MD Jean-Pierre Frat
email: jean-pierre.frat@chu-poitiers.fr

PhD Jean-Damien Ricard

PhD Jean-Pierre Quenot

MD Nicolas Pichon

PhD Alexandre Demoule

MD Jean-Marie Forel

PhD Jean-Paul Mira

MD Rémi Coudroy

MD Guillaume Berquier

MD Benoit Voisin

MD Gwenhaël Colin

MD Bertrand Pons

MD Pierre Eric Danin

MD Jérome Devaquet

MD Gwenael Prat

MD Raphaël Clere-Jehl

MD Franck Petitpas

MD Emmanuel Vivier

MD Keyvan Razazi

MD Mai-Anh Nay

MD Vincent Souday

PhD Jean Dellamonica

PhD Laurent Argaud

PhD Stephan Ehrmann

MD Aude Gibelin

MD Christophe Girault

MD Pascal Andreu

PhD Philippe Vignon

MD Laurence Dangers

PhD Stéphanie Ragot

PhD Arnaud W Thille

Non-invasive Ventilation versus High-Flow Nasal Cannula Oxygen Therapy with apnoeic oxygenation for Pre-Oxygenation before Intubation of Patients with Acute Hypoxemic Respiratory Failure. A randomised multicentre open label trial

HAL is

Key words: preoxygenation, non-invasive ventilation, high-flow oxygen, respiratory failure.

Funding: Financial support was provided by the "Programme Hospitalier de Recherche Clinique National" 2015 of the French Ministry of Health.

The firm "Fisher & Paykel HeathCare" provided the equipment for some of the participating centers.

Research in context panel

Evidence before this body

We searched PubMed from Jan 1, 2000 to oct 1, 2018 using the following search terms: "preoxygenation" or "pre-oxygenation" and "apneic oxygenation" and "non-invasive ventilation". The literature shows one small randomised control study reporting better efficacy of non-invasive ventilation in preventing severe hypoxemia before intubation as compared to standard oxygen using valve-bag mask in patients with acute hypoxemic respiratory failure. Another study showed a lower incidence of severe hypoxemia with highflow oxygen therapy as compared to standard oxygen in a prospective before-after study, however these results were not confirmed in the different randomized controlled trials carried out to date. It therefore raises the question whether NIV as compared to high-flow oxygen therapy could better prevent severe hypoxemia during the intubation procedure of patient with hypoxemic respiratory failure.

Added value of this study:

This multicentre randomised controlled trial shows that preoxygenation with non-invasive ventilation or high-flow oxygen therapy carried out in patient ongoing intubation for acute hypoxemic respiratory failure did not change the risk of oxygen severe desaturation or other complications. However, episodes of severe oxygen desaturation were less frequent after preoxygenation with non-invasive ventilation in the prespecified stratum of the subgroup of patients with severe-moderate hypoxemia whatever the previous treatment carried out before randomization. Additionally, the lowest pulse oximetry was significantly higher after preoxygenation with non-invasive ventilation than high-flow oxygen.

Implications of all the available evidence:

The findings of the FLORALI-2 trial lead to carry out non-invasive ventilation during preoxygenation before intubation of patients with severe hypoxemic respiratory failure. This subgroup of patients represents more than three quarter of patients with acute hypoxemic respiratory failure. In view of these results and previous studies, preoxygenation with valvebag facemask should be replaced by high-flow nasal cannula oxygen therapy or non-invasive ventilation in ICU before intubation of patients with acute mild hypoxemic respiratory failure.

However, further study should be conducted to investigate whether non-invasive ventilation might be carried out in all patients whatever their level of hypoxemia in a larger population of patients with acute hypoxemic respiratory failure during preoxygenation before intubation.

INTRODUCTION

Tracheal intubation is one of the most commonly performed procedures in intensive care units (ICUs). [START_REF] Roux | Acquiring procedural skills in ICUs: a prospective multicenter study*[END_REF] Unlike the operating room, intubation procedure in ICU carries a high risk of lifethreatening complications including severe hypoxemia, neurological or cardiac ischemia, and cardiovascular collapse. [START_REF] Griesdale | Complications of endotracheal intubation in the critically ill[END_REF][START_REF] Jaber | Clinical practice and risk factors for immediate complications of endotracheal intubation in the intensive care unit: a prospective, multiple-center study[END_REF] Severe hypoxemia occurs in 20-25% of cases especially in hypoxemic patients intubated for acute respiratory failure. [START_REF] Griesdale | Complications of endotracheal intubation in the critically ill[END_REF][START_REF] Jaber | Clinical practice and risk factors for immediate complications of endotracheal intubation in the intensive care unit: a prospective, multiple-center study[END_REF][START_REF] Jaber | An intervention to decrease complications related to endotracheal intubation in the intensive care unit: a prospective, multiple-center study[END_REF] Cardiac arrest is the ultimate catastrophic complication, which can occur in 2-3% of intubation procedure in ICU, and is strongly related to hypoxemia or absence of preoxygenation before intubation. [START_REF] Jong | Cardiac Arrest and Mortality Related to Intubation Procedure in Critically Ill Adult Patients: A Multicenter Cohort Study[END_REF][START_REF] Mort | The incidence and risk factors for cardiac arrest during emergency tracheal intubation: a justification for incorporating the ASA Guidelines in the remote location[END_REF] Consequently, optimization of pre-oxygenation may help to secure the procedure by mitigating the risks of severe hypoxemia and subsequent complications.

Non-invasive ventilation and high-flow nasal cannula oxygen therapy (high-flow oxygen) are two oxygenation devices largely used in ICU that provide higher fraction of inspired oxygen (FIO 2 ) than standard oxygen. [START_REF] Sim | Performance of oxygen delivery devices when the breathing pattern of respiratory failure is simulated[END_REF][START_REF] Frat | Sequential application of oxygen therapy via high-flow nasal cannula and noninvasive ventilation in acute respiratory failure: an observational pilot study[END_REF][START_REF] Her | Physiologic effects of noninvasive ventilation during acute lung injury[END_REF][START_REF] Frat | High-flow oxygen through nasal cannula in acute hypoxemic respiratory failure[END_REF] High-flow oxygen enables delivery of continuous high gas flow via nasal prongs resulting in higher FiO 2 than with standard oxygen. [START_REF] Sim | Performance of oxygen delivery devices when the breathing pattern of respiratory failure is simulated[END_REF] Another theoretical advantage of high-flow oxygen may consist in maintaining oxygenation during the apnoeic phase of intubation after anaesthetic induction, thereby avoiding hypoxemia whereas noninvasive ventilation is removed at this phase. Only one randomised controlled study including a small sample of patients with acute hypoxemic respiratory failure has shown better efficacy of non-invasive ventilation in preventing severe hypoxemia as compared to standard oxygen using valve-bag mask. [START_REF] Baillard | Noninvasive ventilation improves preoxygenation before intubation of hypoxic patients[END_REF] Another study showed a higher rate of severe hypoxemia with standard oxygen than with non-invasive ventilation in the subgroup of patients previously treated with non-invasive ventilation. [START_REF] Baillard | Effect of preoxygenation using non-invasive ventilation before intubation on subsequent organ failures in hypoxaemic patients: a randomised clinical trial[END_REF] High-flow oxygen has also shown a lower incidence of severe hypoxemia during intubation procedure as compared to standard oxygen in a prospective before-after study. [START_REF] Miguel-Montanes | Use of High-Flow Nasal Cannula Oxygen Therapy to Prevent Desaturation During Tracheal Intubation of Intensive Care Patients With Mild-to-Moderate Hypoxemia*[END_REF] However, these encouraging results were not confirmed in the different randomised controlled trials carried out to date. [START_REF] Semler | Randomized Trial of Apneic Oxygenation during Endotracheal Intubation of the Critically Ill[END_REF][START_REF] Simon | High-Flow Nasal Cannula Versus Bag-Valve-Mask for Preoxygenation Before Intubation in Subjects With Hypoxemic Respiratory Failure[END_REF][START_REF] Vourc'h M, Asfar | High-flow nasal cannula oxygen during endotracheal intubation in hypoxemic patients: a randomized controlled clinical trial[END_REF] We conducted a prospective, multicentre, randomised, controlled trial involving patients admitted to the ICU with acute hypoxemic respiratory failure and undergoing tracheal intubation with the hypothesis that non-invasive ventilation could be associated with a lower rate of severe hypoxemia during the procedure as compared to high-flow oxygen therapy.

METHODS

Trial Design and Oversight

The high-FLow oxygen for pre-Oxygenation as Respiratory support during Acute Lung Injury before Intubation (FLORALI-2) trial was a non-blinded, multicentre, open-label, 2 parallelgroup randomised, controlled clinical trial (RCT). In the trial, patients from 28 ICUs in France were randomly assigned (1:1) to receive either non-invasive ventilation or high-flow oxygen therapy during pre-oxygenation.

The study protocol was approved for all centres by the ethics committee at Poitiers University Hospital (Ethics Committee Ouest III, Poitiers, France, registration number 2015-A00530-49). According to the French low and decision of the ethics committee, no safety committee was required, because the interventions used in the study were strategies of preoxygenation that are usually carried out in clinical practice. Inclusions were performed after having obtained informed consent from all patients or next of kin. The trial was overseen by a steering committee that presented information regarding the progression and monitoring of the study at Réseau Européen de Recherche en Ventilation Artificielle (REVA) Network meetings to all the investigators and/or research assistants of the participating centres every 4 months. Moreover, the role of the steering committee included making decisions, endorsing actions of the clinical research team and working with the public funder (University hospital of Poitiers). Members of the steering committee were not independent and were also members of the scientist committee, who designed the study. Members checked all relevant publications on the field of the study to ensure consistency in continuing the study. However, they had no access to the data collected or database until it was locked after the monitoring of centres. Research assistants regularly monitored all centres on-site to check adherence to the protocol and accuracy of the data recorded in accordance with the Good Clinical Practice

Guidelines. An investigator at each centre was responsible for enrolling patients in the study, ensuring adherence to the protocol and completing the electronic case-report form.

Patients

Consecutive patients older than 18 years admitted to the ICU and requiring intubation could be enrolled if they had acute hypoxemic respiratory failure according to the following criteria: a respiratory rate above 25 breaths/min or signs of respiratory distress, and a ratio of partial pressure of arterial oxygen to fraction of inspired oxygen (PaO 2 :FIO 2 ratio) equal to or below 300 mm Hg regardless of oxygenation strategy. For the calculation of PaO 2 :FIO 2 ratio, FiO 2 was measured under non-invasive-ventilation or high-flow oxygen and estimated under standard oxygen as follows: FIO 2 = 0.21 + oxygen flow rate x 0.03. [START_REF] Frat | High-flow oxygen through nasal cannula in acute hypoxemic respiratory failure[END_REF] The main exclusion criteria were as follows: intubation for cardiac arrest, altered consciousness defined by a Glasgow coma score <8 points, other contraindications to noninvasive-ventilation (recent laryngeal, oesophageal or gastric surgery, and significant facial fractures), pulse oximetry not available, pregnant or breast-feeding women, and refusal to participate.

Randomization and Allocation Concealment

Randomization was computer-performed in permuted blocks of four (unknown to investigators), with stratification according to centre and level of the PaO 2: FIO 2 ratio (equal to or below 200 and above 200 mm Hg). Within one hour after the validation of inclusion criteria, patients were randomly assigned in a 1:1 ratio, with the use of a centralised Webbased management system (G-ERDC, Clinfile, France), to one of the two following strategies: high-flow oxygen therapy or non-invasive ventilation.

Blinding

Although individual patient assignments could not be masked, the coordinating centre and all the investigators remained unaware of the study-group outcomes until the data were locked in October 2017. An adjudication committee unaware of the study groups reviewed all the data of pulse oximetry recorded and stored so as to analyse the events occurring during the intubation procedure, after an independent biostatistician, who was unaware of the study group outcomes collected data of patients from the recordings, with extraction of pulse oximetry curves and values. All analyses were performed by the study statistician in accordance with both the International Conference on Harmonization and Good Clinical Practice Guidelines. The complete methodology of the study has been previously published. 17

Interventions

Preoxygenation was carried out in a semi-recumbent position at 30° for 3-5 min with the technique assigned by randomization whatever the previous technique used for oxygenation.

In the non-invasive ventilation group, preoxygenation was delivered with a face mask connected to an ICU ventilator. Pressure-support ventilation was adjusted to obtain an expired tidal volume between 6 and 8 mL/kg of predicted body weight with a positive end-expiratory pressure (PEEP) level of 5 cm H 2 O and a FIO 2 of 1.0. Consequently, non-invasive ventilation provides oxygenation and ventilation during preoxygenation, between induction and laryngoscopy, but neither oxygenation nor oxygenation during laryngoscopy.

In the high-flow oxygen group, preoxygenation was delivered by applying oxygen continuously via bi-nasal prongs, with a gas flow of 60 L/min through a heated humidifier (MR 850, Fisher & Paykel, Auckland, New Zealand) and a FiO 2 of 1.0. Clinicians were instructed to perform a jaw thrust so as to maintain patent upper airway and to maintain the high-flow oxygen therapy during all the procedure of laryngoscopy until the endotracheal tube was placed into the trachea. Consequently, high-flow oxygen provides oxygenation but with little ventilation during preoxygenation, between induction and laryngoscopy, and also during laryngoscopy.

Bundle management including the following measures was proposed to all the participating centres for the intubation procedure as previously described [START_REF] Jaber | An intervention to decrease complications related to endotracheal intubation in the intensive care unit: a prospective, multiple-center study[END_REF] : the presence of two operators, systematic fluid loading before intubation (isotonic saline or balanced crystalloids at the discretion of the physician in charge of the patient) in absence of cardiogenic pulmonary oedema and rapid-sequence induction using etomidate (0.2-0.3 mg/kg) or ketamine (1.5-3 mg/kg) combined with rocuronium (0.6-1 mg/kg) or succinylcholine (1 mg/kg). In case of unsuccessful intubation, the following algorithm according to a given centre's procedure was proposed: an introducer first (intubating stylet or Eschmann introducer), then videolaryngoscopy, an intubation laryngeal mask airway, and finally fiberscopy and rescue percutaneous or surgical tracheostomy. After endotracheal intubation, patients were mechanically ventilated with a tidal volume of 6 ml/kg of predicted body weight, a respiratory rate of 25-30 breaths/min, a PEEP of 5 cm H 2 O and a FiO 2 set to maintain a pulse oximetry above 90%.

Study Outcomes

The primary outcome was the occurrence of an episode of severe hypoxemia defined by a decrease in pulse oximetry below 80% for at least 5 seconds between the beginning of rapidsequence induction (end of preoxygenation) and 5 min after confirmation of the tracheal intubation by capnography. To ensure that all centres have equivalent monitoring of pulse oximetry, a dedicated portable pulse oximetry monitor (Covidien, Nelcor DS 100A) and single-patient-use digital sensors (Covidien, Max-A-I) were provided to all the participating centres. All values of pulse oximetry were recorded with a 1 Hz frequency (one value of pulse oximetry by second) during the procedure and stored for subsequent analysis.

Secondary outcome variables were collected at the bedside in a separate form by physicians, residents or nurses and included the value of pulse oximetry at the end of preoxygenation and the lowest value during intubation procedure. Other prespecified outcomes included feasibility of preoxygenation evaluated by a 4-points scale (easy, quite easy, quite difficult, difficult), Cormack grade, [START_REF] Cormack | Difficult tracheal intubation in obstetrics[END_REF] intubation difficulty scale, [START_REF] Adnet | The intubation difficulty scale (IDS): proposal and evaluation of a new score characterizing the complexity of endotracheal intubation[END_REF] difficulty for intubation (>2 laryngoscopic attempts to place the endotracheal tube into the trachea or as lasting more than 10 minutes using conventional laryngoscopy, MACOCHA score), [START_REF] Jong | Early identification of patients at risk for difficult intubation in the intensive care unit: development and validation of the MACOCHA score in a multicenter cohort study[END_REF][START_REF]Practice guidelines for management of the difficult airway. A report by the American Society of Anesthesiologists Task Force on Management of the Difficult Airway[END_REF] immediate complications (arterial hypotension, sustained cardiac arrhythmia, bradycardia, cardiac arrest, death, oesophageal intubation, regurgitation, gastric distension, dental injury and new infiltrate on chest radiograph,) and late complications (occurrence of ventilator-associated pneumonia, worsening of SOFA score [START_REF] Vincent | The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine[END_REF] from day 1 to day 7, duration of mechanical ventilation, ICU length of stay and mortality at day 28).

Sample Size

Assuming a rate of severe hypoxemia episodes of 25% in patients having preoxygenation with high-flow oxygen, [START_REF] Simon | High-Flow Nasal Cannula Versus Bag-Valve-Mask for Preoxygenation Before Intubation in Subjects With Hypoxemic Respiratory Failure[END_REF][START_REF] Vourc'h M, Asfar | High-flow nasal cannula oxygen during endotracheal intubation in hypoxemic patients: a randomized controlled clinical trial[END_REF] we calculated that enrolment of 320 patients would provide the study with 95% power to show an absolute difference of 15% percentage points in the primary outcome between the high-flow oxygen therapy and non-invasive-ventilation group [START_REF] Jaber | An intervention to decrease complications related to endotracheal intubation in the intensive care unit: a prospective, multiple-center study[END_REF][START_REF] Baillard | Noninvasive ventilation improves preoxygenation before intubation of hypoxic patients[END_REF] at a two-sided alpha level of 0.05.

Statistical Analysis

Analyses were performed in the intention-to-treat population and in pre-specified subgroups determined by the stratification variable, moderate-to-severe hypoxemic patients with a PaO 2 :FiO 2 ratio equal to or below 200 mm Hg vs. mild hypoxemic patients with PaO 2 :FIO 2 ratio above 200 mm Hg. Baseline characteristics in each study group were analysed as frequencies and percentages for categorical variables and as means and SDs for continuous variables, as appropriate. The primary analysis compared the incidence of severe hypoxemia between the non-invasive ventilation group and the high-flow oxygen group using an unadjusted Chi-square test.

Heterogeneity of treatment effects across these pre-specified subgroups was examined by testing for treatment-covariate interaction with the logistic regression model. Adjustment on baseline level oxygenation was performed using logistic regression.

The secondary analyses compared secondary outcomes using an unadjusted Chi-square test for categorical variables and the Student's t-test or the Mann-Whitney test for continuous variables.

A two-tailed P value of less than 0.05 was considered to indicate statistical significance. No allowance for multiplicity has been performed, all secondary outcomes should be considered exploratory.

We used SAS software, version 9.4 (SAS Institute), for all the analyses.

Role of the funding source

Financial support was provided by the following organizations, which had no other involvement in the study: the French Ministry of Health supported the study through the grant "Projet Hospitalier de Recherche Clinique National" grant of year 2015; the "Fisher & Paykel Heath Care" firm provided the equipment for all the participating centers.

The corresponding author had full access to all the data and the final responsibility to submit for publication. The biostatistician (SR) had access to the raw data.

RESULTS

Recruitment

From April 2016 to January 2017, a total of 2079 patients were intubated in the 28 participating ICUs, and 322 underwent randomization. After the secondary exclusion of five patients having no recorded data, two patients having withdrawn consent or being under legal protection, one not intubated and one having cardiac arrest, 313 patients were included in the analysis (Figure 1). Among them, 142 patients were assigned to non-invasive ventilation and 171 to high-flow oxygen therapy. The median interval between ICU admission and randomization was 1 day (interquartile range (IQR), 0 to 2).

Baseline Data

The characteristics of patients at enrolment did not differ between the two groups (Table 1 and Table S1 in

Primary Outcome

In the overall study population, the proportion of patients with severe hypoxemia was 23% (33 of 142 patients) after preoxygenation by non-invasive ventilation and 27% (47 of 171) after high-flow oxygen (absolute difference -4•2%, 95% confident interval (CI) -13•7 to 5•5; p=0•39) (Table 2). Significant interaction was found between PaO2:FIO2 level at enrollment and the treatment group with respect to status regarding the primary outcome (Figure S2 in appendix). Consequently, results were split in two subgroups: patients with moderate-to-severe hypoxemia (PaO 2 :FiO 2 equal to or below 200), and patients with mild hypoxemia (PaO 2 :FiO 2 ratio above 200 mm Hg).

In the subgroup of patients with moderate-to-severe hypoxemia, the severe hypoxemia rate occurred in 28 (24%) of 117 patients in the non-invasive ventilation group and 44 (35%) of 125 patients in the high-flow oxygen group (absolute difference estimate -11•3%, 95% CI -22•3 to 0•3, p=0•055) (Table 3). The risk of severe hypoxemia was significantly lower with non-invasive ventilation than with high-flow oxygen after adjustment for PaO 2 at randomization, adjusted odds ratio 0•56 (95% CI, 0•32-0•99, p=0•0459) (Table 3).

In the subgroup of patients with mild hypoxemia, there was no difference in severe hypoxemia rates, 5 (20%) of 25 patients and 3 (7%) of 46 in non-invasive ventilation and high-flow oxygen therapy group, respectively (absolute difference estimate 13•4%, 95% CI -2•2 to 33•1, p=0•12) (Table 3).

Secondary Outcomes

In the overall study population, pulse oximetry values, duration of laryngoscopy or procedure of tracheal intubation were not different between the two strategies of preoxygenation (Table 2). There was also significant interaction between PaO2:FIO2 level at enrollment and the treatment group with respect to status regarding the lowest pulse oximetry during the intubation procedure (Figure S2 in appendix).

In the subgroup of patients with moderate-to-severe hypoxemia, the lowest pulse oximetry during intubation procedure was significantly higher in the non-invasive ventilation group as compared with the high-flow oxygen group, 86±12% versus 81±17% (p=0•02) (Table 3, Figure 2 and Figure S1 in appendix). Similarly, the pulse oximetry at the end of preoxygenation was higher in the non-invasive ventilation group as compared with the highflow oxygen group.

In the subgroup of patients with mild hypoxemia, there was no difference in pulse oximetry at the end of preoxygenation and during intubation procedure (p=0•31) (Table 3, Figure 2 and

Figure S1 in appendix).

Feasibility, complications and clinical outcomes

Preoxygenation during intubation procedure was perceived by practitioners as "easy" or "quite easy" in 94% of cases (134 of 142 patients) with non-invasive ventilation and 94% of cases (161 of 171 patients) with high-flow oxygen (Table S2

in appendix).

Preoxygenation was aborted in 3 patients during non-invasive ventilation and in 6 during high-flow oxygen therapy, mainly due to severe hypoxemia (5 of 9 patients). Rates of immediate and late complications did not differ between the two treatment groups (Table 2).

Cumulative probability of survival was not different between both strategies of preoxygenation whatever the subgroup of patients (Table 2 and Figure S3 in appendix).

DISCUSSION

In this multicentre, randomised, open-label trial including patients with acute hypoxemic respiratory failure (PaO 2 :FIO 2 ratio of 300 mm Hg or less), preoxygenation with non-invasive ventilation as compared with high-flow oxygen therapy did not change the risk of severe hypoxemia during intubation procedure or the occurrence of late complications. Baseline PaO 2 :FiO 2 ratio appeared to modify the effect of preoxygenation strategies on the risk of severe hypoxemia, with secondary analyses suggesting a possible benefit of non-invasive ventilation among patients with moderate-to-severe hypoxemia.

When planning the study, we assumed a reduction rate of severe hypoxemia from the usual 25% to 10%. This reduction could seem optimistic, however most of the studies reported a rate of severe hypoxemia of 25%, with high-flow oxygen or standard oxygen preoxygenation. [START_REF] Jaber | Clinical practice and risk factors for immediate complications of endotracheal intubation in the intensive care unit: a prospective, multiple-center study[END_REF][START_REF] Semler | Randomized Trial of Apneic Oxygenation during Endotracheal Intubation of the Critically Ill[END_REF][START_REF] Simon | High-Flow Nasal Cannula Versus Bag-Valve-Mask for Preoxygenation Before Intubation in Subjects With Hypoxemic Respiratory Failure[END_REF][START_REF] Vourc'h M, Asfar | High-flow nasal cannula oxygen during endotracheal intubation in hypoxemic patients: a randomized controlled clinical trial[END_REF] In the non-invasive ventilation group, we based our estimation of severe hypoxemia on two previous studies reporting a rate of 10% or less. [START_REF] Jaber | An intervention to decrease complications related to endotracheal intubation in the intensive care unit: a prospective, multiple-center study[END_REF][START_REF] Baillard | Noninvasive ventilation improves preoxygenation before intubation of hypoxic patients[END_REF] Our results showed a rather high rate around 25% in both groups. Accurate off-line analysis of pulse oximetry recordings during the whole intubation procedure period using a dedicated monitor may have identified otherwise unrecognised events and subsequently increased the rates of severe hypoxemia. Intubation procedure is sometimes urgent, difficult and confusing and it may be difficult to detect all episodes of severe hypoxemia, which could lead to an underestimation of events.

Otherwise, it seems there was an imbalance in stratification factors especially in the subgroup of patients with mild hypoxemia. In fact, the observed imbalance was 21 patients in this stratum. This can be explained by the stratification according to the PaO 2 :FiO 2 ratio level and to centres with randomisation performed in permuted blocks of four. The maximum theoretical imbalance between the two groups of treatment for a given stratum in a given centre was two patients. As 21 among the 28 participating centres have included patients in this stratum, the final imbalance could have reached up to 42 patients, In order to explore effects of pre-oxygenation strategies in hypoxemic patients, we planned subgroups analysis according to the severity of hypoxemia based on the classification of the acute respiratory distress syndrome (moderate-to-severe versus mild). [START_REF]The ARDS Definition Task Force. Acute Respiratory Distress Syndrome: The Berlin Definition[END_REF] These subgroups included strata determined by PaO 2 :FiO 2 ratio level (equal to or below 200 and above 200 mm Hg) at randomization regardless the oxygen device applied before pre-oxygenation. Our results showed qualitative effect of treatment across these two predefined subgroups for severe hypoxemia supported by a significant test of interaction. [START_REF] Wang | Statistics in medicine--reporting of subgroup analyses in clinical trials[END_REF] Finally, the impact of noninvasive ventilation on severe hypoxemia was different according to the prespecified subgroup, and appeared to be beneficial during pre-oxygenation only in patients with moderate-to-severe hypoxemia.

Previous studies have never compared effects of non-invasive ventilation with high-flow oxygen. In three randomized controlled studies, high-flow oxygen usually set with a gas flow of 50L/min and a FiO2 of 100% was compared to standard oxygen preoxygenation, but highflow oxygen had never been found to be superior to standard oxygen preoxygenation. [START_REF] Semler | Randomized Trial of Apneic Oxygenation during Endotracheal Intubation of the Critically Ill[END_REF][START_REF] Simon | High-Flow Nasal Cannula Versus Bag-Valve-Mask for Preoxygenation Before Intubation in Subjects With Hypoxemic Respiratory Failure[END_REF][START_REF] Vourc'h M, Asfar | High-flow nasal cannula oxygen during endotracheal intubation in hypoxemic patients: a randomized controlled clinical trial[END_REF] One previous pilot study including 53 patients found that non-invasive ventilation was superior to valve-bag mask during preoxygenation in avoiding risk of severe hypoxemia and obtaining higher pulse oximetry. [START_REF] Baillard | Noninvasive ventilation improves preoxygenation before intubation of hypoxic patients[END_REF][START_REF] Baillard | Effect of preoxygenation using non-invasive ventilation before intubation on subsequent organ failures in hypoxaemic patients: a randomised clinical trial[END_REF] However, none of these studies evaluated effects of preoxygenation strategies according to the level of oxygenation in patients with respiratory failure. A recent study showed that non-invasive ventilation during preoxygenation was more efficient than bag valve-mask oxygen to prevent severe hypoxemia in a subgroup of patients previously treated with non-invasive ventilation. [START_REF] Baillard | Effect of preoxygenation using non-invasive ventilation before intubation on subsequent organ failures in hypoxaemic patients: a randomised clinical trial[END_REF] One explanation might be that these patients were more severe than those treated with standard oxygen. In our study, non-invasive ventilation was also beneficial for preoxygenation of the most severe patients, although the oxygen device applied prior to inclusion, i.e. non-invasive ventilation, standard oxygen or high-flow oxygen, was not independently associated with severe hypoxemia.

The significantly higher pulse oximetry at the end of preoxygenation with non-invasive ventilation may explain its potential positive effect during preoxygenation among moderateto-severe hypoxemic patients. Physiologic effects of non-invasive ventilation include the ability to rapidly improve 11 oxygenation similarly to invasive ventilation, [START_REF] Antonelli | A comparison of noninvasive positive-pressure ventilation and conventional mechanical ventilation in patients with acute respiratory failure[END_REF] through delivery of high levels of FiO 2 and intrathoracic positive pressures favouring the increase of lung volumes or alveolar recruitment. [START_REF] Her | Physiologic effects of noninvasive ventilation during acute lung injury[END_REF] High-flow oxygen may have similarly rapid effect, i.e. positive end-expiratory pressure effect with an increased end-expiratory lung volume, [START_REF] Mauri | Physiologic Effects of High-Flow Nasal Cannula in Acute Hypoxemic Respiratory Failure[END_REF] but with a lower magnitude than non-invasive ventilation. [START_REF] Frat | Sequential application of oxygen therapy via high-flow nasal cannula and noninvasive ventilation in acute respiratory failure: an observational pilot study[END_REF][START_REF] Frat | High-flow oxygen through nasal cannula in acute hypoxemic respiratory failure[END_REF] In fact , high-flow oxygen may generate a positive end-expiratory ranging from 1 to 3cm of water in patient with respiratory failure, a level lower than that obtained with non-invasive ventilation. [START_REF] Parke | The effects of flow on airway pressure during nasal high-flow oxygen therapy[END_REF] Consequently, the effect of apnoeic oxygenation during laryngoscopy under high-flow oxygen does not seem as efficient as high positive pressures delivered by non-invasive ventilation as a mean of preventing hypoxemia.

There was no impact on mortality of pre-oxygenation strategies whatever the subgroup of patients. In fact, the intubation procedure is at risk of mortality during the procedure or immediately after starting the mechanical ventilation, especially in severe hypoxemic patients.

However, it seems that there is no specific risk of delayed mortality within the following days.

Our trial had several strengths that suggest that the results are applicable for most of patients with acute hypoxemic respiratory failure requiring intubation in ICUs. These strengths included the multicentre design and sealed randomization to the assigned technique of preoxygenation, subgroups analysis enabling to detect differences across strata, a well-defined protocol that included the adjudication of downloaded pulse oximetry recordings of each patient with the same dedicated portable pulse oximetry monitor among centres, complete follow-up at 28 days, and an intention to treat analysis.

Limitations of our study could be first, to have not considered a strategy of preoxygenation with valve-bag facemask in the control group. As high-flow oxygen therapy has showed efficacy in the management of patients with acute respiratory failure, [START_REF] Frat | High-flow oxygen through nasal cannula in acute hypoxemic respiratory failure[END_REF] and at least as efficient as valve-bag facemask for pre-oxygenation, [START_REF] Miguel-Montanes | Use of High-Flow Nasal Cannula Oxygen Therapy to Prevent Desaturation During Tracheal Intubation of Intensive Care Patients With Mild-to-Moderate Hypoxemia*[END_REF][START_REF] Vourc'h M, Asfar | High-flow nasal cannula oxygen during endotracheal intubation in hypoxemic patients: a randomized controlled clinical trial[END_REF] consequently most of investigators were concerned about switching to a preoxygenation with valve-bag facemask, which could be potentially less effective to improve oxygenation. [START_REF] Miguel-Montanes | Use of High-Flow Nasal Cannula Oxygen Therapy to Prevent Desaturation During Tracheal Intubation of Intensive Care Patients With Mild-to-Moderate Hypoxemia*[END_REF] Second, a high proportion of patients who were intubated in ICUs during the study period were not included that may limit generalization of findings. Many of the patients excluded from the study were not hypoxemic, underwent urgent intubation or had cardiac arrest or coma. However, these two last situations are not frequent reasons of intubation during acute hypoxemic respiratory failure and represent contraindications to non-invasive ventilation. [START_REF] Frat | High-flow oxygen through nasal cannula in acute hypoxemic respiratory failure[END_REF] Third, our primary outcome was not a patient-important outcome, [START_REF] Gaudry | Patient-important outcomes in randomized controlled trials in critically ill patients: a systematic review[END_REF] however most of previous randomised, controlled studies [START_REF] Baillard | Noninvasive ventilation improves preoxygenation before intubation of hypoxic patients[END_REF][START_REF] Baillard | Effect of preoxygenation using non-invasive ventilation before intubation on subsequent organ failures in hypoxaemic patients: a randomised clinical trial[END_REF][START_REF] Semler | Randomized Trial of Apneic Oxygenation during Endotracheal Intubation of the Critically Ill[END_REF][START_REF] Simon | High-Flow Nasal Cannula Versus Bag-Valve-Mask for Preoxygenation Before Intubation in Subjects With Hypoxemic Respiratory Failure[END_REF][START_REF] Vourc'h M, Asfar | High-flow nasal cannula oxygen during endotracheal intubation in hypoxemic patients: a randomized controlled clinical trial[END_REF][START_REF] Jaber | Apnoeic oxygenation via high-flow nasal cannula oxygen combined with non-invasive ventilation preoxygenation for intubation in hypoxaemic patients in the intensive care unit: the single-centre, blinded, randomised controlled OPTINIV trial[END_REF] assessed hypoxemia as primary outcome, which should be considered as a surrogate endpoint [START_REF] Wittes | Surrogate endpoints in clinical trials: cardiovascular diseases[END_REF] for hypoxia-driven cardiac arrests. Last, because treatments allocation could not be blinded, pulse oximetry curves were recorded and downloaded to be evaluated by blinded adjudicators in order analysing the events occurring during intubation procedure.

In conclusion, preoxygenation with non-invasive ventilation or high-flow oxygen therapy during intubation procedure did not change the risk of severe hypoxemia and other immediate or late complications. However, non-invasive ventilation may prevent severe hypoxemia among patients with severe-moderate hypoxemia as compared with high-flow oxygen. This The other authors declared no conflicts of interest. The data are means ± SD or absolute numbers (%). Reason for ICU admission was compared in 3 classes with chi 2 test: respiratory primary failure, non-respiratory primary failure, post-operative.

Abbreviations: SD, standard deviation; NIV, non-invasive ventilation; HFOT, high flow nasal cannula oxygen therapy; COPD, chronic obstructive pulmonary disease; ARDS, acute respiratory distress syndrome. a The body-mass index is the weight in kilograms divided by the square of the height in meters.

  appendix).The mean settings were as follows: in the non-invasive ventilation group, a pressure support level of 9±4 cm H 2 O, a PEEP of 5±0•5 cm H 2 O, and a FiO 2 of 0•99±0•06, resulting in a tidal volume of 8•3±2.6 ml per kilogram of predicted body weight; in the high-flow oxygen group, a gas flow of 58±9 litres per minute with a FiO 2 of 0•99±0•08. The duration of preoxygenation lasted 5±2 minutes with non-invasive ventilation and 5±4 with high-flow oxygen (p=0•45).

  finding calls for confirmation in future research. the conduct of the study; personal fees from Fisher & Paykel Healthcare and non-financial support from Resmed, outside the submitted work. Dr. Thille reports travel and accommodation expense coverage to attend scientific meetings from Covidien, General Electric Healthcare, Fisher&Paykel, Maquet -Getinge, and reports no income or personal fees from no company

Figure 1 : 2 :

 12 Figure 1: Flow of participants through the high-flow oxygen for pre-oxygenation as respiratory support during acute lung injury before intubation (florali-2) randomised clinical trial.Figure 2: Variations in mean values of minimal pulse oximetry from baseline to the end of the intubation procedure, (A) in the overall population, (B) in patients with moderate to severe hypoxemia (PaO 2 :FiO 2 ≤200 mm Hg) and (C) in patients with mild hypoxemia (PaO 2 :FiO 2 >200 mm Hg), in the non-invasive ventilation group (red line) and high-flow nasal cannula oxygen therapy group (blue line). Points and squares represent mean values with the 95% confident interval. At baseline (defined as values obtained at time of randomization), pulse oximetry values were similar. Values of pulse oximetry at the end of preoxygenation and lowest pulse oximetry values during intubation procedure were higher after preoxygenation with non-invasive ventilation than with high-flow oxygen (p=0•02) in patients with moderate-to-severe hypoxemia.Figure S1:

  Rates of severe hypoxemia during intubation procedure after preoxygenation with noninvasive ventilation (NIV, red bar) and high-flow oxygen therapy (HFOT, blue bar), in the overall population and subgroups of strata. (B) Lowest pulse oximetry values during intubation procedure after preoxygenation with non-invasive ventilation (NIV, red points) and high-flow nasal cannula oxygen therapy (HFOT, blue points) in the overall population and subgroups of strata.

  Figure S2: (A) Forest plot showing primary outcome (severe oxygen desaturation) broken down by the PaO 2 :FIO 2 stratum, the vertical line set at 15% corresponds to the expected difference between the two treatments. The value 15% is within the 95% confidence interval for the stratum PaO 2 :FiO 2 ≤200. (B) Restricted cubic spline regression between PaO 2 :FiO 2 ratio at baseline and lowest SpO 2 value for noninvasive ventilation (red line) and high-flow oxygen (blue line) showing the qualitative interaction between the level of PaO 2 :FIO 2 baseline and treatment (p=0.047). The cut point of PaO2:FiO 2 level effect is nearly 200 mmHg at which the sign is swapped for the treatment.

Figure S3 :

 S3 Figure S3: Kaplan-Meier Plot of the Probability of Survival from Randomization to Day 28,

Table 1 : Baseline Characteristics of the Intention-to-Treat Population according to Study Group

 1

	NIV (n=142)	HFOT (n=171)	p value

Acknowledgement:

The authors wish to thank Jeffrey Arsham for reviewing and editing the original Englishlanguage manuscript.

The data are means ± SD, or absolute numbers (%).

Abbreviations: NIV, non-invasive ventilation; HFOT, high flow nasal cannula oxygen therapy; SpO2, pulse oximetry; CI, confident interval; SD, standard deviation; SOFA, Sepsis-related Organ Failure Assessment; ICU, intensive care unit.

Data sharing:

Data collected for the study are not available.

Author contribution:

JPF: investigator of the study, study design, member of the steering committee, data acquisition and collection, data analysis and interpretation, drafting the manuscript. JDR, AWT: study design, data acquisition and collection, data analysis and interpretation, draft of the manuscript, critical revisions of the manuscript for important intellectual content. JPQ, NP, AD, JMF, JPM, RC, GB, BV, GC, BP, PED, JD, GP, RCJ, FP, EV, KR, MAN, VS, JD, LA, SE, AG, CG, PA, PV, and LD: data acquisition and collection, critical revisions of the manuscript for important intellectual content.

SR: data analysis and data interpretation.

Conflict of interest


Cristiana Pistono
email: pistono.cristiana@gmail.com

Amandine Zimmermann

Chloé Morel

Karine Herbeaux

Céline Héraud

Stéphanie Dumont-Kientzy

Paul Pevet

Marie-Paule Felder-Schmittbuhl

Chantal Mathis

A I Virtanen

Major role of MT2 receptors in the beneficial effect of melatonin on long-term recognition memory in C57BL/6J male mice

Keywords: melatonin, MT1 deficiency, MT2 deficiency, long-term recognition memory, object recognition, object location

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

INTRODUCTION

Melatonin regulates the circadian rhythms of several biological functions such as sleep or memory [START_REF] Comai | Unveiling the role of melatonin MT2 receptors in sleep, anxiety and other neuropsychiatric diseases: a novel target in psychopharmacology[END_REF]. Chronic administration of melatonin and its analogs has been proposed to have therapeutic potentials in brain diseases such as Alzheimer's disease (AD) [START_REF] Shukla | Mechanisms of Melatonin in Alleviating Alzheimer's Disease[END_REF].

Brain melatonin acts mainly through G-protein-coupled MT1 and MT2 receptors [START_REF] Dubocovich | Effect of MT1 melatonin receptor deletion on melatonin-mediated phase shift of circadian rhythms in the C57BL/6 mouse[END_REF]. Clear differences in their neuroanatomic distribution suggest an involvement in distinct brain processes [START_REF] Klosen | MT1 and MT2 melatonin receptors are expressed in nonoverlapping neuronal populations[END_REF][START_REF] Lacoste | Anatomical and cellular localization of melatonin MT1 and MT2 receptors in the adult rat brain[END_REF]. Restoring melatonin's levels affected by aging improves cognitive status in the elderly and in early stages of AD [START_REF] Jean-Louis | Melatonin effects on sleep, mood, and cognition in elderly with mild cognitive impairment[END_REF][START_REF] Peck | Cognitive effects of exogenous melatonin administration in elderly persons: a pilot study[END_REF]. Chronic melatonin supplementation in drinking water also had beneficial effect on cognition in rodent models of normal and pathological aging [START_REF] Mukda | Melatonin administration reverses the alteration of amyloid precursor protein-cleaving secretases expression in aged mouse hippocampus[END_REF][START_REF] O'neal-Moffitt | Prophylactic melatonin significantly reduces Alzheimer's neuropathology and associated cognitive deficits independent of antioxidant pathways in AβPP(swe)/PS1 mice[END_REF].

However, both pharmacological and genetic approaches have not yet provided a clear picture of the respective involvement of MT1 and MT2 in the positive effects of melatonin on learning and memory because most studies used MT1/MT2 double knock-out mice and mixed MT1/MT2 agonists [START_REF] Bertaina-Anglade | Memory facilitating effects of agomelatine in the novel object recognition memory paradigm in the rat[END_REF][START_REF] He | A novel melatonin agonist Neu-P11 facilitates memory performance and improves cognitive impairment in a rat model of Alzheimer' disease[END_REF][START_REF] O'neal-Moffitt | Genetic deletion of MT₁/MT₂ melatonin receptors enhances murine cognitive and motor performance[END_REF]. In order to progress in therapeutic approaches based on melatonin analogs to improve memory disorders it is important to know the respective role of MT1 and MT2 receptors in memory processes. Here, we used single receptor-(MT1 -/- and MT2 -/-) deficient mice created with a "knock-in" strategy to insert a LacZ reporter instead of the receptor coding sequences to determine whether these receptors are involved in recognition memory [START_REF] Klosen | MT1 and MT2 melatonin receptors are expressed in nonoverlapping neuronal populations[END_REF]. This form of memory is known to be affected in early stages of Alzheimer's disease [START_REF] Mathis | The Value of the Object Recognition Paradigm in Investigating Animal Models of Alzheimer's Disease[END_REF]. We first confirmed that chronic melatonin treatment improves recognition memory performance in C57BL/6J (B6) mice tested in object recognition (OR) and object location (OL) tasks.

Then, the treatment was given to MT1 -/-and MT2 -/-mice to determine the possible involvement of MT1 and/or MT2 in the memory-enhancing effect of melatonin.

MATERIAL AND METHODS

The B6 strain is well adapted to test the effects of exogenous melatonin because it produces very low amounts of melatonin with functional melatonin receptors [START_REF] Dubocovich | Effect of MT1 melatonin receptor deletion on melatonin-mediated phase shift of circadian rhythms in the C57BL/6 mouse[END_REF]. The experiments were first conducted in male B6 mice (Charles River Laboratories, L'Arbresle, France) and then in MT1 - /-and MT2 -/-mice and their wild type (WT) littermates. The two lines were maintained separately on a B6 background (INCI, Strasbourg, France; see [START_REF] Klosen | MT1 and MT2 melatonin receptors are expressed in nonoverlapping neuronal populations[END_REF] The effect of chronic melatonin treatment on recognition memory was first assessed in 12 6-month-old B6 males, and then in MT1 -/-(n = 13) and their WT (n = 12), and also MT2 -/-(n = 14) and their WT (n = 12) mice at the age of 6-7 months. They were single-housed with a plastic tunnel (5 cm Ø x 15 cm long) added to their cage. This tunnel was used to transport the mouse during testing in order to reduce anxiety levels. A 16-day chronic treatment was launched with melatonin in drinking water. Melatonin (80 mg) progressively dissolved in 1L stirred tap water was freshly prepared and dispatched in opaque bottles every 8 days (O'Neal-Moffit et al., 2015;[START_REF] Moussaoui | The Influence of Storage Conditions on Melatonin[END_REF]. Since mice are active and drink mostly during the night, our chronic treatment tends to mimic the natural night-time increase in melatonin observed in mice as in humans [START_REF] Comai | Unveiling the role of melatonin MT2 receptors in sleep, anxiety and other neuropsychiatric diseases: a novel target in psychopharmacology[END_REF]. All mice were tested twice under different treatments in two successive series (Figure 1A): one half received water and the other half received melatonin in the first series, then after a 5-day washout period, the treatment assignment was reversed for the second series. This protocol was designed to reduce the number of mice (3R reduction rule) and it was validated in the first experiment. Mice were tested the last 5 days of each series between 9.00 a.m. and 1.00 p.m. Bottles and mice were weighed at the start and at the end of each 8-day period to calculate daily fluid intake (approximately 0.45 mg/day). This was within the range of doses used in studies reporting a promnestic effect of melatonin in the drinking water of mouse models of AD (Mukda O'Neal-Moffitt et al., 2016;[START_REF] O'neal-Moffitt | Prophylactic melatonin significantly reduces Alzheimer's neuropathology and associated cognitive deficits independent of antioxidant pathways in AβPP(swe)/PS1 mice[END_REF].

Object based recognition tasks were performed in an open field (92.5 x 92.5 x 35 cm; Ugo Basile, Italy).

After a familiarization trial of 8 minutes, mice received a 9 minutes acquisition trial on the next day with two identical objects. After 24 hours, they had a 6-minutes retention trial with one object replaced by a new one in the OR task or with one object moved 30 cm away from the fixed one in the OL task. A discrimination ratio was calculated as the time spent exploring the novel or displaced object minus time spent exploring the familiar or fixed object/total time spent exploring both objects. The performance is higher than chance level (0) if the mouse detects novelty. Different sets of object were used for each series and the open field and objects were all cleaned with 30% alcohol between each mouse. We verified that there was no bias due to side, object or series. Two criteria excluded mice for exaggerated neophobic behavior during the retention trial (total exploration below 4 s: 1 MT1 -/-, 1 MT2 -/-and 1 WT (MT2 line) mice in the OL task and 1 other WT (MT2 line) mouse in both tasks; strong novel object avoidance with ratio below -0.45: 1 MT1 -/-mouse in the OR task).

The light/dark test (LD) was performed to evaluate any possible effect of melatonin on anxiety level because this can be a major confounding factor in OR tasks. The light/dark apparatus was composed of two opaque and transparent PVC boxes (18.5×18.5×15 cm 3 ), connected with an opaque tunnel (5x5x5.5 cm 3 ). Illumination of the transparent box was set at 1000 lux. The mice were placed in the dark box and time spent in the lit compartment was recorded for 5 minutes.

Recognition performance was compared to chance level (0) using a one-sample t-test for each group to evaluate novelty detection in OR and OL tasks (reported in the text). A two-way ANOVA for genotype and repeated measures on treatment was applied for recognition performance of MT1 -/-and MT2 -/-mice.

We had no expectation on which and how much each receptor would be involved, so between group multiple comparisons were performed with the Student Newman-Keuls test (NK) to gain power in detecting effects. The latter were then analyzed using the more conservative Tuckey HSD to confirm the most robust effects [START_REF] Sauder | An Updated Recommendation for Multiple Comparisons[END_REF][START_REF] Wilcox | New Designs in Analysis of Variance[END_REF]. Recognition performances in the B6 experiment and acquisition exploration were compared using a two-tailed paired t-test. Finally, LD performance was analyzed with a two-way ANOVA on genotype and treatment factors. Effect sizes were calculated as Cohen's d for significant t-tests and eta squared (η2) for significant ANOVAs.

Normality and variance equality were assessed by means of the D'Agostino-Pearson normality test and the Levene's test, respectively. Differences were considered significant at p < 0.05.

RESULTS

Effects of melatonin in C57BL/6J mice

Chronic melatonin treatment improved OR performance of B6 mice compared to water condition (Figure 1B; paired t = 4.98, df = 11, p = 0.0004, d = 2.29) and chance level (t = 9.61, df = 11, p < 0.0001).

It also improved OL performance when compared to water condition (Figure 1C; paired t = 3.18, df = 11, p = 0.009, d = 1,23) and chance level (t = 6.76, df = 11, p < 0.0001). Melatonin in drinking water did not affect object exploration during acquisition trials in OR (water: 17.9 ± 1.8 sec and, melatonin: 15.8 ± 1.8 sec; paired t = 1.16, df = 11, p = 0.27) and OL tasks (water: 20.5 ± 1.9 sec and, melatonin: 21.0 ± 2.4 sec, respectively: paired t = 0.18, df = 11, p = 0.86). Similar results were obtained in the following experiments. The validity of the 2-phase protocol was confirmed in this first experiment (OR main effect of phase: F1,10 = 2.13, p = 0.17 and phase x treatment interaction F1,10 = 0.39, p = 0.55; OL main effect of phase: F1,10 = 0.09, p = 0.77 and phase x treatment interaction F1,10 = 1,68, p = 0.22). We verified in the following experiments that there was no significant phase effect and interactions with phase.

Effects of melatonin on recognition performance in MT1 -/-mice

The melatonin treatment significantly increased OR memory of MT1 -/-mice and their WTs when compared to water condition (treatment F1,22 = 21.44, p = 0.0001, no genotype or treatment x genotype interaction) (Figure 1D). Both genotypes performed higher than chance level when treated with melatonin (t > 5.0, df = 11, p < 0.005). The significant NK effect of melatonin was confirmed by the Tuckey HSD test mainly for WT mice (WT p = 0.003; MT1 -/-p = 0.08). There was significant genotype (F1,22 = 8.26, p = 0.009, η 2 = 0.002) and treatment effects (F1,22 = 6.78, p = 0.016, η 2 = 0.49) on OL performances, but no treatment x genotype interaction (Figure 1E). Melatonin barely improved performance in MT1 -/-mice (NK only MT1 -/-vs WT; vs chance level, t = 1.33, df = 11, p= 0.21) whereas performance of WTs improved when compared to water condition (NK only) and to chance level (t = 5.36, df = 11, p= 0.0002). Thus, only OR performance in MT1 -/-mice were modestly improved by melatonin.

MT1 deficiency did not affect objects exploration during acquisition (OR: water WT 23.5 ± 3.6 sec, water MT1 -/-20.7 ± 2.0 sec; OL: water WT 23.3 ± 3.0 sec, water MT1 -/-18.9 ± 2.7 sec; for all comparisons: t < 1.09, df = 22, p > 0.05). The level of anxiety was similar among groups (time in lit box: water WT 114.4 ± 10.4 sec; melatonin WT 117.6 ± 4.7 sec; water MT1 -/-111.3 ± 5.8 sec; melatonin MT1 -/-100.2 ± 7.4 sec, F1,21 < 2.21, p > 0.05, no NK significance).

Effects of melatonin on recognition performance in MT2 -/-mice

MT2 -/-mice did not show the increase in OR performance observed in their WTs under melatonin treatment (treatment F1,23 = 9.03, p = 0.006, η 2 = 0.27; treatment x genotype interaction F1,23 = 7.33, p = 0.012; no genotype effect; vs chance level, WT melatonin: t = 6.85, df = 11, p < 0.0001 and MT2 -/- melatonin: t = 2.18, df = 13, p = 0.05; Figure 1F). The melatonin effect detected in WT (NK) was confirmed by the Tuckey HSD test (WT water vs melatonin: p = 0.005; melatonin WT vs MT2 -/-: P=0.0504; MT2 -/-water vs melatonin: p=0.996). Melatonin improved OL performance to a greater extent in WT mice than in MT2 -/-mice as confirmed by the Tuckey HSD test (water vs melatonin in WT only p = 0.049 and melatonin WT vs MT2 -/-p = 0.04; treatment F1,21 = 5.66, p = 0.027, η 2 = 0.19; genotype F1,21 = 5.08, p = 0.035, η 2 = 0.09; no treatment x genotype interaction; vs chance level, melatonin WT and MT2 -/-: t ≥ 3.3, df = 9,13, p < 0.006) (Figure 1G). Thus, MT2 deficiency affected the beneficial effect of melatonin in the OR task and to a lesser extent in the OL task. MT2 -/-deficiency did not affect object exploration during acquisition trials (OR task: water WT 19.3 ± 1.8 sec, water MT2 -/-18.4 ± 1.4 sec; OL task: water WT 17.0 ± 1.8 sec, water MT2 -/-16.8 ± 1.8 sec; for all comparisons: t < 0.17, df = 21/23, p > 0.05). Anxiety level was similar among groups (time in lit box: water WT: 116.8 ± 11.0 sec; melatonin WT: 116.8 ± 12.5 sec; water MT2 -/-: 117.7 ± 5.7 sec; melatonin MT2 -/-: 110.1 ± 7.7 sec, F1,22 < 0.16, p > 0.05, no NK significance).

DISCUSSION

Our aim was to explore the role of MT1 and MT2 in recognition memory using MT1 -/-and MT2 -/-deficient mouse lines on a B6 background. The key result is that melatonin lost its memory improving effect on OR performance in MT2 -/-mice, and to a lesser extent on OL performance in both MT1 -/-and MT2 -/- mice. These results are in favor of a strong involvement of MT2 in the effects of melatonin on long-term OR, whereas both receptors seem to have a less critical role in OL memory.

Handling, treatments and genetic manipulations can affect memory performance in object-based recognition tasks through changes in anxiety levels or motivation to explore novelty (Mathis, 2019). It is noteworthy that we handled our mice by tunnel to reduce anxiety levels throughout the experiment.

Here, we show that chronic melatonin treatment and melatonin receptor deletion did not affect novel objects exploration and anxiety levels. A lack of effect on locomotion, object exploration and anxiety levels during the light phase has also been reported in other studies using chronic melatonin treatment and other MT1 and/or MT2 deficient mouse lines [START_REF] Comai | Dysfunction of serotonergic activity and emotional responses across the light-dark cycle in mice lacking melatonin MT 2 receptors[END_REF][START_REF] Olcese | Protection against cognitive deficits and markers of neurodegeneration by long-term oral administration of melatonin in a transgenic model of Alzheimer disease[END_REF][START_REF] O'neal-Moffitt | Genetic deletion of MT₁/MT₂ melatonin receptors enhances murine cognitive and motor performance[END_REF], 2015). The lack of effect on object exploration and anxiety levels strongly suggests that the chronic melatonin treatment promoted mechanisms underlying long-term recognition memory.

Although mice had continuous access to melatonin, it was most likely ingested during the night when mice are the most active and feed [START_REF] Possidente | Circadian rhythms for food and water consumption in the mouse, Mus musculus[END_REF]. Given our interest in melatonin receptors as potential targets to treat memory disorders, mice were tested during day light when memory performance must be improved in humans. However, the limit of this method is that we cannot conclude on which stage of information processing benefited from the treatment. Previous studies using single peripheral injection of melatonin (2.5-50 mg/kg) indicate that melatonin can improve both acquisition/consolidation and recall processes [START_REF] Bertaina-Anglade | Memory facilitating effects of agomelatine in the novel object recognition memory paradigm in the rat[END_REF][START_REF] He | A novel melatonin agonist Neu-P11 facilitates memory performance and improves cognitive impairment in a rat model of Alzheimer' disease[END_REF].

The differential involvement MT1 and MT2 could be related to a direct action on different sets of brain structures involved in long-term recognition memory. Long-lasting OR memory requires a brain network which includes hippocampal CA1, medial prefrontal cortex and perirhinal cortex [START_REF] Cohen | The rodent hippocampus is essential for nonspatial object memory[END_REF][START_REF] Tanimizu | Brain networks activated to form object recognition memory[END_REF]. CA1 and prefrontal regions are also critically involved in the formation of long-lasting OL memory, but not perirhinal cortex [START_REF] Barker | Recognition Memory for Objects, Place, and Temporal Order: A Disconnection Analysis of the Role of the Medial Prefrontal Cortex and Perirhinal Cortex[END_REF][START_REF] Tuscher | Chemogenetic inactivation of the dorsal hippocampus and medial prefrontal cortex, individually and concurrently, impairs object recognition and spatial memory consolidation in female mice[END_REF]. MT1 and MT2 are expressed in all these hippocampal and cortical regions, except in perirhinal cortex for MT1 [START_REF] Klosen | MT1 and MT2 melatonin receptors are expressed in nonoverlapping neuronal populations[END_REF][START_REF] Lacoste | Anatomical and cellular localization of melatonin MT1 and MT2 receptors in the adult rat brain[END_REF][START_REF] Musshoff | Melatonin receptors in rat hippocampus: molecular and functional investigations[END_REF]. Thus, brain distribution of the MT2 is coherent with a role in long lasting OL and OR memories. On the other hand, the lack of MT1 in perirhinal cortex may explain negative results in the OR task, whereas its expression in CA1 and prefrontal cortex regions is in accordance with an involvement in long-term OL memory. Alternatively, MT1 and MT2 could have played an indirect effect on memory consolidation through their implication in the regulation of sleep episodes and/or architecture [START_REF] Gobbi | Differential Function of Melatonin MT1 and MT2 Receptors in REM and NREM Sleep[END_REF]). An increase in NREM sleep due to MT2 activation may favor OL memory consolidation [START_REF] Binder | Sleep enhances memory consolidation in the hippocampus-dependent object-place recognition task in rats[END_REF]. On the other hand, sleep fragmentation has a deleterious effect on OR memory consolidation [START_REF] Rolls | Optogenetic disruption of sleep continuity impairs memory consolidation[END_REF]. In depth exploration of these hypotheses would certainly clarify the role of MT1 and MT2 in sleep features that modulate each form of recognition memory.

Our study suggests that MT2 is required for the beneficial effect of melatonin on long-term OR memory, whereas both MT1 and MT2 activation seem to modulate long-term OL memory. These results should encourage further research to elucidate the respective roles of these two receptors in mediating the regulating effect of melatonin on memory. It would be worth combining the use of single receptor deficient mice and selective agonists of each receptor. Understanding the contribution of MT1 and MT2 to recognition memory process is crucial in designing melatonin-based drugs for the treatment of pathologies associated to cognitive disorders such as Alzheimer's disease.

Figure 1 .

 1 Figure 1. Effect of a chronic treatment of melatonin on recognition memories. A) Schema showing the testing schedule used for the whole study. B) Melatonin significantly improved OR performance and C) OL performance in C57BL/6J mice (n=12). Paired t test, melatonin-vs water-treated mice: # p < 0.05, ## p < 0.01, ### p < 0.001. D) Melatonin improved OR performance in WT and MT1 -/-mice

ACKNOWLEDGEMENTS

The authors thank Olivier Bildstein and Georges Egesi for taking care of the mice.

FUNDING SOURCES

This work was funded by an Idex 2018 postdoctoral award of the University of Strasbourg and the Centre National de la Recherche Scientifique (CNRS). These funding sources had no involvement in any aspect of the study design, collection, analysis and interpretation of data, writing of the report and decision to submit the article for publication.

CONTRIBUTORS

Cristiana Pistono: data analyses and and presentation, manuscript writing. Amandine Zimmermann, Chloé Morel, Karine Herbeaux, Céline Héraud and Stéphanie Dumont-Kientzy: technical support and data collection. Marie-Paule Felder-Schmittbuhl: creation and breeding of receptor deficient mice, critical revision of the manuscript. Paul Pevet: conceptualization, critical revision of the Chantal Mathis: conceptualization, funding acquisition, writing review & editing.

DECLARATION OF COMPETING INTEREST

The authors declare that they have no competing financial interests in relation to the work described.

(n=12/group), E) as well as in the OL task for WT, but not MT1 -/-mice (n=12/group). Melatonin improved OR performance in WT mice (n=11), but not in MT2 -/-mice (n=14), D) as well as in the OL task for WT mice (n=10), but not MT2 -/-mice (n=13). Post-hoc NK : melatonin-vs water-treated mice: # p < 0.05, ## p < 0.01, ### p < 0.001. Significances confirmed by the Tukey HSD test are indicated in the text.

HIGHLIGHTS

Melatonin improves performance in long-term object recognition and location tasks MT2 is required for the effect of melatonin on long-term object recognition memory Both MT1 and MT2 activation seems to modulate long-term object location memory


Julie Gueudry
email: julie.gueudry@chu-rouen.fr

François Majo

Agnès Delcampe

Marc Muraine

Panorama of limbal changes

Keywords: corneoscleral limbus, cornea, conjunctiva, limbal epithelium, stem cell, ocular surface tumors

The corneal limbus is a privileged region on the border between two quite different microenvironments, where corneal epithelial stem cells, numerous melanocytes, and antigenpresenting cells are all concentrated within a richly vascularized and innervated stroma. This situation within the ocular surface confers on it the key functions of barrier, epithelial renewal and defense of the cornea. As an immunological crossroads and since the corneoscleral limbus is directly exposed to external insults such as caustic agents, ultraviolet radiation, microbial agents, and allergens, it is the potential site of many tumoral, degenerative or inflammatory pathologies and may progress under certain conditions to limbal stem cell deficiency.

INTRODUCTION

In astronomy, the word "limbus" represents the edge of the disc-shaped image of a star. Its parallel use in ophthalmology is easily understood, since the limbus defines the border between the transparent cornea and the opaque sclera. Its privileged location on the ocular surface confers to it key functions as a barrier, source of epithelial renewal and defense of the cornea. It is thus easy to understand that it is also the site of numerous pathologies, which we will present in this chapter in the form of a panorama, with emphasis on visual recognition.

We refer the reader to various reference articles in which each specific pathology is detailed, particularly in the various treatises of the Medical-Surgical Encyclopedia (EMC) of Ophthalmology.

THE LIMBUS

Anatomy of the limbus

The limbus designates the anatomic border between the transparent, avascular cornea and the white sclera covered with richly vascularized conjunctiva. For surgeons, it corresponds to the macroscopic zone of color change from the sclera to the cornea, which goes from white to blue or gray, over a length of about 1.2 mm before reaching the transparent cornea [START_REF] Van Buskirk | The anatomy of the limbus[END_REF]. This is where the transition between the bulbar conjunctival and corneal epithelium occurs.

Histology and functional anatomy

Limbal epithelium

The limbal epithelium is a transitional epithelium between the conjunctival and cornea epithelium, in other words between two fundamentally different biologic micro-environments -the vascularized, lymphoid conjunctival environment on one side, and the very innervated, avascular corneal environment on the other side. The limbal epithelium is a multi-layered squamous epithelium, non-keratinized and without mucous cells, although they are present throughout the rest of the conjunctival epithelium. It is thicker than the central corneal epithelium and the rest of the conjunctival epithelium with ten to fifteen cell layers.

The underlying stroma contains a dense vascular network and is organized in a series of radial elevations centered about a fibrovascular axis known as the palisades of Vogt. These elevations are oriented radially, perpendicular to the limbus. These palisades form undulations on the limbal surface, especially visible superiorly and inferiorly, more easily seen in more darkly pigmented subjects (Fig. 1). Between the palisades of Vogt, the crypts are home to the corneal stem cells, more deeply embedded [START_REF] Grieve | Three-dimensional structure of the mammalian limbal stem cell niche[END_REF].

Melanocytes are present in the basal layer of the conjunctival epithelium, especially at the limbus. Just as with epidermal melanocytes, they transfer their melanin granules to the epithelial cells [START_REF] Hu | Isolation, purification and cultivation of conjunctival melanocytes[END_REF]. Their primary role is to protect tissues from ultraviolet radiation. More recently, a role has been identified within the "limbal stem cell niche;" through their interaction, they are said to permit maintenance of the stem cell character of certain limbal cells as well as their multipotent character [START_REF] Dziasko | Anatomical Features and Cell-Cell Interactions in the Human Limbal Epithelial Stem Cell Niche[END_REF].

The limbus is a richly vascularized zone. Its arterial supply is achieved through anterior conjunctival arteries which arise from the anterior ciliary arteries, which in turn arise from the ophthalmic artery. These anterior conjunctival arteries head radially toward the cornea within the episcleral tissue and insure vascularization of the limbal conjunctiva via a direct branch, which ends in a terminal meshwork, forming the limbal vascular arcades. From the terminal meshwork, venous return occurs through the corresponding conjunctival veins [START_REF] Mouillon | Anatomie de l'angle iridocornéen[END_REF].

The anterior ciliary nerves, coming from the ophthalmic branch of Willis of the trigeminal, collect sensory information from the limbal conjunctiva. The limbus is the site of passage of the long ciliary nerves, which radiate toward the center of the cornea. There is also vegetative innervation to the limbus, notably parasympathetic [START_REF] Kantelip | Histologie et anatomie de la surface oculaire[END_REF].

Renewal of the corneal epithelium

The corneal epithelium is a tissue with rapid renewal, which occurs, according to the predominant theory, from corneal epithelial stem cells situated within the limbus. The basal epithelial cells migrate continuously from the limbus toward the central cornea like an escalator from the basal layer. The more superficial cells then migrate vertically by "pushing off" from the basal "escalator." This model is the basis of our current understanding of corneal pathologies known as "limbal stem cell deficiency." According to this pathologic model, if there are no more stem cells within the limbus, or if the limbus is destroyed, there are no more epithelial cells left to renew the corneal epithelium, and this is then replaced by the nearest epithelium -the conjunctival epithelium.

LIMBAL PATHOLOGIES

As indicated in the introduction, we have taken the liberty to present here all of the limbal pathologies in the form of a rich panorama of photographs. For each of them, we cite references to specific corresponding chapters in the EMC treatise. Diseases of the trabecular meshwork, which belongs to the anatomic limbus, will not be addressed in this chapter.

Limbal tumors

The limbus as a specific site for carcinogenesis

The presence of epithelial stem cells might in part explain the preferential presence of conjunctival carcinomas within the limbus. In effect, the protective process of desquamation is less active here. On the other hand, rapidly renewing epithelial cells desquamate rapidly and would theoretically be protected against phenomena of carcinogenesis, which may explain the rarity of carcinomas in the central cornea. In addition, the limbus is one of the ocular surface zones most exposed to ultraviolet rays, which constitute and additional risk factor. Finally, one hypothesis suggests that transitional epithelia might be specific sites for the occurrence of carcinomas due to their location between several different microenvironments and their reception of possibly conflicting biologic signals. This phenomenon might induce a deregulation of the cellular machinery, with a secondary increased risk of metaplasia and carcinoma [START_REF] Majo | Physiologie de la conjonctive[END_REF].

Limbal tumors are varied, with various prognoses. The clinical appearance is often insufficient to characterize these various lesions as benign, precancerous or malignant [START_REF] Maschi | Benign conjunctival tumors[END_REF][START_REF] Maschi | Tumeurs conjonctivales malignes EMC -Ophtalmologie[END_REF].

It is thus recommended to be relatively systematic when in the slightest doubt, by performing an excisional biopsy so as to obtain a histologically certain diagnosis and thus be able to appropriately customize management.

The evaluation of 5002 conjunctival tumors by Shields et al, in an American tertiary care center for ophthalmologic oncology, has shown that the limbal location was the most frequent localization, since 2370 lesions were localized to the limbus, i.e. 47.4% of cases. The large number of melanomas and squamous cell cancers in this study is very probably related to the specific recruitment at this referral center (Table 1) [START_REF] Shields | Conjunctival Tumors in 5002 Cases. Comparative Analysis of Benign Versus Malignant Counterparts[END_REF].

Benign lesions

We typically distinguish acquired benign tumors from congenital tumors, in particular dermoids, located mostly at the limbus. Histologically, dermoids are choristomas, defined as congenital proliferations of tissue of normal structure, but in a site where they should not be located. Their presence must trigger an attentive examination looking for a congenital malformation syndrome, in particular Goldenhar syndrome. Surgical excision may be considered in the case of functional, visual or aesthetic compromise (Fig. 2). Acquired tumors may be melanotic (Fig. 3,4), epithelial, some of which are considered precancerous (Fig. 5), or stromal (Fig. 6) (Table 1).

Malignant lesions

In this situation, one must distinguish between melanocytic lesions, epithelial or conjunctival stromal lesions and metastases. These malignant lesions have a tendency to occur in elderly patients; the majority of conjunctival lesions in children, whether limbal or not, are benign [START_REF] Shields | Conjunctival Tumors in 5002 Cases. Comparative Analysis of Benign Versus Malignant Counterparts[END_REF][START_REF] Shields | Clinical Features Differentiating Benign From Malignant Conjunctival Tumors in Children[END_REF]. Primary acquired melanosis is a flat pigmentation of variable extent, most often unilateral, appearing relatively late between 40 and 60 years of age, and with a progressive course. We distinguish primary acquired melanosis without cytologic atypia from primary acquired melanosis with cytologic atypia, which are considered precancerous. All areas of the conjunctiva may be affected, but in the case of limbal localization, the pigmentation is particularly visible and may result in diffusion of pigment into the corneal epithelium. (Fig. 7). Classically, melanomas have no specific conjunctival localization, but their localization at the limbus is not rare (Fig. 8). Malignant epithelial tumors, essentially intra-epithelial (or in situ) or invasive squamous cell tumors are difficult to diagnose clinically, which justifies surgical excision to obtain a confirmed diagnosis (Fig. 9, 10, 11).

Pterygium and pinguecula

Pterygium is a benign conjunctival fibrovascular lesion, spreading progressively onto the cornea, in a potentially disabling fashion [START_REF] Sarda | Ptérygions EMC -Ophtalmologie[END_REF]. Exposure to ultraviolet rays represents the main risk factor. The limbal location is explained by the Coroneo effect, corresponding to the focusing of reflected light rays coming from the temporal region onto the nasal limbus.

Pinguecula is a yellowish degenerative lesion of conjunctival elastic tissue, adjacent to the nasal or temporal limbus, which does not invade the cornea. It has the same pathophysiologic origin as a pterygium (Fig. 12).

Immune and infectious lesions

The limbus is also the site of immune inflammatory and infectious pathologies [START_REF] Borderie | Pathologie immunitaire cornéenne[END_REF]. In effect, the corneal-conjunctival epithelium constitutes the final barrier to the entry of pathogenic agents into the deep ocular structures after the lids and the tear film. The conjunctiva possesses a network of intra-epithelial immunologic cells, in particular the Langerhans cells.

These cells are antigen-presenting cells, which enables them to be recognized and then eliminated by subepithelial immunocompetent cells. In the case of ocular surface inflammation, their density increases considerably [START_REF] Becquet | Immunophénotype des cellules dendritiques de la conjonctive humaine[END_REF]. The conjunctival stroma is extremely rich in immunocompetent cells. Lymphatic drainage proceeds to the parotid, pretragal and submaxillary nodes [START_REF] Mouillon | Anatomie de l'angle iridocornéen[END_REF]. Due to its abundant supply of vessels and lymphatics, the limbus is a veritable immunologic crossroads. It is indeed possible to imagine that the limbus plays a role as guardian of the cornea, which is very poorly defended by the immune system, since such a reaction would be harmful to its integrity and transparency. Thus, in the case of keratitis, limbal inflammation occurs, with dilation of the vessels and migration of inflammatory cells into the extracellular space, which then migrate into the normally avascular cornea.

An auto-amplification of the inflammatory process through the involvement of secreted pro-inflammatory substances favors its extension to the entire ocular surface [START_REF] Majo | Surface oculaire[END_REF].

A limbal location of an ulcer is very suggestive of an inflammatory cause, although this is not a given (Table 2) [START_REF] Soukiasian | Peripheral ulcerative keratitis[END_REF]. Thus, in rosacea or Meibomian gland dysfunction, the infiltrates are probably a type III hypersensitivity reaction (immune complex deposits) and phlyctenular keratoconjunctivitis and type IV hypersensitivity reaction (cell-mediated immunity) to staphylococcal antigens colonizing the lids in these situations (Fig. 13). In the limbal form of vernal keratoconjunctivitis, a limbal gelatinous bead-like appearance is seen, within which one may sometimes see white nodules known as Trantas dots, which represent the accumulation of eosinophils (fig. 14). This form is most frequently encountered in black subjects, thus sometimes known as tropical endemic limbo-conjunctivitis. A limbal bead may also be identified in atopic keratoconjunctivitis [START_REF] Mortemousque | Conjonctivites allergiques[END_REF].

One must be aware that an autoimmune peripheral ulcerative keratitis may be the first manifestation of a systemic disease. Mooren's ulcer, an idiopathic peripheral ulcerative keratitis, is a diagnosis of exclusion which requires other diseases causative of peripheral corneal ulcers to be ruled out (Fig. 15). An underlying systemic disease is found in about one half of cases, and a fourth of predisposing conditions are unknown at the time of ophthalmologic symptoms [START_REF] Tauber | An analysis of therapeutic decision making regarding immunosuppressive chemotherapy for peripheral ulcerative keratitis[END_REF]. Rheumatoid arthritis is the main cause, found in 38%, then, next most frequently, Wegener's granulomatosis or granulomatosis with polyangiitis [START_REF] Sainz De La Maza | Ocular characteristics and disease associations in scleritis-associated peripheral keratopathy[END_REF].

Other systemic conditions associated with peripheral ulcerative keratitis include other ANCA positive vasculitis (Churg-Strauss syndrome and microscopic polyangiitis), polyarteritis nodosa, chronic atrophic polychondritis and systemic lupus erythematosus. Certain ocular surface infections may present in a manner which mimics an autoimmune condition, particularly herpetic limbal epithelial keratitis (Fig. 16). In the case of chronic conjunctivitis and a limbal bead, chlamydia must also be considered.

Limbal stem cell deficiency

Regardless of the theory of epithelial renewal, this can only produce an epithelium with a corneal phenotype (transparent, non-keratinized) if the environment is healthy (quality of the tears and lids, absence of conjunctival inflammation). In the case of a chronic alteration of this micro-environment, the corneal epithelium transforms and takes on the phenotypical appearance of conjunctiva or epithelial metaplasia [START_REF] Kocaba | Limbal stem cell deficiency management. A review[END_REF]. The corneal epithelium is invaded by the conjunctival epithelium or changes phenotype to appear vascularized with the presence of goblet cells. This situation occurs in several pathologic conditions and results in limbal stem cell deficiency (LSCD), which occurs not only by destruction of the limbal cell reserve in a strict sense (Table 3) [START_REF] Majo | Pathologies épithéliales cornéennes et insuffisance en cellules souches limbiques[END_REF]. In the largest series published to date, a retrospective series of 1512 eyes, the most frequent causes are, in decreasing order, chemical or thermal burns (54%), atopic and vernal keratoconjunctivitis (18%), Stevens-Johnson syndrome and toxic epidermal necrolysis (15%), congenital aniridia (5%), and mucous membrane pemphigoid (3%) [START_REF] Vazirani | Limbal Stem Cell Deficiency-Demography and Underlying Causes[END_REF]. Clinically, one of the first signs may be the disappearance of the normal limbal architecture (palisades of Vogt). Then, the corneal surface neovascularizes and opacifies.

This syndrome may be complicated by chronic or recurrent corneal ulcers which may eventually progress to perforation. In the end stage, the corneal surface may keratinize, be invaded by a fibrovascular pannus and develop calcium deposits (Fig. [START_REF] Mortemousque | Conjonctivites allergiques[END_REF][START_REF] Tauber | An analysis of therapeutic decision making regarding immunosuppressive chemotherapy for peripheral ulcerative keratitis[END_REF][START_REF] Sainz De La Maza | Ocular characteristics and disease associations in scleritis-associated peripheral keratopathy[END_REF][START_REF] Kocaba | Limbal stem cell deficiency management. A review[END_REF].

Miscellaneous

Terrien's marginal degeneration

Terrien's marginal degeneration is a rare bilateral peripheral corneal limbal degeneration of unknown cause. It is characterized by a non-inflammatory thinning of the peripheral cornea (Fig. 21).

Corneal Dellen

The corneal Dellen is characterized by a corneal thinning typically at the limbus, in association with an elevation, most often conjunctival, causing a reduction in the spreading of the tear film at its base, which results in stromal dehydration and thus visible thinning of the limbus or peripheral cornea (Fig. 22).

CONCLUSION

The anatomic and physiologic configuration of the corneoscleral limbus explains the specificity and rich panorama of pathologies which may affect it. A more precise knowledge of the phenomena of cellular regeneration and the various intercellular interactions or those with the extracellular matrix may be able to improve medical and surgical management of these apparently definitive changes in the limbal epithelium [START_REF] Gueudry | Panorama des altérations limbiques cornéennes[END_REF].

Disclosure of interest

The authors have no conflicts of interest to disclose in relation to this article.

Further reading

See this article, unabridged, illustrated and detailed, with electronic enhancements, in EMC- Figure 11. Appearance of an invasive squamous cell carcinoma of the limbus. On histology, the edges of the tumor have crossed the epithelial basement membrane with a resultant risk of distant metastasis, which is not the case for carcinoma in situ.

  Ophtalmologie : Gueudry J, Majo F, Delcampe A, Muraine M. Panorama des altérations limbiques cornéennes. EMC -Ophtalmologie 2018;15(4):1-13 [Article 21-200-C-20].

Figure 1 .

 1 Figure 1. Palisades of Vogt superiorly (a), better seen in black patients due to the

Figure 2 .

 2 Figure 2. Limbal dermoids (a, b).

Figure 3 .

 3 Figure 3. Conjunctival nevi are the most frequent tumors of the ocular surface. They are

Figure 4 .

 4 Figure 4. Ethnic melanosis. This is a flat, bilateral pigmentation predominantly at the limbus

Figure 5 .

 5 Figure 5. Actinic keratosis is an epithelial lesion considered precancerous and having a

Figure 6 .

 6 Figure 6. Corneal xanthoma is a deposition of cholesterol-rich material and appears as a

Figure 7 .

 7 Figure 7. Primary acquired melanoses with cytologic atypia (a, b). Primary acquired

Figure 8 .

 8 Figure 8. Invasive limbal conjunctival melanomas of variable pigmentation (a,b).

Figure 9 .

 9 Figure 9. Squamous cell carcinoma in situ with gelatinous invasion of the cornea (a,b,c,d).

Figure 10 .

 10 Figure 10. Squamous cell carcinoma in situ in its papillomatous form (a). Squamous cell

Figure 12 .

 12 Figure 12. Characteristic triangular appearance of pterygium. Note the limbal location of

Figure 13 .

 13 Figure 13. Rosacea and Meibomian gland dysfunction. Catarrhal infiltrates (a). Phlyctenular

Figure 14 .

 14 Figure 14. Vernal keratoconjunctivitis. Limbal bead (a). Trantas dots (b).

Figure 15 .

 15 Figure 15. Peripheral ulcerative keratitis. Perforated Mooren's ulcer (a). Peripheral

Figure 16 .

 16 Figure 16. Peripheral or limbal epithelial herpetic keratitis. Diagnostic delay in this form is

Figure 17 .

 17 Figure 17. Syndrome of limbal stem cell deficiency and aniridia keratopathy (a). Appearance

Figure 18 .

 18 Figure 18. Syndrome of limbal stem cell deficiency and chemical burn of the ocular surface.

Figure 19 .

 19 Figure 19. Syndrome of limbal stem cell deficiency and Stevens-Johnson syndrome and toxic

Figure 20 .

 20 Figure 20. Syndrome of limbal stem cell deficiency and mucous membrane pemphigoid.

Figure 21 .

 21 Figure 21. Terrien's marginal degeneration. Peripheral corneal thinning and lipid deposits,

Figure 22 .

 22 Figure 22. Corneal Dellen caused by an adjacent limbal lesion.

Table 2 . Etiologies of peripheral ulcerative keratitis according to [16] Non-infectious ocular causes

 2

	• Idiopathic or Mooren's ulcer
	• Ocular rosacea
	• Exposure, post-traumatic, toxic keratopathy
	Local infectious causes • Bacterial (Staphylococcus, Streptococcus, Gonococcus)
	• Viral (Herpes simplex or varicella-zoster virus)
	• Parasitic (Acanthamoeba)
	• Fungal
	General non-infectious causes
	Connective tissue/vascular disease
	• Rheumatoid arthritis (frequent)
	• Granulomatosis with polyangiitis (Wegener's Granulomatosis)
	• Periarteritis nodosa
	• Microscopic polyangiitis
	• Churg-Strauss syndrome
	• Chronic atrophy polychondritis
	• Systemic lupus
	• Scleroderma
	• Giant cell arteritis (very rare)
	Other autoimmune diseases
	• Chronic inflammatory bowel disease
	• Cicatricial pemphigoid
	• Sjögren's syndrome
	• Leukemia, sarcoidosis (very rare)

Table 1 . The 12 most frequent limbal tumors according to [10]

 1

	Total number (n) of	Limbal
	patients	localization

Table 3 . Possible etiologies of limbal stem cell deficiency (LSCD) according to [21] LSCD secondary to chemical, physical or mechanical destruction

 3

	• Chemical or thermal burns
	• Radiotherapy
	• Surgical trauma: history of surgery (peritomy) or cryotherapy involving the limbus,
	surgical excision of limbal neoplasm
	• Mechanical trauma: trichiasis, prolonged contact lens wear
	LSCD of infectious origin
	• Severe bacterial keratitis, trachoma, herpes, mycoses
	LSCD of inflammatory origin
	• Stevens-Johnson syndrome and toxic epidermal necrolysis Mucous membrane
	pemphigoid Atopic keratoconjunctivitis, ocular rosacea, Sjögren's syndrome
	LSDC of genetic origin
	• Congenital aniridia
	• Hereditary epidermolysis bullosa
	• Congenital erythrokeratodermia
	• Hereditary multiple endocrinopathy
	LSCD and surface dysplasia or dystrophies
	• Pterygium
	• Extensive limbal neoplasms
	Iatrogenic LSCD
	• Antimitotic agents
	LSCD by destruction of a component of the corneal matrix
	• Neurotrophic keratitis (herpes, diabetes)
	Idiopathic LSCD