Aurore Delvart
email: aurore.delvart@inrae.fr

Céline Moreau
email: celine.moreau@inrae.fr

Angélina D'orlando
email: angelina.dorlando@inrae.fr

Xavier Falourd
email: xavier.falourd@inrae.fr

Bernard Cathala
email: bernard.cathala@inrae.fr

Dextran-based polyelectrolyte multilayers: effect of charge density on film build-up and morphology

Keywords:

We have studied the growth process of thin polyelectrolyte (PE) films fabricated by the layer-bylayer assembly (LbL) and composed of Dextran sulfate with high (DexS H) and low (DexS L) sulfation rate and poly(allylamine hydrochloride) (PAH). Film growths were monitored by combining Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), Surface Plasmon Resonance (SPR) and Atomic Force Microscopy (AFM). Even though, the two films growth up to 10 bilayers, QCM-D showed that polyelectrolyte pairs do not display similar behaviours.

(PAH/DexS H) systems lead to linear growth, i.e. amounts deposited increase both for PAH and DexS H, while the PAH/DexS L pair generated zig-zag shaped asymmetric growth. Film water contents were determined by QCM-D solvent exchange and SPR experiments. DexS L contains less water than DexS H and in agreement with the QCM-D dissipation values that suggest the formation of more rigid films in the case of DexS L than DexS H. Surface morphology investigated by AFM display distinct surface patterns since DexS H form thin films with fibril-like morphology covering all the surface while heterogeneous films with "puddle-like" aggregates were imaged in the case of DexS L. Difference of charge compensation and charge neutralisation between both systems likely lead to dissimilar growth mechanisms that are tentatively proposed in this paper.

INTRODUCTION 3

Dextrans are a family of α-glucans naturally produced by bacteria, whose main chain consists of D-glucose units linked by α-(1→6) type glycosidic bonds and which presents backbone with various size and ramifications, depending on the bacterium involved during production. [START_REF] Díaz-Montes | Dextran: Sources, Structures, and Properties[END_REF] Within the increasing interest in the elaboration for biobased materials, dextrans present many interests such as their wide availability and their diversified structure differing in branching patterns or molar mass, but also with their functionalization ability due to the occurrence of hydroxyl groups on the surface of dextran main chain and branches. [START_REF] Heinze | Functional Polymers Based on Dextran[END_REF][START_REF] Antonini | Studies on dextran and dextran derivatives. I. Properties of native dextran in different solvents[END_REF] Dextrans, partially degraded-dextrans and dextran derivatives find numerous applications in various areas as for instance in biomedical field, food science, cosmetics formulation and environmental field. [START_REF] Naessens | Leuconostoc dextransucrase and dextran: production, properties and applications[END_REF][START_REF] Leathers | Dextran[END_REF][6][START_REF] De Belder | Polysaccharides in Medicinal Applications[END_REF] Low molecular weight dextrans can be used as a therapeutic agent for replacing moderate blood losses as blood-plasma substitute, as well as improving blood flow as blood volume expander. [START_REF] Shoemaker | Comparison of the relative effectiveness of whole blood transfusions and various types of fluid therapy in resuscitation[END_REF][START_REF] Richter | Normalizing effect of low molecular weight dextran fractions on the reduced suspension stability of human erythrocytes in vitro[END_REF] Dextran polymers and derivatives have also be used in treatment involving the supply of iron in anaemic patients and as antiviral agent selective of various viruses. [START_REF] Kaisi | Evaluation of tolerance of and response to iron dextran (Imferon ® ) administered by total dose infusion to pregnant women with iron deficiency anemia[END_REF][START_REF] Baba | Sulfated polysaccharides are potent and selective inhibitors of various enveloped viruses, including herpes simplex virus, cytomegalovirus, vesicular stomatitis virus, and human immunodeficiency virus[END_REF][START_REF] Mitsuya | Dextran sulfate suppression of viruses in the HIV family: inhibition of virion binding to CD4+ cells[END_REF] They are great candidates for formulation of skin products and for food transformations, as they can be used as emulsifier, thickening agent and moistening agent. [START_REF] Dickinson | Emulsifying properties of covalent protein-dextran hybrids[END_REF][START_REF] Mccurdy | Rheological properties of dextran related to food applications[END_REF] Moreover, dextran derivatives provide a large range of charged or neutral polymers with different structures which present potential uses in chemistry and in environmental chemistry. [START_REF] Gelotte | Studies on gel filtration[END_REF][START_REF] Jellum | Mercaptodextran, a metal-chelating and disulphide-reducing polythiol of high molecular weight[END_REF] Charged modified dextrans, such as dextran sulfates, have also been used for coatings and thin films elaboration. Their charged groups promotes interactions with other polyelectrolytes (PEs) and thus, their inclusion in polymers assemblies such as polyelectrolyte multilayers (PEMs). [START_REF] Serizawa | Enzymatic hydrolysis of a layer-by-layer assembly prepared from chitosan and dextran sulfate[END_REF][START_REF] Balabushevich | Loading the multilayer dextran sulfate/protamine microsized capsules with peroxidase[END_REF][START_REF] Pescosolido | Hyaluronic Acid and Dextran-Based Semi-IPN Hydrogels as Biomaterials for Bioprinting[END_REF][START_REF] Matsusaki | Controlled release of vascular endothelial growth factor from alginate hydrogels nano-coated with polyelectrolyte multilayer films[END_REF] Due to their availability and biocompatibility, dextrans have been already successfully incorporated in PEMs with various positively charged PEs for biological and biomedical applications. Among them, dextran sulfates are the most common ones integrated in PEMs formation : alternating pro-and anti-coagulant films, [START_REF] Sakaguchi | Layer-by-layer assembly on hydrogel surfaces and control of human whole blood coagulation[END_REF] antimicrobial and antifouling multi-layered coating [START_REF] Xu | Dextran-and Chitosan-Based Antifouling, Antimicrobial Adhesion, and Self-Polishing Multilayer Coatings from pH-Responsive Linkages-Enabled Layer-by-Layer Assembly[END_REF] or high loading efficiency systems and immobilization of biomacromolecules for drug delivey. [START_REF] Hashide | Insulin-containing layerby-layer films deposited on poly(lactic acid) microbeads for pH-controlled release of insulin[END_REF] Films of PEs can be typically formed using layer-by-layer (LbL) technique, introduced by Decher et al. in the 1990, [START_REF] Decher | Buildup of ultrathin multilayer films by a self-assembly process: III. Consecutively alternating adsorption of anionic and cationic polyelectrolytes on charged surfaces[END_REF][START_REF] Decher | Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites[END_REF] based on the alternative adsorption of cationic and anionic charged PEs, via the contribution of various driving forces. 35 Among all interactions, electrostatics between oppositely charged PEs was thought to be solely the main driving force for multilayer, [START_REF] Decher | Buildup of ultrathin multilayer films by a self-assembly process: III. Consecutively alternating adsorption of anionic and cationic polyelectrolytes on charged surfaces[END_REF] and the film build-up is explained by charge density of the adsorbing PEs and associated charge matching between poly-ions. [START_REF] Schoeler | Investigation of the Influence of Polyelectrolyte Charge Density on the Growth of Multilayer Thin Films Prepared by the Layer-by-Layer Technique[END_REF][START_REF] Voigt | Charge Effects on the Formation of Multilayers Containing Strong Polyelectrolytes[END_REF] Schlenoff et al. have relayed studies on charge balance in PEMs based on the charge overcompensation and distribution in films and suggested a modelling method to determine growth mechanism of PEMs. [START_REF] Schlenoff | Charge and Mass Balance in Polyelectrolyte Multilayers[END_REF][START_REF] Schlenoff | Mechanism of Polyelectrolyte Multilayer Growth: Charge Overcompensation and Distribution[END_REF] They discussed the amount of individual polymer as well as salt content and charges within individual layers and demonstrated the impact of charge overcompensation on the growth of layers. Furthermore, the authors suggest that polymer charges are responsible of intrinsic compensation while salt counterions are responsible of extrinsic compensation. However, defining a precise critical level of charge is not obvious since multilayer growth have been obtained with very low charged group amounts [START_REF] Crouzier | Ion Pairing and Hydration in Polyelectrolyte Multilayer Films Containing Polysaccharides[END_REF] and several works reveal the contribution of other driving non-electrostatic forces, such as hydrophobic forces or hydrogen bonds, especially when polysaccharides are involved. [START_REF] Crouzier | Ion Pairing and Hydration in Polyelectrolyte Multilayer Films Containing Polysaccharides[END_REF][START_REF] Kotov | Layer-by-layer self-assembly: The contribution of hydrophobic interactions[END_REF][START_REF] Cerclier | Elaboration of Spin-Coated Cellulose-Xyloglucan Multilayered Thin Films[END_REF] Linear growth of the film thickness with the number of deposited layers is commonly obtained with strong PEs leading to stable PEMs. On the other hand, PEMs constructed from weakly charged PEs, polysaccharides or polypeptides are often characterized by a non-linear growth processes, due to the fast diffusion of both PE types into PEM films. [START_REF] Picart | Molecular basis for the explanation of the exponential growth of polyelectrolyte multilayers[END_REF][START_REF] Picart | Buildup Mechanism for Poly(L-lysine)/Hyaluronic Acid Films onto a Solid Surface[END_REF][START_REF] Boulmedais | Buildup of Exponentially Growing Multilayer Polypeptide Films with Internal Secondary Structure[END_REF][START_REF] Elzbieciak | Nonlinear growth of multilayer films formed from weak polyelectrolytes[END_REF] In fact, the internal structure of the PEM is governed by several parameters such as the degree of charge of the polyelectrolyte, [START_REF] Schoeler | Investigation of the Influence of Polyelectrolyte Charge Density on the Growth of Multilayer Thin Films Prepared by the Layer-by-Layer Technique[END_REF] the pair of polyelectrolytes, [START_REF] Dubas | Factors Controlling the Growth of Polyelectrolyte Multilayers[END_REF][START_REF] Klitzing | Polyelectrolyte Membranes[END_REF] the ionic strength, the type of salts, [START_REF] Salomäki | The Hofmeister Anion Effect and the Growth of Polyelectrolyte Multilayers[END_REF] and the pH of the solution. Charge density can be tuned either by varying the pH in the case of weak polyelectrolytes or by adjusting the polymer structure in the case of strong charged moieties. In both cases, evaluation of the charge density of the films structure and properties represent a challenging investigation as well as real opportunity to tune and control the thin films structure. Moreover, LbL assemblies generally entrap significant amount of water [START_REF] Schmitt | Internal structure of layer-by-layer adsorbed polyelectrolyte films: a neutron and x-ray reflectivity study[END_REF] and swelling/deswelling character of PEMs can be controlled by : modulation of PEs chain conformation in solution or introduction of hydrophobicity in the system [START_REF] Kittle | Enhanced Dewatering of Polyelectrolyte Nanocomposites by Hydrophobic Polyelectrolytes[END_REF][START_REF] Höök | Variations in Coupled Water, Viscoelastic Properties, and Film Thickness of a Mefp-1 Protein Film during Adsorption and Cross-Linking: A Quartz Crystal Microbalance with Dissipation Monitoring, Ellipsometry, and Surface Plasmon Resonance Study[END_REF] by selecting counterion, [START_REF] Salomäki | Specific Anion Effect in Swelling of Polyelectrolyte Multilayers[END_REF] decreasing the ionic strength [START_REF] Dubas | Swelling and Smoothing of Polyelectrolyte Multilayers by Salt[END_REF] or reducing the polyions charge density. [START_REF] Shiratori | pH-Dependent Thickness Behavior of Sequentially Adsorbed Layers of Weak Polyelectrolytes[END_REF] In 2012, Kittle et al. demonstrated that water content of nanocomposites based on various dextrans can be modified by changing the degree of substitution and the hydrophobic content of absorbed charged dextrans derivatives. [START_REF] Kittle | Enhanced Dewatering of Polyelectrolyte Nanocomposites by Hydrophobic Polyelectrolytes[END_REF] In the present study, we report the investigation of PEMs growth containing Poly(allylamine hydrochloride) (PAH) and Dextran Sulfates (DexS) with different charge densities determined by 1 H NMR. We investigate the films growth mode by Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) and Surface Plasmon Resonance (SPR), with special focus on the water content. Those two techniques permit quantitative, real-time, in situ, non-invasive and highly sensitive detection and determination of the adsorbed molecules on various type of surfaces, including biopolymers and polysaccharides. [START_REF] Davantès | Adsorption of Hyperbranched Arabinogalactan-Proteins from Plant Exudate at the Solid-Liquid Interface[END_REF][START_REF] Stokes | Lubrication, Adsorption, and Rheology of Aqueous Polysaccharide Solutions[END_REF][START_REF] Sedeva | Reduction of Surface Hydrophobicity Using a Stimulus-Responsive Polysaccharide[END_REF] By accessing the variation of the shear rate of an oscillating piezoelectric sensor, QCM-D probes variations in total mass and viscoelastic properties of the absorbed species coupled with solvent i.e. the "wet" film formed on a surface. SPR allows to determine the "dry" mass of the adsorbed film from change of refractivity of the surface caused by the adsorption of molecules. A combination of those methods supplies significant information of the layer-by-layer adsorption mechanism, structural changes and the extent of hydration. The morphologies of the layer were investigated with Atomic Force Microscopy (AFM). In this work, we describe how charge density of DexS affects film growth and morphology of (PAH/DexS) PEMs. These experimental investigations should lead to a better understanding of the construction of PEMs based on dextrans derivates as well as allow to control the surface patterning of films by variation of the dextran charge content.

EXPERIMENTAL SECTION

Materials. Poly(allylamine hydrochloride) (PAH) with a molecular mass in the range of 120 -200 000 g.mol -1 was purchased from Polysciences (Germany) and was used as a polycation.

Dextran sulfates (MW ~40 000 g.mol -1 ) obtained from Sigma Aldrich (Germany), one noted as high sulfate content (DexS H) and the another one as low sulfate content (DexS L), were employed as polyanions. Chemical structures of DexS are described on Figure 1a. All polymers were dissolved in deionized water (18.2 mΩ, Millipore Milli-Q purification system) containing 25 mM NaCl at 0.1 g.L -1 for PAH and at 0.5 g.L -1 for DexS during 12 hours at room temperature under agitation. All solutions were stocked at 4°C. The pH of PAH solution and both DexS solutions was measured between pH 6 -6.5. Deuterium oxide D2O (98%) used for D2O/H2O exchange was purchased from Euriso-Top (France).

Characterization of Dextran Sulfate

Molar masses were determined using High-Performance Size-Exclusion Chromatography (HPSEC) with OMNISEC Reveal and OMNISEC Resolve (Malvern Panalytical Ltd., Malvern, UK). DexS were dissolved at 5 g.L -1 in deionized water (18.2 mΩ, Millipore Milli-Q purification system) and filtrated at 0.45 μm before measurement. The DexS samples were eluted at a range of 0.7 mL/min with 50mM NaNO3 containing 0.02% NaN3. The column used was a polymethylmethacrylate (A-series) A5000 column (300 mm x 7.8 mm) and intrinsic viscosity determinations were performed using a viscometer included in multi angle light scattering detector Viscotek SEC-MALS 9 (Malvern Panalytical Ltd., Malvern, UK). Intrinsic viscosity data were recorded using OMNISEC 11.01 software (Malvern Panalytical Ltd., Malvern, UK). Molar masses were obtained using Mark-Houwink equation:

(1) = ×
where [η] (dL.g -1 ) is intrinsic viscosity obtained with HPSEC, Mv (g.mol -1 ) is the polymer viscosity-average molar mass and K (dL.g -1 ), α are viscosity-molecular weight constants for particular polymer-solvent combination. Values of K and α were assumed to be respectively 7.337×10 -4 dL.g -1 and 0.533 according to value for dextrans at 20°C reported by Güner. [START_REF] Güner | Unperturbed dimensions and the theta temperature of dextran in aqueous solutions[END_REF] Molar masses obtained were consistent with supplier data.

1 H nuclear magnetic resonance (NMR) experiments were carried out on a Bruker AvanceIII 400 NB spectrometer operating for 1 H, equipped with a double-resonance H/X BBo 5-mm probe.

Between 90 and 95 mg of dextran sulfates were solubilized in 1 g of 99.96% D2O. All spectra were recorded at 30°C using a 1 H 90° pulse of 10.2 µs and 128 accumulation with a recycle delay of 10 s. Acquisitions parameters including a water signal pre-saturation applied to decrease the HDO signal in the same order of magnitude than the others peaks. NMR spectra deconvolution was performed using the PeakFit® software (Systat Software, Inc., US). Peak chemical shifts and relative contribution were assigned according to the study report by Neville et al. [START_REF] Neville | Characterization and differentiation of some complex dextran sulfate preparations of medicinal interest[END_REF] (PAH/DexS) film deposition by dipping method. Multilayer films were deposited on silicon wafers as solid substrates for AFM analysis. Surfaces were cleaned in piranha bath (mixture of H2O2/H2SO4 at 70%/30% v/v) for 30 minutes for silicon wafers, then they were extensively rinsed in pure deionised water and dried under a N2 stream. (PAH/DexS) multilayer films were also formed by LbL method using dipping procedure. Silicon substrates were alternately immersed in PAH solution and DexS solution for 5 minutes. Between each adsorption step, the surfaces were rinsed in 3 water baths containing no NaCl. The dipping sequence was repeated n times to form (PAH/DexS)n with n the number of bilayers on the substrate where a (PAH/DexS) bilayer (n = 2) is a double layer composed for a single PAH layer and a single DexS layer. All surfaces were dried under N2 stream once 10 bilayers, (PAH/DexS)10, were assembled.

Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D).

A QCM-D equipment (E4 Q-Sense instrument, AB, Sweden) was used to investigated the adsorption process of PAH and DexS in the formation of (PAH/DexS) PEMs. Films were deposited on gold-coated quartz crystals (QSX301, Q-Sense) which were cleaned in piranha bath for 5 minutes. The quartz crystals used, AT-cut quartz crystal coated with a nominal resonance frequency of 5 MHz (f0), were introduced into the cell and water was introduced at a flow rate of 50 µL.min -1 at 20°C for at least 1 hour until a stable baseline was obtained. First, PAH solution was injected at the same flow rate for 5 minutes, followed by 5 minutes of water rinsing. Then DexS solution was injected at 50 µL.min -1 for 5 minutes, once again followed a 5-minutes rinsing step. The variations in frequency (Δf) and dissipation (ΔD) signals were recorded at the fundamental resonance frequency and its overtones (from 3 rd overtone to 7 th overtone). All experiments were repeated at least 3 times.

Normalized shifts in frequency (Δfn/n) due to additional mass on the sensor surface can be linked to the adsorbed mass (Δm) using Sauerbrey equation for rigid films (dissipation ΔDn/n < 1×10 -6 ) or using viscoelastic modeling for non-rigid films (dissipation ΔDn/n > 1×10 -6 ). [START_REF] Sauerbrey | Verwendung von Schwingquarzen zur Wägung dünner Schichten und zur Mikrowägung[END_REF][START_REF] Höök | Energy Dissipation Kinetics for Protein and Antibody-Antigen Adsorption under Shear Oscillation on a Quartz Crystal Microbalance[END_REF] In this study, the interpretation was done with Dfind Smartfit model on QSense Dfind® Software (version 1.2.6, Biolin Scientific, Q-Sense, Sweden), which integrates both frequency and dissipation assuming a film density ρ of 1.200 g.cm -3 for all films. [START_REF] Serizawa | Enzymatic hydrolysis of a layer-by-layer assembly prepared from chitosan and dextran sulfate[END_REF][START_REF] Serizawa | Alternating Bioactivity of Polymeric Layer-by-Layer Assemblies: Anti-vs Procoagulation of Human Blood on Chitosan and Dextran Sulfate Layers[END_REF] The film built under LbL process was assumed to have a uniform thickness and a uniform density. The model, based on Kelvin-Voigt model, was fitted with at least four overtones with a good signal-to-noise S/N ratio and gave access to film thickness, shear elastic modulus and shear viscosity. [START_REF] Höök | Variations in Coupled Water, Viscoelastic Properties, and Film Thickness of a Mefp-1 Protein Film during Adsorption and Cross-Linking: A Quartz Crystal Microbalance with Dissipation Monitoring, Ellipsometry, and Surface Plasmon Resonance Study[END_REF] Only values from fit with quality indicator higher than 0.70 were used. The adsorbed mass ΓQCM-D (ng.cm -2 ) of the polymer layer in hydrated state was calculated as follows:

(2) = .

where dQCM-D is the film thickness obtained with QCM-D technique (nm) and ρ (g.cm -3 ) is the assumed density of the film.

Surface Plasmon Resonance Measurement (SPR).

Building of (PAH/DexS) PEMs was followed by SPR on a Biacore ×100 instrument (Sweden) using gold-coated chips (SIA kit Au, GE Healthcare, France) cleaned in piranha bath for 5 minutes. The sensor was slide into the equipment flow cell, where desionised water was injected at a flow rate of 30 µL.min -1 at 20°C until baseline stabilization. Then PAH solution and DexS solution was alternately introduced for 5 minutes, followed by 5-minutes injection of deionized water at 30 µL.min -1 flow rate.

SPR measurements reports changes in the refractive index (n) of a surface, caused by the adsorption of molecules on it. A change in the refractive index induces a variation in the SPR angle ∆θ, which is detected by SPR equipment. [START_REF] Kittle | Enhanced Dewatering of Polyelectrolyte Nanocomposites by Hydrophobic Polyelectrolytes[END_REF][START_REF] Höök | Variations in Coupled Water, Viscoelastic Properties, and Film Thickness of a Mefp-1 Protein Film during Adsorption and Cross-Linking: A Quartz Crystal Microbalance with Dissipation Monitoring, Ellipsometry, and Surface Plasmon Resonance Study[END_REF][START_REF] Stenberg | Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proteins[END_REF] The SPR angle is related to the thickness and mass concentration of the absorbed layers without associated water and the thickness dSPR (nm) was determined using the following equation derivatized from Feijter et al. equation: [START_REF] Feijter | Ellipsometry as a tool to study the adsorption behavior of synthetic and biopolymers at the air-water interface[END_REF] (3)

= ∆ ( )
where ld is a characteristic decay length of the evanescent electromagnetic field estimated at 0.37 of the light wavelength (λd = 281.2 nm), m is a sensitivity factor for the sensor obtained after calibration of the SPR (101.93°/RI), ns is the refractive index of the bulk solution (ns = 1.33371), na is the refractive index of the adsorbed species in rigid and compact film. In this study, the refractive index na was assumed to be 1.49 and 1.48 respectively for PAH and DexS, which are values for rigid dry films entirely made of the polymer. The adsorbed mass ΓSPR (ng.cm -2 ) was calculated as follows:

(4) = .

where dSPR is the film thickness (nm) and ρ is the density of the film.

Determination of Water Content of films.

Calculated QCM-D mass ΓQCM-D represents the hydrated layers, i.e. the absorbed polymer and the coupled water while calculated SPR mass ΓSPR is related to the "dry" mass, i.e. the polymer without water. [START_REF] Davantès | Adsorption of Hyperbranched Arabinogalactan-Proteins from Plant Exudate at the Solid-Liquid Interface[END_REF][START_REF] Stokes | Lubrication, Adsorption, and Rheology of Aqueous Polysaccharide Solutions[END_REF] Using both techniques, the water content of (PAH/DexS) films, H2OSPR/QCM (%) is calculated as follow:

(5)

" # / (%) = & '()*+ & ,-. & '()*+ × 100%
The water content by switching the solvent from water to deuterium oxide D2O was calculated using QCM-D. [START_REF] Kittle | Enhanced Dewatering of Polyelectrolyte Nanocomposites by Hydrophobic Polyelectrolytes[END_REF][START_REF] Kittle | Water Content of Polyelectrolyte Multilayer Films Measured by Quartz Crystal Microbalance and Deuterium Oxide Exchange[END_REF][START_REF] Villares | Effect of xyloglucan molar mass on its assembly onto the cellulose surface and its enzymatic susceptibility[END_REF] The H2O/D2O solvent exchange was performed with one (PAH/DexS) bilayer constructed in the same conditions as described in the section "QCM-D". After stabilization in H2O at a flow rate of 50 µl.min -1 , D2O is injected for 5 minutes followed by rinsing with H2O during 30 minutes. The same experiment was done using the bare quartz substrate as reference system.

The changes in frequency between bare quartz crystal (∆fn/n)bare and quartz crystals covered by layers (∆fn/n)film allow to determine the frequency shift (∆fn/n)water caused by solvent exchange with the following equation: [START_REF] Kittle | Enhanced Dewatering of Polyelectrolyte Nanocomposites by Hydrophobic Polyelectrolytes[END_REF][START_REF] Kittle | Water Content of Polyelectrolyte Multilayer Films Measured by Quartz Crystal Microbalance and Deuterium Oxide Exchange[END_REF] (6)

1 ∆2 3 45678 = 1 ∆9 : 3 9;<= 1 ∆9 : 3 > ?@ A B +CD B ECD F G
where n the overtone number and ρ the densities of solvent, 0.998 g.cm -3 for H2O and 1.106 c.cm -3 for D2O at 20°C. The water content from H2O/D2O exchange, H2Oexchange (%), can then calculated as:

(7) " # 7HIJ5 K7 (%) = Fabrication of the LbL films. We focused on poly(allylamine hydrochloride) (PAH) as polycation and two dextran sulfates (DexS) as polyanions since this polyion pair has demonstrated its ability to build LbL films as well as layered nanocapsules. [START_REF] Anandhakumar | A facile route to synthesize silver nanoparticles in polyelectrolyte capsules[END_REF][START_REF] Sripriya | Laser receptive polyelectrolyte thin films doped with biosynthesized silver nanoparticles for antibacterial coatings and drug delivery applications[END_REF] Figure 1a presents the structure of the dextran sulfate used in this study. DexS have the same molar mass and the same D-glucose backbone linked by α(1→6) glycosidic linkages (Figure 1a). They differ only by their charge densities and sulfate substitution patterns as determined by 1 H NMR (Table in Figure 1a and Figure S1). DexS with high sulfate content (DexS H) is mainly disubstituted since NMR spectra displays peaks for simultaneous sulfation at C-2/C-3 (76%) and -or monosubstituted either at C-2 or C-3 (14 and 9 %, respectively). On the contrary, DexS with low sulfate content (DexS L) shows peaks for monosulfation at C2 and C3 (24 and 17%) and little amount of disulfation (9%) (Supporting Information, Figure S1). A majority of unsulfated glucose units were also detected (51%) for DexS L. In summary, all glucose units of DexS H bear at least on charge, a majority being double charged leading to an average charge density of 1.75 charges per glucose unit, while, in the case of DexS L, only 0.6 glucose groups in average are charged. In the case of poly(allylamine hydrochloride) (PAH), the polymer contains 1 amino group by monomer unit taking into account the neutral pH of the polymer solution so that all the amino groups are charged leading to the presence of 1 charge per monomer. This estimation gives a rough overview of potential electrostatic interaction between DexS and PAH since other parameters such as the molar mass of the monomers, the stiffness of the chains and the chains conformation will also influence the polymer adsorption. Nevertheless, the difference of charge density between the two DexS is significant and relevant to explain their adsorption behaviours.

Adsorptions of PEs solutions were monitored by QCM-D and carried out in 25 mM NaCl followed by a rinsing step in pure water to remove all loosely bound polymers or salt excess as in classical LbL process (Figure 1b). These conditions are in agreement with previous studies on salt concentration effect, as it is well known that salt can affect the thickness, surface roughness and the structure of the films. [START_REF] Lourenço | Counterions in Poly(allylamine hydrochloride) and Poly(styrene sulfonate) Layer-by-Layer Films[END_REF][START_REF] Moreau | Tuning the Architecture of Cellulose Nanocrystal-Poly(allylamine hydrochloride) Multilayered Thin Films: Influence of Dipping Parameters[END_REF][START_REF] Elosua | Comparative study of layer-by-layer deposition techniques for poly(sodium phosphate) and poly(allylamine hydrochloride)[END_REF][START_REF] Elżbieciak-Wodka | Comparison of permeability of poly(allylamine hydrochloride)/and poly(diallyldimethylammonium chloride)/poly(4-styrenesulfonate) multilayer films: Linear vs. exponential growth[END_REF] For similar films with PAH, NaCl concentration used is between 10 mM NaCl and 1 M NaCl. [START_REF] Lourenço | Counterions in Poly(allylamine hydrochloride) and Poly(styrene sulfonate) Layer-by-Layer Films[END_REF][START_REF] Moreau | Tuning the Architecture of Cellulose Nanocrystal-Poly(allylamine hydrochloride) Multilayered Thin Films: Influence of Dipping Parameters[END_REF][START_REF] Elosua | Comparative study of layer-by-layer deposition techniques for poly(sodium phosphate) and poly(allylamine hydrochloride)[END_REF][START_REF] Elżbieciak-Wodka | Comparison of permeability of poly(allylamine hydrochloride)/and poly(diallyldimethylammonium chloride)/poly(4-styrenesulfonate) multilayer films: Linear vs. exponential growth[END_REF]. Polymer concentrations can also influence the multilayer film assembly and its adsorption kinetics; PAH concentration (0.1 g.L -1 ) and DexS concentration (0.5 g.L -1 ) were chosen according to our previous studies one QCM-D monitored LbL growth. [START_REF] Dubas | Factors Controlling the Growth of Polyelectrolyte Multilayers[END_REF][START_REF] Moreau | Tuning the Architecture of Cellulose Nanocrystal-Poly(allylamine hydrochloride) Multilayered Thin Films: Influence of Dipping Parameters[END_REF] and values are reported in Figure 2b. 54 Films based on DexS H result in a final frequency shift of -320 Hz for a total mass absorbed of about 8000 ± 870 ng.cm -2 while films based on DexS L result in a final frequency shift of -165 Hz for a total mass absorbed of 3600 ± 1770 ng.cm -2 for 10 bilayers. For both dextran two regimes were observed (Figure 1b). The first one between bilayer 1 and bilayer 3 and a second one after bilayer 3, as previously observed for others systems during multilayer assembly. [START_REF] Caruso | Ultrathin Multilayer Polyelectrolyte Films on Gold: Construction and Thickness Determination[END_REF][START_REF] Pinto | Mechanism of Formation and Construction of Self-Assembled Myoglobin/Hyaluronic Acid Multilayer Films: An Electrochemical QCM, Impedance, and AFM Study[END_REF] The first one, i.e. before the bilayer 3, is mainly driven by the substrate surface properties while the second one is related to the formation of a surface charge excess only related to bilayer structure. Thus the adsorption behaviour of the PE couple (PAH/DexS) becomes independent from the surface chemistry of the substrate. [START_REF] Caruso | Ultrathin Multilayer Polyelectrolyte Films on Gold: Construction and Thickness Determination[END_REF][START_REF] Villiers | Introduction to nanocoatings produced by layer-by-layer (LbL) self-assembly[END_REF] In the case of DexS H, the second regime highlights also a linear growth with an average mass increase of 858 ng.cm -2 per bilayer.

Whatever the PE deposited, PAH or DexS H, the adsorbed mass increases. However, it may be noted that PAH deposition yields to lower value (338 ng.cm -²) while DexS H deposition yields to higher value (520 ng.cm -2 ). The amount being evaluate by QCM-D, associated water must be also considered, and will be discussed after. The (PAH/DexS L)10 PEMs also exhibited a linear regime after bilayer 3 based with, however, a lower average mass increment of 375 ng.cm -2 for bilayer deposition in average. Remarkably, the growth pattern displays non-monotonous zig-zag shaped growth (Figure 1b). PAH layer adsorption on DexS L layer presents an increase of average mass deposition of 835 ng.cm -2 while adsorption of DexS L layer on PAH layer displays an average mass loss of 460 ng.cm -2 . This behaviour has been already observed and was associated to an adsorption-desorption process of hydrated polymers that can either removed from the surface due to loose attachment or expulsion of water and/or counter-ions from the film. [START_REF] Calvo | Effects of the nature and charge of the topmost layer in layer by layer self assembled amperometric enzyme electrodes[END_REF][START_REF] Ghostine | Asymmetric Growth in Polyelectrolyte Multilayers[END_REF] Moreover, even though both PEs allow to build stable films, they display different adsorption behaviours (symetric linear growth vs asymmetric linear growth) as well as a higher amount of polymer adsorbed for (PAH/DexS H)10 films. of splitting between frequencies for films based on DexS L while a difference of 50 Hz between the 3 rd and the 7 th overtones is observed. In addition, as illustrated on Figure 3b and 3d, (PAH/DexS H) films present an average final dissipation of 30×10 -6 while (PAH/DexS L) PEMs display an average final dissipation of 2×10 -6 , respectively. This indicates different viscoelastic properties with a more rigid film for (PAH/DExS L) as compared to (PAH/DexS H) film. These properties can be qualitatively compared through the -∆D/∆f ratio calculated from final frequencies and dissipation values reported on Figure 3b,d since the ratio correspond to a normalization of dissipation variation per unit of mass adsorbed. [START_REF] Jaafar | Meaning of xylan acetylation on xylan-cellulose interactions: A quartz crystal microbalance with dissipation (QCM-D) and molecular dynamic study[END_REF][START_REF] Towle | Impact of molecular weight on polyelectrolyte multilayer assembly and surface properties[END_REF] Final ratio -∆D5/∆f5 (after rising of bilayer 10) for (PAH/DexS L) films is an order of magnitude lower (0.015×10 -6 Hz -1 ) than (PAH/DexS H) value (0.122×10 -6 Hz -1 ) which confirms that the PAH/DexS L film is more rigid than the PAH/DexS H. From literature, value of about 0.3×10 -6 Hz -1 have been measured for poly(acrylic acid)/poly-L-lysine pairs [START_REF] Towle | Impact of molecular weight on polyelectrolyte multilayer assembly and surface properties[END_REF] and values varying from 0.1 to 0.5×10 -6 Hz -1 have been found for PEMs based on cellulose nanocrystals/hemicellulose, i.e. non-electrostatic based systems. [START_REF] Jaafar | Meaning of xylan acetylation on xylan-cellulose interactions: A quartz crystal microbalance with dissipation (QCM-D) and molecular dynamic study[END_REF] This clearly indicates that (PAH/DexS) PEMs are quite rigid and especially (PAH/DexS L) systems after DexS L adsorption step. Besides information provided by QCM-D on hydrated absorbed mass on substrate, SPR allows to calculate a "dry" polymer mass, i.e. the mass of polymer contained in the films without the contribution of the water bound. From equation (4) and equation ( 5), SPR absorbed masses are determinate to be 130 ng.cm -2 for (PAH/DexS H)2 PEMs and 120 ng.cm -2 for (PAH/DexS L)2

PEMs. Meanwhile, hydrated masses from QCM-D are 1100 ng.cm -2 for (PAH/DexS H)2 PEMs and 570 ng.cm -2 (PAH/DexS H)2 PEMs, which suggest that (PAH/DexS H)2 films contain more water than (PAH/DexS L)2. Based on QCM-D and SPR data, water content H2OSPR/QCM(%) was found to be about 72% in the case of DexS H and 50% in the case of DexS L (Figure 4a).

Water content has also be evaluated from H2O/D2O exchange experiment as monitored by QCM-D, i.e. H2Oexchange(%). [START_REF] Kittle | Water Content of Polyelectrolyte Multilayer Films Measured by Quartz Crystal Microbalance and Deuterium Oxide Exchange[END_REF] Figure 4b shows the change in frequency (∆fn/n) versus time for the (PAH/DexS)2 PEMs switched from H2O to D2O and back to H2O. The calculated water content of films was lower for (PAH/DexS L) films (44.5 %) than for the (PAH/DexS H) films (66.8%). Both analyses give consistent values of water content around 66 -72% of water for (PAH/DexS H)2

PEMs and 44 -50% of water for (PAH/DexS L)2 PEMs (Figure 4b). As discussed in the previous section, PEMs build from DexS H are more hydrated than PEMs build from DexS L consistent with a less rigid film structure. PEMs present a more granular surface with "puddle-like" aggregates. Similar aggregates in the dry state were previously observed for porous PEMs based on synthetic and natural polymers [START_REF] Picart | Buildup Mechanism for Poly(L-lysine)/Hyaluronic Acid Films onto a Solid Surface[END_REF][START_REF] Elzbieciak | Nonlinear growth of multilayer films formed from weak polyelectrolytes[END_REF][START_REF] Ghavidel Mehr | Chitosan surface modification of fully interconnected 3D porous poly(ε-caprolactone) by the LbL approach[END_REF][START_REF] Barroso | Self-healable hyaluronic acid/chitosan polyelectrolyte complex hydrogels and multilayers[END_REF], proteins and complex of biopolymers and proteins [START_REF] Davantès | Adsorption of Hyperbranched Arabinogalactan-Proteins from Plant Exudate at the Solid-Liquid Interface[END_REF][START_REF] Liu | Porous star-star polyelectrolyte multilayers for protein binding[END_REF] and well-known microporous surfaces [START_REF] Hollman | Pore assembled multilayers of charged polypeptides in microporous membranes for ion separation[END_REF][START_REF] Fu | Hydrogen-Bonding-Directed Layer-by-Layer Multilayer Assembly: Reconformation Yielding Microporous Films[END_REF]. For both dextran sulfate, increasing the number of bilayers seems to lead to a better surface coverage, as AFM images for PAH10 and DexS10 shows lower contrast than images for PAH5 and DexS5. Moreover, in the case of (PAH/DexS L), surface morphology seems to changes between PAH adsorption step and DexS adsorption step. Images on PAH10

Surface

show better surface coverage than images on DexS10, i.e. adsorption of DexS10 on PAH10 seems to reduce covered surface. to the formation of polyelectrolyte-complexes [START_REF] Feng | Self-assembly and characterization of polyelectrolyte complex films of hyaluronic acid/chitosan[END_REF], that may indicate the complexation of PAH and DexS L onto the surface. This complexation can be due to charge neutralisation in the films and thus lead to water removal and an increases of hydrophobic content of the film as it was previously observed by Kittle et al. [START_REF] Kittle | Enhanced Dewatering of Polyelectrolyte Nanocomposites by Hydrophobic Polyelectrolytes[END_REF] From all information collected in this study, Figure 6 presents tentative mechanisms of film building explaining differences observed in the two systems. In the case of (PAH/DexS H) films, illustrated on Figure 6a, the amount of negative charges from DexS are likely high enough to largely overcompensate charges coming from previous PAH layer that might provide high surface charge density after DexS H adsorption (Figure 6a). [START_REF] Glinel | Influence of Polyelectrolyte Charge Density on the Formation of Multilayers of Strong Polyelectrolytes at Low Ionic Strength[END_REF] In the Step 1, the adsorption of PAH on adsorbed DexS layer leads to an increase in mass and in dissipation as hydrated PAH is adsorbed. During the Step 2, water rising does not seem to induce any mass loss which indicate that previous layers were charged enough to be strongly linked to the surface undernear. Thus, (PAH/DexS) system leads to the formation of stable and charged layers.

Then, the adsorption of DexS H on previous PAH layer (Step 3) followed by the rinsing step (Step 4) leads to surface charge inversion and no significant DexS and counter-ion excess removing.

During these four steps, the amount of water in the layer is stable and PE form stable and highly charged layers.

For (PAH/DexS L) systems (Figure 6b), the adsorption mechanism is more complex. At the Step 1, adsorption of PAH induces an increase of film hydration and a decrease of the film rigidity followed by an internal film restructuration. As DexS L presents a number of charges per monomer For (PAH/DexS H) systems, the high amount of charges on surface due to charges overcompensation leads to the construction of (PAH/DexS H) PEMs with typical symmetric linear growth and homogenous thin films. In contrast, (PAH/DexS L) PEMs present an asymmetric growth between polycation and polyanion adsorption, which suggests a restructuration and a loss of water during LbL process, as it was observed with previous PE couples including synthesized dextrans by Kittle et al. [START_REF] Kittle | Enhanced Dewatering of Polyelectrolyte Nanocomposites by Hydrophobic Polyelectrolytes[END_REF] As DexS L presents a number of charges per monomer comparable to the number of charges presents on PAH monomer, the restructuration must be due to the partial neutralisation of films and internal diffusion of PEs with adsorption, which contribute to the PEs complexation and dehydration of the system. The effect of restructuration on film surface morphology also influences the film morphology as the formation of PEMs with different pattern is obtained. (PAH/DexS L) PEMs surfaces display "puddle-like" aggregates with pores similar to ones obtained with PE-complex films and microporous membranes based on polysaccharides. [START_REF] Liu | Porous star-star polyelectrolyte multilayers for protein binding[END_REF][START_REF] Hollman | Pore assembled multilayers of charged polypeptides in microporous membranes for ion separation[END_REF][START_REF] Fu | Hydrogen-Bonding-Directed Layer-by-Layer Multilayer Assembly: Reconformation Yielding Microporous Films[END_REF] This work highlights the impact of charge density on the PEMs building and properties at each adsorption step as well as on final aspect of the surface. The possibility to design the surface pattern reminds nano-printing and nano-lithography processes used to fabricate patterns of nanometre scales. [START_REF] Vigneswaran | Recent Advances in Nano Patterning and Nano Imprint Lithography for Biological Applications[END_REF] In this context, patterning by modulation of PE charge density and LbL process is believed to be a simple method for micro-and nano-patterning especially with the possibility to play with both charge density to build mix-structures with controlled internal and surface morphology based on dextrans sulfates. As it is excepted to obtain different porosity as well as different colours with the various nanostructures, those systems could help in the development of colour-based sensors and permeability-controlled nano-objets.

Figure 1 .

 1 Figure 1. (a) Chemical structures of dextran sulfate with high sulfate content DexS H (at left) and low sulfate content DexS L (at right); sulfation patterns of both DexS are indicated. (b) Demonstrative scheme of the LbL formation of films from cationic PAH (orange chains), and negatively charged DexS (green chains) in solid substrate (grey bar).

Figure 2 .

 2 Figure 2. (a) Graph of Δf5 vs time for (PAH/DexS)10 multilayer with DexS H (dark green) and DexS L (light green) building on gold coated surface. (b) Adsorbed mass evaluated with Voigt model for (PAH/DexS)10 multilayer with DexS H (dark green) and DexS L (light green). Bn corresponds to the n th (PAH/DexS) bilayer of the multilayer, the full symbols are representatives of PAH adsorption while the open symbols are representatives of the DexS adsorption.

Figure 3 .

 3 Figure 3. (a), (c) ∆fn and ∆Dn as function of time for (PAH/DexS)10 multilayer on gold coated quartz crystal using QCM-D. The frequency (blue) and dissipation (red) changes for the third, fifth and seventh overtone are depicted. (b), (d) Zoom on the bilayer 10 build-up with adsorption of PAH (orange), rising with pure water (grey), adsorption of DexS (green).

Figure 4 .

 4 Figure 4. (a) Water content (%w/w) of the films calculated from the ratio of adsorbed mass obtained by SPR and adsorbed mass obtained by QCM-D on the 2 nd bilayer and the value calculated from D2O-H2O exchange on the 2 nd bilayer. (b) ∆f5 vs time for D2O-H2O exchange on

  Morphology and Roughness. AFM was performed to analyse the surface morphology and roughness of the (PAH/DexS) adsorbed layers on the silicon wafer after adsorption, in a dry state, and images are reported on Figure 5. After drying of 5 (PAH/DexS) and 10 (PAH/DexS) adsorbed bilayers, different patterns are observed on both films as illustrated on Figure 5. (PAH/DexS H) PEMs exhibit a surface displaying fibrillar network pattern while (PAH/DexS L)

Figure 5 . 10 DexS 5 (

 5105 Figure 5. AFM topography images from (a) (PAH/DexS H) multilayer and (b) (PAH/DexS L) multilayer of (on the top) the PAH 5 (bilayer 4.5 i.e. outermost layer of PAH before bilayer 5), the

Figure 6 .

 6 Figure 6. Proposed mechanisms for the build-up of (a) (PAH/DexS H) PEMs and (b) (PAH/DexS L) PEMs starting from bilayer 4 on PEMs previously ending with DexS.

  comparable to the number of charges presents on PAH monomer, a partial neutralisation of sublayers with PE adsorption occurred, which contributes to the diffusion of the adsorbed PAH into the (PAH/DexS L) PEMs. During the rising in Step 2, the film swells again that may promote further diffusion internal and restructuration of PEMs. Charge neutralisation itself generates a water removal as shown by[START_REF] Kittle | Enhanced Dewatering of Polyelectrolyte Nanocomposites by Hydrophobic Polyelectrolytes[END_REF].[START_REF] Kittle | Enhanced Dewatering of Polyelectrolyte Nanocomposites by Hydrophobic Polyelectrolytes[END_REF] During Step 3, as soon as DexS L diffusion starts after adsorption, charge compensation between PAH and DexS L occurred inside the films that likely promote charge neutralisation then complexation between PAH and DexS L. Formation of PEs-complexes decreases electrostatic repulsion between PEs and the affinity with bondingwater of highly hydrated PAH/DexS decreases resulting in the water expulsion from the film. This film dehydration, also named deswelling or dewatering, favour short range interactions within the final system[START_REF] Höök | Variations in Coupled Water, Viscoelastic Properties, and Film Thickness of a Mefp-1 Protein Film during Adsorption and Cross-Linking: A Quartz Crystal Microbalance with Dissipation Monitoring, Ellipsometry, and Surface Plasmon Resonance Study[END_REF][START_REF] Kittle | Water Content of Polyelectrolyte Multilayer Films Measured by Quartz Crystal Microbalance and Deuterium Oxide Exchange[END_REF] and induce PAH/DexS L aggregation onto the surface leading to the "puddlelike" pattern (Step 3).[START_REF] Feng | Self-assembly and characterization of polyelectrolyte complex films of hyaluronic acid/chitosan[END_REF] CONCLUSIONS Dextran sulfate (PAH/DexS) PEMs films with linear growth were successfully constructed and we demonstrated that difference of charge density between DexS H and DexS L resulted in distinct adsorption capacity and influence the type of adsorption as well as the film surface morphology.

ACKNOWLEDGMENT

The authors gratefully acknowledge Nadège Leray for her technical support during all the project and Luc Saulnier for carrying HPSEC experiments. We would also like to acknowledge the financial support for this study from French National Agency (ARN).

Funding

This work has been supported by the French National Agency (ANR) as part of the program ANR-18-CE43-0007 (GRαFTING).

All authors have given approval to the final version of the manuscript.

Declaration of Competing Interest

The authors declare no competing financial interest or personal relationships that could have appeared to influence the work reported in this paper.


A De Carbonnières

; A Challine

; A S Cottereau

; R Coriat

; P Soyer

; E Abou

; F Prat

; B Terris

; J Bertherat

; B Dousset

; S Gaujoux

Sébastien Pr

MD Gaujoux

Surgical management of insulinoma over three decades

Keywords: Insulinoma, Pancreas, Pancreatectomy, Laparoscopy, Tomography, X-Ray computed, Parenchyma-sparing pancreatectomy, Enucleation, Neuroendocrine tumours, Postoperative complications

Introduction: This paper reports our experience of the perioperative management of patients with sporadic, non-malignant, pancreatic insulinoma.

Methods:

A retrospective monocentric cohort study was performed from January 1989 to July 2019 using a prospectively maintained database, including all the patients who had been operated on for pancreatic insulinoma as of the time of the study. The preoperative work-up, surgical management, and postoperative outcome were analyzed.

Results: Eighty patients underwent surgery for sporadic pancreatic insulinoma, 50 of which were female (62%), with a median age of 50 (36-70) years. Preoperatively, the tumors were localized in 76 patients (95%). Computed tomography (CT) and magnetic resonance imaging allowed exact preoperative tumor localization in 76% of the patients (64-85 and 58-88 patients, respectively), increasing to 96% when endoscopic ultrasonography was performed in addition to CT. Forty-one parenchyma-sparing pancreatectomies (including enucleation, caudal pancreatectomy, and uncinate process resection) and 39 pancreatic resections were performed, 32% via laparoscopy. The mortality rate was 6% (n = 5, 4 after pancreaticoduodenectomy), with a morbidity rate of 72%, including 24 severe complications (30%) and 35 pancreatic fistulas (44%). No differences were found between formal pancreatectomy and parenchyma-sparing pancreatectomy in terms of postoperative outcome procedures. The surgery was curative in all the patients, with long-term postoperative normalization of the glycemia and insulin blood levels.

Conclusion:

CT used in combination with endoscopic ultrasonography allows accurate localization of insulinomas in almost all patients. When possible, a parenchyma-sparing pancreatectomy should be proposed as the first-line surgical strategy.

Introduction

Insulinoma is the most common functional pancreatic neuroendocrine tumor. In most patients, it is a small, sporadic, solitary, and intrapancreatic benign tumor, [START_REF] Crippa | Surgical management of insulinomas: shortand long-term outcomes after enucleations and pancreatic resections[END_REF][START_REF] Tucker | The management of insulinoma[END_REF][START_REF] Davì | Pancreas: Insulinoma--new insights into an old disease[END_REF] but its diagnosis can be difficult and is often delayed because of the highly variable and nonspecific symptoms of hypoglycemia. When a diagnosis of organic hypoglycemia with inappropriately elevated endogenously produced insulin after a 48-72 h fasting test [START_REF] Guettier | The role of proinsulin and insulin in the diagnosis of insulinoma: a critical evaluation of the Endocrine Society clinical practice guideline[END_REF] is confirmed, the next issue is to accurately localize the insulinoma to anticipate the surgical approach, which has long been exclusively based on intraoperative ultrasonography and thorough pancreatic palpation. This is often challenging because the tumors distributed throughout the pancreatic gland are usually small. The combination of cross-sectional imaging techniques, nuclear medicine, and endoscopic ultrasonography, however, has dramatically changed the preoperative assessment of insulinomas. [START_REF] Gouya | Endoscopic Sonography, and a Combined Protocol for Preoperative Evaluation of Pancreatic Insulinomas[END_REF][START_REF] Mehrabi | A Systematic Review of Localization, Surgical Treatment Options, and Outcome of Insulinoma[END_REF][START_REF] Nockel | Localization of Insulinoma Using 68 Ga-DOTATATE PET/CT Scan[END_REF] Thus, blind pancreatectomy is no longer recommended. [START_REF] Zhao | Surgical management of patients with insulinomas: Result of 292 cases in a single institution: Surgical Management of Insulinoma[END_REF] Surgery remains the cornerstone of insulinoma treatment. [START_REF] Placzkowski | Secular trends in the presentation and management of functioning insulinoma at the Mayo Clinic, 1987-2007[END_REF] As it is benign in most patients, it should ideally be done through parenchymasparing pancreatectomy [START_REF] Goudard | Reappraisal of central pancreatectomy a 12year single-center experience[END_REF] ideally enucleation [START_REF] Cherif | Enucleation of pancreatic lesions through laparotomy[END_REF][START_REF] Faitot | Reappraisal of pancreatic enucleations: A singlecenter experience of 126 procedures[END_REF] to preserve both the exocrine and endocrine functions. [START_REF] Crippa | Surgical management of insulinomas: shortand long-term outcomes after enucleations and pancreatic resections[END_REF] Nevertheless, these procedures often expose patients to substantial postoperative morbidity. [START_REF] Strobel | Risk of pancreatic fistula after enucleation of pancreatic tumours[END_REF] Reported herein are the evolution in the pre-and perioperative management of patients with sporadic non-malignant insulinoma and our experience in relation to such, with specific attention given to preoperative localization and postoperative outcome following pancreatectomy and the development of the laparoscopic approach.

Methods

Patients and methods

Data collection

All the patients who underwent pancreatectomy for sporadic non-malignant insulinoma within the period from 1989 to 2019 at Cochin Hospital, Paris, France, a tertiary referral center for endocrine diseases, were included in this study. Demographic, clinical, radiological, pathologic, and follow-up data were obtained from a prospectively maintained database, with additional retrospective review. For each patient, the diagnosis was confirmed by an experienced pathologist. Malignant (i.e., metastatic insulinoma) and multiple endocrine neoplasia type 1 (MEN1) insulinoma were excluded from the analysis. Postoperative mortality included all the deaths that occurred within 90 days from the surgery. Morbidity included all the complications that occurred within 90 days from the surgery and was graded according to the Clavien-Dindo classification classification [START_REF] Strobel | Risk of pancreatic fistula after enucleation of pancreatic tumours[END_REF] . Postoperative pancreatic fistula, hemorrhage, and delayed gastric emptying were defined according to the International Study Group of Pancreatic Surgery [START_REF] Core | R: A language and environment for statistical computing[END_REF][START_REF] Okabayashi | Diagnosis and management of insulinoma[END_REF][START_REF] Thoeni | Detection of Small, Functional Islet Cell Tumors in the Pancreas: Selection of MR Imaging Sequences for Optimal Sensitivity[END_REF][START_REF] Kulali | Role of diffusionweighted MR imaging in predicting the grade of nonfunctional pancreatic neuroendocrine tumors[END_REF] . The follow-up was based on clinical, radiological, and laboratory assessments and was updated on the basis of outpatient reviews and correspondence. All the patients were followed up at least 1 year after surgery. The year 2000 was considered a time landmark because it marked the beginning of the evolution of imaging, endoscopic, and laparoscopic techniques. The study protocol was approved by the hospital's medical ethics committee.

Preoperative work-up and surgical indications

The preoperative work-up (all performed in Cochin Hospital) included a clinical evaluation with patient questioning and a complete hormonal and radiological evaluation with triphasic computed tomography (CT) examination (unenhanced, arterial, and portal phases) and endoscopic ultrasound (EUS) since it became available. Additional investigations with magnetic resonance imaging (MRI) including standard sequences and cholangiopancreatography images and somatostatin receptor scintigraphy or positron emission tomography (PET)/CT either with 111 In-DTPA-octreotide or more recently 68 Ga-DOTATOC or EUS with fine needle aspiration (FNA) were left to the surgeon's discretion.

The policy regarding the use of nuclear medicine imaging has evolved over time. 111In-DTPA-octreotide, despite its low sensibility, was initially used but was later replaced by 68 Ga-DOTATOC when it became more easily available. All surgical indications were discussed in a multidisciplinary tumor board that included surgeons, radiologists, nuclear physicians, endocrinologists, pathologists, and gastroenterologists.

Surgical techniques

The type of surgical resection that was performed was based on the tumor location and the distance to the main pancreatic duct. Distal pancreatectomy was defined as a pancreatectomy with pancreatic resection at the level of the isthmus. Caudal pancreatectomy, on the other hand, which is considered parenchyma-sparing pancreatectomy, was defined as a pancreatectomy with pancreatic resection on the left side of the isthmus. Enucleation and parenchyma-sparing pancreatectomy (central pancreatectomy, resection of the uncinate process, and caudal pancreatectomy) was preferred whenever possible. During surgery, pancreatic exploration after mobilization of the transverse colon root was first performed through visual inspection and surgical ultrasonography to confirm the tumor location.

Statistical analysis

The variables were expressed as medians, interquartiles, or numbers and percentages of patients, as appropriate. The continuous variables were evaluated for normal distribution using the Komogorov-Smirnov test. Chi square or Fisher's exact test was used to evaluate the categorical variables. The continuous variables were analyzed using Student's t-test or Mann-Whitney test, as appropriate. The capability of each imaging test to determine the exact tumor location was calculated in terms of the true location rate, using the surgical and histopathological findings as the standards of reference. The McNemar test was used to compare the diagnostic tests. All the tests were two-tailed, and significance was set at p < 0.05. The analysis was conducted with the R software. 14

Results

Characteristics of the population and preoperative imaging

The final study population included 80 patients who underwent surgery for pancreatic insulinoma, 50 (62%) of which were women and 30 (38%) men, with a median age of 50 (36-70) years (Table 1). Most of the patients were ASA 1 or 2 (n = 76; 95%) and presented neuroglycopenic symptoms (n = 74; 92%), including fainting (40%; n = 32). The median time interval between the first symptoms and the surgery was 13 months (4-26 months).

The proportions of tumors that were correctly localized using the different preoperative imaging tests are shown in pancreatectomy. In all the cases, the pancreas was considered soft.

Postoperative outcomes

The postoperative course is summarised in Table 3. The mortality rate was 6% (5/80), including 1 death from septic shock from the venous central catheter, 2 from grade C pancreatic fistula (bleeding and septic shock), 1 from liver abscesses due to hepatic artery thrombosis, and 1 from fungal septicemia. Four of these deaths occurred after pancreaticoduodenectomy. The morbidity rate was 72% (n = 58), including 24 severe (grade III/IV or V) complications (30%), and a pancreatic fistula occurred in 35 patients (44%), with 19 (24%) grade B or C. No significant differences were found between the patients who underwent parenchyma-sparing pancreatectomy and those who underwent formal pancreatectomy. The surgery was successful in all the patients, with long-term postoperative normalization of the glycemia and insulin blood levels. One patient presented a distant liver recurrence 6 years after the initial pancreatic resection of a 2 cm insulinoma.

Evolution over time

The use of MRI increased after 2000 (< 2000: 24% vs. > 2000: 71%; p < 0.001) along with the rate of detected lesions (< 2000: 93% vs. > 2000: 97%; p = 0.6). The rate of laparoscopy also increased after 2000 (< 2000: 22% vs. > 2000: 41%; p = 0.1). The severe complications (death, severe complication, or severe leakage) did not significantly differ between the two study periods.

Discussion

Reported herein is the pre-and perioperative management of patients with sporadic non-malignant insulinoma over three decades, and several questions were raised. Once the hormonal diagnosis of organic hypoglycemia has been confirmed by a 72 h fasting test, the main challenge for the surgeon is to accurately determine the tumor location. The second challenge is to define the best surgical strategy with regard to the type of resection (formal pancreatectomy vs. parenchyma-sparing pancreatectomy) and the surgical approach (laparotomy vs. laparoscopy). The final objective is to decrease the perioperative morbidity and minimize the late functional consequences of surgery.

Accurate preoperative localization of insulinoma is one of the main issues in the management of this tumor. Indeed, insulinomas are small lesions (with 84% typically < 2 cm) randomly distributed in a fragile retroperitoneal organ [START_REF] Mehrabi | A Systematic Review of Localization, Surgical Treatment Options, and Outcome of Insulinoma[END_REF] . Our study reported high accuracy in the detection and localization of insulinoma. We previously reported that dual-phase thinsection multidetector CT showed 93% localization sensitivity [START_REF] Gouya | Endoscopic Sonography, and a Combined Protocol for Preoperative Evaluation of Pancreatic Insulinomas[END_REF] . The performances of MRI reported in our study are also in line with those reported in previous studies [START_REF] Okabayashi | Diagnosis and management of insulinoma[END_REF][START_REF] Thoeni | Detection of Small, Functional Islet Cell Tumors in the Pancreas: Selection of MR Imaging Sequences for Optimal Sensitivity[END_REF] and had been increased by the addition of diffusion-weighted MRI sequences [START_REF] Okabayashi | Diagnosis and management of insulinoma[END_REF][START_REF] Kulali | Role of diffusionweighted MR imaging in predicting the grade of nonfunctional pancreatic neuroendocrine tumors[END_REF] . EUS has also been a major improvement in insulinoma location, with very high sensibility and specificity [START_REF] Okabayashi | Diagnosis and management of insulinoma[END_REF][START_REF] Goh | Accurate preoperative localization of insulinomas avoids the need for blind resection and reoperation: analysis of a single institution experience with 17 surgically treated tumors over 19 years[END_REF] .

Nevertheless, as the results of the other investigation methods are usually available when EUS is done, the investigator is often helped by such results.

EUS also allows contrast-enhanced ultrasonography, which began to be used in our department only after 2010, improving the localization of the pancreatic mass to approximately 8-10% [START_REF] Gincul | Contrast-harmonic endoscopic ultrasound for the diagnosis of pancreatic adenocarcinoma: a prospective multicenter trial[END_REF][START_REF] Fusaroli | Contrast harmonic echo-endoscopic ultrasound improves accuracy in diagnosis of solid pancreatic masses[END_REF] and providing additional information about the vascularization of the tumor, FNA or biopsy (uncommonly needed for insulinoma), or EUS fiducial placement for an accurate intraoperative location of smaller lesions [START_REF] Okabayashi | Diagnosis and management of insulinoma[END_REF][START_REF] Goh | Accurate preoperative localization of insulinomas avoids the need for blind resection and reoperation: analysis of a single institution experience with 17 surgically treated tumors over 19 years[END_REF][START_REF] Gaujoux | Insulinoma enucleation after echoendoscopic fiducial placement[END_REF] . Nuclear medicine has also improved the location and characterization of insulinoma since the disappointing sensitivity of methods, such as 68 Ga-DOTATATE PET/CT, have shown sufficiently high sensitivity for identifying most insulinomas, and are considered by some adjunct imaging studies when the previous imaging findings were negative [START_REF] Nockel | Localization of Insulinoma Using 68 Ga-DOTATATE PET/CT Scan[END_REF] . [START_REF] Goh | Accurate preoperative localization of insulinomas avoids the need for blind resection and reoperation: analysis of a single institution experience with 17 surgically treated tumors over 19 years[END_REF] F-FDOPA PET/CT with carbidopa premedication has also shown interesting results, accurately locating more than 80% of lesions when combined with carbidopa premedication and early pancreatic acquisitions [START_REF] Imperiale | 18F-FDOPA PET/CT imaging of insulinoma revisited[END_REF][START_REF] Leroy-Freschini | Early 18F-FDOPA PET/CT imaging after carbidopa premedication as a valuable diagnostic option in patients with insulinoma[END_REF] . The policy regarding the use of nuclear-medicine imaging has evolved over time, and more recently, 68 Ga-NOTA-exendin-4 PET/CT scan has shown high sensitivity and specificity for the detection and localization of insulinoma [START_REF] Nockel | Localization of Insulinoma Using 68 Ga-DOTATATE PET/CT Scan[END_REF][START_REF] Luo | Glucagon-Like Peptide-1 Receptor PET/CT with 68Ga-NOTA-Exendin-4 for Detecting Localized Insulinoma: A Prospective Cohort Study[END_REF] . With modern imaging tools combining CT, MRI, EUS, and nuclear medicine, insulinoma detection and localization can be adequately achieved preoperatively in nearly 100% of patients. In this setting, blind pancreatectomy or exploratory laparotomy is no longer advised 8 .

When the lesion has been identified, the next step for the surgical management of insulinoma is to define the best surgical procedure (parenchyma-sparing pancreatectomy vs.

formal pancreatectomy) and surgical approach (laparoscopy vs. laparotomy). It is important to

emphasize that the open approach to pancreatectomy remains the standard of care, and that there is no clear consensus regarding the indications for a minimally invasive approach in pancreatic surgery. Regarding pancreaticoduodenectomy, the laparoscopic approach did not show any clear advantage over open pancreaticoduodenectomy [START_REF] Drymousis | Laparoscopic versus open pancreas resection for pancreatic neuroendocrine tumours: a systematic review and meta-analysis[END_REF] . Additionally, a Dutch multicentric randomized controlled study comparing laparoscopic and open pancreaticoduodenectomy was prematurely stopped because of increased mortality in the laparoscopic arm [START_REF] Han | Laparoscopic versus open distal pancreatectomy for nonfunctioning pancreatic neuroendocrine tumors: a large single-center study[END_REF] . In contrast, pancreatectomy without reconstruction (i.e., distal or caudal pancreatectomy and enucleation) remains the best procedure for insulinoma when feasible.

Several studies and meta-analyses have reported that laparoscopic distal pancreatectomy is a safe and effective procedure associated with a lower rate of complications and a shorter length 13 of hospital stay compared to open surgery [START_REF] Drymousis | Laparoscopic versus open pancreas resection for pancreatic neuroendocrine tumours: a systematic review and meta-analysis[END_REF][START_REF] Han | Laparoscopic versus open distal pancreatectomy for nonfunctioning pancreatic neuroendocrine tumors: a large single-center study[END_REF] , but very few of these studies specifically focused on pancreas neuroendocrine tumors (PNETs). Similarly, laparoscopic enucleation is safe and effective for the treatment of benign and borderline pancreatic neoplasms [START_REF] Song | Enucleation for benign or low-grade malignant lesions of the pancreas: Single-center experience with 65 consecutive patients[END_REF] , as confirmed by a meta-analysis of more than 400 patients, which showed that a minimally invasive approach does not induce a higher risk of pancreatic fistula and offers numerous advantages over the conventional surgery for pancreatic enucleation [START_REF] Guerra | Minimally invasive versus open pancreatic enucleation. Systematic review and meta-analysis of surgical outcomes[END_REF] . This, along with the increasing relevant technical skills and experience, explains the more frequent use of the laparoscopic approach for parenchyma-sparing pancreatectomy. The choice of surgical technique between parenchyma-sparing pancreatectomy and formal pancreatectomy for the treatment of insulinoma is a crucial issue. The decision should be made according to anatomical considerations (size and location) and should take into account the distance between the lesion and the main pancreatic duct, which should be greater than 2-3 mm 8 .

Nevertheless, parenchyma-sparing pancreatectomy has a similar postoperative mortality rate as and a higher risk of developing pancreatic fistula than formal pancreatectomy, especially when performed on a soft pancreas [START_REF] Cherif | Parenchyma-Sparing Resections for Pancreatic Neuroendocrine Tumors[END_REF] . Moreover, it is associated with excellent long-term functional results [START_REF] Cherif | Parenchyma-Sparing Resections for Pancreatic Neuroendocrine Tumors[END_REF] . In the present study, its overall morbidity rate was high, with 30% severe complications, 24% grade B or C pancreatic fistulas, and a 6% postoperative death rate, mainly observed after pancreaticoduodenectomy, with an unusually high mortality rate. This underlines the risk posed by pancreatic surgery on a soft pancreas without main-pancreaticduct dilatation even if performed in young and fit patients. The high morbidity in recent years might have also been the consequence of the greater use of laparoscopic parenchyma-sparing pancreatectomy.

The major challenge for pancreatic surgeons is the significant rate of postoperative complications. In large surgical series for insulinoma, the reported mortality rate is 0-6%, with a 33-56% morbidity rate, whereas the mortality rate of pancreatic surgery ranges from 1% to 3% in high-volume centers [START_REF] Cameron | One thousand consecutive pancreaticoduodenectomies[END_REF][START_REF] Cameron | Two thousand consecutive pancreaticoduodenectomies[END_REF] , increasing to 6-10% when nationwide centers are considered [START_REF] Swanson | The 90-Day Mortality After Pancreatectomy for Cancer Is Double the 30-Day Mortality: More than 20,000 Resections From the National Cancer Data Base[END_REF][START_REF] El Amrani | Should all pancreatic surgery be centralized regardless of patients' comorbidity? HPB[END_REF][START_REF] El Amrani | Specificity of Procedure volume and its Association With Postoperative Mortality in Digestive Cancer Surgery: A Nationwide Study of 225,752 Patients[END_REF] . Considering these heterogeneous results, being treated in expert-organized high-volume centers presents a clear benefit for patients [START_REF] Swanson | The 90-Day Mortality After Pancreatectomy for Cancer Is Double the 30-Day Mortality: More than 20,000 Resections From the National Cancer Data Base[END_REF][START_REF] El Amrani | Should all pancreatic surgery be centralized regardless of patients' comorbidity? HPB[END_REF][START_REF] El Amrani | Specificity of Procedure volume and its Association With Postoperative Mortality in Digestive Cancer Surgery: A Nationwide Study of 225,752 Patients[END_REF] . Considering that the mortality rate of pancreatectomy is mainly related to pancreaticoduodenectomy, other therapeutic options have emerged in the last few years. From a technical view point, the prophylactic endoscopic placement of a pancreatic stent for "deep" pancreatic enucleations [START_REF] Giuliani | Endoscopic placement of pancreatic stent for "Deep" pancreatic enucleations operative technique and preliminary experience at two high-volume centers[END_REF] for years as the only curative option for insulinoma, local destruction of tumors through radiofrequency ablation (RFA) may be proposed as an alternative despite the fact that very few such procedures have been performed to date. In the literature, only about 60 patients with PNETs have been treated with RFA [START_REF] Rossi | Radiofrequency ablation of pancreatic neuroendocrine tumors: a pilot study of feasibility, efficacy, and safety[END_REF][START_REF] Pai | Endoscopic ultrasound guided radiofrequency ablation, for pancreatic cystic neoplasms and neuroendocrine tumors[END_REF][START_REF] Limmer | Radiofrequency ablation of solitary pancreatic insulinoma in a patient with episodes of severe hypoglycemia[END_REF][START_REF] Armellini | Endoscopic ultrasound-guided radiofrequency ablation of a pancreatic neuroendocrine tumor[END_REF][START_REF] Barthet | Endoscopic ultrasound-guided radiofrequency ablation for pancreatic neuroendocrine tumors and pancreatic cystic neoplasms: a prospective multicenter study[END_REF][START_REF] Waung | Successful management of a sporadic pancreatic insulinoma by endoscopic ultrasound-guided radiofrequency ablation[END_REF][START_REF] Lakhtakia | EUS-guided radiofrequency ablation for management of pancreatic insulinoma by using a novel needle electrode (with videos)[END_REF][START_REF] Choi | Endoscopic ultrasound-guided radiofrequency ablation for management of benign solid pancreatic tumors[END_REF][START_REF] Bas-Cutrina | Small pancreatic insulinoma: Successful endoscopic ultrasound-guided radiofrequency ablation in a single session using a 22-G fine needle[END_REF][START_REF] Procházka | Laparoscopic radiofrequency ablation of functioning pancreatic insulinoma: video case report[END_REF] , but it has a clinical success rate of 83.3-100% [START_REF] Rossi | Radiofrequency ablation of pancreatic neuroendocrine tumors: a pilot study of feasibility, efficacy, and safety[END_REF][START_REF] Pai | Endoscopic ultrasound guided radiofrequency ablation, for pancreatic cystic neoplasms and neuroendocrine tumors[END_REF][START_REF] Limmer | Radiofrequency ablation of solitary pancreatic insulinoma in a patient with episodes of severe hypoglycemia[END_REF][START_REF] Armellini | Endoscopic ultrasound-guided radiofrequency ablation of a pancreatic neuroendocrine tumor[END_REF][START_REF] Barthet | Endoscopic ultrasound-guided radiofrequency ablation for pancreatic neuroendocrine tumors and pancreatic cystic neoplasms: a prospective multicenter study[END_REF][START_REF] Waung | Successful management of a sporadic pancreatic insulinoma by endoscopic ultrasound-guided radiofrequency ablation[END_REF][START_REF] Lakhtakia | EUS-guided radiofrequency ablation for management of pancreatic insulinoma by using a novel needle electrode (with videos)[END_REF][START_REF] Choi | Endoscopic ultrasound-guided radiofrequency ablation for management of benign solid pancreatic tumors[END_REF][START_REF] Bas-Cutrina | Small pancreatic insulinoma: Successful endoscopic ultrasound-guided radiofrequency ablation in a single session using a 22-G fine needle[END_REF][START_REF] Procházka | Laparoscopic radiofrequency ablation of functioning pancreatic insulinoma: video case report[END_REF] . Nevertheless, the morbidity rate, technical constraints, and long-terms results of this emerging treatment should be investigated in depth. Considering the experience with RFA so far, we believe that local ablation of tumors through it may be considered a suitable alternative for insulinomas located in the head of the pancreas.

The main limitation of this study is its retrospective design spanning over three decades due to the low incidence of insulinoma. This long inclusion period saw improvements in imaging techniques and perioperative management with the development of the laparoscopic approach. Even though this study's sample consisting of 80 patients operated on for insulinoma may be viewed as restricted, this study was one of the largest monocentric studies that have been conducted to date, providing an interesting overview of the management of the rare disease of insulinoma [START_REF] Zhao | Surgical management of patients with insulinomas: Result of 292 cases in a single institution: Surgical Management of Insulinoma[END_REF][START_REF] Thoeni | Detection of Small, Functional Islet Cell Tumors in the Pancreas: Selection of MR Imaging Sequences for Optimal Sensitivity[END_REF][START_REF] Goh | Accurate preoperative localization of insulinomas avoids the need for blind resection and reoperation: analysis of a single institution experience with 17 surgically treated tumors over 19 years[END_REF][START_REF] Cunha | Laparoscopic versus open approach for solitary insulinoma[END_REF][START_REF] Daneshvar | Preoperative tumor studies using MRI or CT in patients with clinically suspected insulinoma[END_REF][START_REF] Morera | Preoperative localization of an insulinoma: selective arterial calcium stimulation test performance[END_REF][START_REF] Sotoudehmanesh | Endoscopic ultrasonography (EUS) in the localization of insulinoma[END_REF][START_REF] Peltola | Characteristics and Outcomes of 79 Patients with an Insulinoma: A Nationwide Retrospective Study in Finland[END_REF] .

In conclusion, the management of insulinoma has evolved dramatically over the recent decades with the emergence of modern imaging techniques, including cross-sectional imaging, EUS, and nuclear medicine. As such, accurate preoperative detection and localization is obtained in most patients. A major challenge remains, however: the significant postoperative morbidity rate, which is high after parenchyma-sparing pancreatectomy, counterbalancing the long-term benefits on both the exocrine and endocrine functions.

Table 2 .

 2 The

	pancreaticoduodenectomy was performed instead. Overall, 25/50 patients (31%) underwent
	surgery via the laparoscopic approach, and 5 (20%) required conversion to accurately locate
	and resect the tumor. Twelve (48%) patients underwent pancreatic enucleation; 6 (40%),
	distal pancreatectomy; 3 (12%), distal pancreatectomy with splenectomy; and 4 (16%), caudal
	pancreatectomy. In addition, an open approach was adopted in 55/80 patients (69%); among
	them, 24/55 (44%) had pancreatic enucleation; 9/55 (16%), pancreaticoduodenectomy; 15/55
	(27%), distal pancreatectomy; 6/55 (11%), distal pancreatectomy with splenectomy; and 1/55
	(2%), resection of the uncinate process. Regarding the surgical approach, 34/80 patients

exact preoperative location was obtained in 76/80 patients (95%), and the preoperative tumor location was accurate in all the patients who underwent CT, MRI, EUS, and somatostatin receptor imaging (19/19; 100%; 95% confidence interval [CI]: 79-100). When CT was used in combination with EUS, an accurate preoperative tumor location was obtained in 65/68 patients (96%; 95% CI: 87-99).

The results of the other combinations are given in Table

2

. CT, MRI, and EUS showed more accurate preoperative tumor locations (p = 0.009, 0.01, and 0.02, respectively) than octreoscan, with only four DOTAPETs realised after the year 2000 and only one positive for a 7 mm lesion. There were no differences in size between the negative and positive octreoscans and before and after the year 2000. No significant differences were found between CT, MRI, and EUS. Four patients were operated on without a preoperative location, all before 1995. For the lesion in the tail of the pancreas detected via intraoperative ultrasonography or palpation, none of the patients had preoperative MRI or EUS.

Surgical procedure

In most cases, the actual surgery follows the preoperative planning. In 2 cases in this study, however, the planned enucleation of a large cephalic lesion was not possible because of the intraoperative lateral wound in the main pancreatic duct; as such, (43%) had a tumor located in the head of the pancreas, 32/34 (94%) of whom were operated on using the open approach whereas 22/46 patients (48%) with tumors in other locations underwent laparoscopy (p < 0.001). In summary, 41 parenchyma-sparing pancreatectomies, including enucleation, caudal pancreatectomy, and uncinate process resection, were realised, with a 41% laparoscopic rate. The rate of laparoscopy was 28% before the year 2000 and increased to 55% after 2000 (p = 0.09) for the patients who underwent parenchyma-sparing

  Controlled studies, although desirable, are unlikely to be feasible due to the low incidence of such lesions. If surgical resection has been advocated

								may help
	extend the surgical indications for pancreatic enucleation, but this advantage may be
	overshadowed	by	the	risk	of	developing	post-endoscopic	retrograde
	cholangiopancreatography pancreatitis.

Table 2 :

 2 Capabilities of imaging tests for preoperative tumour detection

	Imaging tests	Number of patients (%)	Number of patients with a correctly localised lesion	True localisation rate (%)	95% Confidence interval
	CT	76 (96%)	58	76%	64-85
	MRI	37 (47%)	28	76%	58-88
	EUS	71 (89%)	64	90%	80-96
	Somatostatin receptor imaging	26 (34%)	10	38%	21-59
	CT and MRI	35 (44%)	32	91%	76-98
	CT and EUS	68 (85%)	65	96%	87-99
	CT and IRM and EUS and somatostatin receptor imaging	19 (34%)	19	100%	79-100
	CT = computed tomography; MRI = magnetic resonance imaging; EUS = Endoscopic
	ultrasonography

In-pentreteotide somatostatin receptor scintigraphy. Other somatostatin receptor imaging