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Participatory monitoring and evaluation of new technologies developed with farmers in Northern Tanzania

Elisabeth Maeda, Juma Wickama

To cite this version:

Elisabeth

Objective

The objective of the study is to illustrate the pattern of farmers' bottom-up participation in monitoring and evaluation processes in soil management and improvements. Specific objectives of the study was to enhance the positive synergies between water, soil and tree management in micro-catchments, improve farmers incomes and system productivity (crops, livestock & trees) and ensuring sustainable nutrient management in the system (integrated production and nutrient management). Information that answered the 5 core evaluation questions on (i) relevance (ii.)Effectiveness (iii.) efficiency, (iv) Effectiveness and (v) impact and sustainability were monitored and evaluated as well as cross cutting issues on gender balance and reaching the poorest and so on. The farmers were principal decision makers, active participants and controllers of the direction of their initiatives in On-farm agriculture research and development in collaboration with research scientists in evaluating new technology. The primary stakeholders are the local People, local women, men and children (pivot to the learning process), grassroots organizations at community and higher levels-facilitate participatory processes during implementation and implementers Partners as Government services NGOs and commercial operators (catalytic and advisory functions). The Responsible is the government Ministry (Ministry of Agriculture).

Description of the project

Results and Impacts

In the procedure used, eight different stages were distinguished: diagnostic, solution identification, trial planning, trial implementation, trial management, monitoring (data collection), data analysis (evaluation) and dissemination. Once developed scientists went about to develop action plan designing adaptive research experiments which were established on farmer's fields managed by farmers' groups and evaluated to select and adapt best bet options to disseminate to farmers. Such a process of participation described ways of building relationships with farmers, and improving communication through choices of various tools used in participatory monitoring and evaluation activities. In addition it covers the selection of farmers for evaluation, and reviews a number of methodologies employed. The process resulted in systematically monitoring and evaluation of outcomes, its challenges, their experiences, lessons and behavioral changes that has taken place as they try to apply the approach. Such participation enhanced farmers' capacity to adapt and develop new and appropriate innovations by encouraging them in learning through experimentation, building on their own knowledge and practices and blending them with new ideas in a participatory learning and action mode. The involvement has resulted in the collective decision which has enable the group to identify , clarify and select the objectives that they wish to observe and analyze, the indicators for measuring the achievements of the project's objectives and the activities needed to reach them. The main impacts were in:

-Facilitating the building of partnerships -Getting the Community involved-somehow, sometime and somewhere -Involving Community in evaluation ensured a sustainable appropriation of their innovations -Empowering local people to control their own development -Providing responsive feedback from primary stakeholders and to changing circumstances -Developing M&E capacity and empowerment oriented initiatives -Downward accountability and stakeholder participation in developing, implementing and improving the M&E process

Prospects

The action provided opportunity for joint learning among scientists and target communities through the identification of specific areas to be monitored, openly discussing challenges in adapting the monitoring and evaluation tools as well as recognizing the benefits of using the tools in project assessments. Such interaction between farmers and scientists definitely improved and both sides were learning from each other.

Keywords: Monitoring, Evaluation, Participation, Joint learning, iIdicators, Capacity enhancement, Feedback mechanisms, Lushoto, Tanzania

  Maeda, Juma Wickama. Participatory monitoring and evaluation of new technologies developed with farmers in Northern Tanzania. APPRI2008 International Workshop. Learning, producing and sharing innovations. Tools for co-construction and sustainable implementation of innovations in dryland Africa. Atelier International APPRI2008. Apprentissage, Production et Partage d'Innovations : quels outils pour le renforcement des capacités, les mesures d'accompagnement, la co-construction et la mise en oeuvre durable d'innovations dans les zones rurales sèches en Afrique?, Oct 2008, Ouagadougou, Burkina Faso. cirad-00406615 Ouagadougou, Hôtel Palm Beach, 20-24 octobre 2008

A

  baseline survey, was conducted before the field action started with the involvement of the target group from Northern Tanzania for the purpose of comparison. A methodology participatory learning action research that involves a team of multidisciplinary scientists from the National Agricultural Research Institutes (NARIs) in collaboration with target group of farmers were involved in initiating the assessment and development of new technologies. Tools used involved (i)sampling methods, (ii) Core M&E methods (stakeholder analysis & Questionnaire), (iii) methods for groups discussion (brainstorming & role plays), (iv) spatially distributed information (maps & transects), (v) time based patterns of change (diaries & photographs), and (vi) analyzing relationships and linkages (impact flow diagrams & problem trees).

  Irish potatoes and beans. The main constraints are the loss of soil fertility from excess runoff resulting in land degradation and poor available water quality and quantity. The definition of Participatory Monitoring and Evaluation (PME) differs greatly from one place to the other depending on the local context and conditions. PME is a process which stakeholders at various levels engage in monitoring and evaluating a project, share control over the content, the process and the results of M&E activity and engage in taking or identifying corrective measures.

	Participatory monitoring and evaluation of new technologies developed with farmers in Northern Tanzania Elisabeth Maeda 1 (elizabeth.maeda@kilimo.go.tz & Juma Wickama Ministry of Agriculture Food Security and Cooperatives, Tanzania
	Background
	The Lushoto district is located in the Eastern Usambara Mountains-Northeast Tanzania at an altitude of 200-2000m with annual rainfall of 1400 to 1800mm. The region have high density population 200-300/km² and 96 % rural main activity is as subsistence farming of vegetables, fruits, maize,

Participatory monitoring and evaluation of new technologies developed

with farmers in Northern Tanzania.

Elisabeth Maeda (elizabeth.maeda@kilimo.go.tz ) & Juma Wickama Ministry of Agriculture food Security & Cooperatives, Tanzania Participatory monitoring and evaluation (PME) was applied in this context where severe soil erosion, decrease ecosystems productivity, declining quality of natural resources base and increasing levels of poverty due to limited income generation alternatives were pregnant.

Ministry of Agriculture, Food Security and Cooperatives, Division of Research and Development, P.O Box 9192 , Dar es Salaam, Tanzania


domain: spi phys

text:
Mathieu Muller
email: mathieu.muller@onera.fr

Etienne Terrenoire
email: etienne.terrenoire@onera.fr

NEAR-FIELD MESH ADAPTATION FOR CONTRAIL MODELING OF A SUPERSONIC AIRCRAFT

The International Civil Aviation Organization (ICAO) needs to define standards for the advent of this new generation of supersonic aircrafts. In this context, the SENECA EU H2020 project aims at quantifying the NO x , H 2 O and contrails climate effects from supersonic aviation. 3D RANS contrails simulations are performed during the jet phase with a realistic supersonic aircraft configuration. A crucial step of the contrail simulation process related to the grid mesh optimization is described and analysed in this paper.

INTRODUCTION

As new configurations of supersonic aircrafts are expected to enter the market in next years, it is important for the International Civil Aviation Organization (ICAO) to define new certification regulations. In this context, the SENECA project aims at developing a good understanding and a detailed modelling of the LTO noise and global climate effects of water vapour, NOx and contrails. Our aim of the project is to model the formation of contrail that could occur at supersonic cruise altitude. The role played by contrail and induced cirrus remains uncertain [START_REF] Lee | The contribution of global aviation to anthropogenic climate forcing for 2000 to 2018[END_REF]. The formation and the evolution of contrail involve many complex processes controlled by aerodynamic, thermodynamic, and microphysics. The jet-phase, which represents the first instants of the contrail formation, is strongly dependant on the engine emissions and the aircraft configuration, due to the interaction between the wing-tip vortex with the exhaust flow. Previous studies have already been performed at ONERA for modelling the contrail formation with a realistic subsonic aircraft configurations [START_REF] Ghedhaïfi | Influence of atmospheric conditions on contrail formation: 3D simulation versus Schmidt-Appleman criterion[END_REF][3] [START_REF] Montreuil | Numerical Simulation of contrail formation on the Common Research Model wing/body/engine configuration[END_REF]. Hence, this work extends those previous studies to a supersonic jet. Three dimensional Reynolds-Averaged Navier-Stokes (RANS) simulations of the flow around the E-19 Aeolus jet are performed with the ONERA Computational Fluid Dynamics (CFD) code CEDRE [START_REF] Refloch | [END_REF]. A mesh adaptation is carried out with the FEFLO software [START_REF] Loseille | Achievement of second order mesh convergence for discontinuous flows with adapted unstructured mesh adaptation[END_REF] from INRIA to refine the mesh in areas of interest for the contrail formation. This paper focuses on the description and analysis of the mesh adaptation process and its validity for the aerodynamic jet-phase modelling. The associated contrail formation simulation is finally presented using the contrail module of CEDRE.

COMPUTATIONAL FLUID DYNAMICS

To simulate the plume behind the E-19 jet during the jet-phase, 3D simulations are carried out using the compressible multi-species Navier-Stokes solver CHARME integrated in the CEDRE numerical code. This code is using numerical methods based on cellcentered finite-volume approach on unstructured grids. The differential Navier-Stokes equations used in CEDRE [START_REF] Refloch | [END_REF] are the 𝑘 mass conservation equations (1), the 3 momentum conservation equations [START_REF] Ghedhaïfi | Influence of atmospheric conditions on contrail formation: 3D simulation versus Schmidt-Appleman criterion[END_REF]

𝜕 𝜕𝑡 (𝜌̅ 𝑒̃𝑡) + 𝜕 𝜕𝑥 𝑗 (𝜌̅ 𝑢 ̃𝑗ℎ ̃𝑡) = 𝜕 𝜕𝑥 𝑗 (𝜌̅ 𝑐 𝑝 𝛼 𝜕𝑇 ̅ 𝜕𝑥 𝑗 + ∑ (𝜌̅ ℎ ̃𝑡𝐷 𝑘 𝜕𝑌 ̃𝑘 𝜕𝑥 𝑗 -𝜌̅ ℎ ̃𝑡𝑢 𝑗 " 𝑌 𝑘 " ̃) 𝑘 -𝜌̅ 𝑢 𝑗 ′ 𝑇′ ̃+ 2µ𝑆 𝑖𝑗 𝑑 ̃-𝜌̅ 𝑢 𝑗 " 𝑢 𝑖 " ̃𝑢 ̃𝑖), (2)
with ρ the density, Y k the mass fraction of the k th species , u the velocity, x the coordinates, 𝐷 𝑘 the diffusion coefficient for the k th species in the gas mixture, 𝜔̅ 𝑘 the mass production rate for the 𝑘 𝑡ℎ species, 𝑔 the gravity constant, 𝛿 the Kronecker delta, µ the dynamical viscosity of the gas mixture, 𝑆 𝑑 the deviator strain-rate tensor, 𝑒 𝑡 the gas mixture total energy, ℎ 𝑡 the gas mixture total enthalpy, 𝑐 𝑝 the gas mixture specific heat capacity at constant pressure and 𝑇 the gas mixture static temperature. The index 𝑗 is used to specify the components, 𝑖 to characterize the momentum equation component, and 𝑘 to identify the species. The Favre average, corresponding to a density-weighted and time average decomposition, is used in those equations. All variables Φ are then decomposed in a mean part Φ ̅ and a fluctuating part Φ " :

Φ = Φ ̃+ Φ " , (4)
with Φ ̃= 𝜌Φ ̅̅̅̅̅ 𝜌 ̅ .

In both momentum and energy equations, the Reynolds stress tensor 𝑢 𝑗 " 𝑢 𝑖 " ̃ appears and needs to be modeled. In this study, the Boussinesq hypothesis and the twoequation 𝐾 -𝜔 with SST correction from Menter [7][8] are used to calculate the Reynolds stress tensor. A specific formulation is used to calculate the production term using vorticity instead of deformation, which is adapted for supersonic cases.

In this study, it is assumed that the complete gas-phase reactions and microphysics phenomena have no impact on the aerodynamics of the flow for the jet-phase. Thus, for the mesh adaptation process study presented in section 4, the atmospheric composition is characterized with the two main atmospheric species: O 2 and N 2 . The molar fraction of O 2 and N 2 are respectively set to 0.21 and 0.79.

RANS CONFIGURATION

Aircraft configuration

In order to accurately model contrail formation processes, a 3D RANS simulation is performed using the E-19 Aeolus's CAD developed by Cranfield University and presented on Figure 1.

Computational Domain

The computational domain contains half of the aircraft geometry. One of the box face is set as a symmetry plane.

Boundary Conditions

For the supersonic cruise, the reference altitude and the Mach number are respectively set to ℎ = 16339 m and 𝑀 = 1.6. The total temperature and total pressure are respectively 𝑇 0,𝑎 = 327.7 K and 𝑃 0,𝑎 = 0.409 atm. Thus, the static conditions for the ambient atmosphere are: 𝑇 𝑎 = 216.73 K and 𝑃 𝑎 = 9750 Pa. The wind velocity is then ‖𝑉 ⃗ 𝑎 ‖ = 472.16 m/s. The Table 2 sums up the boundary conditions of the computational domains. The angle of attack needs to be calculated for the E-19 Aeolus jet. For a supersonic aircraft, the angle of attack can be calculated with the following relation lift coefficient is defined by:

𝛼 = 𝐶 𝐿 4 √𝑀 2 -1, (5)
with 𝐶 𝐿 the lift coefficient. The lift can be calculated by:

𝐹 𝐿 = 1 2 𝜌 𝑎 ‖𝑉 ⃗ 𝑎 ‖ 2 𝑆 𝑟𝑒𝑓 𝐶 𝐿 , (6)
with 𝜌 𝑎 the ambient density and 𝑆 𝑟𝑒𝑓 the reference surface. The reference surface is the projection of the aircraft surface in the plane along the flow which is around 203 m² for the E-19 Aeolus jet. For cruise regime, the lift is balancing the weight of the aircraft, thus the lift coefficient becomes using (6):

𝐶 𝐿 = 2𝑚𝑔 𝜌 𝑎 ‖𝑉 ⃗ 𝑎 ‖ 2 𝑆 𝑟𝑒𝑓 , (7)
with 𝑚 the aircraft mass. The angle of attack thus becomes:

𝛼 = 𝑚𝑔√𝑀 2 -1 2𝜌 𝑎 ‖𝑉 ⃗ 𝑎 ‖ 2 𝑆 𝑟𝑒𝑓 . ( 8
)
The empty weight of the aircraft is 20228 kg and the full fuel weight is 45000 kg, which gives a range of 𝐶 𝐿 between 𝐶 𝐿 = 0.056 and 𝐶 𝐿 = 0.125 and a range of angle of attack between 𝛼 = 1.00° and 𝛼 = 2.23°. Thus, an angle of attack 𝛼 = 2° is chosen to be imposed, which implies that around 4.6 tons of fuel (18.7% of total fuel mass) where used before the studied cruising point. The corresponding value for the lift coefficient is thus 𝐶 𝐿 = 0.112. To respect the angle of attack, as the aircraft is oriented along the x axis, same conditions are imposed on the bottom face of the box and on the inlet. Figure 2 shows the boundary conditions for the computational domain.

Figure 2. Boundary conditions for the computational domain

For the engine, a wall condition is imposed except for the entry for which a supersonic outlet condition is applied and for the exit for which the static temperature, the static pressure and the velocity are imposed. Figure 3 illustrates the boundary conditions for the engine. A wall condition is also imposed for the other parts of the aircraft.

MESH ADAPTATION PROCEDURE AND ANALYSIS

To avoid large computational time and to correctly capture the interaction between the wing tip vortex and the exhaust flow during the jet phase which is of interest for contrails modelling, the mesh needs to be optimised in the areas of interest, where detailed physical must be captured. To distinguish those zones, a mesh adaptation technique using FEFLO [START_REF] Loseille | Achievement of second order mesh convergence for discontinuous flows with adapted unstructured mesh adaptation[END_REF] is performed. This software uses a surface and volume anisotropic re-meshing based on a prescribed Riemannian metric field. The mesh adaptation aims to reduce the interpolation error in the field, which means the difference between calculated and exact solutions. The algorithm for steady simulations is described by the following steps:  Discretization of the field by generating an initial coarse mesh  Computation of the flow field on the mesh.  Estimation of the metric-based error  Modification of the mesh with respect to these metric fields.  Projection of the surface mesh onto the true geometry using the CAD data  Interpolation of the flow solution on the new adapted mesh  Go back to step 2

The parameter 𝑉 𝑡 used for optimization is defined by:

𝑉 𝑡 = √ ∑(𝑉 𝑗 -𝑉 𝑗,𝑎 ) 𝑗 2 + max(|𝐾 -𝐾 𝑎 |, 𝐾 𝑎 ), ( 9
)
with 𝑉 𝑗,𝑎 the 𝑗 component of the upwind velocity and 𝐾 𝑎 the turbulent kinetic energy of the ambient atmosphere. This parameter takes into account two terms. The first one characterizes the velocity regarding the upwind flow. The second measures the impact of the turbulent kinetic energy in the plume compared to the ambient one. The mesh refinement depends on another parameter, the complexity 𝑐. This parameter characterizes the number of cells. When using FEFLO, it is advised to make 3 mesh refinements for each chosen complexity. Gradually increasing the complexity is then a good strategy to get more and more details on areas of interest. In this study, three complexities are chosen: 20000, 40000 and 70000. The grid mesh optimization is stopped after 3 refinements for the complexity of 70000. This choice is motivated by a compromise between the computational cost and vortices details. The Table 3 shows the mesh characteristics for each step of the mesh adaptation. For the first mesh (top left) refinement, the number of tetrahedrons decreases significantly despite a better characterization of the flow because the initial mesh far from the aircraft is too fine. The global mesh of the box is presented in Figure 4. For the initial mesh, a small area around the aircraft is refined. After the first mesh adaptation steps, two refined zones appear. The first one corresponds to the plume behind the aircraft. Another refined zone corresponds to the different shockwaves that are created due to the aircraft moving at supersonic speed. Indeed, the velocity flow is strongly impacted by the shockwave which explains the mesh adaptation in those areas. The higher the complexity, the more refined the mesh. In other regions inside the box, the mesh is coarser when the mesh adaptation is used than for the initial mesh. A zoom on the shockwaves on lateral cuts is presented in Figure 5. For the initial mesh, the area around the aircraft is refined. After 3 refinements for a complexity of 20000, the mesh starts to follow the corresponding shockwaves. For the complexity of 40000, the plume starts to be refined enough to be seen just with the mesh. Finally for the complexity of 70000, both the plume and the shockwaves are refined enough to be distinguishable. A multitude of shockwaves appears due to the geometry of the aircraft. On Figure 6 are presented transversal cuts of the mesh behind the aircraft. The vortex starts to visually appear on the mesh 20 spans behind the aircraft for a complexity of 40000. For lower complexities and for the initial mesh, the mesh is not capturing the vortex this far its formation. For the latest complexity, the interaction between the exhaust flow and the wing tip vortex on the mesh is appearing clearer.

Figure 5. Lateral cut of the mesh for the grid meshes 1, 4, 7 and 10 as defined in Table 3 a) b) c) Figure 6. Transversal cut of the mesh for the grid meshes 1, 4, 7 and 10 as defined in Table 3 On Figure 7 are presented transversal cuts of the temperature field behind the aircraft. For the initial mesh, the temperature seems homogeneous 5 spans behind the aircraft and for higher distances, which is due to a strong numerical dissipation, illustrating a bad mesh quality. For other meshes, the high temperature is concentrated in a small area at 5 spans behind the aircraft. The vortex starts to make an impact on the plume as it extends this area, which is noticeable for the complexities 40000 and 70000. For higher distances, the temperature decreases due to dissipation but remains higher on the boundary of the wing tip vortex, which is due to the interaction between the exhaust flow and the vortex.

Circulation study

To check that the vortex is correctly estimated, some criteria must be verified. First the vortex must be spatially discretized enough far from the aircraft. This is verified for the last mesh as noticed in Figure 6: there are more than 15 cells to characterize the velocity profile along any radius inside the vortex. The second criterion concerns the conservation of the circulation. If the mesh is correctly adapted, the circulation behind the aircraft must be conserved until the end of the domain.

The circulation due to the aircraft is calculated in several planes orthogonal to the flow direction by integrating the vortex vector defined by:

𝜔 ⃗ ⃗ = 𝑟𝑜𝑡 ⃗⃗⃗⃗⃗⃗ 𝑉 ⃗ . (10)
For an upwind flow following the 𝑥 axis, the circulation is then defined by:

𝛤 = ∬ ( 𝜕𝑉 𝑧 𝜕𝑦 - 𝜕𝑉 𝑦 𝜕𝑧 ) 𝑑𝑦𝑑𝑧 𝑦,𝑧 . ( 11
)
Figure 8. Circulation versus the normalized distance behind the aircraft for all complexities Figure 8 presents the circulation versus the distance behind the aircraft. For the initial mesh, a fast decrease of the circulation is observed as the vortex gets far from the aircraft, which shows that the mesh is not refined enough to correctly model the jet phase. Some fluctuations are observed for all the meshes whose amplitudes decrease with the number of mesh adaptation. Those fluctuations may be due to some interpolation errors as the circulation is calculated in planar cuts in a strongly anisotropic mesh, but remain small for the last mesh, with a maximum of 1% regarding the maximum amplitude. Ignoring the slight fluctuations, a conservation of the circulation is observed as the wing tip vortex is convected downstream. The circulation is around 345.8 m²/s which can be compared to the theoretical value deduced from the lift 𝐹 𝐿 :

𝛤 = 𝐹 𝐿 𝑏𝜌 𝑎 ‖𝑉 ⃗ 𝑎 ‖ , (12)
with 𝑏 the wing span. By combining the relations (5)( 6) and ( 12), the circulation becomes:

𝛤 = 2𝛼‖𝑉 ⃗ 𝑎 ‖𝑆 𝑟𝑒𝑓 𝑏√𝑀 2 -1 . ( 13
)
The theoretical value is then 𝛤 = 366.5 𝑚²/𝑠. The value of the circulation obtained with the simulation is thus close to the theoretical value, with an error lower than 6%. Another estimation of the circulation is possible by assuming that the aircraft in cruise regime can be approximated as an elliptically loaded wing [START_REF] Abegglen | Effective density and mass-mobility exponents of particulate matter in aircraft turbine exhaust: Dependance on engine thrust and particle size[END_REF][10] [START_REF] Jacquin | The properties of a transport aircraft wake in the extended near field: an experimental study[END_REF]:

𝛤 = 𝑚𝑔 𝐵 0 𝜌 𝑎 ‖𝑉 ⃗ 𝑎 ‖ , ( 14
)
with 𝑚 the mass of the aircraft and 𝐵 0 the vortex spacing defined by:

𝐵 0 = 𝜋 4 𝑏. ( 15
)
with 𝑏 the aircraft span. The empty weight of the aircraft is 20228 kg and the full fuel weight is 45000 kg, which gives respectively 𝛤 = 234 𝑚²/𝑠 and 𝛤 = 520 𝑚²/𝑠. The value of 𝛤 = 367 𝑚²/𝑠 is then within the permissible circulation range.

Vortex study

The third criterion concerns the vortex evolution. As the wing vortex is brought downstream, it goes down and its descent speed can be theoretically calculated with the following equation [START_REF] Paoli | Contrail Modeling and Simulation[END_REF]:

𝑉 𝑑𝑒𝑠𝑐𝑒𝑛𝑡 = 2𝛤 𝜋 2 𝑏 , ( 16
)
As the circulation is close to 350 m²/s, the wing tip vortex descent speed is close to 4.8 m/s. The simulated vortex descent speed is around 5.2 m/s, which is close to the theoretical value. The difference can be caused by the additional effect of the tailplane that is taken into account by the simulation. The temporal evolution of the wing tip vortex center position 𝑧 𝑟 is represented in Figure 9 with red crosses and its linearized evolution with the black line. Finally, the last criterion concerns the radius growth of the wing-tip vortex as it is transported far behind from the aircraft. This radius must remain lower than the mean chord of the aircraft to be assured that the numerical dissipation due to the mesh remains negligible. For the E-19 Aeolus jet, the mean chord length of the wing is 11.2 m. The evolution of the vortex radius for all complexities is presented in Figure 10. For each case, the vortex radius is calculated in transversal cuts every meter behind the aircraft. For each planar cut, the maximum of the vorticity magnitude is spotted and corresponds to the vortex center. The radius is calculated by measuring the distance between this vortex center and the position of the maximum of azimuthal velocity. For the initial mesh, the calculated vortex radius is fluctuating a lot and is higher than the mean chord, which shows that the initial mesh is not able to correctly model the wing-tip vortex transport. When 𝑐 = 20000, the growth of the wing tip vortex seems slowed. There are still some fluctuations because of interpolation approximation. For 𝑥/𝑏 < 1, the wing-tip vortex radius as a Lamb-Oseen is overestimated and should not be taken into account. For 𝑐 = 40000 and 𝑐 = 70000, the wing-tip vortex radius linearly increases with the distance behind the aircraft with the same slope. This vortex radius is still lower than the mean chord for large distance behind the aircraft (𝑟 𝑐 = 3.15𝑚 for 𝑥/𝑏 = 23), which indicates that the vortex is not strongly dissipated by numerical effects. One can notice than even for adapted meshes, some fluctuations appear which are due to interpolation errors in the transversal cuts.

𝑇 𝑟 = 𝑇 -𝑇 𝑎 𝑇 𝑚𝑎𝑥 -𝑇 𝑎 , 𝑉 𝑟 = 𝑉 -𝑉 𝑎 𝑉 𝑚𝑎𝑥 -𝑉 𝑎 , (17)
where the index "max" refers to the maximum value along the streamline coming from the center of the engine exit. The physical time from the point of view of the exhaust flow is defined by:

𝑡 𝑐𝑜𝑟𝑒 = ∫ 1 𝑉 𝑐𝑜𝑟𝑒 (𝑥) 𝑑𝑥 𝑥 0 , ( 18
)
with 𝑉 𝑐𝑜𝑟𝑒 the velocity along the streamline from the engine exit center. For the initial mesh, the normalized temperature decreases fast because of the numerical dissipation due to a coarse mesh. As the complexity increases, the mesh gets finer in the region where the vortex is transported, which explains higher normalized temperatures for longer times. After around 1s, the normalized temperature and normalized velocity get below 0.05. The dilution ratio 𝜏, defined by:

𝜏 = 1 𝑇 𝑟 = 𝑇 𝑚𝑎𝑥 -𝑇 𝑎 𝑇 -𝑇 𝑎 , (19)
is one of the characteristic parameter of the plume. The dilution ratio for the last mesh is presented in Figure 13. Two areas appear. First, a plateau can be noticed until around 10 ms where the temperature remains quasiconstant. Then, the dilution increases following a power law:

𝜏~𝑡 𝑐𝑜𝑟𝑒 𝑛 . ( 20
)
with 𝑛 ≈ 0.74 for this particular case. This value is close to the empirical value of 0.8 from Schumann et al. [START_REF] Schumann | Dilution of aircraft exhaust plumes at cruise altitudes[END_REF]. The dilution ratio follows a similar law in supersonic cruise when comparing with the subsonic data from [START_REF] Schumann | Dilution of aircraft exhaust plumes at cruise altitudes[END_REF]. When the plume age is below 20 ms, the dilution ratio is higher for the E-19 Aeolus jet than for the data from [START_REF] Schumann | Dilution of aircraft exhaust plumes at cruise altitudes[END_REF], which is due to a low temperature at the engine exit, characteristic for this supersonic cruise.

Contrail model

Once the latest mesh is obtained, a gas-phase reaction scheme based on [START_REF] Kärcher | Smallscale chemical evolution of aircraft exhaust species at cruising[END_REF][15] and already used in previous studies [START_REF] Ghedhaïfi | Influence of atmospheric conditions on contrail formation: 3D simulation versus Schmidt-Appleman criterion[END_REF][4] [16][17] is implemented in CEDRE. This scheme consists of 23 species and 60 reactions, including NO x , SO x , and HO x chemistry, which is important to characterize the soot activation process. The soot particles are assumed to be spherical and monodispersed in size. Gaseous species and particles (soot and induced ice crystals) are assumed to be in dynamic and thermal equilibrium [START_REF] Khou | Spatial Simulation of Contrail Formation in Near-Field of Commercial Aircraft[END_REF]. The soot particles are transported using an Eulerian approach. In this model, the soot particles are assumed to initially be hydrophobic. Thus, to get ice crystals, the soot coating with SO 3 and H 2 SO 4 by adsorption is modelled. The activation of the soot particles is based on [START_REF] Wong | Microphysical Modeling of Ground-Level Aircraft-Emitted Aerosol Formation: Roles of Sulfur-Containing Species[END_REF].

Additional parametrisation

The Dalton's law gives the relation between the molar fraction and the partial pressure:

𝑋 𝐻 2 𝑂 = 𝑃 𝐻 2 𝑂 𝑃 𝑎 . ( 23
)
By combining relations ( 21) and ( 23), the relative humidity is thus:

𝑅𝐻 = 𝑋 𝐻 2 𝑂 𝑃 𝑎 𝑃 𝑣𝑎𝑝 𝑠𝑎𝑡 𝑙𝑖𝑞 ⁄ , (24)
which gives a relative humidity around 0.6%, which correspond to a dry ambient atmosphere.

For the engine exhaust, typical emission indices for Jet-A1 are taken for H 2 O and SO 2 : 𝐸𝐼 𝑆𝑂 2 = 1 g/kg fuel, 𝐸𝐼 𝐻 2 𝑂 = 1233 g/kg fuel. For the soot particle, the emission index in terms number is fixed to 9.64 10 14 #/kg fuel with a radius of 𝑅 𝑠 = 27 nm. Figure 14 shows slices of the ice crystals radius at different positions behind the aircraft. As the exhaust flow moves downstream, the concentration of particle spreads horizontally. On the pictures, the maximum of the radius ice particle appears when 𝑥 𝑏 ⁄ = 5, which means the flow is already cold enough to allow the formation of ice crystals. This formation happens on the top of the exhaust flow. For larger distances behind the aircraft, the ice crystals stop growing. The ice crystals remain small regarding the soot radius, with a global maximum for the radius around 30 nm. After a distance of 20 spans behind the aircraft, the ice crystals location starts to be driven by the wing-tip vortex. The growth of ice crystals may be limited by several factors. First, the water content in the ambient atmosphere is very low for the chosen conditions. Indeed, with 𝑃 𝑎 = 9750 Pa and 𝑇 𝑎 = 216.73 K, the Schmidt-Appleman criterion gives a minimum value of 𝑅𝐻 = 33% for contrail persistency [START_REF] Schumann | A contrail cirrus prediction model[END_REF]. Thus, it is not surprising that with a value of 𝑅𝐻 = 0.6%, the growth of ice crystals is stopping fast. A higher water concentration would likely allow the growth of ice crystals during a longer time. Another factor is the number of exhausted soot particles. A lower number will allow fewer nucleation sites which could promote a higher growth of the ice crystals radius. This will be prioritized in the future work.

Results

CONCLUSION

In the present study, 3D RANS simulations of the flow around the E19-Aeolus jet in cruise phase were performed with the in-house CFD code CEDRE from ONERA. A mesh adaptation procedure using the FEFLO tool from INRIA was carried out. After several iterations of mesh refinement, the obtained solution was analysed to verify if the mesh is correctly refined in the area of interest for the contrail modelling. The criteria that have been checked are: the conservation of the circulation, the mesh discretization inside the wing tipvortex, the evolution of the radius of the vortex versus the distance behind the aircraft and its descent velocity. A preliminary test was performed to model contrail formation. A first perspective to be explored in the future is the influence of ambient specific humidity and soot number emission index on the ice crystals growth rate. A second perspective is to model the contrail aging. Indeed, the RANS 3D simulations that were performed in this study are modelling a physical time of around 0.7 s after the exhaust. Now that an adapted mesh was obtained for the jet-phase, the next step will be to simulate the vortex and dissipation phases with a temporal Large Eddy Simulation method (LES) using the results from the jet-phase as initial conditions. This work will be conducted in the context of the SENECA project.

Figure 1 .

 1 Figure 1. CAD of the E-19 Aeolus supersonic jet

Figure 3 .

 3 Figure 3. Boundary conditions for the engine

Figure 4 .

 4 Figure 4. Global mesh of the box

Figure 7 .

 7 Figure 7. Transversal cut of the temperature field behind the aircraft for the grid meshes 1, 4, 7 and 10 as defined in Table 3: 5 spans (a), 10 spans (b) and 20 spans (c)

Figure 9 .

 9 Figure 9. Wing tip vortex center position versus physical time

Figure 10 .

 10 Figure 11 represents the streamlines behind the E-19 Aeolus jet. A black dashed line represents the direction of the upwind flow from the wing tip. In both figures, the exhaust flow is initially following the direction of the plane and spreads then due to its interaction with the wing tip vortex. After a distance of 20 spans, the exhaust flow, driven by the vortex, makes less than 1 turn.

Figure 11 .

 11 Figure 11. Streamlines behind the E-19 Aeolus jet coloured by origin: orange for the engine exit, blue for the trailing edge

Figure 12 .

 12 Relative temperature (a) and relative velocity (b) versus physical time along a streamline from the engine exit

Figure 13 .

 13 Figure 13. Dilution ratio versus physical time from the engine exit for the last mesh 5. APPLICATION TO CONTRAIL MODELLING

For

  the ambient atmosphere, 21 species are added to the O 2 and N 2 species already present, including H 2 O. A H 2 O background mixing ratio representative of cruise altitude (16 to 18 km in this case study) of 3 ppm taken from [19][20]. The relative humidity RH is defined by: With 𝑃 𝐻 2 𝑂 the partial pressure of water vapor in ambient atmosphere and 𝑃 𝑣𝑎𝑝 𝑠𝑎𝑡 𝑙𝑖𝑞 ⁄the saturation vapor pressure of water, which can be estimated by the Rankine formula:

Figure 14 .

 14 Figure 14. Particle radius for different distance behind the aircraft: 𝑥 𝑏 ⁄ = 5 (𝑎), 𝑥 𝑏 ⁄ = 10 (𝑏), 𝑥 𝑏 ⁄ = 15 (𝑐), 𝑥 𝑏 ⁄ = 20 (𝑑)

  To limit the effects of the boundary on the flow, the other boundaries of the box are set far from the aircraft. The extension of the domain is around 10 spans ahead of the aircraft nose and 25 spans behind the aircraft. Both lower and upper boundaries of the box are set to 20 spans. Inside the box, the aircraft is oriented along the 𝑥 axis. The Table1presents the computational domain dimensions for the simulations carried out for this configuration. The span 𝑏 of the E-19 Aeolus jet is 14.6 m.

	Table 1. Computational domain dimensions
	𝐿 𝑥	[-10𝑏, 25𝑏]
	𝐿 𝑦	[0, 20𝑏]
	𝐿 𝑧	[-20𝑏, 20𝑏]

Table 2 .

 2 Boundary conditions of the box

	Boundary	Position	Condition
	Inlet	𝑥 = -10𝑏	V,T,P imposed
	Outlet	𝑥 = 20𝑏	Supersonic outlet
	Left	𝑦 = -20𝑏	Slip
	Symmetry	𝑦 = 0𝑏	Symmetry
	Bottom	𝑧 = -20𝑏	V,T,P imposed
	Top	𝑧 = 20𝑏	Supersonic outlet

Table 3 .

 3 Grid mesh characteristics Grid Mesh

		Complexity	Number of
			tetrahedrons
	1	-	5,817,083
	4	20,000	2,986,988
	7	40,000	3,943,205
	10	70,000	5,064,171

ACKNOWLEDGMENTS

The authors would like to acknowledge all the partners participating to the SENECA Project. This project has received funding from the European Union's Horizon 2020 research and innovation program under grant No 101006742.


Clélia Allioux

Laëtitia Achaintre

Fatima Cheataini

Baptiste Balança

Stéphane Marinesco
email: stephane.marinesco@univ-lyon1.fr

Animal welfare assessment after severe traumatic brain injury in rats

Keywords: Traumatic brain injury, rat welfare scale, humane endpoint

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

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. In humans, the Glasgow Coma Scale is used to classify patients' state of consciousness and TBI severity, ranging from mild to moderate and severe. Among the 69 million people affected by TBI each year worldwide, an estimated 5.48 million suffer from severe TBI. [START_REF] Dewan | Estimating the global incidence of traumatic brain injury[END_REF] Such patients suffer from complex and multifactorial injuries and they have a high mortality rate. [START_REF] Roozenbeek | Changing patterns in the epidemiology of traumatic brain injury[END_REF] Therefore, modelling this pathology in biomedical research is a priority.

Primary injuries are apparent immediately after a TBI has been sustained, and then evolve over hours, weeks, or even years. [START_REF] Kaur | Recent Advances in Pathophysiology of Traumatic Brain Injury[END_REF][START_REF] Prasetyo | The primary, secondary and, tertiary brain injury[END_REF] In addition to brain changes, TBI can also affect respiratory, cardiovascular, and immune functions. [START_REF] Masel | Traumatic brain injury: a disease process, not an event[END_REF] Consequently, TBI is difficult to model in vitro, in cell cultures, or in brain explants, and in vivo animal research is warranted. [START_REF] Bonder | Modeling Traumatic Brain Injury In Vitro[END_REF] The ethical use of laboratory animals requires that the benefits of the approach be carefully weighed against potential harm to animals. In TBI research, ethical concerns are especially relevant to severe TBI, after which animals may experience suffering. Animal research laboratories worldwide have committed to the three Rs-replacement, reduction, and refinement [START_REF] Russell | The Principles of Humane Experimental Technique[END_REF] -and they are additionally required to abide by national regulations. [START_REF] Bayne | Chapter 6 -Laboratory Animal Welfare: International Issues[END_REF][START_REF] Vasbinder | Chapter 3 -Regulations, Policies, and Guidelines Impacting Laboratory Animal Welfare[END_REF] Moreover, animal welfare can impact on scientific outcomes in terms of reliability and reproducibility . [START_REF] Loss | Combining Animal Welfare With Experimental Rigor to Improve Reproducibility in Behavioral Neuroscience[END_REF] Thus, for both ethical and scientific reasons, it is important to assess animal welfare after TBI accurately. The first attempts to understand and evaluate general animal welfare in research laboratories were developed in the 1980s based on behavioral and physiological criteria, such as body weight, cardio-respiratory function, and general appearance. [START_REF] Montgomery | Control of animal pain and distress in cancer and toxicologic research[END_REF][START_REF] Db | Guidelines on the recognition of pain, distress and discomfort in experimental animals and an hypothesis for assessment[END_REF] Since then, scoresheets have become increasingly specific to species and the pathology being modeled. Severity scoring rubrics have been developed for use in welfare assessment and to define humane endpoints in toxicology or cancer research. Although a brain injury-specific scoresheet has been proposed, [START_REF] Pinkernell | Severity assessment and scoring for neurosurgical models in rodents[END_REF] specific scoring grids adapted to TBI research are lacking.

Here, we introduce a scoresheet designed to monitor animal welfare following TBI that includes both general welfare indices and TBI-associated characteristics. This scoresheet was used to describe animal welfare for a period of 160 days after severe TBI induced by the lateral fluid percussion (LFP) method (5-6 months and up to 1 year is considered a long-term timeframe for TBI consequences in rodents). [START_REF] Bramlett | Long-Term Consequences of Traumatic Brain Injury: Current Status of Potential Mechanisms of Injury and Neurological Outcomes[END_REF] Following this descriptive approach, observations documented via the scoresheet provided a basis for the definition of humane endpoints, and refinement of animal care after severe TBI.

Materials and methods

Animals

Experimental protocols were approved by the CELYNE (CEA-042) committee on animals in research. APAFIS#10019-201704211705870), according to European Directive 2010/63/UE and abode to the ARRIVE guidelines. Forty-two female and male adult (10-week-old) Sprague-Dawley rats (Envigo, Gannat, France) were randomized into four groups: 10 sham females; 9 sham males; 11 TBI females; and 12 TBI males (the TBI group was designed to be larger than the sham group because of possible deaths that could be expected following severe TBI). There was no significant difference in body weight between the sham and TBI groups (females 219 [212.5-223.5] g, males 264.0 [251.5-279.5] g). The rats were housed in pairs, a sham and TBI rats, on a 12-h light/dark cycle with controlled temperature (23 ± 3°C) and hygrometry (50 ± 5%). Enrichment included one wooden stick and a sizzle nest. Rats had ad libitum food and water access. After one week of acclimatization, the animals were handled gently for 5 min/day for one week to habituate them to the laboratory conditions and the experimenter.

Surgery and TBI induction

TBI was induced using the LFP model. [START_REF] Balança | Altered hypermetabolic response to cortical spreading depolarizations after traumatic brain injury in rats[END_REF][START_REF] Mcintosh | Traumatic brain injury in the rat: characterization of a lateral fluid-percussion model[END_REF] Rats were anesthetized with isoflurane (induction, 4.5%; maintenance, 1.8-2% in 30% oxygen/70% air) and received a subcutaneous infiltration of buprenorphine (0.02 mg/kg, Buprecare ® , Axience, Pantin, France) and glycopyrronium bromide (0.1 mg/kg, Robinul-V ® , Centravet, Lapalisse, France). After 25 min, Animals were intubated with an endotracheal tube coated externally with pramocaine (Thronothane ® , Biocodex, Gentilly, France) and anesthesia was maintained via the artificial ventilation system (Ispra MA1 55-7058, Harvard Apparatus R , Edenbridge, UK; tidal volume = 6 ml/kg, breath rate = 60/min, positive end expiratory pressure = 5 cm H2O). Animals received an additional subcutaneous injection of ropivacaine (3.75 mg/mL, Naropeine, AstraZeneca LP, Courbevoie, France) and lidocaine (5 mg/mL, Xylovet ® , Ceva, Libourne, France) in the scalp 10 min prior to incision. A circular craniotomy (4 mm diameter) centered 3.8 mm caudal to Bregma and 3.00 mm lateral to the midline was performed over the right hemisphere. A luer-lock syringe tip was attached to the skull and over the cranial window with cyanoacrylate and dental acrylic cement.

Tubing (100 cm in length and 2.5 mm inner diameter, model TU0425, SMC) filled with saline was connected on one side to the LFP device (Custom Design and Fabrication, Virginia Commonwealth University, Richmond, VA), and on the other side, to the luer-lock tip cemented to the cranial window. Anesthesia was stopped and TBI was induced upon recovery of the hindpaw withdrawal reflex. This lighter anesthesia corresponded to the transition from plane 2 to plane 1 of stage 3 (moderate) anesthesia as defined by Siddiqui and Kim (2021), [START_REF] Siddiqui | Anesthesia Stages. StatPearls. Treasure Island (FL)[END_REF] and was shown to limit the loss animals by respiratory arrest after TBI. The pendulum was released, and stroke the water column, inducing a single 20 ms-5 atm fluid percussion pulse.

The pressure of the shockwave usually decreased in intensity when it reached the dura mater, and was also measured at that location using an MLT0699 Disposable Blood Pressure Transducer (AD Instruments, Oxford, UK). A few drops of doxapram hydrochloride (Dopram-V ® , Centravet) were applied on the tongue before injury, and mechanical ventilation was resumed without anesthesia. The scalp was sutured and the rat received 2 mL of saline in the flank subcutaneously. The same procedure, including a scalp incision and cranial window, was used for the sham group, but without TBI induction. Body temperature was set at 37 °C with a temperature-controlled heating pad (LSI Letica, Barcelone, Spain). Breathing and heart rate were monitored with a MouseOx (STARR Life Sciences Corp., Oakmont, PA, USA) or Etisense system (Etisense, Lyon, France). After surgery, animal consciousness was assessed with the Tübingen-Boston Rat Coma Scale (RCS). [START_REF] Pais-Roldan | Multimodal assessment of recovery from coma in a rat model of diffuse brainstem tegmentum injury[END_REF] The RCS was completed every five minutes for a total score ranging from 0 (coma) to 16 (normal full consciousness).

Rats were injected with buprenorphine at 8h and 16h after surgery, and during the first 24h they were heated with a red lamp. Moistened food was given for 1-2 weeks to facilitate food intake.

Post-TBI welfare scoresheet

The 11-item welfare scoresheet (Figure 1) was completed daily (8:00 to 9:00 a.m., 3 min by cage) for the first 28 days post-TBI by three experimenters, then by one, until 160 days. The experimenters were one PhD student with six years of laboratory experience with rodents, a zootechnician with 10 years' experience, and a master's degree student with one year of experience. The three scorers examined the animals independently and were alone in the room during scoring. Blinding was not possible because the difference in animal behavior after TBI was obvious. Animals were first observed remotely through transparent cages to determine their general condition, grimace score, breathing rate, and spontaneous behavior. The experimenter then opened the cage and scored each animal's reaction to handling.

General condition, physical appearance included coat (presence of piloerection, unkempt fur), eyes (presence of porphyrin), forelimbs and nose (clean or stained). We should note that adult animals have a tendency for a rougher, less silky coat than adolescent animals, and this should be differentiated from suffering.

Grimace scale: pain and discomfort were indexed based on facial expressions with the Rat Grimace Scale developed by Sotocinal et al. [START_REF] Sotocinal | The Rat Grimace Scale: a partially automated method for quantifying pain in the laboratory rat via facial expressions[END_REF] ; rated expressions include orbital tightening, nose/cheek flattening, and ear and whisker changes.

Breathing rate was observed visually, with attention to potential breathing problems (open mouth, wheezing, vocalizations or jerking) and cyanosis (paleness or blue coloration of the limbs, tail, ears, or nose).

Spontaneous behavior: animals must be housed in pairs to enable observation of social behavior and note any isolation from the congener. Subsequently, the experimenter moved each cage to a bench and opened it while watching the animal's behavior.

Reaction to handling: prostration, isolation, absence of movement or escape from the experimenter were considered signs of poor welfare.

Locomotion/gait were scored while the animal was on a table, with specific attention to the signs of atonia, loss of balance, and/or weakness.

Interaction with environment, enrichment: evidence of interaction with enrichment items, such as nibbling of the wooden stick or nesting.

Injury or sequelae: the surgical scar and incisor growth were examined daily. Because malocclusion can reduce food intake and lead to weight loss, incisor filing was performed after isoflurane induction (4.5%, 2-3 min procedure) with miniature saw (Dremel, France) whenever necessary until resumption of normal wear.

Hydration: hydration was determined by pinching the upper back skin and measuring its relaxation time. [START_REF] Burkholder | Health Evaluation of Experimental Laboratory Mice[END_REF] Body condition and weight: each animal was observed visually and palpated on its back and flanks to feel the prominence of its bones. [START_REF] Hickman | Use of a body condition score technique to assess health status in a rat model of polycystic kidney disease[END_REF] Four score-based welfare stages were defined: 0-10, good physical condition and absence of pain; 11-60, possible discomfort or pain, triggering increased monitoring and adapted corrective measures, including subcutaneous 50-mg glucose injection and/or appetitive food 3-4 times/day (if weight loss > 5% or dehydration), buprenorphine injection (0.02 mg/kg, subcutaneous) for pain or injury; 61-89, substantive pain and probable general suffering, triggering veterinarian advice; and ≥90, humane endpoint triggering euthanasia. Grades assigned on the scoresheet increased exponentially as the symptoms worsened, following a similar approach to Pinkernell et al. [START_REF] Pinkernell | Severity assessment and scoring for neurosurgical models in rodents[END_REF] Pinkernell et al. [START_REF] Pinkernell | Severity assessment and scoring for neurosurgical models in rodents[END_REF] proposed a scoresheet for both TBI and subarachnoid hemorrhage models, that was used here to evaluate animal welfare independently of our own scoresheet for comparison during 20 days.

[Insert Figure 1] Endpoints Two peri-operative endpoints were defined: dural tear; and respiratory failure requiring mechanical ventilation for >45 min. Subsequently, a veterinarian indicated if euthanasia was warranted based on daily interaction. When necessary and at the end of the experiments, euthanasia was performed with 5% isoflurane anesthesia followed by lethal intraperitoneal injection of barbiturates (100 mg/kg, Euthasol ® , Centravet). Finally, the brain was removed for further histological analysis.

Data analysis

Data are expressed as median [interquartile range]. Data were analyzed in Prism ® version 8.4.3 (Graphpad software LLC, San Diego, CA, USA) or R software 4.0.3. [START_REF] Core | R: A Language and Environment for Statistical Computing[END_REF] Raw data were first analyzed for normality with the Shapiro-Wilk test. For two-group comparisons, normally and non-normally distributed values were analyzed with Student's unpaired t-tests and Mann-Whitney U tests, respectively. Including both male and female animals in the study warranted statistical analysis with a two-way ANOVA for sex and TBI. Before the two-way ANOVA was performed, data samples were tested for homoscedasticity with the Levene test and normality of residues. For non-normally distributed residues or heterogeneity, raw data were transformed with the Box-Cox power transformation. [START_REF] Box | An Analysis of Transformations[END_REF] If the two-way ANOVA revealed an interaction between sex and TBI, a Mann-Whitney U post-hoc test adjusted by the Bonferroni method was applied to identify specific differences. If not, no post-hoc tests were performed. Differences were considered significant at p < 0.05 for ANOVAs and at p < 0.025 for repeated Mann-Whitney U tests (two repetitions for males and females).

Inter-experimenter variability was studied by calculating intraclass correlation coefficient, expressed with 95% confidence intervals based on a mean-rating (k = 3) absoluteagreement/two-way mixed-effects model, and interpreted based on that of Koo et al. [START_REF] Koo | A Guideline of Selecting and Reporting Intraclass Correlation Coefficients for Reliability Research[END_REF]

Results

Severe TBI induction and acute recovery

Although LFP induction produced a similar 5 atm shockwave in the water column, dural peak pressure diminished to a lower value in males (2.5 [2.4-2.6] atm) than in females (2.8 [2.6-2.8] atm; t(21) = 3.04; p = 0.006). Eights rats placed in the TBI group were not included in the analysis: five were euthanized due to a dural tear; one died spontaneously in the first hour following TBI; and two did not show evidence of TBI from RCS. Welfare worsening following TBI was attributable to virtually all scoresheet items. Compared to sham animals, TBI animals took longer to recover a silky coat (female, p < 0.001; male, p = 0.004) and had more days with probable pain (grimace scale 10; F, p < 0.001 and M, p = 0.008), dyspnea (F, p < 0.001 and M, p = 0.012), abnormal responses to handling (F, p < 0.001 and M, p = 0.002), and impaired locomotion (both p < 0.001). TBI rats had greater weight loss than sham rats (F, p < 0.001 and M, p = 0.005), and took several days to normalize (female 16 days, p < 0.001 and male 8 days, p = 0.016) (Figure 3 (c)). Finally, dehydration paralleled the changes observed in body weight, lasting 8-16 days in TBI rats, versus 0-1 day in sham animals (F, p < 0.001 and M, p < 0.005). Detailed results for each scoresheet item are shown in Table 1.

TBI female rats took longer than males to recover a normal coat (10. 1). At 160 days post-surgery, no significant differences between sham and TBI animals were evident. Notwithstanding these sex differences suggesting a greater impact on female well-being, our scoresheet detected suffering or pain during less than two weeks in both sexes. Welfare scores were then similar between sham and TBI animals, indicating that suffering, if present at these later time points, was not detectable by our scoring method. Results of score from each item can be found in Supplementary Table S1.

The scoresheet was also used to decide on animal care. For example, analgesic treatment with buprenorphine was administered during the first 24 h after injury, and was prolonged or reinstated if reaction to handling, grimace scale and/or mouth injury scored ≥10. This happened during the first week for 12/15 animals with severe TBI (all female, 4 males), but only for one sham female. In addition, subcutaneous saline or glucose injections were decided if dehydration or body weight scored ≥10, which happened in 12/15 TBI animals (all females, 4 males) and never for sham animals in the two weeks after surgery. Rats in which visual inspection revealed abnormally long incisors and displaying a welfare score ≥5 had their incisors filed. This happened in 13/15 animals (all females, 5 males) at 4 days, 9/15 animals (6 females, 3 males) at 1 week, 7/15 animals (4 females, 3 males) at 28 days and 3/15 (1 female, 2 males) at 160 days after the TBI.

[Insert Figure 3]

[Insert Table 1]

Inter-rater reliability and an alternative scoresheet

Our scoresheet was validated across three evaluators with different levels of education and lab experience. Scoring took about 3 min per cage of two animals, and was very consistent among the three experimenters for 22/23 TBI animals, with only one female receiving high scores >90 by two experimenters, but not the third during the first four days post-surgery. Overall, even taking this animal into account, the intraclass correlation coefficient was 0.89 (0.88-0.91) (Figure 4(a) and supplementary figure S1). [START_REF] Koo | A Guideline of Selecting and Reporting Intraclass Correlation Coefficients for Reliability Research[END_REF] We also applied an alternative scoresheet used for brain injury. [START_REF] Pinkernell | Severity assessment and scoring for neurosurgical models in rodents[END_REF] Using this scale, 13/15 TBI animals (8 females, 5 males) that recovered a normal condition within two weeks reached a total score that would have triggered euthanasia from 1-4 days after surgery (Figure 4(b-c)) due to maximal score for spontaneous behavior (1 female), and weight loss (2 females, 2 males), and/or maximal overall score (12 animals, 7 females, 5 males). Among these animals, all but two recovered normal scores ≤10, 28 days after TBI (except one male that scored 12, and one female at 30), and all were ≤5 after 160 days, indicating that suffering was no longer detected in these animals in the long-term. Results from each item can be found in Supplementary Table S2.

[Insert Figure 4]

Discussion

The presently introduced welfare scoresheet was developed specifically for assessing animal suffering after severe TBI. It includes general wellbeing items, such as weight, hydration, fur condition, and interactions with conspecifics, experimenters and toys, as well as TBI-associated items, such as respiratory function, gait, and the presence of wounds. The LFP method often induces transient respiratory dysfunction and mimics systemic complications in human patients.

Gait deficits in TBI models parallel motor deficits [START_REF] Song | Differences in pathological changes between two rat models of severe traumatic brain injury[END_REF][START_REF] Hausser | Detecting Behavioral Deficits in Rats After Traumatic Brain Injury[END_REF][START_REF] Yu | Severity of controlled cortical impact traumatic brain injury in rats and mice dictates degree of behavioral deficits[END_REF] and immobility/paralysis 26 in human patients. These deficits are not apparent in many rodent models of human disease, and should be specifically scored after TBI. In addition, TBI model rats should be monitored for the presence of wounds, excessive incisor growth, reduced food intake, weight loss, and a poor general condition. This scoresheet took only a few minutes to complete, and was implemented with a high degree of correlation across evaluators, even with different backgrounds in education and lab experience, suggesting high robustness.

Welfare scores took longer to normalize for females than males, including a longer lasting impact on grooming, dehydration, and interaction with environmental enrichment products. In humans, a meta-analysis suggested women may fare worse than men following mild-moderate TBI, but may fare better than men following severe TBI. [START_REF] Gupte | Sex Differences in Traumatic Brain Injury: What We Know and What We Should Know[END_REF] Females have been reported to fare better than males in some animal models. [START_REF] Gupte | Sex Differences in Traumatic Brain Injury: What We Know and What We Should Know[END_REF][START_REF] Rubin | Sex Differences in Animal Models of Traumatic Brain Injury[END_REF][START_REF] Spani | Sex-related responses after traumatic brain injury: Considerations for preclinical modeling[END_REF] Prior examination of sex differences in outcomes of moderate-injury LFP model rats have also revealed higher mortality but smaller contusions in females. [START_REF] Bramlett | Neuropathological protection after traumatic brain injury in intact female rats versus males or ovariectomized females[END_REF][START_REF] Emerson | Increased mortality in female rats after brain trauma is associated with lower free Mg2+[END_REF][START_REF] Suzuki | The importance of gender on the beneficial effects of posttraumatic hypothermia[END_REF] Here, worse outcome in females was observed, but was also associated with a higher pressure applied to the dura. This result suggests that biomechanical factors, such as a smaller intracranial volume in females, [START_REF] Hughes | A longitudinal radiographic study of the growth of the rat skull[END_REF] should be taken into account when assessing sex differences in TBI outcome, for example by comparing female and male rats of similar body weight (but different ages). Further studies are therefore required to better understand the sexual dimorphisms that may contribute to differences in TBI outcome.

To our knowledge, there are no specific studies applying a welfare scoresheet to a severe TBI model. An alternative scoresheet by Pinkernell et al. was designed for brain injury in general, but led to high scores and would have led to euthanasia in most of our animals, due to weight loss (>20%), immobility scores, and/or epileptic seizures. [START_REF] Eakin | Modeling Fluid Percussion Injury: Relevance to Human Traumatic Brain Injury[END_REF][START_REF] Pitkänen | Epileptogenesis After Traumatic Brain Injury[END_REF] Unspecific scoresheets and poorly adapted endpoints therefore have the potential of leading to unnecessary euthanasia of the animals. Notably, we observed rapid recovery to normal welfare scores within two weeks of LFP. Severe TBI was demonstrated by loss of consciousness for >1 h, respiratory arrest lasting 10-30 s with cyanosis, and heart rate modifications lasting ~5 min. The transience of effects on welfare scores in our rats differs from the long-term cognitive problems and disability found in 60-65% of patients with severe TBI, 2, 37 perhaps due to a higher degree of neuroplasticity and neurogenesis [START_REF] Rosa | Brain Plasticity in Mammals: An Example for the Role of Comparative Medicine in the Neurosciences[END_REF][START_REF] Paredes | Brain size and limits to adult neurogenesis[END_REF] as well as accelerated timescales of metabolic, inflammatory, and axonal dysfunctions in rodents. [START_REF] Agoston | How to Translate Time: The Temporal Aspects of Rodent and Human Pathobiological Processes in Traumatic Brain Injury[END_REF] Our study was limited to welfare assessment, and thus we did not track behavioral deficits or neurological lesions that might persist beyond 160 days.

Most importantly, data collected with this welfare scoresheet can provide information relevant to the ethical justification of animal use in TBI research. Careful rating of procedures in terms of animal welfare and injury severity is needed to clarify benefits to society versus hardship for animals. The present data demonstrate that LFP-induced severe TBI does not induce longlasting distress or suffering that could be detected by our scoresheet. By principle, any unnecessary suffering should be avoided in animal research, and when suffering is inevitable, its impact should be carefully weighed against expected benefits to society. Careful characterization of animal welfare using a scoresheet precisely adapted to TBI is therefore a pre-requisite for accurate judgement of the costs of animal research on severe TBI by ethical committees, and supports a justified use of rodents in such research projects. Our scoresheet is also helpful for defining precise humane endpoints for animals with severe TBI in accordance with the 3R criteria. [START_REF] Russell | The Principles of Humane Experimental Technique[END_REF] We propose that a weight loss trigger for euthanasia be 30% rather than the common 20% limit [START_REF] Pinkernell | Severity assessment and scoring for neurosurgical models in rodents[END_REF][START_REF] Morton | A systematic approach for establishing humane endpoints[END_REF] because 20~30% weight loss is common in moderate-to-severe TBI [START_REF] Eakin | Modeling Fluid Percussion Injury: Relevance to Human Traumatic Brain Injury[END_REF] , and in our study, animals that lost up to 30% of their body weight recovered their normal weight within two weeks if other welfare indicators were improving. This higher limit would prevent unnecessary euthanasia and thus could reduce the total number of animals used in TBI projects. This study can provide experimental evidence for discussions with ethical committees worldwide, and possible adjustments of endpoints for animals with severe TBI. Other humane endpoints are respiratory dysfunction, total paralysis, irreversible infection, untreatable wounds, and a total score of ≥90 on our welfare scoresheet. Optimal adjustment of humane endpoints is key to the reduction of the number of animals included in experimental groups, as well as the refinement of procedures that may induce suffering.

Finally, welfare scores obtained during the first week following TBI could also provide an indication of the severity of the trauma, in addition to shockwave intensity, loss of consciousness indicated by RCS, withdrawal and righting reflexes recovery, and possible neurological lesions apparent on MRI or histological images. Mild, moderate and severe TBI are still poorly defined in animals, and it is possible that this scoresheet could participate in a better characterization of TBI severity, by taking into account animal welfare.

Study limitations

This study was performed using a limited sample size, and the scoresheet presented here has not been validated by multiple research centers. Blinding the scorers was not possible due to the large difference in welfare between sham and TBI animals during the first weeks following surgery. This could have potentially impacted the reliability of our scoring at later time points.

We should also mention possible neurological effects on repeated anesthesia in animals requiring frequent incisor filing. We are aware that some but not all ethical committees might allow an endpoint definition at 30% weight loss, depending on national regulations and local ethical sensitivities.

Conclusion

This study proposes a reproducible and easy-to-use welfare scale adapted to severe TBI in rodents. In addition to providing semi-quantitative estimates of animal welfare to refine TBI models, this scoresheet demonstrated that suffering is apparent during a transitory period following TBI, but no longer detected in the long term, with animals recovering normal welfare within less than two weeks.

  Relative to sham animals (N = 19; 10 females), the remaining TBI animals (N = 15; 8 females) required an extended surgery-recovery time, as indicated by hindpaw withdrawal reflex (36.0 [18.0-72.0] min vs. 0 min in sham animals), righting reflex recovery (84.0 [51.0-90.0] min vs. 3.0 [0.0-30.0]; F(1,30) = 134.40; p < 0.001) and RCS normalization (100.0 [75.0-115.0] vs. 5.0 [5.0-10.0] min; F(1,30) = 161.4; p < 0.001) (Figure 2). Immediately after TBI, we observed a 10 to 30-s respiratory arrest and abnormal heart rhythms (tachycardia and bradycardia) with cyanosis for about 5 min. Female rats took more time to recover their righting reflex (100.0 [82.5-145.0] min) than males (85.0 [40.0-150.0]; F(1,30) = 7.44; p = 0.011), and showed a trend toward a longer hindpaw withdrawal reflex recovery (females, 57.0 [26.25-73.50] min; males, 34.0 [10.0-46.0] min; t(13) = 1.91; p = 0.088). Finally, 11/15 TBI rats (4 females and 7 males) had a subdural hematoma. These observations indicated severe TBI. [Insert Figure 2] Applying our scoresheet to rats with severe TBI Score of TBI animals worsened dramatically during the first 1-2 days postoperatively and then improved gradually thereafter until reaching stable basal values (< 0.001; post-hoc Mann-Whitney U sham vs. TBI female p < 0.001) with an interaction of sex and TBI variables (F(1, 30) = 7.19; p = 0.02) but no significant difference between female and male groups (F(1, 30) = 1.577) (Figure 3(b)).

  5 [7.0-26.8] vs. 4.0 [3.0-8.0] days, p = 0.019), were dehydrated longer (15.5 [9.0-23.3] vs. 7.0 [4.0-8.0] days, p = 0.007), and took longer to recover their normal body condition (12.5 [9.0-23.5] vs. 6.0 [3.0-9.0] days, p = 0.01). Body condition (thinness, 12.5 [9.0-23.5] days) and interaction with the environment (1.0 [0.3-2.8] day) were significantly impacted compared to sham animals in females only (p < 0.001 and p = 0.002, respectively) (Table

Figure 1 .

 1 Figure 1. Scoresheet for severe traumatic brain injury. 0-10 normal physical condition, no pain;

Figure 2 .

 2 Figure 2. Return to consciousness after severe traumatic brain injury (TBI). Hindpaw

Figure 3 .

 3 Figure 3. Animal welfare after severe TBI. (a) Evolution of welfare scores in sham and TBI

Figure 4 .

 4 Figure 4. Inter-rater reliability and excess mortality with alternative welfare scoresheet. (a)

Table 1 .

 1 Return to basal levels of scoresheet criteria and minimum weight after the severe traumatic brain injury (TBI). Values are presented as median [inter-quartile range]. Repeated Mann-Whitney U tests (p < 0.025).

Females

Figure 4

 4 Figure 4

Table 1

 1

Acknowledgements

CA is the recipient of a doctoral fellowship by Région Auvergne-Rhône-Alpes. Foundation Gueules Cassées "Sourire Quand Même" (grants FGC57-2020 and FGC34-2018) supported this research. We thank Mélodie Borel, Ellora Sequeira and Jenya Dursun for help with experiments and for their assistance in data collection. Thanks to Yannick Tholance for the discussions on statistics, Sophie Picavet and Manon Dirheimer for veterinarian advice.

Author contributions

CA and SM designed the welfare scoresheet. CA performed in vivo experiments. CA, LA and FC scored animals and retrieved data. CA, SM and BB contributed to the design of statistical analyses. CA and SM wrote the manuscript, with contributions made by all of the other authors.

Disclosure statement

No competing financial interests exist.