Alexandra Basilio Lopes

Mickael Choury

Patrick Wagner

Mihaela Gulea

Tandem Double-Cross-Coupling/Hydrothiolation Reaction of 2-Sulfenyl Benzimidazoles with Boronic Acids

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Tandem Double-Cross-Coupling/Hydrothiolation Reaction of 2-Sulfenyl Benzimidazoles with Boronic Acids

Alexandra Basilio Lopes, Mickael Choury, Patrick Wagner, and Mihaela Gulea* Université de Strasbourg, CNRS, Laboratoire d'Innovation Thérapeutique, LIT UMR 7200, 67412 Illkirch, France Supporting Information Placeholder A new tandem palladium-catalyzed reaction involving a Suzuki-Miyaura coupling, a desulfenylative coupling, and a hydrothiolation of a triple bond is reported. Notably, the desulfenylative coupling occurs without copper(I)-assistance and the hydrothiolation is totally regio and stereoselective. The overall process results in the double incorporation of the boronic acid and the reincorporation of the sulfenyl moiety into the product structure. Starting from 2-(bromobenzylsulfenyl)-1-propargyl benzimidazoles the transformation led to variously substituted benzimidazoles bearing a stereodefined alkenyl sulfide.

Due to the important role of organosulfur compounds in synthetic chemistry and in applied fields such as materials science and pharmaceutical industry, the research of efficient methods for the C-S bond formation is constantly advancing. Among these methods, transition-metal-catalyzed crosscoupling of S-nucleophiles with aromatic or alkenyl halides and hydrothiolation of double or triple bonds were widely studied and developed in recent years. [START_REF] Beletskaya | Reviews on metal-catalyzed C-S formation[END_REF]2 In parallel to this, metalcatalyzed reactions involving a C-S bond cleavage with release of the sulfur-function of the S-electrophilic partner, emerged as a complementary and practical alternative to more classical C-C cross-couplings using organohalides. [START_REF] Hea | Reviews on desulfurative cross coupling[END_REF] One well-known method based on C-S cleavage was developed starting with the publication in 2000 by Liebeskind and Srogl 4 of a new reaction consisting of the Pd-catalyzed coupling of thioesters with boronic acids, in the presence of a stoichiometric amount of thiophene-2-carboxylate (CuTC) as an activator. This method found a huge amount of synthetic applications and was extended to other nucleophilic coupling partners and particularly to heteroaromatic sulfides substrates. [START_REF] Prokopcova | Reviews on the Liebeskind-Srogl coupling[END_REF] In 2012, Weller and Willis reported the Rh(I)-catalyzed carbothiolation of alkynes by aryl methyl sulfides, involving an Ar-S bond cleavage. 6 This represents a very interesting atom economical process, as the methylsulfenyl leaving-group released in the course of the reaction is integrated in the final product as an alkenyl methyl sulfide. Due to the widespread occurrence in biologically active compounds and drugs of benzazole moiety, 7 it was not surprisingly that 2-sulfenyl derivatives of these heteroaromatics have been used as substrates in the above-mentioned metal-catalyzed cross-coupling reactions. Thus, 2-sulfenyl benzoxazoles and benzothiazoles have been successfully coupled with aryl or heteroaryl boronic acids under the Liebeskind-Srogl reaction conditions (Scheme 1, Eq 1). 8 Furthermore, the alkynes carbothiolation was applied to 2sufenyl benzoxazoles and benzothiazoles by Nishihara and coworkers using Pd-catalysis (Scheme 1, Eq 2). 9 Surprisingly, we did not find examples involving benzimidazole substrates. In an approach inspired by these two methodologies and by an unexpected finding obtained in a previous study (see below for details), we report here a Pd-catalyzed tandem one-pot transformation which consists of a classical Suzuki-Miyaura coupling and a desulfenylative cross-coupling of 2benzylsulfenyl benzimidazole with aryl boronic acids, followed by an intermolecular regio and stereoselective hydrothiolation of the triple bond of the N-propargyl benzimidazole (Scheme 1, Eq 3). As the result of the overall transformation, the sulfenyl moiety is reincorporated into the product structure as an alkenyl benzyl sulfide, and the aryl substituent provided by the boronic acid partner is introduced twice, once in the 2-position of the benzimidazole and second on the benzene ring of the benzylsulfenyl group. It was possible to achieve the two types of coupling with the boronic acid under the same reaction conditions as in our case the desulfenylative coupling occurred efficiently without use of copper(I) carboxylate. Thus, substrates bearing an aryl bromide (present in the form of a bromobenzyl substituent on the S-atom) were used. This choice could be considered as complementary to the strategies using orthogonal reactivity, 10 increasing the molecular complexity and diversity of the final products. 18 examples (up to 82%) Br -tandem Pd-catalyzed C-C/C-S bond formation -copper(I)-free desulfenylative coupling -thiol reincorporation by selective hydrothiolation

R 1 B(OH) 2 R 1 B(OH) 2
Scheme 1. Pd-catalyzed processes involving desulfenylative cross-coupling of 2-sulfenyl benzazoles.

Initially, substrate 1a was designed to produce thiazocine 3aaA via a Pd-catalyzed 8-exo-dig cyclization/ Suzuki coupling domino reaction (Scheme 2), based on a methodology that was developed previously in our group to access five and sixmembered sulfur-heterocycles. 11 In a preliminary experiment, by reacting 1a with phenyl boronic acid 2a, a very small amount of the desired product 3aaA was detected (2%), [START_REF] Basilio Lopes | Another approach was then developed to access thiazocines[END_REF] together with a majority of product 3aaB resulting from the competing direct coupling (58%), but also with two other products which were identified as the desulfenylative coupling product 3aaC (19%), and the unexpected alkenyl sulfide 3aa (11%). Each product was isolated from the crude mixture and characterized by NMR and mass spectroscopy. We noted that the hydrothiolation of the triple bond took place with a remarkable regio and stereoselectivity, as alkenyl sulfide 3aa was present as a single stereoisomer, resulting from the syn-addition of the thiol 2,[START_REF] Zhang | The Pd-catalyzed syn-addition of a thiol to a triple bond has been already established; see a proposed mechanism studied by DFT[END_REF] on the carbon bearing the ethyl substituent. The structure of 3aa and the stereochemistry of its double bond have been unambiguously assigned by a 2D-Noesy experiment (see spectra in SI). In this context it should be noted that hydrothiolations of internal disymmetric alkynes are rather rare. Intrigued by this result and considering the originality of the tandem transformation, we decided to optimize the reaction conditions to obtain 3aa as the main product. Many experiments have been done varying all parameters (Pd-source, ligand, base, solvent, temperature, reaction time) and selected results are summarized in Table 1 (see more details in SI). Compared with the initial reaction conditions (Scheme 2), we increased the amount of the boronic acid (as 2 equiv are anyway necessary to obtain 3aa), of the base, and the catalyst loading (from 10% to 15% mol Pd), and a significant improvement (43% yield, entry 1) was obtained by removing TBAI, using K2CO3 as the base, changing the reaction concentration from 0.05 M to 0.1 M, and prolonging the reaction time from 3 h (at 130 °C, MW) to 19 h (at 130 °C, in oil bath). Keeping the same catalyst and base but heating only for 8 h, at 140 °C, in oil bath, the yield turned low again (13%, entry 2). Changing the base to K3PO4 did not modify the result, while the use of LiOH decreased slightly the yield (entries 3 and 4). The conventional Pd(OAc)2/PPh3 catalytic systems was found to be less effective (entry 5). Then, Pd2(dba)3 was used in combination with different phosphine ligands, such as tri(2-furyl)phosphine (TFP), tricyclohexyl phosphine (PCy3), or di(1-adamantyl)-n-butylphosphine (catCXiumA) (entries 6-8). The best result was obtained with the Pd2(dba)3/PCy3 combination (entry 7) and the yield was even improved with less catalyst loading (entry 9). The amount of catalyst used in this reaction remains high compared in particular to domino reactions involving carbopalladation, however, the presence of sulfur species represents a difficult case due to the catalyst deactivation caused by the thiophilicity of palladium. Thus, the use of 5 mol% of Pd2(dba)3 enabling a satisfactory yield of 66% with respect to three one-pot Pdcatalyzed process can reasonably be accepted. Thus, these last reaction conditions were kept for the further stages of the study. Scheme 2. Initial Pd-catalyzed reaction of benzimidazole 1a with boronic acid 2a. Table 1. Optimization of the reaction of 1a with 2a to access 3aa. [a] entry Pd-catalyst ligand L base yield (%) [b] 1 Pd(PPh3)4 -K2CO3 43 2 [c] Pd(PPh3)4 -K2CO3 Pd2(dba)3 cataCXium K2CO3 33 9 [d] Pd2(dba)3 PCy3 K2CO3 66 [a] Reaction conditions: 1a (1 equiv), 2a (3 equiv), catalyst loading (15 mol% Pd), ligand (30 mol%), base (3 equiv), dioxane/H2O 8/2 or 7/3 (0.1 M), 130 °C, 19 h. [b] Isolated yield. [c] Performed at 140 °C, for 8 h. [d] Pd2(dba)3 (5 mol%), ligand (20 mol%).

The sequence of this Pd-catalyzed tandem reaction would reasonably consist of three steps: direct Suzuki coupling on the phenyl bromide moiety of 1a with boronic acid 2a leading to 3aaB, desulfenylative coupling of the latter with 2a to give 3aaC and thiol 4, and hydrothiolation by 4 of the triple bond of 3aaC to afford the alkenyl sulfide 3aa as the final product (Scheme 3, I). To confirm this, we decided to perform these different steps independently under the same Pd-catalyzed reaction conditions (Scheme 3, II, III). First, compound 3aaB was prepared separately from commercial reagents, by Sbenzylation with 2-(bromomethyl)-1,1'-biphenyl and subsequent N-propargylation with 1-bromopent-2-yne of 2mercaptobenzimidazole. Its reaction with boronic acid 2a under the established reaction conditions led to the expected product 3aa in 63% yield, via a tandem desulfenylative crosscoupling/hydrothiolation (Scheme 3, II). To examine the triple bond hydrothiolation, we prepared substrate 3aaC by Npropargylation of 2-phenyl-benzimidazole, as well as the appropriated thiol 4 in two steps from 2-(bromomethyl)-1,1'biphenyl and potassium thioacetate. Under the same reaction conditions, the hydrothiolation led to the expected product 3aa (36% yield), which was separated from the recovered starting materials, 3aaC and the disulfide formed from thiol 4 (Scheme 3, III). the same Pd-catalyst and reaction conditions were used, the absence of the boronic acid and the different nature of the precursor of the active Pd-S species could explain the lower yield in product 3aa obtained in this way. Scheme 3. Proposed mechanism and control experiments.

We also examined the importance of the presence of the aryl bromide and of the triple bond on the reaction (Scheme 3, IV, V). Suppressing the bromine on the benzene ring of the substrate, the desulfenylative coupling/hydrothiolation process worked in a satisfactory manner, leading to the expected alkenyl sulfide 6 in 48% isolated yield (Scheme 3, IV). Remarkably, all these hydrothiolations have occurred with total regio and stereoselectivity, as only one stereoisomer of the alkenyl sulfide is formed. For comparison, we also reacted substrate 5 with 2a under classical Liebeskind-Srogl conditions (1.3 equiv CuTC, 4 mol% Pd2(dba)3, 16 mol% TFP, in THF, 50 °C, 18 h). 8 The result obtained in this case showed the particularity of our substrate, as the crude mixture contained the starting product 5, the desulfenylative coupling product 3aaC, and the hydrothiolation product 6 in a ratio of 67:20:13. Finally, we checked if the presence of the triple bond is beneficial to the desulfenylative coupling process, which has no need of Cu(I)carboxylate in our case. We prepared for this purpose 2-(benzylthio)-1-methyl-benzimidazole 7 in which the propargyl chain was replaced by a methyl substituent. Its reaction with PhB(OH)2 under the same catalytic conditions did not afford the coupling product 8, only the starting material was recovered (Scheme 3, V). The latter result, together with the low yield obtained in the hydrothiolation of 3aaC with thiol 4, may suggest that the triple bond is ideally placed to coordinate the palladium-thiolate species, thereby facilitating the hydrothiolation step in the tandem process. The regioselectivity could be explained by the fact that the sp-hybridized carbon bearing the 1-methylene-2-phenyl benzimidazole is more sterically hindered than the ethyl-substituted one. The validation of such hypotheses needs however additional experimental and theoretical studies. We next investigated the scope and limitations of the reaction by varying the substituents on the substrate and the boronic acid partners (Table 2). [Pd] tandem process

Table 2. Tandem reaction of various substrates 1 and aryl boronic acids 2.

(II) 3ba (70%) N N S Et Ph N N S R Ph Ph Ph R = Et, 3ca (82%) R = Ph, 3da (3%) N N S Et X = OMe: 3cb (62%) X = OCF 3 : 3cc (64%) X = F: 3cd (57%) X = CF 3 : 3ce (46%) reaction conditions N N S R R = Et, R 2 = H: 1a (2-Br), 1b (3-Br), 1c (4-Br) R = Ph, R 2 = H: 1d (4-Br) R = Et, R 2 = OMe: 1e (2-Br, 5-MeO) 3 Br N N S R R 1 R 1 X X N N S Et R 1 B(OH) 2 2a-h X = H: 3ea (42%) X = OMe: 3eb (56%) X = F: 3ed (43%) N N S Et OMe X X X = NO 2 : 3cf (+ 3cfC, 18%) X = CF 3 : 3cg (66%) X = OMe: 3ch (74%) R 2 R 2 3aa (66%) N N S Et Ph X Ph X 1 [Pd]
[a] Reaction conditions: 1 (1 equiv), 2 (3 equiv), Pd2(dba)3 (5 mol%), PCy3 (20 mol%), K2CO3 (3 equiv), dioxane/H2O 4/1 (0.1 M), 130 °C, 19 h; isolated yields are given.

Thus, we prepared two other 2-(bromobenzylsulfenyl)-1propargyl benzimidazole derivatives 1b and 1c, bearing the bromide in meta and para position, respectively. Their reaction with PhB(OH)2 2a under the optimized reaction conditions, led to the expected products 3ba and 3ca, in higher yields (70% and 82%, respectively) than that obtained with the ortho derivative 1a. In the case of substrate 1d we changed the substituent on the triple bond, from ethyl to phenyl. The reaction of 1d with 2a afforded a complex mixture, in which only a very little amount of the expected alkenyl sulfide 3da was isolated and characterized by 1 H NMR and mass analyses. We then decided to pursue the study by reacting the substrate 1c with various boronic acids. By using electron-rich or electron-poor arylboronic acids substituted either in para or in meta position, the reaction worked well, leading to the desired alkenyl sulfides 3 with yields ranging from 46% to 74%. In the case of product 3cc the procedure was scaled up on 1 mmol (50% isolated yield). Only in the case of the use of 3-(nitrophenyl)boronic acid 2f the crude reaction mixture contained unreacted substrate 1c, both of coupling products 3cfB and 3cfC, and the desired compound 3cf (ratio 2c/3cfB/3cfC/3cf 2:1:1:2). After column chromatography, an inseparable mixture of 3cf and 3cfC in a 3/1 ratio was obtained. Finally, we prepared also substrate 1e having bromine in ortho-position and a methoxy group in metaposition, and involved it in reactions with three boronic acids, 2a, 2b and 2d. The corresponding desired products were obtained in satisfactory yields, up to 56%. Other modifications of the substrate were made to determine their influence on the efficiency of the reaction and to identify its limitations. Thus, the heterocyclic part of the substrate was modified by replacing the benzimidazole by an imidazole and by an indole (Scheme 4). Starting from the 4,5-diphenylimidazole derivative 1f and phenyl boronic acid 2a, the tandem process still worked well, leading to the expected alkenyl sulfide 3fa in this case the use of Pd(PPh3)4 furnished a better yield than the use of Pd2(dba)3/PCy3 catalyst (53% vs 39%). By contrast, the indole-based substrate 1g in which the sulfenyl group led under the same conditions only to the direct coupling product 3gaB, isolated in 43% yield. Scheme 4. Scope and limitations: replacement of benzimidazole by another heterocycle.

Finally, we investigated the challenging possibility to perform this reaction one-pot, keeping the same reaction conditions, by using two different boronic acids, one for the debrominative coupling and another for the desulfenylative coupling (Scheme 5). Therefore, substrate 1c was placed in the reaction conditions with 1 equiv of boronic acid 2b. After 4 h the starting material was consumed, and the main product detected by HPLC was the Suzuki coupling product (3cbB). At this stage 1.5 equiv of boronic acid 2a was added and the reaction continued for 15 h. The desired product 9 was identified in the crude mixture as the major component (80% by 1 H NMR), together with other products which were difficult to assign. The separation by column chromatography was difficult and although pure 9 was isolated with only 35% yield, we succeeded to validate the proof of concept. Scheme 5. One-pot Suzuki-Miyaura cross-coupling and desulfenylative cross-coupling/hydrothiolation.

In summary, we have developed a Pd-catalyzed tandem process involving 2-(bromobenzylsulfenyl)-1-propargyl benzimidazoles and boronic acids. It consists of three reactions: first a classical Suzuki debrominative cross-coupling, then a desulfenylative coupling in 2-position of the benzimidazole that does not need copper(I)-assistance, and finally an intermolecular regio and stereoselective hydrothiolation of the triple bond of the N-propargyl benzimidazole. The overall process results in the double incorporation of the boronic acid partner and the reincorporation of the sulfenyl moiety into the product structure, as an alkenyl benzyl sulfide. Further studies will be conducted to extend the scope of the Pd-catalyzed desulfenylative coupling/hydrothiolation tandem reaction to other related heterocycles. Moreover, the alkenyl sulfenyl pendent resulting from hydrothiolation can be converted into other functionalities by known methods.

ACKNOWLEDGMENTS

This work has been published under the framework of the IdEx Unistra and benefits from a funding from the state managed by the French National Research Agency (postdoctoral grant for A.B. Lopes). The CNRS is acknowledged for financial support. We thank Dr. D. Garnier and E. Oliva (PACSI, GDS 3670) for NMR and mass analyses. One Reviewer is acknowledged for interesting suggestions.

ASSOCIATED CONTENT Supporting Information

Detailed experimental procedures and spectra data for new compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author

*E-mail: gulea@unistra.fr ORCID: 0000-0002-2945-0078

Notes

The authors declare no competing financial interest.


Bernard Lanz

Alireza Abaei

Olivier Braissant

Cristina Cudalbu

Phil Lee

CRMBM Norbert W Lutz

Małgorzata Marjańska

Vladimir Mlynarik

Volker Rasche

Lijing Xin

Julien Valette

Preclinical magnetic resonance spectroscopy: Experts' consensus recommendations

In vivo magnetic resonance spectroscopy (MRS) is a non-invasive measurement technique used not only in humans, but also in preclinical models, in particular in rodents, using high field horizontal MR magnets. MRS enables the measurement of metabolite concentrations as well as metabolic rates and their modifications in healthy animals and disease models. Such data open the way to a deeper understanding of the underlying biochemistry, related disturbances and mechanisms taking place during or prior to symptoms and tissue changes. In this work, we focus on the main preclinical 1 H, 31 P and 13 C MRS approaches to study brain metabolism in rodent models, with the aim to provide general experts'consensus recommendations (animal model, anesthesia, acquisition protocols). An overview of the main practical differences in preclinical MRS compared to clinical studies as well as the additional biochemical information and the technical limitations are also described.

1 Introduction: (Olivier Braissant) The use of rodents as experimental models provides a great opportunity to increase our understanding about human tissue development, function and metabolism which is relevant to better understand pathologies and to develop treatment strategies. While basic cellular or metabolic questions can be studied using in vitro models (e.g., cell cultures of different levels of complexity, from monotypic monolayers to complex organotypic pluricellular cultures), understanding more complex traits of the living organism (e.g., interactions between different cell types, organ systems and behavior) requires the use of in vivo experimental models, for which mice and rats have been the most widely used for many reasons such as their proximity to the human genome, their short generation time and small size.

In vivo magnetic resonance imaging and spectroscopy (MRI/MRS) have become tools of choice to study and better understand CNS activity and metabolism as well as human brain diseases, both in human patients and through the use of animal models, including mice or rats.

They have the great advantages of being non-invasive and allowing an easy longitudinal follow-up of brain development and activity, disease evolution, or treatment efficacy [START_REF] Cudalbu | In vivo studies of brain metabolism in animal models of Hepatic Encephalopathy using (1)H Magnetic Resonance Spectroscopy[END_REF][START_REF] Oz | Clinical proton MR spectroscopy in central nervous system disorders[END_REF][START_REF] Rackayova | Creatine in the central nervous system: From magnetic resonance spectroscopy to creatine deficiencies[END_REF] .

While human patients are usually followed in clinics using magnetic fields of 1.5 or 3 T, the recent development of new technologies allowing MRI/MRS at much higher magnetic fields (≥ 9.4 T) enables the study of CNS in rodent models with unprecedented resolution, allowing for example the deciphering of up to 20 different neurochemicals by [START_REF] Kjell | Rat models of spinal cord injury: from pathology to potential therapies[END_REF] H-MRS [START_REF] Braissant | Longitudinal neurometabolic changes in the hippocampus of a rat model of chronic hepatic encephalopathy[END_REF] . Moreover, combining different types of MRS (e.g., [START_REF] Kjell | Rat models of spinal cord injury: from pathology to potential therapies[END_REF] H-, [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C-, [START_REF] Albrecht | Influence of repeated anaesthesia on physiological parameters in male Wistar rats: a telemetric study about isoflurane, ketamine-xylazine and a combination of medetomidine, midazolam and fentanyl[END_REF] P-MRS) broadens the range of brain metabolic and neurotransmission pathways available to study, including the precise follow-up along time of metabolic fueling within these pathways (in particular with [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C-MRS). This consensus recommendation paper aims at providing specific technical recommendations for MRS in preclinical studies of brain function, metabolism and disease using animal models, including a broad overview of the characteristics and specificities of MRS. For MRS aspects and challenges common to clinical and preclinical studies, the reader is referred to the other chapters of this special issue.

2 Animal physiology and anesthesia aspects: (Firat Kara and Kejal Kantarci)

Choice of animal models

For several reasons discussed below, rats have been considered for decades as better models than mice to tackle human pathology, and have been used for a long time as models for surgery, exposure to toxins or pathological vectors, or development of treatments by exposure to various agents (e.g. drugs, vaccines, viral vectors) [START_REF] Kjell | Rat models of spinal cord injury: from pathology to potential therapies[END_REF] . In contrast, since 1980 and the availability of genetically modified mice (and particularly homologous recombination technology) [START_REF] Thomas | Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells[END_REF] , the mouse has been the most used preclinical model to investigate normal gene functions as well as human genetic diseases. While historically generating genetically modified rats was much more difficult than mice, the recent development of new technologies (in particular Crispr/CAS9) [START_REF] Zan | Production of knockout rats using ENU mutagenesis and a yeast-based screening assay[END_REF][START_REF] Geurts | Generation of gene-specific mutated rats using zinc-finger nucleases[END_REF][START_REF] Tong | Generating gene knockout rats by homologous recombination in embryonic stem cells[END_REF][START_REF] Shao | CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos[END_REF] makes the development of genetically modified rats easier and provides new and valuable rat models to better understand both basic gene functions and human diseases.

Important differences exist between mice and rats, the two most used in vivo experimental models [START_REF] Francis | Divergence of RNA localization between rat and mouse neurons reveals the potential for rapid brain evolution[END_REF] . While mice have, as advantages, their size (for economy of costs and space) and their facility of gene modifying, they are less easily handled for brain studies (due to small size of brain), and are less similar to human in terms of metabolism (most probably due to vegetarian-adapted metabolism) and behavior. In contrast, while rats are more expensive in terms of costs and space used, they have long been recognized, especially in neuroscience and behavioral research, as better models to study basic functions and metabolism of the central nervous system (CNS) as well as human brain diseases (larger brain size; more similar to human in terms of metabolism most probably due to omnivorous-adapted metabolism; behavioral tests more comparable to human, see below). In particular, while rat and mouse brains develop in a very similar manner, several major differences have been identified in terms of circuitry and functions of their brain, as well as of their respective behaviors, making rats often better models for preclinical studies of human brain pathologies (examples: rats show a more social behavior, and are more prone to addictions; levels and spatial distribution of 5-HT6 serotonin receptors are very similar between rat and human CNS while different in mice) [START_REF] Ellenbroek | Rodent models in neuroscience research: is it a rat race?[END_REF] . On the practical side however, one disadvantage of working on rats is the large change in body size over the lifetime of the animals. Mice are easier in that respect for MRS experiments.

Effect of anesthesia on brain metabolism

Effective and standardized mouse MRI/MRS studies require attention to many aspects of experiment design. One of the important aspects of the experimental design is the anesthesia.

Anesthesia is essential for in vivo preclinical MRI and MRS. It decreases the stress (and potential pain in case of surgical intervention) of the animals and restricts biological motion (e.g., physical activity, head movement, as well as respiratory and cardiac activities) [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF][START_REF] Tremoleda | Anaesthesia and physiological monitoring during in vivo imaging of laboratory rodents: considerations on experimental outcomes and animal welfare[END_REF][START_REF] Lukasik | Animal anaesthesia for in vivo magnetic resonance[END_REF] . This section aims to provide a brief overview of preanesthetic considerations, a review of the existing literature regarding the effect of anesthesia on neurochemical profile in rodents (i.e., rat and mice), and a practical guideline for selecting an appropriate anesthetic protocol for MRS studies of small animals.

Preanesthetic considerations for in vivo MRS studies

The effect of anesthesia depends on a variety of factors including stress, strain, gender, circadian cycles, weight and age of the animals [START_REF] Lukasik | Animal anaesthesia for in vivo magnetic resonance[END_REF][START_REF] Hildebrandt | Anesthesia and other considerations for in vivo imaging of small animals[END_REF] . These factors may not only change the effectiveness of anesthesia and required dosage but may also have a direct impact on MRS measures of the neurochemical profile in the rodent brain [START_REF] Bresnen | Brain high-energy phosphates and creatine kinase synthesis rate under graded isoflurane anesthesia: An in vivo (31) P magnetization transfer study at 11.7 tesla[END_REF] .

Stress: Transporting, handling, restraining and injection of anesthetics may cause acute stress, which leads to alterations in physiological parameters, such as corticosteroid and epinephrine levels, glucose levels, respiration and heart rate [START_REF] Hildebrandt | Anesthesia and other considerations for in vivo imaging of small animals[END_REF] . Some period of acclimatization (between 24 h-48 h) is necessary after transporting the animals to the imaging facility to decrease the stress level. The experimenter should be well trained to handle the animals (and to acclimatize the animals for handling) properly before putting the animal into the anesthesia in order to prevent high level of acute stress. Stress-induced alterations in physiological parameters such as corticosteroids may increase the required dosage for proper anesthesia and alter the cerebral metabolic concentrations. Increased glutamate levels were reported in a rat group with high corticosteroid levels relative to those with physiological corticosteroid level [START_REF] Schubert | Effects of altered corticosteroid milieu on rat hippocampal neurochemistry and structure -An in vivo magnetic resonance spectroscopy and imaging study[END_REF] . Strain: Genetic variation (i.e., background) among mice is well known. Earlier studies demonstrated that genetic background of the mouse strain has a substantial effect on physiological parameters such as the sleep time of anesthesia and stress [START_REF] Hildebrandt | Anesthesia and other considerations for in vivo imaging of small animals[END_REF][START_REF] Lovel | Variation in pentobarbitone sleeping time in mice 1. Strain and sex differences[END_REF][START_REF] Lovel | Variation in pentobarbitone sleeping time in mice 2. Variables affecting test results[END_REF][21] . For example, Lovell reported that there were variations in pentobarbitone sleeping time in mice with different strain [START_REF] Lovel | Variation in pentobarbitone sleeping time in mice 1. Strain and sex differences[END_REF] . Strain-specific differences in neurochemical levels have also been demonstrated [START_REF] Schwarcz | Localized proton MRS of cerebral metabolite profiles in different mouse strains[END_REF] , i.e. elevated levels of N-acetylaspartate, creatine, and phosphocreatine, choline containing compounds, glucose and lactate in C57BL/6 strain relative to BALB/c mice [START_REF] Schwarcz | Localized proton MRS of cerebral metabolite profiles in different mouse strains[END_REF] measured by [START_REF] Kjell | Rat models of spinal cord injury: from pathology to potential therapies[END_REF] H MRS.

Gender: The gender effect should be well taken into account in collecting and analyzing MRS data. Few rodent studies reported substantial effect of gender differences on the neurochemical profile in rodents [START_REF] Roy | Sex-and age-specific modulation of brain GABA levels in a mouse model of Alzheimer's disease[END_REF][START_REF] Van Duijn | Longitudinal monitoring of sex-related in vivo metabolic changes in the brain of Alzheimer's disease transgenic mouse using magnetic resonance spectroscopy[END_REF] . The presence of menstrual cycle effect on neurochemical profile in human brain has been demonstrated [START_REF] Hjelmervik | Sex-and sex hormone-related variations in energy-metabolic frontal brain asymmetries: A magnetic resonance spectroscopy study[END_REF] . However, the estrous cycle effect on the brain neurochemistry of female rodents has not been thoroughly studied.

Circadian Cycles: Rodents have circadian rhythms which provide rhythmic variations in their many physiological functions, including hormones [START_REF] Aschoff | Circadian timing[END_REF] . A recent in vivo MRS study reported that diurnal changes occurred in the neurochemical profile in rats when MRS data were collected one hour after light onset (7 am) or offset (7 pm) [START_REF] Liachenko | 1H-MRS changes in the rat brain due to circadian cycle[END_REF] . The results of this study suggest that recording the time of the experiment may be an important parameter while assessing the data collected from randomly assigned experimental groups. It was recommended that timing of the experiments and treatment should be controlled and reported in order to increase reproducibility between studies [START_REF] Hildebrandt | Anesthesia and other considerations for in vivo imaging of small animals[END_REF] .

Age: The age of the animals has an important effect on the neurochemical profile in the rodent brain [START_REF] Duarte | Longitudinal neurochemical modifications in the aging mouse brain measured in vivo by 1H magnetic resonance spectroscopy[END_REF] and also on anesthetic variability. For example, young mice (<8 weeks) cannot metabolize anesthetics as effectively as adults can [START_REF] Gargiulo | Mice anesthesia, analgesia, and care, Part I: anesthetic considerations in preclinical research[END_REF] . Therefore, the impact of same levels of anesthetics on cerebral metabolites might be different in young mice relative to mature mice (>3 months). In a longitudinal setting, the brain size of rodents may also change with age. The MRS voxel size may, therefore, need to be adjusted according to age-dependent changes of the brain.

Guidelines and recommendations for anesthesia protocols

A variety of inhaled and injectable anesthetics are available for MRS studies of rodents [START_REF] Makaryus | The metabolomic profile during isoflurane anesthesia differs from propofol anesthesia in the live rodent brain[END_REF] .

The appropriate choice of the anesthetic procedure is essential, as anesthetics may have variable effects on the neurochemical profile in rodents, as well as other side effects, as summarized in Table 1 and described with more details in Supplementary Materials. A detailed discussion on the influence of inhaled and injectable anesthetics on the physiology of animals and more information about the properties of these agents can be found elsewhere [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF][START_REF] Tremoleda | Anaesthesia and physiological monitoring during in vivo imaging of laboratory rodents: considerations on experimental outcomes and animal welfare[END_REF][START_REF] Gargiulo | Mice anesthesia, analgesia, and care, Part I: anesthetic considerations in preclinical research[END_REF][START_REF] Albrecht | Influence of repeated anaesthesia on physiological parameters in male Wistar rats: a telemetric study about isoflurane, ketamine-xylazine and a combination of medetomidine, midazolam and fentanyl[END_REF][START_REF] Furtado | Comparison of the beneficial and adverse effects of inhaled and injectable anaesthetics: A mini-review[END_REF][START_REF] Gargiulo | Mice anesthesia, analgesia, and care, Part II: anesthetic considerations in preclinical imaging studies[END_REF] .

The anesthesia type, administrative method, duration and dosage should be optimized carefully according to the aim of the experiment [START_REF] Makaryus | The metabolomic profile during isoflurane anesthesia differs from propofol anesthesia in the live rodent brain[END_REF][START_REF] Lei | Deep thiopental anesthesia alters steady-state glucose homeostasis but not the neurochemical profile of rat cortex[END_REF][START_REF] Du | In vivo proton MRS to quantify anesthetic effects of pentobarbital on cerebral metabolism and brain activity in rat[END_REF][START_REF] Choi | Effect of deep pentobarbital anesthesia on neurotransmitter metabolism in vivo: on the correlation of total glucose consumption with glutamatergic action[END_REF][START_REF] Horn | Lactate levels in the brain are elevated upon exposure to volatile anesthetics: A microdialysis study[END_REF] . Overall, an inhaled anesthetic is easier to handle for MRI/MRS experiments due to its way of administration, possibility to adapt the dose inside the magnet during the experiments, fast kinetics and ease of use for longitudinal studies. Information about the drug doses of anesthetics which are commonly used in rodents in MRI and MRS studies can be found elsewhere [START_REF] Lukasik | Animal anaesthesia for in vivo magnetic resonance[END_REF] . The right dosage should be optimized for the desired length of experiment, provide adequate sedation but also adequate analgesia and less variability in physiological parameters during MRI and MRS experiments [START_REF] Lukasik | Animal anaesthesia for in vivo magnetic resonance[END_REF] . Therefore, monitoring and recording the respiration rate and temperature of animals under anesthesia is essential (if available, pulse oximetry and electrocardiography can provide further control). If the experiment requires repeated exposure to the anesthetic, the one having a quick recovery phase and fewer side effects should be chosen. Providing an adequate environment for the animals not only during exposure to anesthesia but also during the recovery phase is vital to prevent complications, such as hypothermia, stress, and respiratory arrest (i.e. recovery on a heating pad or under a heating lamp). Ocular protection should likewise be provided, as rodents may keep their eyes open under anesthesia. The correct application of anesthesia is essential, as inappropriate use of these agents may cause physiological instability and deleterious effects, including pain, fear, distress, hypothermia, and hypoxia [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] .

MRS in awake rodents

Performing MRS with awake rodents is challenging and requires relatively long time to train the animals to stay still during scans. Restraining awake animals without proper training may induce stress and affect MRS results. For awake-rodent MRS studies, monitoring serum cortisone levels and heart rate of the animals are recommended [START_REF] Xu | In vivo high-resolution localized (1) H MR spectroscopy in the awake rat brain at 7 T[END_REF] . If awake MRS study needs to be performed, new methodologies should be developed to train and acclimate the animals to the MRI environment in a shorter time period.

3 Hardware (Małgorzata Marjańska and Alireza Abaei)

The small size of the brain and a strong B 0 inhomogeneity induced in the brain by air/tissue interface are two major differences distinguishing preclinical studies of small animals from clinical studies and leading to different hardware requirements. The small volumes of interest (VOI) (rats: 50 -150 μL, mice: 2 -15 μL) necessitated by the small size of the brain and significant regional differences in neurochemical concentrations benefits from ultra-high magnetic fields (> 9.4 T), where the increased sensitivity compensates for the reduced signalto-noise ratio (SNR) due to the small size of VOI. The small VOIs at ultra-high fields put higher requirements on the gradient strength (ideally ≥400 mT/m) compared to human systems (human 7 T: typically 70 mT/m). It is known that rapid switching of magnetic field gradients stimulates impulses in peripheral nerves, known as peripheral nerve stimulation (PNS), though these effects have hitherto not been reported to be of concern in preclinical research. Another advantage of using the ultra-high field is the increased chemical shift dispersion which helps in resolving overlapped resonances and simplifies strongly coupled spin systems. However, to take the advantage of increased chemical shift dispersion and spectral resolution, B 0 inhomogeneity needs to be minimized by using an efficient shimming method and shim system, strong enough to compensate for the field gradient induced in the brain. Stronger shims are required for mouse brain than for rat brain. In addition, the required strength of the shims in the mouse brain is region-dependent [START_REF] Tkac | Highly resolved in vivo 1 H NMR spectroscopy of the mouse brain at 9.4 T[END_REF] . At 9.4 T, shim strengths of up to 47 μT/cm 2 for XZ, YZ, Z 2 and 23.5 μT/cm 2 for XY and X 2 Y 2 are needed for mouse brain spectroscopy [START_REF] Tkac | Highly resolved in vivo 1 H NMR spectroscopy of the mouse brain at 9.4 T[END_REF] , while the strength of the shims scales linearly with the field strength, since the amplitudes of susceptibility-induced B 0 inhomogeneity scales with the B 0 field strength [START_REF] Lei | Neurochemical profile of the mouse hypothalamus using in vivo 1 H MRS at 14.1[END_REF] .

Automatic shimming methods such as FAST(EST)MAP [START_REF] Gruetter | Automatic, localized in vivo adjustment of all first-and second-order shim coils[END_REF][START_REF] Gruetter | Field mapping without reference scan using asymmetric echo-planar techniques[END_REF] or 3D B 0 mapping 48 can be used efficiently for shimming on preclinical systems. Regarding the B 0 shim quality expressed as water linewidths (full width at half maximum, FWHM) in a specific brain region, the lowest linewidths can be achieved from more homogeneous regions, such as hippocampus and striatum (i.e. 10-12 Hz in the rat brain at 9.4T for a voxel of 2x2.8x2mm 3 for hippocampus and 2.2x2x2.5mm 3 for striatum using FAST(EST)MAP). In the cerebellum, the water linewidth is increased (i.e. 15 -17 Hz at 9.4T in the rat brain for a voxel of 2.5x2.5x2.5 mm 3 ) due to intrinsic properties of the tissue (i.e. microscopic heterogeneity).

The power of the radiofrequency (RF) amplifiers used in preclinical systems is lower than for human systems, since much smaller and more efficient coils (either volume or surface) are used on preclinical systems. Volume RF coils provide uniform images of the whole brain due to the homogenous B 1 field. However, when used for signal reception in MRS, they can lead to increased contamination from areas outside the VOI and collect more subject thermal noise from their larger field of view. Surface coils provide much higher SNR from regions close to the RF coil and higher B 1 efficiency than volume coils, but the B 1 field is spatially inhomogeneous. The usage of adiabatic RF pulses can mitigate B 1 inhomogeneities. It is also worth to mention that, in contrast with human studies, legally unlimited B 1 and strong gradients enable to choose the optimum RF coil/combination of RF coils for a specific experiment (from the point of view of SNR and CSD error).

To maximize sensitivity, most [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C and 31 P studies have used surface coils. The most commonly used arrangement is a combination of a single loop coil for [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C or [START_REF] Albrecht | Influence of repeated anaesthesia on physiological parameters in male Wistar rats: a telemetric study about isoflurane, ketamine-xylazine and a combination of medetomidine, midazolam and fentanyl[END_REF] P and quadrature coil for 1 H 77 or vice-versa [START_REF] De Graaf | Vivo NMR Spectroscopy -2nd Edition: Principles and Techniques[END_REF][79][START_REF] Evelhoch | 31P spin-lattice relaxation times and resonance linewidths of rat tissue in vivo: dependence upon the static magnetic field strength[END_REF][START_REF] Remy | In vivo 31P nuclear magnetic resonance studies of T1 and T2 relaxation times in rat brain and in rat brain tumors implanted to nude mice[END_REF] . Alternate coil arrangements have been proposed to further increase sensitivity with quadrature detection [START_REF] Van Der Kemp | P T2 s of phosphomonoesters, phosphodiesters, and inorganic phosphate in the human brain at 7T[END_REF][START_REF] De Graaf | B1-insensitive, single-shot localization and water suppression[END_REF][START_REF] Connelly | Outer volume suppressed image related in vivo spectroscopy (OSIRIS), a high-sensitivity localization technique[END_REF] .

Cryogenically cooled RF coils can be used for further noise reduction. For small sample volumes, the thermal noise in the coil and the receive pathway is the dominant noise source.

By cooling the respective components [START_REF] Kovacs | Cryogenically cooled probes -a leap in NMR technology[END_REF] , in direct comparison to a room temperature coil, a reduction of the overall noise by a factor of 2-3 has been reported [START_REF] Baltes | Micro MRI of the mouse brain using a novel 400 MHz cryogenic quadrature RF probe[END_REF] . This enables a remarkable reduction of the acquisition time or acquisitions from smaller volumes within a reasonable acquisition time. A limiting factor for the general usage of cryogenically cooled coils results from the requirement of dominating coil noise, which restricts its application to small animal applications [START_REF] Mispelter | NMR Probeheads for Biophysical and Biomedical Experiments: Theoretical Principles & Practical Guidelines[END_REF][START_REF] Junge | Cryogenic and superconducting coils for MRI[END_REF] . Basic pulse sequences for localized spectroscopy were designed a long time ago and they are still used in most preclinical studies. The most popular localization methods are based either on a stimulated echo (e.g., STEAM [START_REF] Frahm | Localized Proton Spectroscopy Using Stimulated Echoes[END_REF][START_REF] Tkac | In vivo 1H NMR spectroscopy of rat brain at 1 ms echo time[END_REF] ) or on a double spin echo (e.g., PRESS [START_REF] Bottomley | Spatial localization in NMR spectroscopy in vivo[END_REF] ).

The STEAM sequence uses three slice-selective 90° pulses to form a stimulated echo.

Although one half of the magnetization is lost with this pulse sequence, it is suitable for short or even ultra-short echo time measurements (TE=??). The magnetization is conserved along the direction of the static magnetic field during the time period between second and third 90° pulses (TM). Consequently, there is no evolution of the J-coupled signals during TM and signal intensity is not affected by T 2 relaxation times. The TM time is also used for suppressing unwanted echoes and for the attenuation of possible eddy current effects. Because of the use of 90° pulses for localization, this sequence is relatively insensitive to the B 1 inhomogeneity.

The PRESS sequence consists of one slice selective 90° pulse and two slice-selective 180° refocusing pulses forming a double spin echo. The advantage of this sequence is that it preserves all the magnetization available in a selected volume of interest. On the other hand, it is quite difficult to suppress all undesired echoes created by pairs of the slice selective pulses.

Thus, this sequence is mainly used for longer echo time measurements.

In recent years, novel methods of localized spectroscopy suitable for preclinical studies have appeared. The LASER (localization by adiabatic selective refocusing) sequence [START_REF] Garwood | The return of the frequency sweep: designing adiabatic pulses for contemporary NMR[END_REF] is a fully adiabatic single-scan 3D localization sequence which does not require outer volume suppression. It consists of a nonselective adiabatic half-passage (AHP) pulse followed by three pairs of slice selective adiabatic full-passage (AFP) pulses (Figure 1A). On preclinical scanners, optimized LASER sequence can result in the TE ranging from 15 to 28 ms, similar to PRESS [START_REF] Oz | Noninvasive detection of presymptomatic and progressive neurodegeneration in a mouse model of spinocerebellar ataxia type 1[END_REF][START_REF] Marjanska | Monitoring disease progression in transgenic mouse models of Alzheimer's disease with proton magnetic resonance spectroscopy[END_REF][START_REF] Santin | In vivo 1H MRS study in microlitre voxels in the hippocampus of a mouse model of Down syndrome at 11.7 T[END_REF][60][START_REF] Deelchand | Effect of Carr-Purcell refocusing pulse trains on transverse relaxation times of metabolites in rat brain at 9.4 Tesla[END_REF] (Figure 1B). Due to the properties of the AFP pulses, clean profiles with sharp transitions are obtained and the CSD error is minimal due to the large bandwidth of the AFP pulses which are typically 10 kHz or higher. At 9.4 T on a preclinical scanner, the CSD error is 2.4%/ppm. The successive application of multiple AFP pulses in LASER suppresses Jevolution in coupled spin systems and prolongs apparent T 2 [START_REF] Deelchand | Effect of Carr-Purcell refocusing pulse trains on transverse relaxation times of metabolites in rat brain at 9.4 Tesla[END_REF][START_REF] Michaeli | Proton T-2 relaxation study of water, N-acetylaspartate, and creatine in human brain using Hahn and Carr-Purcell spin echoes at 4T and 7T[END_REF][START_REF] Deelchand | Properties of localization by adiabatic refocusing (LASER) sequence[END_REF] resulting in much smaller signal loss for LASER than what is observed for other sequences at the similar TE. For TE between 15 and 28 ms at 9.4 T, the loss due to J-evolution and T 2 is minimal 61 (Figure 1C).

One-dimensional ISIS [START_REF] Ordidge | Image-Selected Invivo Spectroscopy (Isis) -a New Technique for Spatially Selective Nmr-Spectroscopy[END_REF] and a slice selective single spin echo have been combined in a technique with the acronym SPECIAL [START_REF] Mlynarik | Localized short-echo-time proton MR spectroscopy with full signal-intensity acquisition[END_REF] . Standard and adiabatic versions of this sequence as well as its advantages and disadvantages of this technique are described in details in another paper of this issue 66 . The method has been successfully used for short echo time localized spectroscopy (TE=2.8ms) in mice and rats [START_REF] Dhamala | Validation of in vivo MRS measures of metabolite concentrations in the human brain[END_REF][START_REF] Heo | On the Utility of Short Echo Time (TE) Single Voxel 1H-MRS in Non-Invasive Detection of 2-Hydroxyglutarate (2HG); Challenges and Potential Improvement Illustrated with Animal Models Using MRUI and LCModel[END_REF][START_REF] Duarte | Impact of Caffeine Consumption on Type 2 Diabetes-Induced Spatial Memory Impairment and Neurochemical Alterations in the Hippocampus[END_REF] . Localization efficiency of all those sequences can be improved by saturation of the magnetization outside the volume of interest using a series of slice-selective saturation pulses.

In all pulse sequences, efficient water suppression is important to eliminate the strong water signal, which can overlap with metabolite signals and cause baseline distortion. Total elimination of the residual water signal is possible with different methods, e.g., VAPOR water suppression [START_REF] Tkac | In vivo 1H NMR spectroscopy of rat brain at 1 ms echo time[END_REF] .

Similar to human neurochemical profile data, the pre-clinical data should be fitted using a software which allows the decomposition of the spectrum into that of particular metabolites, using a metabolite basis set, such as LCModel [START_REF] Provencher | Estimation of Metabolite Concentrations from Localized in-Vivo Proton Nmr-Spectra[END_REF][START_REF] Provencher | Automatic quantitation of localized in vivo H-1 spectra with LCModel[END_REF] , jMRUI / QUEST 72 or others [START_REF] Bottomley | Handbook of Magnetic Resonance Spectroscopy In Vivo: MRS Theory, Practice and Applications[END_REF] . In addition to metabolites, the basis sets should include the experimentally acquired macromolecules spectrum. It has been reported that the macromolecules content and spectral pattern does not change in healthy rodents in hippocampus, cortex and striatum, mainly due to the fact that the rodent brain contains mostly grey matter. Thus, assuming a constant shape for the macromolecules is a practical approach when fitting metabolite concentrations. Additionally, the macromolecules content was shown to change during development [START_REF] Tkac | Developmental and regional changes in the neurochemical profile of the rat brain determined by in vivo H-1 NMR spectroscopy[END_REF] with no change in the macromolecular pattern in normal brain. Note that one recent study reported variation in MM patterns during astrocyte reactivity in mice [START_REF] Ligneul | Diffusion-weighted magnetic resonance spectroscopy enables cell-specific monitoring of astrocyte reactivity in vivo[END_REF] , suggesting that group-specific MM spectra might be necessary in some cases. More information on the spectrum of macromolecules can be found in Ref. [START_REF] Cudalbu | Contribution of macromolecules to magnetic resonance spectra: Experts' consensus recommendations[END_REF] .

5 Sequences and acquisition protocols: 31 P MRS: (Julien Valette) 31 P is the 100%-natural abundance, NMR-visible isotope of phosphorus. [START_REF] Albrecht | Influence of repeated anaesthesia on physiological parameters in male Wistar rats: a telemetric study about isoflurane, ketamine-xylazine and a combination of medetomidine, midazolam and fentanyl[END_REF] P MRS allows noninvasive measurement of the concentration of phosphorylated metabolites such as adenosine triphosphate (ATP), inorganic phosphate (Pi) or phosphocreatine (PCr), which are involved in energy metabolism. Furthermore, [START_REF] Albrecht | Influence of repeated anaesthesia on physiological parameters in male Wistar rats: a telemetric study about isoflurane, ketamine-xylazine and a combination of medetomidine, midazolam and fentanyl[END_REF] P MRS combined with magnetization / saturation transfer can be used to quantify reaction rates of key metabolic enzymes, such as creatine kinase or ATP synthase [START_REF] Du | Efficient in vivo P-31 magnetization transfer approach for noninvasively determining multiple kinetic parameters and metabolic fluxes of ATP metabolism in the human brain[END_REF] . In addition, pH can be determined from the chemical shift difference between Pi and PCr, while [Mg 2+ ] can be determined from the chemical shift difference between the -and β-ATP resonances [START_REF] De Graaf | Vivo NMR Spectroscopy -2nd Edition: Principles and Techniques[END_REF] . [START_REF] Albrecht | Influence of repeated anaesthesia on physiological parameters in male Wistar rats: a telemetric study about isoflurane, ketamine-xylazine and a combination of medetomidine, midazolam and fentanyl[END_REF] P MRS hence has an enormous potential to probe metabolic features that cannot be assessed with other non-invasive techniques and is a complementary technique to 1 H MRS and [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C MRS. However, despite the abundance of 31 P nuclei in vivo, [START_REF] Albrecht | Influence of repeated anaesthesia on physiological parameters in male Wistar rats: a telemetric study about isoflurane, ketamine-xylazine and a combination of medetomidine, midazolam and fentanyl[END_REF] P MRS is facing three major hurdles compared to 1 H MRS:

i) The gyromagnetic ratio is low (~2.5-fold lower than 1 H's), thus resulting in intrinsically low sensitivity. Going to higher magnetic fields as generally available for preclinical studies is beneficial to increase 31 P MRS sensitivity 79 , especially considering the fact that metabolite T 1 decreases with the field, possibly due to increased contribution of chemical shift anisotropy to relaxation [START_REF] Evelhoch | 31P spin-lattice relaxation times and resonance linewidths of rat tissue in vivo: dependence upon the static magnetic field strength[END_REF] .

ii) T 2 relaxation time constants are short for some important metabolites such as Pi (<80 ms)

and ATP (<40 ms) [START_REF] Remy | In vivo 31P nuclear magnetic resonance studies of T1 and T2 relaxation times in rat brain and in rat brain tumors implanted to nude mice[END_REF][START_REF] Van Der Kemp | P T2 s of phosphomonoesters, phosphodiesters, and inorganic phosphate in the human brain at 7T[END_REF] , so that signal loss during the echo time can be significant when using conventional single-shot localization sequences (e.g., STEAM, PRESS or LASER).

iii) The frequency-range spanned by metabolites is large (~25 ppm, i.e., ~5000 Hz at 11.7 T), thus requiring broadband pulses, in particular to avoid CSD error.

In a clinical context, the last two points are usually circumvented by using either chemical shift imaging (CSI), or no spectroscopic localization at all (e.g., just exploiting the sensitivity profile of a surface coil). These approaches are possible in humans because the contribution from skin, muscles and fat surrounding the brain is small due to their small volume fraction, and because the large brain size allows CSI with decent SNR. However, this can hardly be translated to a preclinical context, because animal brains are smaller (making CSI quite inefficient) and in general surrounded by a significant amount of muscles (in rodents but even more in primates). Hence for 31 P MRS in a preclinical context it is recommended to use one of the two following localization approaches:

Image-Selected In vivo Spectroscopy (ISIS) combines several advantages: it is basically a zero echo time sequence although some T2 relaxation occurs during the RF pulses, thus reducing signal loss; localization can be achieved solely by adiabatic inversion pulses, which ensures efficient inversion throughout the volume even when surface coils are used, provided high enough transmit B 1 can be reached, thus avoiding signal loss due to incorrect flip angles;

and the large bandwidth that can be achieved with adiabatic pulses (with less constraint on maximal transmit B 1 than with conventional pulses) reduces CSD error. Because it relies on the combination of signals collected over 8-scan cycles, ISIS is unfortunately less robust to motion and drifts than single-shot localization techniques.

In Outer Volume Suppression (OVS) based localization, where the magnetization surrounding the VOI is destroyed by trains of RF pulses and crusher gradients. Because the magnetization within the VOI is (ideally) not perturbed, such localization limits signal loss due to relaxation.

Also, as RF pulses used for OVS are not meant to perform large flip angles, their bandwidth is less constrained by transmit B 1 than that of 180° pulses, thus reducing CSD. If surface coils are used, OVS can be made largely insensitive to B 1 inhomogeneities using BISTRO-type OVS trains [START_REF] De Graaf | B1-insensitive, single-shot localization and water suppression[END_REF] .

Note that OVS and ISIS can be combined to further improve localization [START_REF] Connelly | Outer volume suppressed image related in vivo spectroscopy (OSIRIS), a high-sensitivity localization technique[END_REF] . In the end, when localization is performed, adequate shimming within the volume of interest will allow observing subtle spectral features, such as resolving two Pi resonances at 4.9 and 5.3 ppm [START_REF] Tiret | Evidence for a "metabolically inactive" inorganic phosphate pool in adenosine triphosphate synthase reaction using localized 31P saturation transfer magnetic resonance spectroscopy in the rat brain at 11.7 T[END_REF] , presumably corresponding to intracellular and extracellular Pi. Distinguishing both Pi resonances in the brain should be considered as a signature of excellent spectral quality (both in terms of shim and SNR). [START_REF] Albrecht | Influence of repeated anaesthesia on physiological parameters in male Wistar rats: a telemetric study about isoflurane, ketamine-xylazine and a combination of medetomidine, midazolam and fentanyl[END_REF] P spectra can be processed with fitting algorithms that include prior knowledge, such as AMARES [START_REF] Vanhamme | Improved method for accurate and efficient quantification of MRS data with use of prior knowledge[END_REF] or LCModel [START_REF] Deelchand | Quantification of in vivo P-31 NMR brain spectra using LCModel[END_REF] .

6 Sequences and acquisition protocols: [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C MRS: (Pierre-Gilles Henry)

In contrast to [START_REF] Kjell | Rat models of spinal cord injury: from pathology to potential therapies[END_REF] H MRS, which measures the concentration of metabolites, [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C MRS allows measurement of metabolic rates in vivo. The low natural abundance of 13 C (1.1%) makes it possible to use it as a non-radioactive tracer and to follow incorporation of [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C label into downstream metabolites after injection of a 13 C-labeled precursor.

Detection of [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C label can be achieved either using direct [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C detection at [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C frequency, or indirect detection via protons attached to [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C via heteronuclear editing (POCE) (see [START_REF] Gruetter | Localized in vivo 13C NMR spectroscopy of the brain[END_REF][START_REF] Lizarbe | In Vivo Heteronuclear Magnetic Resonance Spectroscopy[END_REF][START_REF] De Graaf | State of the art direct 13C and indirect 1H-[13C] NMR spectroscopy in vivo. A practical guide[END_REF][START_REF] Henry | In vivo 13C NMR spectroscopy and metabolic modeling in the brain: a practical perspective[END_REF] for reviews).

Factors to consider for successful experiments include:

Pulse sequences: Direct detection gives the most biochemical information, but requires larger detection volumes due to the lower sensitivity of [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C detection. Indirect detection is more suitable for small volumes (higher sensitivity), but greater spectral overlap makes detection of certain resonances difficult (e.g., resolved detection of glutamate and glutamine C2, or of glutamate and glutamine C3).

For direct detection, many recent studies have used [START_REF] Kjell | Rat models of spinal cord injury: from pathology to potential therapies[END_REF] H localization (e.g. ISIS) followed by polarization transfer [100][101][102] , which provides better sensitivity and smaller chemical-shift displacement error than [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C localization and excitation + NOE, as well as excellent localization with small CSD error 100-102 . Polarization transfer, however, cannot be used for carbons that have weak coupling to nearby protons (e.g., carbonyl/carboxyl carbons), or those with very short T 2 (glycogen) 103,104 .

For indirect detection, virtually every 1 H MRS sequence can be modified for heteronuclear editing by adding a [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C inversion pulse every other scan. Examples are: ACED-STEAM [START_REF] Pfeuffer | Localized in vivo 1H NMR detection of neurotransmitter labeling in rat brain during infusion of [1-13C] D-glucose[END_REF] , POCE with ISIS 105 , POCE-PRESS [START_REF] Kumaragamage | In vivo proton observed carbon edited (POCE) (13) C magnetic resonance spectroscopy of the rat brain using a volumetric transmitter and receive-only surface coil on the proton channel[END_REF]106 , POCE-LASER 107 or BISEP-SPECIAL 108 .

Adiabatic pulses mitigate the effects of inhomogeneous B 1 with surface coils. Semi-selective pulses can be used in the [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C channel to separate overlapping resonances 109,110 .

Heteronuclear decoupling: Excellent coil design and additional RF filters are necessary to achieve sufficient electrical isolation between 1 H and [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C channels and avoid injection of unwanted noise during decoupling. WALTZ-16 is the most commonly used decoupling scheme in preclinical [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C studies. Adiabatic decoupling can be used to further improve performance 111 . Unlike in humans, power deposition is generally not an issue in preclinical studies.

Data processing: Both direct [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C MRS and indirect 1 H- [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C MRS spectra should be processed with fitting algorithms that include prior knowledge, such as LCModel or jMRUI/AMARES 112,113 .

Metabolic modeling: The vast majority of in vivo [START_REF] Wolfensohn | Handbook of Laboratory Animal Management and Welfare[END_REF] C MRS studies have been done in the brain. Brain metabolic models can be divided into so-called one-compartment models and two (or more) compartment models (see for [START_REF] Gruetter | Localized in vivo 13C NMR spectroscopy of the brain[END_REF][START_REF] De Graaf | State of the art direct 13C and indirect 1H-[13C] NMR spectroscopy in vivo. A practical guide[END_REF][START_REF] Henry | In vivo 13C NMR spectroscopy and metabolic modeling in the brain: a practical perspective[END_REF]114,115 for reviews). One compartment models comprise one (primarily neuronal) TCA cycle rate. More complex two-and threecompartment models allow determination of neuronal-glial metabolic rates such as glial TCA cycle rate, pyruvate carboxylase, or glutamate-glutamine cycle.

More recently, models have also been developed to take into account the additional information from 13 C-13 C isotopomers [116][117][118] .

General consensus and recommendations:

 The anesthesia protocol should be carefully chosen and optimized considering the biological question to be addressed.

 It is essential to monitor and record physiological parameters under anesthesia.

 Surface coils are recommended to maximize transmit B 1 (and thus maximize RF pulse bandwidth) and increase SNR for MRS.  The preclinical MRS data should be quantified using a software that allows for a robust decomposition of the spectrum into a combination of individual metabolites. Alireza Abaei, Core Facility Small Animal Imaging, Ulm University, Ulm, Germany Volker Rasche, Core Facility Small Animal Imaging, Ulm University, Ulm, Germany The localization achieved with the STEAM (TR/TE = 5000/3.5 ms, 384 accumulations) sequence. While a cryogenically cooled 2-element phased-array transmit/receive coil was employed for excitation and signal reception, a 72 mm birdcage quadrature volume resonator was used for excitation and a receive-only rat brain 2x2 element surface coil array was used for signal reception. Here a 5.2-fold higher SNR in CP was realized.

4

  Sequences and acquisition protocols: 1 H MRS (Małgorzata Marjańska and Vladimir Mlynarik) In preclinical studies, short TE (≤ 10ms) localization sequences are usually possible to achieve and preferentially used because they provide the maximum information content in a 1 H MR spectrum by minimizing the J-evolution in coupled spins systems and reducing T 2 losses. The use of long TR eliminates possible T 1 effects and helps in quantification. The localization performance of the sequence is very important and the following properties should be considered when choosing the localization sequence: 1. ability to detect signals originating from the VOI; 2. ability to suppress the signals from outside of the VOI; 3. minimal chemical shift displacement (CSD) error related to the bandwidth of the localization pulses; and 4. insensitivity to the B 1 inhomogeneity especially when using surface coils.

FiguresFigure 1 :

 1 Figures

Figure 2 :

 2 Figure 2: Example 1 H MR spectra obtained in rodent brains at 9.4 T with (A) STEAM, (B) SPECIAL, and (C) LASER sequences. (A) STEAM spectrum: rat brain, 2.3 x 1.3 x 2.5 mm 3 voxel placed in the hippocampus, TR = 5 s, TE = 2 ms, TM = 20 ms, number of averages = 448. Spectrum is shown with Gaussian factor = 0.15. (B) SPECIAL spectrum: rat brain, 2 x 2.8 x 2 mm 3 voxel placed in the hippocampus, TR = 4, TE = 2.8 ms, number of averages = 160. (C) LASER spectrum: mouse brain, 1.7 x 2.25 x 2.25 mm 3 voxel placed in hippocampus, TR = 4 s, TE = 27 ms, number of averages = 384.

Figure 3 :

 3 Figure 3: 1 H MRS spectra of 4.4 μL voxel placed in the mouse cingulate cortex at 11.7T using cryogenically cooled RF coi (solid line: CP) and room temperature coil (dashed line: RT).

Figure 4 :Figure 5 : 13 C 2 ] 13 C 6 ] 13 C

 4513213613 Figure 4: 1 H MRS spectra of 4.4 μL voxel placed in the mouse cingulate cortex at 11.7T using LASER (solid line) and STEAM (dashed line) sequences. In comparison to STEAM (TR/TE = 5000/3.5 ms, 384 accumulations) the use of LASER (TR/TE = 5000/16.75 ms, 384 accumulations) yields a better SNR. Here a 2.3-fold higher SNR in LASER was realized.(174.6 vs 74.9)

Figure 6 : 31 P

 631 Figure 6: Example of in vivo localized (A upper spectrum) and unlocalized (A lower spectrum and B) 31 P spectra in the rat brain at 9.4T. Voxel size 5x9x9 mm 3 , TR=8s, 512 averages.

Table 1 :

 1 Characteristics of commonly used anesthetics and their impact on brain metabolites

		Physiological		Side effects		Effect on brain	Type	References
		Effects				metabolites
						↓↑*
						statistically
						significant
						changese
						(p<0.05)
	Propofol	Rapid-and short-	Muscle twitching,	Lactate	↓,	Injectable	15,30
		acting anesthesia	apnea,		glutamate ↓*
		effect,	fast	hypotension,		*(compared
		recovery time		decreased cardiac	with isoflurane)
				output
	Isoflurane	rapid-and short-	Respiratory		Lactate	↑,	Inhaled
		acting anesthesia	depression, dose	GABA	↑,
		effect,	fast	dependent		Choline-
		recovery time		hypotension,		containing
				increased cerebral	compounds ↑,
				blood	flow,	myo-inositol ↑,
				immune		glucose ↓, N-
				suppression		Acetylaspartate
						↑,	total
						creatine	↑,
						creatine	↑,


Cyril Gorlla
email: cyril.m.gorlla@jacobs.ucsd.edu

Prediction of Responses To Binary Customer Questions With Answer Data

Keywords: CCS Concepts:, Computing methodologies → Machine learning; Artificial intelligence neural networks, natural language processing, machine learning, artificial intelligence

Within the context of online shopping, it is often imperative to respond to customer queries promptly. In this paper, we explore and evaluate various methods of responding to binary customer questions with machine learning algorithms. Specifically, we implement models based on logistic probabilities as well as collaborative filtering in order to predict the response to binary questions on Amazon. We find that probabilistic models fare the best on our validation set (which includes unseen data), with the logistic model trained with vector embeddings achieving 67.6% accuracy.

Dataset

The proliferation of online shopping has naturally led to a large amount of questions being asked by customers on various websites, the most notable being Amazon. Here, we Permission to make digital or hard copies of part or all of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page. Copyrights for thirdparty components of this work must be honored. For all other uses, contact the owner/author(s). , , © 2021 Copyright held by the owner/author(s). . utilize a subset of a dataset [START_REF] Mcauley | Addressing Complex and Subjective Product-Related Queries with Customer Reviews[END_REF][10] consisting of Amazon products, along with questions and answers related to the products.

We selected a subset of the dataset that concerned electronics, amounting to 231,449 questions and 867,921 answers. The dataset contains information on the product ID (ASIN), the questions asked about that product, and answers for each question. A feature denoting whether the question is binary or open-ended also exists, as well as polarity scores for answers in the former category. We then refined the dataset to only include binary questions, defined as being able to be answered by a "yes" or "no".

After this, we were left with 61,222 samples. First, we explored the breakdown of the answers (Fig. 2). As we have a 2 : 1 imbalance of "yes" to "no" answers, we may either represent this as an accurate distribution of answers in the real world, or account for the differences in building our model. Next, we looked at the length of the questions (Fig. 3). The distributions of length were fairly identical. Finally, we examined the polarity score (Fig. 4). As the binary label was based on the polarity scores, it is sensible that the "yes" and "no" scores are respectively very high and low.

Predictive Task

Having now filtered the dataset to include only binary questions and binary responses to those questions, we proceed with formalizing the predictive task. Given some features for a binary question 𝑖 : {𝑖 1 ...𝑖 𝑛 }, we wish to predict the answer to this question in {𝑌 |𝑁 }. Features that are relevant for use in such a task consist of the product that the question is being asked about, as well as features pertaining to the question itself, such as the length and text of the question. It is also necessary to determine how to generate a proper label for each question. We use the most frequent binary label that appears in a question's answers to determine the "ground truth" label for that question. In cases of a tie, the label is randomly selected. This occurs in .06% of the data.

As the data was "nested", that is, each product contained a list of questions, and each question contained a list of answers, it was necessary to "flatten" the data such that we had a single row that contained the product ID, a binary question about that product, and the labelled response for that question. We also assigned a unique number to the products Figure 4. Polarity Distributions and questions in order to create valid training vectors. Word vector embeddings may also be predictive in this task, and so we utilize Word2Vec [START_REF] Mikolov | Distributed Representations of Words and Phrases and their Compositionality[END_REF] in order to train two word vector models: one trained on "yes" questions, and the other trained on "no questions". If there are semantic differences within these questions, the higher of the probability scores when inputting a question into the two models may be a valid signal for our model.

Two main models were used: a logistic model and a collaborative filtering neural network model. For the logistic model, we can utilize a standard train-validation split of 75%-25%, and due to the randomized nature of the split, we can utilize accuracy as our evaluation metric. For the neural network model, we utilize the same split but use binary cross-entropy as an evaluation metric to rectify the imbalance. The logistic model allows us to have a simple model with the notion of probability, with the sigmoid function scaling the output from 1 to 0, where 1 is "yes" and 0 is "no". The neural network allows us to utilize patterns of overlap in products and questions.

For a baseline for our model, we can utilize a naive input of the products and questions encoded as integers into the collaborative filtering model, with no additional processing.

Model

The final logistic model was trained with scores generated from the Word2Vec models. Word2Vec tries to find [START_REF] Mostafachatillon | mostafachatillon/word2vec[END_REF] 𝑅 : Words = {𝑤 1 , ..., 𝑤 𝑁 } → Vectors = {𝑅(𝑤 1 ), ..., 𝑅(𝑤 𝑁 )} ⊂ R 𝑑 such that:

𝑤 𝑖 ≈ 𝑤 𝑗 (meaning of words)
is equivalent to:

𝑅(𝑤 𝑖 ) ≈ 𝑅(𝑤 𝑗 ) (distance of vectors)
If a particular question had a higher score from the "no" model, the feature for that question would be 1 -𝑠𝑐𝑜𝑟𝑒, or just the score if the positive model was higher. Unlike the collaborative filtering model, the logistic model does not capture notions of product-question overlap, but is able to more directly represent patterns of probability in the data. Other features and combinations of features like length were implemented, but did not function as well.

The final collaborative filtering model utilized the length of the question and the product ID to predict a response to the question. Similarly to the logistic model, other combinations of features proved less useful. A batch size of 8 constituted the best performance in training the model. We define the notion of similarity in this model with the dot product of the vectors, that is

𝑠𝑖𝑚(𝑖, 𝑗) = - → 𝑖 • - → 𝑗 (1)
. The work that this dataset originated from [START_REF] Wan | Modeling Ambiguity, Subjectivity, and Diverging Viewpoints in Opinion Question Answering Systems[END_REF] [6] dealt with addressing customer queries from available product data.

The authors chose to predict responses to questions based on review data using a mixture-of-experts (MoE) model, factoring in product reviews and giving each review a "vote". We use a similar notion, although we only use question data and focus on binary questions. Our process of generating ground truth labels is similar in that we examine the binary responses and choose the mode of them, as the most frequent answer is likely to be the correct response to the query.

Other research [START_REF] Clark | BoolQ: Exploring the Surprising Difficulty of Natural Yes/No Questions[END_REF] has focused more generally on answering binary questions, not in the specific context of online shopping. They concluded that predicting responses to binary questions was a difficult task, with their final model scoring 10% worse than human performance at the same task. This general pattern was also replicated in our work, with prediction accuracy generally being meager compared to human performance. BERT, a large language model, was used in that paper to derive meaning from questions. In comparison, the Word2Vec implementation we use is limited and lightweight, only being trained on the test of the questions with each label. Using a model trained on much more general data like BERT may yield performance gains at the expense of significantly increased model complexity.

A pertinent application of predicting question responses is to facilitate chatbots on websites that assists customers with their queries. When this has been implemented [START_REF] Cui | SuperAgent: A Customer Service Chatbot for E-commerce Websites[END_REF], parsing text both from the product page as well as user-generated content like reviews and questions/answers has proven to be useful in accurately answering customer queries. While we only look at the ASIN to provide a unique identifier for each product, incorporating details about the product may be a valuable area of exploration.

Results

The best model was the logistic model that incorporated Word2Vec similarity scores, with a validation accuracy of 67.7%. Our baseline model had a validation loss of .68, which was improved to .65 when replacing the direct question encoding with the question length instead. However, it is apparent that the collaborative filtering model is generally unconvincing when compared to the validation accuracy of the logistic model. This denotes that the product and question vector embeddings are not predictive within the context of the collaborative filtering model. Probabilistic methods, such as our logistic regression model, fared the best on our validation set, indicating that the proportion of "yes" and "no" answers in general is predictive, along with word vector embeddings of the two groups. Further development in this area may find the incorporation of further information about the product and larger language models to be valuable in predicting answers.

Figure 2 .Figure 3 .

 23 Figure 2. Answer Distribution

Figure 5 .

 5 Figure 5. Collaborative Filtering Model

Figure 6 .

 6 Figure 6. Final Logistic Model Predictions

Table 1 .

 1 Length Statistics

	Mean	50.198
	Std. Dev. 38.56
	25%	29
	75%	59


Albert Leroy

Alexandre Navet

Thierry Schweitzer

Lionel Aranda

Patrice Berthod
email: patrice.berthod@univ-lorraine.fr

Effect of the presence of water vapour on the high temperature oxidation of Co-10Ni-30Cr and Co-10Ni-30Cr-0.5C alloys. Part 1: Results in oxidation in dry air 1

Water vapour is known to influence the general be haviour of refractory a lloys and superalloys in oxidation at high temperature [1], in terms of isothermal ox idation rate, volatilization of the protective scale formed (alumina, chr omia or silica), and of oxide scale spallation in thermal cycling. After having studied the double kinetic of both mass gain of oxidation and mass loss by chr omia volatilization in the case of chromia-forming nickel-based alloys [2], it was wished here to characterize the effect on oxidation of water vapour in the case of c obalt alloys, with thermogravimetry recording followed, here too by using the {m x dm/dt = Kp -Kv x m} method [3] for the isothermal part, and by examining their heating and cooling parts. Cobalt-based superalloys are ge nerally ric h in chromium (several tens weight percents) and are strengthened by carbides. Many of them additiona lly contain several weight pe rcents of nickel to stabilize the FCC network of the matrix. These are the reasons why the two simple m odel a lloys considered in this work are: first a carbide-free Co-10Ni-30 Cr ternary alloy to study the specific beha viour of the matrix, and second a Co-10Ni-30Cr-0.5C to examine the case of the same alloy but containing significant fraction in chromium carbides. These two alloys were elaborated by high frequenc y induction melting fr om pure elements, in inert gas. The parallelepiped samples were cut and ground with smoothing of e dges a nd corners, then subjected to thermogravimetr y runs performed in dr y s ynthetic air and in humidified air (temperatures of test: 1000, 1100 and 1200°C, duration: 50 hours. The a nalys is of the three parts (heating, isothermal oxidation, cooling) of the mass gain files, as well as the post-mortem metallographic characterization of the oxidize d samples, will allow e videncing the effect of water vapour on numerous characteristics of the high temperature oxidation of these cobalt-based alloys. In this first part only the results of oxidation in dry air will be presented.

With these two simple alloys, typical of many of commercial cast cobalt-based alloys and their 10 wt.%Ni for austenite stabilizing and their 30 wt.%Cr, and 0.5 wt.%C for the second alloy, this characterization of their high temperature oxidation behaviour will be used for comparison basis for the same alloys but oxidizing in wet air. Differences concerning the start of oxidation and the rates of mass gain, but also the rate of chromia volatilization and the effect on scales spallation at cooling or during thermal cycling oxide adherence will be particularly expected.