X Zhou

V Schmitt

P Bertet

D Vion

W Wustmann

V Shumeiko

D Esteve

High-gain weakly nonlinear flux-modulated Josephson parametric amplifier using a SQUID-array

We have developed and measured a high-gain quantum-limited microwave parametric amplifier based on a superconducting lumped LC resonator with the inductor L including an array of eight superconducting quantum interference devices (SQUIDs). This amplifier is parametrically pumped by modulating the flux threading the SQUIDs at twice the resonator frequency. Around 5 GHz, a maximum gain of 31 dB, a product amplitude-gain × bandwidth above 60 MHz, and a 1 dB compression point of -123 dBm at 20 dB gain are obtained in the non-degenerate mode of operation.

Phase sensitive amplification-deamplification is also measured in the degenerate mode and yields a maximum gain of 37 dB. The compression point obtained is 18 dB above what would be obtained with a single SQUID of the same inductance, due to the smaller nonlinearity of the SQUID array.

Although superconducting parametric amplifiers based on Josephson junctions have been known and understood for decades [START_REF] Barone | Physics and applications of the Josephson effect[END_REF][START_REF] Yurke | [END_REF], they have recently received an increased attention [3] because of their ability to measure single quantum objects and engineer quantum fluctuations of a microwave field. They are extensively used to readout superconducting quantum bits [4,6,7] or mechanical resonators [8] at or near the quantum limit, i.e, with minimum back-action imposed by quantum mechanics for the given amount of information taken on the system. They have permitted, for instance, the measurement of quantum trajectories [4] and the implementation of quantum feedback schemes [5,9]. In the field of quantum microwaves, they are also used to squeeze quantum noise and produce itinerant squeezed states for encoding quantum information [10,11] or demonstrating fundamental effects such as the reduction of the radiative decay of an artificial atom [12].

Compared to the noisier high electron mobility transistor (HEMT) based amplifiers, these Josephson parametric amplifiers (JPAs) suffer from limited bandwidth and from gain saturation at extremely low input power. A strong effort is thus made to increase the bandwidth and to mitigate saturation of JPAs by varying their design and mode of operation [13][14][15][16].

In all cases, parametric amplification of a signal at angular frequency ω S occurs by transfer of energy from a pump at frequency ω P to the signal and to a complementary idler frequency ω I . For amplifiers based on resonators, one distinguishes the case of intrinsically nonlinear resonators with bare frequency ω R that are pumped at ω P ≃ ω S ≃ ω R directly on their signal line, from the (possibly linear) resonators whose frequency ω R is parametrically modulated with a pump tone at ω P ≃ 2ω S ≃ 2ω R on a dedicated line separated from the signal port. In the first case the intrinsic nonlinearity of the resonator is usually obtained by implementing all or part of its inductance by Josephson junctions (or superconducting weak links). The pumping at ω P at sufficiently high amplitude modulates this nonlinear inductance at 2ω P , and is responsible for a four-wave mixing such that 2ω P = ω S + ω I . In the present work, we are interested in the second case [17,18], for which the nonlinearity is due to an externally imposed parametric modulation of the frequency and is responsible for a three-wave mixing such that ω P = ω S + ω I . The interest of this 3 wave mixing is that no pump mode propagates along the input and output signal lines and can blind a detector or spoil a squeezed field, at a close frequency. In practice, the true parametric modulation is usually obtained by embedding in the resonator inductance one or several superconducting quantum interference devices (SQUIDs), the Josephson inductance of which is modulated by an ac magnetic flux. The nonlinearity of the resonator inherited from the SQUID(s) is in this case an unwanted feature, which leads to saturation of amplification, and should thus be kept low. So besides the advantage of getting rid of the pump along the signal lines, a truly parametrically pumped amplifier can also be made more robust against saturation by reducing its nonlinearity without having to pump it more strongly. In this work, we test this idea and demonstrate a weakly nonlinear JPA with high gain, made of a lumped LC resonator with the inductor L terminated by a SQUID array. This paper first summarizes the theoretical description of such a JPA, then describes the device implemented and its characterization setup, and finally presents the experimental data and a comparison between measured and calculated gain, bandwidth, and saturation.

I. THEORETICAL SUMMARY AND DESIGN CHOICES

The specificity of the JPA presented here (pure three-wave mixing with spurious nonlinearity) makes the standard classical description of parametric amplifiers [START_REF] Yurke | [END_REF] not directly applicable to it. This is why a comprehensive theoretical summary is given here, based on the theoretical work [21] (note that a similar theoretical treatment can be found in [19]).

The equivalent circuit of the JPA is shown in the bottom-right corner of Fig. 1(b). For a dc flux bias Φ DC , a parametric modulation δL A cos(ω P t) of its array inductance L A , and a microwave input signal V S cos(ω S t + χ), the JPA equation of motion at the lowest nonlinear order in phase ϕ = 1/ϕ 0 ´V dt across the total inductance

L = L g + L A (Φ DC ) or the capacitance C R is ϕ ′′ + 2Γ a ϕ ′ + ω 2 R [1 + a P (Φ AC ) cos(ω P t)]ϕ + α 1 ϕ 3 = ϕ S cos(ω S t + χ), (1)
where

ϕ 0 = /2e = Φ 0 /2π is the reduced flux quantum, ω R = 2πf R = 1/ L(C R + C c ) is
the frequency of the resonator, ω P = 2πf P is the pumping frequency, Γ a = ω R /2Q is its amplitude decay rate, a P = δL A (Φ AC )/L is the relative pumping amplitude, α 1 = -ω 2 R p 3 /2N 2 the Josephson nonlinearity coefficient with N the number of SQUIDs and p = L J /L the so-called participation ratio of the total Josephson inductance L J to the total inductance L, and ϕ S is the drive amplitude proportional to V S . Taking into account the finite ratio β of each SQUID loop inductance to the inductance L J1 of a single junction, the SQUID array inductance is L A = N L J1 β/4 + L J with L J = N L J1 / 2 cos (x) + β/2 sin 2 (x) [20] and

x = πΦ/Φ 0 .
Equation (1) contains the parametric nonlinearity cos(ω P t)ϕ and the intrinsic Josephson nonlinearity α 1 ϕ 3 mentioned in the introduction. We rewrite it in the frame rotating at ω P /2 using the slow complex internal amplitude A(t) defined by ϕ(t) = A(t) √ Z R e -iω P t/2 + cc, as well as the constant complex amplitude B S of the input signal and the slow output amplitude

C(t)
     iA ′ + (iΓ a + δ + α |A 2 |)A + ǫA * = √ 2Γ a B S e -i∆t C = -i √ 2Γ a A + B S , (2)
where δ = ω P /2 -ω R is the pump to resonator detuning, ∆ = ω S -ω P /2 the signal to pump detuning, α = -p 3 Z R ω R /16N 2 is the new nonlinear coefficient, and ǫ = ω R a P = 2ω R κΦ AC /Φ 0 is the pumping strength with κ ∝ pQ the relative frequency change per flux quantum deduced from the slope of the modulation curve ω R (Φ DC ).

Although the most general stationary solution of Eq. ( 2) is a sum k∈Z A k e -ik∆t of all harmonics at frequencies ω P /2 + k∆, only the signal A S = A 1 and the idler A I = A -1 contributions happen to be non negligible at not too high pumping strength. In this case, they obey

                   δ r + α r |A S | 2 + 2 |A I | 2 + ∆ r + i A S + ǫ r A * I = 2/Γ a B S δ r + α r 2 |A S | 2 + |A I | 2 -∆ r + i A I + ǫ r A * S = 0 C S = -i √ 2Γ a A S + B S C I = -i √ 2Γ a A I (3)
with δ r = δ/Γ a , ∆ r = ∆/Γ a , α r = α/Γ a = -p 3 Z R Q/8N
2 and ǫ r = ǫ/Γ a the dimensionless detunings, nonlinear coefficient, and pumping strength, respectively.

Our goal is to make the nonlinearity α r as small as possible and benefit from the linear signal and idler complex gains given by system (3) when α r = 0, i.e.

     G S = C S B S = δ 2 r -∆ 2 r -1-ǫ 2 r -2iδr δ 2 r -∆ 2 r +1-ǫ 2 r -2i∆r G I = C I B S = 2iǫr δ 2 r -∆ 2 r +1-ǫ 2 r +2i∆r e i2χ , (4)
yielding the power gains

|G S | 2 = 1 + |G I | 2 = 1 + 4ǫ 2 r [1 -ǫ 2 r + δ 2 r -∆ 2 r ] 2 + 4∆ 2 r . (5)
In the degenerate case corresponding to ∆ = 0, the signal power gain becomes phase χ dependent and is given by

|G S,deg | 2 (χ) = 1 + 4ǫ r 2 [ǫ r -δ r cos(2χ)] + (1 + ǫ 2 r -δ 2 r ) sin(2χ) (1 -ǫ 2 r + δ 2 r ) 2 . ( 6
)
Equations (4-6) are valid only below the onset of parametric oscillations, that is of pumpinduced auto-oscillations at zero signal B S for ǫ r > 1+δ 2 r . For sufficiently large pumping strength ǫ r > 0.42 the power gain |G S | 2 is larger than 2 at small ∆ r and δ r , and a gain bandwidth ∆ω = 2π∆f at -3 dB can be defined. For the optimal pumping frequency δ r = 0 we find

∆ω 2Γ a = (1 + ǫ 2 r ) 2ǫ r ǫ 4 r -6ǫ 2 r -1 -1 , (7)
which yields a gain bandwidth product

|G S | ∆ω/2Γ a ≃ 1 (8)
that is constant within 10% for gains larger than 7 dB. Then, saturation can be evaluated approximately in an easy way by noticing that as the internal amplitudes of oscillation A S and A I increase with the pumping strength and gain, they tend to the same value when 5)]. Consequently, the terms in α r in the first two equations of system (3) also converge to close values and play the very same role as the pump to resonator detuning δ r , which is itself responsible for a gain drop given by Eq. ( 6). Equating

|G S | 2 ≃ |G I | 2 ≫ 1 [see Eq. (
α r |A 2 S + 2A 2 I | at δ r = 0 to the value δ r,sat = 0.35 1 -ǫ 2 r that produces a -1 dB drop of |G S | 2 -1, leads to the following equivalent values for A S and B S (so called 1 dB compression point):      A 2 S,sat ≃ 0.35 |α R | √ 1-ǫ 2 r 1+2ǫ 2 r B 2 S,sat Γa ≃ 0.17 |α R | (1-ǫ 2 r ) 5/2 1+2ǫ 2 r (9)
In addition, saturation at large gain |G S | has to occur when the peak current i in the junctions is still well below their critical current i c . Since at δ r ∼ 0,

i/i c = (1 + ǫ r ) |A S | p √ Z r /N , keeping i/i c < 0.5 yields the design rule pQ > 21/ |G S,max | + 1, (10)
which imposes a minimum p for low Q and wide bandwidth JPAs.

In this work, we choose to implement a tunable amplifier in the 5-6 GHz range with a quality factor Q of order 100, which should have a product gain×bandwidth of ∼ 50 MHz according to Eq. ( 8). To reduce the maximum microwave pumping power corresponding to ǫ r = Qa P /2 = 1, i.e. to a modulation a P ∼ 1% of the total inductance, a high participation ratio p ≃ 0.5 is chosen. On the other hand, in order to keep the nonlinearity α r weak and to increase the 1 dB compression point B S,sat , the tunable inductance is implemented with N = 8 SQUIDs. In this case, the saturation power is increased by N 2 or 18 dB, compared to the case of a single SQUID with the same total Josephson inductance. Finally, as Z r plays only a minor role in the nonlinearityα r (in comparison with p 3 and N -2 ), its value will be simply chosen at the best convenience for implementing the lumped element resonator.

II. SAMPLE AND MEASUREMENT SETUP

An optical micrograph of the JPA and its equivalent circuit are shown in Fig. 1(a).

This JPA is made of an interdigitated coplanar capacitor to ground (split in two parts) uncertainty. The reflected and amplified signal is routed to the output line by a cryogenic circulator with -18 dB isolation. This output line includes an isolator for protecting the sample from higher temperature noise, a 4 -8 GHz filter, a cryogenic high electron mobility transistor (HEMT) amplifier at 4K with 38 dB gain and a calibrated noise temperature of 3.8 K, as well as additional room temperature amplifiers. The output signal is finally analyzed using a spectrum analyzer or a homodyne demodulator followed by a digitizer.

Microwave generators for the input signal and pump are precisely phase locked.

III. EXPERIMENTAL RESULTS

Measurements were performed in a dilution refrigerator at the temperature of 30-40 mK.

As a preliminary characterization, the resonance was measured with a vectorial network analyzer by recording the phase of the reflected signal at zero pumping and at a nominal input power P S,n = -126 dBm that is small enough to avoid any nonlinear effects (all nominal powers mentioned here and below refer to powers at the sample ports given the calibration of the lines). The inset of Fig. 2 shows this resonance at zero flux with a fit of the curve yielding a maximum frequency f R0 = 5.97 GHz. The comparison with the f R1 = 8.06 GHz resonance frequency of a similar resonator with shorted junctions yields p = 1-(f R0 /f R1 ) 2 = 0.45, close to the 0.42 design value. Fitting the expression -2 arctan[2Q(f S /f R -1)] to the measured resonance curve also gives the quality factor Q 0 ≃ 70, with, however, limited accuracy due to a setup imperfection yielding spurious multiple wave interferences (see shoulders in inset of Fig. 2). The main graph of Fig. 2 shows the variation of f R as a function of the applied flux Φ and its comparison with the theoretical prediction from Sec. I. The agreement is only qualitative, especially above 0.35 Φ 0 , where f R decreases faster than predicted by our simple model that does not include either the flux inhomogeneity in the different SQUIDs or the possible penetration of the flux through the junctions.

For characterizing amplification, the input and output lines are then connected as shown in Fig. 1. The signal and idler gains are measured with the spectrum analyzer by comparing the output powers of the signal and idler without and with parametric pumping at f P = 2f R .

The gains increase with |Φ DC | and the slope of the modulation curve at fixed absolute pumping power. In the following measurement, signal amplification is fully characterized at the working point (Φ 1 /Φ 0 = -0.32, f R1 = 5.17 GHz), i.e. point B on Fig. 2, where the slope κ 1 = 1.62 is at the same time large and in agreement with the predicted value. At this point the SQUID array model predicts a participation ratio p 1 = 0.59 and a quality factor

Q 1 = 81.
The non-degenerate (∆ = 0) signal power gain |G S | 2 is measured with the pump frequency

f P = 2f R (δ = 0
) as a function of the signal frequency f S for increasing nominal pump power P P,n , at an input power P S,n = -142.5 dBm that is sufficiently low to avoid the saturation at the highest gain. Close to the resonance, a minimum detuning δ/2π = 5 kHz is used to avoid operation in the degenerate mode. In order to check that the amplifier operates close to the quantum limit, i.e., with a noise temperature of order T N = hf R /2k B ≃ 125 mK [22], the variation of the signal and noise powers are compared when switching on and off the pump: From the 2.9 dB increase of the noise when switching on a 18.4 dB gain, from the calibrated 3.8 ± 0.3 K noise temperature of the HEMT amplifier alone in a separate run, and from the 1.7±0.2 dB attenuation of elements placed below 250 mK between the sample and the HEMT amplifier, we deduce an apparent noise temperature of only 80 ± 10 mK. This value is smaller than the expected quantum limit of 125 mK, a discrepancy that shows that modeling the line by a simple attenuator is not sufficient, as supported by our observation of the setup imperfection already mentioned.

This result, nevertheless, indicates that our JPA is not far from the quantum limit. A more precise determination of T N would require a much more precise control and calibration of the low temperature part of the measurement line, as well as a switch to connect the detection chain either to the JPA or to a low-temperature reference noise source [10].

The phase-dependent gain in the degenerate case (δ = 0 ) was then measured with f S = 5.17 GHz by varying the phase χ of the signal with respect to the pump; it is shown in Fig. 3(b) for the two values of the pump power that correspond to a 20 dB gain and to the maximum gain in the non-degenerate mode. As expected, the maximum degenerate gain is 6 dB larger than the non degenerate gain at almost the same frequency. At the highest degenerate gain of 36.9 dB, it was checked using the IQ demodulator [see inset of Fig. 3(b)] that the phase of the amplified signal is stable over minutes and that the output signal drops down to zero (no parametric oscillation) when the input signal is switched off.

As the phase χ is varied, the measured degenerate gain varies as expected, i.e., the lowest value of -25 dB resulting from the uncontrolled interference between the deamplified signal and the ∼ -18 dB leak of input signal through the circulator (see Fig. 1). This strong deamplification and the low noise temperature indicate that our JPA could also be used as a vacuum squeezer. In the inset of Fig. 3, the elongation of the Gaussian spot along the amplified quadrature shows that after parametric amplification, the noise coming from the sample at f S = 5.17 GHz overcomes the noise of the cryogenic amplifier placed at 4 K, the size of which is given by the spot size in the perpendicular direction. In this latter direction, we observe that the spot size is reduced by 1.1% when switching on the parametric pumping.

This reduction is twice as small as the 2.2% expected from deamplification of vacuum noise, which is again related to the difficulty to determine the noise temperature of the whole setup.

Finally, the saturation of the JPA is measured by recording the non degenerate signal power gain as a function of the signal input power P S,n for the same series of pump powers P P,n as before [see Fig. 4(a)]. The signal gain is almost constant at low input power and then decreases above a P P,n dependent threshold in P S,n (however with a small bump of up to 1 dB just before saturation possibly due to higher orders in non-linearity). In practice, the 1 dB compression point is defined as the input power P S,sat at which the gain is 1 dB lower than at P S,n = -150 dBm; it is plotted in Fig. 4(c). The set of measurements of Figs. The performances of the present device are comparable to those of other truly parametric amplifiers recently made. Due to our choice of a rather large Q ∼ 70, the gain bandwidth product is smaller than what was obtained, for instance, in [23] with Q ∼ 10. In [15], the direct coupling of the resonator to a cleverly engineered, frequency-dependent external impedance yielded an even lower Q and a bandwidth above 500 MHz. Despite the use of N = 8 SQUIDs, the 1dB compression point obtained here is not very high due to its scaling as N 2 Q -2 p -3 and to the large participation ratio and quality factors chosen to minimize the pump power. It is, however, about 12 dB above a similar amplifier made of a single SQUID with about the same critical current [18] and only a few dB below another one [23] with smaller participation ratio p (three times larger critical current Ic) and Q.

In terms of perspectives, Eqs ( 8)- (10) predict that with a similar geometry N∼ 10, a smaller Q ∼ 10, and higher critical currents yielding p ∼ 0.25, a bandwidth of ∼ 50 MHz, and a compression point ∼ -100 dBm should be obtained at 20 dB gain. This would require a larger pump power, i.e. a larger flux modulation Φ AC ∝ 1/pQ at constant gain, which would reach 0.1Φ 0 . Such a large modulation could be technically difficult to achieve. Increasing the number of SQUIDs is also an obvious optimization axis: If theoretically the array length has just to be kept much smaller than the pump wavelength so that all SQUIDS are pumped in phase, the practical difficulty is to dc flux bias and modulate all of the SQUIDs homogeneously.

In summary, a lumped element, truly parametric Josephson amplifier has been designed and characterized. Its inductance is implemented by a SQUID array to limit its nonlinearity and increase the maximum allowed input power. With a quality factor of 70-80, this simple device provides a gain of up to 30 dB, a product amplitude gain×bandwidth of 61 MHz, and a 1 dB compression point of -123 dBm at 20 dB gain. Although its behavior is in agreement with theory and demonstrates the advantage of using a SQUID array, it can still be optimized by reducing both its quality factor and its Josephson participation ratio to the inductance and/or by increasing the number of SQUIDs in the array. Operated close to the quantum limit, this parametric amplifier could also be used as a quiet and strong squeezer in degenerate mode or as the first stage of amplification in a superconducting quantum bit readout. Note that given the ±1 dB precision on the calibration of the pumping and signal lines, +0.8 dB and -1.0 dB were added to the nominal P P,n and P S,n values to match the data to the theoretical curves at low pumping strength. The vertical dotted line indicates the frontier between parametric amplification and parametric oscillation (infinite gain) for the linear model.

4 -

 4 with capacitance C R = 0.40 pF , in parallel with an inductance to ground L combining in series a meander of inductance L g = 0.80 nH with an array of eight SQUIDs of total Josephson inductanceL J = 0.88 nH at zero magnetic flux Φ. Being designed to be operated in reflection, this LC circuit is coupled to a single input-output signal line [50 Ω coplanar waveguide (CPW) ] through a C c = 55 fF capacitance yielding a characteristic impedance Z r ⋍ 65 Ω and a quality factor Q ≃ 70 at Φ = 0. On the other side of the device, a 50 Ω CPW line shorted to ground by two loops coupled inductively to four SQUIDS each, serves both for their dc flux biasing and for parametric pumping. Note that after compensation of any global dc flux offset, the magnetic fluxes Φ are exactly opposite in the left and right four SQUID sub-arrays, which yields the same inductance modulation. The device was fabricated on a thermally oxidized Si chip by sputtering 170 nm of niobium and patterning the whole structure (except the SQUID array) by optical lithography and CF Ar reactive ion etching. The SQUID array was then fabricated by electron-beam lithography and double-angle evaporation of aluminum with oxidation of the first Al layer. Each SQUID has a loop area of 8 µm × 15 µm and two junctions with nominal area 2.2 µm × 0.7 µm and tunnel resistance 141 Ω, yielding β ≃ 0.1. The active antenna wires of the pump line are positioned 16 µm away from the SQUID centers. The measurement setup is schematized in Fig. 1b. A small superconducting coil is used to compensate the global dc flux offset. The dc flux biasing and ac pumping of the SQUIDs are obtained by two attenuated and filtered lines combined with a bias tee. The input line includes attenuators at various temperatures and a 4 -8 GHz bandpass filter. The -71.5 dB and -51 dB transmissions of the input and pumping lines are calibrated with a ±1 dB

Figure 3 (

 3 a) shows the gain increase up to 31.8 dB (dashed top curve, for which parametric oscillations are about to start) and the corresponding bandwidth decrease. The maximum power gain |G S | 2 and the corresponding -3 dB bandwidth ∆f deduced from Fig. 3 are plotted in Fig. 4(b) together with the amplitude gain × bandwidth product |G S | ∆f . This product happens to be almost constant around 61 MHz over the whole 7 dB -30 dB gain range. Besides, the idler gain (data not shown) approaches the signal gain at large values.

4 4 .

 4 (b) and 4(c) is then compared to the linear model of Sec. I: the power gain, bandwidth, product amplitude gain × bandwidth, as well as the 1 dB compression point of Eqs. (6-9) are calculated by using the values of f R1 , p 1 , κ 1 and Q 1 indicated above and are plotted in Fig.Giventhe ±1 dB uncertainty on the calibration of the signal and pump lines, the nominal input and pump powers were shifted by +0.8 dB and -1.0 dB to match the theory at the lowest pumping power. The agreement between the overall measured data and the model is surprisingly good given the crudeness of the linear model. This fair agreement validates the idea of increasing the number N of SQUIDs to increase the saturation power that scales with N 2 . With a single SQUID having the same total inductance as the array implemented here (about 1.7 nH), the saturation would have been N 2 = 18 dB lower, as indicated by the dashed line of Fig.4(c). The discrepancy between experimental data and the model increases with P p as the nonlinearity plays a more important role, and the actual parametric amplification region extends a bit over the theoretical parametric oscillation region of the linear model (dotted line of Fig.4).

Figure 1 :

 1 Figure 1: Experimental setup. (a) Optical micrograph of the tested parametric amplifier showing its 50 Ω coplanar waveguide (CPW) signal input port (top), its coupling capacitor C c , its capacitor C R (left and right), its inductor L g (middle) terminated by an eight SQUID array with total inductance L A (Φ), and its split magnetic flux line coupled to a 50 Ω CPW (bottom). The dc current in the flux line sets the dc flux Φ DC and the resonance frequency f R of the resonator, whereas the ac current parametrically pumps the resonator at ω P ≃ 2ω R . The black arrow points to the equivalent circuit. (b) Electrical circuit diagram showing (from left to right) the pump line, the dc flux line added to the pump with a bias tee, an additional flux line feeding a coil for compensating any flux offsets, the signal line, and a circulator routing the reflected and amplified signal to a measurement line through an isolator protecting the sample from the noise of the first amplifier placed at 4 K.Feeding lines are attenuated and filtered. The output signal is split after amplification and analyzed both with a spectrum analyzer and by homodyne demodulation.

Φ DC / Φ 0 Figure 2 :Figure 4 :

 024 Figure 2: DC flux modulation. Experimental (dots) and calculated (line) resonator frequency f R as a function of the dc current in the on-chip flux line [see Fig.1(a)] measured by fitting the phase of a weak signal reflected on the resonator in the absence of parametric pumping, as shown in the inset for zero flux bias (point A). We attribute the two shoulders on the sides of the measured resonance to multiple wave interferences due to an imperfect impedance matching somewhere in the setup. Parameters used for calculation of the modulation curve are f R0 , p = 0.45, and β = 0.1 . Full characterization in the next figures are done at working point B.

  related to the input and output voltages V in,out by V in (t) = iϕ 0 √ Z 0 ω S B S e -iω S t /2 + cc

	and V out (t) = iϕ 0	√	Z 0 ω S C(t)e -iω S t /2 + cc. Here, Z 0 is the impedance of the line, Z R =
	L/(C R + C c ) the characteristic impedance of the resonator, and cc stands for the complex
	conjugate of the previous term. Neglecting fast oscillating terms (rotating wave approxima-
	tion), one obtains [21]

Acknowledgment

We gratefully acknowledge discussions within the Quantronics group, technical support from P. Orfila, P. Senat, J.C. Tack and Dominique Duet, as well as financial support from the European Research Contract SCALEQIT.


Renaud Tissier
email: rtissier@vet-alfort.fr

PhD, Research associate Sebastien Giraud

PhD, Research associate Nathalie Quellard

PhD, Research associate Béatrice Fernandez

BSc, Research assistant Fanny Lidouren

PhD Lys Darbera

Matthias Kohlhauer

Sandrine Pons

Mourad Chenoune

MD Patrick Bruneval

PhD, Professor Renaud Tissier Dvm

MSc, PhD student Matthias Kohlhauer Dvm

PhD Sandrine Pons Pharmd

PhD, Post- Mourad Chenoune Dvm

MD Jean-Michel Goujon

MD Bijan Ghaleh

MD Alain Berdeaux

MD Thierry Hauet

Virginie Ameteau

Kidney protection by hypothermic total liquid ventilation after cardiac arrest in rabbits

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

Institution of therapeutic hypothermia has been well demonstrated to improve both survival and neurological outcome in patients resuscitated after out-of-hospital cardiac arrest 1,2 . Beyond this neuroprotective effect, it is also important to investigate the effect of hypothermia on the multivisceral dysfunction and the so-called "post-cardiac syndrome" [START_REF] Adrie | Postresuscitation disease after cardiac arrest: A sepsis-like syndrome?[END_REF] . As example, acute kidney injury affects ~12% of the survivors after cardiac arrest and could worsen the prognosis [START_REF] Domanovits | Acute renal failure after successful cardiopulmonary resuscitation[END_REF] . In this setting, Susantitaphong et al. recently analyzed clinical studies reporting kidney-related outcomes and demonstrated that therapeutic hypothermia neither prevented the development of acute kidney injury nor dialysis requirement [START_REF] Susantitaphong | Therapeutic hypothermia and prevention of acute kidney injury: A meta-analysis of randomized controlled trials[END_REF] . In animal models of cardiac arrest, the benefit afforded by hypothermia however directly depends upon its rapidity of institution after cardiopulmonary resuscitation [START_REF] Kuboyama | Delay in cooling negates the beneficial effect of mild resuscitative cerebral hypothermia after cardiac arrest in dogs: A prospective, randomized study[END_REF] . This benefit was investigated regarding cardiac and neurological outcomes [START_REF] Kuboyama | Delay in cooling negates the beneficial effect of mild resuscitative cerebral hypothermia after cardiac arrest in dogs: A prospective, randomized study[END_REF][START_REF] Yu | Survival and neurological outcomes after nasopharyngeal cooling or peripheral vein cold saline infusion initiated during cardiopulmonary resuscitation in a porcine model of prolonged cardiac arrest[END_REF][START_REF] Yannopoulos | Intra-cardiopulmonary resuscitation hypothermia with and without volume loading in an ischemic model of cardiac arrest[END_REF][START_REF] Abella | Intra-arrest cooling improves outcomes in a murine cardiac arrest model[END_REF][START_REF] Chenoune | Ultrafast and wholebody cooling with total liquid ventilation induces favorable neurological and cardiac outcomes after cardiac arrest in rabbits[END_REF] while renal function was not precisely investigated. Therefore, we hypothesized that hypothermia can also exert a renalprotective effect after cardiac arrest if applied very rapidly after the no-flow episode.

An original strategy providing ultrafast cooling is total liquid ventilation (TLV) of the lungs with temperature-controlled perfluorocarbons [START_REF] Shaffer | Physiological effects of ventilation with liquid fluorocarbon at controlled temperatures[END_REF] . TLV can indeed use the lung as a heat exchanger and cool the body while maintaining gas exchanges [START_REF] Chenoune | Ultrafast and wholebody cooling with total liquid ventilation induces favorable neurological and cardiac outcomes after cardiac arrest in rabbits[END_REF][START_REF] Chenoune | Rapid cooling of the heart with total liquid ventilation prevents transmural myocardial infarction following prolonged ischemia in rabbits[END_REF][START_REF] Tissier | Rapid cooling preserves the ischaemic myocardium against mitochondrial damage and left ventricular dysfunction[END_REF][START_REF] Tissier | Total liquid ventilation provides ultra-fast cardioprotective cooling[END_REF] . As compared to conventional cooling with combined cold blankets and fluid administration, TLV provided a potent protection on brain and heart in rabbits submitted to equal or less than 10 min of cardiac arrest [START_REF] Chenoune | Ultrafast and wholebody cooling with total liquid ventilation induces favorable neurological and cardiac outcomes after cardiac arrest in rabbits[END_REF][START_REF] Darbera | Hypothermic liquid ventilation prevents early hemodynamic dysfunction and cardiovascular mortality after coronary artery occlusion complicated by cardiac arrest in rabbits[END_REF] . Here, we propose to use more severe experimental conditions inducing kidney dysfunction after 15 min of cardiac arrest in rabbits. We hypothesised that ultrafast cooling with TLV can increase kidney resistance to the post-cardiac arrest syndrome. Our endpoints were kidney function biomarkers, morphological appearance and transcriptomic responses.

Materials and Methods

The experiments were conducted in accordance with French official regulations, after approval by the institutional Animal Care Committee (ComEth "Anses/ENVA/UPEC" n°16, Maisons-Alfort; protocol 13/12/11-5). It conformed with the guidelines laid out in the Guide for the Care and Use of Laboratory Animals from the National Academy of Science.

Animal preparation

New Zealand rabbits (3.0-3.5 kg) were anesthetized using zolazepam, tiletamine and pentobarbital (all 20-30 mg/kg i.v.). They were intubated and mechanically ventilated (FiO2=100%). After administration of pancuronium bromide (200 µg/kg i.v.), two electrodes were implanted upon the chest and inserted into the esophagus for subsequent induction of ventricular fibrillation. Rectal, esophageal and tympanic temperatures were continuously monitored using thermal probes (Harvard Apparatus, Paris, France). Throughout the protocol, external electrocardiogram was recorded, as well as arterial blood pressure from a catheter implanted into the ear artery. Data were digitalized and analyzed using the data acquisition software HEM v3.5 (Notocord, Croissy-sur-Seine, France).

Experimental protocol

As illustrated in Figure 1, the animals were randomly assigned after a period of stabilization to the Sham group or two groups submitted to cardiac arrest (Control and TLV groups). In these two groups, cardiac arrest was induced by ventricular fibrillation by passing an alternating current (10 V, 4 mA) between the implanted electrodes. After 15 min of untreated fibrillation, cardiopulmonary resuscitation was started using cardiac massage (~ 200 compressions/min), electric attempts of defibrillation (5-10 J/kg) and intravenous administration of epinephrine (15 µg/kg i.v.). After resumption of spontaneous circulation (ROSC), administration of epinephrine was still permitted with an infusion pump to maintain mean arterial pressure at ~80 mmHg. In the TLV group, the animals were cooled to 32°C using TLV after ROSC. The lungs were filled with 10 ml/kg of perfluorocarbon (Fluorinert, 5/18 3M, Cergy, France) and the endotracheal tube was connected to our prototype of liquid ventilator (tidal volume = 7-10 ml/kg; respiratory rate = 6 breaths/min). The temperature of the perfluorocarbon was adjusted to maintain esophageal temperature at a target temperature of ~32°C. After 20 min of TLV and achievement of the hypothermic target temperature, the perfluorocarbon was evacuated from the lungs and the endotracheal tube was again connected to a conventional mechanical ventilator. Hypothermia was maintained externally at 32°C during the subsequent entire follow-up. In all groups, the animals were followed during a total duration of 6 hours after cardiac arrest. Blood samples were withdrawn at baseline, 15, 60, 180 and 360 minutes for the assessment of blood creatinin levels and blood gases partial pressure. Urine production was also measured, as well as urinary creatinin concentration for calculation of the corresponding clearance. We also assessed blood and/or urinary osmolarity (Freezing point depression osmometer; Roebling osmometer, Burladingen, Germany) and levels of sodium, glucose, creatin phosphokinase (Hitachi/Roche Cobas, Roche Diagnostic, Meylan, France), N-acetyl-β-(D)-glucosaminidase (proximal tubule lysosomal enzyme; Roche Diagnostic, Mannheim, Germany), γglutamyltransferase (marker of acute renal injury; Roche Diagnostic, Mannheim, Germany) and β2-microtubulin (marker of proximal tubule dysfunction; Roche Diagnostic, Mannheim, Germany). Fractional sodium excretion was calculated from blood and urinary sodium levels.

At the end of the follow-up, animals were euthanized and kidneys were sampled for electron microscopy, conventional histology and molecular biology.

Electron microscopy and histological analyses

Kidneys samples were processed for transmission electron microscopy, as previously described [START_REF] Goujon | Histological evaluation of proximal tubule cell injury in isolated perfused pig kidneys exposed to cold ischemia[END_REF] . Briefly, tissue sections of 1 mm 3 were fixed in glutaraldehyde (3%; 2h at 4°C), washed and postfixed in osmium tetroxide (1%; 1h at 4°C). They were dehydrated in graded series of aceton and embedded in araldite. Ultrathin sections were cut and stained with uranyl acetate and lead citrate and were examined under an electron microscope (JEOL 6/18 1010, Tokyo, Japan). Mitochondria integrity (membrane damage and crest reduction), cellular oedema, loss of brush bordure, cellular vacuolization, lyses of intracellular organelles were evaluated. The degree of histological lesions was determined in the cortex and medulla using a semi-quantitative graded scale from 0 to 10 according to lesions extension among the kidney samples (0: no alterations; 1, mild lesions < 10% of the kidney; 2, lesions affecting 11-20%; 3, 21-30%; 4, 31-40%; 5, 41-50%; 6, 51-60%; 7, 61-70%; 8, 71-80%; 9, 81-90%; 10, >91%). Scores were blindly attributed by two independent observers after examination of at least 10 different sections.

Kidneys slices were also fixed in formaldehyde (4%) for conventional histology after hematoxylin-eosin-saffron staining. We used a 0-5 score system to blindly quantify the lesions severity in the cortex and the medulla (0: normal appearance; 5: extensive necrosis).

For each animal, the sum of these two scores (cortex and medulla) led to an overall score from 0 to 10. Detection of macrophages was performed on paraffin tissue sections using the RAM11 antibody against rabbit macrophages (Dako, Trappes, France), as previously described [START_REF] Aouam | Preconditioning of salvaged myocardium in conscious rabbits with postinfarction dysfunction[END_REF] . The brush border integrity was also evaluated by immunochemistry staining using the CD10 antibody (Dako).

Real-Time Quantitative polymerase chain reaction

In all animals, kidney samples were fixed immediately after organ removal using liquid nitrogen. For cortical tissue RNA extraction, we used a commercial kit (Macherey Nagel, Hoerdt, France). Genomic DNA was removed using DNA-free kit (Applied Biosystems, Saint Aubin, France) and first-strand reverse transcription (Applied Biosystems) was performed.

Real-Time polymerase chain reaction assays were performed on a RotorGene Q (Qiagen,Courtaboeuf, France) following the manufacturer's recommendations. Rabbit DNA primers were designed using OligoPerfect™ (Invitrogen, Carlsbad, NM, USA), QuantPrim (Universität Potsdam, Max-Planck-Gesellschaft) and OligoAnalyser (Integrated DNA Technologies, Coralville, IO, USA), with the sequences detailed in Supplemental Digital

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Content 1 (Table ). Finally, messenger RNA expression level, relative to expression in healthy kidneys, was quantified with the Pfaffl method (expressed as Relative Fold Change), using ribosomal L19, β Actine and RPLPO as gene references.

Statistics

Data were expressed as mean±SEM. Statistical analyses were performed using a statistical software (SigmaStat 3.5, Systat Software Inc., Chicago, IL, USA). Hemodynamic and biochemical parameters were compared between groups using a two-way ANOVA for repeated measures. Post-hoc analyses were performed between groups at each time-point using a Student t-test with Bonferonni correction (two-tailed). Values were not compared between the different time-points in order to avoid multiple comparisons. Histological scores and molecular biology markers were compared between groups using a Kruskall-Wallis non parametric test. Significant differences were determined at P≤0.05.

Results

Twenty-four animals were included in the different groups (n=8 / group), with no missing data. In Control and TLV groups, ROSC was obtained in 4.1±0.7 and 3.7±0.5 min after cardiac arrest, respectively.

TLV affords a very rapid cooling and preserves hemodynamic

As illustrated in Figure 1B, a mild and reversible decrease in esophageal and rectal temperatures was observed in the Control group after cardiac arrest. In comparison, temperatures decreased very rapidly in the TLV group and achieved 32°C within 5 min after the onset of TLV in the esophagus. As shown in Table 1, this was associated with a strong decrease in heart rate in the TLV group as compared to Control and Sham groups (e.g, -32% in TLV vs Control groups at t=360 min after cardiac arrest). Only minor changes were observed regarding mean blood pressure as our goal was to support values of ~80 mmHg using epinephrine infusion. The total doses administered to achieve this goal was significantly higher in Control vsTLV groups (990±179 and 361±23 µg/kg, respectively), showing a favourable hemodynamic effects of hypothermia. Despite epinephrine administration, blood pressure was moreover significantly decreased in the Control group at the end of the follow-up (t=360 min after cardiac arrest) as compared to the TLV group. In these two groups, we also observed a dramatic increase in glucose and lactate blood levels, as well as acidosis and decrease in blood oxygen partial pressure (Table 1). During the TLV episode (15 min after cardiac arrest), blood carbon dioxide partial pressure was also significantly higher in the TLV group as compared to Control and Sham. Creatinin phosphokinase blood levels were similarly increased in TLV and Control vs Sham.

TLV limits kidney injury after cardiac arrest

As shown in Table 2, blood creatinin levels were significantly increased at the end of the follow-up (360 min) in both groups submitted to cardiac arrest as compared to the Sham group. They were significantly higher in the Control group as compared to TLV. Creatinin 9/18 clearance was also significantly reduced in both Control and TLV groups when compared to Sham. This reduction tended to be more important in the Control vs TLV group (+50%) but this did not achieve statistical significance. Total urine output also non-significantly decreased in Control as compared to Sham and TLV groups. As shown in Table 3, the beneficial effect of TLV on tubule function was evidenced by preserved fractional sodium excretion and urine concentration capacity. We also observed a limited glucose urinary excretion in TLV as compared to Control animals, despite similar blood glucose levels (Table 1). The urinary concentrations of selective markers of tubular damages, i.e., N-acetyl-β-(D)glucosaminidase, β2-microtubulin and γ-glutamyl transferase, were also significantly decreased in TLV vs Control despite not completely normalized as compared to Sham animals.

These functional alterations were supported by kidney lesions in animals submitted to cardiac arrest as compared to Sham animals (Figures 2 and3). As shown in Figure 2, electron microscopy revealed altered microvilli (brush border) and loss in cytosolic and mitochondrial crest density in the cortex and medulla in the Control group (Figures 2C and2G). In comparison, cortex and medulla appearance was better preserved in the TLV group (Figure 2D and2H). In Sham animals, the appearance was normal at electron microscopy (Figures 2B and2F). This led to a significant decrease in lesion score in TLV vs Control group with a significant increase for both groups when compared to Sham (Figure 2A). The glomerular apparatus was preserved in all groups (Figure 2E). These differences were confirmed using conventional histology (Figure 3A). The injuries were indeed particularly marked in the cortex of the Control group as illustrated by a tubular necrosis in Figure 3C. In the TLV group, lesions were attenuated with "only" dilation of the proximal tubes (Figure 3D).

Virtually no macrophages infiltrations were detected using the RAM-11 antibody (Figure 3E-3H), even in territories with extensive necrosis in the Control group (Figure 3F). Figure 3G illustrates one of the rare macrophages in a territory with a normal appearance in this same 10/18 group. Immunochemistry marking of the brush border membrane showed extensive degradation in Control animals (Figure 3J) as compared to Sham animals (3I). These alterations were not prevented in all animals in the TLV group. Some kidneys indeed showed normal appearance (Figure 3K) while others had clear brush border membrane alteration (Figure 3L).

Cardiac arrest strongly upregulates hypoxia and inflammation markers in both Control and TLV groups

As shown in Figure 4A, we observed a dramatic increase in the expression of hypoxia markers in both Control and TLV groups as compared to Sham (Heme Oxygenase-1 (HO-1), Erythropoïetin, Hypoxia-Inducible Factor 1α and Vascular Endothelial Growth Factor. The expression of the apoptotic marker Fas and the mobility marker RhoA were also similarly expressed in TLV and Control groups (Figure 4B). As illustrated in Figures 4C and4D

Discussion

In the present study, we demonstrate that ultrafast cooling induced by TLV protects kidneys in a severe model of cardiac arrest in rabbits. This was strongly supported by improved renal function and preserved morphology using electron microscopy and conventional histology. Transcriptomic profiles were not much affected by TLV regarding numerous genes involved in innate immunity and hypoxic responses.

We previously showed that ultrafast hypothermic TLV can strongly prevent both cardiovascular and neurological dysfunctions after cardiac arrest [START_REF] Chenoune | Ultrafast and wholebody cooling with total liquid ventilation induces favorable neurological and cardiac outcomes after cardiac arrest in rabbits[END_REF] . These investigations were conducted in rabbits after shorter duration of cardiac arrest comprised between 5 and 10 min [START_REF] Chenoune | Ultrafast and wholebody cooling with total liquid ventilation induces favorable neurological and cardiac outcomes after cardiac arrest in rabbits[END_REF] . In these conditions, we observed dramatic cardiac and neurological dysfunctions while renal dysfunction was very mild. Here, we used a prolonged episode of 15 min of noflow to obtain acute renal dysfunction and likely very poor neurological prognosis. TLV preserved urine output and significantly limited the rise in blood creatinin levels. The latter benefit was importantly not related to a decreased release of creatinin with hypothermia since creatinin phosphokinase blood levels were similarly enhanced in TLV and Control groups. Surprisingly, we did not observe any significant improvement in creatinin urinary clearance in TLV vs Control. It is rather difficult to analyze this result according to the complex effect of hypothermia on urine production. After out-of-hospital cardiac arrest, Zeiner et al. also showed a delayed recovery in creatinin clearance with therapeutic hypothermia (32-34°C) [START_REF] Zeiner | The effect of mild therapeutic hypothermia on renal function after cardiopulmonary resuscitation in men[END_REF] . One could also argue that hypothermia directly decreased urine production or conversely increased the production of diluted urine as previously showed in hypothermic patients treated for acute ischemic stroke [START_REF] Guluma | Therapeutic hypothermia is associated with a decrease in urine output in acute stroke patients[END_REF] or with profound hypothermia [START_REF] Gil-Rodriguez | Renal function during profound hypothermia[END_REF][START_REF] Knight | Urinary responses to cold temperature during water immersion[END_REF] . This was not the case in our experimental conditions as hypothermia preserved urine production and osmolarity in the TLV group as compared to Control. Importantly, the functional benefit afforded by TLV was also supported by a significant improvement of all markers of tubular damage, including N-acetyl-β-(D)-glucosaminidase, β2-microtubulin and γ-glutamyl 12/18 transferase. It would also be relevant to investigate original and specific biomarkers such as Neutrophil gelatinase-associated lipocalin (NGAL) and Kidney injury molecule-1 [START_REF] Arthur | Evaluation of 32 urine biomarkers to predict the progression of acute kidney injury after cardiac surgery[END_REF][START_REF] Mishra | Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury[END_REF] .

Morphological data definitely confirm the renal-protective effect of TLV in our study using either conventional histology or electron microscopy. Such a beneficial effect of moderate hypothermia has also been shown in animal models of regional renal ischemiareperfusion in rats [START_REF] Delbridge | The effect of body temperature in a rat model of renal ischemia-reperfusion injury[END_REF] . In the latter study, body temperature during ischemia dramatically affected the severity of injury, again showing the importance of per-ischemic and early cooling. Intra-ischemic hypothermia is however difficult to achieve during cardiac arrest, especially regarding abdominal and renal temperatures. In a recent meta-analysis including 19 studies and 2218 patients, Susantitaphong et al. showed that therapeutic hypothermia neither prevented the development of acute kidney injury nor dialysis requirement but was associated with lower mortality [START_REF] Susantitaphong | Therapeutic hypothermia and prevention of acute kidney injury: A meta-analysis of randomized controlled trials[END_REF] . The present study suggests that the benefit of hypothermia could be still evidenced in very severe experimental conditions when it is induced very rapidly.

In the present study, the severity of the ischemic insult was also supported by kidney transcriptomic alterations. As an example, hypoxic stress led to a 30-50 fold increase in HO-1 expression, as well as a 10 and 6 fold increases for Erythropoïetin and Hypoxia-Inducible Factor 1α, respectively. These expressions were not different between TLV and Control groups, suggesting that the renal-protective effect of TLV was not directly mediated through these pathways. However, one could speculate that the potent upregulation of Erythropoïetin and HO-1 could be associated with a facilitation of tissue repair, as previously suggested after tubulointerstitial injury and progressive nephritis [START_REF] Tanaka | Induction of protective genes by cobalt ameliorates tubulointerstitial injury in the progressive Thy1 nephritis[END_REF] . We also did not observe any significant difference in transcriptomic profiles of cellular mobility, endothelial activation and innate immunity markers between Control and TLV groups. A non-significant decrease in TLR2 and pro-inflammatory Tumor Necrosis Factor-α and Interferon-γ markers was however observed in the TLV group, as well as a mild and non-significant upregulation of the 13/18 regulatory IL-10 cytokine. Interestingly, the latter cytokine was shown to mediate delayed protection afforded by remote ischemic preconditioning against myocardial ischemiareperfusion injury [START_REF] Cai | Remote ischemic preconditioning confers late protection against myocardial ischemia-reperfusion injury in mice by upregulating interleukin-10[END_REF] . Conversely, TLR2 and TLR4 expression are well known to mediate kidney ischemia-reperfusion damages [START_REF] Leemans | Renal-associated TLR2 mediates ischemia/reperfusion injury in the kidney[END_REF][START_REF] Wu | HMGB1 contributes to kidney ischemia reperfusion injury[END_REF] . The lack of differences between groups could be a consequence of the early time-point of organ removal (6 h after cardiac arrest). This also suggests that the protective effect of TLV is at least in part related to other mechanisms, likely an early anti-necrotic effect through ultrafast hypothermia. Improvement in the hemodynamic status could also participate in the renal-protective effect.

The main limitation of the present study was therefore probably the short duration of follow-up before organ sampling and analyses after cardiac arrest. Animals were followed during only 6 h as it was difficult to maintain animals alive for much longer duration (e.g., 24 h) according to the severity of the cardiac arrest insult (15 min of no-flow). In future studies, it could be relevant to analyse kidney function at later time-points, e.g. after transplantation in recipient animals. Hypothermic TLV could indeed offers quite new and promising therapeutic perspectives for uncontrolled organ donation after cardiac arrest or for controlled organ donation after brain death in initially resuscitated patients [START_REF] Rodriguez-Arias | Protocols for uncontrolled donation after circulatory death[END_REF] . It was not possible to conduct transplantation experiments in rabbits in the present experimental conditions but we currently are working on a new technology of liquid ventilation that could be used in a relevant porcine model for a definitive proof-of-concept using organ transplantation [START_REF] Giraud | Contribution of large pig for renal ischemia-reperfusion and transplantation studies: The preclinical model[END_REF] . There is indeed a high degree of proximity between human and pig kidneys with multilobular, multipapillary architecture, while mice, rats, dogs and rabbits have unilobular, unipapillary kidneys [START_REF] Simmons | Surgical renal ischemia: A contemporary overview[END_REF] . In dogs and rodents, segmental arteries are bypassed due to the lack of multiple medullary pyramids, while in humans and pigs an elaborated system of interlobar and segmental arteries is present to supply the numerous kidney lobes [START_REF] Giraud | Contribution of large pig for renal ischemia-reperfusion and transplantation studies: The preclinical model[END_REF] .

  , the expression of endothelial activation and innate immunity markers was also not different between these two groups, including E-selectin, Vascular Cell Adhesion Molecule 1, Interleukin (IL)-10, IL-1β, Interferon-γ, Tumor Necrosis Factor-α, IL-18, Monocyte Chemoattractant Protein-1 and Toll-Like Receptors (TLR) 2 and 4.

Table 1 : Heart rate, mean arterial pressure and blood biochemical parameters

 1 TLV, total liquid ventilation; *, p<0.05 vs corresponding Sham group; †, p<0.05 vs corresponding Sham and Control groups.

		Baseline		After cardiac arrest (min)
			15	60	180	360
	Heart rate (beats/min)
	Sham	253±12	234±8	239±12	244±11	235±8
	Control	254±8	204±15*	184±9*	187±5*	203±11*
	TLV	250±7	151±6 †	131±3 †	137±2 †	139±5 †
	Mean blood pressure (mmHg))
	Sham	75±3	72±3	80±3	87±3	83±5
	Control	81±4	84±4*	80±5	84±3	63±5*
	TLV	78±3	98±3 †	84±4	74±2 †	74±5 †
	Lactates blood levels (mmol/L)
	Sham	3.0±0.3	2.8±0.5	2.6±0.3	2.2±0.3	2.2±0.5
	Control	2.3±0.4	13.0±1.7*	12.9±1.3*	13.1±0.8*	11.6±1.3*
	TLV	2.7±0.2	9.6±0.9 †	12.1±1.0*	10.6±0.8*	9.8±1.0*
	Glucose blood levels (mg/dL)
	Sham	183±23	180±19	130±15	133±6	126±4
	Control	184±31	480±27*	536±35*	700±28*	550±47*
	TLV	153±58	375±64*	460±76*	477±88 †	474±82*
	Creatin phosphokinase blood levels (mg/Dl)
	Sham	42±1	-	-	-	56±1
	Control	43±1	-	-	-	69±2*
	TLV	44±1	-	-	-	67±1*
	Blood pH
	Sham	7.41±0.02	7.42±0.03	-	7.44±0.02	7.45±0.02
	Control	7.42±0.02	7.05±0.05*	-	7.06±0.08*	7.05±0.07*
	TLV	7.42±0.05	6.92±0.06 †	-	7.11±0.05*	7.04±0.05*
	Blood pCO2 (mmHg)
	Sham	46±3	37±4	-	37±3	38±2
	Control	46±5	44±3	-	43±3	43±6
	TLV	42±3	77±8 †	-	34±7	32±4
	Blood pO 2 (mmHg)
	Sham	532±31	558±34	-	550±29	559±38
	Control	504±34	185±41*	-	269±72*	250±65*
	TLV	520±57	245±61*	-	310±58*	201±71*

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Table 2 : Blood creatinin levels, urine production and creatinin clearance TLV

 2 . total liquid ventilation; *, p<0.05 vs corresponding Sham group; †, p<0.05 vs corresponding Sham and Control groups.

		After cardiac arrest
	Baseline	T=360 min

Table 3 :

 3 Urinary markers of renal function TLV. total liquid ventilation; *, p<0.05 vs corresponding Sham group; †, p<0.05 vs corresponding Sham and Control groups.

	Baseline	After cardiac arrest T=360 min

N-acetyl-β-(D)-glucosaminidase urinary levels (Unit / mol creatinin)

Institution from which the work was received : INSERM, Unité 955, Equipe 3 Source of Funding: This study was supported by a grant (ABYSS-R12031JJ) from the "Agence Nationale pour la Recherche" (Paris, France). It was also suported by Inserm, Université de Poitiers, Région Poitou-Charentes, Conseil général de la Vienne, Région Ile-de-France (CODDIM), University Paris Est Créteil and CHU de Poitiers. R. Tissier was a recipient of a "Contrat d'Interface Inserm-ENV". M. Kohlhauer was supported by a doctoral fellowship from the "Region Ile-de France".

In conclusion, ultrafast cooling with TLV is renal-protective after cardiac arrest and resuscitation. Further experiments with organ transplantation might be relevant to afford a proof-of-concept in this setting.


Matthias Kohlhauer

Lys Darbera

Fanny Lidouren

Mourad Chenoune

Bijan Ghaleh

Benoit Vivien

Pierre Carli

Hubert Dabire

Alain Berdeaux

DVM Renaud Tissier
email: rtissier@vet-alfort.fr

Comparative effect of hypothermia and adrenaline during cardiopulmonary resuscitation in rabbits

Keywords: Cooling, Cardiac arrest, Ventricular fibrillation, Fluid, Cardiac massage, Animal Study

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INTRODUCTION

It is well admitted that therapeutic hypothermia (32°C-34°C) improves the prognosis and the neurologic recovery of comatose survivors after cardiac arrest [START_REF] Bernard | Treatment of comatose survivors of out-of-hospital cardiac arrest with induced hypothermia[END_REF][START_REF]The Hypothermia After Cardiac Arrest Study Group: Mild therapeutic hypothermia to improve the neurologic outcome after cardiac arrest[END_REF][START_REF] Nolan | European Resuscitation Council Guidelines for Resuscitation 2010 Section 1. Executive summary[END_REF] . A maximal neurological [START_REF] Abella | Intra-arrest cooling improves outcomes in a murine cardiac arrest model[END_REF][START_REF] Nozari | Critical time window for intra-arrest cooling with cold saline flush in a dog model of cardiopulmonary resuscitation[END_REF][START_REF] Menegazzi | Effects of pre-arrest and intra-arrest hypothermia on ventricular fibrillation and resuscitation[END_REF][START_REF] Chenoune | Ultrafast and wholebody cooling with total liquid ventilation induces favorable neurological and cardiac outcomes after cardiac arrest in rabbits[END_REF] and cardiovascular 8-10 protection is obtained when hypothermia is started as soon as possible, e.g., using cold saline infusion during cardiopulmonary resuscitation (CPR). The so-called "intra-arrest" hypothermia was shown to facilitate resumption of spontaneous circulation (ROSC) in rodents and porcine models [START_REF] Menegazzi | Effects of pre-arrest and intra-arrest hypothermia on ventricular fibrillation and resuscitation[END_REF][START_REF] Boddicker | Hypothermia improves defibrillation success and resuscitation outcomes from ventricular fibrillation[END_REF] . Two recent observational studies corroborate these results by the demonstration of a higher frequency of ROSC in patients receiving hypothermia through cold saline infusion during CPR as compared to standard care [START_REF] Freese | Hypothermia Working Group: Abstract 2: Intra-arrest Induction of Therapeutic Hypothermia via Large-Volume Ice-Cold Saline Infusion Improves Immediate Outcomes for Out-of-Hospital Cardiac Arrest Circulation 124 A[END_REF][START_REF] Garrett | The association between intra-arrest therapeutic hypothermia and return of spontaneous circulation among individuals experiencing out of hospital cardiac arrest[END_REF] .

To our knowledge, the exact mechanism underlying the effect of therapeutic hypothermia on ROSC remains still unknown but several hypotheses have been made. In pigs, hypothermia was shown to improve the response to defibrillation attempts [START_REF] Menegazzi | Effects of pre-arrest and intra-arrest hypothermia on ventricular fibrillation and resuscitation[END_REF][START_REF] Boddicker | Hypothermia improves defibrillation success and resuscitation outcomes from ventricular fibrillation[END_REF] and decrease the amount of adrenaline required to achieve ROSC [START_REF] Menegazzi | Effects of pre-arrest and intra-arrest hypothermia on ventricular fibrillation and resuscitation[END_REF] . Hypothermia was also reported to improve systemic hemodynamics by increasing arterial resistances [START_REF] Polderman | Mechanisms of action, physiological effects, and complications of hypothermia[END_REF] . In animal models, the influence of hypothermia on vessel function and hemodynamics was however investigated when combined to adrenaline, which could alter the exact role played by therapeutic hypothermia [START_REF] Menegazzi | Effects of pre-arrest and intra-arrest hypothermia on ventricular fibrillation and resuscitation[END_REF][START_REF] Riter | Intra-arrest hypothermia: both cold liquid ventilation with perfluorocarbons and cold intravenous saline rapidly achieve hypothermia, but only cold liquid ventilation improves resumption of spontaneous circulation[END_REF][START_REF] Mustafa | Cooling-induced carotid artery dilatation: an experimental study in isolated vessels[END_REF][START_REF] Schmoker | Cerebrovascular response to continuous cold perfusion and hypothermic circulatory arrest[END_REF] . The aim of the present study was to investigate the intrinsic vascular effect of hypothermia during CPR. Accordingly, we investigated the effect of therapeutic hypothermia induced by cold saline infusion on cardiac massage efficiency in rabbits without adrenaline administration. In order to determine the real effect of hypothermia versus that of fluid loading, we compared cold saline to warm saline infusion. We finally included a group with adrenaline administration alone, as a positive control of efficient CPR. Our end-points were cardiac massage efficiency assessed by hemodynamic parameters, rate of defibrillation success SHOCK-D-13-00373-R1 5 and ROSC occurrence. In addition, we also examined the direct effect of hypothermia (32°C vs 38°C) on the vascular response of isolated vessels after cardiac arrest in rabbits.

SHOCK-D-13-00373-R1

6

MATERIALS AND METHODS

The animal instrumentation and the ensuing experiments were conducted in accordance with French official regulations, after approval by the local ethical committee (ComEth AnSES/ENVA/UPEC n°16).

Animal preparation and cardiac arrest procedure

Male New Zealand rabbits (2.5-3.0 kg) were anaesthetized using zolazepam, tiletamine and pentobarbital (all 20-30 mg/kg i.v.). After intubation and initiation of mechanical ventilation (FiO2 = 30%), two central catheters were inserted in the carotid artery and jugular vein for measurements of central arterial and venous pressures, respectively. Two electrodes were implanted upon the chest and inserted into the oesophagus. After a period of stabilization, ventricular fibrillation was induced by an alternative current (10 V, 4 mA) between the two electrodes. Mechanical ventilation was stopped throughout the cardiac arrest period. After 15 min of untreated cardiac arrest, CPR was initiated using cardiac massage (~200 beats/min) and restoration of a continuous oxygen flow (Fi02=100%). Electric attempts of defibrillations (10 J/kg) were started at the 3 rd min of CPR and repeated every 2 min until ROSC, which was considered as an organized cardiac rhythm with a systolic arterial pressure above 40 mmHg during at least 1 min.

Experimental protocol

In addition to basic life support and electric attempts of defibrillation, rabbits were randomly assigned to one experimental group (Figure 1A). The Control group did not receive any additional procedure. In the "Saline 4°C" and "Saline 38°C" groups, the animals received 20 ml/kg of NaCl (0.9% at 4°C or 38°C, respectively) from the 1 st to the 3 rd min of CPR. The last group received bolus administrations of adrenaline (15 µg/kg i.v.) every 2 min until occurrence of ROSC. In the Control group, as well as in "Saline 4°C" SHOCK-D-13-00373-R1 and "Saline 38°C" groups, no any vasopressor drug was used. Resuscitation efforts were stopped after 10 min of unsuccessful CPR or in case of haemoptysis.

Throughout the protocol, rectal, oesophageal and tympanic temperatures were monitored using thermal probes (Harvard Apparatus, Paris, France). Hemodynamic parameters were also continuously recorded using external electrocardiogram, arterial and venous blood pressures in the right carotid and jugular vein, respectively. The difference between arterial and venous pressures was calculated with the data acquisition software HEM version 3.5 (Notocord, Croissy-sur-Seine, France). End-tidal CO2 concentration in the expired air (EtCO2) and blood oxygen saturation (SpO2) were continuously assessed. The primary end-point of the study was the percentage of animals achieving ROSC in each group. Defibrillation success and hemodynamic parameters were secondary end-points.

In vitro analysis of vascular function

Additional rabbits were anaesthetized and intubated as described above. They were randomly submitted to a "Sham" procedure without any cardiac arrest or to 15 min of untreated ventricular fibrillation as previously described. In the latter case, animals were resuscitated using cardiac massage, electric attempts of defibrillation and adrenaline administration. After ROSC, the animals were monitored during 6 hours with constant adrenaline infusion in order to avoid hypotension if necessary. If necessary, anesthesia was maintained using pentobarbital administration. Animals were then euthanized and the descending thoracic aorta was removed and cleaned of connective tissues. Aorta rings were mounted in isolated vessels chambers, as previously described [START_REF] Dabire | Vascular endothelial dysfunction in Duchenne muscular dystrophy is restored by bradykinin through upregulation of eNOS and nNOS[END_REF] . After 120 min of equilibrium under resting tension of 2 g, the chamber temperature was randomly adjusted at either 32°C or 38°C. Thirty minutes later, the response to increasing concentrations of noradrenaline was evaluated (0.3 , 1 and SHOCK-D-13-00373-R1 8 3 µmol/L). The endothelial-dependent and independent relaxation was then assessed using acetylcholine (0.1 mmol/L) and sodium nitroprusside (0.1 mmol/L), respectively.

The experiments were repeated at two levels of temperature (32 or 38°C).

Statistical analysis

Data were expressed as mean±SEM. Temperatures, hemodynamic and in vitro parameters were compared between the different groups using a two-way ANOVA for repeated measures followed by a Fisher LSD post-hoc analysis. The time to achieve successful defibrillation were compared between groups using a log-rank test. A similar analysis was used for the time to ROSC. The rate of successful defibrillation and ROSC were compared using a Chi-square test. The corresponding Kaplan-Meier curves were drawn. Significant differences were determined at p≤0.05.

SHOCK-D-13-00373-R1

9

RESULTS

In vivo investigations

Thirty-two rabbits were randomly included in the different groups (n=8 in each group). As illustrated in Figure 1B, the esophageal, tympanic and rectal temperatures were not significantly different among groups at baseline. During CPR, a significant and expected decrease was observed for oesophageal and tympanic temperatures in the "Saline 4°C" group as compared to all other groups. The rectal temperature was still not significantly different among the 4 groups.

As shown in Table 1, hemodynamic parameters, SpO2 and EtCO2 were not different among groups at baseline. After the onset of CPR, cardiac massage efficiency was greater in the "Adrenaline" group as compared to the 3 other groups as evidenced by a significant increase in arterial blood pressure and in the maximal difference between arterial and venous pressures. These parameters were conversely not significantly modified in the two "Saline" groups as compared to the "Control" group.

As illustrated in Figure 2, electric attempts of defibrillation led to a high and similar rate of successful defibrillation in all groups (7/8 animals in the "Control", "Saline 4°C" and "Adrenaline" groups; 6/8 in the "Saline 38°C" groups, respectively). However, no animal elicited successful ROSC in the "Control", "Saline 4°C" and "Saline 38°C" whereas 5/8 rabbits achieved ROSC in the "Adrenaline" group. In the "Saline 38°C" group, resuscitation efforts were interrupted in 3 rabbits after occurrence of haemoptysis.

In vitro investigations

Experiments were conducted in 15 and 14 aorta rings sampled from 3 rabbits under sham condition and 4 others after cardiac arrest, respectively. In the rabbits submitted to cardiac arrest, the total dose of adrenaline administered in vivo before euthanasia was 990±179 µg/kg. As illustrated in Figure 3A, the noradrenaline administration induced a SHOCK-D-13-00373-R1 10 concentration-dependent contraction in vitro in all groups. This effect was however significantly attenuated after cardiac arrest as compared to sham condition but this was not modified by temperature (32 vs 38°C). The endothelium-dependent relaxation in response to acetylcholine was also significantly altered after cardiac arrest but remained unchanged regardless the chamber temperature (figure 3B). Endothelium-independent relaxation after sodium nitroprusside administration was maximal and identical in all conditions.

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11

DISCUSSION

The present study demonstrates that hypothermia induced by cold saline infusion neither affected defibrillation success, ROSC frequency nor cardiac massage efficiency in rabbits. Pure fluid loading through warm saline infusion was also inefficient whereas adrenaline administration improved cardiac massage efficiency and rate of ROSC. To our knowledge, this is the first study to specifically address the effect of cold or warm fluid loading with no concomitant administration of adrenaline during CPR in animals.

Previous studies rather investigated the role of hypothermia "on top of" adrenaline administration [START_REF] Menegazzi | Effects of pre-arrest and intra-arrest hypothermia on ventricular fibrillation and resuscitation[END_REF]8,[START_REF] Boddicker | Hypothermia improves defibrillation success and resuscitation outcomes from ventricular fibrillation[END_REF][START_REF] Riter | Intra-arrest hypothermia: both cold liquid ventilation with perfluorocarbons and cold intravenous saline rapidly achieve hypothermia, but only cold liquid ventilation improves resumption of spontaneous circulation[END_REF][START_REF] Scolletta | Intra-arrest hypothermia during cardiac arrest: a systematic review[END_REF][START_REF] Staffey | Liquid ventilation with perfluorocarbons facilitates resumption of spontaneous circulation in a swine cardiac arrest model[END_REF][START_REF] Cho | Early selective trans-nasal cooling during CPR improves success of resuscitation in a porcine model of prolonged pulseless electrical activity cardiac arrest[END_REF][START_REF] Wang | Intra-arrest selective brain cooling improves success of resuscitation in a porcine model of prolonged cardiac arrest[END_REF] . In our experimental conditions, mild hypothermia did also not affect vessel reactivity in isolated aorta after cardiac arrest.

The most important finding of this study is the lack of effect of hypothermia on ROSC occurrence when applied alone, i.e., without adrenaline administration. In a recent review, Scolletta et al. showed that hypothermia conversely facilitates ROSC in pigs and rodents when it was combined to adrenaline administration [START_REF] Scolletta | Intra-arrest hypothermia during cardiac arrest: a systematic review[END_REF] . The rate of ROSC was for example improved with cold blanket cutaneous application [START_REF] Boddicker | Hypothermia improves defibrillation success and resuscitation outcomes from ventricular fibrillation[END_REF] , cold saline infusion [START_REF] Menegazzi | Effects of pre-arrest and intra-arrest hypothermia on ventricular fibrillation and resuscitation[END_REF] , hypothermic liquid ventilation [START_REF] Riter | Intra-arrest hypothermia: both cold liquid ventilation with perfluorocarbons and cold intravenous saline rapidly achieve hypothermia, but only cold liquid ventilation improves resumption of spontaneous circulation[END_REF][START_REF] Staffey | Liquid ventilation with perfluorocarbons facilitates resumption of spontaneous circulation in a swine cardiac arrest model[END_REF] , trans-nasal evaporation cooling [START_REF] Cho | Early selective trans-nasal cooling during CPR improves success of resuscitation in a porcine model of prolonged pulseless electrical activity cardiac arrest[END_REF][START_REF] Wang | Intra-arrest selective brain cooling improves success of resuscitation in a porcine model of prolonged cardiac arrest[END_REF] or endovascular cooling 8 in pigs. Interestingly, this was often attributed to defibrillation facilitation during hypothermia [START_REF] Menegazzi | Effects of pre-arrest and intra-arrest hypothermia on ventricular fibrillation and resuscitation[END_REF][START_REF] Boddicker | Hypothermia improves defibrillation success and resuscitation outcomes from ventricular fibrillation[END_REF] . As example, Boddicker et al. demonstrated that hypothermia dramatically improved the chance of defibrillation in a swine model of refractory ventricular fibrillation [START_REF] Boddicker | Hypothermia improves defibrillation success and resuscitation outcomes from ventricular fibrillation[END_REF] . Menegazzi et al. also demonstrated that hypothermic CPR reduced the decay of ECG waveforms and subsequently improved the rate of successful defibrillation [START_REF] Menegazzi | Effects of pre-arrest and intra-arrest hypothermia on ventricular fibrillation and resuscitation[END_REF] . In our study, it was not possible to show a similar benefit since the rate of successful defibrillation was virtually maximal in Control conditions.

SHOCK-D-13-00373-R1

12 Another apparent discrepancy is the lack of effect of hypothermia on cardiac massage efficiency, as observed in a previous pig study [START_REF] Riter | Intra-arrest hypothermia: both cold liquid ventilation with perfluorocarbons and cold intravenous saline rapidly achieve hypothermia, but only cold liquid ventilation improves resumption of spontaneous circulation[END_REF] . Indeed, neither cold saline infusion nor normothermic fluid loading were able to affect hemodynamic parameters during CPR. This could be in part related to the proper effect of fluid loading which could compromise the direct effect of cardiac hypothermia. Indeed, Riter et al. showed that a load-independent cooling strategy (hypothermic liquid ventilation) could improve coronary perfusion pressure during CPR as compared to cold saline infusion. In our study, the appearance of several cases of haemoptysis also suggests a poor tolerance of fluid loading after warm saline infusion. It was not possible to directly assess coronary perfusion pressure in the present study since this is technically challenging in the rabbit model [START_REF] Riter | Intra-arrest hypothermia: both cold liquid ventilation with perfluorocarbons and cold intravenous saline rapidly achieve hypothermia, but only cold liquid ventilation improves resumption of spontaneous circulation[END_REF][START_REF] Derwall | Hydrogen sulfide does not increase resuscitability in a porcine model of prolonged cardiac arrest[END_REF][START_REF] Ristagno | Post-Resuscitation Treatment With Argon Improves Early Neurological Recovery in a Porcine Model of Cardiac Arrest[END_REF] . The difference between arterial and venous central pressures could however be considered as an indirect evaluation. In accordance with the previous findings of Riter et al. [START_REF] Riter | Intra-arrest hypothermia: both cold liquid ventilation with perfluorocarbons and cold intravenous saline rapidly achieve hypothermia, but only cold liquid ventilation improves resumption of spontaneous circulation[END_REF] in pigs, it was not modified by hypothermic fluid loading, this is actually in accordance.

In the present study, we did not investigate the combined effect of hypothermia and adrenaline in vivo as the latter was too efficient by itself. Any improvement in the rate of ROSC would therefore be hard to evidence in such experimental conditions. We proposed to address this issue in vitro through the determination of the temperature effect on the vascular response to adrenergic stimulation. These experiments clearly showed a lack of effect of temperature (32 vs 38°C) on the vascular response to noradrenaline, while cardiac arrest dramatically altered vascular function. The latter result could be either attributed to tachyphylaxis and desensitization after adrenaline administration or to an actual vascular dysfunction, as described regarding microcirculation in patients after cardiac arrest [START_REF] Omar | Sublingual microcirculation is impaired in post-cardiac arrest patients[END_REF] . With lower temperatures, Mustafa and Thulesius interestingly showed that profound hypothermia (27°C and 10°C) altered SHOCK-D-13-00373-R1 13 the response to noradrenaline of isolated carotids arteries [START_REF] Mustafa | Cooling-induced carotid artery dilatation: an experimental study in isolated vessels[END_REF] . The cutaneous application of low temperatures (e.g., ice packs) is also well known to induce superficial vasoconstriction 14 , 26 .

In conclusion, cold saline infusion in the absence of adrenaline administration did not improve ROSC occurrence in rabbits. One could speculate that fluid loading hidden the beneficial effect of temperature reduction in this model.

Figure 2 :

 2 Figure 2: Rate and frequency of successful defibrillation and resumption of spontaneous

Figure 3 :

 3 Figure 3: In vitro investigations at two different temperatures (32 vs 38°C) of the

Table 1 :

 1 Hemodynamic parameters

			Baseline		Cardiopulmonary resuscitation (t=3 min)
		Control	Saline 4°C	Saline 38°C	Adrenaline	Control	Saline 4°C	Saline 38°C	Adrenaline
	Heart rate (beats/min)
		256±7	260±9 254±8	260±8	244±7	242±8 245±11	251±7
	Arterial pressure (mmHg)
	Max	103±7	104±4 102±3	107±4	62±2	57±4	51±3	92±11 *
	Mean	83±10	84±7	89±4	90±5	26±2	30±7	25±1	51±6 *
	Min	72±10	70±4	78±4	79±5	2±2	6±2	6±1	19±9 *
	Venous pressure (mmHg)
	Max	6±1	6±1	4±1	5±1	57±10	38±9	54±13	60±11
	Mean	4±1	5±1	3±1	3±1	22±3	18±4	17±4	16±4
	Min	2±1	3±1	1±1	1±1	-1±1	2±2	-4±3	-3±2
	Maximal difference between arterial and venous pressure (mmHg)
		101±6	98±4	99±3	106±4	36±4	28±8	33±5	69±11 *
	End-tidal CO2 concentration in the expired air (mmHg)
		43±7	38±2	40±2	35±5	21±7	19±3	18±2	19±2
	Blood oxygen saturation (%)
		100	99±1	100	99±1	76±9	71±5	79±4	78±6
	*, p<0.05 vs all other groups

Funding sources: Grant (ABYSS-R12031JJ) from the "Agence Nationale pour la Recherche" (Paris, France). M. Kohlhauer was supported by a grant from the "Région Ilede-France" (CORDDIM). R. Tissier was recipient of a "Contrat d'Interface INSERM-ENV".

No conflict of interest

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