Lilian Moumaneix

Sébastien Fontana

Claire Hérold

François Lapicque

Dr Francois Lapicque
email: francois.lapicque@univ-lorraine.fr

Metal-free nitrogen-doped graphenic materials as cathode catalysts for the oxygen reduction reaction in polymer electrolyte membrane fuel cells

Keywords: N-doped graphenic materials, oxygen reduction reaction, cathode, PEM fuel cells, metal-free catalysts

Exhibiting a very high surface area, a good electrical conductivity and a high density of active sites, nitrogendoped graphenic materials are considered as promising catalysts for the oxygen reduction reaction (ORR).

Seldom studied in acidic media, N-doped graphenic foams were tested here as the cathode catalysts of a polymer electrolyte membrane fuel cell (PEMFC). The materials were prepared via a solvothermal-based process, by reacting either cyclohexanol and ethanolamine or 1-(2-hydroxyethylethyl)piperidine with metallic sodium, under high pressure and temperature. Membrane electrode assemblies were prepared with a Pt/C anode, 212 Nafion membrane, and an 8 mm disk cathode based on the graphenic materials. The performance exhibited by the PEMFC was evaluated using chronopotentiometry and impedance spectroscopy, depending on the synthesis conditions. The kinetic parameters of the ORR were estimated by interpretation of the experimental data: the high Tafel slope found might express the partial control of oxygen diffusion through the graphenic microporous structure. Relationships between the electrochemical behavior of the materials and their structural properties are discussed. Moderately crystallized materials with a low oxygen content showed the highest catalytic properties, with a current density larger than 30 mA cm -2 and a maximum power density at 2.3 mW.cm -2 .

Introduction

In order to meet the demands of the European Union for strong reductions of emissions to combat global warming, the need for more efficient energy systems is critical. H 2 fuel cells based systems could provide higher energy efficiency with nearly zero carbon emissions for stationary or transport applications [START_REF] Gröger | Review-Electromobility: Batteries or Fuel Cells?[END_REF]. Up to now, the most efficient catalyst for the sluggish oxygen reduction reaction (ORR) are platinum or platinum-alloy catalysts [START_REF] Karan | Catalytic Activity of Platinum Monolayer on Iridium and Rhenium Alloy Nanoparticles for the Oxygen Reduction Reaction[END_REF][START_REF] Zhang | One-pot synthesis of Ir@Pt nanodendrites as highly active bifunctional electrocatalysts for oxygen reduction and oxygen evolution in acidic medium[END_REF][START_REF] Zhu | Synthetic control of FePtM nanorods (M = Cu, Ni) to enhance the oxygen reduction reaction[END_REF][START_REF] Mani | Dealloyed Pt-Cu Core-Shell Nanoparticle Electrocatalysts for Use in PEM Fuel Cell Cathodes[END_REF]. High market penetration of polymer electrolyte membrane fuel cells (PEMFCs) suffer from their need in platinum-based catalysts. For instance, the 114 kW fuel cell stack in Toyota's Mirai contains approx. 30 g Pt . The availability of platinum is subject to environmental, geopolitical and economic factors, carrying a high-risk of primary source disruption [START_REF] Robinson | Sustainability of platinum production in South Africa and the dynamics of commodity pricing[END_REF][START_REF]Mineral commodities summary 2018[END_REF]. Development of alternative critical metal-free catalysts is thus essential for the expansion of H 2 fuel cells. Various directions have been explored provide alternative materials e.g. transition metal catalysts and metal-free catalysts.

Thanks to their high stability in agressive environment, their high electrical properties and their adaptable morphology, particular carbon-based materials appear promising metal-free catalysts for ORR. In particular, nitrogen-doped graphenic materials have drawn researchers' attention since their high catalytic activity towards the ORR was demonstrated using density functional theory (DFT) calculations and experimental works [START_REF] Zhang | Manageable N-doped Graphene for High Performance Oxygen Reduction Reaction[END_REF][START_REF] Zhang | Mechanisms of Oxygen Reduction Reaction on Nitrogen-Doped Graphene for Fuel Cells[END_REF][START_REF] Zhang | Effect of Microstructure of Nitrogen-Doped Graphene on Oxygen Reduction Activity in Fuel Cells[END_REF][START_REF] Duan | Heteroatom-Doped Graphene-Based Materials for Energy-Relevant Electrocatalytic Processes[END_REF].

N-doped graphenic materials can present higher stability than Pt-based catalyst during operation [START_REF] Ma | Novel synthesis of N-doped graphene as an efficient electrocatalyst towards oxygen reduction[END_REF] and the active sites are less subject to poisoning, one of the main limitations of current Pt-based catalysts [START_REF] Shao | Nitrogen-doped graphene and its electrochemical applications[END_REF][START_REF] Song | Fuel processing for low-temperature and high-temperature fuel cells Challenges, and opportunities for sustainable development in the 21st century[END_REF]. These recent years, focus on N-doped graphenic materials for ORR has been put on alkaline media, where their catalytic activity can exceed those of platinum-based catalysts [START_REF] Shao | Nitrogen-doped graphene and its electrochemical applications[END_REF][START_REF] Huang | Metal-free nitrogen-doped carbon nanoribbons as highly efficient electrocatalysts for oxygen reduction reaction[END_REF][START_REF] Geng | High oxygen-reduction activity and durability of nitrogen-doped graphene[END_REF][START_REF] Qu | Nitrogen-Doped Graphene as Efficient Metal-Free Electrocatalyst for Oxygen Reduction in Fuel Cells[END_REF]. However, because of the growing use of PEMFCs for transportation applications, studies in acidic media are also needed, even if the ORR remains rate-controlling in such conditions. Upon further progress, N-doped graphenic materials could ultimately provide an alternative to Pt-based catalysts in PEMFCs.

Mechanisms responsible for the ORR activity in N-doped graphenic materials have been highly debated since several critical parameters play a role in this reaction. Nitrogen atoms can be present in five main functions in Ndoped carbon materials. First, pyrrolic-N and pyridinic-N are both bonded to two carbon atoms within pentagonal or hexagonal cycles, respectively. Graphitic-N substitutes one carbon atom and is therefore bonded to three carbon atoms inside the graphenic network. Surface functions such as amine or nitrile can also be found, mostly at the edges of the layers. Finally, N-oxides are also present, in the form of pyridine-N-oxide or pyridonic-N [START_REF] Inagaki | Nitrogen-doped carbon materials[END_REF][START_REF] Quílez-Bermejo | Effect of carbonization conditions of polyaniline on its catalytic activity towards ORR. Some insights about the nature of the active sites[END_REF]. DFT calculations showed that mainly graphitic, pyrrolic and pyridinic-N are susceptible to provide catalytic activity towards ORR [START_REF] Zhang | Manageable N-doped Graphene for High Performance Oxygen Reduction Reaction[END_REF][START_REF] Zhang | Mechanisms of Oxygen Reduction Reaction on Nitrogen-Doped Graphene for Fuel Cells[END_REF][START_REF] Zhang | Effect of Microstructure of Nitrogen-Doped Graphene on Oxygen Reduction Activity in Fuel Cells[END_REF][START_REF] Duan | Heteroatom-Doped Graphene-Based Materials for Energy-Relevant Electrocatalytic Processes[END_REF]. Besides, Wu et al. [START_REF] Wu | Microscopic effects of the bonding configuration of nitrogen-doped graphene on its reactivity toward hydrogen peroxide reduction reaction[END_REF] demonstrated by DFT that hydrogen peroxide H 2 O 2an undesired intermediate in ORR-can chemically adsorb on the surface of N-graphenic materials containing graphitic, pyrrolic or pyridinic-N in the presence of protons, thus indicating its possible reduction to water. The hypothesis was experimentally confirmed [START_REF] Ma | Novel synthesis of N-doped graphene as an efficient electrocatalyst towards oxygen reduction[END_REF][START_REF] Shao | Nitrogen-doped graphene and its electrochemical applications[END_REF][START_REF] Song | Fuel processing for low-temperature and high-temperature fuel cells Challenges, and opportunities for sustainable development in the 21st century[END_REF][START_REF] Huang | Metal-free nitrogen-doped carbon nanoribbons as highly efficient electrocatalysts for oxygen reduction reaction[END_REF][START_REF] Geng | High oxygen-reduction activity and durability of nitrogen-doped graphene[END_REF], showing that O 2 is mainly reduced to water through a four-electron reaction.

However, no experimental consensus on the most effective N-function has yet emerged because of the difficulty to obtain comparable materials containing very different proportions of nitrogen functions. Moreover, the chemical nature of the nitrogen-containing groups cannot be controlled during the synthesis.

Common mechanisms suggest that the introduction of nitrogen atoms into the graphenic network modifies the global distribution of spin and charge densities of neighboring carbon atoms, influencing the oxygen adsorption and further electrochemical reactions around nitrogen atoms. While spin density would act on the positional selectivity of radical adsorption, charge density would be responsible for the attractive force between charged atoms. Zhang et al. [START_REF] Zhang | Manageable N-doped Graphene for High Performance Oxygen Reduction Reaction[END_REF] demonstrated that carbon atoms with a charge density larger than 0.15 were likely to serve as active sites. Guo et al. [START_REF] Guo | Active sites of nitrogen-doped carbon materials for oxygen reduction reaction clarified using model catalysts[END_REF] experimentally established a linear relationship between the current density and the concentration of pyridinic-N. According to the authors, carbon atoms close to pyridinic-N behave as Lewis bases owing to the possible electron pair donation. Since O 2 is susceptible to adsorb on Lewis bases, neighboring pyridinic-N carbon atoms would be the ORR active sites. Another study [START_REF] Geng | High oxygen-reduction activity and durability of nitrogen-doped graphene[END_REF] suggested that pyridinic-N facilitates O 2 adsorption and eliminates intermediate H 2 O 2 formation. In contrast, according to Lai et al. [START_REF] Lai | Exploration of the active center structure of nitrogen-doped graphene-based catalysts for oxygen reduction reaction[END_REF], graphitic-N species reduce the electron density on adjacent carbon atoms, improving electron transfer from carbon to nitrogen atoms: electron backdonation from nitrogen to adjacent C pz orbitals out of the graphenic plan actually facilitates O 2 dissociation.

In addition to the direct electronic influence of nitrogen atoms on neighboring carbon atoms, nitrogen species are presumably markers of structural and microstructural changes in the carbon matrix. Parameters such as the size of the π-conjugated system [START_REF] Guo | Active sites of nitrogen-doped carbon materials for oxygen reduction reaction clarified using model catalysts[END_REF], the pore diameter distribution [START_REF] Florent | Oxygen Electroreduction on Nanoporous Carbons: Textural Features vs Nitrogen and Boron Catalytic Centers[END_REF] or the nature of carbon defects [START_REF] Jiang | Significant Contribution of Intrinsic Carbon Defects to Oxygen Reduction Activity[END_REF][START_REF] Ikeda | Carbon Alloy Catalysts: Active Sites for Oxygen Reduction Reaction[END_REF] have been shown to affect the catalytic activity, independent of heteroatom doping. The debate to elucidate which is the most active nitrogen specie could be biased without considering the intrinsic carbon defects present in the material due to the insertion of nitrogen or to the synthesis method. As a matter of fact, the pyrolysis temperature could have a direct impact on the carbon defect content and the π-conjugated system size. Florent et al. [START_REF] Florent | Oxygen Electroreduction on Nanoporous Carbons: Textural Features vs Nitrogen and Boron Catalytic Centers[END_REF] observed that the hydrophobic ultramicropores in the carbon matrix, withdraw oxygen from the electrolyte and promote oxygen bond splitting and ORR. Moreover, pentagon and zigzag edge defects thanks to their specific electronic structures, were shown to be good candidates for ORR from a thermodynamic point of view [START_REF] Jiang | Significant Contribution of Intrinsic Carbon Defects to Oxygen Reduction Activity[END_REF].

Coupling nitrogen-containing catalytic centers to a controlled carbon matrix microstructure seems to be the key to obtain highly performing nitrogen-doped graphenic materials for the ORR.

The present work aims to study the performances for ORR of nitrogen-doped graphenic foams, a material that exhibits very large surface area and various nitrogen functions, in fuel cell operation. Prepared after solvothermal route, the materials have been tested under H 2 and air supply in a proton membrane fuel cell, which has not yet been reported in the literature to our state of knowledge. The catalytic efficiency of various N-doped graphenic materials on ORR in a complete fuel cell was measured in terms of voltage versus current density relationship. EIS measurements and voltage monitoring at a fixed current level allowed the kinetic parameters of the cathode reaction with these materials to be estimated. These parameters were then discussed depending on the operating conditions for their synthesis and the physico-chemical properties of these materials.

Experimental

Material synthesis

Elaboration of the N-doped graphenic materials: Two sample series were synthesized, differentiated by the initial reagents employed for their synthesis, either a mixture of cyclohexanol and ethanolamine (abbreviated CE series), or 1-(2-hydroxyethyl)piperidine (abbreviated HEP series).

o Elaboration of the CE series materials:

A mixture of 0.1 mol of cyclohexanol (Sigma Aldrich, 99%), 0.1 mol of ethanolamine (Aldrich chemistry, >99.5%) was put in contact with 0.26 mol of metallic sodium (Merck Millipore) under inert atmosphere (nitrogen Air Liquide, alphagaz 2) in a 0.5 L Parr autoclave. Cyclohexanol and ethanolamine were used as received. Sodium rods were washed in petroleum ether to remove the paraffin oil then cut in small pieces and stored under nitrogen atmosphere. Sodium pieces were scrapped before use to remove the passivation layer that is formed even under protective atmosphere. The autoclave was sealed and nitrogen (Air Liquide, alphagaz 2) was introduced so that the pressure attained 70 bar. The reactor was then heated up to 350 °C at 8 °C.min -1 , the internal pressure reached 200 bar, and the mixture was let to react for 72 h. Afterwards, the reaction was allowed to cool down naturally to room temperature, and the solvothermal product was collected under nitrogen atmosphere. About 5 g of solvothermal product were placed in an Inconel® 600 crucible and then introduced in a vertical tubular oven. Depending on the planned conditions, the oven was heated at a heating rate of 20 °C.min -1 up to the reaction temperature in the range 750 °C -900 °C for 4 h, then cooled down to room temperature naturally.

The crucible was permanently kept under a nitrogen flow (Air Liquide, alphagaz 1) to avoid combustion of the carbonaceous phase and to eliminate pyrolysis gases. The pyrolysis product consisted of a greyish black powder, with in some cases sodium carbonate and sodium hydroxide white crystals.

The pyrolysis product was crushed then sonicated 10 min in 200 mL of distilled water. The powder was washed alternatively with 1 L 6 mol.L -1 hydrochloric acid (Sigma Aldrich, ≥37%) and 1 L distilled water. The initially grey powder turned to black due to the removal of sodium carbonate and sodium hydroxide formed during the pyrolysis. The washed sample was finally dried at 100 °C for 24 h, and stored under air.

o Elaboration of the HEP series materials:

The elaboration process of the HEP series follows the one described for the CE series, except for the quantities of reagents initially introduced in the solvothermal reactor. 0.17 mol of 1-(2-hydroxyethyl)piperidine (ACROS organics, 99%) was introduced inside the solvothermal reactor with 0.34 mol of metallic sodium (Merck Millipore) under nitrogen (Air Liquide, Alphagaz 2). 1-(2-hydroxyethyl)piperidine was used as received. The pressure of nitrogen injected into the reactor and the other experimental parameters remained strictly identical as the CE series.

Material characterizations

Physisorption of nitrogen at 77 K was performed with a Micromeritics ASAP2020 adsorption apparatus. The samples were outgassed during 12 h at 300 °C before analysis. Specific surface areas were calculated using the BET (completed with the Rouquerol correction) and 2D-NLDFT models, micropore volumes and pore size distributions were obtained using 2D-NLDFT model: this model can actually avoid overestimation of the specific surface area by the BET model for microporous solids. 2D-NLDFT calculations were carried out using SAIEUS software, with a corrugation parameter λ=4.25 and an integration range from 0.4 nm to 29.3 nm. X-ray photoelectron spectroscopy (XPS) analysis was achieved with a Kratos Axis Ultra DLD, using an Al K1 X-ray source (1486.6 eV). C-C and C-H bounds were taken as references at 284.6 eV. The analyzed area was of 700 x 300 µm 2 with a 5-10 nm depth. Deconvolution of the N1s spectra was carried out considering five contributions, namely pyridinic-N at 398.3 eV, aminic-N at 398.9 eV, pyrrolic-N at 400.0 eV, graphitic-N at 401.4 eV and N-oxides at 402.4 eV. Deconvolution of O1s spectra revealed the presence of chemisorbed O at 531.4 eV, carbonyl-O at 532.9 eV and ether or alcohol O at 535;2 eV. Raman spectroscopy investigations were conducted with a Renishaw inVia Quontor equipped with a 532 nm laser operating in the spectral range 100-3600 cm -1 in synchroscan mode. Samples were first dispersed in absolute ethanol then deposited on a glass slide.

Preparation of the ink and MEAs:

16 mg of N-doped graphenic foam were mixed with 50 µL of distilled water, 20 µL of Nafion ® Solution (Nafion ® perfluorinated resin, 10 wt.% in H 2 O, Aldrich) and 560 µL of 2-propanol (Sigma-Aldrich). The mixture was then stirred at 20,000 rpm for 30 s, before being sonicated for 15 min. The resulting ink was carefully deposited on a 8 mm disk of Sigracet 34BC GDL. 35 µL of ink were deposited on the GDL to prepare a GDE with a catalyst loading of 1.77 mg cm -2 . The prepared electrodes were finally dried in an oven at 50 °C for at least 4 hours. The thickness of the deposit has been estimated around 18 µm, considering an overall density for the carbon material near 1 g cm -3 , corresponding to a porosity in the order of 50%.

MEAs were prepared by assembling a 40 x 35 mm 2 Nafion ® membrane (PaxiTech, NR212) , a 30 x 25 mm 2 piece of Pt-based GDE (Quintech, Sigracet 10BC, BC-H225-05S, 0.5 mg cm -2 ) on the anodic side, and 30 x 25 mm 2 piece of GDL Sigracet 34BC. An 8 mm circular hole had been punched in the rectangular GDL piece, allowing insertion of the above prepared disk for the cathode. The different MEA components were assembled by brushing the coated surface with Nafion solution (Ion Power, water based at 10 wt.%) before pressing them onto the membrane at 20 bars. The MEAs formed and still under pressure, were heated up at 100 °C for 4 min. The resulting MEA and its expanding view are shown in Figure 1.

Catalysts electrochemical characterization

The prepared MEAs were inserted into the 5 cm 2 Paxitech fuel cell and tests were carried out at ambient temperature: dry hydrogen was fed at 50 mL min -1 and fully humidified air was injected at 100 mL min -1 into the cathodic chamber. Hydration and maturation of the MEAs were conducted with increasing gradually the current density for at least two hours for steady, reproducible measurements with a Bio-Logic VSP3 potentiostat.

Steady value of the cell voltage was measured in 30 min long tests at a given current density. Then, galvanostatic electrochemical impedance spectroscopy (GEIS) measurements were carried out at this current density level within the frequency range 10 kHz -100 mHz with ten points per decade, the amplitude being fixed at 10% of the steady current. As shown in Figure 2, EIS spectra usually consisted of one very small loop for frequencies larger than 100 Hz corresponding to the response of the Pt/C anode, and a far larger, incomplete loop below 100 Hz being for the cathode. The profile of spectra led us to model the cell as two (R-CPE) circuits for the anode and the cathode (subscripts a and c respectively), in series with the overall ohmic resistance of the cell, R cell . As a matter of fact, EIS spectra did not allow to consider more complex models, with mass transfer control. Fitting of the spectra led to estimates for charge transfer resistances R a and R c , CPE parameters Q a , n a , Q c , n c , and R cell .

The cell voltage V was expressed as follows:

(1) Where V 0 is the equilibrium cell voltage, near 1.23 V at the considered conditions neglecting the contributions of partial pressures in Nernst equation, I the cell current,  a and  c being the anode and cathode overpotentials respectively. Because the charge transfer resistance of the anode R a has been found nearly constant at 1 Ω within 0.1 Ω for all current values,  a was approximated by the product of this resistance and the cell current. The cathode overpotential was expressed after Tafel's law, involving the cathode exchange current density, i 0 , and the Tafel slope, b. However, because the cell voltage at open circuit was measured at a value far lower than V 0 , Tafel's law has been modified by addition of "crossover" current density I FCO as follows:

(2) Therefore, the cell voltage was approximated as:

(3)

The specific cathode resistance, r c in Ω.cm -2 , equal to (R c .A) is defined as the derivative function on of the cathode overpotential with respect to the current density: [START_REF] Zhu | Synthetic control of FePtM nanorods (M = Cu, Ni) to enhance the oxygen reduction reaction[END_REF] The interpretation of chronopotensiostatic measurements and impedance spectroscopy led to kinetic parameters b, i 0 and i FCO to be estimated for all the materials tested. For discussion, parameter b was related to the Tafel slope, m, by: (

Finally the equivalent double layer capacitance of the cathode was estimated from parameters Q and n c , using the approximate relationship [START_REF] Franck-Lacaze | Ageing of PEMFCs due to operation at low current density: investigation of oxidative degradation[END_REF]: [START_REF] Robinson | Sustainability of platinum production in South Africa and the dynamics of commodity pricing[END_REF] where  c is the pulsation corresponding to the top of the charge transfer loop in Nyquist plot.

Results and discussions

Before presenting the results of the present work, properties of CE materials extensively characterized [START_REF] Moumaneix | Nitrogen-doped graphenic foam synthesized by solvothermal-based process: Effect of pyrolysis temperature on the material properties[END_REF] are summarised below. CE materials obtained by pyrolysis at 850°C or more, exhibited a high crystallinity, as expressed by narrow bands D and G in Raman spectra. Specific surface area was in the range 1600-2240 m 2 .g -1 , with microporous volume varying from 0.68 to 1.05 cm 3 .g -1 , both properties having higher values at 750 and 800°C. In all CE materials, pyrrolic-N functions were found to be the most abundant fraction of nitrogen atoms, whereas carbonyl functions were the predominant O-containing groups.

Electrochemical measurements

In a previous work [START_REF] Moumaneix | Nitrogen-doped graphenic foam synthesized by solvothermal-based process: Effect of pyrolysis temperature on the material properties[END_REF], the experimental conditions used for the elaboration of the N-doped graphenic materials have been shown to have a great impact on their textural, structural, microstructural, and chemical properties. In particular, changing the temperature for the pyrolysis treatment allows tailoring of the material properties. In the present study, the catalytic properties of the N-doped graphenic materials have been investigated in regard of the pyrolysis temperature. Moreover, the influence of the change in reagents have also been studied by examining the performance exhibited by CE and HEP series materials.

As a reference, measurements have been carried out on an N-free graphenic material, elaborated under experimental conditions established in a previous study [START_REF] Speyer | Multi-scale characterization of graphenic materials synthesized by a solvothermal-based process: Influence of the thermal treatment[END_REF]. The maximum power density has been measured at around 0.05 mW.cm -2 , the cell voltage being close to 0.74 V at i=0 but dropping rapidly to reach 0.03 V at i=0,8 mA.cm -2 (data not shown). This sample will help in understanding the influence of the nitrogen atoms insertion inside the graphenic network on the catalytic activity. Four pyrolysis temperatures have been studied for the CE series, from 750 °C to 900 °C, and three for the HEP series, from 800 °C to 900 °C. Higher temperatures have not been investigated because of the decreasing variation of the reaction yield with this temperature, found below 1 wt.% at 900 °C. At 750 °C, the materials were considered too poorly ordered, i.e. with insufficient crystallinity [START_REF] Moumaneix | Nitrogen-doped graphenic foam synthesized by solvothermal-based process: Effect of pyrolysis temperature on the material properties[END_REF] to justify lower pyrolysis temperatures. Moreover, the electric conductivity of samples pyrolyzed at low temperatures was found much lower than that of samples treated at medium/high temperatures in relation to their compared crystallinity, being for instance at 0.010 ± 0.001 and 0.40 ± 0.04 S.cm -1 at 800 and 875 °C respectively [START_REF] Moumaneix | Nitrogen-doped graphenic foam synthesized by solvothermal-based process: Effect of pyrolysis temperature on the material properties[END_REF].

Figure 3 shows the variations of the cell voltage and the power density with the current density, for CE (a) and HEP (b) series materials. Comparable trends can be observed for the two series, i.e. with the highest performances obtained with materials pyrolyzed at the lowest temperatures. In particular, CE-750 appears to be the most promising material with a maximum power density near 2.3 mW.cm -2 and tolerating a current density larger than 30 mA.cm -2 . HEP series shows lower catalytic activity with a maximum power density at 0.9 mW.cm -2 , and a current density always below 10 mA.cm -2 . The two maximum power densities are respectively

The ohmic resistance R cell deduced from the impedance spectra was always in the range 0.15-0.25 Ω: this value although significant is mainly due to the small area of the cathode (approx.0.5 cm 2 ). Nevertheless, considering the current produced by the cell, the ohmic drop was always below 2.5 mV. Moreover, the anode overpotential estimated from R a was below 15 mV in all cases, thus the deviation of the cell voltage to the equilibrium voltage corresponds fairly well to the cathode overpotential, including the effect of fuel crossover. The overall cathode resistance R c varied from 7 to 380 , depending on the current density and the material evaluated. Finally, exponent n of the constant phase element lay in the range 0.69-0.75, a range slightly lower than that usually observed with Pt-based catalysts. The equivalent capacitance C deduced from rel. ( 6) for the various materials at their maximal performance, varied from 0.015 to 0.23 F.cm -2 . Interestingly, the max power of the cell appears to be a linear function of the equivalent capacitance within 25% as shown in Figure 4.

The fuel crossover, expressed as its corresponding current density i FCO as in relation [START_REF] Karan | Catalytic Activity of Platinum Monolayer on Iridium and Rhenium Alloy Nanoparticles for the Oxygen Reduction Reaction[END_REF], was in the order of 0.01 -0.1 mA.cm -2 . The higher value found for CE-800 near 1 mA.cm -2 , may be due to a defect in the membrane electrode assembly which drastically limited its open circuit voltage at 0.42 V.

The values for the exchange current density i 0 , obtained from combined interpretation of impedance spectra and the V vs. i curves using relations ( 2) and ( 3), are in accordance with the cell performances, varying from 2.10 -3 mA.cm -2 for sample CE-750, to 4.10 -5 mA.cm -2 for sample CE-900 (cf. Figure 5 (a)). Similar trend is observed for the HEP series. The compared reactivity of the materials are to be linked to their physicochemical properties: these aspects are further discussed in Section 3.2.

Tafel parameter b was found to be an increasing function of the pyrolysis temperature (Figure 5 (b)) for both series of materials, being near 8.3 V -1 at 750°C, and 10.8 V -1 at 900°C. Kinetic parameters i 0 and b follow opposite variations with the pyrolysis temperature, parameter b dampening the very strong effect of i 0 values exhibited by CE-750 and CE-900 materials. Using relation [START_REF] Karan | Catalytic Activity of Platinum Monolayer on Iridium and Rhenium Alloy Nanoparticles for the Oxygen Reduction Reaction[END_REF], the current density to be delivered by the cell for an overpotential chosen arbitrarily at 800 mV varies only by a factor 7 between CE-750 and CE-900 materials (Table 1). The maximal power density allowed by the N-doped graphenic materials followed comparable decreasing trends with the increase in pyrolysis temperature (Figure 5 (c)).

Besides, values for b correspond to Tafel slopes varying from 277 to 213 mV.dec -1 , values approximately two times larger than those usually accepted for oxygen reduction on Pt surfaces in acidic media, ideally near 118 mV.dec -1 at 25°C. The strong difference may originate from the very different nature of the catalytic sites; however, Liu et al. [START_REF] Liu | Electrochemical oxygen reduction on metal-free nitrogen-doped graphene foam in acidic media[END_REF] reported a 120 mV.dec -1 Tafel slope for the reduction of dissolved oxygen in 0.1 M HClO 4 on their N-graphene. Mass transfer control from dioxygen transfer in the catalytic layer could provide another explanation: when diffusion becomes the rate-determining step inside this layer, the Tafel slope was shown to be doubled from the calculated one without diffusion control [START_REF] Perry | Mass Transport in Gas-Diffusion Electrodes: A Diagnostic Tool for Fuel-Cell Cathodes[END_REF][START_REF] Tant | An algorithm for diagnosis of proton exchange membrane fuel cells by electrochemical impedance spectroscopy[END_REF]. At ambient temperature, the theoretical Tafel slope for the ORR on Pt surfaces under mass transfer control would be near 236 mV.dec -1 .

The resistance at the cathode determined by impedance spectroscopy would then cover combined controls of the reduction rate by diffusion of oxygen to the N-functions and by charge transfer. Improvement of the MEA preparation, in particular through thinner and more regular catalytic layer and optimized addition of PTFE solution may allow substantial enhancement of the cathode performance by hindering mass transfer control.

Correlation with physicochemical properties

The electrochemical properties of the two material series described above, were compared to other physicochemical properties, e.g. textural properties, crystallinity or chemical composition, to extract pertinent correlations helping to understand which critical parameters should be optimized in order to increase the catalytic activity towards the ORR. Only the CE series has been treated here, since these materials have been extensively characterized [START_REF] Moumaneix | Nitrogen-doped graphenic foam synthesized by solvothermal-based process: Effect of pyrolysis temperature on the material properties[END_REF] and because the electrochemical behavior of the two series appeared similar.

Textural properties

If the evolution of the maximum power density allowed by one material appears correlated with its specific surface area (Figure 6), the data corresponding to CE-750 diverges. In their work, Byambasuren et al. [START_REF] Byambasuren | The particle size effect of N-doped mesoporous carbons as oxygen reduction reaction catalysts for PEMFC[END_REF] showed that in the case of N-doped mesoporous carbon materials promoted with 1.5% of iron, the smallest pores allowed highest mass transfer rates due to the higher surface area, and therefore leading to highest MEA performances. However, in their materials, pores were mainly wider than 4.1 nm, for which gas diffusion although hindered by Knudsen effect, can be effective. In the present case, the pore sizes reported in [START_REF] Moumaneix | Nitrogen-doped graphenic foam synthesized by solvothermal-based process: Effect of pyrolysis temperature on the material properties[END_REF], are far smaller, with bimodal distributions, being in the ranges 0.5-1.1 and 1.2-2.0 nm for CE-750 and CE-800, and 0.7-1.2 and 1.3-2.8 nm for CE-900 material. Because the overall cell performance depends on both the specific area and effectiveness of gas transfer, it might be postulated that diffusion in very small pores, i.e. below 1 nm becomes more rate controlling, the beneficial effect of the pyrolysis temperature on the specific area being annihilated by the more sluggish diffusion in materials CE-750 and CE-800. This hypothesis appears to be in accordance with the lower b values i.e. the larger Tafel slopes, observed with these materials than with CE-850 or CE-900. Nevertheless, the above attempts in interpreting the data in Figure 6 do not explain the significantly higher performance of CE-750, so the effects of other factors have to be investigated.

Carbon phase crystallinity

Crystallinity of graphenic materials is an uneasy parameter to quantify. Nonetheless, Raman spectroscopy analysis can provide general trends on this property. Figure 7 (a) reports the variation of the maximum power density with the intensity of the defects bands D', T and F. The D' band at 1620 cm -1 is associated with defects in the graphene basal plans and can be clearly identified in the case of a limited amount of defects [START_REF] Eckmann | Probing the Nature of Defects in Graphene by Raman Spectroscopy[END_REF]. Bands T and F observed near 1100 cm -1 and 1500 cm -1 respectively [START_REF] Piscanec | Ab initio resonant Raman spectra of diamond-like carbons[END_REF][START_REF] Bhaumik | Reduced Graphene Oxide Thin Films with Very Large Charge Carrier Mobility Using Pulsed Laser Deposition[END_REF] can be attributed to the amorphous carbon fraction, hydrocarbon molecules or aliphatic molecules bound to the graphitic material [START_REF] Smith | Structural analysis of char by Raman spectroscopy: Improving band assignments through computational calculations from first principles[END_REF][START_REF] Bokobza | Raman Spectra of Carbon-Based Materials (from Graphite to Carbon Black) and of Some Silicone Composites[END_REF]. These observations are perfectly consistent with Jiang's [START_REF] Jiang | Significant Contribution of Intrinsic Carbon Defects to Oxygen Reduction Activity[END_REF] conclusions on the positive effect of pentagons and zigzag edge defects on graphene efficiency for ORR.

The increase in the intensity of the defects bands goes along with an increase in the catalytic performance of the MEA. This trend could be explained by a higher reactivity of the amorphous regions or by an augmentation of the tortuosity inside the carbon materials, sp 3 carbon atoms disrupting the linearity of the sp 2 carbon sheets due to the different angle between atoms, or by a higher reactivity of the amorphous regions. These observations tend to be confirmed by the variation of G and G' bands intensities, emerging around 1580 cm -1 and 2700 cm -1 respectively (data not shown) and respectively linked to the presence of sp 2 carbon atoms and high order in the graphene network [START_REF] Dresselhaus | Characterizing Graphene, Graphite, and Carbon Nanotubes by Raman Spectroscopy[END_REF]. Moreover, G' band is often considered as a marker for local order, its low significance expressing poor order in stacking of single graphene sheets. The highest power densities of the fuel cell were measured for the lowest intensities of G and G' bands (Figure 7 (b)), confirming that amorphous carbon fractions can increase the catalytic properties.

Chemical composition

The chemical composition of the elaborated materials is also an important parameter to be considered in examining the electrochemical properties. The atomic amounts of carbon, nitrogen and oxygen elements measured by XPS are presented in Figure 8. Since the penetration depth of the light beam is estimated around 5-10 nm, the results can be considered as corresponding to the "bulk" graphenic material, assuming a thickness of the graphenic walls in the range of 3-6 nm, corresponding to the stacking of 10 to 20 graphene layers, as found for N-free graphenic foams [START_REF] Speyer | Multi-scale characterization of graphenic materials synthesized by a solvothermal-based process: Influence of the thermal treatment[END_REF].

The power density is clearly correlated with the oxygen atomic amount in the samples, the highest power density being measured for the lowest amount of oxygen, namely CE-750 (Figure 8 (a)). This observation shows that oxygen-containing groups have a detrimental influence on the ORR with our N-doped graphenic foams. The evolution of power density also tends to follow the carbon atom concentration (Figure 8 (b)). It appears logical to link the increase in carbon atoms with an increase in the number of active sites, resulting in higher performances for the MEA. Nevertheless, since oxygen is the second most abundant element in the studied materials, high carbon quantities correspond to lower oxygen quantities, and therefore to higher power densities.

On the other hand, nitrogen atomic quantity does not seem to have a clear influence on the measured power density (Figure 8 (c)). In the domain studied, ranging from 0.8 to 2.3 at.%, high doping rates do not appear as necessary to allow good catalytic properties. However, it must be noted that the presence of nitrogen atoms inside the graphenic network is required to observe significant catalytic activity towards the ORR since the N-free graphenic material presents only extremely low activity. The atomic quantity of the three main nitrogen functions, i.e. pyridinic-N, pyrrolic-N and graphitic-N, have been extracted from deconvolutions of XPS N1s spectra. Their variations with the power density are plotted in Figure 9.

None of the N-function contents shows a correlation with the power density, since these parameters change with the oxygen content and the density of defects in the carbon network, which have a greater impact on the electrochemical performances. However, the synthesis method used to obtain the 3D N-doped graphenic materials does not yet allow efficient control of nitrogen doping, so that separate benefits from one specific Nfunction could not be evidenced in the present study.

Conclusion

The catalytic activity towards the ORR of N-doped carbon materials, although widely studied in the literature, is rarely evaluated in real condition tests inside a PEMFC. In this work, 3-dimensional N-doped graphenic materials have been inserted inside MEAs as cathodic catalysts for investigation of their intrinsic electrochemical properties. Electrochemical impedance spectroscopy measurements have been modeled using a Randles equivalent circuit in order to extract pertinent information yielding estimates for the exchange current density and Tafel slopes, by using voltage vs. current density variations. Moreover, the max. power allowed by the fuel cell appears to vary linearly to the equivalent capacitance of the cathode.

Comparison between N-doped and N-free graphenic materials clearly shows the beneficial impact of the chemical modification on the ORR catalytic activity, with a nearly 46-fold higher activity with N-doped materials. Regardless of the solvothermal precursor, the electrochemical performances are closely linked to the pyrolysis temperature, the lowest temperatures, 750-800 °C, leading to the highest catalytic activities. The differences observed between the CE and HEP series let us think that significant improvements could be obtained from the research of an optimal solvothermal precursor.

Even if the catalytic activity of N-doped graphenic materials in acidic fuel cells remains far below that of commercial Pt-based catalysts, several routes for improvement have been identified. Electrochemical performances could be linked to the textural properties of the materials since these exhibiting the highest surface areas possess the highest amount of active sites. The structural defects in the graphenic network seem to play an important part in the catalytic properties, even if their exact nature has still to be found. Nitrogen doping has been revealed necessary to bring out the catalytic properties towards the ORR, although its quantity seems to have no clear influence on the MEAs performances. On the other hand, the quantity of oxygen atoms has been shown to influence negatively the ORR reaction. Chemical reduction of the N-graphene materials by hydrazine [START_REF] Park | Chemical structures of hydrazine-treated graphene oxide and generation of aromatic nitrogen doping[END_REF] or ammonia [START_REF] Zhu | Low temperature reduction of graphene oxide film by ammonia solution and its application for high-performance supercapacitors[END_REF] similar in its principle to the reduction of graphene oxidecould be used to improve this parameter, and increase the quantity of nitrogen doping. Besides, the protocol for MEA preparations could be greatly improved, with thinner but more regular catalyst layers, and more effective dispersion of Nafion on the catalyst to reduce the significance of internal oxygen diffusion control, resulting in larger b parameter values, then in higher power density. Finally, for other applications, other MEAs could be prepared with alkaline ionomers, for which higher cell performances could be expected. HEP-900 1 10 -5 10.9 0.1 1 1

Figure caption

Fig. 1 Fig. 2 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7

 12234567 Fig. 1 View of a typical MEA, left with the disk cathode

Fig. 8 Fig. 9

 89 Fig. 8 Variation of the maximum power density with the atomic quantities of oxygen (a), carbon (b) and nitrogen (c) Fig. 9 Variation of the maximum power density with the atomic quantities of N-Pyridinic (a), N-Pyrrolic (b) and N-Graphitic (c)

Table 1 :

 1 Calculated values of the i 0 /i 0(900 °C) and i/i (900 °C) ratios for an 800 mV overvoltage

	Sample ID	i 0 (mA cm -2 )	Tafel's factor b (V -1 )	i at |η C | = 800 mV (mA cm -2 )	i 0 /i 0(900 °C) ratio	i/i (900 °C) ratio
	CE-750	2 10 -3	8.3	1.5	55	7
	CE-800	6 10 -4	9.1	0.8	15	4
	CE-850	1 10 -4	10.4	0.4	3	2
	CE-900	4 10 -5	10.9	0.2	1	1
	HEP-800	2 10 -4	9.6	0.4	18	7
	HEP-850	3 10 -5	10.7	0.1	3	2

Acknowledgements

This work was partly supported by the French PIA project "Lorraine Université d'Excellence", reference ANR-15-IDEX-04-LUE.


MD Brigitte Girard

Saint Sauveur G De

Tear Osmolarity

de SAINT SAUVEUR Guy

Osmolarity and BEB

come

Tear osmolarity, dry eye syndrome, blepharospasm and botulinum neurotoxin

Conclusions:

The results clearly demonstrate that poor tear secretion seems present in BEB patients even prior to treatment,. This poor lacrimal secretion was not correlated with hyperosmolarity. Clinicians should proactively treat dry eye syndrome in conjunction with management of the blepharospasm.

Financial disclosure :

Brigitte Girard is Consultant for Merz Pharma France, Allergan, Beaufour Ipsen Pharma SAS.

Résumé

Objet: Évaluer la sévérité de la sécheresse oculaire chez les patients atteints de blépharospasme et si celle-ci s'aggrave lors de l'injection de toxine botulique.

Design : Étude transversale

Methodes : Analyse des patients atteints de blépharospasme essentiel sans antécédents connus de syndrome sec oculaire précédant l'apparition du blépharospasme, avant (patients témoins) et après traitement par injection de

Les ophtalmologues prenant en charge ces pathologies devraient traiter

Page 5 sur 16 activement cette insuffisance lacrymale lors de la prise en charge des dystonies palpébrales.

INTRODUCTION

Benign essential blepharospasm (BEB) is a neurological condition that is defined by uncontrolled and abnormal eyelid closure from a central cerebral disorder, located in basal ganglia. Diagnosis is based on clinical observation, and MRI remain normal. BEB is a functional central dystonia, that typically manifests as frequent and sporadic blinking or eyelid twitching, which can affect an individual's eyesight and daily functioning [START_REF] Hallett | Update on blepharospasm: report from the BEBRF International Workshop[END_REF]. Dry eye symptoms, such as burning and grittiness, are known to occur in 40 to 85% of BEB patients [START_REF] Hallett | Update on blepharospasm: report from the BEBRF International Workshop[END_REF][START_REF] Martino | Relationship between eye symptoms and blepharospasm: a multicenter case-control study[END_REF].

Although dry eye disease (DED) has been linked with eyelid spasms, whether dry eye symptoms trigger BEB or if BEB leads to dry eye symptoms still remains uncertain.

Dry-eye disease could induced modification of the trigeminal blink reflex, because of disrupted tear film stability [START_REF] Hallett | Blepharospasm: recent advances[END_REF][START_REF] Evinger | Dry eye, blinking, and blepharospasm[END_REF]. The botulinum neurotoxin A (BoNT/A) inhibits acetylcholine release from neuron axon terminals. The BoNT/A is clinically used to relax muscular hyperactivity, including blepharospam [START_REF] Durand | Botulism toxin in practice[END_REF]. When injected into the orbicularis oculi, BoNT/A causes orbicularis oculi muscle paralysis preventing spasmodic blinking. This treatment can reduce lacrimal duct pump outflow and improve ocular surface lubrication, offering potential relief from blepharospasm and dry eye symptoms [START_REF] Sahlin | Eyelid botulinum toxin injections for the dry eye[END_REF]. Despite Page 6 sur 16 treatment with botulinum toxin, the majority of patients with blepharospasm describe severe and persistent dry eyes. In the other hand, injection of BoNT/A in principal lacrimal gland reduce lachrymal lacrimal secretion [START_REF] Girard | Botulinum neurotoxin A injection for the treatment of epiphora with free lacrymal ducts[END_REF]. Increased tear osmolarity is a recognized feature of dry eye disease [START_REF]Report of the International Dry Eye Workshop (DEWS)[END_REF]. Previous studies suggested that hyperosmolar tears, at least in part, underlie the pathogenesis of dry eye [START_REF]Report of the International Dry Eye Workshop (DEWS)[END_REF][START_REF] Messmer | Hyperosmolarity of the tear film in dry eye syndrome[END_REF]. Further, severity of the dry eye condition has been shown to positively correlate with increased tear osmolarity [START_REF] Sullivan | An objective approach to dry eye disease severity[END_REF][START_REF] Mastman | The total osmotic pressure of tears in normal and various pathologic conditions[END_REF][START_REF] Mathers | Model for ocular tear film function[END_REF][START_REF] Liu | A link between tear instability and hyperosmolarity in dry eye[END_REF]. Schirmer's I test is a conventional dry eye diagnostic tool known to assist in the identification of dry eye, including in patients with BEB.

The purpose of this study is to assess the severity of dry eye disease in patient with blepharospasm and. The second aim of this study was to compare the results obtained with Schirmer I and tear osmolarity between four groups (not treated with BoNT/A or treated with one of the three types available of BoNT/A). This study analysed data or biologic samples already collected as a routine examination. Patients were asked to discontinue all topical eye medications the evening prior to their appointment, as usual in our department. All were nonrandomly assigned in one of the four groups as described : not being injected yet 1). For the Schirmer test, the strip was placed in the conjunctival lower fornix, right eye tested first.

Patients and methods

Blepharospasm

No anesthetic eyedrop was used and the strip was left in place for five minutes.

Treated BEB patients were placed on a therapeutic eye drops regimen for treatment of dry eye consisting of 0.18% sodium hyaluronate eyedrops four times a day and carboxymethylcellulose sodium eyedrops three times a day.

Patients were asked to stop artificial tears the day before the appointment.

Statistical Analysis

Statistical analysis was performed using the Microsoft Excel 2010 software package and Biosta TGV software (UPMC Jussieu). The Student's t-test was used to compare the mean tear osmolarity and Schirmer's I test measurements captured among the treated and untreated groups. A p-value less than 0.05 was statistically significant.

Page 9 sur 16

Results

The mean age was 67.7 years ± 12 (range 30-86 years), 101 patients patients were included. All were non-randomly assigned to receive no BoNT/A injection (n=17; mean age 74.0 years ± 9), or injected with onabotulinumtoxinA (n= 26; mean age 66.8 years ±12), abobotulinumtoxinA (n= 23; mean age 67.9 years ±10) or incobotulinumtoxinA (n= 35; mean age 71.8 years ±10).

In the untreated blepharospasm group we observed a mean normal tear osmolarity at the first examination: right mean osmolarity at 289,29 mosml/ml ±15,48; left mean osmolarity at 293,12mosml/ml ±12.9; bilateral mean osmolarity at 296,67 mosm/ml±19. The mean tear osmolarity was normal in the Botox® injected blepharospasm group: right mean osmolarity at 294,31 mosml/ml ±19.09; left mean osmolarity at 270mosml/ml ±25.22; bilateral mean osmolarity at 295,46 mosm/ml±31. The mean tear osmolarity was normal in the Dysport® injected blepharospasm group: right mean osmolarity at 287,61 mosml/ml ±60; left mean osmolarity at 302,7mosml/ml ±26.49; bilateral mean osmolarity at 296,54 mosm/ml±35. The mean tear osmolarity was normal in the Xeomin® injected blepharospasm group: right mean osmolarity at 302mosml/ml ±21.89; left mean osmolarity at 300mosml/ml ±20.67; bilateral mean osmolarity at 301mosm/ml±21. In the three BoNT/A injected groups, the tear osmolarity do not revealed a hyperosmolarity in mean data (Table 1).

Page 10 sur 16

Statistical analysis of inter-group osmolarity (Table 2, and fig 4) showed no statistically significant difference between the mean osmolarity of all three

BoNT/A groups (onabotulinumtoxinA, abobotulinumtoxinA, incobitulinumtoxinA) and the group not injected (p=0.65, p=0.92, p=0.15 respectively). There was no statistically significant difference between the different groups (Tables 3,4,5).

Schirmer's I test results did not vary significantly between the different groups (fig. 5). Specifically, mean (right and left eyes) Schirmer's I test was 3.00±6mm among untreated BEB patients, 3,48±6,01 mm among onabotulinumtoxinA patients, 3±5,72 mm among abobotulinumtoxinA patients and 4,19±7.14 mm among incobotulinumtoxinA patients, with no statistical differences (p>0.05 for all groups). Schirmer's I test results (<10 mm) was observed among all of untreated (94%) blepharospasm patients except one up to 13mm, 88% of onabotulinumtoxinA patients, 91% abobotulinumtoxinA patients and 88% of incobotulinumtoxinA patients.

Discussion

This study suggest that dry eye is present in BEB patients, prior to neurotoxin injection. Because dry eye can affect vision, accurate and early identification, as well as treatment of dry eye is advisable in blepharospasm [START_REF] Lemp | Management of dry eye disease[END_REF]. Martino BEB [START_REF] Martino | Relationship between eye symptoms and blepharospasm: a multicenter case-control study[END_REF]. In the study of 165 patients with blepharospasm and 180 -age and gender-matched controls with hemifacial spasms found that ocular symptoms started in the year preceding onset of the disease.

Previous studies have demonstrated that dry eye syndrome is a side effect of botulinum toxin type-A injection [START_REF] Shorr | The use of botulinum toxin in blepharospasm[END_REF][START_REF] Horworth-Winter | Botulinum toxin A treatment in patients suffering from blepharospasm and dry eye[END_REF][START_REF] Dutton | Botulinum toxin in ophthalmology[END_REF]. In this study, we found 88% up to 94% of the BEB patients present Schirmer I test < 10mm, confirming that dry eye was present prior to BEB treatment by BoNT/A. This result suggests that dry eye is related to the disease and not to the botulinum toxin treatment [START_REF] Martino | Relationship between eye symptoms and blepharospasm: a multicenter case-control study[END_REF].

Other studies exploring ocular symptoms in idiopathic blepharospasm patients found that 86% have ocular surface symptoms suggestive of DED [START_REF] Girard | Dry eye syndrome in benign essential blepharospasm[END_REF][START_REF] Elston | The significance of ophthalmological symptoms in idiopathic blepharospasm[END_REF].

A recently published prospective case series from China also found that DED was a factor related to BEB and that botulinum toxin improved the condition of the ocular surface by reducing inflammation due to the reduction in blinking [START_REF] Lu | The Influence of Benign Essential Blepharospasm on Dry Eye Disease and Ocular Inflammation[END_REF]. This is another study to exonerate BoNT/A in dry eye syndrome of the BEB treated patients [START_REF] Lu | The Influence of Benign Essential Blepharospasm on Dry Eye Disease and Ocular Inflammation[END_REF].

Studies have shown that tear osmolarity correlates positively with dry eye disease severity and may diagnose dry eye with greater discrimination than Schirmer's I test [START_REF] Sullivan | Response to « Variability of Tear Osmolarity Measurements with a Point-of-Care System in Healthy Subjects-Systematic Review[END_REF][START_REF] Tomlinson | Tear film osmolarity: determination of a referent for dry eye diagnosis[END_REF][START_REF] Lu | The Influence of Benign Essential Blepharospasm on Dry Eye Disease and Ocular Inflammation[END_REF].

Although our results found a severe decrease of principal lacrimal gland secretion by the Schirmer I test in the four blepharospasm groups, osmolarity Page 12 sur 16 tests are unexpectedly in the normal limits in all these blepharospasm groups (Table 1, 2, 3, 4, 5).

One bias of the present study was the absence of randomization of patients between the three toxin's injected groups and the naïve group; however, as dry eye was present prior to treatment (naïve group), this should not have impacted the results presented here.

Another bias consists in lacrimal supplementary eyedrop treatment that was stopped the night before the examination. Extending the washout period did not seem appropriate given the severe ocular surface discomfort it would have caused. This study may consider the normality of osmolarity due to its its variability in the time and circumstances. The difference between right and left eye at the same time is usually the main criteria for unstable lacrimal secretion more than a gold number limit. Hyperosmolarity reflect more or less a dry eye

  To assess the severity of dry eye disease in patients with blepharospasm (BEB) and whether it worsens upon injection of botulinum neurotoxin A (BoNT/A). Design: Cross-sectional study Methods: Patients with clinically diagnosed BEB and no known history of dry eye syndrome before the onset of the disease, received not yet Botulinum neurotoxin A injection (controls) or Botulinum neurotoxin A (BoNT/A) injection with one of the three types availables of BoNT/A: onabotulinumtoxinA (Botox®; Allergan, Irvine, CA, USA), abobotulinumtoxinA (Dysport®; Beaufour Ipsen Pharma SAS, Paris, France) or incobotulinumtoxinA (Xeomin®; Merz Pharma GmbH, Frankfurt, Germany) every 3 months. Tear osmolarity and Schirmer's I test control were performed at the first visit for non-treated BEB patients as control patients or 3 months after the injection for BoNT/A injected BEB patients. Results: The study consisted of 101 BEB patients (86 females, 15 males) with a mean age of 67 years (range 30-86 years). Seventeen patients were untreated, 26 treated with onabotulinumtoxinA, 23 treated with abobotulinumtoxinA and 35 treated with incobotulinumtoxinA. Postinjection mean tear osmolarity was Page 3 sur 16 not significantly higher among patients treated with onabotulinumtoxinA, abobotulinumtoxinA, incobotulinumtoxinA (p=0.65 , p=0.92, and p= 0.15 respectively) compared with controls and remain under 308mosm/ml. Mean Schirmer's I test results remain under 5mm and did not vary between the four groups.

  patients aged at least 18 years were included in this transversal study. Inclusion criteria included previous diagnosis of blepharospasm, informed patient's consent, an initial osmolarity test (fig 1 and 2) and Schirmer's I test (fig 3) performed at the first examination for the naïve blepharospasm patients and the same examinations before the usual BoNT/A injection for the treated patients. Exclusion criteria included secondary blepharospasm, spasmodic Page 7 sur 16 entropion, eyelid fluttering, facial nerve paralysis and facial dystonia other than essential blepharospasm.

(

  first visit), or already receiving BoNT/A injection of onabotulinumtoxinA (Botox; Allergan, Irvine, CA, USA), abobotulinumtoxinA (Dysport; Beaufour Ipsen Pharma SAS, Paris, France) or incobotulinumtoxinA (Xeomin; Merz Pharma GmbH, Frankfurt, Germany) into the orbicularis oculi muscle. The trademark BoNT/A used was not changed during this study. The BoNT used depend on the severity of the BEB, the more severe receiving abobotulinumtoxinA (Dysport; Beaufour Ipsen Pharma SAS, Paris, France, and the others equally incobotulinumtoxinA (Xeomin; Merz Pharma GmbH, Frankfurt, Germany), and onabotulinumtoxinA (Botox; Allergan, Irvine, CA, USA) for the oldests patients. Based on the report from the Tear Film & Ocular Surface Society International Dry Eye Workshop, dry eye syndrome is defined as a tear osmolarity >308 mOsm/L and a Schirmer's I test result of <10 mm/5 minutes [9]. Schirmer's I test results of <5 mm/5 minutes were interpreted as strongly positive, indicating severe dry eye [15]. All patients underwent bilateral tear osmolarity measurement using the TearLab Osmolarity System (TearLab Corp., San Diego, CA) followed by Schirmer's I test one hour after but always before botulinum toxin injection. Tear osmolarity testing was performed first when the patient arrived at the ophthalmologic department, approximately one hour before Schirmer's I test, to allow the tear film environment to stabilize prior to Schirmer's I testing. Osmolarity was measured first in the right and then in the left eye (Table

  et al suggested that ocular surface diseases may contribute to the pathogenesis of Page 11 sur 16

  by lacrimal insufficiency and by tear evaporation induced by Meibomius gland disease. High variability of lacrimal osmolarity in the study need to be confirmed in further studies. The results of this study indicate that clinicians should be screening for dry eye in all BEB patients prior to BoNT/A treatment. Tear osmolarity in BEB could be analyzed to prescribe the optimal artificial tears. These BEB patients should be treated with artificial tear adapted to their specific lacrimal disease to reduce pain and symptoms of dry eye. Ophthalmologists should be aware of the Page 13 sur 16 frequent association of blepharospasm and severe dry eye, which appears to occur early in the onset of the disease. Page 16 sur 16

Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :

 12345 Figure 1 : TearLab Osmolarity System (TearLab Corporation, San Diego, CA)

Fig 1

 1 Fig 1

Figure 1

 1 Figure 1 Click here to access/download;Figure;fig 1.docx

Figure 2

 2 Figure 2 Click here to access/download;Figure;fig 2.docx

Figure 3

 3 Figure 3

Figure 3

 3 Figure 3 Click here to access/download;Figure;fig 3.docx

Figure 4 :

 4 Figure 4 : Osmolarity (mosm/ml) of untreated BEB versus BoNT/A BEB

Figure 4

 4 Figure 4 Click here to access/download;Figure;fig 4.docx

Figure 5 :

 5 Figure 5 : Schirmer (mm) in untreated BEB versus BoNT/A BEB

Figure 5

 5 Figure 5 Click here to access/download;Figure;fig 5.docx

Table 1 :

 1 Schirmer I results and the osmolarity in the 4 blepharospasm patient groups.

Table 2 :

 2 Statistics between all groups and untreated blepharospasm group (0)

Table 3 :

 3 Statistics between all groups and onabobotulinumtoxinA group (B)

Table 4 :

 4 Statistics between all groups and abobotulinumtoxinA group (D)

Table 5 :

 5 Statistics between all groups and incobotulinumtoxinA group (X)

	Figures

TABLE 1

 1

	Tableau 1
		N	Age	RE	LE	Mean	RE	LE	Mean
			years	Osmolarity	osmolarity	osmo	Schirmer I	Schirmer I	Schirmer
				mosm/ml	mosm/ml	mosm/ml	mm	mm	mm
	Patient groups	101	67 ±
			12
	Untreated	17	74 ± 9	289,29 ±	293,12 ±	296,67 ±	2,53 ± 4,08	3,59 ±	3,05 ±
				15,48	12,9	19		6,62	6,05
	onabotulinumtoxinA	26	67±12	294,31 ±	270 ± 25,22	295,46±	3,85 ±6,04 3,12±5,63	3,48 ±6
	Botox®			19,09		31,92
	(Allergan, USA)
	abobotulinumtoxinA	23	68±10 287,61 ± 60	302,7 ±	296,54 ±	2,35 ± 5,05	3,04 ±	2,96 ±
	Dysport ®				26,49	35,14		6,10	5,72
	(Ipsen Pharma,
	France)
	incobotulinumtoxinA	35	72±10	302.02 ±	300,47 ±	301,25 ±	3,53 ± 6,68	4,85 ±	4,19 ±
	XEOMIN®			21,89	20,67	21,14		7,61	7,14
	(Merz Pharma GmbH
	Germany)

TABLE 2 :

 2 statistics between the 3 treated groups compared with the untreated group (0)

	-	Mean osmo	p	IC95%	Mean	p	IC95%
					Schirmer
		mosml/ml			mm
	Untreated (0)	296,67 ±			3,05 ±
		19,17			6,05
	OnabotulinumtoxinA	295,46±31,92	0,65	-7,78;	3,48 ±6	0,73	-2,06; 2,90
	(B)			12,38
	AbobotulinumtoxinA	296,54 ±	0,92	-15 ; 16,6	2,96 ±	0,91	-2,51;2,79
	(D)	35,14			5,72
	incobotulinumtoxinA	301,25 ±	0,15	-2,60 ;	4,19 ±	0,38	-1,4; 3,66
	(X)	21,14		16,39	7,14

TABLE 3 :

 3 Student test comparison between all groups and OnabotulinumtoxinA injected group (B)

	Tableau 3
	Compared with B	Mean osmo	p	IC95%	Mean	p	IC95%
		mosm/ml			Schirmer
					(mm)
	Untreated (0)	296,67 ±	0,65	-7,78;	3,05 ±	0,73	-2,06; 2,90
		19,17		12,38	6,05
	OnabotulinumtoxinA	295,46±31,92			3,48 ±6
	(B)
	AbobotulinumtoxinA	296,54 ±	0,84	-16,50;	2,96 ±	0,81	-2,18; 2,75
	(D)	35,14		13,49	5,72
	incobotulinumtoxinA	301,25 ±	0,25	-3,36;	4,19 ±	0,54	-1,62; 3,05
	(X)	21,14		12,55	7,14

TABLE 4 :

 4 statistics Student test between all groups and AbobotulinumtoxinA injected patients(D)

	Compared with D	Mean osmo	p	IC95%	Mean	p	IC95%
		mosm/ml			Schirmer
					(mm)
	Untreated (0)	296,67 ±	0,92	-15 ;	3,05 ±	0,91	-2,51 ; 2,79
		19,17		16,60	6,05
	OnabotulinumtoxinA	295,46±31,92	0,84	-16,50;	3,48 ±6	0,81	-2,18 ; 2,75
	(B)			13,49
	AbobotulinumtoxinA	296,54 ±			2,96 ±
	(D)	35,14			5,72
	incobotulinumtoxinA	301,25 ±	0,40	-8,52;	4,19 ±	0,44	-1,55; 3,54
	(X)	21,14		20,72	7,14

TABLE 5 :

 5 statistics Student test between all groups and IncobotulinumtoxinA injected patients (X)

	Compared with D	Mean osmo	p	IC95%	Mean	p	IC95%
		mosm/ml			Schirmer
					(mm)
	Untreated (0)	296,67 ±	0,15	-2,60 ;	3,05 ±	0,38	-1,4 ; 3,66
		19,17		16,39	6,05
	OnabotulinumtoxinA	295,46±31,92	0,84	-16,50;	3,48 ±6	0,54	-1,63 ; 3,05
	(B)			13,49
	AbobotulinumtoxinA	296,54 ±	0,40	-	2,96 ±	0,44	-1,55 ; 3,54
	(D)	35,14		8,52;20,72	5,72
	IncobotulinumtoxinA	301,25 ±			4,19 ±
	(X)	21,14			7,14

Cher Monsieur, Chère Madame, Nous souhaitons vous remercier pour le temps passé à la relecture de notre étude et la clarté des demandes de corrections.

Voici ci-après les différents corrections réalisées :

1)Encore une fois on a une erreur dans le type d'étude annoncé.

Changement de type d'étude fait.

2) Dans le style on retrouve encore beaucoup de lourdeur et d'anglais francisé. Diminution de la longueur de certaines phrases. Modifications de certaines tournures de phrases.

3) il faut redéfinir le but principal de l'étude (question posée) en gardant à l'esprit qu'il s'agit d'une étude transversale et non longitudinale: A-t-on une sécheresse oculaire (Schirmer I + Osmolarité) chez ces patients pris en charge pour belpharospasme? Changement en ce sens du but principal de l'étude.