F Mezei

M T Caccamo

F Migliardo
email: fmigliardo@unime.it

S Magazù

Resolution-Elastic Neutron Scattering by Correlation Techniques

Keywords: Resolution-Elastic Neutron Scattering, quasi-elastic neutron scattering, statistical chopper, correlation spectroscopy, instrumental resolution, numerical simulation

Introduction

Neutron probe constitutes a powerful tool in condensed matter investigations because neutrons have proper wavelengths (Angstroms) and kinetic energies (μeV to meV) to probe both the structural and dynamical properties of material systems. X-rays interact with matter via electromagnetic interactions through atomic electron clouds (atoms have sizes comparable to the wavelength of the probing radiation). Electron beams interact with matter via electrostatic interactions, and light interacts with matter through polarizability. In contrast, neutrons interact through nuclear interactions (scattering nuclei are point particles on the scale of atomic dimensions) and have a low absorption (high penetration capability) for most elements, hence representing a unique probe for bulk. Furthermore, neutrons do not heat up or destroy samples, and the atomic neutron scattering lengths vary in a non-systematic way with atomic number. Thus, neutrons offer a series of advantages, such as the possibility of isotopic labelling and the possibility of designing sample environments with high atomic number materials.

However, neutron sources are expensive to build and maintain, are characterised by relatively low fluxes compared to synchrotrons, are not effective in investigations of rapid time-dependent processes, and require relatively large amounts of samples, which is difficult when using biological specimens.

For these reasons, instrumentation improvement and enhanced computational modelling are key components for future neutron scattering development. The latest generation neutron sources, such as the European Spallation Source (ESS), offer new opportunities to combine higher beam intensities with innovative approaches, stimulating the design of flexible neutron spectrometers to span a wide range of experimental conditions. As a rule, Quasi Elastic Neutron Scattering (QENS) corresponds to energy transfers smaller than the incoming neutron energy, whereas Inelastic Neutron Scattering (INS) corresponds to larger energy transfers.

In Elastic Incoherent Neutron Scattering (EINS) the scattered intensity is collected at neutron energy transfer =0 with a given "energy resolution window" ∆ω. Compared with inelastic scattering, this contribution is higher by two or three orders of magnitude, hence providing better quality data to study small amounts of samples or small-sized samples as well as strongly absorbing samples. In this framework, the RENS technique which ultimately consists of the analysis of the EINS intensity collected at varying, as an external parameter, the instrumental energy resolution δω provides an effective and innovative tool of investigation [START_REF] Magazù | Elastic incoherent neutron scattering operating by varying instrumental energy resolution: Principle, simulations, and experiments of the Resolution Elastic Neutron Scattering (RENS)[END_REF][START_REF] Magazù | A neutron spectrometer concept implementing RENS for studies in life sciences[END_REF] for characterising the blurred boundary between elastic and quasi-elastic scattering. In an upgraded and revised work [START_REF] Magazù | Upgrading of Resolution Elastic Neutron Scattering (RENS)[END_REF], an increasing and a decreasing sigmoidal curve described the elastically scattered intensity vs increasing instrumental energy resolution and vs increasing temperature values. Summarising, energy resolution scans at a fixed temperature and temperature scans at a fixed instrumental energy resolution of the elastic intensity furnish a complementary approach for the characterisation of system dynamical properties. The RENS approach has been experimentally tested on glycerol and sorbitol [START_REF] Migliardo | Contrasting dynamics of fragile and non-fragile polyalcohols through the glass, and dynamical, transitions: A comparison of neutron scattering and dielectric relaxation data for sorbitol and glycerol[END_REF], trehalose/glycerol mixtures and hydrated lysozyme [START_REF] Magazù | Protein dynamics as seen by (quasi) elastic neutron scattering[END_REF]. Following Van Hove, the time dependent pair correlation function ) , ( t r G [START_REF] Hove | Correlations in Space and Time and Born Approximation Scattering in Systems of Interacting Particles[END_REF], indicates the probability to find a particle at distance r after a time t when a particle was at

ω ω ω Q R Q S Q S t R ⊗ = (1)
In the general case, the resolution function in ω-space has a full width

ω ω πω ω ω ω ω ω ω ω ω ω ω ω d Q S d Q R Q S Q S RES M R RES RES RES RES         ∆ - = = - = ∫ ∫ ∆ + ∆ - ∆ + ∆ - (2)
For the measured elastic scattering

) ( ) 0 , ( Q S Q S M R M R = = ω , one obtains =         ∆ = ∫ ∞ ∞ - ' ' cos ) ' , ( ) ( ω ω πω ω d Q S Q S RES M R ) , ( t Q I = with RES t ω π ∆ = (3)
Here, the Riemann lemma (as also used in Fresnel zone construction) that the integral of the product of a smooth function f and a sine or cos function averages to zero is used; furthermore outside the

      ∆ ∆ - 2 , 2 RES RES ω ω domain ) , ( ω Q S
is assumed to be a smooth function in the ω variable.

Furthermore, ) ,

( ω Q S is assumed to be an even function of ω .

In RENS, the variable energy window as the scan parameter fundamentally corresponds to the physical time in the intermediate scattering function

RES RES t ω π τ ∆ = = / .
Q S
that is consistent with the measured spectra.

Fig. 1 shows the variable energy window RENS approach achieved by integrating a measured, good energy resolution spectrum over different domains: For a given fixed energy resolution, the EISF contribution refers to system dynamics on a time scale longer than the instrumental time resolution. System dynamics occurring on a time scale shorter than the instrumental time resolution is reflected in the quasi-elastic and inelastic contributions [START_REF] Magazù | QENS study of trehalose/water/acrylamide-acrylic acid[END_REF][START_REF] Triolo | QENS from polymeric micelles in supercritical CO2[END_REF][START_REF] Jannelli | Transport properties of liquid alcohols investigated by IQENS, NMR and DLS studies[END_REF][START_REF] Magazù | IQENS -Dynamic light scattering complementarity on hydrogenous systems[END_REF][START_REF] Magazù | Quasielastic neutron scattering study on disaccharide aqueous solutions[END_REF] Notably, a complementary way to extract quantitative information from the EINS spectra is the thermal restraint approach, which consists of maintaining fixed the instrumental energy resolution while varying the system temperature; in this case, a sigmoid behaviour whose inflection point occurs when the instrumental energy resolution matches the system relaxation time [START_REF] Migliardo | Thermal Analysis on Bioprotectant Disaccharides by Elastic Incoherent Neutron Scattering[END_REF][START_REF] Migliardo | Elastic Incoherent Neutron Scatterings Wavevector and Thermal Analysis on Glass-forming Homologous Disaccharides[END_REF][START_REF] Magazù | Investigations of Homologous Disaccharidesby Elastic Incoherent Neutron Scattering and Wavelet Multiresolution Analysis[END_REF], as shown in Fig. 2.

∫ ∆ + ∆ - = 2 2 ) , ( ) ( RES RES d Q S Q S R M R

Statistical chopper and correlation spectroscopy

Neutron correlation spectroscopy has been extensively studied in the 1960s and 1970s [START_REF] Rosenkranz | CORELLI: Efficient single crystal diffraction with elastic Discrimination[END_REF][START_REF] Gordon | Correlation type time offlight spectrometer with magnetically pulsed polarized neutrons[END_REF][START_REF] Mezei | Enhanced Performance Neutron Scattering Spectroscopy by Use of Correlation Techniques[END_REF][START_REF] Pal | Correlation-Type Time-Of-Flight Spectrometer with Magnetically Chopped Polarized Neutron Beam[END_REF][START_REF] Gompf | The Use of a Pseudostatistical Chopper for Time-of-Flight Measurements[END_REF][START_REF] Crawford | Test of a correlation chopper as a pulsed spallation neutron source[END_REF][START_REF] Magazù | Correlation spectrometer for filtering of (quasi) elastic neutron scattering with variable resolution[END_REF].

Mechanical statistical choppers were first tested and then implemented as in the case of the now Statistical choppers are characterised by slits and absorbing wings occupying the chopper disc perimeter [START_REF] Mezei | Enhanced Performance Neutron Scattering Spectroscopy by Use of Correlation Techniques[END_REF][START_REF] Jan | The statistical chopper for neutron time-of-flight spectroscopy[END_REF][START_REF] Mook | Search for Bose-Einstein condensation in superfluid 4He[END_REF][START_REF] Doster | Elastic resolution spectroscopy: a method to study molecular motions in small biological samples[END_REF][START_REF] Skjold | Neutron scattering study of elementary excitations in liquid helium-3[END_REF][34]. The slit and wing widths are multiples of the perimeter fraction 1/n that defines the narrowest (unit) slit in the pattern (e.g., for n= 255, it is 1.4177°). The time corresponding to this angle is the chopper sequence time unit (e.g. for n= 255 at 18000 RPM chopper rotation speed, it is 13.123 µs). In the evaluation of the correlation function, the statistical error occurring in the calculation of the correlation function is the origin of the delicate features of the method. The most relevant feature of the RENS technique is that it offers very significant gains in the data collection rate when the intensity of the elastic or quasi-elastic contribution of interest gives the major part of the integral of the whole scattering spectrum [START_REF] Mezei | Enhanced Performance Neutron Scattering Spectroscopy by Use of Correlation Techniques[END_REF].

Since the beam intensity in correlation spectroscopy is independent on the resolution achieved via the random beam modulation (the time average transmission of the statistical chopper is fixed, commonly to 50 %), correlation choppers offer a very flexible way of changing instrumental resolution.

To prove the feasibility and obtain a realistic estimate of the gain in efficiency achievable by crosscorrelation for energy discrimination, experimental simulations have been performed by using MATLAB software.

The simplest modulation function would be a sequence of rectangular pulses with ) (t m equal to one or zero during equidistant time intervals (Fig. 3a). For example, the mechanical chopper uses a rotating disc with transparent slits corresponding to a pattern of type Fig, 3a. Since the slit width must be equal to the beam width for the optimum intensity, the single pulses become triangles of half width θ and multiple pulses become trapezoids (Fig. 3b). In Fig. 3, an example of a periodic pseudo-random function is shown. With mechanical disc choppers, only periodic pseudo-random pulse sequences can be produced, while truly random sequences should be aperiodic. This A binary sequence (BS) is a sequence

∑ - = + = 1 0 ) ( N j v j j a a v C ( 4
)
has two values:

   = , , ) ( mc m v C otherwise v 0 = ( 5
)
where

1 1 - - = N m c ( 6
)
is the PRBS duty cycle. The PRBS is 'pseudorandom' because, although it is deterministic, it seems to be random in a sense that the value of an j a element is independent of the values of any of the other elements, similar to real random sequences. The correlation function decreases due to the decrease of the number of sums; therefore, the sequence length can be prolonged to avoid this influence, e.g., by duplicating it.

With a statistical chopper, a pseudo-random sequence must be considered as the sequence reported in the table on the right of Fig. 4 constituted by 255 elements, equal to 2 n -1, where n= 8. This sequence gives rise to the chopper design on the left in Fig. 4 [START_REF] Mezei | Enhanced Performance Neutron Scattering Spectroscopy by Use of Correlation Techniques[END_REF].

Fig. 4 Statistical chopper (on the left) realised on the basis of the sequence reported in the table (on the right).

Fig. 5 shows the "ideal" rectangular linear pattern of a pseudo-random statistical chopper. [1 0 0 0 0 0 1 0 1 1 0 0 0 1 0 0 0 1 1 1 1 0 1 0 1 0 1 0 0 1 0 0 1 1 1 0 1 1 1 1 1 0 1 1 1 0 0 0 1 0 0 1 1 1 0 1 1 1 0 0 0 0 0 1 0 0 1 1 0 1 0 0 1 0 0 0 0 0 1 0 1 0 1 1 0 0 0 0 1 0 1 0 1 1 0 1 0 0 0 1 1 1 1 0 0 1 0 1 1 0 0 0 0 0 0 1 0 0 0 0 1 1 0 1 1 0 1 1 0 0 0 1 0 0 1 1 0 1 0 1 0 1 1 1 1 1 1 0 1 1 0 1 1 0 0 1 1 1 0 0 1 0 0 1 0 1 1 1 0 0 0 0 1 1 1 0 1 0 0 1 1 0 1 1 1 1 1 1 1 0 0 0 1 0 1 0 1 1 0 1 0 1 0 0 1 1 0

0 0 0 1 1 1 1 1 0 0 1 1 1 1 1 1 1 0 1 0 1 0 1 0 0 0 0 1 0 0 1 0 0 1 1 1 0 0 1 1 1 0 0 0 1 1 0 0] 14

Simulation results

To examine the basic mathematical features of this approach, the process on a schematic model construction has been simulated.

In Fig. 8, these three functions are shown in panels a), b) and c); it can be observed that the assumed detector spectrum ) (t z looks like random fluctuations and has no resemblance to the signal function ) (t s . However, the signal function can be fully recovered if the cross-correlation function between the modulation and detector signal [START_REF] Mezei | Enhanced Performance Neutron Scattering Spectroscopy by Use of Correlation Techniques[END_REF] is computed: In order to test the validity of such an approach a set of new simulations, taking into account only one spectral contribution with an increased linewidth, have been performed. In particular, the above pseudo-random sequence ) (t m and three Lorentzian functions ) (t s centered at t=128 and linewidth 0.7, 1.8 and 3.2, respectively, have been considered: Fig. 9 reports the simulations performed with three Lorentzian functions with different amplitude. In Fig. 10, a sketch of the flowchart of the simulation process leading to Fig. 8 Furthermore, as shown in a previous study [START_REF] Magazù | Correlation spectrometer for filtering of (quasi) elastic neutron scattering with variable resolution[END_REF], simulation results have been obtained for the measured conventional and statistical chopper spectra and for the calculated correlation spectra for a quasi-elastic model function with long tails in the presence of a uniform, "sample independent" background of 100 times the sample peak intensity. During the same measuring time, the signal remains unobservable next to the background created by statistical noise in conventional spectroscopy, and clearly revealed in some detail by using a statistical chopper, that provides 100 times higher incoming beam intensity on the sample for correlation based data collection. In the mentioned study, the obtained curves also could be part of a RENS experiment, with changing the quasi-elastic resolution by adding the contents of a certain number of channels. The simulation data well illustrate the decisively enhanced data collection rate obtained by the statistical chopper in the case of high intensity background compared to the sample scattering, which can e.g. be a very practical situation for samples only available in small quantities.

) ( ) ( ) ( t s t m t z = * (9)

Conclusions

In this work, the RENS spectroscopy is described, providing evidence of the main important features. Then, it is demonstrated that the correlation neutron spectroscopy based on a statistical chopper offers significant advantages, including significant gains in beam intensity and enhanced opportunities for variable resolution studies requiring flexibility in selecting the proper balance between resolution, intensity, and sensitivity to external background.

A formulation of neutron correlation spectroscopy, which makes it possible to reconstruct neutron scattering spectra from time-modulated detected beam intensity data, is also presented. The numerical simulation results confirm the perfect reconstruction of a model scattering function for a schematic example of a beam modulation algorithm. The prominent efficiency of the correlation method in the case of the presence of very high intensity sample independent background is also shown by further simulation results.

  towards high RENS energy resolutions, i.e., with increasing RES RES ω π τ ∆ = / . In particular, on the left column of the figure, a fixed scattering law , with a Gaussian profile, and different instrumental energy resolution windows (coloured rectangles) are reported. On the right column of the figure, the integrated resolution-elastic intensity as a function of the logarithm of the instrumental resolution is shown; furthermore, the red circle with a black contour, corresponding to the fourth point, represents the curve inflection point.

Fig. 1

 1 Fig. 1 Left column: fixed scattering law

Fig. 2

 2 Fig. 2 Left: Thermal restraint approach consisting of maintaining fixed instrumental energy resolution

  decommissioned time-of-flight (TOF) spectrometer IN7 at Institute Laue Langevin (ILL) and the recently commissioned CORELLI spectrometer at Spallation Neutron Source (SNS). Correlation techniques that were initially developed in connection with steady-state neutron sources have been adapted to pulsed spallation neutron sources, allowing for the analysis of TOF spectra through their fine time modulation without corresponding monochromatisation of the beam[START_REF] Tomiyasu | Modified cross-correlation for efficient white-beam inelastic neutron scattering spectroscopy[END_REF][START_REF] Ye | Implementation of cross correlation for energy discrimination on the time-of-flight spectrometer CORELLI[END_REF]. More specifically, the scattering law in such a case is calculated by evaluating the cross-correlation between the measured signal and the used beam modulation sequence, which latter ideally should be of a random type[START_REF] Mezei | Enhanced Performance Neutron Scattering Spectroscopy by Use of Correlation Techniques[END_REF]. Comparisons to current state of the art direct time-of-flight instruments at a steady state source, rep-rate multiplication (multiplexing) and inverted time-of-flight are reported in refs.[START_REF] Mezei | Enhanced Performance Neutron Scattering Spectroscopy by Use of Correlation Techniques[END_REF][START_REF] Russina | Upgrade project NEAT′2016 at Helmholtz Zentrum Berlin -What can be done on the medium power neutron source[END_REF][START_REF] Russina | First implementation of novel multiplexing techniques for advanced instruments at pulsed neutron sources[END_REF][START_REF] Sivia | Optimization of resolution functions for neutron scattering[END_REF][START_REF] Fedrigo | VESPA: The vibrational spectrometer for the European Spallation Source[END_REF][START_REF] Price | A detailed evaluation of the mechanical correlation chopper for neutron time of-flight spectrometry[END_REF]

Fig. 3

 3 Fig. 3 Example of a periodic pseudo-random function: a) "ideal" rectangular pattern and b) "true" trapeze

Fig. 5 "

 5 Fig. 5 "Ideal" rectangular pattern of the pseudo-random function of the statistical chopper.

Fig. 6 "

 6 Fig. 6 "True" trapeze pattern of the pseudo-random function of the statistical chopper.

Fig. 7

 7 Fig. 7 shows a diagram of a simplified correlation technique-based instrument; the neutron beam is time-modulated by a statistical chopper following the law ) (t m , being 1 ) ( 0 ≤ ≤ t m , while the

Fig. 7

 7 Fig. 7 Sketch of a correlation technique-based schematic instrument where a neutron beam produced by a

Fig. 8 a

 8 Fig. 8 a) Function representing the pseudo-random sequence ) (t m ; b) signal ) (t s constituted by the sum of

Fig. 9

 9 Fig. 9 Simulations performed with three Lorentzian functions with different amplitude: on the left the signal

Fig. 10

 10 Fig. 10 Sketch of the simulation procedure. The function ) (t m represents the statistical chopper, and ) (t s is

Acknowledgements

Salvatore Magazù and Federica Migliardo gratefully acknowledge financial support from Elettra -Sincrotrone Trieste in the framework of the PIK project "Resolution-Elastic Neutron Scattering Time-of-flight Spectrometer Operating in the Repetition Rate Multiplication Mode".

Graphical abstract

A neutron beam is intercepted by a statistical chopper, with sequence m(t), before the sample while the scattered beam is registered by a detector, z(t); the crosscorrelation between this latter and the chopper sequence provides the scattering law, s(t).


Jan Holub
email: jholub@iciq.es

Antonio Santoro

Jean-Marie Lehn

Electronic absorption and emission properties of bishydrazone [2 x 2] metallosupramolecular grid-type architectures

Keywords: Hydrazone, Metallosupramolecular grids, Coordination architecture, Photoluminescence of coordination compounds

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

Introduction

In the last two decades, photoactive organic compounds gained significant attention for their application in organic light-emitting diodes (OLED) [1], organic solar cells [START_REF]Grid Metallosupramolecular Architectures from Preformed Ditopic Bis(acylhydrazone) Ligands and through Component Self-Assembly)[END_REF] or fluorescent chemosensors [START_REF] Li | Ru(bpy)2+ and other remarkable metal-to-ligand charge transfer (MLCT) excited states); c) Electrochemiluminesence of ruthenium complex and its application in biosensors[END_REF]. Nowadays, many of the promising fluorescent and luminescent 2 compounds still rely on the properties of expensive precious metals such as Ir(II) [4][5], Ru(II) [4][6], Pt(II) [4][7] or expensive lanthanides (Eu, La,) [8]. Therefore, recently much effort was put into their substitution with less expensive common metals such as Al(III), Cu(II), Zn(II). [9]. Among these, zinc appears especially appealing for its accessibility and simple chemistry.

Many fluorescent and phosphorescent organo-zinc complexes are showing potential in biological studies [10] or as emitters for organic light-emitting diodes [START_REF] Dumur | [END_REF]. Extensive use has been made of a tridentate NNN terpyridine-type design for the coordination subunits of the ligands in these complexes, which in some cases present a photoluminescence quantum yield exceeding 90%. [12] In addition, bisterpyridine ligands (L Tpy , Figure 1 left) are widely recognized supramolecular building blocks capable of a formation of highly ordered square-shaped metallo-supramolecular arrays further referred to as [2x2] grids. [13] Initial studies carried out with the bisterpyridine ligand L Tpy (Figure 1 left) showed that an acetonitrile solution of its Zn(II) grid L Tpy 4Zn4 displays a bluish fluorescence with an emission maximum at 450 nm (λexc = 335 nm) and a moderate quantum yield of 11% ‡ . [14] Coincidentally, the hydrazone generated by the condensation of pyridine-2-carboxaldehyde with 2-hydrazinopyridine represents an easily accessible isosteric analogue of the terpyridine motif with well-described coordination behaviour [15]. By extension, double hydrazone isosteric analogues of the bisterpyridine ligand L Tpy such as L1 (Figure 1 right) and L2 (Figure 2) have been implemented for the assembly of hydrazone-based [2x2] grid complexes [13,[START_REF] Schmitt | [END_REF]. One may also note that the presence of hydrazone functional groups confers dynamic covalent features to these ligand entities [17].

Experimental:

2.1.General information

Commercially available chemicals and solvents were purchased from Sigma-Aldrich, Alfa Aesar, and STREM and were used without any additional purification. The 4,19], 4,6-bis(1-methylhydrazinyl)-2-phenyl-1-pyrimidine [16a],

methyl-1-(2-pyridyl)hydrazine [20], 2-phenyl-4,6-pyrimidinedicarboxaldehyde [16a, 21] and ligands L1 [START_REF] Schmitt | [END_REF], L2 [START_REF] Schmitt | [END_REF] and L1 H [START_REF] Schmitt | [END_REF] were prepared according the procedures described in the corresponding literature references.

NMR spectra were recorded on Bruker Avance 400 (400.

2.2.General procedure for ligand synthesis (Figure 2)

The 4,6-bis(1-methylhydrazinyl)-2-phenylpyrimidine (1 eq., 2.05x10 -4 mol) was dissolved in EtOH (20 ml) followed by the addition of an appropriate aldehyde (2.1 eq, 4.3x10 -4 mol).

Reaction mixture was then heated under reflux for 5-12 h. The reaction proceeds well even in case when the reaction components are not completely soluble in EtOH. For such cases, the longer reaction time is recommended. In all cases, an appearance of the precipitate was observed during the reaction period. After the reaction was cooled down to the room temperature this precipitate was filtered off and washed with ethanol yielding the desired bishydrazone ligands (L1 XX ) in high yields (70-95 %). The analytical descriptions and accompanying spectra of individual ligands are provided in the accompanying electronic supporting info (ESI).

The same procedure was applied for the preparation of the other constitutional isomer (L2), only this time using 2-phenyl-4,6-pyrimidinedicarboxaldehyde (1 eq., 2.05x10 -4 mol)

and 1-methyl-1-(2-pyridyl)hydrazine. [START_REF] Schmitt | [END_REF] The ligand L2 was isolated as a bright orange solid in >80 % yield.

2.3.General procedure for complex preparation L4M4

To the preformed ligand (L1 XX , L2, 1 eq., 4.73 x 10 -4 mol) in acetonitrile (5 ml) was added M(OTf)2 (Zn(OTf)2 or Cd(OTf)2 x 6 DMSO, 1 eq., 4.73 x 10 -4 mol) and the reaction mixture was heated at 70 ºC overnight. Afterwards, the reaction mixture was cooled down to the room temperature and filtered through the cotton pad. The assembly is clean and almost quantitative as checked by NMR of the reaction mixture. The desired grid complex could be precipitated/crystalised from the acetonitrile solution by the vapour diffusion of diethyl ether into the reaction solution. The analytic descriptions and accompanying spectra of individual complexes are provided in the accompanying electronic supporting info (ESI).

2.4.UV-VIS, fluorescence and quantum yield measurements

To obtain UV-Vis absorption spectra the ligands were dissolved in spectrophotometric or HPLC grade CHCl3 or DMSO regarding their individual solubility. Typically, 1 ml of 1 mM solution was prepared beforehand from which 50 μl were transferred into the quartz UV-Vis cuvette and diluted to the 3 ml (1.7 x 10 -5 M) with the solvent of choice. This concentration was chosen so the absorbance of all the measured compounds falls into the range of 0.6 -1.In all cases, the background spectrum of the blank solution was collected first and then subtracted from the sample. All UV-Vis absorption spectra of the complexes were measured in spectroscopic grade acetonitrile by the same procedure using the working concentration of 2.5 x 10 -6 M of the L4M4 grid complex.

To obtain the fluorescence spectra of the metal complexes, 1 ml of the 1 mM acetonitrile solution was prepared from which typically 2-10 μl equivalents were transferred to the quartz cuvettes, diluted to 3 ml volume and the fluorescence spectra recorded. The reported spectra were typically measured at the concentration of 6 x 10 -7 M of the .L4M4.

The solid-state fluorescence spectra were measured by introducing several crystal grains of the sample compound between two quartz slides. These slides were fixed together and introduced at the angle of 45º to the slot in the measuring machine.

The quantum yield was measured by integrating sphere in quartz tubes, using the previously prepared acetonitrile solution of the ligand or solid powdered sample.

Results and Discussion:

3.1.Synthesis and structural features of hydrazone ligands and their[2x2]grid type complexes

The fluorescence of Zn(II)-hydrazone complexes has been known for a long time [22], but only few studies were performed on oligonuclear hydrazone complexes. The complementary aldehydes were either obtained commercially (L1, L1 5Me , L1 6Me ) or synthesised according to the available literature (L1 5OH [START_REF] Seredyuk | [END_REF], L1 COOMe [START_REF] Ch | Anticancer Efficacy of Unique Pyridine-Based Tetraindoles[END_REF], L1 OAcOMe

[adapted from 24], L1 PhtIm [adapted from 24], See ESI). Due to the significantly inferior spectroscopic performance (weak emission and low quantum yield) of the L2 isomer, only the basic derivative was studied and its spectroscopic behaviour is discussed further below.

Subsequently, the addition of 1 eq. of Zn(OTf)2 (or Cd(OTf)2x6 DMSO) in acetonitrile to the ligand L1 XX or L2 provides the desired [2x2] grid in close to quantitative yields in all cases.

All the complexes have tetrameric [2x2] square geometry of the general formula [L4M4] 8+ .

This structure is in particular indicated by the presence of corresponding fragments in the HR-MS spectra as well as by distinctive doublets in the 1 H NMR spectrum (5 -6.5 ppm, Figure 5 top, see also ESI of respective grids), characteristic for this family of grid complexes. [14, 16d] In the case of L1 5OH 4Zn4, its highly ordered grid-like structure was directly ascertained by the structural X-ray analysis of a suitable single crystal (obtained by vapour diffusion of the Et2O into an ACN solution, Figure 3, see also ESI L1 5OH 4Zn4). Its crystal structure illustrates the highly ordered and rigid assembly of the grids based on the bishydrazone coordination motif. [13,14,[START_REF] Schmitt | [END_REF] Noteworthy are the parallel layout of the ligands with the defined distance between them (6.58 Å) and the almost perpendicular angles (91.49º) around the metal centers. Furthermore, the spatial positioning of the eight hydroxyl groups evenly distributed around the periphery could make these grids an interesting building block for the bottom up assembly of the higher 2D structures.

[16f]

3.2.Photophysical properties of bishydrazone ligands and grids: Absorption behaviour of ligands

The absorption spectra of the ligands L1 XX cm -1 ), 300 nm (44 000 M -1 cm -1 ) and 347 nm (22 900 -1 cm -1 ) and for L2 at 259 nm (22 800 M -1 cm -1 ), 340 nm (40 000 M -1 cm -1 ) and 378 nm (21 700 M -1 cm -1 ).

In generall, the UV-Vis absorption spectra of L1 XX and L2 ligand familly (Figure 2, Table 1) is dominated by π-π* spin allowed transitions. [14,23] The presence of the different weakly electron-donating groups in L1 XX plays a marginal role on the absorption behaviour, by varying the maxima peaks of few nm compared to the L1 model (Tab. 1, for absorption spectra see respective ESI). The only anomaly is presented by ligand L1 COOMe (ESI L1 COOMe ) whose absorbance peaks are much broader with the second maximum at 336 nm being stronger in comparison to the first at 313 nm and also with a visible shoulder around 375 nm. In a case of L1 Phtim , its low solubility in tested solvents prevented a measurement of the relevant spectroscopical data.

Absorption properties of the grids

In the case of the L24Zn4 grid the formation of the complex in acetonitrile is accompanied by the appearance of a characteristic orange colour while solutions of the L1 xx 4Zn4 are generally colourless, displaying the marked differences of their absorption spectra. (Figure 5 Left, Tab. 2, also see respective ESI) Especially interesting in the case of L24Zn4 absorption, is the complete loss of the vibrational structure, the consequential broader band and the pronounced red shift of its visible region maximum (λmax = 464 nm, ε = 152 700 M -1 cm -1 ,) in comparison to the L14Zn4 complex. (Figure 5 Left)

The shape of the double maxima peak (373 nm, ε = 228 300 M -1 cm -1 and 390 nm, ε = 209 400 M -1 cm -1 ) observed in electronic spectrum of L14Zn4 (Figure 5) is characteristic for almost all the derivatives of L1 and closely resembles that of related complexes already described in a literature. [14,[START_REF] Schmitt | [END_REF] The only exception to this is the grid L1 6Me 4Zn4 (ESI Figure 61) which displays a partial loss of structure with a broad peak with only a single maximum at 379 nm and which may be associated with a distortion of the coordination centres caused by the steric interactions introduced by the methyl groups.

All the L1 XX 4Zn4 grids from substituted ligands show a bathochromic shift between 6-14 nm (424-969 cm -1 ) for first maxima and 15-17 nm (949-1071 cm -1 ) when compared with L14Zn4 (Table 2, see also ESI of the respective grids). It seems that the steric distortion influences the shift and shape of an absorption spectrum (L1 5Me ~12 nm, 835 cm -1 vs. L1 6Me ~7 nm, 424 cm -1 , Table 2) more than the electron donating or withdrawing nature of the substituents (L1 5Me ~12 nm, 835 cm -1 vs. L1 COOMe ~12 nm, 835 cm -1 , Table 2). grids from substituted ligands show a bathochromic shift between 6-14 nm (424-969 cm -1 ) for first maxima and 15-17 nm (949-1071 cm -1 ) when compared with L14Zn4 (Table 2, see also ESI of the respective grids). It seems that the steric distortion influences the shift and shape of an absorption spectrum (L1 5Me ~12 nm, 835 cm -1 vs. L1 6Me ~7 nm, 424 cm -1 , Table 2) more than the electron donating or withdrawing nature of the substituents (L1 5Me ~12 nm, 835 cm -1 vs. L1 COOMe ~12 nm, 835 cm -1 , Table 2).

Regarding the L4Cd4 grids, in general these complexes display properties similar to those of their Zn(II) counterparts. With a comparison to L14Zn4, the electronic spectrum of L14Cd4 shows a stronger absorption with the maximum in the same regions of 373 nm (ε = 527 200 M -1 cm -1 ), however, with the second maximum reduced to a shoulder at 386 nm (ε = 432 700 M -1 cm -1 ). (Figure 5 Left, ESI Figure 18)

Emission behaviour of ligands and grids

Name

UV-Vis maxima/nm (ε/ M -1 cm -1 )

UV-Vis maxima/nm (ε/ M -1 cm -1 )

UV-Vis maxima/nm (ε/ M -1 cm -1 ) L1

259 (22 800) 300 (44 000) 347 ( 22 S12) and ~4 % for L24Zn4. The increase in quantum yields upon complexation can be explained by a decrease in non-radiative decay rates. This decrease is caused by straightening of the bent geometry/conformation with inhibited rotational motion of the central phenyl substituent (Figure 1 Right) [14, 16d] as well as an inhibition of the photoinduced electron transfer (PET) via binding a lone electron pair of the nitrogen to the metal cation. [START_REF] Konar | spectroscopic and photoluminescence studies of manganese(II), cobalt(II), cadmium(II), zinc(II) and copper(II) complexes with a pyrazole derived Schiff base ligand)[END_REF] The observed 95 nm red shift of the emission maxima between the L2 and L24Zn4. (Figure 6) may be related to the influence of metal ion binding on the levels involved in the intraligand charge transfer absorption. A contribution can also come from π-π stacking between ligands and intercalated phenyl rings in the complex. The overall result is a remarkable difference in the emission wavelengths and intensities between the ligands and grids.

Previous work on terpyridine based compounds [14] indicated that the fluorescence of this family of grids is closely related to the d 10 nature of the Zn(II) cation. In confirmation of these findings, excitation of the complexes of L1 with different metal cations (Cu(I), Cu(II), Pb(II), In a same way as for the electronic spectra, the peripheral substitution at L1 induces a small red shift (Figure 8). Almost all emission spectra display two peak maxima except for L1 H 4Zn4 (ESI Figure 22) and L1 COOMe 4Zn4 (ESI Figure 48) where there is a partial loss of the vibrational structures and the second maximum is reduced to a shoulder. The L1 COOMe 4Zn4 maximum (at 428 nm) also shows the largest red shift of ~20 nm in the comparison to the maximum displayed by L14Zn4 (at 408 nm). A summary of the emission maxima and quantum yields measured for the [2x2] grid complexes studied herein is given in Table 3. The quantum yields listed in Table 3 show that the strongest emission and highest quantum yield (φ) correspond to the simplest bishydrazone grid L14Zn4. On the opposite side of the series is the unsubstituted derivative with ionisable hydrazone N-H L1 H 4Zn4 [17, 18b], which has a low quantum yield of approximately ~2%. The reason for this is possibly the quenching via hydrogen abstraction in the excited state. [30] A similar effect may be present in the derivative L1 5OH 4Zn4 whose quantum yield falls to ~8 %. The substitution of the hydroxyl substantially increases φ as could be observed L1 Phtim 4Zn4 (φ = ~37 %) and L1 OAcMe 4Zn4 (φ = ~36 %). Unfortunately, in the cases of L1 OAcMe 4Zn4 and L1 COOMe 4Zn4 the acetonitrile solution shows dissociation of the grid structure with formation of some amount of a "corner" complex L1 COOMe 2Zn3, which was also identified by single-crystal X-ray diffraction (see ESI). This complex is most likely also fluorescent thus making it difficult to discuss the quantum yields of these two compounds.

Regarding the position of substituents on the pyridine moieties it can be concluded that the quantum yields of "5"-substituted pyridines (L1 5Me 4Zn4) are much higher (~40 %) than those of the "6"-substituted ones (L1 6Me 4Zn4, ~5 %), possibly due to the steric hindrance from methyl groups when forced into its grid form in the latter case.

Conclusion

Herein, we presented a study of spectroscopic features of different Zn(II) and Cd(II) [2x2] grid-type complexes. Interestingly, the best performer is also the simplest ligand (L14Zn4).

The grids which were built from 4,6-bis(1-methylhydrazino)-2-phenylpyrimidine (L1 ligands) had substantially more intense emission than those built from 2-phenyl-4,6pyrimidinedicarboxaldehyde (L2 ligand). It was observed that the electronic absorption, emission spectra and quantum yields of both ligands and grids were very sensitive to the sequence direction of the central hydrazone group. The peripheral substitution by (weakly) electron donating/withdrawing groups was observed to play only a minor influence on the absorption and emission spectra but the nature of the substituent has a strong impact on the quantum yields of individual grid complexes. The results described demonstrate the effects of constitutional isomerism, metal cation and peripheral substitution on spectroscopic features of bishydrazone ligands and grid-type complexes.

Footnotes

Figure 1 :

 1 Figure 1: Illustration of the structural and [2x2] grid assembly analogies between bisterpyridine L Tpy ligand (left) and double hydrazone (pyridine=N-N-pyrimidine) L1 ligand (right).

  [23] Regarding the bishydrazone-based ligands L1 and L2 investigated herein, some interesting spectroscopic aspects involving pH dependent colour change were already mentioned in one of the first reports on this family of grids [16b]. However, despite these ligands being known for a rather long time, their photoluminescence properties were so far largely overlooked and only briefly mentioned in the literature [13a, 14 ‡]. It was thus of interest to perform a spectroscopic study of the differently functionalized bishydrazone ligands L1 and L2 and of their [2x2] grid-type complexes formed with Zn(II) and Cd(II) cations. Special attention has been given to the effects of the imine bond position

Figure 2 .

 2 Figure 2.: Top: Synthesis of the two constitutional isomers of bishydrazone: L1 derived from a bis-hydrazine core and L2 from a bis-aldehyde core; schematic representation of the square [2x2] grid-type complexes formed by these ligands. Bottom: Molecular structures of differently functionalized ligands studied in this work.

and

  Figure 4, see ESI for the rest). The model ligands L1 and L2 display three bands with maxima for L1 at 259 nm (ε = 22 800 M -1

Figure 3 :

 3 Figure 3: The crystal structure of octahydroxyl grid [L1 5OH 4Zn4](OTf)8.

Figure 4 :

 4 Figure 4: UV-VIS absorption spectra of the ligands L1 and L2 in CHCl3. (1.7x10 -5 M in acetonitrile)

Figure 5 :

 5 Figure 5: Left: Absorption spectra of L14Zn4 (blue), L14Cd4 (green) and L24Zn4 (red). Right: Absorption spectra of L14Zn4 at various concentrations.

Figure 6 :

 6 Figure 6: Left: Powder emission spectra of ligands L1 (Blue) and L2 (orange) under λex = 365 nm. Middle: Colours of individual ligands and grids under white light and UV light. Right: The powder emission of grids L14Zn4 (blue) and L24Zn4 (orange) and at λex = 365 nm. (ISIS -Institut de Science et d'Ingénierie Supramoléculaires)

Figure 7 :Figure 9 )

 79 Figure 7: Left: Emission spectra of L14Zn4 (6x10 -7 mol/dm 3 , acetonitrile) and L14Cd4 (6x10 -7 mol/dm 3 , acetonitrile) at λex = 365 nm; Middle: Visual comparison of the L14Zn4 (Blue), L24Zn4 (Orange) and L14Cd4 (blue) emissions under λex = 365 nm; Right: The emission spectra of L24Zn4 (4x10 -6 mol/dm 3 , acetonitrile) at λex = 365 nm;

Figure 8 :

 8 Figure 8: Emission spectra of selected L1 XX 4Zn4 grid complexes in acetonitrile. (Each c = 6x10 -7 mol/dm 3 of grid)

  14 MHz for 1 H and 100.62 MHz for 13 C) and Bruker Avance III plus 400 (400.34 MHz for 1 H and 100.67 MHz for 13 C) NMR

	Melting points were measured on a Buchi Melting Point B-540 apparatus and temperature
	data are uncorrected.
	The X-ray crystallography studies were performed at the Service de Radiocrystallographie,
	University of Strasbourg and at the Karlsruhe Institute of Technology. At the University of
	Strasbourg crystallographic data were collected on a Bruker Kappa Apex II diffractometer
	using graphite-monochromated Mo-Ka radiation (λ = 0.71073 Å). Crystallographic CIF data
	can be found deposited in the Cambridge Crystallographic Data Centre database under the
	CCDC 1897491 (L1 5OH 4Zn4) and CCDC 1897489 (L1 (COOMe) 2Zn3) codes.

spectrometers. All the collected spectra were referenced on residual solvent signal according to Nudelman et al.

[START_REF] Gottlieb | [END_REF]

Mass spectra were obtained on Bruker MicroTOF and HRMS on Bruker MicroTOF-Q, both with electrospray ionization. Nominal precision of the HRMS analysis is 10 ppm.

UV-VIS spectra were recorded on a JASCO V670 dual beam spectrophotometer. The fluorescence spectra were recorded on Horiba Jobin-Yvon IBH FL-322 Fluorolog 3 spectrometer equipped with 450 W xenon arc lamp, double grating excitation and emission monochromators (2.1 nm x mm -1 of dispersion; 1200 grooves mm -1 ) and a TBX-04 single photon-counting detection. Emission and excitation spectra were corrected for source intensity (lamp and grating) and emission spectral response (detector and grating by standard correction curves. The photoluminescence quantum yield is a Quantaurus C11347 (Hamamatsu, Japan).

Elemental analysis was performed at the Service de Microanalyse, University of Strasbourg at ThermoFisher Scientific Flash 200 with absolute precision of 0.3 %.

Table 1 :

 1 UV-Vis absorption maxima and extinction coefficients (ε) of the ligands L1 xx and L2 in chloroform

Table 2 :

 2 UV-Vis absorption maxima and extinction coefficients (ε) of the [2x2] grids of

	L1 xx 4Zn4 and L24Zn4 in acetonitrile
	Name	UV-Vis	UV-Vis	UV-Vis	UV-Vis
		maxima/nm (ε/ M -1 cm -1 )	maxima/nm (ε/ M -1 cm -1 )	maxima/nm (ε/ M -1 cm -1 )	maxima/nm (ε/ M -1 cm -1 )
	L14Zn4	-	-	373 (228 300)	390 (209 400)
	L24Zn4	-	-	-	464 (152 700)
	L14Cd4	-	-	372 (527 200)	386 (432 700)
	L1 5OH 4Zn4	222 (90 200)	279 (83 500)	385 (234 800)	405 (250 300)
	L1 Phtim 4Zn4	219 (390 500)	286 (103 300)	387 (297 700)	407 (317 800)
	L1 OAcMe 4Zn4 -	284 (101 200)	385 (313 100)	405 (334 800)
	L1 COOMe 4Zn4 247 (97 600)	-	385 (257 700)	402 (260 600)
	L1 5Me 4Zn4	222 (9 000)	279 (83 500)	376 (234 800)	394 (250 300)
	L1 6Me 4Zn4	237 (87 000)	269 (74 600)	379 (195 600)	-
	All the L1 XX 4Zn4

Table 3 :

 3 Quantum yields of chosen grids

	Name	First Emission	Second Emission	Fluorescence
		Maximum (λex =	Maximum (λex =	quantum yield Φ(%)
		360 nm, ACN)	360 nm, ACN)
	L1 (solid state)	409 (s)*	518 (s)*	<1
	L2 (solid state)º	503(s)	-	2.4
	L14Zn4	409	429	60
	L14Zn4 (solid state)	440	-	~3
	L24Zn4	585	-	6
	L24Zn4 (solid state)	598	-	~4
	L14Cd4	409	429	8
	L1 5OH 4Zn4	419	445	8
	L1 H	407	-	2
	L1 Phtim 4Zn4	421	447	37
	L1 OAcMe 4Zn4	420	445	36
	L1 COOMe 4Zn4	428	-	41
	L1 5Me 4Zn4	410	434	40
	L1 6Me 4Zn4	415	436	5
	*(s) -solid state

‡ I. Manet, V. Balzani, D. Volkmer, J.-M. Lehn, unpublished results. Related species gave emission quantum yields of < 1x10 -3 and 0.11 for the ligand and the Zn(II) grid complex, respectively. [14]

Acknowledgements

We thank Dr. Olaf Fuhr, Prof. Dieter Fenske, Lydia Brelot and Corinne Bailly for the crystal structure determinations. We thank the ANR (grant DYNAFUN), the ERC (Advanced We also thank Karlsruhe Nano Micro Facility (KNMF) for financial support of the Karlsruhe X-ray facility.

Appendix A. Supplementary data

Electronic supplementary information to this article can be found online… Crystallographic CIF data can be found deposited in the Cambridge Crystallographic Data Centre database under the CCDC 1897491 (L1 5OH 4Zn4) and CCDC 1897489 (L1 (COOMe) 2Zn3) numbers.

indoles by excited-state proton transfer); b) K. K. Kalninsh, Opt. Spectrosc. 103 (2007) 549-567 (Electron-proton effect in excited molecular complexes: a review) ---------TOC


Alexandru Szathmari

M Zerah

M Vinchon

C Dufour

E Gimbert

F Di

S Chabaud

C Conter

C Mottolese

D Frappaz

Affiliations

Ependymoma of the spinal cord in children. A Retrospective French study

Keywords: ependymoma in children, spine surgery, radiation therapy

Background: Ependymoma is the most frequent spinal tumor in adults but it is rather uncommon in children.

The aim of this study is to summarize retrospectively clinical and therapeutic experience in the treatment of pediatric spinal ependymomas in France.

Material and methods: In this retrospective multicenter study, data from patients younger than 18 years old treated between 2000 and 2010 for spinal ependymomas were collected. Epidemiologic, tumor-and treatment-related data were analysed. Prognostic factors for progression free survival (PFS) were assessed.

Results: Twenty-eight patients (22M, 6F) were identified. Median age at surgery was 13.67 (0.7 to 17.6 years). Initial gross total removal was achieved in 22 children and subtotal removal in 6. Histologically, 15 were myxopapillary ependymomas, 11 grade II and 2 grade III ependymomas. Adjuvant initial radiotherapy (RT) was performed in 6 patients. Median follow-up was of 40 months (2.3 to 127.5 months). The 5-year PFS is 51% (IC95% = 26.3 ;71.2) and the overall survival (OS) is of 100%. In univariate analysis, only the gross total removal (GTR) had significant influence on the PFS (p=0.0013). A subgroup analysis showed a benefit of RT delivered to patients with GTR but it failed to prevent relapse in the group with initial subtotal removal (STR).

Introduction

Primary spinal cord ependymomas (SpEp) are rare in children. They account for less than 13% of all CNS ependymomas in the group between 0-19 years of age [START_REF] Mcguire | Incidence patterns for ependymoma: a Surveillance, Epidemiology, and End Results study[END_REF] . Population based registries show an incidence rate as low as 0.06 per 100.000 people per year in US with predominance in male gender and in non-Hispanic whites [START_REF] Schellinger | Descriptive epidemiology of primary spinal cord tumors[END_REF] . Age at diagnosis usually lies between 10 and 14 years. Histologically, spinal ependymomas comprise grade I myxopapillary ependymomas (MPE) that predominantly affect the conus-cauda equina region and WHO grade II/III tumours that may be found at any level of the central nervous system [START_REF] Louis | The 2007 WHO classification of tumours of the central nervous system[END_REF] .

There is no current prospective trial for the management of children with primary spinal ependymoma. The current SIOP ependymoma II study includes these patients in a registry and may provide useful information in the future. Meanwhile, better understanding of such an entity is necessary in order to improve the management of SpEp. The aim of this study is to summarize the retrospective clinical and therapeutic experience of children with a spinal ependymoma in France.

Methods

Between 2000 and 2010 medical records of patients younger than 18 years old with a primary SpEp treated in one of the following French Pediatric Neurosurgery and Oncology institutions (Institute Gustave Roussy Villejuif, Paris-Necker, Lille, Lyon, Bordeaux) were analysed. Epidemiologic (gender, age, potential predisposing status and symptoms at diagnosis, delay from initial signs to diagnosis), tumor-and treatmentrelated data (site and extension of the tumor, histology, extent of initial resection and number of relapse and complementary treatments) were retrospectively analysed. Two patients with cervical location were excluded as study focused on the most frequent location of spinal ependymomas of the child, thus at the distal part of the spinal cord. Most recent data were obtained from the follow-up files of the participating centers according to the French Data Protection Authority (CNIL 2211717 v 0). Progression-free survival (PFS) was calculated from the date of surgery to the date of recurrence/progression for patients who relapsed/progressed or until the date of latest news otherwise (censored data). The PFS was estimated using the Kaplan-Meier method. A Cox proportional hazard regression model was used to identify prognostic factors that can influence recurrence/progression such as gender, age at surgery, level of lesion(s), histological grade (I, II or III), quality of initial surgical removal (gross total removal GTR versus subtotal removal STR) and whether adjuvant RT was administered or not. Due to the small sample size, only univariate analysis was considered. A subgroup analysis according on removal status with or without RT was performed.

Results

Twenty-eight patients were identified. The main clinical characteristics are reported in Table 1. The median age at diagnosis was 13.67 year (7 month to 17.6 years). Sex ratio M:F was of 3.6:1. The median time interval between symptoms and diagnosis was 4 months (6 days to 3 years). Back pain was present in 26 of 27 evaluable patients (92.5%) and another patient had no symptoms as the diagnosis of spinal ependymoma was made during systematic screening for NF2 disease. In the youngest patient, diagnosis was made at postnatal ultrasound for skin abnormalities (lateralized sacral dimple) and controls showed cystic progression at 7 months. In 7 cases (25%), the back pain progressed to sciatica. Other contemporary symptoms of back pain were: partial motor (n=8) and/or sensitive (n=3) deficit, bladder (n=8) and bowel movement dysfunction (n=3). The ependymoma was localized at the level of cauda equina in 15 patients, at the conus level in 4 or both at the same time in 9 patients. One presented associated bilateral vestibular schwannomas (underlying neurofibromatosis type 2) on cranial MRI. Metastatic seeds at the level of the inferior part of the lumbar cistern were present at diagnosis in 6 (21%) cases: 4 were MPE's, 1 grade II and 1 grade III.

Preoperative spinal MRI was performed in all patients with additional cranial MRI in 5 patients for cranial extension research. All 28 patients underwent surgery with a laminotomy approach. Initial gross total resection (GTR) was obtained in 22 patients (78,6%) and subtotal resection (STR) in 6 patients (21,4%).

Histopathologic diagnosis was determined according the 2007 WHO classification by the local pathologist.

No central review of histology was conducted since all participating institutions were ranked as reference centers in France. Fifteen were grade I (MPE), 11 grade II and 2 grades III. An initial GTR was obtained in 10 of 15 patients with grade I ependymoma, in 10 of 11 patients with grade II and in all patients (n=2) with grade III. Initial STR was reported in 5 patients with grade I MPE and one with grade II. Two children of the MPE group who were in STR had an elective attempt of second look surgery resulting again in STR. Among 6 patients with seeding 4 patients had GTR (2 MPE, 1 gr II and 1 gr III) and 2 STR (both MPE) at initial surgery.

Adjuvant Radiotherapy

Six patients (21%) received adjuvant RT after initial surgery: 4 in GTR (1 MPE, 2 Grade II and 1 Grade III) and 2 in STR (both MPE's) (Table2). The median dose used was of 46.6Gy with a range from 45 to 50.4Gy

Clinical course and status of remission

All patients were alive at the end of the study with a median follow-up period of 40 months (4 to 124 months).

Group with no relapse/progression

Eighteen patients (64%) did not progress. The median follow-up was 23 months (2.3 to 127.5 months). There was a male predominance with 17 males (M) and 1 female (F): ratio M:F of 17:1. All patients had an initial GTR. Regarding histology, 7 were MPE's (46% from all MPE's), 9 were grade II (81% from all grade II patients) and 2 were grade III (100% of grade III). Four patients had received adjuvant RT (1 MPE, 2 grades II and 1 grade III) and 14 patients had not (6 MPE; 7 grades II, and 1 grade III).

Group with relapse/progression (Table 3) Ten patients (37.5%) relapsed or progressed. The median time from surgery to relapse was 24.9 months (range 11.2 to 70 months). The M:F ratio was 1:1: Five out of 6 (83.3%) female versus 5 out of 22 (22.7%) male presented a relapse/progression. Eight out of 10 patients were MPE (3 GTR and 5 SRT) and two were grade II (one GTR and one STR). Thus, altogether in the MPE group, 8/15 patients ultimately relapsed/progressed, 2 despite adjuvant RT: 3/9 GTR and 3/3 STR relapsed/progressed when no adjuvant radiation was delivered, whereas 0/1 GTR and 2/2 STR did so when adjuvant RT was delivered. In the grade II cohort, 1/8 patients with GTR and 1/1 with STR relapsed/progressed when no radiation was delivered, while 0/2 (Table 2) relapsed after receiving adjuvant RT for GTR.

The treatment of relapses included surgery with a laminotomy (6) or laminectomy (1): in 5 MPE's, 1 grade II and 1 grade III. In the latter, no tumoral tissue was found and the child remains in first remission. Two patients had further surgeries for progression of their residue: one with MPE with stable disease at follow-up after 2 more surgeries followed by salvage RT and one with grade II ependymoma in remission at last follow-up after 3 more surgeries followed by salvage RT (Table 4).

Radiotherapy was given at time of relapse or progression in 5 MPE's and one grade II. One of the MPE patient received RT twice: at initial diagnosis and at relapse. Chemotherapy was administered only in 1 MPE patient with residue after first and second look surgeries, and received adjuvant RT. He received chemotherapy at time of further progression.

The follow-up of the group with relapse/progression (n=10) shows complete remission in 3 patients (30%), stable residual MRI images in 3 patients (33%) and progression in 4 patients (44%).

Survival analysis

With a median follow-up of 40 months (min=2.3; max=127.5), the 5-year PFS is 51% (IC95% = 26.3 ;71.2) and the overall survival (OS) is 100% (Figure 1). In univariate analysis, only the quality of the initial surgical removal has a significant influence on the PFS, the GTR being superior to STR (p=0.0013). The details of the univariate analysis are shown in the table 5.

Complementary analysis was done on 4 subgroups according to the quality of the resection and adjuvant radiotherapy. Although the sample size is small, the subgroup analysis shows that RT may improve PFS in case of GTR (p=0,006) (Figure 2).

Discussion

Ependymoma is the most frequent spinal tumor in adults but it is rather uncommon in children. There appears to be a relationship between the age and location of ependymomas in children with a more frequent posterior fossa location in infants compared to spinal tropism in older children [START_REF] Mcguire | Incidence patterns for ependymoma: a Surveillance, Epidemiology, and End Results study[END_REF] . In recent decades, studies on the molecular signatures of ependymomas revealed distinct genetic expressions in the respect of age of the patients and the localization of these tumors. These differences may also explain the specifics of clinical evolution and response to treatment for each site [START_REF] Korshunov | Gene expression patterns in ependymomas correlate with tumor location, grade, and patient age[END_REF][START_REF] Palm | Expression profiling of ependymomas unravels localization and tumor grade-specific tumorigenesis[END_REF][START_REF] Pajtler | Molecular Classification of Ependymal Tumors across All CNS Compartments, Histopathological Grades, and Age Groups[END_REF] . The prognosis differs from MPE in children compared to adults because they may have initial spinal spread [START_REF] Landriel | Multicentric extramedullary myxopapillary ependymomas: Two case reports and literature review[END_REF] and may show a greater tendency to recur [START_REF] Merchant | Pediatric lowgrade and ependymal spinal cord tumors[END_REF][START_REF] Fassett | The high incidence of tumor dissemination in myxopapillary ependymoma in pediatric patients. Report of five cases and review of the literature[END_REF][START_REF] Lin | Treatment of spinal cord ependymomas by surgery with or without postoperative radiotherapy[END_REF][START_REF] Pica | Ep RCN Myxopapillary: The results of surgery, with or without radiotherapy, for primary spinal myxopapillary ependymoma: a retrospective study from the rare cancer network[END_REF][START_REF] Feldman | Tumor control after surgery for spinal myxopapillary ependymomas: distinct outcomes in adults versus children: a systematic review[END_REF] .

Moreover, the paediatric population appears to have a median recurrence time shorter than that of adults 12 .

Relation between histology and prognosis is debated for grade II and III spinal ependymomas [START_REF] Louis | The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary[END_REF] . Recent studies on the molecular classification of ependymomas in children have shown common genetic alterations of the NF2 gene between WHO grade II and III spinal ependymomas [START_REF] Pajtler | Molecular Classification of Ependymal Tumors across All CNS Compartments, Histopathological Grades, and Age Groups[END_REF] .

We observed a male predominance (78.6%) already reported by McGuire et al 1 , but also à tendency for a higher recurrence rate for girls (66.7%) but no statistical difference in PFS could be found. Only the GTR was statistically significantly associated with a better PFS. All other tested parameters (age, tumor location, histology, initial adjuvant RT) did not show a significant difference in PFS.

Role of surgical treatment

In spinal ependymoma, as for its intracranial counterpart, the quality of resection was identified as the main prognostic factor [START_REF] Sonneland | Myxopapillary ependymoma. A clinicopathologic and immunocytochemical study of 77 cases[END_REF][START_REF] Pollack | Intracranial ependymomas of childhood: Longterm outcome and prognostic factors[END_REF][START_REF] Chang | Surgical outcome and prognostic factors of spinal intramedullary ependymomas in adults[END_REF][START_REF] Merchant | Three-dimensional conformal radiation therapy for ependymoma[END_REF][START_REF] Boström | Surgery for Spinal Cord Ependymomas[END_REF] . Several studies support that quality of surgery is associated with a good survival [START_REF] Lucchesi | Primary spinal myxopapillary ependymoma in the pediatric population: a study from the Surveillance, Epidemiology, and End Results (SEER) database[END_REF][START_REF] Benesch | Ependymoma of the spinal cord in children and adolescents: a retrospective series from the HIT database[END_REF] .

Our study also supports these findings, as PFS was clearly associated with GTR (p=0,0013). In this regard, the difficulty in managing spinal ependymomas is due to their location with high neurological risk associated to the surgery at the level of cauda equina, but also because of the initial loco-regional extension of the disease.

The myxopapillary ependymomas are slow growing tumors with no significant trend towards histologic dedifferentiation. Surgery alone is the usual treatment and in more than 50% of the cases complete removal has been reported [START_REF] Sonneland | Myxopapillary ependymoma. A clinicopathologic and immunocytochemical study of 77 cases[END_REF][START_REF] Gagliardi | Ependymomas of the filum terminale in childhood: report of four cases and review of the literature[END_REF] . Long-term recurrence is not unusual, even after complete removal or with more aggressive treatment, requiring long follow-up [START_REF] Sonneland | Myxopapillary ependymoma. A clinicopathologic and immunocytochemical study of 77 cases[END_REF][START_REF] Akyurek | Spinal myxopapillary ependymoma outcomes in patients treated with surgery and radiotherapy at M.D. Anderson Cancer Center[END_REF] . Regarding the quality of the tumour removal for MPE's, some authors have found no differences in the PFS between GTR and STR [START_REF] Akyurek | Spinal myxopapillary ependymoma outcomes in patients treated with surgery and radiotherapy at M.D. Anderson Cancer Center[END_REF][START_REF] Safaee | Surgical outcomes in spinal cord ependymomas and the importance of extent of resection in children and young adults[END_REF] and some studies have suggested the need for radiotherapy to improve local control even after GTR [START_REF] Feldman | Tumor control after surgery for spinal myxopapillary ependymomas: distinct outcomes in adults versus children: a systematic review[END_REF][START_REF] Akyurek | Spinal myxopapillary ependymoma outcomes in patients treated with surgery and radiotherapy at M.D. Anderson Cancer Center[END_REF] .

Concerning grade II ependymomas, complete surgery is associated with good outcome [START_REF] Benesch | Spinal cord ependymomas in children and adolescents[END_REF][START_REF] Tarapore | Pathology of spinal ependymomas: An institutional experience over 25 years in 134 patients[END_REF] . The GTR rate varies between 50-100% in the literature [START_REF] Lin | Treatment of pediatric Grade II spinal ependymomas: a population-based study[END_REF] and is 91% in our series. Gross total resection (GTR) seems to be associated with a favourable outcome with a PFS of 90 to 100% at 5 years [START_REF] Lonjon | Intramedullary spinal cord ependymomas in children: treatment, results and follow-up[END_REF][START_REF] Kukreja | Cumulative survival analysis of patients with spinal myxopapillary ependymomas in the first 2 decades of life[END_REF] . Hence, a GTR is advocated even in the presence of functional risk as the OS was found significantly superior after GTR versus STR plus RT [START_REF] Lucchesi | Primary spinal myxopapillary ependymoma in the pediatric population: a study from the Surveillance, Epidemiology, and End Results (SEER) database[END_REF] . Although there is no strong evidence for initial adjuvant radiotherapy (RT) for completely removed grade II ependymomas, this hypothesis has been advocated following subtotal resection [START_REF] Benesch | Spinal cord ependymomas in children and adolescents[END_REF][START_REF] Lin | Predictors of survival in patients with spinal ependymoma[END_REF] .

For the grade III ependymomas the most reasonable strategy is surgery (GTR or STR) followed by radiotherapy [START_REF] Benesch | Spinal cord ependymomas in children and adolescents[END_REF] .

Other paediatric studies showed no difference of the PFS across histological grades (I, II and III) and that OS was better after GTR than STR regardless of tumor grade [START_REF] Safaee | Surgical outcomes in spinal cord ependymomas and the importance of extent of resection in children and young adults[END_REF][START_REF] Benesch | Spinal cord ependymomas in children and adolescents[END_REF] . Likewise, our study found no statistical difference between the PFS related to the histological grade of the tumor.

With regard to the extension of the disease, the initial multifocal ependymoma is not unusual [START_REF] Landriel | Multicentric extramedullary myxopapillary ependymomas: Two case reports and literature review[END_REF] . In our series, metastatic seeds at the lower part of the lumbar cistern were present at diagnosis in 6 (21%) cases and not only in MPE. Therefore, even in cases with apparent complete removal, some residual synchronous lesions located on the roots may be left behind and may be targeted by complementary radiotherapy.

Role of radiation therapy

The role of the adjuvant radiotherapy in pediatric spinal ependymomas is still debated as relevant data are not available due to the rarity of these lesions in children. Some authors advocate the use of adjuvant radiotherapy only after STR in grade II-III ependymomas [START_REF] Benesch | Spinal cord ependymomas in children and adolescents[END_REF][START_REF] Lin | Predictors of survival in patients with spinal ependymoma[END_REF] and others also in MPE's [START_REF] Merchant | Pediatric lowgrade and ependymal spinal cord tumors[END_REF][START_REF] Al-Halabi | Adjuvant Radiotherapy in the Treatment of Pediatric Myxopapillary Ependymomas[END_REF] . Nevertheless, multivariate analysis of radiotherapy in different grade ependymomas showed no difference for PFS [START_REF] Safaee | Surgical outcomes in spinal cord ependymomas and the importance of extent of resection in children and young adults[END_REF] or OS 31 .

8

Regarding complementary treatment for MPE, some series showed good results with STR and adjuvant RT [START_REF] Merchant | Pediatric lowgrade and ependymal spinal cord tumors[END_REF][START_REF] Fassett | The high incidence of tumor dissemination in myxopapillary ependymoma in pediatric patients. Report of five cases and review of the literature[END_REF] or after GTR and RT [START_REF] Ross | Myxopapillary ependymoma. Results of nucleolar organizing region staining[END_REF] . In contrast, Bagley et al. [START_REF] Bagley | Resection of myxopapillary ependymomas in children[END_REF] found no significant role in the disease control for adjuvant RT in 14 pediatric patients with MPE.

Analysis of the PFS of our total population shows an improvement in outcome when the radiation was delivered to completely removed tumors (p=0,006). Like other studies [START_REF] Constantini | Radical excision of intramedullary spinal cord tumors: surgical morbidity and long-term follow-up evaluation in 164 children and young adults[END_REF] , we find no advantage of use of RT in STR. Another recent study of child MPE's showed no benefit of RT on OS after GTR or STR but PFS analysis could not be done [START_REF] Lucchesi | Primary spinal myxopapillary ependymoma in the pediatric population: a study from the Surveillance, Epidemiology, and End Results (SEER) database[END_REF] .

However, for the grade 3 ependymomas radiotherapy is advocated as a complementary treatment in several studies [START_REF] Boström | Surgery for Spinal Cord Ependymomas[END_REF][START_REF] Benesch | Spinal cord ependymomas in children and adolescents[END_REF] .

In the adults, the role of adjuvant radiation is also debated: some studies show a benefit of irradiation after STR only [START_REF] Lin | Treatment of spinal cord ependymomas by surgery with or without postoperative radiotherapy[END_REF][START_REF] Marco A Di | Postoperative management of primary spinal cord ependymomas[END_REF][START_REF] Lee | Surgical treatment of spinal ependymoma and post-operative radiotherapy[END_REF][START_REF] Wahab | Long term outcome with post-operative radiation therapy for spinal canal ependymoma[END_REF][START_REF] Whitaker | Postoperative radiotherapy in the management of spinal cord ependymoma[END_REF] , others recommend postoperative radiation even after GTR [START_REF] Boström | Surgery for Spinal Cord Ependymomas[END_REF][START_REF] Akyurek | Spinal myxopapillary ependymoma outcomes in patients treated with surgery and radiotherapy at M.D. Anderson Cancer Center[END_REF] Role of chemotherapy Chemotherapy for spinal ependymomas has been rarely used and only in refractory cases [START_REF] Madden | Experience with tamoxifen/etoposide in the treatment of a child with myxopapillary ependymoma[END_REF] . The use of high dose Tamoxifen has been reported in a child with recurrent spinal MPE after STR and radiotherapy, but results were only transient [START_REF] Madden | Experience with tamoxifen/etoposide in the treatment of a child with myxopapillary ependymoma[END_REF] . In our study, the combination of Cisplatin and Temozolomide in a child with progressive MPE after STR and RT did not stop the evolution of the disease. In a mixed series, Chamberlain et al. [START_REF] Chamberlain | Salvage chemotherapy for recurrent spinal cord ependymona[END_REF] reported the prospective use of etoposide in 10 patients with recurrent intramedullary ependymoma with a stable or partial response following a single cycle of etoposide in 70% of cases for a period of 17 months. More recently, the bevacizumab has been showed to be effective in reducing the cystic forms of spinal ependymomas in NF2 patients but not the solid ones. For high grade ependymomas, chemotherapy may be used as an adjuvant treatment combined with radiotherapy [START_REF] Aghakhani | Intramedullary ependymomas: A French retrospective multicenter study of 221 cases[END_REF] .

Considering new therapeutic alternatives for SpEp, a study identified the PDGFR alfa as a potential target.

However, the use of PDGFR alfa inhibitor (Inatinib) did not show a consistent effect in the course of the disease [START_REF] Fakhrai | Recurrent spinal ependymoma showing partial remission under Imatimib[END_REF] .

Analysis of the outcome

In order to achieve GTR, there is a balance between the neurological risk associated to a radical surgery of ependymomas at the level of cauda equina and a potential increase in PFS. This is true especially for the MPE's as they show more tendency to recur in children [START_REF] Bagley | Long term outcomes following surgical resection of myxopapillary ependymomas[END_REF] . Therefore, the neurological risk has to be considered especially at the level of infiltration around the nerve roots. Meanwhile, the risk in case of repeated surgery can be even higher as dissection is usually more difficult because of fibrotic attachments.

Secondary radiotherapy, advocated in case of progression, is also at risk for delayed neurological impairment, secondary malignancies, growth and spinal deformity [START_REF] O'sullivan | Spinal cord tumors in children: long-term results of combined surgical and radiation treatment[END_REF] .

The limitations of this study include the bias of a retrospective study with information's gathered from local data charts. We must also consider the bias of a collaborative multicentre study as the diagnosis, and the quality of the surgical and complementary treatment are heterogeneous.

Conclusion

Despite recent advances in the management of child CNS tumours, surgery remains the main core in the management of the MPE and grade II spinal ependymomas in children. Our data suggest that initial adjuvant radiotherapy may improve the PFS after GTR. Further studies with larger collaborative cohorts are necessary to confirm our findings of the need for more aggressive treatment in SpEp in children.

Figure1.

  Figures legend:

Figure 2 .

 2 Figure 2. Analysis of progression free survival for 4 groups depending on the quality of resection (GTR/STR)

Table . 1

 . . Patients characteristics. GTR: gross total removal, STR: subtotal removal, RT: radiotherapy. First complete remission=remission obtained after 1 surgery +/-complementary treatment. Further complete remission=remission obtained after more than one surgery +/-complementary treatment.

	Patients characteristics Case no Initial surgery Grade GTR/STR	Adjuvant RT (Y/N)	Number (%) Salvage RT Clinical (Y/N) status
	Total					28 (100)
	16 Age at surgery (yrs.) I Mean (Std.) Median [min-max] 17 I 26 I 5 II	STR STR GTR GTR	Y Y Y Y	N Y N N	PD SD CR 13.67 [0.71-17.64] 12.5 (3.6) CR
	Sex 25	II	GTR	Y	N		CR
	19	Male III	GTR	Y	N	22 (78.6)	CR
		Female				6 (21.4)
	Level
		Cauda equina				15 (53.6)
		Conus				4 (14.3)
		Cauda equina + conus			9 (32.1)
	Metastatic seeds (lumbar cistern)			6 (21)
	Removal
		GTR				22 (78.6)
		STR				6 (21.4)
		Adjuvant RT				6 (214%)
	Grade
		I				15 (53.6)
		II				11 (39.3)
		III				2 (7.1)
	No recurrence				18 (64.2)
	Recurrence				10 (35.7)
	Current status
		First complete remission			18 (64.2)
		Further complete remission		3 (10.7)
		Stable				3 (10.7)
		Progression				4 (14.3)

Table 2 .

 2 List of patients who received radiotherapy in adjuvant setting. PD=progressive disease, SD=stable disease, CR=complete remission

	No Grade	Initial	Adjuvant RT	Relapse (1)	Treatment at	Salvage RT	Clinical
			Surgery	(Y/N)	Progression	recurrence or	(Y/N)	status
			GTR/STR		(2)	progression
	21	I	GTR	N	1	Surg	N	CR
	3	I	GTR	N	1	Surg	Y	PD
	18	I	GTR	N	1	Surg	Y	SD
	1	I	STR	N	2	Surg	Y	PD
	16	I	STR	Y	2	CT	N	PD
	17	I	STR	Y	2	RT	Y	SD
	14	I	STR	N	2	RT	Y	CR
	15	I	STR	N	2	Surg	Y	SD
	7	II	GTR	N	1	0	N	PD
	12	II	STR	N	2	Surg	Y	CR

Table 3 .

 3 Patients with recurrence or progression. Surg=surgery, RT=radiotherapy, CT=chemotherapy, 0=no treatment, CR=complete remission, PD=progressive disease, SD=stable disease.

	No Grade	Initial	Adjuvant	Relapse (1)	Further	Quality of	Salvage RT	Last know
			surgery GTR/STR	RT (Y/N)	Progression (2)	Surgery x1, 2, 3	further resection(s)	(Y/N)	Clinical status
	21	I	GTR	N	1	1	GTR	N	CR2
	3	I	GTR	N	1	1	GTR	Y	PD
	18	I	GTR	N	1	1	GTR	Y	SD
	1	I	STR	N	2	1	STR	Y	PD
	15	I	STR	N	2	2	STR	Y	SD
	12	II	STR	N	2	3	GTR	Y	CR2
	10	III	GTR	N	1 (negative	1	GTR	N	CR
					histology)		(negative
							histology)

Table 4 .

 4 List of patients with further surgeries. CR2=secondary complete remission, PD=progressive disease, SD=stable disease.