus callosum -CC, fimbria -Fi, cerebellum -Ce, external capsule -EC), brain gray matter (colliculi -Co, striatum -CPU, cortex -cGM), brain mixed structure (brain stem -BS, thalamus -Tha) and spinal cord gray and white matter (SCGM, SCWM). See Fig 6 for the ROI localization.

  The variations of ihMTR with changes in ∆f (Fig 4), B 1,RMS (Fig 5c) and τ (Fig 5d) in the different mouse brain structures are in good agreement with theoretical expectations. In particular, both the ihMTR bell-shape curves shifting to higher ∆f values when increasing B 1,RMS, the trend of ihMT signal saturation at high B 1,RMS and the decrease of ihMTR values for long saturation time τ, were predicted by the theoretical model (14). However, experimental data showed that the saturation of ihMT signal started at lower B 1,RMS values (B 1,RMS >6µT) than those predicted by the simulations (B 1,RMS ≥10µT). B 1,RMS values at which saturation occurred in experiments corresponded to root-mean-square pulse powers B 1,RMS(p) of ~7µT and higher. These results are in agreement with those previously reported on humans with a similar ihMT sequence (Fig 6 in (3)), which showed saturation of the ihMT signal in human WM/GM for B 1,RMS(p) values as high as ~8µT. Further support for ihMT signal saturation at high RF pulse power was highlighted in Fig 5a, which showed little difference in ihMTR for fixed B 1,RMS when varying PW, and hence upon varying B 1,RMS(p) . The apparent mismatch between the current theoretical model prediction and the experimental results strongly suggests that the instantaneous RF power deposition occurring during PW should be taken into account to refine the ihMT model, and should lead to more accurate prediction of the ihMT signal behavior vs. sequence parameters. Consideration of this factor should also allow investigating new strategies of power deposition in order to further optimize the ihMT sensitivity. The variations of ihMTR with ∆f values obtained in this study (Fig 4) providedadditional support for an effect of the angular orientation of white matter relative to the main magnetic field. This was suggested in our previous studies and illustrated by an ihMT signal only visible in the corpus callosum (axons lying mainly perpendicular to the magnetic field) at high frequency offset (∆f=21kHz)[START_REF] Varma | Magnetization transfer from inhomogeneously broadened lines: A potential marker for myelin[END_REF][START_REF] Varma | Interpretation of magnetization transfer from inhomogeneously broadened lines (ihMT) in tissues as a dipolar order effect within motion restricted molecules[END_REF]. Here, the steeper decrease of ihMTR with ∆f in IC

  values. This may be seen similar to T 2 -weighting imaging achieved with increase of the echo time of spin-echo based sequences and used to filter the signal of short T 2 components. Here, the short-T 1D muscle signal was filtered by means of increase of the repetition time between consecutive RF pulses of the dual saturation, thereby optimizing the contrast for longer T 1D myelinated WM and GM structures (Fig3a).One important finding derived from experiments using alternating pulses for dual saturation is that, whereas absolute ihMTR values in CNS changed with saturation parameters, the relative WM/GM ihMTR contrasts were particularly robust to them, as illustrated by the little differences observed between the ratios of WM/GM ihMTR values (~2) measured over the whole range of investigated saturation parameters. Of particular interest, this relative ihMTR contrast of ~2 associated with a low sensitivity to T 1D -filtering when increasing ∆t (Fig 3c, red curve), supports the close WM and GM T 1D values estimated in vivo (T 1D IC =6.1±0.8ms, T 1D cGM =5.6±1.2ms) (20) and is consistent with associated fractions of bound pool, f, almost doubled in WM compared to that in GM (14). In other words, the bound pool fraction associated to the long T 1D of brain tissue is the dominant source of ihMTR contrast between WM and GM. These results however differ with measurement from a recent study, which found shorter T 1D values in GM compared to WM using a Jeener-Broekaert echoes (18) technique applied on a fixed bovine spinal cord. T 1D in fixed tissue specimens may differ from in vivo values. One should also note that unlike the Jeener-Broekaert echoes technique, which estimates T 1D values based on the superposition of decay of all components, the fitting technique used for in vivo measurements (20) is biased towards long values as components with short T 1D are filtered before signal acquisition. GM T 1D could be composed of a long T 1D component correctly estimated by the fitting technique, and a short T 1D component filtered by the fitting technique. The robustness of relative ihMTR contrasts in brain to experimental settings may be particularly advantageous as assessment of the

(

  and/or ihMTR values) and gold standard myelin density measurements such as assessed by quantitative histology. In this context, the strong similarities between the ihMTR maps obtained in this study(Fig 6) and the bound pool fraction maps in rat brain, derived from qMT experiments, and which has been shown to correlate with myelin density maps[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], are very promising.More generally, the reinforced contrast for WM structures obtained with ihMT at 11.75T are of very significant importance in the context of high-magnetic field preclinical imaging, where more common myelin-sensitive techniques such as T1-weighted imaging or standard-MT based imaging (32) tend to lose contrast[START_REF] Deloire-Grassin | In vivo evaluation of remyelination in rat brain by magnetization transfer imaging[END_REF][START_REF] Lemaire | High-field quantitative transverse relaxation time, magnetization transfer and apparent water diffusion in experimental rat brain tumour[END_REF][START_REF] Natt | Magnetization transfer MRI of mouse brain reveals areas of high neural density[END_REF] due to uniform increases in T1

Fig 1 .

 1 Fig 1. Pulsed ihMT saturation preparation. (a) Single frequency-offset (+∆f) saturation, (b) Cosine-Modulated pulses for simultaneous dual frequency-offset (+∆f/-∆f) saturation. (c) Frequency alternating pulsed approach for dual frequency-offset saturation.

Fig 2 .

 2 Fig 2. In vitro T 1D -filtered ihMT imaging. a) ihMT images (in a.u.) obtained in long-T 1D hc (up) and short-T 1D agarose 4% (bottom) for dual frequency-offset saturation achieved with cosine-modulated pulses (CM, first image from the left) and for frequency alternating pulses with increasing values of ∆t. b) Corresponding ihMTR values measured in hc (red curve, scale ranging from 0-50%) and agarose 4% (black curve, scale ranging from 0-5%). Error bars correspond to in-ROI standard deviation. Other saturation parameters were PW=1.0ms, τ=0.9s, B 1,RMS =6.7µT and ∆f=10 kHz.

Fig 3 .

 3 Fig 3. In vivo T 1D -filtered ihMT imaging. a) M 0 image including location of IC, cGM and Mu structures (up left). Typical mouse brain T 1D map (bottom left) obtained using the method described in (20) and showing long T 1D values in WM, GM structures and short T 1D values in muscle. IhMTR images (in %) obtained for dual frequency-offset saturation achieved with cosine-modulated pulses (CM) and for frequency alternating pulses with increasing values of ∆t are shown on the right. b) Quantitative ihMTR values measured in IC (red curve), cGM (orange curve) and Mu (green curve) and c-d) corresponding ihMTR contrasts and CNR (between IC and Mu -blue curve, cGM and Mu -black curve and IC and cGM -red curve). Error bars correspond to group (N=3) standard deviation. Other saturation parameters were PW=1.0ms, τ=0.9s, B 1,RMS =6.7µT and ∆f=10 kHz.

Fig 4 .

 4 Fig 4. Sensitivity optimization (effect of frequency offset). Variation of ihMTR values (in %) with frequency offset ∆f (in kHz) in IC (a), CC (b), cGM (c) and Mu (d) for different values of B 1,RMS . Error bars correspond to group (N=3) standard deviation. Other saturation parameters were PW/∆t=3/3.3ms and τ=0.9s.

Figure 1 :Fig 2 :Fig 3 .Figure 4 :Figure 5 :Figure 6 :Fig

 123456 Figure 1:Pulsed ihMT saturation preparation. (a) Single frequency-offset (+∆f) saturation, (b) Cosine-Modulated (CM) pulses for simultaneous dual frequency-offset (+∆f/-∆f) saturation. (c) Frequency alternating pulsed approach for dual frequency-offset saturation. 177x234mm (300 x 300 DPI)

  ). The first approach may be considered as a genuine dual frequency-offset saturation agarose 4% were estimated to T 1D =16.1±6.0ms and to T 1D =1.6±0.3ms respectively, by fitting experimental data to an extended ihMT normalized model as outlined in[START_REF] Varma | In vivo measurement of a new source of contrast, the dipolar relaxation time, T 1 D , using a modified inhomogeneous magnetization transfer (ihMT) sequence: In Vivo Measurement of T 1D Using ihMT[END_REF]. A single ∆t value (∆t =1.3ms) was used for dual frequency-offset saturation performed with cosinemodulation for which no T 1D filtering is expected, and ∆t =1.3ms, 2.3ms, 3.3ms, 4.3ms, 5.3ms and 7.3ms were used for dual frequency-offset saturation performed with alternating pulses. Other ihMT saturation parameters were: ∆f=±10kHz, PW=1ms, B 1,RMS =6.7µT and τ=0.9s leading to E TR =40µT2 .s. Experiments were conducted at 25°C.

	for which the dipolar order is actually decoupled from the Zeeman order (14), hence
	providing the largest signal difference with the single frequency saturation experiment, and
	leading to strong ihMT signal. Alternatively, the second approach, which approximates a dual
	frequency-offset saturation, adds an extra degree of freedom corresponding to the time
	between two consecutive pulses (∆t) at alternated frequency offsets. In the following, ∆t is
	referred to switching time. When the switching time increases, the ihMT signal decays (Fig 5f
	of (19)) because of T 1D relaxation. For the sake of illustration, in the limiting case where this
	P e e r R e v i e w switching time increased towards infinity, the "dual-frequency" saturation scenario would become equivalent to the single-frequency one and the ihMT signal would naturally vanish. In a separate work, we reported a theoretical model for this decay, which combined with a modified version of the dual saturation, based upon the insertion of several pulses at one frequency before alternating to the opposite one, allowed in vivo estimations of T 1D values (20). Here instead, for T 1D -filtered ihMT imaging, we used a dual saturation scheme with e e r single pulse at alternating frequency (Fig 1c) and adapted switching P R e v i e w
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time, ∆t. Increasing the switching time, ∆t, increases the time at which the dual-offset saturation could be considered effective. Hence, fixing ∆t should allow T 1D -weighting of the ihMT signal, by reducing the ihMT signal of components with T 1D <∆t. Conversely, when the dual-frequency saturation is achieved by means of cosine-modulated pulses (length PW, Fig 1b), the ihMT signal is sensitized to shorter T 1D value components and minimal T 1D -filtering is achieved.

T 1D -filtered ihMT imaging was illustrated in vitro on a phantom composed of hair conditioner (Le petit Marseillais®) (hc) and agarose 4% (ag4%). Hair conditioners, whose major components are suspensions of fatty alcohols, which form lamellar structures of methylene chains

[START_REF] Malyarenko | Magnetization transfer in lamellar liquid crystals: Lamellar LC as Model MT Systems[END_REF]

have shown strong ihMT effects. Whereas no conclusion regarding neuroimaging can be derived from ihMT experiments on hair conditioners, these latter can be advantageously used for technical set up and adjustment purposes

[START_REF] Varma | Magnetization transfer from inhomogeneously broadened lines: A potential marker for myelin[END_REF]

. T 1D values of hc and

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	Principle of T 1D -filtered ihMT imaging is illustrated in vitro in Figure 2, which shows ihMT
	images (Fig 2a) and variations in ihMTR (Fig 2b) measured in hc (long T 1D hc ~16ms) and
	ag4% (short T 1D ag4% ~1.6ms) for different values of ∆t. For short and long T 1D components, the
	use of cosine-modulated pulses (i.e. simultaneous dual frequency-offset saturation) resulted in
	the highest ihMTR values (Fig 2b, ihMTR hc =42% and ihMTR ag4% =3.4%). T 1D -filtering of the
	ihMT signal was evidenced in the dynamics of ihMTR with increasing ∆t when using
	alternating pulses for dual frequency-offset saturation: ihMTR values of the short-T 1D ag4%
	reduced to ihMTR ag4% =2.7% at ∆t=1.3ms, were further divided by a factor greater than 2 at P e r e ∆t=3.3ms (ihMTR
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Page 12 of 70 http://mc.manuscriptcentral.com/ag4% =1.1%) and tended towards zero for longer ∆t values (ihMTR ag4% <0.5% for ∆t>4.3ms). Conversely, for the long-T 1D hc, the studied variation of ∆t had lower effect on the ihMTR values: ihMTR hc =41% at ∆t=1.3ms, ihMTR hc =38% at ∆t=3.3ms and ihMTR hc =32% at ∆t=6.3ms.

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  pulses yielding to ihMTR IC =7.5%, ihMTR cGM =4.2% and ihMTR Mu =3.8%. Fig 3c), as a consequence of stronger T 1D -weighting introduced with longer T 1D values (Fig 3a). In particular, ∆t values ≥3.3ms allowed filtering of the ihMT signal in muscle (ihMTR Mu ≤1%, T 1D Mu =2.2±0.6ms), hence reinforcing the relative IC/Mu and cGM/Mu contrasts (Fig 3a, blue and black curves of Fig 3c). The relative contrast between IC and cGM was relatively insensitive to ∆t variations, as illustrated by little changes in the IC/cGM ihMTR ratios (~ 2) with increasing ∆t (Fig 3c, red curve). Contrast-to-noise ratios measuredbetween the same structures (Fig.3d) showed moderate decrease for ∆t≤3.3ms, and a big drop for ∆t>3.3ms. These results suggested that when T 1D -filtering is necessary, ∆t=3.3ms

	enhanced (
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	This corresponded to relative ihMTR contrast ratios of ~1.8 between IC and cGM, of ~2.0
	between IC and Mu and of ~1.1 between cGM and Mu (Fig 3c). For the alternating dual

map (Fig 3a), ihMTR maps for different ∆t values (Fig 3a) and the corresponding ihMTR values measured in IC, cGM and Mu (Fig 3b). Similarly to in vitro experiments, higher ihMTR values were obtained when the dual frequency-offset saturation was performed with cosine-modulated frequency saturation implementation, ihMTR values decreased with increasing ∆t (Fig 3b), but the relative ihMTR contrast between myelinated and non-myelinated structures was Page 13 of 70 http://mc.manuscriptcentral.com/

represents a good configuration for enhancing the contrast between myelinated structures and others. With these settings relative ihMTR contrast ratios of ~5.0 for IC/Mu and ~2.3 for cGM/Mu were obtained with a reduced loss of sensitivity in mouse brain (ihMT IC =(5.0±0.1)%, ihMT cGM =(2.3±0.2)%). If one is interested by WM/GM contrast analysis and not concerned with muscle signal, then ∆t of 1.3ms may be more appropriate, offering higher CNR and higher ihMTR values.

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  RMS =6.7µT, ihMTR max =5.2% at ∆f =10kHz and for B 1,RMS =8.4µT, ihMTR max =5.3% at ∆f =12kHz). These results suggested that ∆f values of ~10-12kHz and saturation power B 1,RMS ≥6.7µT can maximize the ihMT signal in WM/GM structures while maintaining a low value in muscle. At fixed B 1,RMS , little difference was observed in ihMTR values for increasing PW and constant ∆t (Fig 5a), whereas ihMTR values clearly decreased when increasing ∆t (Fig 5b), again confirming the role of ∆t as a determinant of T 1D filtering.

	B 1,
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1,RMS =6.7µT, maximum ihMTR values were obtained at ∆f =10kHz for IC, at ∆f =10-12kHz for cGM, and at ∆f =12kHz for CC. For muscle (Fig

4d

), the maximum occurred for ∆f>18kHz. Interestingly, for all structures, the frequency offset corresponding to the maximum ihMTR value tended to shift to higher values when the saturation power increased (e.g. in IC: for B 1,RMS =5.1µT, ihMTR max =4.5% at ∆f=8-10kHz, for Page 14 of 70 http://mc.manuscriptcentral.com/In WM and GM, ihMTR values tended to saturate for B 1,RMS ≥6µT, whereas in Mu, they kept increasing with increasing B 1,RMS (Fig 5c). For increasing τ and fixed B 1,RMS =6.7µT, ihMTR values reached their maximum for τ~900ms and further slightly decreased for τ>1000ms (Fig 5d).

Reference T 1D -filtered ihMTR measurements in mouse CNS

Figure 6 shows typical MTR maps (left column) and ihMTR maps (right column) (NEX=180) obtained in axial, coronal and sagittal directions with optimized parameter settings: ∆f=±10kHz, PW=3ms, ∆t=3.

3ms, B 1,RMS =6.7µT, E TR =40 µT 2 .s and τ=0.9s. A reference database of ihMTR and MTR values in CNS structures measured in such images (location indicated in MT + images, Fig 6 middle column) is presented in table 2 and the relative ihMTR contrasts calculated between all structures are reported in table 3.

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	e e r P e e r P e e r P e e r
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	O O O O
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Table 1 . Saturation parameters varied in optimization experiments.

 1

	Parameter Value

Table 2 . Quantitative ihMTR and MTR values in mouse CNS.

 2

	IhMTR and MTR values measured in CNS structures indicated in Figure 6. Values are
	reported as mean±group standard deviation (N=4 animals)
	(a) measured on axial images;
	(c) measured on coronal images;
	(s) measured on sagittal images;
	(a-c) mean of measurements on axial and coronal images;
	(c-s) mean of measurements on coronal and sagittal images;
	(a-c-s) mean of measurements on axial, coronal and sagittal images;
				mean ihMTR (%)	mean MTR (%)
			± group standard deviation	± group standard deviation
		SCWM (s)	5.4±0.2	40.3±3.2
	White matter	IC TN Fo Fi CC Ce	(a) (a) (s) (c) (a-c-s) (c-s)	4.9±0.3 4.8±0.2 4.1±0.4 4.0±0.2 3.9±0.2 3.4±0.1	44.5±0.7 43.6±1.0 40.9±1.3 39.3±0.7 40.9±0.4 43.6±1.3
		EC	(a-c)	3.3±0.2	42.6±1.0
	Mixed	BS Tha	(s) (s)	5.2±0.3 3.3±0.2	43.6±1.7 41.7±1.0
	Gray Matter	SCGM Co CPU cGM	(s) (c-s) (c) (a)	4.1±0.2 3.6±0.2 3.4±0.2 2.4±0.2	39.8±3.3 42.6±1.2 41.1±1.0 41.4±0.4
		Muscle (a)	1.3±0.2	60.3±0.6

Table 3 . Relative ihMTR contrasts and error estimate in mouse CNS

 3

						NMR in Biomedicine -For Peer Review Only
		Relative ihMTR contrasts between 2 structures are defined as the ratio of ihMTR values of 2 structures. E.g. (in bold): ihMTR contrast between
		IC and Mu is 3.8; 2.0 between IC and cGM; 1.9 between cGM and Mu and 1.3 between SCWM and SCGM.
				GM			Mixed					WM
		Muscle	cGM	CPU	Co	SCGM	Tha	Bs	EC	Ce	CC	Fi	Fo	TN	IC	SCWM
		Muscle	1.9	2.6	2.8	3.2	2.6	4.0	2.5	2.6	3.0	3.1	3.2	3.7	3.8	4.2
			± .3	± .3	± .3	± .3	± .3	± .3	± .3	± .3	± .3	± .3	± .3	± .3	± .3	± .3
		cGM		1.4	1.5	1.7	1.4	2.2	1.4	1.4	1.6	1.7	1.7	2.0	2.0	2.3
				± .1	± .1	± .1	± .1	± .2	± .1	± .1	± .1	± .1	± .2	± .1	± .2	± .1
		CPU			1.1	1.2	1.0	1.5	1.0	1.0	1.1	1.2	1.2	1.4	1.4	1.6
	GM	Co			± .1	± .1 1.1	± .1 0.9	± .1 1.4	± .1 0.9	± .0 0.9	± .1 1.1	± .1 1.1	± .1 1.1	± .1 1.3	± .1 1.4	± .1 1.5
						± .1	± .1	± .1	± .1	± .0	± .1	± .1	± .1	± .1	± .1	± .1
		SCGM					0.8	1.3	0.8	0.8	1.0	1.0	1.0	1.2	1.2	1.3
							± .1	± .1	± .1	± .0	± .1	± .1	± .1	± .1	± .1	± .1
	Mixed	Tha Bs						1.6 ± .1	1.0 ± .1 0.6 ± .0	1.0 ± .0 0.7 ± .0	1.2 ± .1 0.8 ± .0	1.2 ± .1 0.8 ± .0	1.2 ± .1 0.8 ± .1	1.5 ± .1 0.9 ± .0	1.5 ± .1 0.9 ± .1	1.6 ± .1 1.0 ± .0
		EC								1.0	1.2	1.2	1.2	1.5	1.5	1.6
										± .0	± .1	± .1	± .1	± .1	± .1	± .1
		Ce									1.1	1.2	1.2	1.4	1.4	1.6
											± .1	± .1	± .1	± .1	± .1	± .1
		CC										1.0	1.1	1.2	1.3	1.4
												± .1	± .1	± .1	± .1	± .1
	WM	Fi Fo											1.0 ± .1	1.2 ± .1 1.2	1.2 ± .1 1.2	1.4 ± .1 1.3
														± .1	± .1	± .1
		TN													1.0	1.1
															± .1	± .1
		IC														1.1
																± .1
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	Fig 5. Sensitivity optimization (effects of RF saturation pulses). Variations of ihMTR
	values in IC (red curves), cGM (orange curves) and Mu (green curves) with PW (a), ∆t (b),
	B 1,RMS (c) and τ (d). The frequency offset was set to 10kHz and other saturation parameters
	were ∆t=3.3ms, τ=0.9s and B 1,RMS =6.7µT in (a), τ=0.9s and B 1,RMS =6.7µT in (b),
	PW/∆t=3.0/3.3ms and τ=0.9s in (c) PW/∆t=3.0/3.3ms and B 1,RMS =6.7µT in (d). Error bars
	correspond to group (N=3) standard deviation.
	Fig 6. Optimized ihMT mouse CNS images. MT + , MTR and ihMTR images obtained with
	PW/∆t=3.0/3.3ms, ∆f=10kHz, B 1,RMS =6.7µT and τ=0.9s in axial, coronal and sagittal
	directions. Brain CNS structures where ihMTR and MTR values were measured are indicated
	in the MT + images.
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. Error bars correspond to group (N=3) standard deviation. Other saturation parameters were PW/∆t=3/3.3ms and τ=0.9s. Page 30 of 70 http://mc.manuscriptcentral.com/http://mc.manuscriptcentral.com/

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Acknowledgments

The authors thank Nathalie Cuge for animal handling.

Grant Support: V.P. received support from the A*MIDEX grant (n°ANR-11-IDEX-0001-02) funded by the French Government "Investissements d'Avenir" program. S.M received support from IRME 2016

Supplementary Material

Fig. S1. Effect of Magnetic field strength on ihMT signal. (a) M 0 and ihMT images obtained at 1.5T (up) and 11.75T (bottom) in the same conditions of temperature (25°C) and saturation parameters on a phantom composed of 2 different hair conditioner samples (hc1, hc2) and gadolinium-doped (2mM) water (H 2 O*). The 2D ihMT RARE sequence described in the Material and Methods section was used at 11.75T, and the sequence optimized for human and described in [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], was used at 1.5T. (b-e) Variations of ihMTR in hc1, hc2 and H 2 0* at 1.5T (plain marks) and 11.75T (open marks) and as a function of ∆f (b), B 1,RMS (c), τ (d) and ∆t (e). Other saturation parameters were PW/∆t=1.0/2.0ms, τ=1s and B 1,RMS =5.5µT in (b), PW/∆t=1.0/2.0ms, τ=1s and ∆f=10 kHz in (c), PW/∆t=1.0/2.0ms and ∆f=10 kHz in (d), PW=1.0ms, B 1,RMS =5.5µT, τ=1s and ∆f=10 kHz in (e). As expected, doped-water did not show any ihMT signal at both field strengths. For all configurations (except hc1, 4kHz data point in b), attributed to noise variance), absolute ihMTR values were systematically slightly higher at 11.75T compared to that at 1.5T. However, the dynamics of ihMTR was mostly independent of the magnetic field as demonstrated by the similar variations of ihMTR with the saturation parameters values.


M Khlat
email: khlat@ined.fr

S Legleye

B Falissard

N Chau • The Lorhandicap

Á S Legleye

Á B Falissard

Á N Chau

Mortality gradient across the labour market core-periphery structure: a 13-year mortality follow-up study in north-eastern France

Keywords: Mortality, Inequalities, Unemployment, Temporary work, Precarious work, Flexibility, Health behaviours

Purpose This study explores mortality related to temporary employment, about which very little is known to date. Methods In 1996, a health survey was carried out in the French region of Lorraine, and all members of 8,000 randomly chosen households were followed up for mortality over a 13-year period. Mortality of subjects in relation to their employment situation at baseline was analysed using a Cox survival regression. Results In comparison with permanent workers, for unemployed men, we found age and occupation-adjusted hazard ratios (HR) of 4.1 for all-causes of death and 3.9 for non-violent causes, and for male temporary workers a HR of 2.2 for both all-causes and non-violent causes of death. Bad health, tobacco smoking and alcohol misuse explained 17 % of the excess risk for the unemployed and 41 % of that for temporary workers. Conclusion The observation of large mortality inequalities across the labour market core-periphery structure has important policy implications, particularly in terms of prevention focused on unhealthy behaviours among male unemployed and temporary workers.

Introduction

The flexible labour market is typically segmented: the centre or core comprises individuals employed on a permanent basis or with no fixed term, and it is surrounded by a band of individuals in various kinds of restricted-term employment. In past decades, there has been a re-composition of the work force, with a steady development of the peripheral segments to the detriment of the core of secure employments. In both Europe and the USA, the number of individuals in temporary employment is now exceeding the number of unemployed, and the different forms of flexible jobs have much in common with unemployment in terms of social and economic disadvantages [START_REF] Benach | The consequences of flexible work for health: are we looking at the right place?[END_REF][START_REF] Virtanen | Temporary employment and health: a review[END_REF].

The excess morbidity and mortality among men and women having experienced unemployment has been extensively described in the literature [START_REF] Bartley | Living in a highunemployment economy: understanding the health consequences[END_REF][START_REF] Roelfs | Losing life and livelihood: a systematic review and meta-analysis of unemployment and all-cause mortality[END_REF]). However, there is no agreement on the consequences of flexible work on health, since available studies are not conclusive. The experience of job insecurity has been associated with psychological morbidity but not with physical morbidity [START_REF] Quesnel-Valle ´e | Temporary work and depressive symptoms: a propensity score analysis[END_REF][START_REF] Virtanen | Health inequalities in the workforce: the labour market core-periphery structure[END_REF][START_REF] Virtanen | Temporary employment and health: a review[END_REF][START_REF] Waenerlund | Is the core-periphery labour market structure related to perceived health? Findings of the Northern Swedish Cohort[END_REF]), and, apart from studies in Finland (Kivima ¨ki et al. 2003;N a ¨tti et al. 2009), there is little evidence of an association between job insecurity and premature mortality.

Yet there are several pathways through which temporary work might have adverse health consequences. Temporary workers are more exposed than permanent ones to work strain and physical hardship (painful and tiring positions, intense noise and repetitive movements) and to hazardous work conditions, because they often have limited on-thejob training and may feel more constrained by their insecure position to complain about working conditions and risks [START_REF] Benach | The health-damaging potential of new types of flexible employment: a challenge for public health researchers[END_REF][START_REF] Benach | The consequences of flexible work for health: are we looking at the right place?[END_REF]. In addition, studies from different countries suggest that they are more prone to tobacco smoking and alcohol abuse [START_REF] De Cuyper | Associations between temporary employment, alcohol dependence and cigarette smoking among Turkish health care workers[END_REF]Jung et al. 2013;[START_REF] Kivima ¨ki | Temporary employment and risk of overall and causespecific mortality[END_REF][START_REF] Legleye | Gender and age disparities in the association of occupational factors with alcohol abuse and smoking in the French working people[END_REF]. Given their general pattern of social and material deprivation, temporary workers are also less likely to be properly treated [START_REF] Iezzoni | The effects of employment conditions on smoking status and smoking intensity: the analysis of Korean Labor &[END_REF][START_REF] Van Beljouwe | Reasons and determinants for not receiving treatment for common mental disorders[END_REF], and therefore, special attention should be given to their healthcare needs.

Selection processes may, however, distort the picture in unpredictable ways. The ''healthy worker effect'' to which temporary workers are subject at selection into employment is one candidate, and this pattern is compounded by poor health hindering transition from temporary employment to permanent employment and favouring transition from temporary employment to unemployment [START_REF] Bardasi | The impact of atypical employment on individual well-being: evidence from a panel of British workers[END_REF][START_REF] De Cuyper | Transitioning between temporary and permanent employment: a two-wave study on the entrapment, the stepping stone and the selection hypothesis[END_REF][START_REF] Virtanen | Selection from fixed term to permanent employment: prospective study on health, job satisfaction, and behavioural risks[END_REF][START_REF] Virtanen | Sickness absence as a risk factor for job termination, unemployment, and disability pension among temporary and permanent employees[END_REF]. Lastly, poor health may lead to disability, which in turn may accelerate exit from the labour market and transition to inactivity.

The purpose of this study was to investigate mortality inequalities in relation to the structure of the labour force from core to periphery. Our data originate from the Lorraine region in north-eastern France, which has been particularly exposed to labour market hardship during the past decades. In France, the unemployment rate has fluctuated around 10 % since the mid-1980s, and limited-term contracts have constantly been on the rise, reaching 12.6 % of salaried workers in 2009 (INSEE 2011). The Lorraine region, a leading industrial region after the Second World War, has undergone massive de-industrialization and tertiarization over the last 50 years, with a significant growth of unemployment and temporary employment [START_REF] Thirion | L'emploi en Lorraine: tertiarisation par de ´sindustrialisation, forte pousse ´e des femmes et des seniors[END_REF]. Currently, Lorraine is particularly disadvantaged in terms of unemployment and poverty compared to the other French regions.

This study uses a unique epidemiological mortality follow-up survey, the Lorhandicap health survey, which was implemented in Lorraine in 1996 and comprised 8,000 randomly chosen households, of which all members were carefully followed up for mortality over a 13-year period, i.e. until 2008. Our research questions were as follows: (1) Is the mortality of temporary workers higher than that of workers with permanent employment, and how does it compare with that of the unemployed, adjusting for age and occupational group? (2) Are the mortality differences, if any, explained by health status and health-related behaviours at baseline?

Materials and methods

Procedure

Several studies based on the Lorhandicap survey data have already been published [START_REF] Baumann | Associations of social and material deprivation with tobacco, alcohol, and psychotropic drug use, and gender differentials: a population-based study[END_REF][START_REF] Chau | Relationship of job demands to initiating smoking in working people: a populationbased study[END_REF][START_REF] Lemogne | Gender differences in the association between depressive mood and mortality: a 12-year follow-up population-based study[END_REF]. The initial sample consisted of subjects aged 15 years or more living in 1996 in 8,000 randomly selected households in the Lorraine region of north-eastern France (2.3 million inhabitants), representing 1/100 of the population. The investigation was approved by the Commission Nationale de l'Informatique et des Liberte ´s (national review board), and a written informed consent was obtained from the respondents.

Measures

All measures were collected using the survey questionnaire administered at the start of the study in 1996. Occupation was chosen as an indicator of adult socioeconomic status, and employment situation was categorized into permanent employment (paid employment with unlimited duration, i.e. civil servants, workers with indefinite-term contracts and self-employed), temporary employment (project employees, workers ''on-call'' and workers from temporary labour agencies) and unemployment. Subjects who were not in the labour force at study baseline were excluded (retirees, students, at-home mothers and other non-working people not looking for employment).

Five occupational categories were considered, following the International Classification of Occupation (ISCO) [START_REF] Kivima ¨ki | Temporary employment and risk of overall and causespecific mortality[END_REF]: professionals/managers (reference category), associate professionals/technicians, service workers/clerks, manual workers and others. We used selfreported health, with a categorization into good (''very good'' and ''good''), average (''average''), and poor or bad (grouping ''mediocre'' and ''bad''). Behavioural factors included smoking status (smoker, ex-smoker or non-smoker), and ''alcohol misuse'' which was measured using the French version of the Cut/Annoyed/Guilty/Eye-opener (CAGE) questionnaire [START_REF] Beresford | Comparison of Cage questionnaire and computer-assisted laboratory profiles in screening for covert alcoholism[END_REF]) and defined by at least two positive responses to four items: consumption considered excessive by the subject, consumption considered excessive by people around the subject, subject wishes to reduce consumption and consumption on waking.

Follow-up

The cohort was followed up for mortality from 1st July 1996 to 31st December 2008 (12.5 years). The vital status of all subjects was ascertained from the national computerized database listing all deceased subjects in France from 1978, by contacting the registry offices of the birth places for people born in France and by contacting the registry office for foreign-born French citizens (Ministry of Foreign Affairs). Causes of death were coded by the French national cause-of-death registry (INSERM) using the current revisions of the International Classification of Diseases. Violent mortality corresponds to codes E800.0-E999 (8th or 9th revision) or V01.0-Y89.9 (10th revision) and comprises suicides, homicides and accidental deaths. Nonviolent deaths comprise all other deaths.

Statistical analysis

The study aimed to explore the 13-year (1996-2008) subsequent mortality of subjects who were temporary workers at baseline and to compare it to that of unemployed and permanent workers. The duration of follow-up for each subject was from 1 July 1996 to either the date of death or 31 December 2008 for those still alive at the end of followup. Cox regression models were used to examine the associations of employment status with all-cause mortality, non-violent mortality and violent mortality.

To assess the roles played by self-reported health and behavioural risk factors in all-cause mortality, three Cox regression models were performed: a basic model (model 1) including age and occupation; then self-reported health was added to model 1 (model 2); and finally, behavioural risk factors were added to model 2 (model 3). The contributions of self-reported health and those of behavioural risk factors were estimated by the changes in the HRs, derived from the formulas: (HR model 1 -HR model 2 )/ (HR model 1 -1) and (HR model 1 -HR model 3 )/(HR model 1 -1), respectively. The proportional hazard assumption was evaluated from Schoenfeld residuals for the global model and for each covariate. The statistical analyses were performed using STATA software (Stata, College Station, TX, USA).

Sample

Of the 8,000 households included in the sample, dispatches to 193 (2 %) were lost (due to addressing error or death). Of the 7,807 households contacted, 3,460 (44.3 %) participated (all eligible members of the family took part in 86 % of these). In total, 6,234 subjects filled in a questionnaire, including 34 failing to provide gender or age data, leaving a population of 6,200 subjects. The composition of the initial sample was found to be close to that of the census population (''Appendix'').

During mortality follow-up, 157 subjects (2.5 %) were lost and excluded, resulting in a study population of 6,043 participants. Only the subjects who were either working or unemployed and aged between 30 and 64 years at baseline (1st July 1996) were retained for this study, i.e. 2,500 subjects, 1,402 men and 1,098 women. In total, 95 deaths (66 men and 29 women) occurred.

Results

The subjects experiencing temporary employment had lower occupational categories, poorer self-reported health, greater exposure to behavioural risk factors (for men) and higher general and cause-specific mortality than permanent workers, but a somewhat more favourable profile than the unemployed (Table 1). Hazard ratios for mortality from all-causes, adjusted for age and occupation, differed between men and women, and a significantly elevated hazard ratio was found for temporary male workers (2.2 times higher risk, p \ 0.05), and, as expected, for unemployed males (4.1 times higher risk, p \ 0.001), compared to permanent workers (Table 2). With regard to non-violent mortality, the analyses highlighted a significant disadvantage among men for both temporary workers and unemployed (2.2 times higher risk, p \ 0.05 and 3.9 times higher risk p \ 0.01, respectively). After subdividing nonviolent mortality into mortality from cancers and mortality from cardiovascular diseases, the gap remained pronounced and significant for cardiovascular mortality (3.6 and 5.5 times higher risk, p \ 0.05, respectively), while the estimates for cancer mortality, although elevated (2.3 and 2.9), did not reach significance (p \ 0.10). For violent mortality, no significant hazard ratio was found for temporary workers, while the estimates were elevated for unemployed men (7.2, close to significance, p \ 0.10) and significantly elevated for unemployed women (11.2, p \ 0.05).

Using nested models, we found that after inclusion of health in a first stage, and health-related behaviours in a second stage, the excess risks of death found for men were substantially reduced by 35 and 41 % for temporary workers and by 20 and 17 % for unemployed, remaining significant only for the latter (3.6, p \ 0.01) (Table 3). Further analyses failed to demonstrate a major role of these factors in explaining mortality from cardiovascular diseases for either the temporary workers or the unemployed. Conversely, the elevated (but non-significant) estimates for cancer mortality were substantially reduced after adjustment for both temporary workers and the unemployed.

Discussion

The mortality of non-permanent employees has received very little attention to date, and the differences that we have uncovered are impressive, with increasing levels when shifting from permanent employment at baseline to temporary employment (2.2 times higher risk) and to unemployment (4.1 times higher risk). Temporary employment and unemployment are in most cases transient situations in employment trajectories, and a study covering the 1990s indicates that two-thirds of the temporary workers reached permanent employment within 3 years [START_REF] Junod | Le CDD un tremplin vers le CDI dans deux tiers des cas…mais pas pour tous[END_REF]. Our findings are all the more striking, as changes during the course of follow-up are bound to dilute differences across groups.

The large mortality inequalities associated with the situation at baseline are likely to reflect both direct and indirect selection into precarious employment and longterm influence of the socioeconomic disadvantage attached to that position on health. In France, it has been demonstrated that the destabilization of workers into flexible employment was not generalized but rather concentrated on the less stable segment of the population [START_REF] Petit | Trends in employment stability and labour market segmentation[END_REF]. In support of this hypothesis, in this study, we have found that, within each occupational category, unemployed and the temporary workers are less educated than permanent workers. For instance, the proportion of male manual workers with primary education only is 38 % among those with indefinite-term contracts, 51 % among those with fixed-term contracts and 61 % among the unemployed. This is in line with the segmentation theory, which states that there are differences in terms of skills between workers in the ''core'' and the ''periphery'' of the labour market [START_REF] Gordon | Segmented work, divided workers: the historical transformation of labor in the United States[END_REF]. A compositional difference of this type is a kind of indirect selection, which certainly contributes to the mortality disadvantage in the peripheral segments of the labour force.

In the literature, there is to our knowledge only one study, from Finland [START_REF] Kivima ¨ki | Temporary employment and risk of overall and causespecific mortality[END_REF], that has investigated mortality among temporary workers, over a 10-year observation period. In that study, the same type of gradient across the labour market core-periphery structure was found, and mortality was found to be 1.2-1.6 times higher among male and female temporary employees compared with permanent employees. More recently, a second analysis of the Finnish data showed that temporary workers were not a homogeneous group and that the mortality disadvantage was limited to those feeling that the insecure situation was unsatisfactory, and to those holding the job on an involuntary basis [START_REF] Na ¨tti | Type of employment relationship and mortality: prospective study among Finnish employed in 1984-2000[END_REF]). Indeed, temporary jobs are not all necessarily disadvantaged, and some may even be advantageous when they enable better control over the working time or are used as a stepping stone into permanent employment.

In a recent review and meta-analysis of unemployment and mortality [START_REF] Roelfs | Losing life and livelihood: a systematic review and meta-analysis of unemployment and all-cause mortality[END_REF], the mean hazard ratio for mortality was 1.63 among HRs adjusted for age and additional covariates. For the unemployed, we observed a larger gap (fourfold higher risk), but this should be related to the nature of the reference group, which in our study represents the core of the work force. Given the heterogeneity within the employed group, the traditional approach of using all employed as a reference, regardless of their type of employment, is bound to attenuate the association between unemployment and mortality.

In terms of causes of death, external causes, and particularly suicide, are at the forefront in the literature on unemployment and mortality (Ma ¨ki and Martikainen 2010). In the Finnish study [START_REF] Kivima ¨ki | Temporary employment and risk of overall and causespecific mortality[END_REF], temporary employment was associated among both men and women, with increased deaths from external causes and from alcohol-related causes and, in men only, from smoking-related cancers. Recently, [START_REF] Lundin | Unemployment and mortality-a longitudinal prospective study on selection and causation in 49321 Swedish middle-aged men[END_REF] reported an increased risk of mortality for unemployed men from both natural and external causes. In line with these findings, among men we observed an association between temporary employment and unemployment at start of the study with non-violent mortality. Our hazard ratios for violent mortality were also very high among the unemployed, but, given the small number of deaths, significance was reached for women, but was borderline for men (p \ 0.10).

For male temporary workers, we find evidence for a role of health, and, to a lesser extent, of tobacco and alcohol at baseline as explanatory factors for the excess mortality, more so for cancers than for cardiovascular diseases. This difference might be due to the fact that cardiovascular diseases are very much influenced by dietary habits, on which no information was collected in the survey. The role played by poor health in the disadvantage of temporary workers provides some support for the reverse causation (or selection) hypothesis, i.e. that the individuals with poor health are less likely to find a permanent job. On the other hand, the role of reverse causation needs to be mitigated, as health in this group of temporary workers may have been altered by cumulative exposures to hazardous working conditions, occupational injury or occupational diseases. In support of this interpretation, further analysis of the study data showed that the prevalence of self-reported functional limitations for daily living activities at baseline was 20 % for permanent employment, 33 % for temporary employment and 41 % for unemployment among men (p \ 0.001) and 14, 21 and 23 %, respectively, among women (p \ 0.001).

For male unemployed, we find a lower contribution of poor health and behavioural factors than for male temporary workers (fully adjusted HR 3.6, p \ 0.01, 17 % reduction in HR). This may suggest that the post-unemployment pathway plays a significant role for the unemployed. According to the ''coping hypothesis'', individuals cope with the stress associated with unemployment by changing their health behaviours in unhealthy ways [START_REF] Hammarstrom | Unemployment and change of tobacco habits: a study of young-people from 16 to 21 years of age[END_REF]. This change is already visible in our data. Further to this, some studies have demonstrated that the experience of joblessness has longterm effects on income [the ''unemployment scar'' [START_REF] Arulampalam | Is unemployment really scarring? Effects of unemployment experiences on wages[END_REF]]. A ''scar'' of this type could contribute to less favourable pathways, particularly because ''unemployment spells do not happen at random, but tend to take place as part of a generally disadvantaged life course'' [START_REF] Bartley | Do we need to worry about the health effects of unemployment?[END_REF].

The health effects of different types of employment situations may differ by gender, and the idea that loss of employment may have less of a negative influence for women has been supported, with the argument that women are more able to cope because they take on roles as mothers and housewives. We found no significant mortality inequalities among women, although the hazard ratios associated with female unemployment were quite elevated. First, certain types of temporary jobs may have positive aspects for women whenever they need to invest more of their time and energy in family life. This might be changing, since women's traditional social roles are evolving, with working status becoming an increasingly important component of women's identity [START_REF] Bambra | Yesterday once more? Unemployment and health in the 21st century[END_REF]. Second, while women are more likely to belong to the more peripheral temporary workforce, they are less involved than men in manual jobs, which are the most exposed to occupational health risks. It is also worth noting that there were no significant differences across employment situations in terms of smoking and alcohol misuse among women and that the differences for poor health were much smaller than among men (Table 2). This finding is consistent with the greater contribution of unhealthy behaviours to the excess mortality of male temporary workers and definitely calls for targeted prevention approaches on these issues.

Before turning to policy implications, it is important to stress the study limitations. First, the response rate was rather moderate (about 44 %). However, this response rate is similar to those of other studies using postal selfadministered questionnaires in France [START_REF] Goldberg | Socioeconomic, demographic, occupational, and health factors associated with participation in a long-term epidemiologic survey: a prospective study of the French GAZEL cohort and its target population[END_REF]. Furthermore, the distributions for gender, age, education, marital status, occupation, unemployment and occupational injury in the initial sample were close to those of the census population (''Appendix'').

Second, given the small number of observations in the exposure groups (unemployed and temporarily employed), our study did not possess sufficient statistical power and used wide confidence intervals for the estimations. The main issue in this case is type II error rather than type I error, and therefore, the significant findings that we report are all the more unlikely to arise by chance. Another reassuring point is that our hazard ratio estimates for the unemployed agree with those found in a five-year mortality follow-up of the French longitudinal samples derived from the 1975, 1982 and 1990 censuses (3.9 for men and 1.9 for women in [START_REF] Mesrine | La surmortalite ´des cho ˆmeurs: un effet catalyseur du cho ˆmage?[END_REF]), compared to 4.1 and 2.7 (nonsignificant) in our study.

Third, our analyses are based on subjects' employment situation, health and behaviours at the start of the study, and we do not have retrospective information, nor do we have updates during the follow-up period. Not knowing to what extent poor health and unfavourable health behaviours are the cause or the consequence of remaining or becoming a temporary worker or unemployed, we cannot give credit to a causality relation and have therefore emphasized both causation and reverse causation (selection) hypotheses in the discussion. Given the sample size and the issues outlined above, our findings definitely need to be replicated and confirmed.

Conclusion

The issue of precarious employment and health is progressively gaining more visibility in the public health arena, but the empirical evidence is scant. Based on a unique mortality follow-up in a region of France subjected to economic hardship, our study uncovers large mortality inequalities and provides evidence for a new form of mortality inequality across the core-periphery structure of the labour market. The evolution of the Lorraine region during the past decades, from a prosperous industrial region to one of the most socioeconomically disadvantaged regions in the country, could partly explain the role of health behaviours as coping mechanisms for the population. Our findings have important policy implications, particularly in terms of prevention focused on unhealthy behaviours among male temporary workers and the unemployed.

Furthermore, job insecurity today is bound to lead to wider health consequences than in the economic recessions of the 1980s, given the progressive weakening of the protection of citizens against labour market risks by the welfare state in many countries (DeNavas-Walt et al. 2011; Observatoire des ine ´galite ´s 2011). It follows that fuller consideration of the new forms of health inequalities in the flexible labour market landscape is required [START_REF] Benach | Precarious employment and health: developing a research agenda[END_REF]. We cannot separate married and single due to differences between our study (living as couple/married and single) and the Lorraine census (married and unmarried) b We cannot separate these subgroups due to differences between our study and the Lorraine census c Non-included in the 2,882 subjects d Concerns the general compensation system, which includes about the two-thirds of the working population in the Lorraine region (the prevalence was similar for France)

Table 1

 1 Sample description: distribution percentages for the explanatory variables in the different employment groups (age 30-64 years)

		Men				Women
		Permanent	Temporary	Unemployed Pearson chi 2 or	Permanent	Temporary	Unemployed Pearson chi 2 or
		workers	workers		Fisher test	workers	workers		Fisher test
					(p value)				(p value)
	Total number of	1,231	130	41		858	166	74
	individuals
	Number of person-	15,295	1,569	459		10,728	2,072	907
	years until 2008
	Sociodemographic characteristics
	Age (years)
	Mean (SD)	42.0 (7.9)	42.9 (9.1)	46.4 (10.0)	0.0021	42.0 (8.0)	43.6 (10.7) 44.8 (9.4)	0.0045
	Range	30-64	30-62	30-64		30-64	30-64	30-63
	Occupation				\0.001				\0.001
	Managers	22	17	20		17	12	4
	Intermediate	21	15	15		9	4	1
	professionals,
	technicians
	Service workers,	19	22	5		57	51	54
	clerks
	Manual workers	34	32	46		11	10	26
	Others	4	14	15		7	23	15
	Risk factors
	Health-related behaviours
	Smoking				0.056				0.781
	Non-smoker	28	21	12		48	49	42
	Ex-smoker	36	43	44		26	25	26
	Smoker	36	36	44		26	25	32
	Alcohol abuse	13	22	15	0.027	4	4	5	0.797
	Health profile (self-reported health)
	Good	65	51	49	\0.001	66	60	53	0.009
	Average	30	35	34		29	33	34
	Poor or bad	4	14	17		5	7	14
	Mortality (crude death rate per 1,000 person-years)
	From all-causes	3.0	7.6	17.4		1.8	2.4	5.5
	Non-violent deaths 2.6	7.0	15.3		1.6	2.4	3.3
	Cancers	1.5	3.8	6.5		1.1	1.0	3.3
	Cardiovascular	0.5	2.5	4.4		0.2	0.5	0
	diseases
	Other causes	0.6	1.3	4.4		0.3	0.9	0
	Violent deaths	0.4	0.6	2.2		0.2	0	2.2

Table 2

 2 Association between employment status and mortality by cause: age and occupation-adjusted hazard ratios (HRs and 95 % confidence intervals)

		Men			Women
		Permanent	Temporary	Unemployed	Permanent	Temporary	Unemployed
		workers	workers		workers	workers
	Number of subjects	1,231	130	41	858	166	74
	Number of person-years 15,295	1,569	459	10,728	2,072	907
	Mortality from all-causes 1.00	2.21* (1.16-4.24)	4.09*** (1.85-9.01) 1.00	1.28 (0.45-3.62)	2.69 (0.97-7.43)
	Non-violent mortality	1.00	2.22* (1.12-4.40)	3.87** (1.66-9.03)	1.00	1.33 (0.46-3.88)	1.80 (0.51-6.32)
	Cancers	1.00	2.25 (0.90-5.65)	2.89 (0.82-10.18)	1.00	0.88 (0.18-4.21)	2.79 (0.76-10.30)
	Cardiovascular diseases 1.00	3.56* (1.02-12.44) 5.46* (1.07-27.84)	1.00	2.72 (0.21-34.95) 0
	Violent mortality	1.00	1.84 (0.22-15.44)	7.20 (0.82-63.34)	1.00	0	11.16* (1.48-84.37)
	Significant estimates are in bold . * p \ 0.05; ** p \ 0.01; *** p \ 0.001

Table 3

 3 Association between employment status and mortality for men: additional adjustment for health, tobacco and alcohol: hazard ratios (HRs and 95 % confidence intervals, reference category: permanent workers)

	Model	Temporary workers		Unemployed
		(130 men, 1,569 person-years)	(41 men, 459 person-years)
		HR (95 % CI)	% reduction in	HR (95 % CI)	% reduction in
			HR a		HR a
	Mortality from all-causes
	Adjusted for age and occupation	2.21* (1.16-4.24)	100	4.09*** (1.85-9.01)	100
	Adjusted for age, occupation and health	1.79 (0.91-3.54)	35	3.46** (1.53-7.84)	20
	Adjusted for age, occupation, health, tobacco and alcohol	1.71 (0.86-3.40)	41	3.56** (1.59-7.95)	17
	Non-violent mortality
	Adjusted for age and occupation	2.22* (1.12-4.40)	100	3.87*** (1.66-9.03)	100
	Adjusted for age, occupation and health	1.76 (0.86-3.62)	38	3.22** (1.34-7.75)	23
	Adjusted for age, occupation, health, tobacco and alcohol	1.69 (0.82-3.49)	43	3.34** (1.40-7.94)	18
	Cancers
	Adjusted for age and occupation b	2.25 (0.90-5.65)	100	2.89 (0.82-10.18)	100
	Adjusted for age, occupation and health	1.79 (0.68-4.72)	37	2.38 (0.65-8.75)	27
	Adjusted for age, occupation, health, tobacco and alcohol	1.78 (0.67-4.74)	38	2.44 (0.67-8.85)	24
	Cardiovascular diseases
	Adjusted for age and occupation	3.56* (1.02-12.44)	100	5.46* (1.07-27.84)	100
	Adjusted for age, occupation and health	3.43 (0.94-12.56)	5	5.39* (1.04-27.94)	2
	Adjusted for age, occupation, health, tobacco and alcohol	3.46 (0.95-12.67)	4	5.54* (1.08-28.51)	0
	Significant estimates are in bold * p \ 0.05; ** p \ 0.01; *** p \ 0.001

a Reduction (%) in HR computed with the following formula: 100 9 (HR age-occupation-adjusted -HR extended model )/(HR age-occupation-adjusted -1) b The age-occupation-adjusted HRs for temporary workers, and unemployed were close to significance (p \ 0.10)

  Standardized mortality ratio (for 1996-2008 period): 0.91 vs. France, 0.86 vs. Lorraine region Source: Institut National d'Etudes De ´mographiques, 1999. France me ´tropolitaine-Enque ˆte annuelle de recensement 1999 a

	Table 4 continued
		Study	General
		sample	population
	Managers, professionals and associate	32.0	32.2
	professionals b
	Farmers, craftsmen, tradesmen, heads of firm 5.6	7.0
	Service workers/clerks	23.7	28.7
	Manual workers	38.7	32.1
	Unemployed among economically active	11.9	11.8
	people c
	Annual prevalence of occupational injury	4.6	4.0 d
	with sick leave d

Acknowledgments Lorhandicap study group: N. Chau, F. Guillemin, J.-F. Ravaud, J. Sanchez, S. Guillaume, J.P. Michaely, C. Otero Sierra, B. Legras, A. Dazord, M. Choquet, L. Me ´jean, N. Tubiana-Rufi, J. P. Meyer, Y. Schle ´ret and J. M. Mur. The authors would like to thank D. Saouag, M. Weiss, M. Depesme-Cuny and B. Phe ´lut for their help during the survey. The work was supported by a Grant from the Po ˆle Europe ´en de Sante

´.

Conflict of interest

The authors declare that they have no conflict of interest.

Appendix

See Table 4.