Sameh Sassi Aydi

Samir Aydi

Rihab Ben

Abdallah Kolsi

Nouha Haddeji

Rami Rahmani

Naourez Ktari

Jalloul Bouajila
email: jalloul.bouajila@univ-tlse3.fr.

Sameh Sassi Aydi

Samir Aydi

CO2 enrichment: Enhancing antioxidant, antibacterial and anticancer activities in Arthrospira platensis

Keywords: Arthrospira platensis, Spirulina, CO2 enrichements, phytochemicals, antibacterial activity, OVCAR, MCF-7

Introduction

Global atmospheric carbon dioxide (CO2) concentration has significantly increased since the industrial revolution (270 to 400 ppm), elevating by about 2 ppm/yr during the past few decades [START_REF] Dlugokencky | Atmospheric composition[END_REF][START_REF] Moreira | Atmospheric CO2 capture by algae: Negative carbon dioxide emission path[END_REF]. Global CO2 emissions are about 30 Gt/yr, which creates an imbalance in the natural carbon cycle leading to its accumulation in atmosphere (IPPC, 2001). This elevated global atmospheric CO2 concentration may not only cause climate warming, but also cause profound impacts on the net primary productivity of agricultural and natural ecosystems (Zheng et al., 2018).

The reduction of greenhouse gases has become a priority issue (Yoshioka et al., 2013).

Photosynthesis by microalgae using atmospheric CO2 is one process that can reduce CO2 in the atmosphere [START_REF] Jeon | Measurement of microalgal photosynthetic activity depending on light intensity and quality[END_REF][START_REF] Kim | Quantitative analysis of Spirulina platensis growth with CO2 mixed aeration[END_REF][START_REF] Tarko | Influence of growth medium composition on synthesis of bioactive compounds and antioxidant properties of selected strains of Arthrospira cyanobacteria[END_REF]. Cyanobacterium are a blue-green algae found in aquatic and terrestrial habitats. Microalgae produce different compounds, many with bioactivity (Abd [START_REF] El-Baky | Production of phenolic compounds from Spirulina maxima microalgae and its protective effects[END_REF][START_REF] Kumari | Carbon dioxide assisted Spirulina platensis cultivation using NPK-10:26:26 complex fertilizer in sintered disk chromatographic glass bubble column[END_REF][START_REF] Wang | Elevated carbon dioxide increases contents of antioxidant compounds in field-grown strawberries[END_REF]. Microalgal production in raceway ponds seems to be the best method for large scale production. A. platensis is a small spiral-coil shaped blue green algae that grows naturally in mineral-rich alkaline waters, and is increasingly farmed around the world due to its many benefits. Many studies have described A. platensis higher productivity with CO2 added to growing medium [START_REF] Gordillo | Effects of increased atmospheric CO2 and N supply on photosynthesis, growth and cell composition of the cyanobacterium Spirulina platensis (Arthrospira)[END_REF][START_REF] Ravelonandro | Improvement of the growth of Arthrospira (Spirulina) platensis from Toliara (Madagascar): Effect of agitation, salinity and CO2 addition[END_REF]Singh & Singh, 2014). However, none has qualitatively evaluated the bioactive compounds with a CO2 enriched culture media.

Generally, outdoor open raceways have been used in the production of microalgae and cyanobacteria at the commercial level [START_REF] Castro | Biomass production by Arthrospira platensis in different culture conditions[END_REF][START_REF] Costa | Spirulina platensis growth in open raceway ponds using fresh water supplemented with carbon, nitrogen and metal ions[END_REF][START_REF] Richmond | Quantitative assessment of the major limitations on productivity of Spirulina platensis in open raceways[END_REF][START_REF] White | Long-term cultivation of algae in open-raceway ponds: lessons from the field[END_REF].

Several reports dealing with the effects of increasing atmospheric CO2 concentration on the growth parameters and biochemical composition have been done with plants [START_REF] Reitz | Effects of elevated atmospheric carbon dioxide on the growth and linear furanocoumarin content of celery[END_REF][START_REF] Wang | Elevated carbon dioxide increases contents of antioxidant compounds in field-grown strawberries[END_REF]Yusuke et al., 2007) and algae [START_REF] Gordillo | Effects of increased atmospheric CO2 and N supply on photosynthesis, growth and cell composition of the cyanobacterium Spirulina platensis (Arthrospira)[END_REF][START_REF] Kumari | Carbon dioxide assisted Spirulina platensis cultivation using NPK-10:26:26 complex fertilizer in sintered disk chromatographic glass bubble column[END_REF]Singh & Singh, 2014). Algae have an important role in the carbon cycle and their response to elevated CO2 are of potential benefit (Xu et al., 2010). However, information about the effects of elevated CO2 on the health-promoting phytochemicals in food algae are limited. This study aims to assess the impact of CO2 enrichment on phytochemical levels in A. platensis extracts: (i) polyphenols, flavonoids and tannins content, and (ii) antioxidant (TAC, DPPH and FRAP assays), anti-bacterial and cytotoxic activities.

Materiel and Methods

Algae strain, growth medium and culture conditions

The A. platensis strain was obtained from Eden Life (a Tunisian company specializing in the production of microalgae) located in the south of the country (Kettana, Gabes). The A. platensis algae were cultivated using the standard Zarrouk medium (containing macronutrients: NaHCO3: 18.0, NaNO3: 2.5, K2HPO4: 0.5, K2SO4: 1.0, NaCl: 1.0, CaCl2: 0.04, Na2EDTA: 0.08, MgSO4.7H2O: 0.20, FeSO4.7H2O: 0.01 and trace elements: H3BO3: 2.86, (NH4)6 Mo7O4: 0.02, MnCl2.4H2O: 1.80, CuSO4.5H2O: 0.08, ZnSO4.7H2O: 0.22 (g/L)) (Zarrouk 1966). A. platensis cultivation was done in 10 m 3 raceway ponds. A raceway is an oblong and superficial recirculating pond with semi-circular ends. A paddlewheel continuously circulates (30 cm/s) and mixes and aerates the algal broth in the channel. Ponds (50 cm depth) were placed in a controlled glass house protected from UV light, using plastic film. In the open raceway ponds, the volume of the medium was maintained unchanged by the replacement of the water lost by evaporation. The cultures were illuminated using halogen spots (Decostar Titan 51, 12V, 35 W; Osram GmbH, Munich, Germany) which provided 52-55 µmol (photon) m -2 s -1 and light/dark cycles of 16/8 h at 25 °C and aerated continuously with liquid CO2 at ~50 bar with a flow rate of 0.3 L min -1 . The high purity CO2 (99.9%) was provided by the Air Liquide Co. (Sfax, Tunisia). The bubbling of the CO2 in the raceway pond is done using an air flow meter diffuser (Air Liquide, HBS 315/8 c7 1206) with a pipe in the form of a T for greater diffusion. The culture was filtered through a fine mesh (about 30 μm) which collects the Spirulina biomass. The fresh biomass was then dried using a self made solar panel hot air drier and vacuum stored at room temperature (23±3 °C), before extractions were carried out for a maximum of 10 wk.

Proximate Analysis

Analyses of A. platensis for ash was done using combustion at 550 °C overnight [START_REF] Woyewoda | Recommended laboratory methods for assessment of fish quality[END_REF]. Moisture content was assayed as follows: about 3 L of the algal culture were collected and filtered through a pre-weighed 30 μm mesh filter (Diatex, Laval, France).

Samples were weighed before and after drying for 15 h at 105 °C. Water content is calculated by the difference between the fresh weight (fw) and the dry weight (dw). Total sugars were determined using the phenol-sulfuric acid method [START_REF] Dubois | Colorimetric method for determination of sugars and related substances[END_REF].

Kjeldahl apparatus and method (using a DK8 digester and UDK 129 distillation unit, Velp-Scientifica, Usmate, Italy) was used to determine the total nitrogen content in A. platensis samples (AOAC, 1996). After total nitrogen content determination, crude protein contents were calculated: N% = V*0.0014*(100/M) P% = N%*6.25 (Where V = sulfuric acid volume and M = dw)

Mineral composition determination

Contents of Ca, Cd, Fe, K, Mg, Na, Pb, and Zn were assayed according to the method of [START_REF] Indrayan | Determination of nutritive value and analysis of mineral elements for some medicinally valued plants from Uttaranchal[END_REF]. Mineral compositions of the sampled were made as follows: 20 to 25 mg of dried samples were ashed using 50 mL 1% hydrochloric acid then filtered using a Whatman ashless filter paper (GE Healthcare, GmbH, Freiburg, Germany). Atomic absorption spectrometry (Perkin Elmer Model AAS 4100, Boston, MA, USA) was used for determination of mineral contents. All the analysis were evaluated in triplicate.

Soxhlet lipid extraction

Lipid content was analyzed according to the method of [START_REF] Ramluckan | An evaluation of the efficacy of using selected solvents for the extraction of lipids from algal biomass by the soxhlet extraction method[END_REF]. Briefly, dried biomass (0.5 g) was extracted in chloroform for 5 h using a solid-liquid extraction system (Soxhlet extractor CG-1374 GPE Scientific, Ltd., Bedford, UK). The extract was then concentrated on a rotary evaporator and weighed.

Preparation of methanol extracts

Dried biomass of A. platensis was extracted using methanol with a constant solid/liquid ratio of 1:10 (w/v). The solid-liquid extraction was done for 2 h, where the suspension was continuously mixed using a magnetic agitator (AREX Velp-Scientifica, Italy). The extraction was carried out in the dark at room temperature and then the liquid was separated by centrifugation (2,200 g) for 10 min at 20°C using a HERMLE Z 513 K centrifuge (Hermle Labortechnik, Wehingen, Germany). Then, a rotary vacuum evaporator R-200 (Büchi Labortechnik, Flawil, Switzerland) was used to evaporate the extract. The supernatant samples were dissolved in methanol (10 mg mL -1 ).

Total polyphenols content (TPC)

TPC was determined spectrophotometrically using the Folin-Cioclateu method [START_REF] Singleton | Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent[END_REF]. Briefly, 0.1 mL of sample extract was mixed with 0.5 mL of 10-fold diluted Folin-Ciocalteu's phenol reagent and allowed to react for 5 min. Then 0.4 mL 7% Na2CO3 solution was added and the final volume was brought to 2 mL with distilled water. After 1 h at room temperature, the absorbance at 760 nm was measured using a UV-VIS Spectrophotometer (2005, JP Selecta S.A., Barcelona, Spain). A calibration curve was prepared using gallic acid (GA) as a positive control. Results were expressed as mg GA equivalents(E)/g of the Spirulina dw.

Total flavonoids content (TFC)

TFC was done using a colorimetric method according to [START_REF] Dewanto | Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity[END_REF]. An aliquot (500 µL) of algae extract or standard solution of catechin (C, 0 to 500 µg mL -1 )was added to 1.5 mL distilled water, 150 µL 5% sodium nitrite solution and mixed for 6 min before addition of 150 µL 10% aluminum chloride. Volume was adjusted with distilled water to 2.5 mL and incubated at room temperature for 6 min. Reaction was completed by adding 500 µL 4% sodium hydroxide. The final absorbance was determined at 510 nm against a blank. The TFC was reported as mg CE/g dw against the calibration curve of catechin, from 0 to 500 µgmL -1 .

Tannins content

Condensed tannins were assayed using the method previously described by [START_REF] Sun | Critical factors of vanillin assay for catechins and proanthocyanidins[END_REF].

A. platensis extract (50 µL) was added to, 3 mL 4% vanillin (w/v in methanol) and 1.5 mL concentrated HCl. Then the absorbance was measured at 500 nm after incubation in the dark (15 min), at room temperature. The content of total condensed tannins in Spirulina extract is expressed as mg CE/g dw.

In vitro antioxidant assays

Total antioxidant activity (TAC)

The TAC was determined using the phosphate molybdate method [START_REF] Prieto | Spectrophotometric quantitation of antioxidant capacity through the formation of a phospho-molybdenum complex: Specific application to the determination of vitamin E[END_REF]. Briefly, 0.1 mL of extract was combined with 1 mL of solution composed of sulfuric acid (0.6 M), sodium phosphate (28 mM) and ammonium molybdate (4mM). The mixture was then incubated at 95 °C for 90 min. The absorbance was measured at 695 nm, after cooling to room temperature. Results were expressed as mg GAE/g dw.

DPPH radical scavenging assay

The stable free radical scavenging activity was evaluated using the DPPH assay [START_REF] Shekhar | Antioxidant activity by DPPH radical scavenging method of Ageratum conyzoides Linn leaves[END_REF]. A methanol extract of A. platensis was mixed, at equal volume, with 2,2diphenyl-1-picrylhydrazyl (DPPH, 100 mM), then incubated at room temperature, for 15 min.

The absorbance was determined at 517 nm. Butylated hydroxytoluene (BHT) was used as a positive control. These measurements were done in triplicate and the percentage of inhibition of DPPH oxidation (Pi) was calculated using the following formula:

Pi = ((Ab-As)/Ab)*100

Where Ab is the absorbance of the control and As is the absorbance of the extract. The 50% inhibitory concentration (IC50) was expressed as the quantity of the extracts to react with a half of DPPH radicals. The IC50 values were calculated using linear regression analysis and used to indicate antioxidant capacity.

Ferric Reducing Antioxidant Power assay (FRAP)

The FRAP assay was done according to the process of [START_REF] Benzie | The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: The FRAP assay[END_REF]. The FRAP reagent was prepared from 0.3 M acetate buffer (pH 3.6), 10 mmol 2,4,6-tripyridyl-s-triazine (TPTZ) solution in 40 mmol hydrochloric acid and 20 mmol iron (III) chloride solution with the ratio 10:1:1 (v/v/v). Briefly, 50 µL of sample of phytochemicals (three replicates) were added to 1.5 mL of the FRAP reagent. Four min after, the absorbance was determined at 593 nm. The standard curve was constructed using FeSO4 solution (from 50 to 200 µg mL -1 ), and the results were expressed as µg mL -1 Fe (II).

Gas Chromatography -Mass Spectrometry (GC-MS)

The chemical identification of volatile compounds from the organic extracts was determined using the method of [START_REF] Kohoude | Chemical composition and biological activities of extracts and essential oil of Boswellia dalzielii leaves[END_REF]. The gas chromatography-mass spectrometry was an Agilent Technologies GC systems with a GC-7890A/MS-5975C model (Agilent Technologies, Santa Clara, CA, USA) equipped with a fused silica capillary DB-5MS column (5% phenyl-methylpolysiloxane, 30 x 0.25 mm, film thickness 0.25 µm, Agilent Technologies). Chromatographic conditions were; 50 °C hold for 1 min, up to 250 °C at the rate of 10 °C min -1 and then held 1 min at 250 °C. After that, a second gradient was used going to 300 °C at 50 °C min -1 and finally a 300 °C hold for 3 min. For analysis reasons, the samples were dissolved in their principal solvents. Ten µL of each extract was injected (into a 200 °C heated injector) with the ratio of 1:10 in the split mode. The carrier gas used was helium with a flow rate of 1 mL min -1 . The mass spectrometer was adjusted for an emission current of 10 µA and electron multiplier voltage 1400 -1500 V. Trap temperature was 250 °C and that of the transfer line was 270 °C. Mass scanning was in the interval of 40 to 650 amu.

The identification of the compounds was done using mass spectra comparison with those obtained in NIST08 (National Institute of Standards and Technology, https://www.nist.gov/), using AMDIS software (Automated Mass Spectral Deconvolution and Identification System, https://chemdata.nist.gov/dokuwiki/doku.php?id=chemdata:amdis).

AMDIS is a software used for GC-MS data interpretation from NIST. The goal was to find the maximum similarity, in terms of spectra, between the compounds detected in extracts and those suggested by the NIST database. Finally, the retention index facilitated certain assignments.

Cytotoxicity evaluation

Cytotoxicity of extracts against human breast (MCF-7), ovarian (OVCAR) and colon (HCT-116) cancer cell lines, ordered from the American Type Culture Collection (ATCC Co., Manassas, VA, USA), was estimated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay of El [START_REF] El Euch | Chemical composition, biological and cytotoxic activities of Cistus salviifolius flower buds and leaves extracts[END_REF]. The HCT-116 cell line was grown in Gibco RPMI 1640 medium (Thermo Fisher Scientific, Paisley, UK),while MCF-7 and OVCAR cell lines were grown in Dulbecco's modified eagle medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% faetal calf serum (Gibco, Langley, OK, USA), air and 5% CO2. Briefly, 100 µL of cells were distributed in 96-well plates at a concentration of 10 4 cells/well and incubated at 37 °C for 24 h. Then, 100 µL of cells in exponential growth phase were incubated in a fully humidified atmosphere at 37 °C for 48 h with the addition of 100 µL of the culture medium, supplemented with 2 mM L-glutamine and 50 μg mL -1 gentamycin, containing extracts at a concentration of 50 mg L -1 . The medium was removed and cells were treated with MTT solution, i.e., 50 µL, 1 mg mL -1 in phosphate buffered saline (PBS) and incubated at 37 °C for 40 min. MTT solution was then discarded and dimethyl sulfoxide (DMSO) (50 µL) was added to dissolve the blue crystals. A microplate reader (Multiskan Go, F1-01620, Thermo Fisher Scientific, Vantaa, Finland) was used to measure the absorbance at 605 nm. Tamoxifen (100 µM) was used as a positive control.

Antibacterial activity

The A. platensis extracts were tested individually 9 human-pathogenic microbial strains: 6

Gram positive bacteria (Enterococcus faecalis; Listeria monocytogenes; Bacillus subtilis; Bacillus cereus; Bacillus thuringiensis; Micrococcus luteus) and 3 Gram-negative ones (Salmonella enterica; Escherichia coli; Klebsiella pneumoniae). The bacteria used were selected for their involvement in the human skin, oral and intestinal tract.

The antimicrobial activities of Spirulina extracts were assayed using the well diffusion method according to NCCLS M2-A5 (NCCLS, 1993). Inoculum containing 10 6 colonyforming units (cfu) mL -1 of each bacterial culture to be tested was spread on Mueller-Hinton (MH) agar plates with a sterile swab moistened with the bacterial suspension. Subsequently, wells of 6 mm diameter were punched, using a sterile agar cutter, into the MH agar medium.

The antimicrobial activity of the Spirulina extract was checked by introducing 50 μL of 100 µg mL -1 concentrations into triplicate wells. An additional wells in each plate was filled with the solvent DMSO, which served as the negative controls while ampicillin (5 µg mL -1 ) was used as positive control. The culture plates were allowed to diffuse for 2 h at room temperature. The plates were then incubated, for 24 h, in the upright position at 37 °C. After that, the antimicrobial activities of the algae extract and the standard antibiotic were determined. Zones of the inhibition around each of the extract and the antibiotic were measured to the nearest mm using a digital caliper (Shanghai Taihai Congliang Ju Co., Ltd., Shanghai, China). Three replicates were carried out for each test microorganism.

Statistical Analysis

Data were expressed as the mean value ± SD of 3 replicate samples. The statistical analyzes were done using the one way analysis of variance (ANOVA) procedure with the Statistical Package for the Social Sciences (SPSS) version 20.0 software (IBM Corp, released 2011, Armonk, NY, USA). When p < 0.05, differences were considered as statistically significant according to Fisher's LSD test.

Results

Proximate analysis

The proximate composition parameters of A. platensis with controlled (-CO2) or carbon dioxide enriched medium (+CO2) is reported in Table 1. Results showed low moisture content with both culture media. Increased CO2 in the culture medium seemed to decrease protein content and to increase carbohydrate, while moisture, ash and fats contents remained unchanged with CO2 treatment (Table 1). 2 show that A. platensis powder contains high amounts of K, Mg and Na (more than 1.1 g/100 g dw) followed with Ca than iron. Enhanced CO2 in the culture medium affected the mineral composition differently: No significant trend was detected for K, Mg, Zn and Na, while a significant increase was seen for Fe (26%) and Ca (21%). Pb and Cd were not detected with both media.

Mineral element composition

Results in Table

Phytochemical

Contents of polyphenols compounds expressed as GAE and flavonoids and tannins contents expressed as CE were measured in A. platensis methanolic extracts (Fig. 1). Elevated CO2 in the culture medium (+CO2) significantly increased the polyphenols, flavonoids and tannins.

Amongst the components detected in the methanol extract of A. platensis, polyphenols showed the most important increase in CO2 enriched medium reaching 54%, while flavonoids reached 36% and tannins only 28%.

Antioxidant activity

The antioxidant activity of A. platensis methanolic extracts was evaluated. Three complementary experiments were used to estimate the antioxidant activity of A. platensis: TAC, DPPH free radical-scavenging activity and FARP. It is important to use different measures of antioxidant capacity, because many factors can influence this capacity. As summarized in Figure 2, TAC ranged from 25 to 35 mg GAE g -1 dw, respectively with CO2 free (-CO2) and CO2 fed (+CO2) medium The half-maximal inhibitory concentration (IC50) is an estimation of the efficiency of a substance in reducing a specific biological and/or biochemical function. It represents the amount of a compound or bioactive molecule that is required for 50% inhibition in vitro. A. platensis methanol extracts had the capacity to reduce DPPH. CO2 enrichment induced a stimulation of antioxidant activities, the IC50 were increased by 25 and 65% for DPPH and FRAP, respectively.

Antibacterial activity

The antibacterial activities of A. platensis methanol extracts was determined against 6 Grampositive bacteria and 3 Gram-negative ones and results are shown in Table 3. A. platensis methanolic extract showed the highest mean ZOI against the Gram-positive L. monocytogenes and B. thuringiensis while it showed lower ZOI against the other Gram-positive studied bacteria. For Gram-negative bacteria, the maximum ZOI was observed against K.

pneumoniae. E. coli showed the minimum ZOI against all the studied bacteria. No ZOI was found in the DMSO blind control whereas in the positive control ampicillin (5 µg) ZOI ranged from 15 to 20 mm against all the studied bacteria. Increased CO2concentration level with culture conditions (+CO2) directly affected responses of A. platensis methanol extract with both E. coli and S. enterica bacteria by increasing ZOI. The other bacteria showed no significant trend with elevated CO2 in the culture medium.

Cytotoxic activity

Anticancer activity of A. platensis methanol extracts with different concentrations were determined using the cell lines viability assay. Table 4 summarizes

GC-MS analysis

The A. platensis methanolic extract using GC-MS analysis showed the presence of 12 volatile compounds. Table 5 shows the compounds with their retention time (RT) and molecular formula. In the CO2 free medium, the methanol extract of A. platensis showed 12 compounds.

The most abundant compound was the hexadecanoic acid, methyl ester. The n-hexadecanoic acid; 8,11-octadecadienoic acid, methyl ester and phytol were also found at intermediate quantities as compared to the other compounds. However in CO2 enriched medium results shown in Table 5 showed only 7 compounds. Glycerin; 4:3,6-dianhydro-α-d-glucopyranose; heptadecane; 4-hydroxy-β-ionone and 3',8,8'-trimethoxy-3-piperidyl-2,2'-binaphthalene-1,1',4,4'-tetrone were not detected. The production of the n-hexadecanoic acid was reduced by 80% with the CO2 enrichment. The quantities of the other compounds are greater than or equal for the CO2 enriched system. The majority of compounds extracted were fatty acids or esters of fatty acids.

Discussion

CO2 enrichment did not alter the mineral composition in A. platensis (Table 2). A. platensis could be considered as good sources of minerals and available carbohydrate with low levels of fats. Previous studies reported different trends of minerals and proximate composition [START_REF] Babadzhanov | Chemical composition of Spirulina platensis cultivated in Uzbekistan[END_REF][START_REF] Branger | Spiruline as a food supplement in case of infant malnutrition in Burkina-Faso[END_REF][START_REF] Oluyemi | Mineral contents of some commonly consumed Nigerian foods[END_REF]. This could be due to different growth conditions, locality and culture medium composition.

Enhanced CO2 in the culture medium increased carbohydrate content (Table 1). A stimulation of growth, photosynthesis rate and biochemical composition with elevated CO2 are shown in most reviews [START_REF] Bazzaz | The response of natural ecosystems to the rising global CO2 levels[END_REF][START_REF] Mattson | CO2 enrichment and carbon partitioning to phenolics: Do plant responses accord better with the protein competition or the growth-differentiation balance models[END_REF]. Exposure of plants to elevated CO2 usually leads to amplified rates of net photosynthesis which often enhances total nonstructural carbohydrates concentration [START_REF] Booker | Influence of carbon dioxide enrichment, ozone and nitrogen fertilization on cotton (Gossypium hirsutum L.) leaf and root composition[END_REF]. Enhanced polyphenols, flavonoids and tannins in A. platensis with CO2 enrichment was found (Table 3). According to the carbon nutrient balance theory, that with higher atmospheric CO2, the increases of the carbon to nitrogen ratio induces the synthesis of secondary metabolites by carbon allocation to the secondary metabolism [START_REF] Tisserat | Essential oils enhanced by ultra-high carbon dioxide levels from Lamiaceae species grown in vitro and in vivo[END_REF]. CO2 enrichment conditions significantly enhanced polyphenols content (20 mg g -1 ) flavonoids (10 mg g -1 ) and tannins (5.4 mg g -1 ) in A. platensis, and these high contents were associated with elevated antioxidant activity (Fig. 2). Elevated CO2 may allow for the removal of free radicals, which are associated with increased antioxidant capacity [START_REF] Wang | Elevated carbon dioxide increases contents of antioxidant compounds in field-grown strawberries[END_REF]. The polyphenols contained different bioactivities, including anti-inflammatory, antiviral, antioxidant, antithrombotic, vasodilatory, and anticarcinogenic properties [START_REF] Singh | Evaluation of phenolic composition, antioxidant, antiinflammatory and anticancer activities of Polygonatum verticillatum (L.)[END_REF]. [START_REF] Wu | Antioxidant and anti-proliferative activities of Spirulina and Chlorella water extracts[END_REF] estimated that the total polyphenols and other metabolites are linked to antioxidant properties.

The role of dietary antioxidants and their potential health and disease benefits have been studied [START_REF] Huang | Dietary antioxidants and health promotion[END_REF]. Components with antioxidant activity are found in only a few species of algae [START_REF] Shanab | Aqueous extracts of microalgae exhibit antioxidant and anticancer activities[END_REF]. Phenolic compounds acquire antioxidant activity in biological systems [START_REF] Lin | An overview of plant phenolic compounds and their importance in human nutrition and management of type 2 diabetes[END_REF][START_REF] Pourreza | Phenolic compounds as potential antioxidant[END_REF]. Although the accumulation of polyphenols in plants is well known, the antioxidant properties of algae are poorly known. These results suggested that the methanol extracts of A. platensis may be a promising new source of natural compounds with antioxidant ability. The antioxidant activity of A. platensis extracts appeared to be related to their high polyphenols content, since the DPPH scavenging activity of the extracts is correlated with the increase of their polyphenols content. The scavenging activity of phenols extracts of Spirulina maxima toward DPPH radicals suggested that the antioxidant activity was carried out by mechanisms that needed electron and hydrogen donors (Abd El-Bakyet al., 2009;[START_REF] El Jemli | Radical-scavenging activity and ferric reducing ability of Juniperus thurifera (L.), J. oxycedrus (L.), J. phoenicea (L.)[END_REF]. The polyphenols of Spirulina extracts can obstruct the chain reactions of the lipid peroxidation process by providing hydrogen atom to the free radicals. The avoidance of the beginning step of the chain reaction by scavenging free radicals is considered to be an important mode action [START_REF] Ramdani | Evaluation of antioxidant activity and total phenol content of Gracilaria bursa-pastoris harvested in Nador lagoon for an enhanced economic valorization[END_REF][START_REF] Ruberto | Antioxidant activity of extracts of the marine algal genus Cystoseira in a micellar model system[END_REF].

Increased CO2 in the culture medium ameliorated cytotoxic effects against human ovary cancer cells (OVCAR) and breast cancer cell lines (MCF-7) while no cytotoxic effects were detected against colon carcinoma cells (HCT-116) (Table 4). Some studies reported that tumors were prevented with algae extracts (Abu [START_REF] Zaid | Antioxidant and anti-cancer activity of Spirulina platensis water extracts[END_REF][START_REF] Farooqi | Algae extracts and methyl jasmonate anticancer activities in prostate cancer: Choreographers of 'the dance macabre[END_REF][START_REF] Martínez | Marine microalgae with anti-cancer properties[END_REF], because of their antioxidant properties [START_REF] Zaid | Antioxidant and anti-cancer activity of Spirulina platensis water extracts[END_REF]. [START_REF] Mohd-Syahril | Screening of anticancer activities of crude extracts of unicellular green algae (Chlorella vulgaris) and filamentous blue-green algae (Spirulina platensis) on selected cancer cell lines[END_REF] described the importance of chemo in treating cancer.

Hence, new anticancer agents should be sought from various resources. An important number of anticancer compounds are natural products or their derivatives, mostly produced by photosynthetic microalgae [START_REF] Khan | The promising future of microalgae: Current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products[END_REF].

Enriching plants with high CO2 levels has been shown to increase the bioactive compounds and their biological activities [START_REF] Idso | Effects of atmospheric CO2 enrichment on the growth and development of Hymenocallis littoralis (Amaryllidaceae) and the concentrations of several antineoplastic and antiviral constituents of its bulbs[END_REF][START_REF] Karimi | Chemical composition, antioxidant and anticancer potential of Labisiapumila variety alata under CO2 enrichment[END_REF]. Variation of secondary metabolites and phytochemicals, for example, total polyphenols, flavonoids, fatty acids and volatile compounds, may result in high anti-cancer activity of algae and or plants.

Both extracts of A. platensis showed different pattern of antibacterial activities. The difference in the response between Gram-negative and Gram-positive bacteria could be due to the difference in their cell wall structure. CO2 enriched medium seems to enhance the antibacterial effect of A. platensis methanolic extracts on S. enterica, E. coli, and K. pneumoniae (Table 3). It appears that extracts with important anti-microbial activities against Gram-negative bacteria do not necessarily exert a high activity against other Gramnegative bacteria compared to Gram-positive bacteria [START_REF] Elisha | The antibacterial activity of extracts of nine plant species with good activity against Escherichia coli against five other bacteria and cytotoxicity of extracts[END_REF].

The mechanisms of antitumoral, antiviral and antimicrobial effects of A. platensis are due to its composition of endonuclease and fatty acids (specially high amount of γ-linolenic acid), respectively, as well as to its phycocyanin pigment, a brilliant blue polypeptide [START_REF] Hoseini | Nutritional and medical applications of Spirulina microalgae[END_REF].

The production of volatile compounds was higher without CO2 than with CO2 enrichment. This trend can be linked to the production of high molecular weight compounds with CO2 enrichment, and therefore fewer volatiles. These points to the possibility of forming other antioxidant, antibacterial and cytotoxic compounds in a CO2 enriched medium, such as polysaccharides [START_REF] Chaiklahan | Polysaccharide extraction from Spirulina sp. and its antioxidant capacity[END_REF] and/or proteins [START_REF] Wang | Isolation and identification of anti-proliferative peptides from Spirulina platensis using three-step hydrolysis[END_REF].

Conclusion

Elevated CO2 is known to have a positive effect on photosynthetic activity, growth, biomass accumulation and yield. Higher photosynthetic activity can result in carbon accumulation, which can affect a wide range of metabolic functions including primary and secondary metabolisms in algae. CO2 enrichment of the culture medium of A. platensis enhanced the accumulation and distribution of bio-active compounds such as polyphenols and flavonoids, fatty acids or esters, volatile compounds as well as their antioxidant, antibacterial and anticancer activities. The results of antioxidant activity of A. platensis indicated a good growth, photosynthesis and nutrient uptake in the marine macroalga Gracilaria lemaneiformis (Rodophyta). Botanica Marina, 53, 123-129. Yoshioka, I., Obata, D., Nanjo, H., Yokozeki, K. Torichigai, T., Morioka, M., & Higuchi T. (2013). New ecological concrete that reduces CO2 emissions below zero level: New method for CO2 capture and storage. Energy Procedia, 37, 6018-6025. Yusuke, O., Tadaki, H., & Kouki, H. (2007). Effect of elevated CO2 levels on leaf starch, nitrogen and photosynthesis of plants growing at three natural CO2 springs in Japan.

Ecological Research, 22, 475-484. Zarrouk, C. (1966) --------------------------------------------------------------------------------------------------------------Ampicillin -CO2 +CO2

------------------------------------------------------------ --------------------------------------------------------------------------------------------------------------

Gram

  cytotoxic effects of A. platensis methanol extracts on the three cell lines viability and IC50 values. With controlled conditions (-CO2), no inhibition was found in the three cancer cell lines. For algae treated with CO2 A. platensis methanol extracts were found to inhibit proliferation of human colon (HCT-116), breast (MCF-7) and ovarian (OVCAR) cancers. The overall results indicated that A. platensis methanolic extracts cultivated with elevated CO2 concentration showed the strongest cytotoxic activities towards cancer cells HCT-116.

Figure 1 :

 1 Figure legend:

Figure 2 :

 2 Figure 2: Antioxidant activities of methanol extract of A. platensis grown in control (without

  Figure 2

Table 5 :

 5 Phytocomponents identified using GC-MS in the methanol extracts of Arthrospira platensis.

Table 1 .

 1 Proximate composition of Arthrospira platensis grown in control (-CO2) or CO2 enriched (+CO2) medium. Moisture, ash, protein, fats and total sugars are expressed in %.Values are shown as mean±standard deviation (n=3). Values with the different superscript letters (a and b), in the same row, are significantly different at p < 0.05.

	%	-CO2	+CO2
	Moisture	3.8±0.8a	3.9±1.5a
	Ash	16±2a	20±5a
	Crude protein (N*6.25)	67±6a	52±4b
	Fats	6.0±1.4a	5.3±0.9a
	Total sugars	19±3b	36±6a

Table 2 .

 2 Mineral composition in g (Mg, K and Na) or mg (Ca, Fe, Zn)/100 g dw of Arthrospira platensis grown in control (-CO2) or CO2 enriched (+CO2) medium values are shown as mean ± standard deviation (n=3). Values with the different superscript letters (a and b), in the same row, are significantly different at p < 0.05.

		-CO2	+CO2
	Mg	1.24±0.01a	1.25±0.02a
	K	1.14±0.01a	1.03±0.02a
	Na	1.12±0.02 a	1.02±0.02 a
	Ca	160±10b	190±10a
	Fe	27±5b	34±4a
	Zn	0.50±0.01 a	0.50±0.02 a
	Cd	*	*
	Pb	*	*

(*: not detected).

Table 3 .

 3 Antibacterial activity of Arthrospira platensis grown in control (-CO2) or CO2 enriched (+CO2) medium. Values are shown as mean ± standard deviation (n=3). Values with different superscript letters (a, b and c), in the same line, are significantly different at p < 0.05.

-negative bacteria

	Salmonella enterica	17.1±0.4a	14.2±0.3b	18.2±0.5a
	Escherichia coli 25922	15.3±0.4b	13.4±0.3c	16.3±0.2a
	Klebsiella pneumoniae WHO24	18.4±0.3a	16.2±0.3b	16.3±0.2b
	Gram-positive bacteria
	Enterococcus faecalis	19.4±0.1a	15.5±0.2b	14.3±0.2b
	Listeria monocytogenes	21±1a	19.3±0.2a	19.4±0.4a
	Bacillus subtilis	19.5±0.1a	15.2±0.3b	14.2±0.2b
	Bacillus cereus	18.2±0.2a	15.4±0.3b	16.5±0.2b
	Bacillus thuringiensis	20.6±0.3a	19.6±0.1a	18.3±0.2b
	Micrococcusluteus	20.5±0.1a	14.4±0.3b	13.2±0.2b

Table 4 .

 4 Cytotoxicity of methanol extracts of Arthrospira platensis on HCT-116, MCF-7 and OVCAR cell lines. IC50 value (µg mL -1 ). Values are shown as mean ± standard deviation (n=3).

	Cell lines	-CO2	+CO2	Tamoxifen
	OVCAR	>50	17±1	0.19±0.03
	MCF-7	>50	14.5±0.5	0.15±0.02
	HCT-116	>50	>50	0.14±0.02

Acknowledgments

This work was supported by the Tunisian Ministry of Higher Education and Scientific Research by funds allocated to 19PEJC05-13 project and to the Laboratory of Biodiversity and Valorisation of Bioresources in Arid Zones (LR18ES36). The authors would like to express their sincere gratitude to Prof. Chedly Abdelly for being available all the time for discussion and help. We are very grateful to Hedi Gazbar, general manager of "Eden Life" for providing Spirulina.

potential, especially when grown with elevated carbon dioxide. A screening fractionation is planned to identify the most active phenolic compounds in Spirulina extracts. The search is in progress to find new safe and effective antioxidants for use in foods and pharmaceutical preparations to replace synthetics. Therefore, the production of algae under CO2 enrichment could be used as one of the solutions of carbon sequestration to achieve permanent removal of the carbon from the atmosphere. Further extensive experiments will be required to confirm this approach.

Conflict of interest

The authors confirm that they have no conflicts of interest with respect to the study described in this manuscript.


Pierre Sabouret
email: pierre.sabouret@action-coeur.org

Denis Angoulvant

Kausik K Ray

the rediscovered risk factor, or how to get "back to the future"

Keywords: apo(a), apolipoprotein(a), ASCVD, atherosclerotic cardiovascular disease, LDL, low-density lipoprotein, Lp

Background

Elevated lipoprotein(a) -Lp(a) -is a known independent and genetically inherited risk factor for cardiovascular disease. Although its causal role in atherosclerosis and aortic valve calcification was established long ago, there is no international consensus regarding the measurement of Lp(a) and its threshold. To date, no pharmacological intervention targeting Lp(a) to prevent cardiovascular disease has been either approved or recommended. Recent clinical data have rekindled the cardiovascular community's interest in Lp(a), which may become a new player in the lipid-related risk reduction strategy in high-risk patients.

Lp(a): The long history of this neglected particle

Lp(a) was discovered more than 50 years ago, in 1963. The geneticist Kare Berg was seeking to define lipoprotein differences between individual human sera. Through an ingenious set of immunological investigations of human sera, he discovered a new antigen that was associated with low-density lipoprotein (LDL) particles [START_REF] Berg | A New Serum Type System in Man--the Lp System[END_REF][START_REF] Berg | Genetics of the Lp System[END_REF]. Berg soon showed that this new antigen was a genetic trait, and proposed that it should be called Lp(a): Lp referring to the lipoprotein, and (a) as this was the accepted terminology at that time for naming antigens in human immunogenetics. Therefore, the term Lp(a) initially referred to the antigenic structure of the new antigen rather than the lipoprotein as it is used today.

Lp(a) is composed of apolipoprotein(a) -apo(a) -covalently bound to the apolipoprotein B100 of an LDL-like particle (Fig. 1). Apo(a) is partly made of triple-looped structures called "kringles" because they are shaped like the Danish pretzel of the same name. Apo(a) contains various copies of kringle IV type 2 as well as a protease domain similar to that of plasminogen, which may partly explain its prothrombotic effect. Because of its variable structure (in terms of numbers of kringle IV), Lp(a) shows significant protein size heterogeneity within patients, and one individual usually carries two different isoforms, each inherited from one parent. Interindividual and intraindividual Lp(a) particle size heterogeneity renders measurement of its concentration complex [START_REF] Rawther | Biology, pathophysiology and current therapies that affect lipoprotein (a) levels[END_REF][START_REF] Tsimikas | A Test in Context: Lipoprotein(a): Diagnosis, Prognosis, Controversies, and Emerging Therapies[END_REF]. In addition, apo(a) polymorphism may affect its metabolism and its effect on Lp(a) concentration. After biosynthesis within the liver, small isoforms have a short maturation time, leading to quicker secretion of Lp(a); this explains the inverse correlation between plasma Lp(a) concentration and apo(a) isoform size.

There are no standardized assays to measure Lp(a) concentration, and cut-offs for risk remain a matter of debate, even among experts. Circulating concentrations of Lp(a) are genetically determined, and a desirable concentration is often proposed as < 50 mg/dL, which has led guidelines from the USA to consider a 50 mg/dL cut-off [START_REF] Grundy | AHA/ACC/AACVPR/AAPA/ABC/ACPM/ADA/AGS/APhA/ASPC/NLA/PCNA Guideline on the Management of Blood Cholesterol: Executive Summary: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines[END_REF]. The recent European Society of Cardiology/European Atherosclerosis Society guidelines [START_REF] Mach | ESC/EAS Guidelines for the management of dyslipidaemias: lipid modification to reduce cardiovascular risk[END_REF] propose a more extreme 180 mg/dL cut-off to identify patients with very high inherited Lp(a) concentration, who may have a lifetime risk of atherosclerotic cardiovascular disease (ASCVD), similar to the risk associated with heterozygous familial hypercholesterolaemia.

Lp(a) promotes atherosclerosis via its LDL moiety, which has a similar proportion of cholesterol content as traditional LDL particles. Furthermore, Lp(a) promotes inflammatory processes [START_REF] Van Der Valk | Oxidized Phospholipids on Lipoprotein(a) Elicit Arterial Wall Inflammation and an Inflammatory Monocyte Response in Humans[END_REF][START_REF] Wiesner | MCP-1 binds to oxidized LDL and is carried by lipoprotein(a) in human plasma[END_REF] via accumulation of oxidized phospholipids [START_REF] Leibundgut | Determinants of binding of oxidized phospholipids on apolipoprotein (a) and lipoprotein (a)[END_REF], and induces prothrombotic cascade effects via the plasminogen-like apolipoprotein(a) moiety. A potential causal role for Lp(a) was first reported in 1974, with the observation that Lp(a) concentrations were higher in patients with coronary artery disease than in primary prevention individuals [START_REF] Berg | Lp(a) lipoprotein and pre-beta1-lipoprotein in patients with coronary heart disease[END_REF]. In addition, a significant correlation between elevated Lp(a) and calcific aortic valve disease was established [START_REF] Kamstrup | Elevated lipoprotein(a) and risk of aortic valve stenosis in the general population[END_REF]. It has been suggested that Lp(a) concentrations may help to identify patients with more progressive aortic valve disease, who may require early intervention [START_REF] Thanassoulis | Genetic associations with valvular calcification and aortic stenosis[END_REF]. Recent observational data from two Danish registries convincingly showed that high Lp(a) concentration was an independent predictor of total and cardiovascular mortality, but not of noncardiovascular mortality. The authors concluded that a 50 mg/dL (105 nmol/L) increase in Lp(a) was significantly associated with a 1.16 hazard ratio for cardiovascular mortality. Interestingly, in this study, patients carrying the isoforms with a low number of repeats of kringle IV type 2 were at higher risk, supporting the previous physiopathological hypothesis [START_REF] Langsted | High lipoprotein(a) and high risk of mortality[END_REF].

Despite convincing data from epidemiological studies, Lp(a) failed to reach "mainstream" status within the medical community in France. Several factors may have contributed to this. Even today, Lp(a) is not widely known as a cardiovascular risk factor among cardiologists, endocrinologists and general practitioners. Few understand its deleterious role and biological properties, or know how it is regulated. Although international guidelines recommend one Lp(a) measurement during adulthood, this test is not currently reimbursed by French social security, which is a significant hurdle against its use for better individual cardiovascular risk assessment. Another hurdle against its widespread use is that Lp(a) measurements are not routinely available in all laboratories, and may need to be performed in referral laboratories. One may acknowledge that the absence of validated and recommended pharmacological intervention targeting elevated Lp(a) to reduce clinical events may not encourage physicians to prescribe its routine measurement.

Lp(a): A spectacular come-back

Over the past few years, new clinical data have promoted a resurgence of interest in Lp(a) in patients at high cardiovascular risk.

First, several publications reported a strong relationship between high Lp(a) concentrations and the incidence of major adverse cardiovascular events (MACE), which was reinforced by a metaanalysis suggesting that Lp(a) has an independent deleterious role in patients, despite statin treatment [START_REF] Clarke | Genetic variants associated with Lp(a) lipoprotein level and coronary disease[END_REF][START_REF] Erqou | Apolipoprotein(a) isoforms and the risk of vascular disease: systematic review of 40 studies involving 58,000 participants[END_REF][START_REF] Kamstrup | Genetic evidence that lipoprotein(a) associates with atherosclerotic stenosis rather than venous thrombosis[END_REF][START_REF] Kamstrup | Genetically elevated lipoprotein(a) and increased risk of myocardial infarction[END_REF][START_REF] Kathiresan | Lp(a) lipoprotein redux--from curious molecule to causal risk factor[END_REF][START_REF] Willeit | Baseline and on-statin treatment lipoprotein(a) levels for prediction of cardiovascular events: individual patient-data meta-analysis of statin outcome trials[END_REF]. Moreover, the thresholds at which risk increases markedly may occur in as many as 10% of the general population, meaning that the population attributable risk of elevated Lp(a) could be large.

These compelling and consistent findings regarding the strong relationship between Lp(a) concentrations and cardiovascular events have generated renewed interest in Lp(a) among clinicians [START_REF] Nordestgaard | Familial hypercholesterolaemia is underdiagnosed and undertreated in the general population: guidance for clinicians to prevent coronary heart disease: consensus statement of the European Atherosclerosis Society[END_REF]. Several studies have reported the benefit of lipoprotein apheresis in high-risk patients with elevated Lp(a) [START_REF] Klingel | Prevention of cardiovascular complications in patients with Lp(a)-hyperlipoproteinemia and progressive cardiovascular disease by long-term lipoprotein apheresis according to German national guidelines[END_REF]. At the same time, therapeutic innovation in lipid-lowering treatments has brought new insights, suggesting that a decrease in Lp(a) concentration by new drugs may produce significant risk reduction mediated through Lp(a) reduction, reinforcing the interest in Lp(a) and its reduction.

New approaches targeting atherogenic lipoproteins include inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) to decrease circulating LDL cholesterol. PCSK9 inhibitors have also been shown to significantly decrease Lp(a) concentrations [START_REF] Croyal | PCSK9 inhibition with alirocumab reduces lipoprotein(a) levels in nonhuman primates by lowering apolipoprotein(a) production rate[END_REF]. New pharmacotherapeutic agents blocking Lp(a) synthesis with antisense oligonucleotide therapy are being developed, with ongoing phase III outcomes trials planned (ClinicalTrials.gov Identifier: NCT04023552) These new agents are promising, and may play a major role in decreasing the "lipid residual risk" in patients with ASCVD, provided that their clinical efficacy and safety are confirmed in large cardiovascular outcome trials.

PCSK9 inhibitors and Lp(a) modulation

PCSK9 is an enzyme encoded by the PCSK9 gene in humans on chromosome 1. This protein plays a major regulatory role in cholesterol homeostasis, mainly by reducing the levels of LDL receptors on the plasma membrane [START_REF] Bergeron | Proprotein convertase subtilisin/kexin type 9 inhibition: a new therapeutic mechanism for reducing cardiovascular disease risk[END_REF]. Drugs that inhibit PCSK9 thus prevent degradation of LDL receptors, which increases the survival time of hepatic LDL receptors and enhances LDL clearance from circulation. Evolocumab and alirocumab are two fully-human anti-PCSK9 antibodies, which induced a sustained prolonged decrease in LDL cholesterol of 50-70% and a reduction in MACE of 15% in two large phase III randomized cardiovascular outcome trials (FOURIER and ODYSSEY OUTCOMES), conducted in secondary prevention patients. PCSK9 inhibitors have also been shown to reduce the concentration of other atherogenic particles, such as Lp(a) [START_REF] Bergeron | Proprotein convertase subtilisin/kexin type 9 inhibition: a new therapeutic mechanism for reducing cardiovascular disease risk[END_REF][24][START_REF] Schwartz | Alirocumab and Cardiovascular Outcomes after Acute Coronary Syndrome[END_REF]. Absolute MACE risk reduction is more pronounced in patients at very high risk, such as those with peripheral artery disease, recent or multiple previous myocardial infarctions or residual severe multivessel coronary artery disease, or with elevated Lp(a) [24][START_REF] Schwartz | Alirocumab and Cardiovascular Outcomes after Acute Coronary Syndrome[END_REF][START_REF] O'donoghue | Lipoprotein(a), PCSK9 Inhibition, and Cardiovascular Risk[END_REF]. A recent publication from the FOURIER trial reported that evolocumab significantly reduced Lp(a) concentration, resulting in more cardiovascular benefits in patients with higher baseline Lp(a) concentrations. In these patients, a 24% relative risk reduction in the combined endpoint (myocardial infarction, stroke, cardiovascular death) was observed, whereas patients with a lower baseline Lp(a) concentration only had a 15% relative risk reduction in the evolocumab group [START_REF] O'donoghue | Lipoprotein(a), PCSK9 Inhibition, and Cardiovascular Risk[END_REF]. Whether higher levels of Lp(a) modulation reflect a higher risk profile or whether specific Lp(a) modulation by PCSK9 inhibitors provides higher benefits remains to be investigated.

The magnitude of cardiovascular benefits directly linked to a decrease in Lp(a) remains a matter of debate. The ANITSCHKOW study [START_REF]Patients With Elevated Lipoprotein(a) (Lp(a))[END_REF], investigating the anti-PCSK9 antibody evolocumab, showed a modest 14% reduction in Lp(a) in a cohort with a baseline Lp(a) concentration of 75 mg/dL. No decrease in arterial wall inflammation measured by positron emission tomography/computed tomography was observed (results as reported on ClinicalTrials.gov) [START_REF]Patients With Elevated Lipoprotein(a) (Lp(a))[END_REF]. These findings highlight the need for more efficient and specific agents to decrease the adverse proinflammatory state associated with Lp(a) elevation. This controversy may be resolved by the ongoing dedicated cardiovascular outcome trials investigating Lp(a) specific inhibitors that reduce Lp(a) by a sufficient magnitude.

New lipid-lowering treatments specifically targeting Lp(a)

Research in the lipoprotein field has led to the recent development of an antisense oligonucleotide against apo(a), AKCEA-APO(a)-LRx, which selectively decreases Lp(a) concentrations by approximately 80% [START_REF] Vogt | Lipoprotein(a)-antisense therapy[END_REF].

Results from a dose-finding phase 2b AKCEA-APO(a)-LRx study were published recently. Five regimens according to dosage and interval of administration of AKCEA-APO(a)-LRx versus placebo (a saline solution administrated subcutaneously) were randomly administered to 286 patients (n = 47 in the placebo group) with ASCVD and Lp(a) concentrations of ≥ 60 mg/dL for a duration of 6-12 months. The percentage of Lp(a) decrease from inclusion to month 6 was the primary biological efficacy endpoint. At baseline, the mean Lp(a) concentration was 100.0 mg/dL in the AKCEA-APO(a)-LRx group and 103.3 mg/dL in the placebo group. A dose response with AKCEA-APO(a)-LRx was observed regarding decrease in Lp(a) concentration. The magnitude of decrease varied from 35% at a dose of 20 mg/month to 72% at a dose of 60 mg/month; it reached 80% in the arm receiving 20 mg/week. The mean decrease was 6% in the placebo group. Lp(a) concentrations < 50 mg/dL were achieved in more than 80% of patients [START_REF] Tsimikas | Lipoprotein(a) Reduction in Persons with Cardiovascular Disease[END_REF]. No safety issue was reported, especially concerning liver and renal function, platelet count, "influenza-like" symptoms and MACE. As reported for PCSK9 inhibitors, the most frequent adverse event was a local reaction at the injection site, with no clinical impact on adherence. randomization, optimal LDL cholesterol-lowering treatment and optimal medical treatment for other cardiovascular risk factors. The spectrum of patients is large, including those who have had a myocardial infarction or ischaemic stroke 3 months to 10 years before, and patients with asymptomatic peripheral artery disease. The primary endpoint is a composite of cardiovascular death, non-fatal myocardial infarction, non-fatal stroke and urgent coronary revascularization requiring hospitalization in patients with Lp(a) ≥ 70 mg/dL at baseline; the same primary endpoint will be reported in the subgroup of patients with Lp(a) ≥ 90 mg/dL at baseline. Other "usual" efficacy and safety endpoints have also been prespecified in this large randomized controlled trial to validate this new lipid-lowering therapy. In this therapeutic class, a phase 1 study with a single dose of another oligonucleotide antisense named AMG 890 has been initiated, mainly to evaluate the safety of this new entity (ClinicalTrials.gov Identifier: NCT03626662).

Conclusions

Healthy lifestyle remains the cornerstone for cardiovascular prevention. Statins are still the first-line lipid-lowering therapy in the pharmacological approach to cardiovascular risk reduction in high-risk patients. Nevertheless, residual risk in established or high-risk patients with ASCVD remains a challenging issue, which has been attributed, in part, to insufficient lowering of LDL cholesterol and other lipid abnormalities, such as high Lp(a) or triglyceride concentrations. Therefore, new lipidtargeting treatments are needed for further risk reduction, especially for high lipid-mediated residual risk. In this rapidly changing field, Lp(a) should be a systematic component in the evaluation of residual lipid risk, as it might explain part of the risk that may not be well controlled by usual lipidlowering therapy. One may argue that recent data advocate the use of PCSK9 inhibitors in high-risk patients with elevated Lp(a). In addition, the development of new agents specifically targeting Lp(a) are on the way, and may soon bring new therapeutic responses to further decrease cardiovascular risk.

  These promising findings have led to an ongoing phase III outcomes trial investigating cardiovascular benefits in high-risk patients with elevated Lp(a) concentrations. This study, named "Assessing the Impact of Lipoprotein (a) Lowering With TQJ230 on Major Cardiovascular Events in Patients With CVD (Lp(a) HORIZON)" (ClinicalTrials.gov Identifier: NCT04023552), will randomize 7680 patients in secondary prevention to AKCEA-APO(a)-LRx (TQJ230) or placebo. Key inclusion criteria combine the need for secondary prevention with an Lp(a) concentration ≥ 70 mg/dL at

Figure 1 .

 1 Figure 1. Lipoprotein(a) structure. Apo: apolipoprotein; EC: endothelial cell; IL: interleukin; KIV: kringle IV; KV: kringle V; LDL: low-density lipoprotein; OxPL: oxidized phospholipids; PAI: plasminogen activator inhibitor; SMC: smooth muscle cell; TFPI: tissue factor pathway inhibitor.

Acknowledgements

Kausik K. Ray acknowledges the support of the National Institute for Health Research (NIHR) Imperial Biomedical Research Centre. Imperial College London is grateful for support from the North West London NIHR Applied Research Collaboration. The views expressed in this publication are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care. We thank Mrs Elena Angoulvant for drawing the original figure depicting Lp(a) structure and Marinos Fysekidis, MD, PhD, for his comments. D. A. Consulting fees, lecture fees or funding for conference travel from the companies Amgen, AstraZeneca, Bayer, Eli Lilly, MSD, Novartis, Pfizer. Sanofi and Servier. K. K. R. Personal fees from the companies Abbvie, Akcea, Aegerion, Algorithm, Amgen, AstraZeneca, Bayer, Boehringer Ingelheim, Cerenis, Cipla, Daiichi Sankyo, Dr Reddys, Esperion, Kowa, Lilly, MSD, Novartis, Pfizer, Resverlogix, Sanofi/Regeneron, Silence Therapeutics, Takeda, The Medicines Company and Zuelling Pharma. Grants from the companies Amgen, MSD, Pfizer and Sanofi/Regeneron.

Sources of funding

There was no specific funding for the generation of this report.


Cécilia Neige
email: cecilia.neige@u-bourgogne.fr

Dylan Rannaud Monany

Cathy M Stinear

Winston D Byblow

Charalambos Papaxanthis

Florent Lebon

Unravelling the modulation of intracortical inhibition during motor imagery: An adaptive threshold-hunting study

Keywords: Transcranial magnetic stimulation, motor cortex, inhibition, motor imagery

Excitatory and inhibitory neural processes interact during motor imagery (MI).

• The current study investigated the extent of SICI modulation during MI.

• SICI increased during

Introduction

Interactions between excitatory and inhibitory neural processes within the primary motor cortex (M1) are crucial in various cognitive and motor functions [START_REF] Reis | Contribution of transcranial magnetic stimulation to the understanding of cortical mechanisms involved in motor control[END_REF]. For example, during motor imagery (MI), the mental simulation of a movement without any apparent muscular contraction, excitatory and inhibitory processes subtly interact as the motor regions are activated but no movement is produced.

Paired-pulse transcranial magnetic stimulation (TMS) protocols provide a quantification of the intracortical processes at the time of the stimulation [START_REF] Bestmann | The uses and interpretations of the motor-evoked potential for understanding behaviour[END_REF].

Short-interval intracortical inhibition (SICI) measurements can be obtained by delivering a subthreshold conditioning stimulus (CS), followed 1 to 6 ms later by a second supra-threshold test stimulus (TS) applied through the same coil over M1 [START_REF] Kujirai | Corticocortical inhibition in human motor cortex[END_REF]. This produces a decrease of motor-evoked potential (MEP) amplitude in comparison to MEP induced by unconditioned TS.

In the conventional paired-pulse TMS paradigm, the peak-to-peak amplitude of the conditioned MEP is expressed as a percentage of the amplitude of the unconditioned MEP, indicating the amount of SICI [START_REF] Kujirai | Corticocortical inhibition in human motor cortex[END_REF]. This measure depends critically on the CS and TS intensities [START_REF] Ilić | Short-interval paired-pulse inhibition and facilitation of human motor cortex: The dimension of stimulus intensity[END_REF][START_REF] Peurala | Interference of short-interval intracortical inhibition (SICI) and short-interval intracortical facilitation (SICF)[END_REF][START_REF] Vucic | The effects of alterations in conditioning stimulus intensity on short interval intracortical inhibition[END_REF]. First, the TS intensity must be sufficient to recruit the later I-waves suppressed by SICI [START_REF] Garry | The effect of test TMS intensity on short-interval intracortical inhibition in different excitability states[END_REF][START_REF] Lazzaro | Noninvasive Stimulation of the Human Brain: Activation of Multiple Cortical Circuits[END_REF]. Moreover, changing the CS intensity for a given TS intensity results in a U-shaped SICI curve. In the descending part of this curve, the amount of SICI increases when increasing CS intensity from 50% of the resting motor threshold (rMT), with a peak of inhibition occurring at CS about 80%rMT [START_REF] Kujirai | Corticocortical inhibition in human motor cortex[END_REF][START_REF] Ilić | Short-interval paired-pulse inhibition and facilitation of human motor cortex: The dimension of stimulus intensity[END_REF]. Then, increasing the CS intensity toward the rMT (i.e., CS intensity >80 rMT [START_REF] Ilić | Short-interval paired-pulse inhibition and facilitation of human motor cortex: The dimension of stimulus intensity[END_REF][START_REF] Kossev | Intracortical inhibition and facilitation in paired-pulse transcranial magnetic stimulation: Effect of conditioning stimulus intensity on sizes and latencies of motor evoked potentials[END_REF]) leads to the progressive decrease of SICI. This decrease is thought to reflect a "contamination" of the neural process involved in SICI by the recruitment of high-threshold excitatory interneurons. The latter have the potential to override the inhibitory system and are known to contribute to the short interval intracortical facilitation (SICF) phenomenon [START_REF] Ilić | Short-interval paired-pulse inhibition and facilitation of human motor cortex: The dimension of stimulus intensity[END_REF][START_REF] Kossev | Intracortical inhibition and facilitation in paired-pulse transcranial magnetic stimulation: Effect of conditioning stimulus intensity on sizes and latencies of motor evoked potentials[END_REF][START_REF] Peurala | Interference of short-interval intracortical inhibition (SICI) and short-interval intracortical facilitation (SICF)[END_REF][START_REF] Vucic | The effects of alterations in conditioning stimulus intensity on short interval intracortical inhibition[END_REF][START_REF] Wagle-Shukla | Effects of short interval intracortical 21 inhibition and intracortical facilitation on short interval intracortical facilitation in human primary motor cortex[END_REF]. Importantly, it must be pointed out that rMT is not a static but rather a state-dependent measure that is subject to the excitability of several cortical and spinal elements excited by the TMS pulse [START_REF] Groppa | A practical guide to diagnostic transcranial magnetic stimulation: Report of an IFCN committee[END_REF][START_REF] Karabanov | The resting motor threshold -Restless or resting? A repeated threshold hunting technique to track dynamic changes in resting motor threshold[END_REF]. For example, MI decreases the rMT [START_REF] Facchini | Focal enhancement of motor cortex excitability during motor imagery: A transcranial magnetic stimulation study[END_REF][START_REF] Li | Movement-specific enhancement of corticospinal excitability at subthreshold levels during motor imagery[END_REF][START_REF] Grosprêtre | Motor imagery and cortico-spinal excitability: A review[END_REF] and enhances MEP amplitude when compared to rest [START_REF] Kasai | Evidence for facilitation of motor evoked potentials (MEPs) induced by motor imagery[END_REF][START_REF] Yahagi | Facilitation of motor evoked potentials (MEPs) in first dorsal interosseous (FDI) muscle is dependent on different motor images[END_REF]Lebon et al., 2012a;[START_REF] Grosprêtre | Motor imagery and cortico-spinal excitability: A review[END_REF]. As suggested by Grosprêtre et al. (2015), these findings bring evidence that cortical cell responsiveness to TMS may increase during MI and this could be mediated, at least in part, by a decrease of inhibitory activity within M1 [START_REF] Grosprêtre | Motor imagery and cortico-spinal excitability: A review[END_REF]. Indeed, some studies found a reduction of SICI during MI in comparison to rest when using the conventional SICI paradigms [START_REF] Abbruzzese | Changes of intracortical inhibition during motor imagery in human subjects[END_REF][START_REF] Patuzzo | Modulation of motor cortex excitability in the left hemisphere during action observation: A single-and paired-pulse transcranial magnetic stimulation study of self-and non-self-action observation[END_REF][START_REF] Stinear | Modulation of corticospinal excitability and intracortical inhibition during motor imagery is task-dependent[END_REF][START_REF] Kumru | Motor cortex excitability changes during imagery of simple reaction time[END_REF][START_REF] Liepert | Motor excitability during imagination and observation of foot dorsiflexions[END_REF]. Conversely, other studies failed to observe SICI modulation [START_REF] Ridding | Afferent input and cortical organisation : a study with magnetic stimulation[END_REF][START_REF] Stinear | Modulation of corticospinal excitability and intracortical inhibition during motor imagery is task-dependent[END_REF][START_REF] Sohn | Suppression of Corticospinal Excitability During Negative Motor Imagery[END_REF]Lebon et al., 2012a), indicating that mechanisms underlying SICI modulation during MI remain poorly understood. Notably, the difference between these contradictory results seems to rely on the CS intensity. It appears that only studies fixing the CS intensity at ≥75 rMT found a reduction of SICI during MI.

The aim of the present study was to unravel the SICI modulation observed during MI through a methodological and conceptual reconsideration of: (i) the importance of CS intensity and (ii) the inhibitory process within M1 as an inherent feature of MI. To do so, we designed a pair of experiments in which we varied the CS intensity and determined the TS intensity required to maintain a fixed MEP amplitude for each condition using an adaptive threshold hunting technique [START_REF] Awiszus | Characterisation of paired-pulse transcranial magnetic stimulation conditions yielding intracortical inhibition or i-wave facilitation using a threshold-hunting paradigm[END_REF][START_REF] Fisher | Two phases of intracortical inhibition revealed by transcranial magnetic threshold tracking[END_REF][START_REF] Awiszus | TMS and threshold hunting[END_REF][START_REF] Samusyte | Short-interval intracortical inhibition: Comparison between conventional and threshold-tracking techniques[END_REF][START_REF] Vucic | Utility of threshold tracking transcranial magnetic stimulation in ALS[END_REF]. This method has been recently developed in order to overcome the potential limitations of conventional paired-pulse TMS protocols, such as large variability in MEP amplitude and a "floor/ceiling effect" when the observed inhibition leads to complete MEP suppression [START_REF] Cirillo | Threshold tracking primary motor cortex inhibition: The influence of current direction[END_REF][START_REF] Cirillo | Conventional or threshold-hunting TMS? A tale of two SICIs[END_REF][START_REF] Van Den Bos | Physiological processes underlying short interval intracortical facilitation in the human motor cortex[END_REF]. The adaptive threshold-hunting technique provides a new opportunity to extend our understanding of physiological mechanisms underlying intracortical inhibition in healthy subjects and it has been recently shown to be more reliable with shorter acquisition time than conventional SICI techniques [START_REF] Samusyte | Short-interval intracortical inhibition: Comparison between conventional and threshold-tracking techniques[END_REF].

Taking advantage of the adaptive threshold-hunting approach, two different experiments were conducted in order to investigate the evolution of SICI during MI as compared to rest. In experiment 1, we used the adaptive threshold-hunting technique in its original form [START_REF] Fisher | Two phases of intracortical inhibition revealed by transcranial magnetic threshold tracking[END_REF] to measure SICI using three CS intensities. We hypothesized a decrease of SICI during MI when compared to rest, as previously observed in the literature with conventional SICI techniques when CS intensity is high. Then, in experiment 2, we optimized the adaptive threshold-hunting technique with individualized MEPtarget and measured SICI using two low CS intensities. We expected an increase of SICI during MI when compared to rest, as MI is thought to suppress neural commands at some level of the motor system by inhibitory mechanisms [START_REF] Jeannerod | Mental motor imagery: a window into the representational stages of action[END_REF][START_REF] Jeannerod | Neural simulation of action: a unifying mechanism for motor cognition[END_REF].

Experimental Procedures

Participants

Twenty healthy volunteers (five females; mean age 24.3 years, range 22-27 years; eighteen right-handed as assessed by the Edinburgh Handedness Inventory [START_REF] Oldfield | The assessment and analysis of handedness: The Edinburgh inventory[END_REF]) participated in the current study after providing written informed consent. All volunteers were screened for contraindications to TMS by a medical doctor. The protocol was approved by the University of Burgundy Committee on Human Research and complied with the Declaration of Helsinki. Thirteen participants were included in Experiment 1 and six of them plus seven other participants were included in Experiment 2.

General procedure

For the two experiments, participants were comfortably seated on a chair with the forearms supported by a pillow and palms facing down. They were instructed to stay at rest throughout the experiments. For MI trials, participants performed explicit and kinesthetic (somatosensory) MI of a right tonic index abduction with a first-person perspective for 3s after an auditory cue [START_REF] Hanakawa | Organizing motor imageries[END_REF]. The TMS pulses were triggered during the execution phase of MI trials. It has been shown that a kinesthetic MI strategy produces a greater musclespecific and temporally modulated facilitation of the corticospinal pathway, as compared with a visual MI strategy [START_REF] Stinear | Kinesthetic, but not visual, motor imagery modulates corticomotor excitability[END_REF]. At the beginning of the experiment, participants performed and practiced actual maximal abduction of the right index finger. They then received the following instructions (in French): "When you hear the auditory cue, imagine making a maximal abduction of the right index finger. Try to feel the movement, imagining the muscle contraction and tension that you would expect to experience in actual action. Be sure not to contract any muscles during the task and keep your eyes open" [START_REF] Lebon | The Neural Specificity of Movement Preparation During Actual and Imagined Movements[END_REF].

TMS and EMG recordings

Electromyographic (EMG) recordings of the right first dorsal interosseous (FDI) muscle were made with surface Ag/AgCl disposable electrodes in a belly-tendon montage. A ground electrode was placed on the styloid process of the ulna. The EMG signals were amplified and band-pass filtered (10-1000 Hz, Biopac Systems Inc.) and digitized at a sampling rate of 2000 Hz for off-line analysis. Background EMG was monitored for the 100 ms preceding every TMS pulse to ensure a complete muscle relaxation throughout the experiments.

Single-pulse and paired-pulse stimulations were applied with a 70-mm figure-of-eight coil connected to a monophasic Magstim BiStim² stimulator (The Magstim Co., Whitland, UK). The coil was placed over the left M1, tangentially to the scalp with the handle pointing backward and laterally at 45° away from the midsagittal line, resulting in a posterior-anterior current flow within M1. The optimal stimulation site on the scalp (hotspot) was defined as the location eliciting the largest MEP amplitude in the FDI and was marked on the scalp. The conventional rMT was determined as the lowest stimulation intensity required to evoke at least 5 MEPs of 50 μV out of 10 stimulations [START_REF] Rossini | Applications of magnetic cortical stimulation[END_REF] and then was used to set CS intensities.

Adaptive threshold-hunting technique

In Experiment 1, based on the corresponding literature, the hunting-threshold was defined as the TS intensity (expressed in percentage of the maximal stimulator output (%MSO)) required to elicit a MEPtarget in the relaxed FDI of 0.2 mV in peak-to-peak amplitude. This 0.2 mV MEP amplitude was chosen in accordance to numerous previous studies using the adaptive threshold-hunting technique [START_REF] Fisher | Two phases of intracortical inhibition revealed by transcranial magnetic threshold tracking[END_REF][START_REF] Awiszus | TMS and threshold hunting[END_REF][START_REF] Vucic | Assessment of cortical excitability using threshold tracking techniques[END_REF][START_REF] Menon | Sensitivity and specificity of threshold tracking transcranial magnetic stimulation for diagnosis of amyotrophic lateral sclerosis: A prospective study[END_REF][START_REF] Cirillo | Threshold tracking primary motor cortex inhibition: The influence of current direction[END_REF][START_REF] Cirillo | Conventional or threshold-hunting TMS? A tale of two SICIs[END_REF][START_REF] Samusyte | Short-interval intracortical inhibition: Comparison between conventional and threshold-tracking techniques[END_REF][START_REF] Van Den Bos | Physiological processes underlying short interval intracortical facilitation in the human motor cortex[END_REF]. This 0.2mV fixed MEP value has been shown to lie on the middle of the steepest portion of the stimulus response curve plotted on a logarithmic scale [START_REF] Fisher | Two phases of intracortical inhibition revealed by transcranial magnetic threshold tracking[END_REF][START_REF] Vucic | Assessment of cortical excitability using threshold tracking techniques[END_REF][START_REF] Vucic | Utility of threshold tracking transcranial magnetic stimulation in ALS[END_REF]. In the current study, the TS intensity required to elicit the MEPtarget at rest corresponded on average to 109% rMT (range 103-118 % rMT), which is similar to a previous study [START_REF] Cirillo | Threshold tracking primary motor cortex inhibition: The influence of current direction[END_REF]. The adaptive threshold-tracking single-pulse TMS technique was used to first compare the unconditioned TS intensity (%MSO) required to maintain this fixed MEPtarget amplitude at rest vs. during MI.

The adaptive threshold-hunting paired-pulse TMS technique was then used to investigate SICI at rest and during MI. To elicit SICI at rest and during MI in Experiment 1, we delivered high CS intensities: 60%, 70% and 80% of the rMT. The TS intensity was adjusted to reach the MEPtarget. A 2 ms interstimulus interval between CS and TS was chosen based on a similar previous study investigating SICI modulation during motor imagery (Lebon et al., 2012a).

In Experiment 2, the hunting threshold was defined as the TS intensity (%MSO) required to elicit a MEP in the relaxed FDI of at least 50% of MEPmax in peak-to-peak amplitude. This MEPtarget amplitude in Experiment 2 has been chosen since a TS delivered at a low intensity (i.e., below 110% rMT, as it was the case in Experiment 1), could fail to evoke late indirect waves, and limit SICI magnitude [START_REF] Garry | The effect of test TMS intensity on short-interval intracortical inhibition in different excitability states[END_REF]. This subjectspecific relative MEP value is half of the individual's maximum MEP amplitude value at rest.

MEPmax was calculated with a stimulus/response curve performed at the beginning of the experiment. We recorded eight MEPs for each stimulus intensity starting at 110% of rMT with incrementing steps of 10% rMT up to MEPmax [START_REF] Kukke | Efficient and reliable characterization of the corticospinal system using transcranial magnetic stimulation[END_REF][START_REF] Pitcher | A comparison of two methods for estimating 50% of the maximal motor evoked potential[END_REF].

In the current study, the TS Intensity required to elicit the MEPtarget at rest (mean MEP amplitude: 1.4 mV ± 0.81) corresponded on average to 124% rMT (range 112-150 %rMT). In the same way as for Experiment 1, the adaptive threshold-hunting single-pulse TMS technique was used to compare the unconditioned TS intensity (%MSO) needed to elicit the MEPtarget at rest vs. during MI.

To elicit SICI at rest and during MI in Experiment 2, we delivered low-intensity CS, i.e. 50% and 60% rMT. The CS was delivered 2 ms prior to TS. TS intensity was adjusted in each condition.

For both experiments, the order of the experimental conditions was randomized across participants. An available online freeware (TMS Motor Threshold Assessment Tool, MTAT 2.0), based on a maximum-likelihood Parameter Estimation by Sequential Testing (PEST) strategy was used with "assessment without a priori information" in line with previous studies [START_REF] Cirillo | Threshold tracking primary motor cortex inhibition: The influence of current direction[END_REF][START_REF] Cirillo | Conventional or threshold-hunting TMS? A tale of two SICIs[END_REF]. The stimulation sequence always began with the TS at 37 %MSO. One experimenter held the coil over M1, while the other indicated whether or not the MEP amplitude was ≥0.2 mV (Experiment 1) or ≥ 50%MEPmax (Experiment 2). The predictive algorithm then determined the next TS intensity to be delivered and was stopped after thirty stimulations, which provides sufficient accuracy for the threshold estimate according to previous studies [START_REF] Awiszus | TMS and threshold hunting[END_REF][START_REF] Awiszus | Using relative frequency estimation of transcranial magnetic stimulation motor threshold does not allow to draw any conclusions about true threshold[END_REF][START_REF] Sen | Active and resting motor threshold are efficiently obtained with adaptive threshold hunting[END_REF].

Data analysis

First, the unconditioned TS intensity required to elicit the MEPtarget, at rest and during MI was quantified and expressed in %MSO in both experiments.

Then, to probe the influence of the different CS on TS intensity, the amount of SICI (expressed in INH%) was quantified for each condition using the following equation [START_REF] Fisher | Two phases of intracortical inhibition revealed by transcranial magnetic threshold tracking[END_REF]: Additional analyses were performed to control for potential methodological biases. The RMS values were compared across conditions in both experiments, using the same analyses described above.

% = (conditioned

Statistical analysis

Results

Methodological considerations

The analysis of the pre-trigger background EMG level for the unconditioned TS ( ------ CS intensity (F(2,24) < 1, p = .859). However, there was a Task by CS intensity interaction (F(2,24) = 9.069, p = .001; ηp 2 = .43). Post-hoc analyses revealed that there was less SICI during MI than at rest only for the CS intensity of 80% rMT (p = .042; Figure 3 for typical raw MEP recordings).

Please insert

------Please insert Fig. 2 about here ------

The one-way ANOVA conducted on the difference between rest and MI for each CS Intensity revealed a main effect of CS Intensity (F(2,24) = 9.069, p = .001; ηp 2 = .430 ------Please insert Fig. 3

Discussion

The main objective of the present study was to investigate the modulation of SICI during MI, with a particular focus on the effect of the CS intensity. For the first time, we reported that the CS intensity chosen to evaluate SICI led to opposite conclusions regarding SICI modulation during MI. When tested with a high CS intensity there is less SICI during MI, as compared to rest. On the contrary, when tested with optimizing low CS intensities there is a greater SICI during MI than at rest. These findings are the first to strength the idea that MI requires motor command inhibition by active processes acting at a cortical level.

Corticospinal excitability increases during MI

While the major objective of this study was to focus on SICI, we also analyzed the level of corticospinal excitability (unconditioned TS intensity) at rest and during MI for the two experiments. By using the adaptive hunting threshold single-pulse TMS technique, we found that the minimum TS intensity required to elicit the MEPtarget was lower during MI when compared to rest. This result replicates and extends earlier findings showing that corticospinal excitability is higher during MI [START_REF] Karabanov | The resting motor threshold -Restless or resting? A repeated threshold hunting technique to track dynamic changes in resting motor threshold[END_REF][START_REF] Grosprêtre | Motor imagery and cortico-spinal excitability: A review[END_REF][START_REF] Ruffino | Neural plasticity during motor learning with motor imagery practice: Review and perspectives[END_REF], regardless of the MEPtarget amplitude.

SICI is lower during motor imagery when tested with high CS intensity

In Experiment 1, SICI decreased during MI as compared to rest only for the highest CS intensity (CS intensity set at 80% rMT), as previously observed in the literature with conventional SICI techniques. This decrease has been suggested to explain the corticospinal excitability increase observed during MI [START_REF] Abbruzzese | Changes of intracortical inhibition during motor imagery in human subjects[END_REF][START_REF] Patuzzo | Modulation of motor cortex excitability in the left hemisphere during action observation: A single-and paired-pulse transcranial magnetic stimulation study of self-and non-self-action observation[END_REF][START_REF] Stinear | Modulation of corticospinal excitability and intracortical inhibition during motor imagery is task-dependent[END_REF][START_REF] Kumru | Motor cortex excitability changes during imagery of simple reaction time[END_REF][START_REF] Liepert | Motor excitability during imagination and observation of foot dorsiflexions[END_REF].

However, there is now compelling evidence that cortical cell responsiveness to TMS increased during MI, as observed by a decrease of the rMT in comparison to rest [START_REF] Grosprêtre | Motor imagery and cortico-spinal excitability: A review[END_REF]. Therefore, for a similar subthreshold CS intensity based on the rMT calculated when the subject is at rest, this same CS intensity for the MI condition is closer to the motor threshold. The investigations of SICI at rest have found that increasing the CS intensity leads to the recruitment of high-threshold excitatory interneurons that may contaminate SICI, motivating us to carry out the current study. The result obtained here does indeed show that SICI was lower during MI when compared to rest only for high CS intensity (80% rMT). We may therefore suggest that this decrease in SICI could be the result of using CS intensities that were too high and that produced an unwanted recruitment of excitatory interneurons. These findings lead us to reconsider the modulation of SICI underlying MI, taking into account the selection of CS intensity.

SICI is higher during motor imagery when tested with low CS intensities

In Experiment 2, we found for the first time that SICI was greater during MI (vs. at rest) when using low CS intensities (i.e. 50% rMT and 60% rMT). With optimal TMS settings, we revealed an important component of neural processes within M1 during MI.

Neuroimaging studies have shown that brain networks underlying MI and actual movement execution extensively overlap, supporting the elaboration of motor commands during MI [START_REF] Hétu | The neural network of motor imagery: An ALE meta-analysis[END_REF]. During MI, however, motor commands may be stopped at some level of the motor system by active inhibitory mechanisms to prevent them from being sent to peripheral effectors [START_REF] Jeannerod | Mental motor imagery: a window into the representational stages of action[END_REF]. It has been hypothesized that the neural activation within motor and pre-motor areas during MI is blocked by inhibitory mechanisms preventing the overt action [START_REF] Jeannerod | Mental motor imagery: a window into the representational stages of action[END_REF][START_REF] Jeannerod | Neural simulation of action: a unifying mechanism for motor cognition[END_REF]. Because intracortical networks within M1 could be considered as the final cortical modulators of motor output [START_REF] Cowie | Proactive modulation of long-interval intracortical inhibition during response inhibition[END_REF], a possible explanation for these findings is that the increased SICI would prevent the production of an overt movement when the mental representation of that movement is activated. These findings strengthen the idea that MI requires motor command inhibition by active processes acting at a cortical level.

One could argue that the SICI increase, i.e. more inhibition, cannot be at play simultaneously to the corticospinal excitability increase, i.e. more facilitation, in the specific effector involved during MI. However, it is important to keep in mind that MEP amplitude results from the balance between inhibitory and excitatory processes along the corticospinal tract including both cortical and spinal-segmental contributions. Moreover, neuroimaging studies provide evidence that MI is also supported by a network involving motor and premotor regions including cortical and subcortical structures [START_REF] Hétu | The neural network of motor imagery: An ALE meta-analysis[END_REF]. Therefore, corticospinal facilitation could possibly originate from these regions, outside M1 and exert their influence via direct or indirect pathways [START_REF] Reis | Contribution of transcranial magnetic stimulation to the understanding of cortical mechanisms involved in motor control[END_REF]. By contrast, the modulation of SICI observed in this study could reflect the crucial role played by cortical interneurons within M1 in the fine-tuning neural processes required during MI.

MEPtarget amplitude considerations

In the current study, different MEPtarget amplitudes were chosen for the two experiments and this deserves discussion. In Experiment 1, based on the existing literature, a fixed MEP amplitude value of at least 0.2 mV was tracked for all participants [START_REF] Fisher | Two phases of intracortical inhibition revealed by transcranial magnetic threshold tracking[END_REF][START_REF] Vucic | Utility of threshold tracking transcranial magnetic stimulation in ALS[END_REF], corresponding to an average TS intensity of 109% rMT (range 103-118 %rMT), a relatively low intensity. It is known that SICI predominately inhibits the late I-waves (I2, I3 and I4) and that a TS delivered at a low TMS intensity (i.e., below 110% rMT) could fail to evoke late I-waves and thus limits the detection of SICI [START_REF] Garry | The effect of test TMS intensity on short-interval intracortical inhibition in different excitability states[END_REF][START_REF] Lazzaro | Noninvasive Stimulation of the Human Brain: Activation of Multiple Cortical Circuits[END_REF].

In order to answer our second hypothesis and to optimize the conditions of SICI measure, a subject-specific MEP amplitude of at least 50% of MEPmax was tracked for all participants in Experiment 2, corresponding on average to 124 % rMT (range 112-150 %rMT), a moderate TS intensity known to generate the greatest measure of SICI [START_REF] Garry | The effect of test TMS intensity on short-interval intracortical inhibition in different excitability states[END_REF][START_REF] Wagle-Shukla | Effects of short interval intracortical 21 inhibition and intracortical facilitation on short interval intracortical facilitation in human primary motor cortex[END_REF][START_REF] Amandusson | Comparison between adaptive and fixed stimulus pairedpulse transcranial magnetic stimulation (ppTMS) in normal subjects[END_REF][START_REF] Van Den Bos | Physiological processes underlying short interval intracortical facilitation in the human motor cortex[END_REF]

Perspectives and limitations

The results obtained in the current study have demonstrated that modulation of SICI during MI depends critically on CS intensity. Importantly, other cognitive conditions that share analogous control mechanisms and neural circuits with overt movements, such as motor preparation or action observation, are known to selectively modulate corticospinal excitability and to affect SICI [START_REF] Naish | Effects of action observation on corticospinal excitability: Muscle specificity, direction, and timing of the mirror response[END_REF][START_REF] Duque | Physiological Markers of Motor Inhibition during Human Behavior[END_REF]. Supporting this view, a recent study measured SICI using a range of CS intensities at rest and during a warned simple reaction time task [START_REF] Ibáñez | SICI during changing brain states: Differences in methodology can lead to different conclusions[END_REF]. The results showed that show that SICI changes that occurred during the task could be either larger or smaller than at rest depending on the intensity of the CS. Together, these findings also confirmed that testing SICI using a wide range of CS intensities provides a more nuanced interpretation of possible GABAergic changes in M1 than testing with a single CS intensity [START_REF] Ibáñez | SICI during changing brain states: Differences in methodology can lead to different conclusions[END_REF]. We believe that the adaptive threshold hunting paradigm could be useful in further studies to assess SICI during various cognitive and motor states.

The current study presents certain limitations that should be noted. Firstly, we acknowledge that the relatively small sample size of this study and the high inter-and intra-individual variability of the TMS measurement compromise generalizability of the current findings and should be considered. Moreover, it is important to remember that a greater SICI during MI is one of the possible mechanisms acting at a cortical level among many other possibilities that prevent muscle contraction [START_REF] Jeannerod | Mental motor imagery: a window into the representational stages of action[END_REF]Lebon et al., 2012b). The inhibition

  TS Intensity)-(unconditioned TS Intensity) (unconditioned TS Intensity) 100 where positive values indicate inhibition and negative values indicate facilitation. Background root mean square (RMS) of the surface EMG was calculated during the 100 ms epoch prior to TMS to ensure the absence of muscle contraction in each condition.

FigFigure 2

 2 Figure 2 illustrates the percentage of inhibition (SICI) obtained at rest and during MI for the three CS intensities. ANOVA revealed no main effects of Task (F(1,12) < 1, p = .446) or

  Figure 4 illustrates SICI results (INH%) obtained at rest and during MI for two different CS intensities. ANOVA revealed a main effect of Task showing that SICI is higher during MI when compared to rest (+35.4 ± 9.3 INH% vs. +30.5 ± 10.3 INH%, F(1,12) = 4,838; p = .048; ηp 2 = .287). However, no significant main effect of CS intensity (F(1,12) = 2,543, p = .137) nor Task by CS Intensity interaction (F(1,12) < 1, p = .467) was found.

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: Mean ± SE for the Unconditioned TS Intensity obtained with the hunting-threshold technique at rest and during motor imagery in the Experiment 1 (A) and Experiment 2 (B). *p < .05

Figure 4 :

 4 Figure 4: Mean ± SE for SICI obtained in Experiment 2 with the hunting-threshold technique and according to the two conditioning stimulus (CS) intensities expressed in percentage of the rMT at rest (black) and during motor imagery (red). *p < .05

Table 1

 1

	) yielded no significant difference between rest and MI neither in Experiment 1 (t(12) =
	0.201; p=.844) nor in Experiment 2 (t(12) = -1.017; p=.329). In addition, the ANOVAs for
	RMS values for conditioned TS revealed no significant main effects or interactions for
	Experiment 1 (all p > .07) and Experiment 2 (all p > .14). Therefore, any changes observed in
	the subsequent measurements cannot be attributed to differences in EMG levels prior to the
	TMS pulse.
		Experiment 1
		Unconditioned TS		Conditioned TS
			CS 60%	CS 70%	CS 80%
	Rest	5.9 ± 4.3	5.96 ± 4.2 6.41 ± 4.7 6.32 ± 4.5
	Motor Imagery	5.84 ± 4.2	6.45 ± 4.5 6.61 ± 4.8 6.59 ± 4.6
		Experiment 2
		Unconditioned TS		Conditioned TS
			CS 50%	CS 60%
	Rest	3.19 ± 1.7	3.09 ± 1.7 3.04 ± 1.6
	Motor Imagery	3.29 ± 1.8	3.48 ± 1.8 3.18 ± 1.7

Table 1 :

 1 Pre-trigger root mean squared EMG expressed in μV (mean ± SD).

	Unconditioned TS Intensity
	Figure 1 illustrates the unconditioned TS Intensity obtained at rest and during MI in
	Experiment 1 (left panel) and Experiment 2 (right panel) in both groups of participants.
	Two-tailed paired sample t-tests revealed that the unconditioned TS Intensity required to
	reach the MEPtarget was significantly lower during MI than at rest in both Experiment 1 (45.84
	± 12.04 vs. 48.84 ± 10.6; t(12) =2.793; p=.016 and Experiment 2 (43.4 ± 8.7 vs. 45.5 ± 13.6;
	t(12) = 2.976; p=.012). This result indicates that MI leads to increase corticospinal
	excitability.

Funding:

This work was supported by the French "Investissements d'Avenir" program, project ISITE-BFC (contract ANR-15-IDEX-0003).

cerebellum, the brainstem and/or the spinal cord [START_REF] Lotze | Activation of cortical and cerebellar motor areas during executed and imagined hand movements: an fMRI study[END_REF][START_REF] Jeannerod | Neural simulation of action: a unifying mechanism for motor cognition[END_REF].

Overall, this study provides initial evidence that the intensity of the CS crucially affects SICI measurement during MI when compared to rest. The previously reported decrease in SICI during MI could be due to inappropriate TMS settings, with high CS intensities leading to the unwanted recruitment of excitatory interneurons. With low CS intensities, we show that SICI is greater during MI than at rest, probably to prevent the production of an overt movement when the mental representation of that movement is activated. Future studies should consider optimizing the SICI stimulation protocol by careful adjustment of the CS intensity.

Declaration/conflicts of interest:

No conflicts of interest, financial or otherwise, are declared by the authors.