Julie Pabois

Tony Durand

Catherine Le Berre

Jacques Gonzales

Michel Neunlist

Arnaud Bourreille

Philippe Naveilhan
email: philippe.naveilhan@univ-nantes.fr

Isabelle Neveu

T cells show preferential adhesion to enteric neural cells in culture and are close to neural cells in the myenteric ganglia of Crohn's patients

Keywords:

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

Introduction

The enteric nervous system (ENS) is the intrinsic nervous system contained within the gastrointestinal tract. Composed of enteric neurons and glial cells (EGC), the ENS is organized into two major ganglionated plexi, the submucosal and the myenteric plexus. It is well established that enteric neurons and EGC are major regulators of motor and barrier functions [START_REF] Bush | Fulminant jejuno-ileitis following ablation of enteric glia in adult transgenic mice[END_REF][START_REF] Furness | The enteric nervous system and neurogastroenterology[END_REF][START_REF] Neunlist | The digestive neuronal-glial-epithelial unit: a new actor in gut health and disease[END_REF][START_REF] Rao | Enteric Glia Regulate Gastrointestinal Motility but Are Not Required for Maintenance of the Epithelium in Mice[END_REF]. Much less is known about their active implication in the regulation of the gastrointestinal immune functions.

Close proximity of immune cells like plasma cells, macrophages, mast cells, eosinophils or T lymphocytes to nerve structures has been reported in patients with Crohn's disease (MC) since decades [START_REF] Dvorak | Crohn's disease: transmission electron microscopic studies. III. Target tissues. Proliferation of and injury to smooth muscle and the autonomic nervous system[END_REF][START_REF] Dvorak | Differentiation between Crohn's disease and other inflammatory conditions by electron microscopy[END_REF][START_REF] Geboes | Major histocompatibility class II expression on the small intestinal nervous system in Crohn's disease[END_REF]. Mast cells and macrophages were also observed in apposition to nerve in the enteric plexus of nonlesioned animals [START_REF] Newson | Suggestive evidence for a direct innervation of mucosal mast cells[END_REF][START_REF] De Schepper | Self-Maintaining Gut Macrophages Are Essential for Intestinal Homeostasis[END_REF] and early accumulation of T cells was observed in enteric ganglia in animal models of colitis [START_REF] Pontell | Structural changes in the epithelium of the small intestine and immune cell infiltration of enteric ganglia following acute mucosal damage and local inflammation[END_REF].

Whether the enteric neural cells have an active role in the invasion of intestinal nerve structures by the immune cells remains to decipher but it has been shown that enteric neurons and glial cells can secrete neurotrophic factors, neuropeptides and neurotransmitters to which immune cells are sensitive [START_REF] Margolis | Enteric Neuronal Regulation of Intestinal Inflammation[END_REF][START_REF] Yoo | The Enteric Network: Interactions between the Immune and Nervous Systems of the Gut[END_REF].

Importantly, the secretion of GDNF by EGC was shown to regulate a subpopulation of innate lymphoid cells (ILC3) that reduces the susceptibility of mice to develop colitis [START_REF] Ibiza | Glial-cell-derived neuroregulators control type 3 innate lymphoid cells and gut defence[END_REF]. Other studies suggest that the ENS can also be a major source of cytokines or chemokines that could contribute to intestinal inflammation either directly or by modulating immune cell functions and phenotypes [START_REF] Rühl | Interleukin-6 expression and regulation in rat enteric glial cells[END_REF][START_REF] Tixier | Human mucosa/submucosa interactions during intestinal inflammation: involvement of the enteric nervous system in interleukin-8 secretion[END_REF][START_REF] Coquenlorge | Modulation of lipopolysaccharide-induced neuronal response by activation of the enteric nervous system[END_REF]. Altogether, these observations emphasize the need of better characterizing and studying the neuro-immune interactions during intestinal inflammation, and in particular in inflammatory bowel diseases (IBD).

IBD are chronic inflammatory bowel diseases encompassing ulcerative colitis (UC) and Crohn's disease [START_REF] Wallace | Immunopathology of inflammatory bowel disease[END_REF]. They are characterized by phases of relapse-remission, and due to severe complications such as fistulae, ulcer and obstruction, most patients undergo one or several surgeries through their life [START_REF] Thia | Risk factors associated with progression to intestinal complications of Crohn's disease in a population-based cohort[END_REF][START_REF] Frolkis | Risk of surgery for inflammatory bowel diseases has decreased over time: a systematic review and meta-analysis of population-based studies[END_REF]. To optimize their medical treatment and limit recurrence, predictive markers of post-operative relapse are currently under investigation. As several studies have identified the presence of plexitis in the proximal margin of intestinal resections as a powerful tool to predict relapse [START_REF] Ryan | Meta-analysis of Histological Margin Positivity in the Prediction of Recurrence After Crohn's Resection[END_REF], the accumulation of immune cells in and around the nerve enteric ganglia or bundle, has been recognized as a marker of post-operative recurrence in case of Crohn's disease [START_REF] Gionchetti | 3rd European Evidence-based Consensus on the Diagnosis and Management of Crohn's Disease 2016: Part 2: Surgical Management and Special Situations[END_REF]. Attempts to characterize the plexitis describe the presence of mast cells, eosinophils and T lymphocytes infiltrating the submucosal and/or myenteric plexus [START_REF] Bressenot | Submucosal plexitis as a predictor of postoperative surgical recurrence in Crohn's disease[END_REF][START_REF] Decousus | Myenteric plexitis is a risk factor for endoscopic 16 and clinical postoperative recurrence after ileocolonic resection in Crohn's disease[END_REF][START_REF] Ferrante | The value of myenteric plexitis to predict early postoperative Crohn's disease recurrence[END_REF][START_REF] Lemmens | Submucosal Plexitis as a Predictive Factor for Postoperative Endoscopic Recurrence in Patients with Crohn's Disease Undergoing a Resection with Ileocolonic Anastomosis: Results from a Prospective Single-centre Study[END_REF][START_REF] Milassin | Analysis of risk factorsespecially different types of plexitis -for postoperative relapse in Crohn's disease[END_REF][START_REF] Misteli | Myenteric plexitis at the proximal resection margin is a predictive marker for surgical recurrence of ileocaecal Crohn's disease[END_REF][START_REF] Sokol | Plexitis as a predictive factor of early postoperative clinical recurrence in Crohn's disease[END_REF]. However, little is known about the enteric neural cells involved in plexitis. Therefore, we explored the capacity of T lymphocytes to interact with enteric glial cells and neurons in vitro and in vivo.

Methods

Animals

Three-month-old and E15 pregnant female Sprague-Dawley rats (Janvier Laboratories, Le Genest-St-Isle, Fr) were housed in a 12-hours light/dark cycle with ad libitum access to food and water. The experiments were carried out in strict accordance with the recommendations of the Animal Care and Use Committee of Nantes (France). Animals were anesthetized with isoflurane and sacrificed by cervical dislocation.

Preparation and staining of T lymphocytes

For each independent experiment, the T lymphocytes were purified from the spleen of one rat as previously described [START_REF] Bonnamain | Expression of Heme Oxygenase-1 in Neural Stem/Progenitor Cells as a Potential Mechanism to Evade Host Immune Response[END_REF]. Briefly, the splenocytes were collected from the spleen by filtration through 100 µm nylon mesh filter, followed by the depletion of red blood cells. Then, splenocytes were counted and T cells were purified by negative sorting using anti-NKR-P1A (0.7 µg/mL, Thermo Fisher (TF), Waltham, US), anti-CD45RC (1 µg/mL, BD Biosciences (BD), San Jose, CA, US) and anti-CD11b/c (0.5 µg/mL, BD) antibodies associated with anti-mouse IgG coated Dynabeads (TF). T lymphocytes were cultivated in complete RPMI medium composed of RPMI 1640 (TF) supplemented with 10% inactivated fetal calf serum (Eurobio, Paris, Fr), 2 mM L-glutamine (TF), 1% non-essential amino acids (TF), 1 mM HEPES buffer (TF), 1 mM sodium pyruvate (TF), 100 U/mL penicillin and 0.1 mg/mL streptomycin (TF). Activation of T lymphocytes was performed by incubating the cells for three days in wells pre-coated with anti-CD3 antibodies (1 μg/mL, BD) in complete RPMI medium supplemented with anti-CD28 antibodies (2.5 μg/mL, BD) and 1 µM of 2-mercaptoethanol (Sigma-Aldrich (SA), St-Louis, US).

Before the co-cultures, T lymphocytes were counted and labeled with carboxyfluoresceinsuccinimidyl-ester (CFSE, TF) according to the manufacturer's protocol. After 5 min of incubation with 5 µM of CFSE at RT, the reaction was stopped by adding fetal calf serum, and the cells were washed two times with RMPI. For the time-lapse experiments, T cells were labeled with Hoechst-33342 (TF) prior their addition to the cultures of ENS cells. The staining was performed by incubating the T cells with 1 µg/mL of Hoechst-33342 during 30 min at 37°C, followed by two washes with RPMI.

Mixed primary cultures of Enteric Nervous System

Primary ENS cultures were prepared from E15 rat embryos as reported previously [START_REF] Chevalier | Activity-dependent regulation of tyrosine hydroxylase expression in the enteric nervous system[END_REF]. Briefly, the intestines were isolated, minced with scissors and digested for 15 min at 37°C with 0.1% (w/vol) trypsin (SA) in -F12 (1:1) (TF). Fetal calf serum (10%) and 0.01% (w/vol) of DNase (SA) was added. After 10 min of incubation at 37°C, the cells were isolated by mechanical dissociation and seeded at 2.4 x 10 5 cells per cm 2 on gelatin pre-coated plates in DMEM-F12 (1:1) supplemented with 10% FCS, 100 U/mL penicillin, 0.1 mg/mL streptomycin. The FCS-supplemented medium was then replaced by a serum-free medium composed of DMEM-F12 (1:1) supplemented with 1% of N2 (TF), 100 U/mL penicillin, 0.1 mg/mL streptomycin. Half of the supernatant was replaced twice per week. Cells were used for the co-culture experiments after twelve days of culture.

Co-cultures

Activated or non-activated CFSE-labeled T lymphocytes (30 000 per well in 48-well plate) were added to the primary ENS cultures and incubated for two hours at 37°C. The supernatant was gently removed. The plates were then carefully washed five times with warmed medium to eliminate the floating cells, fixed by 4% paraformaldehyde in PBS (vol/vol, ROTH Sochiel E.U.R.L, Lauterbourg, Fr) for 10 min and stored in PBS-azide at 4°C until use.

Immunocytochemistry

Fixed co-cocultures were incubated for 2 hrs at room temperature (RT) in PBS supplemented with 5% (vol/vol) horse serum (TF) and 0.1% (vol/vol) triton X-100 (SA). Cells were then stained with mouse IgG2a anti-SMA (1/500, Abcam, Paris, Fr), mouse IgG2b anti-HuC/D

(1/500, TF), mouse anti-PGP9.5 (1/1000, TF) or rabbit anti-S100β (1/1000, Dako Agilent, Santa Clara, US) antibodies in PBS supplemented with 5% horse serum. After 12 hrs at 4°C, the cells were washed and incubated for 2 hrs at RT with conjugated goat anti-mouse IgG (1/500, Jackson ImmunoResearch, JIR, Baltimore, US), goat anti-mouse IgG2a (1/500, JIR), goat anti-mouse IgG2b (1/500, JIR) and goat anti-Rabbit (1/500, JIR). Nuclei were stained with DAPI (1µg/mL, SA).

Micrographs were taken using confocal microscope Nikon A1 RSi (Nikon SAS, Champigny sur Marne, Fr) or IN Cell Analyzer 2200® (GE Healthcare, Velizy-Villacoublay, Fr).

The surface covered by the enteric neurons and glial cells was estimated automatically by the Developper Toolbox software (GE Healthcare Lifesciences) based on the intensity of PGP9.5 and S100β staining, respectively. As neuronal and glial staining often overlap, the total neural cell surface was calculated as followed: (surface covered by PGP9.5 + staining + surface covered by S100β + staining) -(surface covered by both PGP9.5 + and S100β + staining).

Immunohistochemistry

Transmural paraffin sections (5 µm) were prepared from the proximal margin of ileocolonic resections of patients with Crohn's disease (n=10) or control (n=5) (Anatomical pathology department, CHU of Nantes, France). For control patients, sections were performed away from the tumor tissue. Sections were deparaffined by incubating the slides in two successive baths of xylene (2 x 7 min), then in three successive baths of ethanol (100% -5 min, 95% -4 min, 70% -3 min). After two baths of distilled water (2 x 2 min), sections were incubated in antigen retrieval solution (10 mM Trizma base, 1 mM EDTA, pH 9) during 90 sec at 110°C in a deckloaking chamber (DC2012-220V, Biocare Medical). The sections were washed three

Cell counting

Co-cultures of rat T cells and enteric neural cells

T cells in close proximity (no black pixel between the different staining and/or on the top of PGP-9.5 + or S100β + cells) were counted on the micrographs (pixel size: 0.324 µm) taken by the INCell Analyzer. Each experiment was performed on independent cultures of ENS and T cells. For each independent experiment, each condition was replicated in three wells and a minimum of 200 T cells was analyzed per well. The median values of the triplicate of independent experiments (n) were used to perform the statistical analyses.

Human transmural sections

The CD3 + T cells observed in close proximity or on the top of Hu + or S100β + cells were counted on micrographs (pixel size: 0.413 µm) taken by the Axio Zoom.V16® (Zeiss, Oberkochen, D). The whole interface between circular and longitudinal muscle cell layers was visualized, and images were taken when stained ENS cells were observed. The number of T cells in close proximity with enteric neurons or EGC was then expressed in mean number of T cells per ganglia or per cells, respectively.

Time-lapse

To analyze the dynamic interactions between T lymphocytes and neural cells, activated T cells were labeled with CFSE or Hoechst-33342 and added (30 000 T cell/well) to ENS primary cultures. Then, the co-cultures were (1) immediately analyzed by time-lapse for two hours (n=3), or (2) incubated for forty minutes, carefully washed four times to remove the non-adherent cells and then analyzed by time-lapse for eighty minutes (giving a total of two hours of co-cultures) (n=3). Time-lapse recording (one image/min) was performed at 37°C under 4% CO2 using the IN Cell 2200® Analyzer. Three fields per well were recorded in three wells for each independent experiment and analyzed.

Statistics

Results were presented as mean +/-standard error of the mean (SEM) and analyzed with the non-parametric Mann-Whitney 2-sided U test using GraphPad Prism (GraphPad Software, San Diego, CA, US). *, p<0.05; **, p<0.01.

Results

T cells are in close proximity to enteric neurons and EGC in mixed primary cultures of ENS.

To analyze the interactions between enteric neural cells and T lymphocytes, mixed primary cultures of ENS were cultured for 12 days and activated T cells were added to the ENS cultures. Dynamic of the cell interactions was analyzed by time-lapse. After the addition of activated T cells to the ENS cultures, bright-field images were immediately captured every minute for two hours. In the movies, we observed that after sedimentation, T cells did not move far from their original position during the two hours of co-cultures, but part of them extend cytoplasmic processes on the underlying cell layer (Movie 1). Interestingly, active "scanning-like" behavior was observed with sometimes apparent attempt to penetrate into the ENS cell layer (movie 2). To show that T cells adhere to ENS cultures, a second set of experiments was performed in which activated T cells stained with Hoechst were cocultivated for 40 min with ENS cultures before four careful washes. Then, the co-cultures were analyzed by time-lapse for 80 min. A lower amount of T cells was observed in these cocultures, but their behavior was similar presenting a "scanning-like" movement rather than an "on/off" adhesion (movie 3).

To characterize these cultures, triple immunocytochemistry was performed using antibodies against S100β (EGC), HuC/D (neurons) and SMA (smooth muscle cells). As illustrated in the figures 1A-D, the entire surface of the wells was covered by a layer of SMA-positive cells.

On the top of this layer, ganglia of Hu-positive cells surrounded by S100-positive cells were clearly observed. Analyzes by confocal microscopy showed that the neurons were positioned on the top of EGC and muscle cell layers (Fig. 1E). These cultures were used to determine whether T lymphocytes were able to interact with enteric neural cells. For this purpose, rat T lymphocytes were stained with CFSE and added to the mixed primary cultures of ENS. After two hours of co-culture, CFSE-positive cells were found to be adherent to the ENS cell layers.

To analyze these interactions, the supernatants were removed and the cultures were carefully washed five times with warmed medium to eliminate the floating cells. The cultures were then fixed and double immunocytochemistry was performed with antibodies against PGP9.5 (neurons) and S100β (EGC). Microscopy analyzes revealed that numerous adherent CFSEpositive cells are located in close proximity to PGP9.5-and/or S100β-positive staining (Fig. 1F). Z-reconstruction of confocal images confirmed the close proximity of T lymphocytes to PGP9.5-positive neurites, S100β-positive cells or both in primary ENS cultures (Fig. 1G-I).

Activation of T lymphocytes increases their capacity to adhere to the ENS cultures.

Activation of immune cells leads to rapid behavioral changes which may modify their capacity to interact with the three types of cells present in the mixed primary ENS culture. To evaluate this point, T lymphocytes were activated or not with anti-CD3/anti-CD28 antibodies for three days, next stained with CFSE and co-cultivated for two hours with mixed primary ENS cultures. After the elimination of floating T lymphocytes, the number of total adherent CFSE-positive cells was counted. As shown in the figure 2A, the number of total adherent activated T lymphocytes per mm2 of culture was significantly increased by 2.28 fold compared to non-activated T lymphocytes indicating that T cell activation increased their general capacity to adhere to mixed primary ENS cultures. Since 30 000 non-activated or activated T cells were added to the ENS cultures, the percentages of adherent T cells after two hours of co-culture were 5.2 % +/-1.01 and 11.88 % +/-1.9, respectively (Fig 2 -B). To further explore the interactions of T lymphocytes with enteric neural cells, the co-cultures were immunostained with antibodies against neural markers and the number of T lymphocytes in close proximity to PGP9.5-or S100β-positive cells was quantified as described in the methods sections. The number of activated T lymphocytes in close proximity to enteric neurons and glial cells was 2.4-fold higher than the number of non-activated T lymphocytes (Fig. 2C).

No obvious sign of ENS degeneration was observed in the presence of non-activated or activated T lymphocytes during time-lapse analyze or in the immunostained co-cultures.

T lymphocytes preferentially adhere to enteric neural cells in mixed primary cultures of ENS.

In the mixed primary ENS cultures, adherent T lymphocytes were often observed close to neural ganglia or interganglionic fibers. Therefore, quantitative analyzes were performed to determine whether T lymphocytes preferentially adhere to neurons and glial cells in cultures composed of enteric neural cells and smooth muscle cells. They revealed that 72.9% +/-2.1 of non-activated T lymphocytes (Fig. 2C) and 76.3% +/-3.9 of activated T lymphocytes (Fig. 2 D) were observed in close proximity to PGP9.5-and/or S100β-positive cells (mean +/-SEM).

These percentages were all the more remarkable that the area occupied by both neural cell types were less than 50 % of the total well surface. Indeed, PGP9.5-and S100β-positive cells occupied 26.8% +/-2.1 and 32.6% +/-2.2 of the surface area, respectively, but the total area covered by both neural cell types was estimated at only 46.5% +/-2.9 due to neuron and EGC overlapping (Fig. 2E). These data suggest privileged proximity of T lymphocytes with neural cells and according to the statistical analyzes, T cell activation had no effect on this preferential adhesion in mixed primary ENS cultures (p=0.67). To determine whether T lymphocytes preferentially interact with one type of neural cells, we quantified the number of T cells in close proximity only to PGP9.5, only with S100β cells or with both cell types. The results indicate that over 90% of the non-activated T lymphocytes (91.3% +/-1.03; Fig. 2F) and activated T lymphocytes (93.9 % +/-1.9; Fig. 2G) were in close proximity to both neurons and EGC.

T cells are in close proximity to both neurons and glial cells in the myenteric ganglia of patients with Crohn's disease.

Finally, investigations were carried out to determine whether T lymphocytes can interact with EGC and enteric neurons in patients during Crohn's disease. For this purpose, transmural paraffin sections were prepared from the proximal margin of ileocolonic resections of patients with Crohn's disease. Anti-CD3 immunohistochemistry combined with hematoxylin-eosin staining showed that T cells were present in and around the myenteric ganglia (Fig. 3A) as previously reported. Serial sections were then stained with anti-CD3 combined with anti-HuD or anti-S100β antibodies. Apotome imaging revealed the presence of T cells in close proximity to EGC (Fig. 3B) and enteric neurons (Fig. 3C) within the myenteric ganglia. In the submucosal plexus, T cells were clearly present around the submucosal ganglia (less than 20 µm) in all the patients, but close proximity of CD3 cells to S100β-or Hu-positive cells was rarely observed (Fig. 3D-E).

To quantify the T cells in close proximity to neurons and/or EGC in the myenteric ganglia, the number of CD3 cells was determined in the intestinal resections from ten patients with Crohn's disease and five patients with colorectal cancer. The mean number of T cells in close proximity to neurons was not significantly different in the myenteric ganglia of patients with Crohn's disease or cancer (Fig. 3-G). However, the mean number of T cells in close proximity with EGC per ganglia was significantly higher in patients with Crohn's disease (3.9+/-0.89) as compared to patients with cancer (1.35+/-0.12, p = 0.026) (Fig. 3-F).

Discussion

Accumulation of immune cells within and around the enteric nervous plexus is observed in

Crohn's disease and the presence of enteric plexitis in the proximal margin of intestinal resection is a marker of post-operative recurrence [START_REF] Ferrante | The value of myenteric plexitis to predict early postoperative Crohn's disease recurrence[END_REF][START_REF] Gionchetti | 3rd European Evidence-based Consensus on the Diagnosis and Management of Crohn's Disease 2016: Part 2: Surgical Management and Special Situations[END_REF]. The localization (submucosal or myenteric) and the type of immune cells described in the enteric plexitis vary but T lymphocytes are regularly observed [START_REF] Bressenot | Submucosal plexitis as a predictor of postoperative surgical recurrence in Crohn's disease[END_REF][START_REF] Decousus | Myenteric plexitis is a risk factor for endoscopic 16 and clinical postoperative recurrence after ileocolonic resection in Crohn's disease[END_REF][START_REF] Ferrante | The value of myenteric plexitis to predict early postoperative Crohn's disease recurrence[END_REF][START_REF] Lemmens | Submucosal Plexitis as a Predictive Factor for Postoperative Endoscopic Recurrence in Patients with Crohn's Disease Undergoing a Resection with Ileocolonic Anastomosis: Results from a Prospective Single-centre Study[END_REF][START_REF] Milassin | Analysis of risk factorsespecially different types of plexitis -for postoperative relapse in Crohn's disease[END_REF][START_REF] Misteli | Myenteric plexitis at the proximal resection margin is a predictive marker for surgical recurrence of ileocaecal Crohn's disease[END_REF][START_REF] Sokol | Plexitis as a predictive factor of early postoperative clinical recurrence in Crohn's disease[END_REF]. In contrast, nothing is known about the enteric neural cells involved in plexitis and whether plexitis play a role in Crohn's disease or are a mere epiphenomenon of T cell infiltration within the gut during inflammation. In order to address these issues, a first major step is to better characterize the nature of plexitis and in particular, whether close proximity exists between enteric neural cells and T cells. In this general context, our study brings novel insights by demonstrating that T lymphocytes are in close proximity to S100β-positive EGC and Hu-positive enteric neurons in the myenteric ganglia of patients with Crohn's disease. Such cell arrangements could be also detected in tumor-free transmural sections of patients with cancer, but the number of T cells in close proximity to EGC in the myenteric ganglia was significantly higher in patients with Crohn's disease. In the present work, the number of T cells in close proximity to enteric neurons was not significantly different in patients with cancer or Crohn's disease, but such an observation should be verified with a larger cohort of patients. T cells were also present around the submucosal ganglia of patients with Crohn's disease but close vicinity of T cells with neural cells in these ganglia was rare in the analyzed sections.

In the present paper, we also demonstrate that T lymphocytes adhere to ENS culture and are preferentially in close proximity to neural cells in mixed primary cultures containing EGC, enteric neurons and smooth muscle cells. The mechanism of these neuro-immune interactions remains to be deciphered but according to different studies, enteric neurons and EGC are capable of producing adhesion molecules and chemoattractant factors. Thus, increased levels of ICAM-1 and eotaxin-3 transcripts were observed in the enteric neural ganglia of patients with inflammatory bowel disease [START_REF] Smyth | Activated eosinophils in association with enteric nerves in inflammatory bowel disease[END_REF]. On the other hand, Burgueño et al. showed that rat enteric neurons produced and released the monocyte chemoattractant protein MCP-1 in the rat ENS cultures [START_REF] Burgueño | TLR2 and TLR9 modulate enteric nervous system inflammatory responses to lipopolysaccharide[END_REF]. An expression of MCP1 mRNA was also reported in primary mouse EGC after IL-1β exposure [START_REF] Stoffels | Postoperative ileus involves interleukin-1 receptor signaling in enteric glia[END_REF].

In organotypic cultures derived from human intestinal biopsies, a production of IL-8 was observed in enteric neurons when ENS cells were co-cultivated with toxin-B pretreated epithelium [START_REF] Tixier | Human mucosa/submucosa interactions during intestinal inflammation: involvement of the enteric nervous system in interleukin-8 secretion[END_REF]. This is of great interest as IL-8 derived from the neuron-like cells NT2-N induces the chemoattraction of peripheral blood mononuclear cells [START_REF] Tixier | Intestinal neuro-epithelial interactions modulate neuronal chemokines production[END_REF]. As IL-1β is implicated in the production of these chemoattractant mediators, future analyzes should take into account the inflammatory environment when studying the interactions between T lymphocytes and enteric neural cells. In this regards, inflammatory conditions are all the more important to consider as they favor the production of metalloproteinases by macrophages or EGC [START_REF] Pender | Role of macrophage metalloelastase in gut inflammation[END_REF][START_REF] Lin | Interleukin-1beta and interleukin-6 stimulate matrix metalloproteinase-9 secretion in cultured myenteric glia[END_REF] which in turn, may participate to extracellular remodeling and favor the penetration of T cells into the myenteric ganglia through the ganglionic sheet.

Studies on glial cells from the central nervous system are also of great interest as they show that interactions between astrocytes and T lymphocytes have an important role in the presentation of antigens [START_REF] Fontana | Astrocytes present myelin basic protein to encephalitogenic T-cell lines[END_REF][START_REF] Barcia | T cells' immunological synapses induce polarization of brain astrocytes in vivo and in vitro: a novel astrocyte response mechanism to cellular injury[END_REF] and in the regulation of T cell activation/proliferation [START_REF] Wekerle | Immune reactivity in the nervous system: modulation of T-lymphocyte activation by glial cells[END_REF][START_REF] Gimsa | Astrocytes protect the CNS: antigen-specific T helper cell responses are inhibited by astrocyte-induced upregulation of CTLA-4 (CD152)[END_REF]. Interestingly, using a mice model of experimental autoimmune encephalomyelitis, Filipello et al. have recently described a contact-dependent bidirectional mechanism between astrocytes and T lymphocytes that favor the differentiation of T cells toward an immunosuppressive phenotype [START_REF] Filipello | Ectonucleotidase activity and immunosuppression in astrocyte-CD4 T cell bidirectional signaling[END_REF]. These observations strengthen the importance of close interactions between glial cells and T lymphocytes in the development and regulation of immune and/or inflammatory responses. Finally, it would be interesting to determine whether these close interactions between T lymphocytes and EGC induce changes and lead to functional modifications in both immune and neural cells as reported for the interactions between muscularis macrophages and enteric neurons [START_REF] Muller | Crosstalk between Muscularis Macrophages and Enteric Neurons Regulates Gastrointestinal Motility[END_REF] as well as for EGC and ILC3 [START_REF] Ibiza | Glial-cell-derived neuroregulators control type 3 innate lymphoid cells and gut defence[END_REF].

Conclusion

The demonstration that T lymphocytes preferentially adhere to neural cells in mixed primary ENS cultures raises new perspectives for understanding the development and establishment of plexitis in patients with Crohn's disease. Identifying the molecular actors and pathways involved in these mechanisms may also serve to determine the functional consequences of T cell -ENS cell interactions, and in particular whether adhesion of T lymphocytes to enteric neural cells could participate to the evolution of inflammatory bowel diseases.

  times for one hour in the antigen blocking solution (Dako) before their incubation during 12 hrs at 4°C with mouse anti-CD3 (1/100, Dako Agilent) associated with rabbit anti-HuD (1/500, Santa Cruz Biotechnology, Texas, US) or rabbit anti-S100β (1/1000, Dako Agilent) diluted in the Dako diluent (Dako Agilent). After several washes, the sections were incubated for one hour with Cy5-conjugated anti-mouse IgG (1/500, Invitrogen) and Cy3-conjugated anti-rabbit IgG (1/500, JIR) at RT and washed. The sections were then counterstained with DAPI, mounted in Prolong (TF) and viewed under a Zeiss AxioZoom.V16 fluorescent microscope with Apotome structured illumination module for optical sectioning (Zeiss, Oberkochen, D).

Figure legends Figure 1 :

 legends1 Figure legends

Figure 2 :

 2 Figure 2: Quantitative analyze of non-activated (T) and activated T lymphocyte (ActT) adhesion on primary ENS cultures after two hours of co-culture. A: Number of total adherent non-activated (T) or activated T lymphocytes (ActT) per mm 2 of cell culture. B: Percentage of non-activated T lymphocytes (T) or activated T lymphocytes (ActT) adherent to all cell types in the ENS cultures after two hours of co-culture. The percentage is expressed relative to the number of T cells added to the ENS cultures. C: Number of non-activated (T) or activated T lymphocytes (ActT) in close proximity with S100β + (EGC) or PGP9.5 + (Neurons) cells per mm 2 of culture. D-E: Percentage of non-activated (D, n=4) or activated T lymphocytes (E, n=5) in close proximity(Cp) or not (NCp) to EGC and/or neurons. Percentage is expressed relative to the total number of adherent T lymphocytes. F: Percentage of the surface covered by PGP9.5 + cells, S100β + cells or both (PGP9.5 + S100β -100% overlay) in primary ENS cultures (n=8). G-H: Percentage of non-activated (F, n=4) and activated T lymphocytes (G, n=5) in close proximity only with neurons (PGP9.5 + ), only with EGC (S100β + ) or with both cell types (Both). Data were expressed as mean +/-SEM. Statistics: Mann-Whitney U test; *, p<0.05; **, p<0.01.

Figure 3 :

 3 Figure 3: T cell plexitis in enteric ganglia of patients with Crohn's disease. A: Visualization of T cells in myenteric ganglia after staining with anti-CD3 antibodies (brown) and hematoxylin-eosin. B-C: Serial sections of the myenteric ganglia double stained with anti-CD3 (green) / anti-S100β (red) antibodies (B) or anti-CD3 (orange) / anti-HuD (blue) antibodies (C). D-E: Serial sections of the submucosal ganglia double stained with anti-CD3 (green) / anti-S100β (red, D) or anti-HuD (blue, E) antibodies. F: Number of CD3 + T cells per myenteric ganglia (ggn) in close proximity to EGC (S100+) or (G) number of CD3 + T cells per neuron (Hu+) in close proximity with the cell body of enteric neurons (G). The quantitative analyzes were performed on intestinal tumor-free resections from five patients with colorectal cancer (ctrl) and ten patients with Crohn's disease (Crohn). Data were

Acknowledgements

J. Pabois was supported by a fellowship from the French Ministry of Higher Education and Research. This work was supported by the region "Pays de la Loire" (2012-13456). The authors thank the MicroPicell platform of SFR Bonamy (Nantes, Fr), in particular Philippe Hulin, for their help in confocal microscopy.

The authors declare no financial nor personal interest.


Pierre-Yves Benhamou
email: pybenhamou@chu-grenoble.fr

Rubrique Quoi de neuf ? Le glucagon nasal, une innovation bien sentie Nasal glucagon, a well-designed innovation

Keywords:

L'hypoglycémie induite par les traitements antidiabétiques demeure une préoccupation importante des patients avec diabète et de leur entourage, par sa fréquence et ses conséquences. Toute innovation permettant sa prévention ou son traitement est attendue. Le traitement non médicalisé des formes les plus sévères d'hypoglycémie fait actuellement appel au glucagon injectable. Le glucagon nasal est une formulation nouvellement développée. Les études, y compris en vraie vie, ont montré que le glucagon nasal n'est pas inférieur au glucagon injectable pour la correction de l'hypoglycémie, et qu'il lui est supérieur pour son usage pratique (vitesse et facilité d'administration) et pour la satisfaction de l'utilisateur aidant.

Mots clés : Hypoglycémie -

Le besoin médical

L'hypoglycémie sévère a été définie de façon consensuelle comme un événement nécessitant l'assistance d'une tierce personne pour l'administration active d'hydrates de carbone ou de glucagon, quel que soit le niveau glycémique atteint [START_REF] Seaquist | Hypoglycemia and diabetes: a report of a workgroup of the American Diabetes Association and the Endocrine Society[END_REF]. Cette urgence métabolique vraie demeure une préoccupation cruciale des professionnels de santé, des patients et de leur entourage familial, professionnel, ou social. Cela est bien évidemment lié à ses conséquences somatiques, traumatiques, cognitives, psychologiques, à court, moyen et long terme, reflétant une morbidité et une mortalité qui interpellent [START_REF] Whitmer | Hypoglycemic episodes and risk of dementia in older patients with type 2 diabetes mellitus[END_REF][START_REF] Mccoy | Increased mortality of patients with diabetes reporting severe hypoglycemia[END_REF]. Cela est aussi attribué à une fréquence non négligeable. On a rapporté la survenue d'hypoglycémies sévères chaque année chez 30-40 % des patients avec un diabète de type 1 (DT1) et 10-30 % des patients avec un diabète de type 2 (DT2) insulino-traité [START_REF] Umpierrez | Diabetic emergencies -ketoacidosis, hyperglycaemic hyperosmolar state and hypoglycaemia[END_REF]. Dans le registre américain T1D Exchange, environ 1 patient sur 10 rapporte une hypoglycémie sévère dans l'année précédente, fréquence qui passe à 1 sur 5 chez les patients ayant un diabète de plus de 40 ans d'ancienneté [START_REF] Weinstock | Severe hypoglycemia and diabetic ketoacidosis in adults with type 1 diabetes: results from the T1D Exchange clinic registry[END_REF]. Dans l'étude observationnelle française DIALOG portant sur plus de 3000 patients, l'incidence de l'hypoglycémie sévère était de 0,2 épisode/patient/mois chez les patients DT1 et 0,1 épisode chez les patients DT2, sur un suivi prospectif de 30 jours ; ces chiffres étaient supérieurs à ceux retrouvés dans l'évaluation rétrospective sur l'année précédente, montrant un taux de 0,08 épisode/patient/mois chez les patients DT1 et 0,05 épisode chez les patients DT2 [START_REF] Cariou | Frequency and predictors of confirmed hypoglycaemia in type 1 and insulin-treated type 2 diabetes mellitus patients in a real-life setting: results from the DIALOG Study[END_REF]. Une telle observation montre que l'hypoglycémie sévère est un phénomène à la fois fréquent et sous-estimé.

Les outils thérapeutiques

Le traitement de référence de l'hypoglycémie sévère en dehors d'un milieu médicalisé repose sur l'administration parentérale de glucagon. En raison de l'instabilité de la solution, les préparations commercialisées de glucagon ne sont disponibles que sous forme d'un lyophilisat en poudre devant être reconstitué à l'aide d'un diluant immédiatement avant l'injection qui sera réalisée par voie intramusculaire ou sous-cutanée à la dose unitaire de 1 mg chez l'adulte (0,5 mg chez l'enfant de poids < 25 kg). Le concept d'administration de glucagon dans la cavité nasale, qui offre un volume de 15 à 20 ml et une surface de 150 cm 2 (figure 1), avait été avancé il y a une trentaine d'années, notamment par l'école parisienne de diabétologie [START_REF] Freychet | Effect of intranasal glucagon on blood glucose levels in healthy subjects and hypoglycaemic patients with insulin-dependent diabetes[END_REF][START_REF] Pontiroli | Intranasal glucagon as remedy for hypoglycemia. Studies in healthy subjects and type I diabetic patients[END_REF][START_REF] Slama | A new non-invasive method for treating insulin-reaction: intranasal lyophylized glucagon[END_REF]. Mais il a fallu attendre la fin de la présente décennie pour disposer de la première forme commercialisée de glucagon nasal. La formulation de ce dernier comporte 3 mg de glucagon, un facilitateur d'absorption lipophile (phospholipid dodecylphosphocholine), complété par de la cyclodextrine, soit un volume total de 30 mg de poudre, dans un dispositif d'administration mono-dose (usage unique). Ce dernier permet la délivrance de 3 mg de glucagon, qui sera absorbé par la muqueuse nasale, en insérant l'extrémité du dispositif dans une narine et en actionnant le piston, sans nécessité d'inhalation ou de coopération du patient. Une ligne colorée en vert sur le piston disparaît en fin d'administration, indiquant que la totalité de la dose a été délivrée (figure 2). Les études pharmacocinétiques et pharmacodynamiques n'ont pas montré d'influence d'une congestion nasale d'origine virale ou autre, ni d'impact de l'usage d'agents décongestionnants [START_REF] Guzman | Effects of common cold and concomitant administration of nasal decongestant on the pharmacokinetics and pharmacodynamics of nasal glucagon in otherwise healthy participants: A randomized clinical trial[END_REF]. D'autres études ont montré que l'effet sur la glycémie atteint un plateau pour une dose de 3 mg de glucagon, en relation avec la saturation de l'absorption par la muqueuse nasale. Enfin, les données pédiatriques (de 4 à 17 ans) montrent que la dose de 3 mg a le même profil d'efficacité et de tolérance chez l'adulte, l'adolescent et l'enfant, tandis que cette étude pharmacodynamique montre une augmentation de glycémie ≥ 25 mg/dL à partir du nadir dans un délai de 20 minutes, que ce soit après administration intramusculaire ou intranasale [START_REF] Sherr | T1D Exchange Intranasal Glucagon Investigators. Glucagon nasal powder: a promising alternative to intramuscular glucagon in youth with type 1 diabetes[END_REF].

Les études de validation

3.1. Efficacité sur l'usage Une étude très édifiante conduite sur mannequin a comparé la facilité d'usage du glucagon nasal et du glucagon injectable chez des aidants formés et des personnes de l'entourage non formées [START_REF] Yale | Faster use and fewer failures with needle-free nasal glucagon versus injectable glucagon in severe hypoglycemia rescue: a simulation study[END_REF]. Avec la forme nasale, la dose totale de glucagon a pu être administrée (en 0,27 et 0,44 minutes, respectivement) par la quasi-totalité des intervenants (seuls deux n'ont pas administré la totalité de la dose en n'appuyant pas suffisamment sur le piston). Avec la forme injectable, 50 % des aidants ont bel et bien injecté le glucagon (dans un délai de 1,89 minutes), mais seuls 13 % ont administré la dose totale. Dans le cas des personnes de l'entourage, 20 % ont injecté une dose partielle (dans un délai de 2,40 minutes) et aucun n'a injecté la dose totale. Les erreurs comportaient l'injection du seul solvant, la coudure de l'aiguille, ou l'injection avec une seringue vide (figure 3). De façon intéressante, dans les deux situations, certaines personnes de l'entourage non formées, mais aussi certains aidants, ont également injecté de l'insuline par erreur, car le « design » de l'étude avait intelligemment mis à disposition l'ensemble des matériels habituellement disponibles dans la trousse des patients… Interrogés sur leur préférence entre les deux traitements, le choix se porte vers le glucagon nasal pour 69 % des patients, 81 % des aidants, et 100 % des personnes de l'entourage ; la préférence résiduelle pour le glucagon injectable s'observe chez les individus ancrés dans la croyance bien classique d'une supériorité d'efficacité en cas d'injection, et non informés des études de non-infériorité disponibles. Elle s'observe aussi chez ceux considérant qu'une seringue permet mieux de s'assurer que la dose a bien été administrée [START_REF] Yale | Faster use and fewer failures with needle-free nasal glucagon versus injectable glucagon in severe hypoglycemia rescue: a simulation study[END_REF]. C'est là une faiblesse habituelle d'un spray nasal, et l'éducation et le mode d'emploi devront s'attacher à bien faire identifier la ligne colorée sur le piston.

3.2.

Efficacité métabolique L'étude de référence chez l'adulte validant l'efficacité du glucagon nasal est une étude de non-infériorité conduite chez 75 patients DT1 randomisés entre 3 mg de glucagon nasal et 1 mg de glucagon intramusculaire après hypoglycémie insulinique induite par voie intraveineuse [START_REF] Rickels | T1D Exchange Intranasal Glucagon Investigators. Intranasal glucagon for treatment of insulininduced hypoglycemia in adults with type 1 diabetes: a randomized crossover noninferiority study[END_REF]. Après un nadir glycémique moyen de 48 à 49 mg/dL, le succès thérapeutique, défini par l'obtention d'une glycémie ≥ 70 mg/dL ou une remontée glycémique ≥ 20 mg/dL dans les 30 minutes, est sans différence significative entre les deux traitements. Le délai pour l'obtention du succès a été de 16 minutes par voie nasale et de 13 minutes par voie injectable (p < 0,001), sachant que ce délai ne comprend pas le temps de préparation du dispositif. La montée glycémique observée après glucagon nasal suit d'environ 3 minutes la montée induite par le glucagon injectable, avec un pic de glucagon observé après 15 minutes avec la forme injectable et après 20 minutes avec la forme nasale [START_REF] Rickels | T1D Exchange Intranasal Glucagon Investigators. Intranasal glucagon for treatment of insulininduced hypoglycemia in adults with type 1 diabetes: a randomized crossover noninferiority study[END_REF]. L'effet glycémique maximal est observé après 40 à 60 minutes avec les deux traitements, et se maintient en plateau au moins jusqu'à 90 minutes. Ces données ont été confirmées dans deux autres études randomisées, et l'analyse poolée de ces trois études, récemment rapportée, portant sur 214 adultes, avec 173 DT1, et notamment 41 DT2, est résumée en figure 4 [START_REF] Seaquist | Nasal glucagon reversed insulin-induced hypoglycemia in adults with diabetes: a pooled analysis[END_REF].

3.3.

Études en vie réelle Une étude prospective multicentrique simple bras a été conduite chez des adultes DT1 dont l'entourage était sollicité pour administrer du glucagon nasal en cas d'hypoglycémie sévère, ou modérée ≤ 60 mg/dL symptomatique [START_REF] Seaquist | Prospective study evaluating the use of nasal glucagon for the treatment of moderate to severe hypoglycaemia in adults with type 1 diabetes in a real-world setting[END_REF]. L'analyse a porté sur 69 patients qui ont présenté 157 évènements. Sous glucagon nasal, 152 épisodes se sont résolus en 30 minutes et cinq épisodes entre 30 et 45 minutes. La totalité des 12 hypoglycémies sévères s'est corrigée avec un retour à la conscience et un statut normal dans les 15 minutes. La facilité d'administration du glucagon nasal et la satisfaction des aidants sont illustrées de façon éloquente en figure 5. Des résultats tout à fait superposables, en termes d'efficacité métabolique et de satisfaction des aidants, ont été rapportés dans une étude pédiatrique prospective en vraie vie conduite chez 14 enfants et adolescents (de 4 à 18 ans), 33 épisodes hypoglycémiques tous résolus en moins de 30 minutes, la moitié en 10 minutes, avec une administration de glucagon nasal effectuée en moins de 30 secondes par la plupart des aidants [START_REF] Deeb | A phase 3 multicenter, openlabel, prospective study designed to evaluate the effectiveness and ease of use of nasal glucagon in the treatment of moderate and severe hypoglycemia in children and adolescents with type 1 diabetes in the home or school setting[END_REF].

3.4. Tolérance Les effets indésirables habituellement rapportés avec le glucagon injectable sont les nausées et les vomissements. Dans l'étude princeps de noninfériorité, la comparaison entre forme injectable et nasale montre une incidence respective de 18 et 26 % pour les nausées, 12 et 17 % pour les vomissements [START_REF] Rickels | T1D Exchange Intranasal Glucagon Investigators. Intranasal glucagon for treatment of insulininduced hypoglycemia in adults with type 1 diabetes: a randomized crossover noninferiority study[END_REF]. À quoi s'ajoutent des sensations de gêne faciale et des céphalées (9 % versus 25 %, respectivement). Les études en vie réelle rapportent des effets indésirables locaux à type d'irritation nasale transitoire, fréquents puisqu'observés chez 82 % des adultes et 93 % des enfants (avec dans la population pédiatrique, également des manifestations d'irritation conjonctivale), mais d'intensité légère à modérée, et résolutifs en moins d'une heure dans 60 % des cas [START_REF] Seaquist | Prospective study evaluating the use of nasal glucagon for the treatment of moderate to severe hypoglycaemia in adults with type 1 diabetes in a real-world setting[END_REF][START_REF] Deeb | A phase 3 multicenter, openlabel, prospective study designed to evaluate the effectiveness and ease of use of nasal glucagon in the treatment of moderate and severe hypoglycemia in children and adolescents with type 1 diabetes in the home or school setting[END_REF].

Les perspectives

Le stylo injecteur d'insuline, apparu il y a plus de 30 ans, est à notre connaissance le premier exemple d'outil de la panoplie diabétologique ayant mis en avant « l'usabilité » par rapport au principe thérapeutique. Le glucagon nasal entre dans cette catégorie de traitement offrant une « expérience utilisateur » de rupture tellement significative qu'elle contribue de façon majeure à améliorer le quotidien. Qu'il s'agisse de l'entourage familial (conjoint, parents, grands-parents) ou social (garde d'enfant, instituteur, collègue), le bénéfice de la sécurité et du confort d'administration est évident, pour eux, et donc pour le patient. On peut en effet considérer que la facilité d'utilisation du glucagon nasal permet de laisser ce traitement à la disposition de non-professionnels, par exemple, le staff enseignant à l'école, les moniteurs de colonies de vacances, les grands-parents, ou bien, pour les adultes, les collègues de travail.

Cet exemple du glucagon nasal est un excellent cas d'école montrant que l'évaluation d'un traitement doit combiner les données d'efficacité métabolique les plus rigoureuses à des informations sur l'usage. Ainsi, si certains commentaires de l'étude princeps de non-infériorité avaient pu, à juste titre, souligner que la forme injectable conserve un avantage cinétique de 3 minutes, la forme nasale rattrape ce retard grâce à sa meilleure disponibilité pratique [START_REF] Rickels | T1D Exchange Intranasal Glucagon Investigators. Intranasal glucagon for treatment of insulininduced hypoglycemia in adults with type 1 diabetes: a randomized crossover noninferiority study[END_REF]. Une nouvelle formulation liquide de glucagon, injectable et prête à l'emploi, est actuellement en développement, ne nécessitant pas de reconstitution extemporanée à partir d'un lyophilisat. Il a été rapporté que des minidoses de cette formulation (150 microgrammes) injectées par voie sous-cutanée permettaient la correction d'hypoglycémies non sévères, tout en évitant la prise calorique d'un resucrage oral [START_REF] Haymond | Nonaqueous, mini-dose glucagon for treatment of mild hypoglycemia in adults with type 1 diabetes: a dose-seeking study[END_REF]. De telles minidoses de glucagon ont été également évaluées avec succès dans la prévention de l'hypoglycémie induite par l'exercice physique [START_REF] Rickels | T1D Exchange Mini-Dose Glucagon Exercise Study Group. Mini-dose glucagon as a novel approach to prevent exercise-induced hypoglycemia in type 1 diabetes[END_REF]. Il pourra être intéressant, à l'avenir : (a) d'évaluer l'intérêt pratique éventuel du glucagon nasal dans la gestion de l'activité sportive ; (b) de comparer, face-à-face, le glucagon nasal et le glucagon injectable liquide ; (c) de développer, en fonction des données précédentes, des formulations moins dosées de glucagon nasal. L'avenir nous dira quel sera l'apport du glucagon nasal, en vie réelle, sur l'évolution pondérale, l'incidence des hypoglycémies sévères, celle des hypoglycémies non ressenties, et son impact médico-économique.

Les points essentiels

 L'hypoglycémie sévère est un objet de préoccupation pour les patients présentant un diabète et pour leurs aidants.  Le glucagon nasal n'est pas inférieur au glucagon parentéral pour la correction d'une hypoglycémie induite, qui est corrigée en une quinzaine de minutes dans la majorité des cas, et en moins de 30 minutes dans la quasi-totalité des cas.  Le retard pharmacocinétique de 3 minutes de la forme nasale sur la forme injectable de glucagon est totalement compensé par la facilité de préparation et d'administration du glucagon nasal.  L'expérience utilisateur rapportée avec le glucagon nasal, en milieu adulte ou pédiatrique, montre que ce produit répond à un besoin médical jusqu'à présent insuffisamment couvert.

Déclaration de liens d'intérêt

L'auteur déclare avoir perçu des honoraires de conférencier de Abbott, Eli Lilly, Novo Nordisk, Orkyn, Roche Diabetes Care, Sanofi, Vitalaire ; avoir participé à des conseils d'experts pour Abbott, Agiradom, Dexcom, Diabeloop, Eli Lilly, Insulet, LifeScan, Medtronic, Novo Nordisk, Roche Diabetes Care, Sanofi.

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Figure 1 .

 1 Figure 1. La zone cible d'un spray nasal (d'après [10]).

Figure 2 .

 2 Figure 1

  Figure 2

Figure 4 .Figure 5 .

 45 Figure 3

Figure 5

 5 Figure 5


Ibrahim Zaaroura

Souad Harmand

Julien Carlier

Malika Toubal

Aurélie Fasquelle

Bertrand Nongaillard

Experimental studies on evaporation kinetics of gold nanofluid droplets: Influence of nanoparticle sizes and coating on thermal performance

Keywords: gold nanoparticles, surfactant, marangoni cells, evaporation rate, droplets evaporation %Cv Volume Concentration, % │R│ Modulus of the reflection Coefficient RLL Longitudinal Waves H Humidity, % Tatm Atmospheric Temperature, °C

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

Introduction

A heat transfer fluid is a fluid medium used in a system for adding to or removing from the system an amount of heat at an adequate rate to ensure the functionality and reliability of the system. For that reason, new nanotechnology is used to enhance and improve the heat transfer in these systems such as Electronic cooling systems, Heat pipes, Nuclear reactors, and so on [1][2][START_REF] Rafati | Application of nanofluids in computer cooling systems (Heat transfer performance of nanofluids)[END_REF][START_REF] Jafarmadar | Thermal analysis and entropy generation of pulsating heat pipes using nanofluids[END_REF].

This nanotechnology is known as Nanofluids where nanoparticles, in nano sizes, are suspended in base fluids (water, minerals oil, binary mixtures) [START_REF] Choi | Enhancing thermal conductivity of fluids with nanoparticles[END_REF][START_REF] Akbar | Copper oxide nanoparticles analysis with water as base fluid for peristaltic flow in permeable tube with heat transfer[END_REF][START_REF] Beck | The thermal conductivity of alumina nanoparticles dispersed in ethylene glycol[END_REF]. These nanoparticles have the advantage of improving the heat transfer by absorbing rapidly the excess heat and transmit it outside the system. This improvement is due to their thermophysical properties that showed special advantages, such as better stability, greater thermal conductivity, and lower pressure drop [START_REF] Simpson | Nanofluid thermal Conductivity and Effect Parameters[END_REF][START_REF] Karimzadehkhouei | The effect of nanoparticles type and nanoparticle mass fraction on heat transfer enhancement in pool boiling[END_REF][START_REF] Xuan | Heat transfer enhancement of nanofluids[END_REF][START_REF] Dongliang | Thermal Conductivity Enhancement of Clathrate Hydrate with Nanoparticles[END_REF]. However, the use of nanofluids substantially depends not only on thermal conductivity but also on their viscosity. Viscosity indeed has a negative effect on the enhancement of heat transfer. [START_REF] Abu-Nada | Effects of variable viscosity and thermal conductivity of Al2O3-water nanofluid on heat transfer enhancement in natural convection[END_REF][START_REF] Ghanbarpour | Thermal properties and rheological behavior of water based Al2O3 nanofluid as a heat transfer fluid[END_REF] Many studies were done to analyze the effect of different types of nanoparticles, such as copper oxide, graphene oxide, and carbon nanotubes on the efficiency of heat transfer [START_REF] Ding | Heat transfer of aqueous suspensions of carbon nanotubes (CNT nanofluids)[END_REF][START_REF] Sandeep | Enhanced heat transfer in liquid thin film flow of non-Newtonian nanofluids embedded with graphene nanoparticles[END_REF][START_REF] Manimaran | Preparation and characterization of copper oxide nanofluid for heat transfer applications[END_REF]. Also, the effect of nanoparticle sizes (in nanoscale) on heat transfer with different average diameters (1 nm to 100nm) [START_REF] Warrier | Effect of particle size on the thermal conductivity of nanofluids containing metallic nanoparticles[END_REF]. Some studies show the enhancement in the coefficient of heat transfer by 25% as we increase the size of nanoparticles whereas others showed the inverse [START_REF] Bhuiyan | Effect of nanoparticles concentration and their size on surface tension of nanofluids[END_REF][START_REF] Yao | Effects of nanoparticles types and size on boiling feat transfer performance under different pressures[END_REF]. So, this variation is due to the type of nanoparticles used. Moreover, the influence of nanoparticle coating and their chemical preparation have also its effect on the performances of heat transfer. These chemical preparation presented by coating the nanoparticles with surfactant is used to stabilize the nanoparticles inside the fluids and to avoid the agglomeration or sedimentation in the system.

Nanofluid droplet evaporation has gained many audience nowadays due to its very important phenomenon and wide applications in fuel and sprays cooling [START_REF] Tanvir | Evaporation Characteristics of Ethanol Droplets Containing Graphite Nanoparticles Under Infrared Radiation[END_REF][START_REF] Grich | Experimental study of the effect of water spray in the air on thermal transfer along a plate of an exchanger[END_REF]. Many researchers work on the drying progress and deposition formation from the evaporative sessile droplets with the suspended insoluble solutes, especially nanoparticles [START_REF] Kim | Evaporation of nanofluid droplet on heated surface[END_REF][START_REF] Sefiane | Nanofluids droplets evaporation kinetics and wetting dynamics on rough heated substrate[END_REF][START_REF] Cieslinski | Sessile droplet contact angle of water-Al2O3, water-TiO2 and water-Cu nanofluids[END_REF][START_REF] Zhong | Sessile nanofluid droplet drying[END_REF]. The main content covers the evaporation fundamental, the particle self-assembly, and kinetic deposition of nanoparticles in a sessile nanofluid droplet. The effects of the surfactant and size of nanoparticles on the evaporative dynamics are elucidated at first and the deposition process which is introduced afterward. Droplet shape, spreading dynamics, evaporation rate, and evaporation regimes are the factors affected by the addition of particles.

Droplets evaporate on surfaces and measured using digital image technique (Kruss System) and with this technique we are able to measure the droplet profile, volume, contact angle, base diameter, and droplet height in time-dependent [START_REF] Chen | Evaporation behavior of PEGylated graphene oxide nanofluid droplets on heated substrate[END_REF][START_REF] Zaaroura | Evaporation of nanofluid sessile drops: Infrared and acoustic methods to track the dynamic deposition of copper oxide nanoparticles[END_REF]. So, with this method, we are able to select directly the better nanoparticles to use later in heat exchanger systems. Many factors play an important role in the enhancement of nanofluids droplet evaporation such as Marangoni convection cells and their stability with the base fluid. Nanoparticles have an effect on Marangoni flow, which causes a capillary flow outward from the center of the drop brings suspended particles to the drop edge as the evaporation process. Hu et al. [START_REF] Hu | Marangoni effect reverses coffee-ring depositions[END_REF] showed that Marangoni flow reversed the coffee-ring phenomenon and produces deposition at the droplet center rather than the edge. Also, in his other research work [START_REF] Hu | Analysis of the effects of Marangoni stresses on the microflow in an evaporating sessile droplet[END_REF] Hu et al. explained the effects of Marangoni on the evaporating sessile droplet.

Thus, the edge or droplet contact line has the highest evaporation as it's a weak zone. [START_REF] Benselama | Thermocapillary effects on steadily evaporating contact line: A perturbative local analysis[END_REF] Nanoparticles deposition has a direct effect on heat and mass transfer since the deposition of nanoparticles loses their effectiveness inside the fluid by absorbing the excess heat and transfer it outside and this led to increasing in the system thermal resistance. So, it is very important to takes into account all the conditions for a better thermal system.

In this study, experiments on the evaporation of nanofluids sessile droplets on heated hydrophobic surfaces are conducted using optical and infrared measurements. The objective of this work is to:

• Study the effect of gold nanoparticle with very interesting small sizes (2.2, 5 and 10 nm).

• The effect of surfactant represented by citrate coating and PBS as reactant free on the droplet evaporation rate of heat transfer.

• Introducing a new method illustrated by acoustic high-frequency waves to give more important details at the solid/liquid interface.

Experimental setup

In this work, the studied nanofluid consists of gold (Au) nanoparticles (Sigma Aldrich, Molecular weight = 196.97 g/mol with a volume concentration of 1% Cv) dissolved in distilled water (as a base fluid) and stabilized through ultrasonication (Elma, S 10/H) for at least 30 min before use. Three sizes of gold nanoparticles particle were used at 2.2, 5, and 10 nm for the citrate-capped PBS solution while 5 and 10 nm for the 0.1mM PBS, reactant free, Table . 1. PBS reactant free solution is used to stabilize the nanoparticle and to prevent any change in the PH of the gold nanofluid solution presented in volume concentration (See Fig. 1). To analyze the droplet evaporation process, two different methods are used, one using optical and infrared thermography measurements and the other one using acoustic reflection coefficient measurements at room temperature.

Nanofluids droplets are deposited, in both methods, on a silicon substrate modified with a perfluorodecyltrichlorosilane (PFTS) layer to achieve hydrophobic surfaces [START_REF] Breisch | Selective Chemical Surface Modification of Fluidic Microsystems and Characterization Studies[END_REF]. So, in that case, both methods have the same substrate characteristics. The optical measurements were achieved by increasing the temperature substrate from ambient to 77 °C. The calibration for the silicon substrate emissivity calibration was done using the emissivity calculator by the infrared camera.

The technique is to measure the surface temperature of a known emissivity material such as a black object attached to the silicon substrate. Form the known real temperature of the black object we can find the emissivity of the silicon substrate. This technique is suitable to find out the wanted substrate surface temperature. Finally, the water droplet analysis will be extracted with its known emissivity (≈0.97).

Infrared and optical measurements

This experimental setup allows us to calculate the evaporation rate of nanofluids droplets for each particle size and to visualize the convection cells caused due to Marangoni effect at high temperatures.

The substrate is heated using a joule effect by a resistance wire connected to the voltage supplier to control the temperature,

Acoustic measurements

Measurements are made under a controlled atmosphere using an air-conditioning system. A Cascade PM8 prober system is used to control the position of the S/G (signal/ground) probe at the microscale level on a piezoelectric transducer as small as 250 μm in diameter to achieve electrical measurements, Fig. 3. The specificity of the probe is the possibility to achieve an electrical contact at the backside of the wafer on which the piezoelectric transducer was fabricated [START_REF] Li | High Frequency Acoustic on-Chip Integration for Particle Characterization and Manipulation in Microfluidics[END_REF]. These probes are connected to a Rhode & Schwarz ZVA8 Vector Network Analyzer from which the modulus of the longitudinal reflection coefficient is deduced. The droplet is deposited on the top side of the silicon substrate via a microfluidic device, then a highfrequency (1 GHz) longitudinal wave is generated by ZnO piezoelectric transducers fabricated on the backside of the classical (100) crystal orientation of the silicon substrate on which the solid-liquid interface is characterized. The diameter of the ZnO transducers is about 250 μm.

During the droplet evaporation, the modulus of the longitudinal reflection coefficient is modified as the particles settle. This method allows a highly sensitive analysis of what occurs at the liquidsolid interface. The study and analysis mechanism is similar to our work in Zaaroura et al. [START_REF] Zaaroura | Evaporation of nanofluid sessile drops: Infrared and acoustic methods to track the dynamic deposition of copper oxide nanoparticles[END_REF].

Experimental Results

Infrared and optical investigations

The optical and infrared techniques are applied to investigate the dynamic evaporation of droplets of gold nanofluid. The CCD camera records the evaporation process and then the software Kruss Drop Shape Analyzer analyzes the profile of droplets and the evolution of droplets, contact angle, volume, and diameter, are accessible. Moreover, the infrared camera records the evaporation process at the top view and provides the gradient temperature distribution on the surface of the droplets.

Au nanofluid (1% Cv; 0.1mM PBS, reactant free) -Water Mixture, Particle Size Effects

For the water-Au mixture of different particle sizes (5, 10 nm), the result was compared to that of pure water on the / plot. Fig. 4 shows the evolution of / for droplets of pure water and 1% Cv Au-water mixture of different particle sizes at different surface temperatures. While the measured contact angle with the silicon substrate showed the same starting value for all the mixtures, Fig. 5. The results showed that at ambient temperature, the evaporation of pure water droplets was faster than the gold nanofluids of the two different sizes. Since reducing the contact angle increase the contact area between the droplet and solid surface and also reduces droplet thickness [START_REF] Chandra | Effect of Liquid-Solid Angle on Droplet Evaporation[END_REF]. As the liquid layer becomes thinner, heat transfer from the solid to the liquid-vapor interface is enhanced. Both effects increases the droplet evaporation rate. The measured contact angle of pure water showed a decrease in its values more than that of droplet gold nanofluid and this validates the enhancement in its droplet evaporation.

When the surface temperature increased to 55 °C, the evaporation process showed a higher evaporation rate of the 5 nm Gold-water mixture with 17% while 3% for the 10 nm Gold-water mixture.

Similarly, at a surface temperature of 77 °C, the evaporation process showed a higher evaporation rate of the 5 nm Gold-water mixture with 35% whereas 2% for the 10 nm Goldwater mixture. Thus, the evaporation rate is found to depend on the size of the nanoparticles, which showed that the smallest particle size (5nm) has the greatest effect on increasing the evaporation proceeds during the droplet lifetime than the 10 nm.

This difference can be linked to different reasons, the effective surface area of 5 nm Au-water mixture is greater than the 10 nm Au-water by a factor 2 and this means a greater amount of water molecules can come into contact with a larger surface surrounding the 5nm nanoparticles, thus affecting reactivity). Furthermore, Infrared Camera images made it possible to detect the convection cells, Fig. 6, due to the Marangoni effect that appeared on the surface of droplets.

These convection cells have the ability to increase the vapor mass transfer at the liquid-air interfaces and to induce a faster evaporation rate [START_REF] Chen | Marangoni Flow Induced Evaporation Enhancement on Binary Sessile Drops[END_REF]. The convection cells created by the thermal Marangoni can be observed on the surface of the droplets when the substrate is heated to high temperatures. The convection cells of the gold nanofluids appeared and remained longer than those of pure water and this is due to the Brownian motion of nanoparticles inside the droplet. The absorption by solid particles of gold and their flow has the ability to stay longer and to accelerate the convection liquid/vapor coefficient. These convection cells improve the evaporation process by inducing the thermal Marangoni number.

For this reason, Table. 2 represent the lifetime of these cells during the evaporation process of the pure and gold-water mixture (5 and 10 nm) at 77 °C surface temperature.

The table has shown that the higher evaporation rate has a higher convection cell life and these results validate the increase in the evaporation rate of the 5nm Gold-water droplet. The same measurements were made for the water-Au mixture of the same particle sizes (2.2, 5, 10 nm-Citrate-Capped-PBS solution) but with synthesis of the surface of gold nanoparticles with citrate in order to study the effect of citrate capping on enhancing the evaporation process. The result was compared to that of pure water on the / plot. Fig. 7 shows the evolution of / for droplets of pure water and 1% Cv Au-water mixture of different particle sizes at different surface temperatures. The results showed that at ambient temperature, the evaporation of pure water droplet was a little faster than the gold nanofluids of the three different sizes, with 2% more. When the surface temperature increased to 33 °C, the evaporation process showed a higher evaporation rate of the 10nm Gold-water mixture with 11% while 5% and 4% for the 2.2 and 5nm Gold-water mixtures respectively.

Similarly, at a surface temperature of 55 °C, the evaporation process showed a higher evaporation rate of the 10nm Gold-water mixture with 10% whereas 8% and 2% for the 2.2 and 5nm Gold-water mixtures respectively. Finally, the 10nm Gold-water mixture showed the highest evaporation rate at the surface temperature of 77 °C with 15% more while 1% and 2% for the 2.2 and 5nm Gold-water mixtures respectively.

Thus, the evaporation rate, Fig. 8, was found to depend on the size of the nanoparticles, which showed that the largest particle size (10nm) has the greatest effect on increasing the evaporation proceeds during the droplet lifetime. In Fig. 10, the kinetics of the longitudinal reflection coefficient modulus for the different preparations, different sizes (2.2, 5 and 10 nm) and types of gold nanofluids (Citrate-capping PBS and PBS-reactant free), during the evaporation of a 4% Cv Gold-water mixture drop, are presented. One can notice a clear difference between the particles with and without citrate capping. Indeed, the reflection coefficient of the gold-water mixture drops with citrate capping (for all nanoparticle sizes) begins to decrease almost instantaneously until the end of the evaporation process. Whereas, for drops of the gold-water mixture without citrate (for all sizes of nanoparticles) the reflection coefficient showed a constant value and began to decrease at the end of the process. As discussed in [START_REF] Zaaroura | Evaporation of nanofluid sessile drops: Infrared and acoustic methods to track the dynamic deposition of copper oxide nanoparticles[END_REF], the variation of the reflection coefficient ascribed to a change of the physical properties of the material interacting with the ultrasounds at the interface.

So, the change in the reflection coefficient is due to the gold nanoparticles deposition on the silicon substrate above the transducer.

Moreover, in the previous study [START_REF] Zaaroura | Evaporation of nanofluid sessile drops: Infrared and acoustic methods to track the dynamic deposition of copper oxide nanoparticles[END_REF], we have also shown that the sedimentation of the nanoparticle is correlated to the lifetime of Marangoni cells. Here, the Marangoni cells observed by an infrared camera, (section 3.1), showed no convection cells for the coated gold nanoparticles, with citrate, during the evaporation process, this would, therefore, mean that the particles settle The acoustic method corroborates the link between dynamic deposition of nanoparticles and its effect on the lifetime of convection cells.

Fig. 11, present the final shape pattern of the evaporated Au-water droplets with and without citrate capping for all sizes, 2.2, 5 and 10 nm at ambient temperature. The change in the shape of the dried gold nanoparticles between citrate coated and non-coated nanoparticles are clearly observed. For the non-coated gold nanoparticles (with PBS), a normal and uniform coverage pattern can be observed whereas a viscoelastic pattern is observed for the citrate nanoparticles.

(a)

(b) (c) (d) (e) (f)
In Fig. 12, a profilometer is used to measure the thickness of the deposited nanoparticles on the silicon substrate. This can be explained, in addition to the thermal Marangoni effect, the importance of the stability of the nanoparticles inside the droplet has an important factor during its evaporation.

Since the nanoparticles begin to settle early during evaporation process, this means that they will lose their effectiveness by absorbing the heat flux and to transporting it to the liquid-air interface.

Conclusion

The evaporation rate of gold Au-water nanofluid sessile droplets of 1% Cv has been measured in a controlled room temperature air (H=50%, Tatm=23 °C) using a Drop Shape Analyzer. The size effect of gold nanoparticle (2.2, 5 and 10 nm) was studied as well as different buffer solvents (Citrate-Capping PBS and no citrate-PBS) on the evaporation characteristics of droplets. The analysis was performed using two different methods, these methods have the ability to give a very important information on the fundamentals of droplet evaporation. An optical analysis, using Kruss system, was used to calculate the droplet evaporation rate of Au-water mixture at h different surface temperatures. While, an acoustic method represented by coefficient reflection waves has shown the ability to understand the deposition phenomenon of nanofluid at the solid/liquid interface. The main conclusion of this work are:

The gold nanofluids droplets, for both solutions, evaporate more rapidly as the surface temperature increases in comparison to the evaporation of distilled water droplets.

The uncapped gold nanofluids with PBS solution showed that the smallest nanoparticle size (5 nm) has the greatest impact on the evaporation rate (+35% more) than the larger nanoparticle, 10nm.

The citrate-capped gold nanofluid showed opposite results, the larger nanoparticle (10nm) has the greatest improvement on evaporation rate (+15%) but still less than the solution with PBS only.

The origin of the different behavior for the gold nanofluid compared to the pure water comes mainly from the thermophysical properties represented by thermal conductivity.

The thermal Marangoni plays an important role on enhancing droplet evaporation. It showed that the convection cells were observed clearly and stayed longer for the 5nm uncapped gold nanoparticle (68%) compared to the 10nm (43%) from total evaporation time.

No convection cells were observed for the citrate-capped gold nanofluid due to nanoparticles deposition. This behavior was proved using the high-frequency waves to the interface.

This stability showed an important factor on droplet evaporation process. 2-2 nm Citrate-PBS gold nanofluid.

14.

The repeatability of the contact angle and base diameter variations of 2nm citrate-PBS gold/ water droplet at 77 °C surface temperature.

3-5 nm Citrate-PBS gold nanofluid.

Fig 2 .

 2 The substrate is placed above a semiconductor material to homogenize the heating medium. Calibration was performed after adjusting the voltage setting for each surface temperature. The evaporation process of droplets is observed and recorded using high-speed camera from the side view (CCD camera, Allied Vision Technologies, 780 x 580 pixels) which connected to backlight lighting. Then a Kruss Drop Shape analyzer is used to measure the droplets profile (Contact Angle, Volume, Base diameter, etc...). At the same time, an infrared camera (FLIR, X6580SC, 640 x512 Pixels, 15 um detector pitch) is installed on the top for the infrared thermal mapping and visualization of thermal instabilities on the surface of the droplets through a sapphire window. The evaporation process was carried out under the same conditions (Initial volume droplet Vi =1.5 µL, Substrate surface temperature: Amb to 77 °C) inside a controlled chamber (H=50%, Tatm=23°C).

Figure 2 .

 2 Figure 2. Schema of the experimental setup

Figure 1 .Figure 4 .

 14 Figure 1. Schema of the experimental setup of acoustic measurements

Figure 5 .

 5 Figure 5. Normalized contact angle for water and gold nanofluids during evaporation, with different sizes (5 and 10 nm), at ambient temperature.

Figure 6 .

 6 Figure 6. Snapshots from an infrared video of the evaporation process of (a) pure water (b) 5 nm Au-water and (c) 10 nm Au-water nanofluids at 50 % relative humidity and 77 °C surface temperature, 1%Cv.

Figure 7 .

 7 Figure 7. Evolutions of the volume for water and gold nanofluid (Citrate capping-PBS), with different particle sizes (2.2, 5 and 10 nm) under substrate temperatures (a) at ambient, (b) 55 °C and (c) 77 °C, versus time.

Figure 8 .Figure 9 .

 89 Figure 8. Evaporation rate of the citrated gold nanoparticle sizes at different surface temperatures

Figure 2 .

 2 Figure 2. Reflection coefficient evolution of pure droplet (black), Au-PBS and Au-Citrate-PBS during evaporation process at 23°C.

Fig. 10 .Figure 3 .Figure 12 .

 10312 Figure 3. Gold Nanofluid Droplet deposited on silicon substrate and above the transducer (a) At starting point (b) Au-10nm-PBS (c) Au-5nm-PBS (d) Au-2.2nm-Citrate PBS (e) Au-5nm-Citrate PBS (f) Au-10nm-Citrate PBS (Nano particles Final shape pattern on the 250 µm transducer diameter)

Annex A 1 -Figure 13 .

 113 Figure 13. The repeatability of the Contact angle and base diameter variations of pure water droplet at 77 °C surface temperature.

Figure 15 .

 15 Figure 15. The repeatability of the contact angle and base diameter variations of 5nm citrate-PBS gold/ water droplet at 77 °C surface temperature. 4-10 nm Citrate-PBS gold nanofluid.

Figure 16 .

 16 Figure 16. The repeatability of the contact angle and base diameter variations of 10nm citrate-PBS gold/ water droplet at 77 °C surface temperature.

Figure 1. Gold

  nanofluids of different particle sizes and surfactants coating (PBS and PBScitrate)

	Nanoparticle, 1% Cv Size (nm)	Buffer solutions
	Gold, Au	2.2	citrate-capped-PBS solution	-
	Gold, Au	5	citrate-capped-PBS solution 0.1mM PBS, reactant free
	Gold, Au	10	citrate-capped-PBS solution 0.1mM PBS,

reactant free Table 1. Properties of prepared nanofluids

Table 2 .

 2 Lifetime of the convection cells at 77 °C substrate temperature of pure water, 5 nm Auwater and 10 nm Au-water nanofluids.

	Fluids/medium	Convection cells lifetime (%)
	Water	5 %
	5 nm Au-water,1% Cv	68 %
	10 nm Au-water, 1% Cv	43 %

3.1.2. Au nanofluid (1% Cv; Citrate-Capped-PBS solution) -Water Mixture, Citrate Effects.

Acknowledgments

The authors would like to acknowledge the financial support from the Jeumont Electric, CE2I-CPER, Renatech, French state and Region 'Hauts de France' for this work.

Author Information

Corresponding Author *Email: souad.harmand@uphf.fr Ibrahim.zaaroura@hotmail.com


C De Maissin

J.-C Leclère

N Roudaut

P Thuillier

P Monguillon

R Marianowski

G Potard

Evaluation of the performance of ultrasonography and 99mTc-sestamibi scintigraphy for primary hyperparathyroidism surgery

Keywords: Primary hyperparathyroidism, ultrasonography, Technetium Tc 99m Sestamibi

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

INTRODUCTION

Primary hyperparathyroidism implicates dysfunction of one or more parathyroid glands, leading to excessive production of parathyroid hormone (PTH) and dysregulation of phosphocalcic metabolism. The literature reports rates of 90% single adenoma, 10% multigland disease (multiple adenoma or hyperplasia) and a few cases of other histology such as parathyroid carcinoma [START_REF] Ruda | A systematic review of the diagnosis and treatment of primary hyperparathyroidism from 1995 to 2003[END_REF]. Radical treatment is surgical, on established indications [START_REF] Crouzeix | Primary hyperparathyroidism: new concepts, new recommendations[END_REF][START_REF] Bilezikian | Guidelines for the Management of Asymptomatic Primary Hyperparathyroidism: Summary Statement from the Fourth International Workshop[END_REF].

Minimally invasive surgery has become the attitude of choice, with bilateral neck exploration reserved to multi-gland pathologies [START_REF] Malinvaud | Parathyroid adenoma: surgical strategy[END_REF][START_REF] Westerdahl | Unilateral versus bilateral neck exploration for primary hyperparathyroidism: five-year follow-up of a randomized controlled trial[END_REF]. However, it requires precise preoperative lesion localization. There is no consensual decision-tree in parathyroid imaging. Ultrasound and 99m Tc-sestamibi scintigraphy are the most widely used modalities; each has its pros and cons, performance varying according to series and biases [START_REF] Kuzminski | Update in Parathyroid Imaging[END_REF]. Multi-gland disease is generally recognized as impairing performance [START_REF] Ruda | A systematic review of the diagnosis and treatment of primary hyperparathyroidism from 1995 to 2003[END_REF][START_REF] Kuzminski | Update in Parathyroid Imaging[END_REF][START_REF] Mortenson | Parathyroid exploration in the reoperative neck: improved preoperative localization with 4D-computed tomography[END_REF].

The aim of the present study was to assess performance factors in ultrasonography and 99m Tcsestamibi scintigraphy in minimally invasive surgery for primary hyperparathyroidism.

MATERIAL AND METHODS

A single-center retrospective study included all consecutive patients operated on in the ENT department of the university hospital of Brest, France, between January 2008 and November 2017, with surgery targeting either 2 ipsilateral parathyroid sites (unilateral neck exploration) or a single site (minimally invasive surgery), or else 4 sites (bilateral neck exploration).

Work-up comprised ultrasonography and 99m Tc-sestamibi scintigraphy. Files were selected from the center's medical information department using the health insurance codes KDFA002 and KDQA001.

Exclusion criteria comprised incomplete records, coding error, multiple endocrine neoplasia (MEN) known ahead of surgery, terminal chronic kidney failure (creatinine clearance <15mL/min) and/or dialysis, and no preoperative diagnosis (incidental discovery of primary hyperparathyroidism) or insufficient diagnostic criteria [START_REF] Crouzeix | Primary hyperparathyroidism: new concepts, new recommendations[END_REF].

Two hundred and seventy-three patients underwent the two imaging modalities and were included in the study. Fifty-six of the initial recruitment of 329 patients were excluded: 22 with incomplete files, 6 with MEN known ahead of surgery, 14 with terminal chronic kidney failure and/or dialysis, 6 with coding error, and 6 with no or uncertain diagnosis.

Table 1 shows patient data. There were 52 male (20%) and 218 female patients (80%); mean age, 61 ± 14 years. One hundred and forty-three (52%) were asymptomatic. Seventeen (6%) had multi-gland involvement. After surgery, 257 patients (94%) were cured.

Gland localization on imaging was compared versus intraoperative and biological reference data.

All patients underwent both modalities. They were grouped as showing concordant, discordant or negative imaging results.

Results were concordant when localization and number of pathological glands were similar between imaging modalities, negative when neither imaging modality identified pathologic glands, and otherwise discordant, with 3 discordant subgroups: ultrasound negative/scintigraphy positive (U-/S+), ultrasound positive/scintigraphy negative (U+/S-), or both modalities positive but differing in gland localization and number (U+/S+).

Patient data comprised age, gender, symptomatic primary hyperparathyroidism (urinary symptoms, malignant hypercalcemia, confusion syndrome, bone pain, chondrocalcinosis, acute or chronic calcifying pancreatitis, and other digestive symptoms) or not, in line with recent guidelines [START_REF] Crouzeix | Primary hyperparathyroidism: new concepts, new recommendations[END_REF][START_REF] Bilezikian | Guidelines for the Management of Asymptomatic Primary Hyperparathyroidism: Summary Statement from the Fourth International Workshop[END_REF], preoperative biological data (calcemia, PTH, 24h calciuria, phosphatemia) and relevant history.

Data for the high-resolution ultrasonography and 99m Tc-sestamibi scintigraphy comprised date of scan, lesion size and localization, and interpretation. For scintigraphy, the protocol was also noted: phase, previous scintigraphy, etc. In 123 patients (45%), ultrasonography was performed after scintigraphy. Surgical data comprised date, uni-or bi-lateral exploration, frozen-section biopsy results, lesion localization, definitive pathology result, and lesion size. Lesion volume was estimated from size on histology, on ellipsoid formula. Data were collected for postoperative calcemia and PTH, and for cure (defined by normalized calcemia).

The methodology of Grimaldi et al. [START_REF] Grimaldi | Challenging pre-surgical localization of hyperfunctioning parathyroid glands in primary hyperparathyroidism: the added value of 18F-Fluorocholine PET/CT[END_REF] was used for gland analysis.

In both modalities, all 4 glands were examined. Sensitivity, specificity and positive (PPV) and negative predictive value (NPV) were calculated for each modality and for the two combined when results were similar.

Demographic data (age), preoperative data (calcemia, PTH, asymptomatic primary hyperparathyroidism, and isolated hypercalcemia), surgical data (concomitant thyroid surgery, gland volume, multi-gland involvement) and cure were then compared.

Statistical analyses used SPSS v25 software (IBM Corp, Armonk, NY).

Quantitative variables (age, calcemia, PTH, gland volume) were analyzed on non-parametric Kruskal-Wallis test and parametric ANOVA.

Qualitative variables were analyzed in chi² (asymptomatic primary hyperparathyroidism) or Fisher exact test (concomitant thyroid surgery, multi-gland involvement, isolated hypercalcemia, non-cure) depending on sample size. The significance threshold was set at p <0.05.

RESULTS

PER-PATIENT ANALYSIS

In the 273 patients, sensitivity was 71% and PPV 89% for ultrasound, and respectively 75% and 91% for scintigraphy. When both were concordant (n=177), sensitivity was 84% and PPV 95%.

ANALYSIS BY GLAND LOCALIZATION

Per-gland analysis considered 1,092 glands, 958 of which showed concordant results (Table 2). All performances were better when the two modalities were concordant. Sensitivity showed the greatest improvement, from 70% on ultrasound and 74% on scintigraphy to 81% on the combination.

Combination reduced the false-positive rate, increasing specificity from 97% on ultrasound and 97% on scintigraphy to 99%, and PPV from 89% and 91% to 96%. False positives concerned 26 glands (2.4%) on ultrasound, 21 (1.9%) on scintigraphy and only 7 (0.6%) on combined examination.

False negatives concerned 93 glands (8.5%) on ultrasound, 74 (6.8%) on scintigraphy and 38 (4%) on combined examination.

ANALYSIS OF DISCORDANCE

One hundred and fifty-six of the 273 patients were classified as "concordant", 99 as "discordant" and 18 as "negative". In the "discordant" group, 42 patients had negative ultrasonography (U-/S+), 37 negative scintigraphy (U+/S-) and 20 positive but discordant results (U+/S+) (figure 1).

Comparisons are shown in Table 3.

Analysis of demographic data (age) and preoperative data (calcemia and PTH, asymptomatic hyperparathyroidism, isolated hypercalcemia) found no significant differences between the 3 groups or 3 subgroups.

Analysis of interventional data (concomitant thyroid surgery, gland volume, multi-gland disease) did find significant differences.

Gland volume distribution differed significantly between the 3 groups (p=0.003) and 3 subgroups (p=0.025). In the "concordant group", glands were larger than in the other 2 groups: mean 1,136 ± 1 728 mm 3 , versus 682 ± 864 mm 3 in the "discordant" group and 281 ± 201 mm 3 in the "negative" group. Likewise, glands were larger in the U+/S+ group than in the other 2 groups: mean 1,144 ± 1,243 mm 3 , versus 515 ± 429 mm 3 in U-/S+ and 595 ± 883 mm 3 in U+/S-.

There was an association between imaging results and associated thyroid pathology requiring concomitant thyroid surgery (p=0.03). Concomitant thyroid surgery accounted for 44% of procedures in the "negative" group, versus only 15% and 23% respectively in the "concordant" and "discordant" groups.

Cure rate showed no significant differences between groups (p=0.34) or subgroups (p=0.07).

Unilateral cervical surgery (minimally invasive plus unilateral exploration) was performed in 140 patients (90%) in the "concordant" group, 59 (59%) in the "discordant" group and 4 (22%) in the "negative" group. Ten patients undergoing unilateral cervical surgery were not cured: 7 in the "concordant" group and 3 in the "discordant" group. In 32 patients, bilateral exploration was performed without associated thyroid procedure: 8 (5.1%) in the "concordant" group, 19 (19%) in the "discordant" group, and 5 (28%) in the "negative" group.

Six of the 17 patients without cure showed uninodular thyroid, and 6 multi-gland involvement. The 7 negative explorations were all in the 17 non-cured patients, 4 due to false positive frozen section biopsy.

DISCUSSION

The present study demonstrated benefit of associating ultrasound and 99m Tc-sestamibi scintigraphy for locating pathologic parathyroid glands, notably in reducing the false-positive rate.

When ultrasound and scintigraphy results were discordant or negative, the pathologic gland or glands tended to be smaller and exploration more complicated. Conversely, larger and more functional parathyroid glands were more easily localized.

Detection of thyroid nodules requiring surgery can modify ultrasound and scintigraphy results, with more painstaking and extensive intraoperative exploration. We adopted broad selection criteria, including all primary hyperparathyroidism cases and excluding secondary hyperparathyroidism. Recurrent or persistent hyperparathyroidism was not differentiated. These criteria provided a large cohort, with results easily extrapolated to everyday practice. In Phitayakorn et al.'s article [START_REF] Phitayakorn | Incidence and localization of ectopic abnormal parathyroid glands[END_REF] on the anatomic distribution of ectopic glands in 231 patients, 37 ectopic glands were detected, 18 of which were mediastinal: 2 in the posterosuperior mediastinum, 7 in the thymus, 5 in the anterosuperior mediastinum and 4 in the thyrothymic ligament (7.8% of the cohort).

In the present study, sensitivity was 70% and PPV 89% on ultrasound and 74% and 91% on scintigraphy, in agreement with the literature [START_REF] Ruda | A systematic review of the diagnosis and treatment of primary hyperparathyroidism from 1995 to 2003[END_REF][START_REF] Cheung | A meta-analysis of preoperative localization techniques for patients with primary hyperparathyroidism[END_REF]. In the meta-analysis by Cheung et al.

[10], ultrasound showed 76.1% sensitivity (95%CI, 70.4-81.4%) and 93.2% PPV (95%CI, 90.7-95.3 ), and scintigraphy 78.9% (95%CI, 64-90.6%) and 90.7% (95%CI, 83.5-96%), respectively, although scintigraphy consisted in sestamibi-SPECT. Over the present study period of January 2008 to November 2017, scintigraphy modalities progressed: simple dualphase, dual-phase plus thyroid scintigraphy, dual-phase plus CT, dual-phase plus SPECT, dual-phase SPECT plus thyroid scintigraphy, dual-phase SPECT plus CT (the most frequent: n=155), and eventually dual-phase SPECT plus CT and thyroid scintigraphy. Numbers precluded analysis per modality, but the differences may explain the poorer diagnostic performance in the present study. Studies using different scintigraphy modalities, such as dual-phase dual-isotope iodine 123/technetium Tc 99m sestamibi scintigraphy in Périé et al.'s study [START_REF] Périé | Usefulness of combination of high-resolution ultrasonography and dual-phase dual-isotope iodine 123/technetium Tc 99m sestamibi scintigraphy for the preoperative localization of hyperplastic parathyroid glands in renal hyperparathyroidism[END_REF], showed interesting results.

Group and subgroup analysis involved very variable sample sizes, with likely lack of power; even so, significant differences emerged for gland size and association to thyroid surgery. Philippon et al. [START_REF] Philippon | Bilateral neck exploration in patients with primary hyperparathyroidism and discordant imaging results: a single-centre study[END_REF], whose study contributed to the present design, included only patients with bilateral neck exploration, and also found significant differences in gland weight and thyroid nodule rate, but without association with discordant or negative results or with multigland disease.

Kuzminski et al. [START_REF] Kuzminski | Update in Parathyroid Imaging[END_REF], in a review of the literature, summed up the advantages and limitations of ultrasonography and scintigraphy. Treglia et al. [START_REF] Treglia | Detection rate of (99m) Tc-MIBI single photon emission computed tomography (SPECT)/CT in preoperative planning for patients with primary hyperparathyroidism: A meta-analysis[END_REF], in a meta-analysis of SPECT/CT scintigraphy, found variably significant impact of calcemia, PTH and lesion size, and a strong negative relation between multi-gland disease and the sensitivity of scintigraphy. Impact on sensitivity was also reported for multi-gland disease by Nichols et [START_REF] Erbil | Impact of gland morphology and concomitant thyroid nodules on preoperative localization of parathyroid adenomas[END_REF][START_REF] Liddy | Preoperative Imaging in Primary Hyperparathyroidism: Literature Review and Recommendations[END_REF].

The present findings validate the surgical strategy advocated by Mihai et al. [START_REF] Mihai | Imaging for primary hyperparathyroidism--an evidencebased analysis[END_REF]: in case of concordance, minimally invasive surgery and, in case of discordance, unilateral neck exploration. The diagnostic and therapeutic attitude in case of negative findings on both examinations remains a matter of debate.

We therefore envisage using other imaging modalities to improve preoperative lesion localization precision. 18F-fluorocholine PET scan seems very promising [START_REF] Michaud | A Pilot Comparison of 18F-fluorocholine PET/CT, Ultrasonography and 123I/99mTc-sestaMIBI Dual-Phase Dual-Isotope Scintigraphy in the Preoperative Localization of Hyperfunctioning Parathyroid Glands in Primary or Secondary Hyperparathyroidism: Influence of Thyroid Anomalies[END_REF]. In the present series, 6 patients with negative results underwent 18F-fluorocholine PET on 24 glands, finding 100% for all 4 performance indicators, although interpretation should be cautious, given sample size. The literature on 18 F-fluorocholine PET is growing; for example, Grimaldi et al. [START_REF] Grimaldi | Challenging pre-surgical localization of hyperfunctioning parathyroid glands in primary hyperparathyroidism: the added value of 18F-Fluorocholine PET/CT[END_REF] assessed its added value in case of inconclusive ultrasound and scintigraphy, and suggested indications when ultrasound and scintigraphy results are discordant or negative and in case of persistent or recurrent primary hyperparathyroidism.

Its role, however, remains to be determined in studies including medico-economic aspects.

CONCLUSION

In surgically managed primary hyperparathyroidism, sensitivity in detecting abnormal parathyroid glands was 72% for ultrasound, 74% for scintigraphy and 81% for the combination. Positive predictive value was 90%, 91% and 96%, respectively. Analysis of factors for performance found significant differences for gland size and concomitant thyroid surgery, at respectively p=0.003 and p=0.003 between the 3 imaging results groups (concordant, discordant and negative).

FIGURE 1 : 1 FIGURE 1 :

 111 FIGURE 1: GROUPS AND SUBGROUPS ACCORDING TO ULTRASOUND AND SCINTIGRAPHY RESULTS

  al. and Scott-Coombes et al. [14,15], lesion size by Scott-Coombes et al., Chiu et al. and Swanson et al. [15-17], and thyroid nodules by Liddy et al. and Erbil et al.

ACKNOWLEDGMENTS

Our thanks to Dr Thuillier (endocrinologist), Dr Conan-Charlet (pathologist) and Dr Cavarec for technical help and constant support.

DISCLOSURE OF INTEREST:

The authors declare that they have no conflicts of interest concerning this article.