Jacques Kaminski

Pierre-Olivier Delpech

Sihem Kaaki-Hosni

Xavier Promeyrat

Thierry Hauet

Patrick Hannaert
email: patrick.hannaert@univ-poitiers.fr

Oxygen consumption by WI-injured porcine kidneys in hypothermic static and machine preservation

Static cold storage (SCS) and hypothermic machine perfusion (HMP) are currently standard methods for renal grafts clinical preservation. Both methods are predominantly implemented without the active delivery of oxygen, even for DCD-like kidneys. However, even under severe hypothermia (4-6°C), kidneys can consume oxygen and produce ATP. What is not established, though, is to what extent and how SCS and HMP compare in terms of oxygen. Using a porcine preclinical model of renal warm ischemia (WI) to compare SCS and HMP methods, we continuously monitored and quantified oxygen level and consumption along preservation; we also determined pre and post-preservation cortical ATP level; values given as median and [min;max] range. One hour WI reduced ATP by ∼90% (from 3.3 [1.7;4.5] mmol/L tissue in Controls). Oxygen consumption (QO2, µmol/min per 100g) was determined from initial solution PO2 decrease (SCS and HMP), and from arterio-venous difference (HMP). In SCS and HMP, PO2 decreased rapidly (t1/2 ∼1h), from atmospheric levels to 52.9 [38.0;65.9] and 8.2 [3.0,16.0] mmHg, respectively.

In HMP, QO2 was 2.7 [0.4;3.9] versus 0.5 [0.0;1.3] in SCS (p < 0.05); post-preservation ATP amounted to 5.8 [3.2;6.5] in HMP versus 0.1 [0.0;0.2] in SCS. Despite hypothermic conditions in SCS or HMP, DCD-like renal grafts require oxygen. Increased oxygen consumption, restored ATP level, and improved histological profile in HMP might explain the established HMP superiority over SCS. These results establish a rational basis for the use of oxygen in hypothermic preservation. Optimal levels required for preservation and graft type variants remain to be determined.

Running title

Oxygen use by suboptimal renal grafts in cold preservation

Key words

Introduction

Despite the progressive emergence of normothermic preservation and reconditioning methods, such as ex vivo NMP (normothermic machine perfusion) and in situ ECMO (extra-corporeal membrane oxygenation), hypothermic preservation remains the dominant strategy in renal transplantation. Hypothermic preservation is implemented either as static cold storage (SCS) or as hypothermic machine perfusion (HMP), the later being particularly efficient for higher-risk donors [START_REF] Jochmans | Kidney perfusion: Some like it hot others prefer to keep it cool[END_REF][START_REF] Golbo | Temperature and oxygenation during organ preservation: Friends or foes?[END_REF][START_REF] Jochmans | Past, Present, and Future of Dynamic Kidney and Liver Preservation and Resuscitation[END_REF] . Biochemical processes, including ATP-producing oxidative phosphorylation and glycolysis, are slowed by low temperature:

for instance, at 7°C, cellular reactions occur at a residual ∼12% of normothermic rates [START_REF] Folkert | Principles of solid-organ preservation by cold storage[END_REF] .

However, under hypothermic conditions, even at temperatures as low as 4-6°C, residual oxygen-dependent (and oxygen-independent) metabolic activities can be significant, oxygen has indeed been shown to be consumed, and ATP can be produced, at least under perfusion [START_REF] Stubenitsky | Regeneration of ATP in kidney slices after warm ischemia and hypothermic preservation[END_REF][START_REF] Weegman | Continuous real-time viability assessment of kidneys based on oxygen consumption[END_REF] . Accordingly, the addition of active oxygenation (100%) to HMP preservation improves post-transplantation outcomes, in preclinical models and in clinical situations, as reviewed [START_REF] O'callaghan | Supplemental oxygen during hypothermic kidney preservation: A systematic review[END_REF] . Using a porcine model of WI-injured renal grafts, our laboratory showed that, as compared to passive oxygenation, active oxygenation (100%) during HMP preservation significantly improved early functional and inflammatory scores, as well as chronic functional and fibrosis scores [START_REF] Thuillier | Benefits of active oxygenation during hypothermic machine perfusion of kidneys in a preclinical model of deceased after cardiac death donors[END_REF] . However, as in most other studies, oxygen levels were not measured, and one must surmise that, in the "passive" arm for instance, oxygen level of the preservation solution started at atmospheric levels (21%).

In SCS preservation, on the other hand, to our knowledge, no dedicated study evaluated the addition of active oxygen, nor specifically determined oxygen consumption [START_REF] Golbo | Temperature and oxygenation during organ preservation: Friends or foes?[END_REF][START_REF] O'callaghan | Supplemental oxygen during hypothermic kidney preservation: A systematic review[END_REF][START_REF] Hosgood | Oxygenated kidney preservation techniques[END_REF] . Of note though, one study from our laboratory showed that the addition of an oxygen carrier (marine extracellular hemoglobin, M101) during SCS preservation significantly improved functional and histological post-transplantation outcomes, with a notable reduction of fibrosis, in a Pig model of autotransplantation of "normal", uninjured kidneys (WI time < 30min, SCS 24h, one month follow-up) [START_REF] Thuillier | Supplementation with a new therapeutic oxygen carrier reduces chronic fibrosis and organ dysfunction in kidney static preservation[END_REF] . This strongly suggests that the provision of additional oxygen can be beneficial to renal grafts, even under SCS conditions.

In current clinical settings, all SCS and most HMP methods share the common feature of being non-actively oxygenated, i.e. relying on whatever oxygen is present, usually atmospheric oxygen (∼158 mmHg); this is the case of the LifePort Kidney Transporter (ORS®). Exceptions which provide for active oxygenation allow for supplementation with either 100% oxygen (760 mmHg, Kidney Assist-Transport from Organ Assist® and IGL® Waves) or atmospheric oxygen ( IGL Waves).

Few mechanisms can be invoked to explain the benefits of active oxygenation (or oxygen carriers) in SCS or HMP. Obviously, the intrinsic ability of oxygen to sustain mitochondrial ATP production, represents a key player by maintaining cellular structures and repair processes. An increased ATP level during organ preservation is expected to lead to better outcomes and reduced risks of organ dysfunction after transplantation, in animals and human studies [START_REF] Hosgood | Oxygenated kidney preservation techniques[END_REF][START_REF] Ravaioli | Strategies to restore adenosine triphosphate (ATP) level after more than 20 hours of cold ischemia time in human marginal kidney grafts[END_REF] . However, both in SCS and HMP preservation protocols, and even in preclinical studies, actual levels of renal ATP remain largely undetermined -much like oxygen. The determination of renal graft oxygen levels and consumption, together with tissue ATP levels, should translate into a better understanding of the graft status and its requirements for optimal preservation. Under this rationale, comparing SCS and HMP, the two current, non-actively oxygenated, preservation standards, we detrmined i) oxygen levels simultaneously in the preservation solution and within the kidney, and ii) ATP levels before and after WI, and after SCS and HMP preservation.

Because the current trend in transplantation is to further extend the donor acceptance criteria, leading to lower quality graft, we performed this experimental study with kidneys pre-injured by one-hour warm ischemia, mimicking DCD grafts (donation after circulatory death) [START_REF] Thuillier | Benefits of active oxygenation during hypothermic machine perfusion of kidneys in a preclinical model of deceased after cardiac death donors[END_REF] .

Methods

Animals and ethics.

Large White male pigs (3 months) were bred at the IBiSA MOPICT platform (INRA, Surgères, Fr), where organ procurement and euthanasia were carried out. All surgical and experimental protocols were performed in accordance with the guidelines of the French Ministry of Agriculture (FMA) for the use and care of laboratories animals, after approval by the FMA, the INRA, and the Poitou-Charentes ethical commitee for animal experimentation.

Surgery and warm ischemia.

Surgery has been described in detail [START_REF] Thuillier | Benefits of active oxygenation during hypothermic machine perfusion of kidneys in a preclinical model of deceased after cardiac death donors[END_REF][START_REF] Favreau | Anti-thrombin therapy during warm ischemia and cold preservation prevents chronic kidney graft fibrosis in a DCD model[END_REF][START_REF] Allain | Development of a preclinical model of donation after circulatory determination of death for translational application[END_REF][START_REF] Tillet | Kidney graft outcome using an anti-Xa therapeutic strategy in an experimental model of severe ischaemia-reperfusion injury[END_REF][START_REF] Mallet | Dose-ranging study of the performance of the natural oxygen transporter HEMO2life in organ preservation[END_REF] . Briefly, after general anesthesia, midline abdominal incision, and pedicle clamping, the right kidney was removed by dissection of the vascular pedicle and ureter and transferred into a sterile bag for 60 min warm ischemia WI, and maintained at 37°C in a water bath. For oxygen monitoring during warm ischemia, pedicle-clamped, n = 6 kidneys were used (see figure 1): they were kept in normothermia via a thermostated bath filled with saline, under continuous temperature monitoring (solution and renal cortex). After WI, the kidney was gravimetrically (1 m height) flushed with 500 mL ice-cold UW® for SCS or KPS® for HMP; all flushings occurred in <10 min and yielded whitened kidneys. Figure 1 summarizes the protocol and the use of kidneys. 10 Hypothermic preservation -Oxygen and temperature measurements.

For SCS (20h), the kidney was placed in 1 L ice-cold UW® (Bridge to Life®, London, UK), and placed itself into a styrofoam box, filled with ice. For HMP (20h) the kidney was connected to a LifePort ® machine, filled with ice (Lifeport Kidney Transporter 1.0, ORS®, Brussels, Be), set at 35 mmHg pressure.

Cortical temperature was monitored, via 200 µm-glass thermocouple microsensor (TP200, Unisens®), inserted 3-5 mm below the capsule (signal was fed to a Unisens® MicroSensor multimeter). For oxygen monitoring in the HMP perfusate, fiber optic oxygen microsensors (IMP-Pst1, Presens®) were adapted into the perfusion circuit, at the arterial inlet and venous outlets. Cortical O2 (SCS and HMP) was monitored with Needle-Type optode Oxygen Microsensor (NTH-PSt1, Presens®), introduced 3-5 mm final under the capsule. Fiber optic oxygen electrodes were connected to a PreSens® oxymeter OXY-4 Mini (4 min -1 sampling). Oxygen signal were converted into pressure and concentration with an Excel® sheet (PreSens®). For O2-sensors, two-points calibration was performed for each experiment (100% N2 and 100% air-saturated saline).

Renal tissue ATP

The ATP content from renal cortical samples, immediately frozen in liquid nitrogen, was measured with the ATP Bioluminescent Assay kit (Sigma®), with 40-70 mg tissue samples. The final ATP concentration is expressed as mmol/L of tissue (assuming a tissue density of 1 g/mL). An additional group of n = 5 kidneys was used for control ATP (i.e. ATP before warm ischemia; see figure 1).

Histological analysis.

For histological observations (control, end-CS, end-HMP, end-WI) and for control, 0.5x0.5x1.0 cm 3 tissue samples (cortex and medulla) were cut and immersed into 4% buffered formalin (VWR ® ) for fixation. Fixed samples were then colored with HES/PAS (Hematoxylin-Eosin-Safran/Periodic Acid Schiff) for expert examination and semiquantitative analysis for i) brush-border loss and endoluminal detachment, with the following scoring scale: 0 = no lesion; 1, <10% area; 2, 11-25% area; 3, 26-50 % area; 4, 51-75% area; 5, >76% area), and ii) vacuolization and tissue infiltration (number of features per optical field). For both stainings and for each parameter, five slides were read, with six different fields for each (n = 30).

Data and statistics.

Arterio-venous oxygen consumption in HMP was calculated as the product of the perfusion rate with the oxygen arterio-venous difference (expressed in µmoles/min per 100 g kidney weight). Oxygen consumption from solution concentration was calculated from oxygen decrease between consecutive time-points, under initial rate conditions (10-30 min). Data are presented as median with range [min, max] of n = 5 values, unless otherwise stated (e.g.for cortical temperature mean ± SD).

Statistical analysis of oxygen time-course was performed as repeated measures

ANOVA (NCSS® v0.7.1.21). For ATP values and histological scoring, statistics were performed as non-parametric one-way ANOVA, with Tukey post-test (Graphpad Prism® v5.01). A p value < 0.05 was considered significant. During HMP, average cortical temperature was 8.2 ± 1.0 °C, dropping from 13.2 [11.1;14.4] °C to 7.8 [7.7;7.9] °C (n = 5), with an apparent half-time for stabilization of ∼5 h to 6.9 ± 0.9 °C. In the cortex, the oxygen pressure started from 0.3-0.4 mmHg (between 1 and 30 min), close to the ischemic cortical PO2 (see figure 2).

Results

Renal oxygen during warm ischemia

Renal oxygenation parameters during SCS.

Since PO2 markedly decreased in the solution, it was possible to calculate (pointto-point) the instantaneous rate of oxygen concentration decrease, i.e. oxygen consumption, QO2. The initial rate of oxygen consumption was extracted from the linear phase of PO2 decrease (10 -30 min time range; see figure 3, main panel), i.e. as the average of the 10-20 and 20-30 minutes intervals: initial QO2 was 0.6 [0.5;0.7] µmol/min per 100g (n = 4 kidneys, one outlier excluded according to Grubbs' test, GraphPad online). Solution-based QO2 is plotted in the inset of figure 2, in the 0 to 400 min range. In the cortex, oxygen levels started at 10.2 [0;18.9] mmHg (first time-point, 5min)and dropped (F = 4.79, p < 0.05) to effectively zero afterwards (0.0-0.3 mmHg) within 60 min.

Renal oxygenation parameters during HMP.

In addition, machine perfusion allows for the determination of the "arterio-venous oxygen consumption", based arterial and venous oxygen concentrations (as reported by respective O2 micro-sensors, see Methods) and on perfusion rate (as reported by the HMP device). In the LifePort machine, the renal perfusion rate rapidly increased from 0 to 40 mL/min (in less than 10 min), to slowly stabilize at ∼50 mL/min after 600 min; the 20haverage perfusion rate was 45.5 ± 4.5 mL/min (mean ± SEM SD, n = 20), corresponding to a renal resistance of ∼0.7 mmHg.min/mL. The main panel of figure 5 ATP level of renal cortical tissue. Figure 6 summarizes ATP results. Renal cortical ATP in the control (non-ischemic) group was 3.3 [1.7;4.6] mmol/L of tissue. After 60 minutes of warm ischemia, the ATP level was 0.3 [0.2;0.6] mmol/L (p < 0.01 vs control). After 20 hours of SCS, the ATP content of the kidneys was 0.06 [0.04;0.09] mmol/L (NS vs WI), whereas it was 5.8 [3.2;6.5] mmol/L in HMP (p < 0.01 vs WI).

Histological analysis.

Figure 7a shows representative HES and PAS-colorations of cortical and medullary tissue sections of the four experimental groups. Figure 7b summarizes the corresponding histological scores. It can be seen that WI60 induced significant histological injuries versus control (brush border loss, endoluminal detachment, vacuolization and infiltration). All four parameters were significantly improved by HMP versus SCS, although not normalized to control (except for tissue infiltration). Finally, brush border loss was potentiated by SCS versus WI60 (by about two-fold), and HMP did not restore control level.

Discussion

Oxygen supplementation during hypothermic preservation is the focus of preclinical and clinical studies [START_REF] O'callaghan | Supplemental oxygen during hypothermic kidney preservation: A systematic review[END_REF] , but, paradoxically oxygenation parameters are often ignored and left unquantified (i.e. oxygen level and/or consumption). In non-actively oxygenated hypothermic methods (where either static solution is enclosed in a sealed recipient, or perfusate solution is recirculated without possible or significant re-equilibration with atmospheric oxygen), oxygen-related parameters are usually not measured, e.g. because graft requirements are assumed to be minimal.

On the other hand, we reported, with our porcine model of renal autotransplantation that i) the addition of an hemoglobin-based oxygen carrier to SCS preservation does improve post-transplant outcomes [START_REF] Thuillier | Supplementation with a new therapeutic oxygen carrier reduces chronic fibrosis and organ dysfunction in kidney static preservation[END_REF][START_REF] Mallet | Dose-ranging study of the performance of the natural oxygen transporter HEMO2life in organ preservation[END_REF] , and ii) active oxygenation (100% O2) in HMP improves post-transplantation function recovery of WI-injured kidneys [START_REF] Thuillier | Benefits of active oxygenation during hypothermic machine perfusion of kidneys in a preclinical model of deceased after cardiac death donors[END_REF] . In other words, as reviewed [START_REF] O'callaghan | Supplemental oxygen during hypothermic kidney preservation: A systematic review[END_REF] , renal grafts can draw benefit from oxygen under hypothermic preservation.

On the other hand, due to the expanding use of suboptimal grafts, HMP preservation has now grown more frequent than the simpler and cheaper SCS preservation, at least in european and north-american countries. But, as for SCS, oxygen-related parameters are most generally 'ignored'. Some rare preclinical studies addressed oxygen issues, though.

For instance, one porcine study promoted the determination of oxygen consumption for viability assessment [START_REF] Weegman | Continuous real-time viability assessment of kidneys based on oxygen consumption[END_REF] . At least in the cortex, oxygen can be considered as a proxy for ATP, the cell energetic currency. However, because of technical and logistical easons, ATP is even more difficult to monitor. Accordingly, very few studies, mostly preclinical, did measure ATP [START_REF] Buchs | DCD Pigs' Kidneys Analyzed by MRI to Assess Ex Vivo Their Viability[END_REF][START_REF] Minor | Hypothermic reconditioning by gaseous oxygen persufflation after cold storage of porcine kidneys[END_REF] .

Thus, the monitoring of renal graft oxygen levels and consumption, together with tissue ATP levels, will help understand graft status and requirements for better, if not optimal, preservation. To address the current clinical context, we used renal grafts injured by 60 minutes warm ischemia (WI) 18,[START_REF] Delpech | Inhibition of complement improves graft outcome in a pig model of kidney autotransplantation[END_REF] , UW for SCS, and KPS and the LifePort ® system for HMP.

Oxygen is consumed and limiting in non-oxygenated SCS and HMP

In SCS and HMP, PO2 of the preservation solution decreased rapidly, from atmospheric level, then stabilized. Decreases of perfusion PO2 in HMP were reported in uninjured (WI < 10min) porcine kidneys [START_REF] Weegman | Continuous real-time viability assessment of kidneys based on oxygen consumption[END_REF] and (45min WI-injured) canine kidneys [START_REF] Lindell | Hypothermic Machine Perfusion Preservation of the DCD Kidney: Machine Effects[END_REF] . However, in the first case data are not presented ('...PO2 of the perfusate approached zero within minutes..') [START_REF] Weegman | Continuous real-time viability assessment of kidneys based on oxygen consumption[END_REF] , while in the second, only initial and final PO2 value are reported (from 170 mmHg at zero time to 25 mmHg after 24h HMP, LifePort) [START_REF] Lindell | Hypothermic Machine Perfusion Preservation of the DCD Kidney: Machine Effects[END_REF] . To our knowledge, oxygen level has not been reported in SCS.

SCS exploratory experiments confirmed that solution PO2 was stable for 24h (150 mmHg), whereas it decreased and stabilized with a WI-injured kidney (not shown). We conclude that, in hypothermia, injured kidneys do consume oxygen, despite the low temperature and the static condition (no perfusion and no transport-related consumption).

Of note, the SCS and the HMP systems used in the presenet study were open to atmosphere (electrodes, holding arm), which suggests that in clinics oxygen becomes limiting even faster than observed here (< 60-90min). Finally, our conclusion that oxygen is limiting in non-oxygenated SCS and HMP agrees with reports that a short, postischemic/pre-transplantation period of active graft oxygenation (1-2h, PO2 > 500 mmHg) improves kidney function recovery [START_REF] Hoyer | Influence of oxygen concentration during hypothermic machine perfusion on porcine kidneys from donation after circulatory death[END_REF][START_REF] Kron | Short, cool, and well oxygenated -HOPE for kidney transplantation in a rodent model[END_REF] . This was also shown in HMP or retrograde persufflation of the liver [START_REF] Minor | Role of temperature in reconditioning and evaluation of cold preserved kidney and liver grafts[END_REF] .

In SCS, solution-based oxygen consumption was 0.6 µmol/min per 100g, whereas in HMP, it was 3.4 µmol/min per 100g, the later being not significantly different from the arterio-venous calculation in HMP (av-QO2 3.7 µmol/min per 100g). Of note, in HMP, the initial decrease of cortical oxygen (from 10-15 to 0 mmHg), occurred simultaneously with the initial decrease in the solution.

To our knowledge, QO2 has not been reported in SCS, likely because this was considered irrelevant. Conversely, in HMP QO2 of Pig kidneys has been reported to amount to ∼1,6 µmol/min per 100g (WI < 10min) [START_REF] Weegman | Continuous real-time viability assessment of kidneys based on oxygen consumption[END_REF] , a value close to the present one (3.7 µmol.min per 100g, WI = 60min). Starting from a total physiological oxygen consumption (transport-related + basal ∼200 µmol.min per kidney [START_REF] Juillard | Validation of renal oxidative metabolism measurement by positronemission tomography[END_REF] ), and applying a 20% basal fraction, one obtains ∼40 µmol/min per 100g for non-transport related costs. After temperature-dependent reduction, hypothermic QO2 would amount to ∼5 µmol/min per 100g, very close to and, supporting our determinations. This further supports the notion that, in hypothermic preservation, normal or injured kidneys do consume oxygen. Finally, it also suggests that warm ischemia may not modify basal consumption, although this remains to be established.

In the preservation solution, the low oxygen steady-state indicates that the transfer capacity, via the solution and the capsule, to the cortical parenchyme is limited (and limiting) with respect to intrarenal consumption. This observation is supported by exploratory experiments from our group, showing that solution PO2 not only decreases with time, but that this decrease is more pronounced if electrodes are positioned "close to" versus "far from" the kidney: for instance, between 10 and 15 h the 'close' electrodes (2-3 cm) reported 6-34 mmHg PO2 lower than the distant'electrodes (5-7 cm; n = 2 healthy kidneys and n = 2 WI-injured kidneys). This is also in line with the report of a kidney 'surface PO2' of 5-10 mmHg in the SCS solution, versus 100-150 mmHg in HMP [START_REF] Minor | Kidney transplantation from non-heart-beating donors after oxygenated low-flow machine perfusion preservation with histidinetryptophan -ketoglutarate solution[END_REF] . Furthermore, we suggest that the significantly higher steady-state values of solution oxygen in SCS (∼52 mmHg) versus HMP (∼8 mmHg) indicates that HMP kidneys consume more oxygen than SCS kidneys. Indeed, at 35 mmHg perfusion pressure, filtration still occurs, proportional to renal flow (since perfusion pressure is below the autoregulatory range), roughly amounting to ∼30% of normal filtration. This is comforted by published reports of urine production during HMP preservation(∼0.1mL/min per 100g 26 ) and our unpublished observations. A crude estimation indicates that under our HMP conditions, oxygen consumption should range 6-7 µmol/min per 100g, relatively close to our 3-4 µmol/min per 100g measurement (see above).

ATP level and histological evaluation in HMP and SCS Beneficial effects of oxygen supplementation in hypothermic preservation are acknowledged, i) in HMP and for high-risk kidneys, and ii) as involving some restoration of ATP [START_REF] Taylor | Current State of Hypothermic Machine Perfusion Preservation of Organs : The Clinical Perspective[END_REF] . More surprisingly, even under SCS conditions, direct (100 % O2 [START_REF] Maathuis | Improved kidney graft function after preservation using a novel hypothermic machine perfusion device[END_REF] ) or indirect (M101 carrier [START_REF] Thuillier | Supplementation with a new therapeutic oxygen carrier reduces chronic fibrosis and organ dysfunction in kidney static preservation[END_REF] ) oxygen supplementation is able to provide improvements as well.

As opposed to the renal medulla, cortical ATP synthesis heavily relies on oxygen and mitochondrial phosphorylation. Normal porcine cortical tissue PO2 is ∼45 mmHg [START_REF] Warner | Regional decreases in renal oxygenation during graded acute renal arterial stenosis: a case for renal ischemia[END_REF] .

Here, shortly after the start of the WI period (< 5 min), cortical oxygen barely exceeded electrode sensitivity (0.4-0.8 mmHg), even though the solution was in contact with atmosphere. Accordingly, end-ischemic ATP was strongly reduced (nb: control ATP, was similar to other reports [START_REF] Minor | Kidney transplantation from non-heart-beating donors after oxygenated low-flow machine perfusion preservation with histidinetryptophan -ketoglutarate solution[END_REF] ). ATP levels (after SCS) of "marginal" kidneys (WI 40min) have been reported to amount to ∼0.16 µmol/g tissue (or ∼0.2 mM), in the Pig [START_REF] Minor | Role of temperature in reconditioning and evaluation of cold preserved kidney and liver grafts[END_REF] ; this represents a 80-90% reduction versus control ATP, as we observed here.

After SCS of WI-injured kidneys, ATP remained very low, indicating that these grafts were unable to resynthetize ATP, whereas ATP reached back control level after HMP. These results suggest that ATP was beeing produced during HMP preservation, in excess to consumption. Few studies addressed the ATP production-consumption balance in hypothermic conditions. A careful bibliographical analysis evidenced a patent lack of information about ATP levels in renal graft preservation. Most generally, neither absolute ATP levels nor healthy control value are given (see Bagul and coworkers for a porcine model [START_REF] Bagul | Experimental renal preservation by normothermic resuscitation perfusion with autologous blood[END_REF] , and Ravaioli et al., for human discarded kidneys [START_REF] Ravaioli | Strategies to restore adenosine triphosphate (ATP) level after more than 20 hours of cold ischemia time in human marginal kidney grafts[END_REF] ). Nevertheless, supporting our present observations, two porcine studies, performed on did show marked benefits of HMP versus SCS in terms of graft ATP levels. First, ATP resynthesis was observed in oxygenated HMP but not in SCS, in a WI-injured graft model [START_REF] Lazeyras | Detection of ATP by 'in line'31P magnetic resonance spectroscopy during oxygenated hypothermic pulsatile perfusion of pigs' kidneys[END_REF] . Second, a 2.5-fold ATP post-HMP increase was reported versus post-SCS in a WI-injured kidneys (≤ 30min) model [START_REF] Manna | An in vivo autotransplant model of renal preservation: Cold storage versus machine perfusion in the prevention of ischemia/reperfusion injury[END_REF] . In HMP, the "steady-state" solution oxygen was lower than in SCS, whereas, end-preservation ATP was higher in HMP.

Finally, considering tissue integrity outcomes, by showing less injured tissues, histological analysis confirmed HMP superiority over SCS, in agreement with the increased oxygen consumption and higher levels of ATP. It is important to note though that the ATP 'restoration' may not be the sole factor involved in renal tissue "repair" during hypothermic preservation since SCS itself was able to partially improve the histological profile.

Conclusion

Grafts under SCS or HMP preservation are rarely biopsied, while three-months and oneyear post-transplantation biopsies are common in clinical practice, allowing for histological analysis and grading. Ex vivo quality assessment of renal grafts is limited to visual inspection, before and after flushing (color, patchiness), and to renal resistance in HMP. Oxygen monitoring requires a dedicated setup, and no current perfusion system is equipped with oxygen sensors [START_REF] O'callaghan | Supplemental oxygen during hypothermic kidney preservation: A systematic review[END_REF] . Even when actively oxygenated, no available system allows for oxygen measurements. Thus, oxygen and ATP are seldom measured, much less comparatively in SCS and HMP. Oxygen measurement (and consumption calculation) one the one hand, and high-resolution NMR spectroscopy or electrode-based determinations of tissue ATP, as alternatives, on the other hand, would constitute significant improvements, widening the currently restricted panel of clinical evaluation criteria with physiologically relevant and quantitative indexes 33 .

Conflict of interest statement

No conflict of interest, financial or otherwise are declared by all authors. Average temperature (20h) was 2.9 ± 2.0 °C (mean ± SD, n = 1,200 values, see Results for details); it was 15-8 °C during the first 60 min (corresponding to a temperaturedependent 5-8 fold reduction of reaction rates, assuming Q10 = 2, see [START_REF] Willford | [END_REF] ). The inset shows the oxygen consumption (based on initial decrease of oxygen in the solution), plotted between 0-420 min (0-7h); initial rate was in the 0.5-0.7 µmol/min per 100g. per group). Control was measured before warm ischemia, while end-ischemic ATP was measured at the end of the 60 minutes period of warm ischemia (End-WI). End-SCS and End-HMP, correspond to renal cortical ATP of kidneys submitted to 60 min WI followed by 20 hours SCS and 20 hours HMP preservation, respectively. ATP is given as mmol/L of tissue (conservatively equivalent to µmoles ATP per g wet tissue).

Figure and legends to figures

Legends to figure 7a and 7b

  Abbreviations:

  Overall, during warm ischemia (WI, 60 min), average cortical temperature was 36.0 ± 0.6 °C (mean ± SD, n = 60 time-points): starting from 32.6[29.1;35.1] °C at zero time (n = 6), cortical temperature equilibrated at values between 36 and 37 °C after 10 min; the final WI time-point (60 min) was 36.3 [35.8;36.6] °C (equilibration reache after 5-7min).Oxygen levels of the solution, remained in the 140-160 mmHg range (atmospheric level) throughout WI.Cortical oxygen level was monitored during the warm ischemic phase, simultaneously with oxygen tension in the solution. Figure2plots the bath and cortical oxygen levels (n = 5 kidneys, see Methods). It shows that oxygen level in the solution remained in the 140-160 mmHg range, corresponding to atmospheric levels (open symbols). On the other hand, cortical oxygen level was close to zero (0.1 [0.0;2.5] mmHg, n = 8 time-points; hatched symbols). This confirms that kidneys were hypoxic, quasianoxic, during the ex vivo normothermic ischemic phase. Renal oxygenation parameters in SCS and HMP After WI, kidneys were preserved for 20 hours under SCS (UW), or HMP (KPS; LifePort ® ). During the SCS period, 20h-average cortical temperature was was 2.9 ± 2.0 °C (mean ± SD, n = 1,200 values): starting and final values were 15.0 [9.3;22.2] °C (n = 5) and 2.7 [2.4;3.0] °C (n = 5), respectively, the stable value beeing reached after 5-6 hours.

Figure 3 (

 3 main panel) shows the oxygen level (left axis: partial pressure, mmHg; right axis: concentration, µmol/L) in the preservation solution (open circles) and in the renal cortex (hatched circles), as monitored throughout the 20h SCS phase (n = 5 kidneys). Oxygen level in the solution, starting from atmospheric-equivalent levels (150-160 mmHg) decreased steadily and significantly (F-ratio = 33.13, p < 0.05) to stabilize at 51.7 [51.4;52.9] mmHg (last n = 5 time-points, between 960 and 1,200 min; half-time of decrease ∼90 min).

  Figure 4 plots oxygen levels in solution (open circles) and in renal cortex (hatched circles), during 20h LifePort machine preservation (n = 5 kidneys, main panel). Under hypothermic machine perfusion, oxygen level in the perfusion solution remained shortly (10 min) at the atmospheric level (∼145 mmHg), then rapidly and significantly (F = 59.44, p < 0.05) decreased, to a final value of 6.8 [6.6;8.2] mmHg (last n = 5 time-points, between 960 and 1,200 min; half-time ∼40 min). As for SCS, the initial rate of QO2 in HMP was extracted from the linear phase of PO2 decrease: solution-based QO2 is plotted in the inset of figure 4, in the 0 to 400 min range: initial rate was 3.4 [2.2;4.6] µmol/min per 100g (n = 5 kidneys).

  shows the timedependent evolution of arterio-venous renal QO2 during 20h HMP: from the [3.4;4.1] µmol/min per 100g range, renal QO2 significantly decreased (F = 38.89, p < 0.05) to reach 0.4 µmol/min per 100g at 120 min (see inset, zoomed over 0-120 min), then further decreased, very slowly, down to 0.2 µmol/min per 100g. Initial value of arterio-venous (HMP) QO2 was 3.4-4.1 µmol/min per 100g, NS versus solution-based initial consumption (3.4 [2.2;4.6] µmol/min per 100g , see above). Conversely, one-way ANOVA comparison of solution-based SCS (0.6 µmol/min per 100g) and HMP (3.4 [2.2;4.6] µmol/min per 100g) consumption indicated a significant difference (p < 0.01).

Figure 1 :Figure 2 :

 12 Figure 1: Protocol flow chart

Figure 3 :

 3 Figure 3: Renal oxygenation during 20 hours SCS -Oxygen consumption.

Figure 4 :

 4 Figure 4: Renal oxygenation during 20 hours HMP preservation -Oxygen consumption

Figure 5 :Figure 6 :Legend to figure 2 :Legend to figure 3 :

 5623 Figure 5: Arterio-venous consumption of oxygen during 20 hours HMP

Legend to figure 4 :Legend to figure 5 :

 45 Figure plots oxygen levels in solution (open circles, n = 5) and in renal cortex (hatched circles, n = 5), during 20 hours HMP (LifePort ® ). The left y-axis represents PO2 (mmHg), while the right one represents corresponding concentration (µM, see Methods). The apparent half-time of the decrease is ∼40 min, and the steady-state value is 6.8 [6.6;8.2] mmHg. Time-averaged temperature (20h) was 8.2 ± 1.0 °C (mean ± SD); it was ∼12 °C during the first 60 min (∼6-fold reduction of reaction rates). The inset plots the oxygen consumption (calculated from initial decrease of oxygen in the solution) between 0-400 min, which is in the range 2.2-4.6 µmol/min per 100g. Figure plots the arterio-venous consumption in HMP preservation, calculated as the product of renal perfusion rate by the arterio-venous oxygen difference (see Methods). Please note that the vertical axis is not oxygen (as is in figure 2, 3 and 4) but oxygen consumption. Initial renal graft consumption in HMP was [3.4;4.1] µmol/min per 100g. The inset represents the same results, zoomed over the first 120 minutes Legend to figure 6: ATP concentration in renal cortical tissue (median and [range], n = 5

Figure 7a :

 7a Figure 7a: Representative HES and PAS colorations of renal tissue, split into cortical (upper lines of each quadrant) and medullary (lower lines) areas. Each quadrant is further split vertically, into left and right columns, respectively showing HES and PAS staining.Arrows exemplify: brush border loss (red), endoluminal detachment (blue), vacuolization (black), and interstitially infiltrated cells (yellow). The black bar represents 100 µm.

Figure 7b :

 7b Figure 7b: Semi-quantitative histological scoring (see Methods). Panel A, Brush border loss scores (upper left); panel B, Endoluminal detachment scores (upper right); panel C, Vacuolization scores (lower left); panel D, Infiltration (lower right). Statistics performed as ANOVA one-way, with Tukey's post-test for multiple comparisons: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Acknowledgments

This work was performed in the U1082 INSERM (Institut National de la Santé et de la Recherche Médicale) research unit (dir. Pr T. Hauet), by the Centre Hospitalo-Universitaire (CHU) de Poitiers, and by the Université de Poitiers. It was supported by the CNRS (Centre National de la Recherche Scientifique) for PH salarial cost. JK was supported by a grant from the ANR (2011, RPIB-013, HemoPerf).

We are indebted to Nicolas Chatauret for the initial setting up of the SCS and HMP oxygen measurements, and for helpful discussion. We acknowledge Abdesalam Kasil's help with animal surgery and kidney withdrawaland expert histological slides preparation by Virginie Ameteau. We are grateful to Dr Jérôme Roumy for interest and discussion. Finally, we are greatly indebted to the surgical platform MOPICT (INRA, Surgères) and members for excellent and expert technical support: William Hébrard and Irène Launay (INRA, Surgères), and Pierre Couturier (CHU, Service de Biochimie).

Authors contributions

J Kaminski performed experiments on isolated kidneys, analyzed data, and contributed to the writing of the paper. S Kaaki-Hosni contributed to oxygen determination experiments. PO Delpech and X Promeyrat performed all surgical procedures. T Hauet initiated the project and contributed to the writing of the paper. P Hannaert analyzed the data, performed bibliographical analysis and wrote the paper.


Lucie Goibier

Christophe Pillement

Julien Monteil

Chrystel Faure

Fernando Leal-Calderon

Julien Monteil Abc

Emulsification of non-aqueous foams stabilized by fat crystals: Towards novel air-in-oil-in-water food colloids

Keywords: fat crystals, nonaqueous foams, multiple emulsions, emulsification, coalescence, Ostwald ripening

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

Introduction

Foams are dispersions of air bubbles in a liquid continuous phase containing surface-active species that ensure the foaming ability and stabilization. These systems can be used for a wide range of applications including foods, cosmetics and personal care products. Whereas aqueous foams have been extensively studied [START_REF] Binks | Aqueous Foams Stabilized Solely by Silica Nanoparticles[END_REF][START_REF] Murray | Stabilization of foams and emulsions by mixtures of surface active food-grade particles and proteins[END_REF], nonaqueous foams still represent an emerging field [START_REF] Binks | Oil foams stabilised solely by particles[END_REF][START_REF] Gunes | Oleofoams: Properties of Crystal-Coated Bubbles from Whipped Oleogels-Evidence for Pickering Stabilization[END_REF][START_REF] Mishima | Formation and Microstructures of Whipped Oils Composed of Vegetable Oils and High-Melting Fat Crystals[END_REF]. These systems are more difficult to obtain due to the lower surface-activity of most emulsifiers at the oil-air interface compared to oil-water or air-water interfaces. Nevertheless, several studies have shown that it is possible to stabilize nonaqueous foams using fluoroethylene particles [START_REF] Binks | Oil foams stabilised solely by particles[END_REF], fluorinated clay platelet particles [START_REF] Binks | Dry oil powders and oil foams stabilised by fluorinated clay platelet particles[END_REF] or mono-and diglyceride fatty acid esters [START_REF] Shrestha | Foaming Properties of Monoglycerol Fatty Acid Esters in Nonpolar Oil Systems[END_REF]. [START_REF] Shrestha | Foaming Properties of Monoglycerol Fatty Acid Esters in Nonpolar Oil Systems[END_REF] studied the influence of mono-and diglyceride fatty acid esters on the foamability and stability of foams obtained from different oils, namely liquid paraffin, squalane and squalene. They showed that the alkyl chain length is of paramount importance for the foam stability, as well as the surfactant concentration and the oil nature. They concluded that the foam stability arises from the shape and size of the dispersed solid particles, stable foams being mainly obtained with β-crystals dispersed in the oil phase.

Foams based on naturally occurring and/or biocompatible components are of special relevance owing to their potential applications as food, cosmetic or pharmaceutical products. Kinetically stable nonaqueous foams have been obtained by dispersing crystallizable surfactants like mono-or diglycerides in vegetable oils [START_REF] Shrestha | Stabilization of nonaqueous foam with lamellar liquid crystal particles in diglycerol monolaurate/olive oil system[END_REF][START_REF] Shrestha | Nonaqueous foam with outstanding stability in diglycerol monomyristate/olive oil system[END_REF]. [START_REF] Mishima | Formation and Microstructures of Whipped Oils Composed of Vegetable Oils and High-Melting Fat Crystals[END_REF] studied the stabilization of non-aqueous foams by high melting fat crystals using X-Ray diffraction, FT-IR spectroscopy and polarized optical microscopy. They were able to follow the crystals polymorphism and revealed that small fibrous β-crystals was a prerequisite for whipping the oil. They also proved that these crystals arranged almost parallel to the air-oil surface in lamellar plane. [START_REF] Brun | Stabilization of air bubbles in oil by surfactant crystals: A route to produce air-in-oil foams and air-in-oilin-water emulsions[END_REF] obtained foams based on mixture of mono and diglycerides. They showed that the air bubbles were protected against coalescence and Ostwald ripening by a dense crystal layer surrounding the gas bubbles. In the same vein, [START_REF] Binks | Whipped oil stabilised by surfactant crystals[END_REF] added myristic acid in a high oleic sunflower oil to obtain a two-phase mixture consisting of fatty acid crystals dispersed in a liquid oil. They obtained non-aqueous foams exhibiting remarkable kinetic stability with no drainage, coalescence nor any type of coarsening within the explored time range. More recently, [START_REF] Binks | Ultra-stable self-foaming oils[END_REF] have prepared foams based on triglycerides in the absence of added emulsifiers. By controlling the temperature, they were able to obtain crystals of high melting triglycerides in a continuous phase of low melting triglycerides. This system was then aerated to produce highly stable oil foams. They proved the existence of an optimum solid fat content of around 20-30% for foaming. [START_REF] Gunes | Oleofoams: Properties of Crystal-Coated Bubbles from Whipped Oleogels-Evidence for Pickering Stabilization[END_REF] characterized the interfacial contribution to oleo-foam stability and they evidenced that monoglycerides form a jammed, closely packed layer of crystals around the air bubbles, referred to as a Pickering-type stabilization.

Here, we examined the possibility to obtain highly stable foams by whipping a crystallizable oil, namely anhydrous milk fat (AMF), without adding any external molecule to the oil phase. The obtained foam was not considered as an end-use product, but as an intermediate product to design a novel type of material: Air-in-Oil-in-Water (A/O/W) emulsions, combining the structure of a foam and of an emulsion. The first proof of concept was established by [START_REF] Brun | Stabilization of air bubbles in oil by surfactant crystals: A route to produce air-in-oil foams and air-in-oilin-water emulsions[END_REF], who developed a two-step protocol to first whip AMF using mono-diglycerides crystals and then to emulsify the nonaqueous foam in an aqueous phase.

So far, most of the studies about multiple emulsions were focused on Water-in-Oil-in-Water (W/O/W) emulsions [START_REF] Lobato-Calleros | Reduced-fat white fresh cheese-like products obtained from W1/O/W2 multiple emulsions: Viscoelastic and high-resolution image analyses[END_REF][START_REF] Muschiolik | Multiple emulsions for food use[END_REF][START_REF] Bonnet | Influence of the oil globule fraction on the release rate profiles from multiple W/O/W emulsions[END_REF][START_REF] Silva | Recent advances in multiple emulsions and their application as templates[END_REF][START_REF] Muschiolik | Double Emulsions Relevant to Food Systems: Preparation, Stability, and Applications[END_REF]. These systems are made of oil globules dispersed in an aqueous phase, the globules containing tiny aqueous droplets. Multiple W/O/W emulsions are suitable materials for the encapsulation of various biologically active compounds such as hormones [START_REF] Cournarie | Insulin-loaded W/O/W multiple emulsions: comparison of the performances of systems prepared with medium-chain-triglycerides and fish oil[END_REF], steroids [START_REF] Laugel | Moisturizing substances entrapped in W/O/W emulsions: analytical methodology for formulation, stability and release studies[END_REF] and antiseptics [START_REF] Tedajo | Release of antiseptics from the aqueous compartments of a W/O/W multiple emulsion[END_REF]. They have been successfully formulated for uses in pharmaceutics [START_REF] Hino | Drug release from w/o/w emulsions prepared with different chitosan salts and concomitant creaming up[END_REF][START_REF] Tedajo | Release of antiseptics from the aqueous compartments of a W/O/W multiple emulsion[END_REF] and cosmetics [START_REF] Laugel | Moisturizing substances entrapped in W/O/W emulsions: analytical methodology for formulation, stability and release studies[END_REF]. These applications rely on their multiple technological assets: high entrapment capacity of hydrophilic compounds within the inner aqueous droplets, efficient protection towards degradation of the encapsulated species [START_REF] Tedajo | Release of antiseptics from the aqueous compartments of a W/O/W multiple emulsion[END_REF], improvement of the performance of active compounds and sustained release (Bonnet et al., 2009a). These compartmented emulsions can also find applications in the food industry owing to their capability to entrap hydrophilic substances like minerals (Bonnet et al., 2009b) and vitamins [START_REF] Benichou | Double emulsions stabilized with hybrids of natural polymers for entrapment and slow release of active matters[END_REF]. The encapsulation of such compounds could be interesting for food enrichment or taste-masking. Some recent applications of W/O/W emulsions were reported in the protection of probiotics against the effects of gastric juices [START_REF] Shima | Effect of the hydrophilic surfactants on the preparation and encapsulation efficiency in course and fine W/O/W type emulsions[END_REF]. Another promising application could be in substituting milk fat by multiple emulsions in order to produce dairy-like products that meet dietary recommendations such as reduction of fats in general and saturated fats in particular [START_REF] Lobato-Calleros | Reduced-fat white fresh cheese-like products obtained from W1/O/W2 multiple emulsions: Viscoelastic and high-resolution image analyses[END_REF].

Nevertheless, the application of W/O/W multiple emulsions is limited by their thermodynamic instability, especially their tendency for unwanted droplet-globule coalescence during the storage period [START_REF] Pays | Double emulsions: how does release occur?[END_REF]. Another inconvenient of this type of emulsion lies in the diffusion of water driven by any osmotic pressure difference between the aqueous compartments and the diffusion of hydrophilic compounds from one aqueous phase to the other one through the oil layer due to the concentration mismatch [START_REF] Herzi | Influence of Diffusive Transport on the Structural Evolution of W/O/W Emulsions[END_REF].

By replacing the inner aqueous droplets by air bubbles, our approach aimed at circumventing some drawbacks of W/O/W double emulsions allowing some strategies to be implemented, like the reduction of the fat intake. Novel applications could also be considered like ultrasound imaging owing to the large acoustical contrast provided by the air bubbles. In A/O/W multiple emulsions, osmotic regulation is not an issue. In addition, our A/O/W double emulsions were formulated without any added lipophilic surfactant. This approach is therefore meeting consumers expectations towards a limitation of additives in foods or cosmetic products. We implemented a two-step process to design multiple emulsions of the A/O/W type based on anhydrous milk fat (AMF) and sodium caseinate as sole emulsifier. In the first step, we determined optimal conditions in terms of solid fat content to incorporate air bubbles in AMF and to reduce their size. We then examined the appropriate rheological conditions allowing the break-up of the oil foam into the aqueous phase and to ensure kinetic stability.

Materials and Methods

Materials

Experiments were carried out with anhydrous milk fat (AMF) as the oil phase, provided by Barry Callebaut (Belgium) and used as received. AMF is a complex mixture of triglycerides with a range of melting temperatures from -40 °C to +40 °C [START_REF] Bayard | Free fatty acids and their esters modulate isothermal crystallization of anhydrous milk fat[END_REF][START_REF] Goibier | The effect of surfactant crystallization on partial coalescence in O/W emulsions[END_REF]. It is composed of approximately 6 % short chain fatty acids (strictly less than 8 carbons), 20 % medium chains and 72 % long chains (strictly more than 14 carbons), including 42 % saturated C16 and C18 chains. Unsaturated chains represented 27 % of the total fatty acids, oleic acid being the most abundant one with 22 %. AMF also contains minor components like sterols (≈0.3wt.%) as well as surface-active species (≈1 wt.%) like mono and diglycerides, free fatty acids and phospholipids.

Sodium dodecyl sulfate (SDS) (C12H25O4SNa), Tween  80, sodium azide (NaN3), sodium caseinate (NaCAS) (Mw ≈ 23 300 g.mol -1 , containing 3 wt.% sodium) and hydroxyethyl cellulose were purchased from Sigma-Aldrich (France). The water used in the experiments was deionized with a resistivity close to 15 MΩ.cm at 20 °C.

Whipping of the crystallized oil phase and bubble refining

AMF was initially stored at 4°C for several weeks to allow the formation of stable crystals. It was then warmed up to 20 °C and sheared using an Ultra-Turrax® T5 mixer operating at 24 000 rpm for 30 seconds. This shearing process was implemented to fluidify AMF by breaking fat crystal aggregates.

Then, 50 g of AMF were introduced into a 100 mL beaker immersed in a thermostatically controlled bath at variable temperatures ranging from 10 to 35 °C. AMF was whipped using an IKA RW20 overhead stirrer equipped with 4-bladed R 1342 propeller, at a rotation rate of 2 000 rpm, for variable durations ranging from 5 to 30 min. With the aim of refining the bubble size, the obtained foams were further sheared within a narrow gap between two concentric cylinders' (Couette's cell; TSR, France;) at 20 °C. The inner cylinder of radius r = 20 mm was moved at a selected angular velocity, Ω. The outer cylinder was immobile, and the gap between the stator and the rotor, e, was fixed at 200 µm. For the maximum angular velocity, Ω=78.5 rad.s -1, we are able to reach shear rates, = rΩ/e, as high as 7 850 s -1 .

Characterization of the oil foam

The oil foam was observed using an Olympus BX51 microscope (Olympus, Germany) equipped with an oil immersion objective and a digital camera (Olympus U-CMAD3, Germany). When necessary, a cross-polarized configuration of the microscope was adopted to visualize AMF crystals.

The overrun is defined as the percent of air relative to the foam volume. To measure it, approximately 15 mL of foam were introduced in a graduated Falcon® tube (Dutscher, France) and the initial level was marked. The sample was then centrifuged at 11 500 g, g being the earth gravity constant, using a Rotanta 460 RF centrifuge (Hettich Lab technology, Germany)) for 15 min at 60 °C.

AMF was fully molten at this temperature, allowing fast creaming of the air bubbles and their release at the top of the tube. The sample was finally cooled at 20 °C for 2 h at room temperature to allow recrystallization of AMF. The volume variation was divided by the initial volume to obtain the overrun.

The size distributions of air bubbles were estimated by image analysis. Micrographs were taken and the dimensions of about 350 bubbles were measured to determine the volume-averaged diameter, DV, and polydispersity, P, defined as:

= 4,3 = ∑ ∑
, and

= ∑ ∑ (1)
where Ni is the total number of bubbles with diameter Di. The median diameter, , is the diameter for which the cumulative undersized volume fraction is equal to 50%. The measured diameters were subdivided into 20 granulometric classes.

Fabrication of multiple A/O/W emulsions

Right after the formation of the oil foam, coarse multiple A/O/W emulsions were prepared by progressively incorporating the foam in an external aqueous phase, under manual stirring, up to 30 wt.%. In dilute emulsions, for droplet deformation and break-up to occur, the applied shear stress, ηc , where ηc is the viscosity of the continuous phase and is the applied shear rate, must be sufficiently high [START_REF] Mabille | Rheological and Shearing Conditions for the Preparation of Monodisperse Emulsions[END_REF]. In order to fragment the drops at relatively low shear rates (laminar regime), large viscosities are required. In our experiments, this was achieved by dissolving a large amount of proteins in the continuous phase. The external phase contained 12 wt.% NaCAS, used as both emulsion stabilizer and thickening agent at such large concentration. Sodium azide was also introduced at 0.02 wt.%, as a bactericide. The coarse emulsions were then submitted to different shear rates in the afore-mentioned Couette's cell, at 20 °C. The obtained multiple emulsions were finally submitted to a two-fold dilution in a gelled solution containing 10 wt.% hydroxyethylcellulose, before storage at 4 °C. This biopolymer was selected because of its gelling properties at a relatively moderate concentration and of its compatibility with NaCAS (the mixture in water remained macroscopically homogeneous).

Multiple emulsions characterization

The globule size distribution of A/O/W emulsions was measured using a Mastersizer 2000 Hydro SM from Malvern Instruments S.A (Malvern, UK). Light-scattering data were transformed into size distribution using Fraunhofer theory, valid for droplets whose size exceeds 5-10 µm. The A/O/W emulsion (1 mL) was diluted in 10 mL of a SDS solution at 8×10 -3 mol.L -1 , corresponding to its critical micellar concentration (CMC). This surfactant was used for its ability to dissociate protein aggregates that may bridge emulsion globules. The obtained sample was then diluted in the dispersion unit containing an aqueous solution of Tween  80 at 1.2×10 -5 mol.L -1 (CMC) to avoid droplet deposition on the optics and foaming [START_REF] Goibier | The effect of surfactant crystallization on partial coalescence in O/W emulsions[END_REF]. The emulsions were characterized in terms of their volume-averaged diameter, Dv, as defined in Eq. ( 1). The obtained results were systematically checked using optical microscopy. Micrographs were recorded and the dimensions of about 100 globules were measured. The volume averaged diameters were always in accord (less than 10% difference) with those obtained from light scattering using Fraunhofer model.

The encapsulation yield of air is defined as the percentage of air remaining in the oil globules after the emulsification step, relative to the volume of air in the primary oil foam, . The fraction of air in A/O/W emulsions was estimated by means of the following procedure. A volume V1 of the emulsions was introduced in 15 mL Falcon® tubes and was centrifuged at 11 500 g for 15 min at 60 °C. The largest globules coalesced and formed a macroscopic oil phase residing at the top of the tubes. The encapsulated air was readily released from it and, in the absence of air bubbles, the molten oil phase became transparent. A thin creamed layer made of non-coalesced globules was residing underneath the oil phase. Such globules were observed under the microscope and did not contain air bubbles. After this process, the new volume, V2, was measured. The encapsulation yield was then straightforwardly obtained:

!"#$ %&' (&)$* = + , + - + . / 0 = + , + - + 1234 ×6 7 89 (2)
where :;<= is the foam volume initially introduced in the double emulsion.

3 Results and discussions

Whipping of AMF

AMF was whipped after prolonged storage at 4 °C and the application of an intense shear to break crystal aggregates (see section 2.2). The fat phase was observed under polarized light microscopy. Figure S1a (supporting information) corresponds to a sample observed at 20 °C, after the shear was applied. Using polarized light microscopy, the isotropic liquid oil appears black and the crystals appear as bright spots. The crystals are present as spherulites, revealed by Maltese crosses, whose size is between 1 and 10 µm.

We first tried to find out optimal conditions to aerate AMF in terms of stirring and temperature.

It turned out that the turbulent shear applied by rotor-stator devices like Ultra-Turrax® provided very low overrun levels (<5%). The very high shear rates and the small rotating head were not suited for the incorporation of the air bubbles in the sample. Therefore, a propeller type device was adopted as well as a relatively moderate rotating rate, namely 2 000 rpm. Figure 1 displays the variation of the overrun measured after 10 min of stirring as a function of temperature. The evolution is not monotonous, with the overrun being noticeably smaller at 10 °C and 30 °C than at 20 °C. The presence of a peak in the plot confirms the existence of an optimum solid fat content for foaming, as stated by Binks & Maranopoulos (2017). In the case of AMF, the optimum temperature of 20 °C corresponds to a solid fat content of about 15-20% [START_REF] Thivilliers | Thermally Induced Gelling of Oil-in-Water Emulsions Comprising Partially Crystallized Droplets: The Impact of Interfacial Crystals[END_REF]. Below 20 °C the viscosity of the oil phase increased considerably, which made air incorporation more difficult. At 30 °C and above, the crystals' content was insufficient (< 10%) [START_REF] Thivilliers | Thermally Induced Gelling of Oil-in-Water Emulsions Comprising Partially Crystallized Droplets: The Impact of Interfacial Crystals[END_REF] to ensure a Pickering-type stabilization of the air bubbles and the overrun decayed dramatically. As the maximum overrun was obtained at 20 °C, this temperature was selected for all subsequent experiments.

The agitation time was varied between 0 and 30 min to probe the kinetics of the whipping process. The evolution of the overrun as a function of time in reported in Figure S2 (Supporting Information). Foams containing around 30-35 % air were obtained by whipping AMF for 10 min and no further evolution of the overrun was noticed when the shearing time was prolonged. Moreover, the refining step did not vary the overrun which remained close to 30% as long as the applied shear rate was lower than about 6 000 s -1 . The final air bubbles had a nearly spheroidal shape and their surface was rough and textured due to adsorbed triglyceride crystals (See figure S1b, Supporting Information). This is due to jamming of adsorbed particles which prevents relaxation of the interface to a smooth spherical geometry [START_REF] Subramaniam | Colloid science: Nonspherical bubbles[END_REF][START_REF] Mcclements | Crystals and crystallization in oil-in-water emulsions: Implications for emulsion-based delivery systems[END_REF][START_REF] Rousseau | Trends in structuring edible emulsions with Pickering fat crystals[END_REF][START_REF] Binks | Ultra-stable self-foaming oils[END_REF].

Their initial size distribution is reported in Figure S3a (Supporting information). The volumeaveraged bubble size was 6.4 µm and the polydispersity was P= 0.22%. The mean diameter achieved for air bubbles in the present study was relatively small compared to the values generally reported in the literature [START_REF] Binks | Ultra-stable self-foaming oils[END_REF][START_REF] Brun | Stabilization of air bubbles in oil by surfactant crystals: A route to produce air-in-oil foams and air-in-oilin-water emulsions[END_REF][START_REF] Gunes | Oleofoams: Properties of Crystal-Coated Bubbles from Whipped Oleogels-Evidence for Pickering Stabilization[END_REF][START_REF] Mishima | Formation and Microstructures of Whipped Oils Composed of Vegetable Oils and High-Melting Fat Crystals[END_REF]. The non-aqueous foams were relatively stable against coalescence and Oswald ripening for several weeks when stored at 4 °C. Figure S4 (Supporting information) shows that the overrun remained constant at 32±3 vol.% over 21 days and that the bubbles shape and size did not undergo any significant variation. The bubble size distribution was measured after 21 days (Figure S3b, Supporting information) and the average volume diameter was found to be 8.2 µm, slightly larger than the initial one. It is likely that crystals formed a jammed layer around the bubbles, providing a

Pickering-type stabilization [START_REF] Binks | Ultra-stable self-foaming oils[END_REF]. The same foam stored at 20 °C for the same period of time exhibited a more pronounced evolution, as the average diameter was close to 15 µm. A coarse foam containing large air bubbles was fabricated in order to facilitate the observation of the interfaces. This foam was obtained after whipping AMF at 20 °C for 5 min at a rotation rate of 1 000 rpm (instead of 2 000 rpm). The micrograph of Figure S1c (Supporting information) shows the presence of bright spots surrounding black voids that correspond to air bubbles. This observation gives a hint that fat crystals tend to accumulate at the oil/air interface.

Dispersion of the whipped oil in an aqueous phase

Emulsification of the oil foam in the external aqueous phase was always carried out less than 30 min after fabrication of the foam to avoid any structural evolution of the fat crystals (aggregation and/or sintering) that would produce fat hardening. In these conditions, the viscosity of the nonaqueous foam was low enough to process the material under laminar flow conditions.

The non-aqueous foams, initially containing about 30 vol.% air, were dispersed at 20 °C in a highly viscous aqueous phase with 12 wt.% NaCAS and 0.02 wt.% sodium azide. The oil foam was progressively introduced in the aqueous phase up to 30 wt.% under manual stirring. The obtained coarse emulsion was then sheared in a Couette's cell at 20 °C to obtain the final multiple A/O/W emulsion. The applied shear was varied between 1 050 and 7 350 s -1 . Figure 3

Influence of the shear rate

In Figure 4a The applied fragmentation method not only successfully produced compartmented globules with a relatively narrow size distribution, but also allowed a fine tuning of the average globule diameter by varying the shear rate. Nevertheless, the applied shear rate had an impact on the final air content. Figure S5 (Supporting Information) shows typical micrographs of multiple emulsion globules obtained at different shear rates. The inner bubble concentration does not seem to vary significantly from 1 050 to 5 250 s -1 . However, a significant decrease can be noticed at 7 350 s -1 . This qualitative observation was confirmed by performing measurements of the air encapsulation yield (EY) using the protocol described in section 2.5. The results are reported in Figure 5. The values of the EY reflects partial delivery of the air bubbles induced by globules' break up. Similar trends have been frequently reported in W/O/W emulsions. Indeed, it has been observed that release of the inner droplets occurred in conjunction with globule fragmentation [START_REF] Muguet | W/O/W Multiple Emulsions Submitted to a Linear Shear Flow: Correlation between Fragmentation and Release[END_REF]. In our systems, the EY remained at a relatively high level, above 74% for shear rates not exceeding 6 000 s -1 .

In this case, globules in the freshly formed emulsion contained about 22 vol.% air. Above this threshold shear rate, the applied stress became clearly detrimental to the persistence of the multiple structure, as reflected by the relatively low EY measured at 7 350 s -1 (44%) corresponding to 13 vol.% air within the globules.

Stability assessment

The stability of the emulsion obtained at 1 050 s -1 was assessed after prolonged storage at 4 °C.

Despite the high viscosity of the aqueous phase, the globules tended to cream at the top of the recipient within a short period of time (several days) because of their large size (Dv=57 µm) and of the significant density mismatch between the air-containing globules and the aqueous phase (Δρ >0,25 g.cm -3 ). In our case, the concentration of sodium caseinate in the aqueous phase was high enough to induce depletion flocculation of the globules by unadsorbed caseinate molecules or casein aggregates [START_REF] Dickinson | Depletion flocculation of emulsions containing unadsorbed sodium caseinate[END_REF]. Such attractive interaction could increase further the buoyance driven compression of the globules and could contribute to form a more densely packed creamed layer. In the creamed layer, globules were in permanent contact and this situation provoked partial coalescence, as revealed by Figure S6 (Supporting Information). To obtain this micrograph, a small amount of cream was collected at the top of the sample and was dispersed in pure water. The micrograph reveals the presence of large aggregates made of remnants of the initial droplets which have coalesced and partially relaxed their shape. This type of instability is frequent in emulsions based on partially crystallized droplets (Boode & Walstra, 1993;[START_REF] Boode | Partial coalescence in oil-in-water emulsions 2. Influence of the properties of the fat[END_REF]. Fat crystals formed nearby the interface can protrude into the aqueous phase and pierce the thin film between neighboring droplets. This phenomenon is referred to as partial coalescence since the shape relaxation process driven by surface tension is hampered by the firmness of the partially solidified droplets.

The emulsion was submitted to a 1:1 w/w dilution with a gelled solution containing 10 wt.% hydroxyethylcellulose. The dilution was carried out right after the emulsification process. The gelled state of the aqueous phase prevented creaming, allowing the system to remain homogeneous for at least 3 weeks at 4 °C. Figure S7 (Supporting Information) shows that the size distributions measured at t=0 and t=21 days superimpose. Moreover, we did not observe any variation of the total volume of the emulsion, indicating that the air content was preserved over that period of time. The emulsions were inspected under the microscope at regular time intervals and the internal structure was apparently the same as that observed in Figure S5 (Supporting information).

Emulsions generally exhibit two types of instabilities, coalescence and Ostwald ripening (OR). In the case of multiple A/O/W emulsions, coalescence may occur at different levels: (1) between oil globules, (2) between air bubbles, and (3) between the bubbles and the globule surface. Coalescence was inhibited essentially because of the gelled state of the respective phases the objects were embedded in. The air bubbles were physically trapped in an oleogel, i.e. a network made of fat crystal aggregates dispersed in liquid oil. The absence of dynamics precluded direct contacts to develop over time, thus allowing to maintain the foamed structure within the oil globules.

The other instability is OR which may potentially occur between the oil globules and between the air bubbles. This instability consists in the molecular diffusion across the continuous phase from the smaller to the bigger colloidal objects, due to differences in Laplace pressure [START_REF] Leal-Calderon | Emulsion Science: Basic Principles[END_REF]. OR between the oil globules was insignificant because of the low solubility of triglycerides in the aqueous phase and of the large globule size. OR between air bubbles in the A/O foam stored at 4 °C (see Figures S3 andS4, Supporting information) was not significant either because of the formation of a rigid layer at the interface which mechanically hindered volume variations of the air bubbles. For the same reason, OR between the air bubbles in the oil globules was not apparent, within the time scale of the observations. A double emulsion was warmed at 50°C under the microscope in order to fully melt AMF crystals and to restore the fluidity of the oil phase. The evolution within the globules was recorded using the digital camera of the microscope and images were taken at regular time intervals (Figure 6). Crystal melting immediately triggered both coalescence and OR. Coalescence occurred between air bubbles in direct contact, leading to a decrease in their number and to an increase of their average diameter.

Coalescence appeared as "sudden" events that almost instantaneously transformed two neighboring bubbles into a single one. Once a channel is formed between two bubbles, a shape relaxation process driven by surface tension takes place until unique spherical bubble is finally formed [START_REF] Leal-Calderon | Emulsion Science: Basic Principles[END_REF]. Assuming that the reduction in interfacial energy is permanently compensated by the viscous dissipation in the oil phase, the characteristic time for shape relaxation, τR, is given by [START_REF] Bonakdar | Rupturing of bitumen-in-water emulsions: experimental evidence for viscous sintering phenomena[END_REF]:

(3) where R is the bubble radius, σ is the Air/AMF interfacial tension (≈8 mN.m -1 ) and ηo is the viscosity of the oil phase (≈10 -2 Pa.s). For R=5 µm, we obtain τR =6.2 µs, well below the time resolution of our camera. Interestingly, we never observed coalescence between an air bubble and the globule surface. This suggests that the thermodynamic conditions for air bubbles to be wetted by the aqueous phase are not favorable. In the absence of applied shear, there is consequently no direct transfer of air bubbles into the aqueous phase.

OR was more easily discernable at advanced stages of the coarsening process, when the number of bubbles was sufficiently reduced to follow them individually. In Figure 6, this was the case for times exceeding 30 s. In the bottom row of this figure, we zoomed in on a single globule whose evolution was representative of the emulsion. The small air bubble located at the top of the image (t > 45 s; indicated by the arrows) was submitted to a shrinking process that lasted about 50 s, until its complete disappearance. Conversely, the large central bubble tended to expand over the same time period. These observations are the fingerprint of Oswald ripening, which involves diffusional transfer of oxygen and nitrogen molecules from smaller to larger bubbles through the surrounding liquid phases (oil and water). The sequential micrographs of Figure S8 (Supporting information) show the progressive disappearance of the last small bubble confined within a globule. This observation indicates that the transfer of air also occurred between the oil globules, through the aqueous phase.

Conclusion

In this paper, we a presented a process to produce A/O/W type emulsions without any added lipophilic surfactant. The two-step preparation process is quite similar to that of W/O/W double emulsions, with the difference that the first step consists in an aeration of the oil phase comprising endogenous crystals. A key issue is the control of the solid fat content which must be high enough to stabilize the air bubbles, but low enough to keep the oil phase fluid during the emulsification step.

Also, the processing conditions must favor the formation of air bubbles whose mean size is as small as possible, ideally below 10 µm.

This work constitutes a real step forward compared to the first proof of concept provided by [START_REF] Brun | Stabilization of air bubbles in oil by surfactant crystals: A route to produce air-in-oil foams and air-in-oilin-water emulsions[END_REF]. Indeed, the globules obtained by these authors were much larger (>100 µm) and not spherical. We hope that this work will pave the way for technological developments, especially in the food arena where this type of material materials is highly sought after. It is within the reach of future work to generalize the fabrication procedure to other types of fats.

Figure 1 :

 1 Figure 1: Variation of the overrun of the foam based on anhydrous milk fat measured after 10 min of stirring as a function of temperature. The dotted line is a guide for the eyes. Dots are mean values of at least 3 sample measurements

Figure 2 :

 2 Figure 2: Micrographs of the foam based on anhydrous milk fat obtained (a) after the whipping step (10 min at 20 °C) and (b) after further processing the foam at 5 250 s -1 in a Couette's cell at 20 °C.

  shows micrographs (a) of the multiple emulsion obtained after applying a shear rate of 3 150 s -1 and the corresponding size distribution deduced from static light-scattering (b). The average globule diameter was around 30 µm. The emulsion exhibited a narrow size distribution, as reflected by the relatively low value of the polydispersity index: P= 0.39. The presence of air bubbles within the oil globules is clearly evidenced owing to the large refractive index mismatch between air and AMF. Not surprisingly, most of the air bubbles are confined within the largest globules, whereas small globules (<10 µm) are frequently devoid of bubbles. At low magnification, large globules appear as dark spheres because light is scattered multiple times by the inner bubbles and, as a result, light is hardly transmitted. (a) micrographs of a multiple A/O/W emulsion sheared in a Couette's cell at 3 150 s -1 ; (b) corresponding size distribution of the oil globules.

Figure 4 :

 4 Figure 4: Influence of the shear rate on the size characteristics of A/O/W emulsions. (a): Globule size distributions. (b): Volume-averaged diameter of the fat globules. The dotted line is a guide for the eyes. (c): Encapsulation yield of air in the fat globules. The dotted line is a guide for the eyes. Dots are mean values of at least 4 sample measurements.

Figure 5 :

 5 Figure 5: Encapsulation yield of air in the fat globules of the A/O/W emulsions after their processing in the Couette's cell. The dotted line is a guide for the eyes. Dots are mean values of at least 4 sample measurements.

Figure 6 :

 6 Figure 6: Evolution of the internal globule structure in a double emulsion warmed at 50 °C. The scale bar represents 20 µm. Time increases from left to right. Top row: global view; bottom row: zoom on a single globule.

Acknowledgements

The authors gratefully acknowledge Häagen Dazs and Yoplait companies (Vienne, France). This work was supported by the Banque Publique d'Investissement (BPI France), as well as Häagen Dazs and Yoplait companies (Vienne, France).

Graphical abstract

Air

Water Oil Air-in-Oil-in-Water (A/O/W) Emulsions