Mathieu Chalouni

Stanislas Pol

Philippe Sogni

Helene Fontaine

Karine Lacombe

Jean-Marc M Lacombe

Laure Esterle

Celine Dorival

Marc Bourliere

Firouze Bani-Sadr

M Bourlière

V De Ledinghen

D Zucman

D Larrey

D Salmon

F Carrat

L Wittkop

P Trimoulet

J Izopet

L Serfaty

V Paradis

B Spire

P Carrieri

M A Valantin

G Pialoux

J Chas

I Poizot- Martin

K Barange

A Naqvi

E Rosenthal

A Bicart-See

O Bouchaud

A Gervais

C Lascoux-Combe

C Goujard

C Duvivier

D Neau

P Morlat

F Bani- Sadr

L Meyer

F Boufassa

B Autran

A M Roque

C Solas

D Costagliola

L Piroth

A Simon

F Boué

P Miailhes

E Billaud

H Aumaître

D Rey

G Peytavin

V Petrov-Sanchez

C Katlama

H Stitou

Médecine Interne

: A Simon

P Cacoub

S Nafissa

; Hépato-Gastro-Entérologie :

Y Benhamou

; Anatomo-Pathologie :

F Charlotte

S Fourati

O Zaegel

H Laroche

C Tamalet

P Callard

F Bendjaballah

C Amiel

C Le

Direct, indirect and total effect of HIV co-infection on the risk of nonliver-related cancer in hepatitis C virus infected patients treated by direct-acting antivirals: a mediation analysis

Keywords: HCV, HIV co-infection, DAA treatment, mediation analysis, non-liverrelated cancers

INTRODUCTION

Hepatitis C virus (HCV) infection can lead to several liver-related complications (e.g. liver decompensation, hepatocellular carcinoma (HCC), or liver-related death) [1], but also to non-liver-related events such as non-liver-related cancer [START_REF] Allison | Incidence of Cancer and Cancer-related Mortality Among Persons with Chronic Hepatitis C Infection, 2006-2010[END_REF]. Direct-acting antivirals (DAA) allow to achieve a sustained virological response (SVR) in more than 90% of treated participants [START_REF] Naggie | Ledipasvir and Sofosbuvir for HCV in Patients Coinfected with HIV-1[END_REF][START_REF] Bang | Impact of antiviral therapy on hepatocellular carcinoma and mortality in patients with chronic hepatitis C: systematic review and meta-analysis[END_REF]. SVR is associated with a decreased risk of liver and non-liver-related events [START_REF] Bang | Impact of antiviral therapy on hepatocellular carcinoma and mortality in patients with chronic hepatitis C: systematic review and meta-analysis[END_REF][START_REF] Nahon | Eradication of Hepatitis C Virus Infection in Patients With Cirrhosis Reduces Risk of Liver and Non-Liver Complications[END_REF][START_REF] Chew | The effect of hepatitis C virologic clearance on cardiovascular disease biomarkers in human immunodeficiency virus/hepatitis C virus coinfection[END_REF][START_REF] Tada | Viral eradication reduces all-cause mortality, including non-liver-related disease, in patients with progressive hepatitis C virus-related fibrosis[END_REF][START_REF] Bruno | Improved survival of patients with hepatocellular carcinoma and compensated hepatitis C virus-related cirrhosis who attained sustained virological response[END_REF][START_REF] Carrat | Clinical outcomes in patients with chronic hepatitis C after direct-acting antiviral treatment: a prospective cohort study[END_REF]. In a previous study, HIV co-infection was associated with a three-times higher risk of non-liver-related cancer in HCV mono-infected participants after DAA treatment [START_REF] Chalouni | Increased mortality in HIV/HCV-coinfected compared to HCV-monoinfected patients in the DAA era due to non-liver-related death[END_REF]. This could be explained by chronic inflammation and immunosuppression induced by HIV infection [START_REF] Franzetti | The Pattern of Non-AIDS-defining Cancers in the HIV Population: Epidemiology, Risk Factors and Prognosis. A Review[END_REF][START_REF] Franzetti | Trends and predictors of non-AIDS-defining cancers in men and women with HIV infection: a single-institution retrospective study before and after the introduction of HAART[END_REF][START_REF] Grulich | Incidence of cancers in people with HIV/AIDS compared with immunosuppressed transplant recipients: a meta-analysis[END_REF][START_REF] Tien | Inflammation and mortality in HIV-infected adults: analysis of the FRAM study cohort[END_REF]. HIV co-infection is associated with higher prevalence of alcohol consumption, tobacco consumption [START_REF] Silverberg | HIV infection and the risk of cancers with and without a known infectious cause[END_REF]16] and metabolic syndrome [START_REF] Krishnan | Metabolic syndrome before and after initiation of antiretroviral therapy in treatmentnaive HIV-infected individuals[END_REF]. These factors have been identified as risk factors for several cancers and could explain the higher risk of non-liver-related cancers observed in HIV co-infected patient [START_REF] Silverberg | HIV infection and the risk of cancers with and without a known infectious cause[END_REF][START_REF] Choi | Abdominal obesity, glucose intolerance and decreased high-density lipoprotein cholesterol as components of the metabolic syndrome are associated with the development of colorectal cancer[END_REF][START_REF] Gathirua-Mwangi | Associations of metabolic syndrome and C-reactive protein with mortality from total cancer, obesity-linked cancers and breast cancer among women in NHANES III[END_REF][START_REF] Gacci | Meta-analysis of metabolic syndrome and prostate cancer[END_REF]. HIV co-infected patients are also more exposed to oncogenic viruses [START_REF]Humans IWG on the E of CR to[END_REF][START_REF] De Martel | Cancers attributable to infections among adults with HIV in the United States[END_REF]. These viruses are responsible for or are risk factors for several cancers which could explain the higher risk observed in HIV infected patients. The main aim of this study was to estimate the direct effect (reflecting chronic inflammation and immunosuppression), indirect effect (reflecting higher alcohol and tobacco consumptions and metabolic syndrome) and total effect of HIV co-infection on the risk of non-liver-related cancer in HCV infected participants treated with DAA.

Sensitivity analyses were carried out to estimate the specific effect of each mediator on the risk of non-liver-related cancers. Finally, to take into account the potential increase of non-liver-related cancers in HIV/HCV co-infected participants due to oncogenic viruses, a sensitivity analysis was carried out focusing on non-liver-related non-oncogenic viruses-related cancers. The primary outcome was time between DAA initiation and the occurrence of a nonliver-related cancer, defined as the occurrence of a cancer which was not an HCC, cholangiocarcinoma or hepato-cholangiocarcinoma. The occurrence of a non-liverrelated cancer was declared by investigator centers, with validation by an adjudication committee on medical records. Participants were followed until the occurrence of a non-liver-related cancer, death or the last follow-up visit.

For the sensitivity analysis on non-oncogenic virus-related cancers, the occurrence of a cervical uterine cancer, anal cancer, Hodgkin or non-Hodgkin lymphoma or Kaposi sarcoma were considered as competing events.

Mediators

All data concerning mediators were measured using a standardized questionnaire during medical follow-up of participants. Data on alcohol and tobacco consumption were collected during participant interviews by a physician. All data were measured at the time of DAA treatment initiation.

Even though HIV infection does not induce alcohol or tobacco consumption, the higher rates of these consumptions in HIV co-infected [START_REF] Silverberg | HIV infection and the risk of cancers with and without a known infectious cause[END_REF]16] could reflect higher risk behaviors in this population. This suggests the need to consider these consumptions as confounding factors. In our population, participants were infected by HIV at baseline. Therefore, the consumptions were measured after HIV infection. The temporality hypothesis needed to define a mediator is thus respected [START_REF] Fasanelli | Marginal Time-Dependent Causal Effects in Mediation Analysis With Survival Data[END_REF]. In addition, due to HIV infection, the reduction of these consumptions is seen as a low priority for both patients and clinicians, leading to lower cessation rates in this population [START_REF] Fredericksen | Patient and provider priorities for self-reported domains of HIV clinical care[END_REF][START_REF] Strauss | HIV care providers' implementation of routine alcohol reduction support for their patients[END_REF][START_REF] Pacek | Adherence to Pharmacological Smoking Cessation Interventions: A Literature Review and Synthesis of Correlates and Barriers[END_REF][START_REF] Shahrir | Receipt and predictors of smoking cessation pharmacotherapy among veterans with and without HIV[END_REF]. Indeed, whereas in HCV mono-infected participants the cessation rates are rather high [START_REF] Proeschold-Bell | A Randomized Controlled Trial of an Integrated Alcohol Reduction Intervention in Patients With Hepatitis C Infection[END_REF], in HIV/HCV co-infected participants the cessation rates are markedly low [START_REF] Strauss | HIV care providers' implementation of routine alcohol reduction support for their patients[END_REF].

Consequently, although the HIV co-infection did not directly increase the alcohol and tobacco consumption, these consumptions should be considered as mediators in the relationship between HIV co-infection and the risk of non-liver-related cancer (Supplementary Figure 1).

Alcohol consumption

Alcohol consumption was classified into four categories: participants declaring no consumption, consumption between 0 and 20, 20 and 60 and more than 60 grams of alcohol per day. Alcohol consumption in grams per day was estimated by multiplying the number of glasses of alcoholic beverages consumed per day by 10 [29].

Tobacco consumption

Tobacco consumption was classified into four categories: participants declaring no tobacco consumption, consumption between 0 and 15, 15 and 25 and more than 25 cigarettes per day.

Metabolic syndrome

Following the definition of the national cholesterol education program [START_REF]Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III)[END_REF], metabolic syndrome was defined by the presence of at least three of the following criteria: high blood pressure, hypertriglyceridemia, reduced HDL-cholesterol, hyperglycemia and obesity. Due to the lack of available data for waist circumference, obesity was defined according to body mass index (BMI) (Table 1).

Statistical analysis

Pure direct effect (PDE), total indirect effect (TIE) and total effect (TE) of HIV coinfection on the risk of non-liver-related cancer three years after the initiation of DAA therapy were estimated using mediation analysis [START_REF] Fasanelli | Marginal Time-Dependent Causal Effects in Mediation Analysis With Survival Data[END_REF]. Exposure was HIV co-infection, seropositivity (in years), HCV genotype and cirrhosis status defined by cohort specific algorithms previously published [START_REF] Chalouni | Increased mortality in HIV/HCV-coinfected compared to HCV-monoinfected patients in the DAA era due to non-liver-related death[END_REF] taking into account liver biopsy, liver stiffness and non-invasive liver markers (Figure 1). TIE reflected the effect of mediators on the risk of non-liver-related cancers, whereas PDE reflected the effect of chronic inflammation and immunosuppression in the absence of other unmeasured mediators [START_REF] Fasanelli | Marginal Time-Dependent Causal Effects in Mediation Analysis With Survival Data[END_REF].

PDE of HIV co-infection (solid pathway) reflects how much the hazard of non-liverrelated-cancer at a given time (here, three years) would change if all participants were HIV co-infected versus if none of the participants were HIV co-infected but mediator values were kept at their observed levels. To estimate PDE, first a flexible Cox proportional-hazards model, estimating the baseline hazard with natural cubic splines [START_REF] Royston | Flexible parametric proportional-hazards and proportional-odds models for censored survival data, with application to prognostic[END_REF], was fitted to estimate the association between HIV co-infection and the risk of non-liver-cancer adjusted for mediators, age, sex, time since first HCV seropositivity, HCV genotype and cirrhosis. Then, for each HCV mono-infected participants the hazard function 𝜆 ! !,# $ was estimated by the ratio of the weighted means of survival and density functions if all those participants had been HIV co-infected, but mediator values were kept at their observed levels. The hazard function 𝜆 ! $,# $ was estimated by the ratio of the weighted means of survival and density functions estimated in the subpopulation of HCV mono-infected patients, and mediator values were kept at their observed values. In the same way, the hazard function 𝜆 ! !# ! was estimated in the HIV co-infected subpopulation. For each participant, the weight for the estimation of weighted means of survival and density functions was equal to the inverse of the probability that the exposure was at the observed level. Confidence intervals at 95% of the effects were estimated using percentile bootstrap.

Participants with missing data were compared to participants with at least one missing data for variables included in the analysis, according to HIV co-infection. Assuming data were missing at random, missing data for the explanatory variables included in the analysis were imputed by multiple imputation methods using chained equations in R 3.6.2 software using mice package [32]. Ten tables were generated by 10 iterations.

Quantitative variables were imputed using linear models, while qualitative variables were imputed by logistic or multinomial regression as appropriate. Imputation models included history of non-liver-related cancer or of liver-related event and of anti-HCV treatment, class and duration of DAA treatment, platelet and albumin values, and diabetes, in addition to variables included in the multivariable models for the analysis.

The normality of the quantitative variables included in the imputation model was graphically checked. We carried out several sensitivity analyses. First, only tobacco consumption, and then only alcohol consumption, were considered as mediators. Then, as for the main analysis, we estimated the direct, indirect and total effects of HIV co-infection on the risk of non-liver-related non-oncogenic virus-related cancers.

RESULTS

Population description

Five-hundred and forty-eight HIV/HCV co-infected and 2016 HCV mono-infected participants were included. Among them, respectively 292 and 1416 had at least a missing data. In HIV/HCV co-infected participants, 164 (39.9%), 154 (28.1%) and 223 (40.7%) had missing data respectively for alcohol consumption, tobacco consumption and metabolic syndrome, whereas in HCV mono-infected participants missing data were observed in 679 (33.7%), 64 (3.2%) and 1037 (51.4%), respectively. In HIV coinfected, participants with at least a missing data consumed more alcohol (51.6% declared no alcohol consumption vs 56.6% and 12.5% declared to consumed between 20 and 60 grams/day vs 7.0%) and more were cirrhotic (32.3% vs 23.5%) compared to participants without missing data. HCV mono-infected participants with at least a missing data consumed more tobacco (48.1% declared to consumed between 0 and 15 cig/day vs 42.2%) and were less frequently with a metabolic syndrome (10.8% vs 15.0%) (Supplementary Table 1).

The median age of the population was 52.9 years [interquartile range (IQR): 49.5; 56.6] and 53.3 years [49.5; 56.9] and 405 (73.9%) and 1478 (73.3%) were men, respectively.

HIV co-infected participants were more often infected by HCV due to intravenous drug use (62.2% vs 37.7%), consumed more alcohol (45.1% declared consuming alcohol vs 0.4%), consumed more tobacco (62.9% declared consuming tobacco vs 46.6%) and had a longer time since their first HCV seropositivity (17.9 years [12. [6.3; 20.8]) but were less frequently cirrhotic (28.1% vs 41.1%) compared to HCV mono-infected participants. The proportion of metabolic syndrome (9.2% vs 13.4%) and SVR rates (93.6% vs 94.5%) were similar between both populations (Table 2).

Risk of non-liver-related cancer

After a median follow-up of 2.4 years [1.2; 3.3] and 2.9 years [1.7; 3.9], a non-liverrelated cancer occurred in 22 (of which 17 were non-related to an oncogenic virus) and 28 (all non-related to an oncogenic virus), respectively, in HIV/HCV co-infected and HCV mono-infected participants. Among HIV/HCV co-infected participants, the more frequently occurring cancers were: non-melanoma skin (n = 4), anal (n = 2), lung (n = 2), melanoma (n = 2), nasopharynx (n = 2) and prostate (n = 2) cancers, 2 were AIDS defining cancers (1 cervical cancer and 1 non-Hodgkin lymphoma). In HCV monoinfected participants, the most observed cancers were non-melanoma skin (n = 5), pancreas (n = 5), colon-rectal (n = 3), lung (n = 3), renal-urethra (n = 3) and prostate (n = 2) cancers (Supplementary Table 2).

The TE of HIV co-infection was significantly associated with an increased risk of more than three times (HR: 3.7 [1.7; 7.0]) of non-liver-related cancer in HCV mono-infected participants. This increased risk in HIV co-infected participants was mainly explained by the PDE of HIV co-infection (HR: 3.4 [1.7; 6.6]). In contrast, the TIE of HIV coinfection, reflecting the effect of alcohol consumption, tobacco consumption and metabolic syndrome, was not significantly associated with the risk of non-liver-related cancer (HR: 1.1 [0.8; 1.5]) in HCV infected participants treated by DAA (Fig 2).

When considering only tobacco consumption as a potential mediator of the association between HIV co-infection and the risk of non-liver-related cancers, similar results were found. With PDE, TIE and TE of HIV co-infection on the risk of non-liver-related cancers 3).

When excluding oncogenic virus-related cancers, HIV co-infection was still associated with a more than three times (HR: 3.1 [1,6; 4.9]) increase in the risk of non-liver-related cancers, mainly due to the direct effect of HIV co-infection (HR: 2.4 [1.3; 1.7]) but not to the indirect effect of HIV co-infection (HR: 1.3 [0.9; 1.7]) (Table 3).

DISCUSSION

In a population of HCV infected participants treated with DAA followed for up to three years after the initiation of the DAA therapy, using a methodology allowing us to estimate the direct and indirect effects of an exposure on an outcome, we found that HIV co-infection was directly associated with a 3.4-fold increased risk of non-liverrelated cancer in HCV infected participants. The indirect effect of HIV co-infection, reflecting the effects of higher alcohol consumption, tobacco consumption and metabolic syndrome, was not significantly associated with the risk of non-liver-related cancer in this population. Overall, the total effect of HIV co-infection was associated with a 3.7-fold higher risk of non-liver-related cancers in HCV infected participants.

To our knowledge, no other study evaluated the potential impact of HIV co-infection on the risk of non-liver-related cancers after DAA treatment in HCV infected participants or the potential mediating effect of alcohol consumption, tobacco consumption and metabolic syndrome on the relationship between HIV co-infection and risk of non-liverrelated cancers. Whereas alcohol consumption, tobacco consumption and metabolic syndrome are known risk factors for several cancers [START_REF] Silverberg | HIV infection and the risk of cancers with and without a known infectious cause[END_REF][16][START_REF] Krishnan | Metabolic syndrome before and after initiation of antiretroviral therapy in treatmentnaive HIV-infected individuals[END_REF], we did not observe here a significant mediating effect of these factors for the relationship between HIV co-F o r P e e r R e v i e w 14 infection and the risk of non-liver-related cancer, despite a higher declared alcohol consumption and tobacco consumption in HIV co-infected participants. This could be explained by the very low alcohol and tobacco consumption declared by HCV monoinfected participants, which led to decreased statistical power and did not allow us to perform an analysis with alcohol and tobacco consumption as continuous variables (expressed in grams per day and number of packs per year) rather than categorial variables or to study the direct and indirect effects of HIV co-infection on the risk of each specific cancer. A classification bias due to under-declaration of alcohol and tobacco consumption could also explain why we did not observe a significant TIE in this study. Nevertheless, higher consumption of alcohol and tobacco in HIV co-infected participants has been observed in previous studies [START_REF] Silverberg | HIV infection and the risk of cancers with and without a known infectious cause[END_REF]16], suggesting that the differences observed in our study for alcohol and tobacco consumption between HIV/HCV co-infected and HCV mono-infected were not due to bias. This study had several limitations. First, due to the design of the study we included participants from two different cohorts. If data on non-liver-related cancers or on alcohol and tobacco consumption were not collected in the same way in the two cohorts, this could induce misclassification of participants and result in biased estimation of PDE, TIE and TE. However, both cohorts used similar procedures to report all serious adverse events occurring during the follow-up, with extensive data quality controls and monitoring. Moreover, all deaths were adjudicated by a dedicated committee, and consistent checks for non-liver-related cancers were carried out, limiting the risk of differential misclassification. In addition, since the participants came from two different cohorts, this could explain the important differences observed, including on alcohol and tobacco consumption. These differences could have made the two populations incomparable and could explain why we did not observe a F o r P e e r R e v i e w 15 significant TIE of HIV co-infection on the risk of non-liver-related cancers. Second, alcohol and tobacco consumption were measured at baseline only. So, we did not take into account past consumption or potential cessation before the initiation of DAA. We cannot exclude that the difference observed between the two populations for alcohol and tobacco consumption was due to higher cessation rates in HCV mono-infected than in HIV/HCV co-infected participants. The measurement of consumption at DAA treatment initiation may therefore not reflect the differences between the two populations concerning lifetime consumption and could explain the absence of a significant indirect effect of HIV co-infection on the risk of non-liver-related cancers.

Third, to estimate the unbiased total, direct and indirect causal effects using mediation analysis, some assumptions have to be insured. These assumptions are: no unmeasured confounding factors between (1) the exposure and the outcome, (2) the mediator and the outcome, (3) the exposure and the mediator and (4) none of the mediator-outcome confounders are affected by the exposure. In addition, the direct effect of HIV co-infection estimated here reflects the effect of chronic inflammation and immunosuppression only if no unmeasured mediators are involved. We identified confounding factors of the different studied relations using DAG methodology, and it does not appear that we did not take into account some factors. Nevertheless, we cannot rule out that some confounding factors were not included in the analysis, resulting in bias in the effects estimated. Fourth, the mediation methodology used did not allow us to estimate the mediator-specific indirect effect of HIV co-infection [START_REF] Fasanelli | Marginal Time-Dependent Causal Effects in Mediation Analysis With Survival Data[END_REF].

The estimation of the total indirect effect of HIV co-infection, rather than the indirect effects specific to each mediator, could lead to a dilution of the mediating effect if one mediator was not associated with the risk of non-liver-related cancer. To take this limitation into account, sensitivity analyses were carried out considering only alcohol 16 and tobacco consumption as potential mediators. The results of these sensitivity analyses were similar to those observed in the main analysis. Fifth, the important amount of missing data for mediator values could lead to a selection bias especially if missing data were missing not a random. To reduce this hazard, in addition to variables included in the mediation models, we included in the imputation models factors related to variables with missing values. In addition, missing data were frequent for metabolic syndrome due to its definition, which depends on 5 criteria. To reduce the impact of this definition, we firstly imputed the 5 criteria and then defined the presence of metabolic syndrome for each participant. Finally, due to differences between both populations, notably concerning the mode of transmission of HCV (299 (62.6%) and 756 (37.7%) infected due to intravenous drug use, respectively, in HIV/HCV coinfected and HCV mono-infected participants) which could affect the estimation of the direct, indirect and total effects of HIV co-infection on the risk of non-liver-related cancers, it could be more suitable to estimate the effects of HIV infection in the general population.

This study also had several strengths. First, the large size of the studied population, including 2564 participants who were all treated by DAA. Importantly, the considerable size of the population allows us to have sufficient statistical power. Second, the mediation methodology we used has several advantages, as this approach does not require models for mediators, it allowed us to study non-independent continuous or categorial mediators without needing the rare outcome assumption [START_REF] Fasanelli | Marginal Time-Dependent Causal Effects in Mediation Analysis With Survival Data[END_REF]. Third, both cohorts are prospective French nationwide cohorts with strict monitoring rules allowing for high quality data. Finally, to our knowledge this is the first study estimating the direct and mediating effects of HIV co-infection on the risk of non-liver-related cancers in HCV infected participants treated by DAA.

CONCLUSIONS

In conclusion, in a population of HIV/HCV co-infected participants with controlled HIV viral load, HIV co-infection was associated with a 3.7-fold higher risk of non-liverrelated cancers at three years after DAA initiation, compared to HCV mono-infected participants. This increased risk was mainly explained by the direct effect of HIV coinfection, reflecting chronic inflammation and immunosuppression, which was associated with a three-fold increase in the risk of non-liver-related cancers. In contrast, no significant association was observed for the indirect effect of HIV coinfection (reflecting the more frequent alcohol consumption, tobacco consumption and metabolic syndrome) with the risk of non-liver-related cancer.

F

  co-infected patients from the ANRS CO13 HEPAVIH cohort (ClinicalTrials.gov Identifier: NCT03324633) and HCV mono-infected patients from the ANRS CO22 HEPATHER cohort (ClinicalTrials.gov Identifier: NCT01953458) were included. Both are multicenter, prospective, nationwide French cohort studies, with follow-up of participants every year with supplementary visits scheduled during anti-HCV treatment. Cirrhotic participants from the ANRS CO13 HEPAVIH cohort had a supplementary visit every six months. Written informed consent was obtained from each participant included in both cohorts. The study followed the ethical principles of the World Medical Association (Declaration of Helsinki) and was approved by an institutional review board (Comité de Protection des Personnes (CPP) Ile de France III, Paris, France -ANRS CO13 HEPAVIH and ANRS CO22 HEPATHER). The ANRS CO13 HEPAVIH cohort was initiated in 2005 and included HIV/HCV coinfected participants. Due to the development of new anti-HCVs, there were several inclusion periods. The last one, between 2014 and 2016, included participants according to DAA treatment initiation. Whereas, the ANRS CO22 HEPATHER cohort was opened in 2012 and included participants infected by Hepatitis B Virus or HCV. Participants treated by DAA with or without ribavirin and/or pegylated-interferon between March 1, 2014 and December 31, 2017, not participating in a clinical trial, without history of liver-transplantation or of non-liver-related cancer and with an available SVR status were eligible. For each HIV/HCV co-infected participant, a maximum of 4 HCV mono-infected participants were matched by age at treatment initiation (3 years, more or less) and sex.Non-liver-related cancer

  mediators were alcohol consumption, tobacco consumption and metabolic syndrome and confounding factors were sex, age (in years), HCV transmission routes (intravenous drug use, sexual, transfusion, unknown and others), time since first HCV

  The probability was estimated by a logistic model explaining HIV co-infection by confounding factors. PDE was estimated by 𝜆 ! !,# $ / 𝜆 ! $,# $ .

10 TIE

 10 of HIV co-infection (dashed pathway) reflects how much the hazard of non-liverrelated-cancer would change if exposure was kept at the observed level but mediators values were changed from the level they would have if participants were HIV coinfected versus value they would have if participants were not HIV co-infected. TIE was estimated by 𝜆 ! !,# ! / 𝜆 ! !,# $ .Finally, TE of HIV co-infection reflects how much the hazard of non-liver-related cancer would change if all participants were HIV co-infected versus no participants being HIV co-infected, and mediator values were also changed from the values they would have if none of the participants were HIV co-infected to the values they would have if all participants were HIV co-infected. TE was estimated by multiplying PDE and TIE.

  3.4 [1.7; 6.6], 1.0 [0.8; 1.3] and3.5 [1.7; 6.7]. The same results were found when considering only alcohol consumption as a potential mediator. PDE, TIE and TE estimation were respectively 3.4 [1.7; 6.7], 1.0 [0.7; 1.3] and3.4 [1.7; 6.7] (Table

Figure 1 .

 1 Figure 1. Assumed causal relationship between HIV co-infection in Hepatitis C Virus

Figure 2 .

 2 Figure 2. Direct, indirect and total effects of HIV co-infection on the risk of non-liver-

Table 1 .

 1 Warda Hadi, Clovis Lusivika-Nzinga, Grégory Pannetier, François Pinot, Odile Stahl, François Téloulé (Sorbonne Université & INSERM U1136, Paris, France). Risk factors and levels for defining a metabolic syndrome following the national cholesterol education program recommendations

	o r
	P e e
	r
	R
	e v i e w

Table 2 .

 2 Description of Hepatitis C Virus infected participants from the ANRS CO13 HEPAVIH and ANRS CO22 HEPATHER cohorts at the time of Direct-Acting Antivirals treatment initiation according to HIV co-infection

		HIV † /HCV ‡ co-infected (n = 548)	HCV ‡ mono-infected (n = 2016)
	Characteristics	n	Median [IQR § ] or n (%)	n	Median [IQR § ] or n (%)
	Age (years)	548	52.9 [49.5; 56.6]	2016	53.2 [49.5; 56.9]
	Men	548	405 (73.9%)	2016	1478 (73.3%)
	HCV ‡ transmission routes	481		2004
	Injecting drug use		299 (62.2%)		756 (37.7%)
	Sexual		85 (17.7%)		21 (1.0%)
	Transfusion		37 (7.7%)		465 (23.2%)
	Unknown		55 (11.4%)		459 (22.9%)
	Other		5 (1.0%)		303 (15.1%)
	Time since first HCV ‡ seropositivity (years) 522	17.9 [12.1; 22.2]	2016	14.4 [6.3; 20.8]
	Cirrhosis	512	144 (28.1%)	1896	779 (41.1%)
	Current alcohol consumption (gram/day)	384	0.0 [0.0; 5.7]	1337	0.0 [0.0; 0.0]
	0		211 (54.9%)		1330 (99.5%)
	]0 ; 20]		133 (34.6%)		3 (0.2%)
	]20 ; 60]		34 (8.9%)		1 (0.1%)
	> 60		6 (1.6%)		3 (0.2%)
	Current tobacco consumption (cig/year)	394	7.0 [0.0; 20.0]	1952	0.0 [0.0; 1.0]
	0		149 (37.8%)		1048 (53.3%)
	]0 ; 15]		136 (34.5%)		903 (46.3%)
	]15 ; 25]		75 (19.0%)		1 (0.1%)
	> 25		34 (8.6%)		0 (0.0%)
	Metabolic syndrome	325	30 (9.2%)	979	131 (13.4%)

Table 3 .

 3 HIV co-infection direct, indirect, and total effects on the risk of non-liverrelated cancer and non-liver-related non-oncogenic virus-related cancers at three years after the initiation of Direct-Acting Antivirals treatment in Hepatitis C Virus infected participants from the ANRS CO13 HEPAVIH and ANRS CO22 HEPATHER

	cohorts
	Effects

related non-oncogenic virus-related cancers

	F o r
	P e e
	r
	R
	Pure direct effect Total indirect effect	e v i e w	2.4 1.3	[1.2; 5.0] [0.9; 1.7]
	Total effect		3.1	[1.6; 4.9]
	† HR: Hazard Ratio
	‡ CI 95%: Confidence interval at 95%
	35

Related Mortality Among HIV/HCV Co-infected Patients: The Importance of Behaviors in the HCV Cure Era (ANRS CO13 HEPAVIH Cohort). AIDS Behav. 2020

ACKNOWLEDGMENTS

MC was supported by a doctoral award from the French national Agency for Research on Aids and Viral Hepatitis (ANRS: France Recherche Nord & Sud Sida-HIV Hépatites) For the ANRS CO13 HEPAVIH cohort study group Patients of the ANRS CO13 HEPAVIH Cohort Scientific Committee of the ANRS CO13 HEPAVIH Study Group: D. Salmon (co-Principal investigator), L.

CONFLICT OF INTEREST STATEMENT


Marie-Helene Canu

Valerie Montel

Julie Dereumetz

Tanguy Marqueste

Patrick Decherchi

Jacques-Olivier Coq

Erwan Dupont

Bruno Bastide

Early movement restriction deteriorates motor function and soleus muscle physiology

Keywords: Disuse Sensorimotor restriction Development Muscle contractile properties Muscle morphology BW, body weight, C, control, CNS, Central nervous system, CP, cerebral palsy, CSA, cross sectional area, DCD, developmental coordination disorder, FI, fatigue index, HRT, half-relaxation time, MHC, myosin heavy chain, MWW, muscle wet weight, P0, peak tetanic tension postnatal day, Pt, twitch peak tension, RH, right hind paw, SMR, sensorimotor restriction, TTP, time-topeak

. In the present paper, our objectives were to evaluate the impact of early movement restriction leading to sensorimotor restriction (SMR) during development on the postural soleus muscle and on sensorimotor performance in rats, and to determine whether changes were reversed when typical activity was resumed. Rats were submitted to SMR by hind limb immobilization for 16 h / day from birth to postnatal day 28 (PND28). In situ isometric contractile properties of soleus muscle, fiber cross sectional area (CSA) and myosin heavy chain content (MHC) were studied at PND28 and PND60. In addition, the motor function was evaluated weekly from PND28 to PND60. At PND28, SMR rats presented a severe atrophy of soleus muscle, a decrease in CSA and a force loss. The muscle maturation appeared delayed, with persistence of neonatal forms of MHC. Changes in kinetic properties were moderate or absent. The Hoffmann reflex provided evidence for spinal hyperreflexia and signs of spasticity. Most changes were reversed at PND60, except muscle atrophy. Functional motor tests that require a good limb coordination, i.e. rotarod and locomotion, showed an enduring alteration related to SMR, even after one month of 'typical' activity. On the other hand, paw withdrawal test and grip test were poorly affected by SMR whereas spontaneous locomotor activity increased over time. Our results support the idea that proprioceptive feedback is at least as important as the amount of motor activity to promote a typical development of motor function. A better knowledge of the interplay between hypoactivity, muscle properties and central motor commands may offer therapeutic perspectives for children suffering from neurodevelopmental disorders.

Introduction

Besides sedentary behavior, which is more and more frequent in our societies, acute and prolonged inactivity has deleterious effects on motor function and represents a public health issue [START_REF] Hermans | Clinical review: intensive care unit acquired weakness[END_REF][START_REF] Koukourikos | Muscle atrophy in intensive care unit patients[END_REF]. This issue is particularly acute when hypoactivity is experienced during development, for example in children who are bedridden due to chronic illness or accident. Similarly, children with either disabling motor disturbances, such as cerebral palsy (CP) or motor difficulties, for example developmental coordination disorder (DCD), are less prone to be physically active and to interact with people and the environment. CP is the most common physical disability in children and involves a group of disorders of movement and posture, including spasticity in 85 to 91% of cases, and is often associated with behavioral and cognitive disorders. CP is mainly caused by a lesion, interference or abnormality originated in the immature brain [START_REF] Coq | From cerebral palsy to developmental coordination disorder: development of preclinical rat models corresponding to recent epidemiological changes[END_REF][START_REF] Rosenbaum | A report: the definition and classification of cerebral palsy April 2006[END_REF]. DCD refers to a developmental disorder of motor function with reduced abilities to produce consistent movements, poor motor coordination and somesthetic acuity, and broad impairments in sensorimotor representations and perception, reflected by disrupted central networks in the absence of apparent brain damage. DCD is usually associated with hyperactivity, and other behavioral and cognitive comorbidities [START_REF] Biotteau | Developmental coordination disorder[END_REF][START_REF] Coq | From cerebral palsy to developmental coordination disorder: development of preclinical rat models corresponding to recent epidemiological changes[END_REF]. Children suffering from either CP or DCD have in common reduced physical activity and interactions with their environment, and thus experience atypical sensorimotor development and maturation [START_REF] Vaivre-Douret | Developmental coordination disorder, an umbrella term for motor impairments in children: nature and co-morbid disorders[END_REF]. Early hypoactivity usually leads to muscle atrophy, altered force production and endurance, spasticity, bone impairment, less complex and fluent movements [START_REF] Jeffries | Description of primary and secondary impairments in young children with cerebral palsy[END_REF][START_REF] Li | Motor coordination and health-related physical fitness of children with developmental coordination disorder: a three-year follow-up study[END_REF], and consequently to atypical motor development [START_REF] Biotteau | Developmental coordination disorder[END_REF][START_REF] Vaivre-Douret | Developmental coordination disorders: state of art[END_REF].

It is well established that physical activity and interactions with the environment are required at the earliest developmental stages for typical and harmonious maturation of the organization and functions of the central nervous system (CNS) [START_REF] Luhmann | Spontaneous neuronal activity in developing neocortical networks: from single cells to large-scale interactions[END_REF]. [START_REF] Khazipov | Early patterns of electrical activity in the developing cortex[END_REF][START_REF] Zuk | Fetal and infant spontaneous general movements as predictors of developmental disabilities[END_REF]. Thus, prolonged hypoactivity might also induce secondary complications that occur over time as a result of primary impairment. In fact, many children who experience atypical sensorimotor activity will retain long-term motor difficulties during adulthood [START_REF] Cousins | Developmental coordination impairments in adulthood[END_REF]. However, it is difficult to determine whether CNS abnormalities are the cause or the result of the motor disturbances, due to the reciprocal interplay between CNS and musculoskeletal system [START_REF] Canu | Interplay between hypoactivity, muscle properties and motor command: how to escape the vicious deconditioning circle?[END_REF]. Thus, we hypothesized that limited and abnormal patterns of somatosensory inputs during development may lead to anatomical and functional disorganization of the sensorimotor circuitry in adults through maladaptive brain plasticity. In return, such disorganization may alter somatosensory and bodily perceptions, motor outputs and muscle structure and physiology (Delcour et al., 2018a;Delcour et al., 2018b).

Based on atypical movements and disrupted sensorimotor development, we developed a rat model of functional brain disorganization induced by early movement deprivation leading to sensorimotor restriction (SMR) during development [START_REF] Coq | Impact of neonatal asphyxia and hind limb immobilization on musculoskeletal tissues and S1 map organization: implications for cerebral palsy[END_REF][START_REF] Strata | Effects of sensorimotor restriction and anoxia on gait and motor cortex organization: implications for a rodent model of cerebral palsy[END_REF]. Briefly, hind limb immobilization from birth to postnatal day 28 (PND28) resulted in adult rats to digitigrade locomotion (i.e., "toe walking" or pes equinus observed in children with CP) related to ankle-knee overextension, degraded musculoskeletal histopathologies (e.g., gastrocnemius atrophy, joint degeneration and tibial bone shortening), and hyperexcitability in the lumbar spinal cord. The postnatal SMR also led to functional disorganization of the hind limb representations in the primary somatosensory and motor cortices, altered cortical response properties and cortical hyperexcitability, but without apparent brain damage, all signs of maladaptive cortical plasticity related to early atypical sensorimotor experiences [START_REF] Coq | From cerebral palsy to developmental coordination disorder: development of preclinical rat models corresponding to recent epidemiological changes[END_REF]Delcour et al., 2018a;Delcour et al., 2018b).

In the present paper, our first objective was to characterize the enduring impact of SMR from birth to PND28 on one postural muscle, the soleus, and on sensorimotor performance in rats. After mating with a male rat, pregnant female rats were transferred into individual cages and were pseudo-randomly assigned to two groups: control (C) or sensorimotor restriction (SMR). At parturition (post-natal day 0, PND 0), litter size was adjusted to 10 pups per dam.

Secondly

Material and methods

Animals

Body weight of pups was assessed daily from PND0 to PND28, then weekly until PND60. To obviate any litter effects, animals were randomly chosen from different litters. We used a total of 37C and 33 SMR rats, obtained from respectively 10 and 9 litters.

Hind limb immobilization

The feet of the SMR pups were first gently bound together with medical tape. Their hind limbs were then immobilized in extended position and taped to a cast, made of hand-moldable epoxy putty sticks as previously described (Delcour et al., 2018a) (Fig. 1).

These casts were well tolerated by the pups and mothers, and allowed the pups to move at the hip, urinate, defecate and to receive maternal care.

After casting, pups were returned to their mother and littermates during the dark phase of day, corresponding to daily peaks of motor activity. During the day's light phase, the casts were removed so that pups could move freely for 8 h/day. After daily uncasting, the hind limb joints were passively moved through their full range of motion. The size of the casts was adapted to the growth of the rats from PND1 to PND28.

To minimize the possible impact of stress induced by the casting, hind limb immobilization and maternal separation, C rats received comparable handling.

Muscle analysis

Muscle evaluation (contractile properties, western blot and immunohistochemistry) was performed on a set of 38 rats, at PND 28 (10C and 8 SMR) and PND60 (10C and 10 SMR) (Fig. 1).

In situ isometric contractile properties of the soleus muscle and muscle removal

At PND28 or PND60, the rats were deeply anesthetized with intraperitoneal injections of ketamine (50 mg⋅kg􀀀 1) and dexmedetomidine (Domitor, 0.25 mg⋅kg􀀀 1), prolonged if necessary by supplementary doses. The dissection protocol was previously described [START_REF] Picquet | Compared effects of hindlimb unloading versus terrestrial deafferentation on muscular properties of the rat soleus[END_REF]. Briefly, all the muscles of the right hind limb were denervated, except the soleus muscle. Then, the limb was fixed in isometric

Western blot

Muscle contractile phenotype was determined through analysis of MHC composition. Myofibrillar proteins were extracted from muscle powder in a buffer containing 6.3 mM EDTA (pH 7) and anti-proteases (Complete EDTA-free, Roche Diagnostic), and centrifuged at 13,000 rpm for 10 min at 4 •C, as previously described [START_REF] Toursel | Alterations in contractile properties and expression of myofibrillar proteins in wobbler mouse muscles[END_REF], and then with a second solution containing 50 mM KCl.

The myofibrillar proteins were resuspended in 500 μL of milliQ water, and their concentration was determined with a protein assay kit (Dc Protein Assay, Bio-Rad). The proteins were then precipitated for 2 h with acetone (8 vol (acetone)/vol (sample)), followed by centrifugation for 1 h at 13,000 rpm. The pellet was dissolved in Laemmli solution and was stored at

Histochemistry

The fiber cross-sectional area (CSA) was examined in 5 rats per group. Immunofluorescent staining was carried out on muscle frozen sections (10 μm). In summary, sections were incubated for 60 min at 37 CSA smaller than 125 μm2 and larger than 3500 μm2 for PND28 rats (350 and 7000 μm2 for PND60 animals respectively) were excluded from this analysis. These values correspond to the smallest and largest fibers found by manual observation.

Rate dependent depression

The Hoffman reflex (H-reflex) was measured in a set of PND28 rats1 from both groups (7C and 5 SMR), as already described (Delcour et al., 2018a). Briefly, rats were maintained under deep anesthesia, induced first with isoflurane and then with ketamine (100 mg⋅kg􀀀 1 i.p. induction) and maintained with supplemental doses of ketamine

Behavioral assessment

Behavioral assessment has been performed in a set of 20 rats (10C and 10 SMR). The same rats were tested once a week from PND35 to PND56 (Fig. 1). The training session was reduced to avoid lack of interest.

The total duration to perform the tests (rotarod, grip test, paw withdrawal and footprint) was estimated about 1 h par session. Tests were adapted from [START_REF] Brooks | Tests to assess motor phenotype in mice: a user's guide[END_REF], [START_REF] Maurissen | Factors affecting grip strength testing[END_REF] and [START_REF] Martinov | Measuring changes in tactile sensitivity in the hind paw of mice using an electronic von Frey apparatus[END_REF].

Rotarod task

The motor coordination of rodents was evaluated by using the rotarod test (Bioseb). Training began when rats were 28-day old. In the training trials, the rat was placed on the rod (7 cm diameter), which was rotating at a constant speed of 15 rpm. Habituation was repeated every day for 3 days before the testing sessions. Testing was conducted once per week from PND28 to PND56. The rod was accelerated from 5 to 40 rpm over 180 s and then was left at constant speed until rats fall down.

The latency to fall was recorded.

Grip test (Bioseb, France)

Rats were habituated to the apparatus for 3 days. The The final value was obtained by averaging the 5 measurements than deviate the least from the median [START_REF] Martinov | Measuring changes in tactile sensitivity in the hind paw of mice using an electronic von Frey apparatus[END_REF].

Footprint analysis

The walking characteristics were analyzed with the

Results

By PND28, no difference in morphologic and forcerelated parameters was noticed between males and females. Thus, both sexes were pooled. By contrast, at PND60, these parameters are known to be highly dependent on sex [START_REF] Drzymala-Celichowska | The content of myosin heavy chains in hindlimb muscles of female and male rats[END_REF][START_REF] Drzymała ; Celichowska | Slow motor units in female rat soleus are slower and weaker than their male counterparts[END_REF]. As a consequence, whenever possible, groups were evaluated separately by using a two-way ANOVA with sex and group as between factors.

Morphological parameters

At birth, the body weight (BW) was comparable in the C and SMR groups (respectively 6.59 ± 0.06 and 6.58 ± 0.20 g). At PND28, SMR rats exhibited growth retardation. Their BW was lower (􀀀 23%, P < 0.001) than that of C rats (Table 1). The difference between groups became significant from PND22 (Fig. 2). The BW recovered by PND60, since no difference was measured between the two groups. However, a great difference was observed between male and female rats in C (P < 0.01) as in SMR group (P < 0.01) at PND60.

A severe atrophy of the soleus muscle was noticed in the SMR group, whatever the age and sex (Table 1). This atrophy was highlighted by a decrease in muscle wet weight (MWW; PND28: -39%, P < 0.001; PND60: 􀀀 38% males, 􀀀 44% female, P < 0.001), which cannot be explained by the growth retardation, since it persisted when MWW was normalized to BW (PND28: -24%, P < 0.001; PND60: 􀀀 38% males, 􀀀 41% female, P < 0.001).

The fiber CSA was studied in a total of 20 muscles at PND28 (5C and 5 SMR) and PND60 (5C and 5 SMR). No difference was detected between males and females (P = 0.68). The mean CSA was decreased in SMR rats with respect to C ones (PND28: -29%, P < 0.01) but recovered partially at PND60 (􀀀 14%, P = 0.09). The χ2 analyses showed that the CSA was lower in SMR animals compared to C ones at PND28 (Fig. 3). At PND60, the profile was quite similar in both groups except that higher values (>3000 μm2) were more represented in the SMR group.

Contractile and kinetic parameters

Twitch tension (Pt) was decreased in SMR at PND28 (􀀀 39%, p < 0.01) compared to controls (Table 2). However, when Kinetic parameters varied from PND28 to PND60, but no change was noticed between C and SMR rats (Table 3). At PND28, the P20/P0 ratio was low in C rats and reached the value of a fast muscle. At PND60, this parameter evolved towards a higher value, characteristic of a slow muscle. Values were similar for the SMR group. In the same way, TTP and HRT increased at PND60 with respect to PND28, but data were comparable in the C and SMR groups.

To sum up, changes in contractile and kinetic properties were moderate or absent after SMR, and can be mainly explained by muscle atrophy.

Muscle myosin heavy-chain composition

The MHC expression of the whole soleus muscle was PND28 and PND60 respectively. The CSA of SMR fibers was lower at PND28 (χ2 = 1751; P < 0.0001). At PND60, large fibers (>3000 μm2) were more represented (χ2 = 410.2; P < 0.0001).

(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

H-reflex and rate-dependent depression

The spinal H-reflex was used to determine whether SMR induced hyperreflexia in the lumbar spinal cord and spasticity within the hind limb. Increasing stimulation frequency was performed to determine the rate-dependent depression, i.e. the decrease in reflex magnitude relative to stimulation rate. M and H waves were expressed with respect to the value obtained at a baseline frequency of 0.2 Hz (Fig. 5).

The statistical analysis did not reveal significant difference between groups for M wave. By contrast, for H-reflex wave, a group effect [F (1,45) = 47.15, P < 0.001], a frequency effect [F(4,45) = 7.02, P = 0.001] and a group x frequency interaction [F(4,45) = 3.18, P = 0.05] were observed. In C rats, the H-reflex was depressed when the stimulation frequency was increased to reach 46% of baseline value at 10 Hz (P < 0.001). In the SMR group, the depression was lower than in the C group: at 10 Hz, the H-wave amplitude was 91% of the value measured at 0.2 Hz and was not different from baseline, suggestive of the presence of hyperreflexia and spasticity in the gastrocnemius after SMR.

Behavioral assessment

Qualitative observation revealed that SMR rats exhibited irregular steps, difficult limb coordination and knee and ankle hyperextension, corresponding to digitigrade locomotion, confirmed by locomotor kinematics (Delcour et al., 2018a).

Muscle strength evaluation with the grip test (Fig. 6) revealed that the force was increased from one week to the other The behavioral response to mechanical stimulation of the hind paw was evaluated by using electronic Von Frey. This test is frequently used to test allodynia and tactile sensitivity. However, when pushing on the hind limb, C as well as SMR rats sometimes seemed to be indifferent to the stimulation as they did not remove their paw, but the tip pushed on the paw so much that the paw lifted off the ground. In addition, paw withdrawal occurred within relatively large delay (370 ± 17 ms in C rats), which suggests that the tactile stimulus was nonnoxious [START_REF] Blivis | Identification of a novel spinal nociceptive-motor gate control for Aδ pain stimuli in rats[END_REF]. The threshold required to evoke paw withdrawal increased with time [+32% from PND35 to PND56 in C rats; +95% in SMR rats; time effect: F(3,54) = 6.58; P < 0.001]. SMR rats were more sensitive than C rats, as the threshold was lower [􀀀 37% at PND35

and 􀀀 8% at PND56; group effect: F(1,18) = 4.74; P < 0.05] (Fig. 8A).

However, when paw withdrawal threshold was plotted against body weight (Fig. 8B), it seems that until a body weight of 180 g, the threshold was proportional to the body weight and thus, the lower threshold could be explained by lower body weight due to growth delay.

Analysis of the locomotor kinematics and patterns recorded using the Catwalk system (Fig. 9) reveals an increase (+20%) in 9B). In C rats, the duty cycle increased from 57% at PND35 to 66% at PND56. Moreover, this parameter was decreased in SMR rats (from 􀀀 17% at PND35 to 􀀀 26% at PND49). Maximum contact (Fig. 9D), which corresponds to the point at which the braking phase turns into propulsion phase during stance, occurred later after the onset of stance phase: the value was ~27% in C rats; whereas, it increased to ~43% in the SMR group.

Concerning the spatial parameter, stride length (Fig. 9C) was near 13 cm in C rat and a small decrease was reported in the SMR group. The footfall pattern was categorized into 3 classes. By PND35, the 'alternate' and 'cruciate' patterns predominated in controls (59% and 39% respectively). The 'rotate' pattern was almost absent (<1%). This repartition was very stable over time. By contrast, the footfall pattern was different in SMR rats, with a predominance of 'alternate' pattern (84%) at the expense of 'cruciate' one (11%). 'Rotate' pattern represented 3%. Female 16.1 ± 0.9 10.0 ± 2.3 116.9 ± 5.5 65.9 ± 13.6 * 0.15 ± 0.01 0.18 ± 0.05 1.12 ± 0.09 1.15 ± 0.28 13.7 ± 0.6 15.2 ± 1.5 PND28: 10C and 8 SMR. PND60: 10C (5 males and 5 females) and 10 SMR (5 males and 5 females).

The difference in

Pt: twitch tension, P0: tetanic tension at 100 Hz, MWW: muscle wet weight. *, ** and *** different from controls at the same age and the same sex (for PND60 rats) (P < 0.05, 0.01 and 0.001, respectively). PND28: unpaired t-test or Mann-Whitney; PND60: two-way ANOVA with Tukey's post-hoc test. Values are mean ± SEM. footfall patterns between C and SMR rats was significant.

Finally, the spontaneous activity was assessed by giving the rat free access to activity wheels (Fig. 10). At PND35, the active [START_REF] Huckstorf | Effects of hindlimb unloading on neuromuscular development of neonatal rats[END_REF][START_REF] Kawano | Role(s) of gravitational loading during developing period on the growth of rat soleus muscle fibers[END_REF][START_REF] Picquet | Differential effects of a six-day immobilization on newborn rat soleus muscles at two developmental stages[END_REF][START_REF] Serradj | Early postnatal motor experience shapes the motor properties of C57BL/6J adult mice[END_REF], bone structure [START_REF] Miranda | A pediatric animal model to evaluate the effects of disuse on musculoskeletal growth and development[END_REF] and changes in the locomotor pattern [START_REF] Walton | Identification of a critical period for motor development in neonatal rats[END_REF][START_REF] Westerga | Development of locomotion in the rat: the significance of early movements[END_REF]. In these studies, rodents were immobilized by casting or hind limb unloading from a few days to 2 months, at different stages of development, but the severity and duration of impairment was related to the age at which the animals were immobilized and the duration of immobilization [START_REF] Walton | Identification of a critical period for motor development in neonatal rats[END_REF]. In addition, muscle properties and motor function were rarely studied together, and whether they can recover is unclear. Interestingly, one study performed in mice suggests that motor performance in adults (9 months) is dependent on motor activity during a defined period of motor development, i.e. PND10 to PND30 [START_REF] Serradj | Early postnatal motor experience shapes the motor properties of C57BL/6J adult mice[END_REF].

SMR induces enduring muscle atrophy and force loss

The decrease in BW occurred only at P22, i.e. at the beginning of weaning. This decrease is likely due to difficulties to retrieve food rather than difficulties to access the mother's nipples and/or to a stress induced by manipulation. Indeed, maternal separation during the early life is believed to be stressful [START_REF] Molet | Naturalistic rodent models of chronic early-life stress[END_REF]. However, the duration of separation did not exceed 15 min per day, and C rats were also manipulated by the experimenter and separated from the mother.

The BW recovered at PND60. Chronic immobilization of hind limbs from birth to PND28 altered the muscle development. A severe muscle atrophy was observed at PND28, which persisted at PND60 in spite of a onemonth period of free movements recovery. When immobilization was endured during adulthood, a decrease in MWW was also observed, but relative value (soleus mass to body mass ratio) returned to normal within a few days [START_REF] Lawler | Biphasic stress response in the soleus during reloading after hind limb unloading[END_REF][START_REF] Widrick | Detrimental effects of reloading recovery on force, shortening velocity, and power of soleus muscles from hindlimb-unloaded rats[END_REF]. Thus, it seems that a critical period exists during development, in which muscle activity is required for typical muscle development.

It is classically assumed that atrophy can be explained by a lower CSA. The CSA of SMR rats was decreased at PND28 compared to C rats.

The reduced CSA is classically reported after disuse, in adults as in young rodents. For instance, hind limb unloading from PND8 to PND17 resulted in a decrease in CSA [START_REF] Huckstorf | Effects of hindlimb unloading on neuromuscular development of neonatal rats[END_REF], but the persistence of this effect after reloading has not been explored. [START_REF] Kawano | Essential role of satellite cells in the growth of rat soleus muscle fibers[END_REF] showed that hind limb unloading from PND4 to PND90, i.e. until adulthood, induced a 69% decrease in soleus fiber CSA, that was not completely reversed after a 3-month period of recovery.

In the present work, the decrease in CSA was abolished, and large fibers were even more frequently encountered at PND60. Thus, CSA recovered after one month of normal sensorimotor activity. [START_REF] Serradj | Early postnatal motor experience shapes the motor properties of C57BL/6J adult mice[END_REF] has also provided evidence for recovery in mice submitted to unloading from birth to P21. On the other hand, the CSA for triceps surae myofibers was still reduced in adult SMR rats compared with controls whereas quadriceps and hamstrings CSA were unchanged [START_REF] Coq | Impact of neonatal asphyxia and hind limb immobilization on musculoskeletal tissues and S1 map organization: implications for cerebral palsy[END_REF]Delcour et al., 2018a;[START_REF] Marcuzzo | Beneficial effects of treadmill training in a cerebral palsy-like rodent model: walking pattern and soleus quantitative histology[END_REF]. This discrepancy suggests that recovery from SMR may depend on the muscle phenotype. In fact, slow postural muscles such as soleus muscle being more likely to return to a normal phenotype when postural function is resumed. In the present study, the two parameters, i.e. MWW and CSA, seem to be disconnected since the decrease in MWW was far higher than the decrease in CSA, in particular at PND60 (~ 􀀀 40% vs 􀀀 14% respectively), and CSA recovered but not MWW. The discrepancy between atrophy and CSA could be the result of a decrease in the number of myofibers. This hypothesis is sustained by many studies showing that short-term immobilization of muscles leads to apoptosis (e.g. [START_REF] Hao | beta-Hydroxy-beta-methylbutyrate reduces myonuclear apoptosis during recovery from hind limb suspension-induced muscle fiber atrophy in aged rats[END_REF][START_REF] Slimani | The worsening of tibialis anterior muscle atrophy during recovery post-immobilization correlates with enhanced connective tissue area, proteolysis, and apoptosis[END_REF][START_REF] Suh | Apoptotic changes in a full-lengthened immobilization model of rat soleus muscle[END_REF]. This effect is most noticeable at PND60, likely to a fiber muscle injury induced by unusual increase in mechanical stress after immobilization [START_REF] Andrianjafiniony | Oxidative stress, apoptosis, and proteolysis in skeletal muscle repair after unloading[END_REF][START_REF] Flück | Reloading of atrophied rat soleus muscle induces tenascin-C expression around damaged muscle fibers[END_REF]. An increase in Pax7 immunostaining has been observed in the soleus agonist (gastrocnemius muscle) of adult rats submitted to SMR during development, confirming the existence of lesional tissue [START_REF] Coq | Impact of neonatal asphyxia and hind limb immobilization on musculoskeletal tissues and S1 map organization: implications for cerebral palsy[END_REF]Delcour et al., 2018a). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

The discrepancy between atrophy and CSA could also be explained by a decrease in the non-muscle fiber component, namely the connective tissue. However, this hypothesis is in contradiction with studies showing that a thickening of perimysium is observed in adult rats submitted to immobilization (Delcour et al., 2018a;[START_REF] J¨arvinen | Organization and distribution of intramuscular connective tissue in normal and immobilized skeletal muscles. An immunohistochemical, polarization and scanning electron microscopic study[END_REF][START_REF] Slimani | The worsening of tibialis anterior muscle atrophy during recovery post-immobilization correlates with enhanced connective tissue area, proteolysis, and apoptosis[END_REF] as well as in CP patients [START_REF] De Bruin | Intramuscular connective tissue differences in spastic and control muscle: a mechanical and histological study[END_REF]. We previously showed that SMR leads to increased connective tissue within the triceps surae, quadriceps and hamstrings. Collagen type I showed a small increase, but connective tissue growth factor (CTGF) increased significantly after SMR [START_REF] Coq | Impact of neonatal asphyxia and hind limb immobilization on musculoskeletal tissues and S1 map organization: implications for cerebral palsy[END_REF][START_REF] Marcuzzo | Beneficial effects of treadmill training in a cerebral palsy-like rodent model: walking pattern and soleus quantitative histology[END_REF].

We have shown that SMR profoundly altered tetanic tension (P0) at PND28 as well as at PND60, especially in female rats. [START_REF] Adams | Time course of myosin heavy chain transitions in neonatal rats: importance of innervation and thyroid state[END_REF][START_REF] Kawano | Role(s) of gravitational loading during developing period on the growth of rat soleus muscle fibers[END_REF]. [START_REF] De-Doncker | Effects of cutaneous receptor stimulation on muscular atrophy developed in hindlimb unloading condition[END_REF][START_REF] Falempin | Influence of brief daily tendon vibration on rat soleus muscle in non-weight-bearing situation[END_REF].

On the other hand, changes in the motor drive, either suppression by tetrodotoxin [START_REF] Michel | Regulation of myosin heavy chain expression in adult rat hindlimb muscles during short-term paralysis: comparison of denervation and tetrodotoxin-induced neural inactivation[END_REF] or activation by electrostimulation with a tonic pattern [START_REF] Dupont | Electrostimulation during hindlimb unloading modulates PI3K-AKT downstream targets without preventing soleus atrophy and restores slow phenotype through ERK[END_REF][START_REF] Leterme | Compensatory effects of chronic electrostimulation on unweighted rat soleus muscle[END_REF][START_REF] Pette | The contribution of neuromuscular stimulation in elucidating muscle plasticity revisited[END_REF] prevent phenotypic changes related to unloading but have no impact on muscle mass. Taken together, it seems that in our developmental model of SMR, the paucity of movements (and subsequently of proprioceptive feedback), in both quality and quantity, are the key factor for the maturation of muscle properties, the role of the decrease in motor drive being less prominent.

The sensorimotor performance is altered in SMR rats

The first postnatal weeks are crucial for the integration of sensory input and maturation of spinal reflexes [START_REF] Brumley | Developmental plasticity of coordinated action patterns in the perinatal rat[END_REF]. [START_REF] Walton | Identification of a critical period for motor development in neonatal rats[END_REF] have reported that the period extending from postnatal days 8-13 is a critical period for motor performance, in which animals appear the most sensitive to hind limb unloading.

Moreover, when pups were unloaded from PND 13-31, abnormal walking patterns persisted into adulthood [START_REF] Walton | Identification of a critical period for motor development in neonatal rats[END_REF]. development [START_REF] Brumley | Developmental plasticity of coordinated action patterns in the perinatal rat[END_REF]. We also examined the withdrawal reflex and showed that its threshold was lowered, but this result may be due to reduced and altered somatosensory feedback, (Delcour et al., 2018a;Delcour et al., 2018b). At the spinal level, our electrophysiological approach has shown that the depression of the Hoffmann reflex, that is classically observed in C rats when stimulation rate increases, disappeared in the gastrocnemius muscle. This hyperreflexia is a sign of spasticity and is indicative of hyperexcitability within the spinal cord network. Such a phenomenon had already been demonstrated in the same model of SMR on the flexor digitorum brevis muscle (Delcour et al., 2018a). In human, spasticity is usually caused by lesions of the CNS, such as spinal cord injury or stroke.

In children, it is a common feature of CP [START_REF] Jeffries | Description of primary and secondary impairments in young children with cerebral palsy[END_REF]. In the present study, spasticity can occur without brain damage. Our results once more emphasize the role of sensorimotor experience in the maturation of the spinal circuitry.

Conclusion

Our results showed that SMR from birth to PND28 delayed muscle maturation and altered soleus muscle properties. SMR led also to hyperexcitability of the spinal cord. Our data support the idea that proprioceptive feedback or reafference is at least as important as the quantity of motor activity in promoting typical

  and evoked movements during maturation, early somatosensory feedback drives electrical activity patterning from the spinal cord to the cortex. This feedback guides

  force development, in vivo or ex vivo, was poorly affected but performance in motor tests which require good limb coordination showed enduring perturbation.

  minimize the number of animals and their suffering. Female and male OFA-Sprague-Dawley rats were purchased from Charles River Laboratories (L'Arbresle, France) and were housed in standard plastic cages in controlled conditions of humidity, temperature and light (22 •C, 51% humidity, 12:12-h light-dark cycle). They had ad libitum access to water and food. Rats underwent at least 1 week of acclimatization after reception into the animal facility.

5 ×

 5 conditions and was immersed in a bath of paraffin oil thermostatically controlled (37 •C). The limb was stabilized by using a combination of bars and pins, and the soleus muscle was maintained in a horizontal position. Afterward, the soleus muscle was isolated from surrounding tissues, and its distal tendon was connected to a force transducer (Grass FT 10; Grass Instruments, West Warwick, RI, USA). The muscle length was adjusted to produce a maximal twitch peak tension (Pt). Contractions were induced by stimulating the sciatic nerve (0.2-ms pulses) through bipolar platinum electrodes at (intensity ≥1.minimum voltage required to obtain the maximal twitch response). The following parameters were recorded: Pt, time-topeak (TTP), halfrelaxation time (HRT); peak tetanic tension obtained at 100 Hz (P0). The ratio of subtetanic tension at 20 Hz relative to P0 (P20/P0) was used as an indicator of muscle type; a low value (0.20-0.30) is characteristic of a fast muscle whereas a high value (0.70-0.80) indicates a slow muscle. The fatigue index (FI) was calculated as the percentage of the initial tension divided by the force at the end of the fatigue protocol in a series of 120 consecutive contractions (330 ms duration, 40 Hz, one train per second). At the end of the recording session, the muscle was removed, weighted and frozen in isopentane precooled to its freezing point by liquid nitrogen and stored at 􀀀 80 •C until histochemical and electrophoretic analyses. The rat then received a lethal dose of sodium pentobarbital (100 mg⋅kg􀀀 1).

  I) and fast (MHC IIA, IID/X, and IIB) isoforms were identified according to their migration and previous reports of the laboratory[START_REF] Dupont | Electrostimulation during hindlimb unloading modulates PI3K-AKT downstream targets without preventing soleus atrophy and restores slow phenotype through ERK[END_REF]. The neonatal MHC isoform was identified using immunoblot analysis with a monoclonal antibody (sc-53,097, Santa-Cruz Biotechnology Inc). After separation, proteins were transferred to 0.45-μm nitrocellulose membranes (Hybond, GE Healthcare) using transfer buffer (20 mM Tris base; 150 mM glycine; 20% methanol), added with 0.025% SDS for the transfer of highmolecular weight proteins. Protein load and quality of transfer were verified by Ponceau staining. The blots were then washed in TBST (15 mM Tris/HCl, pH 7.6; 140 mM NaCl; 0.05% Tween-20) and blocked in 5% nonfat dry milk in TBST. Membranes were then blotted with primary antibody against neonatal MHC overnight at room temperature (RT) at 4 •C. Membranes were washed 3 × 10 min in TBST, and then incubated with HRP labelled secondary antibodies in blocking solution for 2 h at RT followed by 5 × 10 min washes in TBST. Chemiluminescence detection was carried out using ECL Clarity (Biorad), and images capture were done with ChemiDoc MP (Biorad). For actin expression, proteins were separated on Criterion 12% Stain-Free precast polyacrylamide gels (Biorad); an internal standard was loaded on each gel. Stain-

•Fig. 1 .

 1 Fig. 1. Experimental timeline showing the design and order of tests and sample sizes. Three sets of animals were used. For the set , animals were submitted to

(

  20 mg⋅kg􀀀 1 i.p.). A transcutaneous pair of stimulating needle wires was inserted around the peroneal branch and a pair of stainless steel recording electrodes was inserted into the gastrocnemius muscle. A reference electrode was placed into the skin. First, we stimulated the peroneal branch for 0.2 ms at 0.2 Hz with increasing current intensities until the maximum M wave stabilized, and determined the intensity required for a maximal H response. The peroneal branch was stimulated with trains of 20 stimulations at 0.2, 0.5, 1, 2, and 5 Hz with 2-min intervals between each train. For each frequency, the responses were averaged, while discarding responses to the first three stimulations required for the depression to occur. The M and H waves were rectified and the areas under the curves were measured. The H responses were expressed as percentages relative to the mean response at 0.2 Hz within the same series of measurements.

  animals were gently supported by the experimenter at armpit level and the hind paw brought into contact with the bar. When they grasped the metal bar, their body was gently moved backwards until they dropped the bar. The dynamometer measured the maximal isometric force before the animal released the bar. Each testing session consisted of 5 successive trials, which duration did not exceed 5 s. The force applied to the apparatus was checked on a computer screen. Trials where the force did not increase regularly, following a ramp force (force/time), were rejected. The results are expressed as the mean of the 5 values in grams (g), and normalized to the body weight (g/g). Because this test requires extensive handling of rats, all tests were performed by the same experimenter.2.5.3. Paw withdrawal threshold (electronic Von Frey hair unit, Ugo Basile, Italy)The animals were placed in 20 × 20-cm Plexiglas boxes equipped with a mesh floor. After a 10-min period of habituation, the withdrawal threshold was evaluated by applying a force ranging from 0 to 1000 g.Stimulus was delivered to the midplantar area of both left and right hind paws. The force was displayed on the computer screen.Only trials with regular linearly increasing force and clear paw withdrawal were taken into account. The measure was repeated until recording 6 successful trials, with an interval of at least 10 s between trials.

  Catwalk system (CatWalk XT, Noldus Information Technology, Netherlands). Rats were trained prior to recording for 3 days. Rats were allowed to freely run across an enclosed walkway illuminated by fluorescent light (width: 5 cm; length: 100 cm). Footprints were digitized with a high-speed colour camera (10 frames⋅s􀀀 1) connected to a computer. Five runs were collected per rat and per day. The right hind paw (RH) was taken as a reference. The following parameters were quantified: step cycle duration (time between two consecutive initial contacts of RH), duration of the stance and swing phases and duty cycle; stride length; body speed; max contact (time that paw makes maximum contact with the glass plate expressed as a percent of the stand phase for RH; footfall patterns (order in which the paws were placed on the walkway). Predefined paw sequences are cruciate (e.g. right forepaw -left forepaw -right hindpawleft hindpaw and so on), alternate (e.g. right forepawright hindpawleft forepaw -left hindpaw) and rotate (e.g. right forepawleft forepaw left hindpawright hindpaw). For each rat and each day, a value was determined, that corresponded to the mean of at least 10 steps. 2.5.5. Running wheel Spontaneous wheel-running activity was recorded for a 2-h session (during the light phase; session started between 10 AM and 12 AM) using activity wheels designed for rats (model BIO-ACTIVW-R, Bioseb). Rats were placed into polycarbonate cages (20 × 35 × 13 cm) with free access to stainless steel activity wheels (diameter 34 cm; lane width 7 cm). The wheel could be turned in both directions. Wheels were connected to a computer that automatically recorded the spontaneous activity by means of ACTIVW-SOFT software (Bioseb). The average and maximum speed and total active time were measured for each rat. The test was conducted at PND35, PND42, PND49 and PND56. 2.6. Data analysis All data are expressed as mean ± SEM. Statistical analysis was performed by using Prism software (GraphPad, USA). Data normality was determined with the Shapiro-Wilk test. Afterward, we applied either parametric or non-parametric tests. Pairs were tested with t-tests or Mann-Whitney. For more than two groups, one-way or two-way ANOVA with Tukey or Sidak post-hoc test was used. The type of statistical test is indicated in each figure or table legend. Values of P < 0.05 were considered statistically significant.

  expressed relative to muscle weight, values were comparable. At PND60, a group effect was still present for Pt [F(1,16) = 9.86; P < 0.01] (males: 􀀀 23%, females: 􀀀 38%) whereas Pt/MWW was unchanged [F(1,16) = 1.61; P = 0.22, ns]. Maximal tetanic tension (P0) was dramatically reduced in SMR rats at PND28 (􀀀 49%, P < 0.001). A group effect was also observed at PND60 [F(1,16) = 11.72; P < 0.01] (males: 􀀀 21%, females: 􀀀 44%). This decrease was due to the muscle atrophy, since relative to muscle weight, the value was unchanged [F(1,16) = 0.96; P = 0.34, n.s.]. As a result, the twitch-to-tetanic ratio (Pt/P0) was increased at PND28 (+20%, p < 0.05) and recovered at PND60 [F(1,16) = 0.24; P = 0.63, ns].

  determined by Western blots and results are presented in Fig. 4. As expected, the MHC pattern of expression in C rats showed a predominance of type I (62%) and IIA (36%) MHC, with only traces of fast IIB isoform for two muscles (0.1%). A small amount of neonatal form was still present (1.7%). In SMR rats at PND28, the proportion of MHC I was lower (49%; P < 0.05) whereas the proportion of fast isoforms was increased almost significantly (MHC IIA + IIB: 48% P = 0.053) compared to controls. Neonatal isoform was still present in one third of SMR subjects at a low level (3.2%). At PND60, the muscle of controls evolved towards a slow-twitch type, with an increase in slow isoform (82%, P < 0.001), a decrease in MHC IIA isoform (18%, P < 0.001) and a total disappearance of MHC IIB and neonatal forms (ns). In SMR rats, no difference was reported with respect to C rats at PND60. The actin expression was also determined. The total expression was increased at PND60 with respect to PND28 [+38%, F(1,24) = 150.5; P < 0.0001], but no difference was noticed between C and SMR rats [F (1,24) = 2.1; P = 0.16, n.s.] (Fig. 4c). The identification of muscle fiber type was also performed by immunohistochemistry. The quantitative data revealed that the soleus muscle of young C rats (PND28) contained 64% of type I fibers, 33% of fast type IIA fibers, and 3% of fibers co-expressing the two MHC isoforms, MHC I/MHC IIA. The amount of fibers expressing MHC I significantly increased at PND60 [Time effect: F(1,13) = 72.98; P < 0.0001] to reach 90%, whereas IIA fibers were reduced at 8%. Values were very close in SMR rats (PND28: 68% of MHC I and 8% of MHC IIA; PND60: 91% and 8% respectively) [Group effect: F(1,13) = 0.19; P = 0.67, n.s.].

  BW (g) MWW (mg) MWW/BW (mg/g) and 8 SMR. PND60: 10C (5 males and 5 females) and 10 SMR (5 males and 5 females). BW: body weight, MWW: muscle wet weight. Values were compared with Mann-Whitney test at PND28 and two-way ANOVA with sex and group as between factors at PND60 (Tukey's multiple comparison test).*** Different from C group at the same age (PND28) or at the same age and sex (PND60) (P < 0.001). Values are mean ± SEM.

Fig. 2 .

 2 Fig. 2. Increases of body weight over time according to group and sex. Values are mean ± SEM. Statistical significance was determined by multiple t-tests with correction for multiple comparisons using the Holm-Sidak method. *: p < 0.001 with respect to C rats.

Fig. 3 .

 3 Fig. 3. Fiber cross-sectional area (CSA) for soleus muscle of control (grey) and SMR (red) rats, at PND28 and PND60. a. Microphotographs showing cross-sections of the soleus muscle after immunostaining of laminin (green) and DAPI (blue). Scale bar: 50 μm. b. Overlay histograms presenting frequency distribution of CSA for C and SMR rats. Bin width are 100 μm2 and 200 μm2 at

[

  time effect: F(3,54) = 5.60; P < 0.01]. In addition, values were always lower for SMR rats than for C rats [group effect: F(1,18) = 21.77; P < 0.001]. However, when normalized to body weight, the force decreased with time [F(3,54) = 14.43; P 〈0,0001] and the group effect disappeared [F(1,18) = 2.63; P = 0.12]. It indicates that the force deficit in SMR rats was mainly due to their growth retardation rather than a deficit in muscle force production. Motor was evaluated by the rotarod test (Fig. 7). Rats were left on the rotating rod until they fall. SMR strongly affected motor coordination as shown by the decrease in the spent on the rotating bar. Fig. 7 presents the mean value for the different days. It clearly appears that the time-to-fall was reduced in SMR rats, compared to controls [group effect: F(1,18) = 22.13; P < 0.001]. In C rats, the duration varied between 15 s and 338 s whereas the maximum duration was only 79 s in SMR animals.

  swing duration in SMR rats but no change in stance duration. Step cycle duration varied with time [F(3,54) = 12.7; P < 0,001]: in C rats, values increased from 0.29 s (PND35) to 0.40 s (PND56). No group effect was detected [F(1,18) = 0.19; P = 0.67]. A time effect was also observed for the duty cycle (corresponding to stand duration expressed as a percentage of step cycle) [F(3,54) = 11.47; P < 0.001] (Fig.

  TTP (ms) HRT (ms) P20/P0 FI (%) C SMR C SMR C SMR C SMR PND28 49 ± 2 51 ± 3 49 ± 3 52 ± 7 46 ± 3 49 ± 2 89 ± 3 97 ± 4 PND60 Male 75 ± 4 64 ± 2 63 ± 3 56 ± 3 68 ± 2 63 ± 1 82 ± 2 88 ± 4 Female 69 ± 4 68 ± 6 61 ± 6 65 ± 7 61 ± 1 63 ± 5 81 ± 3 86 ± 4 PND28: 10C and 8 SMR. PND60: 10C (5 males and 5 females) and 10 SMR (5 males and 5 females). TTP: time-to-peak, HRT: half relaxation time, P0: tetanic tension at 100 Hz, P20: tetanic tension at 20 Hz, FI: fatigue index. PND28: unpaired t-test or Mann-Whitney; PND60: two-way ANOVA with Tukey's post-hoc test. Values are mean ± SEM.

Fig. 4 .Fig. 5 .

 45 Fig. 4. Electrophoretic determination of myosin heavy chain (MHC) isoforms in the soleus muscle of C and SMR rats at two ages (PND28 and PND60). (a) Representative western blot showing the MHC isoforms. (b) Distribution histogram of MHC isoforms. (c) representative Western blot and quantification of actin (normalized to the whole proteome, i.e., Stain Free signal). Data show a delay in muscle maturation with persistence of neonatal isoform at PND28. Control: white bars. SMR: red bars. Dots are individual data points. * P < 0.05, Mann-Whitney. Values are mean ± SEM. a.u. arbitrary unity, MW: molecular weight. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 5. Amplitude of H waves in C and SMR rats, at PND28. H waves are expressed with respect to the value obtained at baseline frequency of 0.2 Hz. The lower rate-dependent depression after SMR is indicative of hyperreflexia. Control: white bars. SMR: red bars. *, **, ***: P < 0.05, 0.01 and 0.001 with respect to C group. § § and § § §: p < 0.01 and 0.001 with respect to baseline value (i.e. 100%). Values were compared with two-way ANOVA, with group as between factor and frequency as within factor, Sidak's post-hoc test. Values are mean ± SEM. Circles are individual data points. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) despite the relevance of this topic, the consequences of disuse during the development of the neuromuscular system and motor performance have been poorly studied. In young rodents, hind limb unloading or restricted range of motion induces altered muscle physiology and phenotype

Fig. 6 .Fig. 7 .

 67 Fig. 6. Performance on the grip test in control and SMR rats. Rats grasped a metal bar, and their body was then gently moved backwards until they dropped the bar. For each animal and each recording day, the final value was obtained by averaging 5 measurements. (a) Histograms present the maximal isometric force. Rats of the SMR group developed less force than C ones [time effect: F(3,54) = 5.60; P < 0.01; group effect: F(1,18) = 21.77; P < 0.001]. (b) When the force was normalized to the body weight (g/g), the force decreased with time [F(3,54) = 14.43; P < 0.0001] and the group effect disappeared [F(1,18) = 2.63; P = 0.12]. Control: white bars. SMR: red bars. Values are mean ± SEM. Circles are individual data points. * P < 0.05; ** P < 0.01; *** P < 0.001, two-way ANOVA with repeated measures and Sidak post-hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 7. Motor coordination was affected in SMR rats. During performance on the accelerated rotarod test (5-40 rpm/3 min), SMR rats demonstrated an

Fig. 8 .Fig. 9 .

 89 Fig. 8. Paw withdrawal threshold was lower in young SMR rats. The tip of an electronic Von Frey was poked on the hind paw through the mesh and the force required to evoke a clear paw withdrawal were recorded. For each animal and each recording day, the final value was obtained by averaging the 5 measurements. (A) The threshold was lower in SMR rats for the early phase of recovery [time effect: F(3,54) = 6.58; P < 0.001; group effect: F(1,18) = 4.74; P < 0.05]. (B) In young rats until 180 g, paw withdrawal threshold was proportional to body weight. Thus, the lower threshold appeared mainly related to growth retardation. Control: white bars. SMR: red bars. Values are mean ± SEM. Circles are individual data points. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 9. SMR induced enduring changes in the locomotor pattern. Locomotor function was evaluated by using the Catwalk system. Swing duration was increased in SMR rats [F(1,18) = 16.16, P < 0.001] with no change in stance duration [􀀀 12%; F(1,18) = 1.19; P = 0.29]. As a consequence, the duty cycle [(stance duration / (stance + swing)) × 100] was lower [Group effect: F(1,18) = 14.69; P < 0.01]. Stride length was reduced in SMR rats [F(1,18) = 8.61; P < 0.01]. Maximum contact is the time that the hind paw makes contact with the floor, expressed as a % of stance phase duration [Max contact = (time of contact -time of stance onset) / stance duration) × 100]. Values were increased [F(1,18) = 15.1; P < 0.01], suggestive of increased braking phase while the propulsion phase was shorter. The footfall pattern was changed durably, towards a higher percentage of alternate pattern (χ2 = 26.47 at PND35, P < 0.001; χ2 = 19.95 at PND42, P < 0.001; χ2 = 8.30 at PND49, P < 0.05; χ2 = 13.42 at PND56, P < 0.01). Control: white bars. SMR: red bars. For each animal and each day, the value was an average for the step cycles. Values are mean ± SEM. Circles are individual data points. * P < 0.05; ** P < 0.01; *** P < 0.001, two-way ANOVA with repeated measures and Sidak post-hoc test).

  Proprioceptive information from muscle afferent fibers appears crucial in the regulation of contraction force. For instance, hind limb unloading in adult rats strongly reduces afferent input from hind limbs and maximal tetanic tension, while activation of Ia afferent fibers by tendon vibration significantly attenuates this decrease (Falempin and Fodili-In-Albon, 1995). The decline in P0 may reflect the muscle atrophy, a selective loss of contractile proteins, thus a decrease in the cross-bridge number, and/or a decrease in the force generated per crossbridge (McDonald and Fitts, 1995). The specific tension, i.e. P0 to MWW ratio, was unchanged at PND28 and PND60, suggesting that force loss was proportional to muscle atrophy, whereas no change was reported in actin content. At a more integrated level also, the force developed on the grip test was decreased but remained unchanged when normalized to body weight. 2. Kinetic parameters are not affected by SMR Muscle phenotype after SMR is characterized by a delay in maturation with persistence of small amount of neonatal isoform of MHC proteins at PND28 and a lower proportion of MHC I. This difference was weak and did not persist at PND60. It should be noted that in some SMR rats, small amounts of MHC IIB were detected while this isoform is usually absent in the control soleus muscle. Other authors have already shown that postnatal disuse resulted in altered muscle phenotype, characterized by a decrease in type I MHC and the persistence of small amounts of neonatal isoform

  [START_REF] Picquet | Differential effects of a six-day immobilization on newborn rat soleus muscles at two developmental stages[END_REF] has shown that immobilization of hind limbs from P6 to P12, i.e. before the appearance of monosynaptic innervation delays the postnatal maturation with the persistence of neonatal isoforms; whereas, when immobilization occurs between PND17 and PND23, i.e. under monosynaptic innervation, the muscle changed towards a faster phenotype. Thus, the maturation of muscle phenotype depends on neural factors. The moderate changes in kinetic parameters and muscle phenotype in our study are striking in view of the persistent changes in contractile properties. It has been shown that afferent input is a major determinant of muscle mass in adult rats whereas phenotype depends on motor command. Indeed, in adult rats submitted to hind limb unloading, activation of proprioceptive fibers by pressure applied on the plantar sole or by tendinous vibration can prevent at least partially muscle atrophy (De

Fig. 10 .

 10 Fig. 10. Spontaneous activity in running wheels increased in SMR rats. (A) The time spent in the running wheel was unchanged in C rats but increased over time in SMR rats [time effect: F(3,42) = 2.55; P = 0.07; group effect: F(1,14) = 4.09; P = 0.06]. (B) Maximum running speed was increased from PND35 to PND56 in SMR rats, but not in C rats [time effect: F(3,42) = 2.50;P = 0.07; group effect: F(1,14) = 5.05; P < 0.05]. Control: white bars. SMR: red bars. Values are mean ± SEM. Circles are individual data points. * P < 0.05; two-way ANOVA with repeated measures and Sidak post-hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

  spinal hyperexcitability or to supraspinal regulation of postural control, which is difficult to established. Our results also suggest that the growth delay, and thus the lower body weight, can contribute to the decrease in withdrawal threshold. Sensorimotor activity plays a crucial role in the formation and refinement of synapses and neural networks (Tiriac and Blumberg, 2016). The discrepancy between the moderate degradation in muscle properties and the striking impairments in motor outputs, particularly on the rotarod, suggests that a functional disorganization of neuronal circuits at the spinal and/or supraspinal level is involved in behavioral changes. In prior studies, we demonstrated that SMR has a strong impact on supraspinal structures, particularly the cerebral cortex where it degraded the topographical organization of the somatosensory hind limb maps, reduced cortical areas devoted to both somatosensory and motor hind limb representations and altered neuronal response properties, such as enlarged tactile receptive fields and reduced electrical thresholds to produce movements, and cortical hyperexcitability as well

  development of motor function. It is also difficult to determine whether muscle atrophy is the cause or the result of the altered sensorimotor experience. The impaired control and coordination of motor activity in the absence of neurological disorder is a characteristic of developmental coordination disorders (DCD) (Vaivre-Douret, 2014). A better knowledge of the interplay between hypoactivity, muscle properties and central motor commands may provide therapeutic perspectives for children with DCD or other related neurodevelopmental disorders.

Table 2

 2 Contractile parameters. ± 2.0 13.5 ± 0.3 116.8 ± 16.1 92.7 ± 2.9 0.14 ± 0.01 0.17 ± 0.01 0.91 ± 0.11 1.21 ± 0.09 15.2 ± 0.4 14.6 ± 0.6

	Pt (mN) P0 (mN) Pt/MWW (mN/mg) P0/MWW (mN/mg) Pt/P0
	C SMR C SMR C SMR C SMR C SMR
	PND28 8.1 ± 0.6 4.9 ± 0.6
	**
	63.6 ± 4.21 32.2 ± 4.77
	***
	0.22 ± 0.01 0.22 ± 0.02 1.71 ± 0.11 1.46 ± 0.19 12.7 ± 0.6 15.3 ± 0.6
	*
	PND60 Male 17.5

Table 3

 3 kinetic parameters.

Acknowledgements

Experiments were performed in the Eurasport facility of Lille University and ISM Marseille.

Funding

This study was funded by Minist`ere de l'Enseignement Sup´erieur et de la Recherche, la Fondation Motrice, Lille University.

Authors' contributions