Dominique Patureau
email: patureau@ensam.inra.fr

Mireille Laforie

Eric Lichtfouse

Giovanni Caria

Laurence Denaix

Jens Ejbye Schmidt

Fate of organic pollutants after sewage sludge spreading on agricultural soils: a 30-years field-scale recording 1

Keywords: biodegradation, bound residues, long term effect, sludge disposal, xenobiotic

Toxic organic compounds, such as the surfactants linear alkylbenzene sulfonates (LAS) and nonylphenol polyethoxylates (NPE), Polycyclic aromatic hydrocarbons (PAH) and residues derived from plastics (PAE-phthalates) end up in sewage sludge. In order to evaluate and quantify the potential environmental risks associated with the xenobiotic introduction into biological life cycle, the EU BIOWASTE project (QLK5-CT-2002-01138) devotes one task to the study of the fate of xenobiotic in a sandy soil after sludge spreading on a 30-year field-scale record experiment. Experimental maize crop fields from Bordeaux (France) have been amended with 100 tons per hectare each 2 years from 1974 to 1992. From 1992 to 2004, the fields were maintained and cropped with maize. This experiment shows that the concentration fluctuations in the sludge amended soil follow the same pattern of those in the sewage sludge showing that there is a real impact of the present xenobiotics in the sewage sludge on the concentration of the xenobiotics in the soil. Nonetheless, 12 years after the last addition of sewage sludge, the residual concentrations remain from 2 to 10 times higher than the content of the control soil, even though these levels are inferior to the Predicted Non Effect Concentration (PNEC). Only LAS level went back to the level in the control soil. However, only the LAS concentration is above the PNEC during all the experiment due to the very high level of LAS in the sludge (20 g/kg dry weight). These results show that even though this compound is much more degradable than NPE and PAE, it may have a long term effect in soil if high quantities are spread. To conclude, this study underlines the importance to fix maximum level for xenobiotic compounds for sewage sludge spreading on agricultural land, and also the central role of the sewage sludge processes in reducing the xenobiotic concentrations before spreading.

Introduction

The environmental burden of organic pollutants contained in sewage sludge, after spreading on soil, depends on numerous factors related to the soil itself, the sludge (nature and way of spreading) and the chemical considered. A review concerning priority organic pollutants has recently been published by the European Union (EU, 2001).

Generally, nonylphenol polyethoxylates (NPE) are degraded in the soil to nonylphenol (NP), which adsorbs to soil particles. Under aerobic conditions, half-lives of these compounds are short. Half-lives for NP have been determined to be between 4.5 and 37 days depending on the conditions (lab scale, lysimeter) and for tested soils (sand, clay) (Staples et al., 1999;Topp and Starratt, 2000;[START_REF] Jacobsen | Degradation and mobility of Linear Alkylbenzene sulfonate and nonylphenol in sludge-amended soil[END_REF]. For [START_REF] Hesselsoe | Degradation of 4-nonylphenol in homogeneous mixtures of soil and sewage sludge[END_REF], the half-life was estimated to 3-6 days using 14 C-NP with a residual 20-60% of radiolabelled compounds in the organic fraction. The authors also showed a correlation between oxygen diffusion and NP mineralization. Indeed, NP mineralization occurs in the soil depending on the oxygen availability and on the type of soil and sludge: it could be as fast as NP is supplied alone, but application of NP spiked sewage sludge delay the NP degradation and favour the adsorption and the formation of bound residues with organic matter. For example, addition of limed sludge increases NP adsorption and persistence in soils [START_REF] Kollmann | Effect of nonylphenol surfactants on fungi following the application of sewage sludge on agricultural soils[END_REF][START_REF] Dubroca | Biotransformation of nonyphenol surfactants in soils amended with contaminated sewage sludges[END_REF]. NPE and NP do not show significant downward transport to underground soil layers [START_REF] Jacobsen | Degradation and mobility of Linear Alkylbenzene sulfonate and nonylphenol in sludge-amended soil[END_REF], and transfer to groundwater, whereas some uptake by higher plants have been observed.

A similar behavior occurs concerning linear alkylbenzene sulfonates (LAS). A wide range of half-lives are reported in the literature from 1 to 87 days [START_REF] Jensen | Fate and effects of linear alkyl benzene sulphonates (LAS) in the terrestrial environnement[END_REF][START_REF] Scott | The biodegradation of surfactants in the environment[END_REF] depending on soil types, LAS formulation, incubation conditions and route of LAS exposure (direct LAS application or LAS-spiked sewage sludge). [START_REF] Gejlsberg | Comparison of biodegradation of surfactants in soils and sludge-soil mixtures by use of C14-labelled compounds and automated respirometry[END_REF], using 14 C-LAS, showed from 48 to 82 % of mineralization of LAS in biometer flasks: the lowest mineralization occurs with sludge amendment where adsorption is higher. In more realistic conditions as lysimeter with non spiked sewage sludge, [START_REF] Jacobsen | Degradation and mobility of Linear Alkylbenzene sulfonate and nonylphenol in sludge-amended soil[END_REF] found that LAS are rapidly degraded firstly with a slower secondary degradation step as already shown by [START_REF] Marcomini | Behavior of aromatic surfactants and PCBs in sludge-treated soil and landfills[END_REF]. They also observed low downward transport of LAS because of strong sorption on the sludge. This also underlines that the fate of LAS in soil is a combination of mineralization and binding to soil particles and LAS may be stabilised as bound residues in soil with time [START_REF] Amundsen | Organic contaminants in some norwegian sludge amended soils.-Specialty conference on management and fate of toxic organics in sludge applied to land[END_REF]. Indeed, LAS has also a potential for uptake by plants.

The plasticizers phthalates have also distinct burdens according to their chemical structures. Di(2-ethylhexyl) phthalate (DEHP) is rapidly degraded in soil under aerobic conditions (EU, 2001). However, Roslev et al. (1998) using 14 C-DEHP applied on sludge amended soil, showed a two phase degradation with a 58 d half-live in the initial phase and 147 d in the late phase : more than 40% is not mineralized after one year and remained in the soil. Numerous esters lead to the formation of phthalic acid. This compound follow the same behavior as the DEHP but is faster degraded [START_REF] Roslev | Degradation of phthalate and di-(2ethylhexyl)phthalate by indigenous and inoculated microorganisms in sludge-amended soil[END_REF]. Another field study indicated that there is no long-term accumulation of dioctylphthalate in soil after following repeated sludge application [START_REF] Rhind | Phthalate and alkylphenol concentrations in soil following aplications of inorganic fertiliser or sewage sludge to pasture and potential rates of ingestion by grazing ruminants[END_REF].

Polycyclic aromatic hydrocarbons (PAH) exhibit distinct behavior in soil according to their molecular weight (number of rings). The lightest are volatile, undergo biodegradation and can be mobile, whereas the heaviest are recalcitrant, strongly adsorb, and poorly transferred. Halflives of month (phenanthrene) to years (benzo(a)pyrene) have been determined [START_REF] Heitkamp | Naphthalene biodegradation in environmental microcosms : estimates of degradation rates and characterization of metabolites[END_REF][START_REF] Goodin | Persistence and fate of anthracene and benzo(a)pyrene in municipal sludge treated soil[END_REF].

To conclude, the behavior of these compounds in soils is a combination of degradationmineralization (from 30 to 80%) and adsorption-bound residues formation (from 20 to 70%) on organic matter, few downward transport are observed as well few plant transfer. A large range of half lives from 1 to 147 days have been determined depending on the experimental conditions showing that real field experiments and long-term experiments are needed to complete all these data. In this way, one task of the EU BIOWASTE project (QLK5-CT-2002-01138) was devoted to the measurement of 4 families of xenobiotics in soil after sludge spreading on a 30-year field-scale record experiment.

Methods

Field set-up

Experimental maize crop fields from Bordeaux (France) have been amended with high load of sewage sludge of up to 100 tons per hectare each 2 years from 1974 to 1992. From 1992 to 2004, the fields were maintained and cropped with maize. Sandy soils were yearly sampled, air-dried, sieved (2mm) and stored at ambient temperature from 1974 to 2004. The following samples were selected for analysis : 1) 0-20 cm sludge amended soils from years 1976, 1978, 1979, 1981, 1986, 1989, 1993, 1997, 2004; 2) 0-20 cm non-amended soils (control soils) from years 1989,1999,2004; 3) four sewage sludge used for amendment from years 1976, 1981, 1989, 1991. The four families of xenobiotics were analysed: PAH, LAS, NPE and PAE. Each sludge and soil sample was analysed in triplicate in 2005.

Xenobiotics analysis

PAE extraction was done adding 1 to 10 g of dried sample in a 250 ml Erlenmeyer flask with 20 ml of ethyl acetate and the flask was placed in an horizontal shaker (30 minutes of extraction on a Yellow line RS 10 basic). After extraction, 1 ml is sampled for analysis. If necessary a purification step may be added. NPE, LAS and PAH are extracted with the ASE-200 procedure (DIONEX TM -Moduleware v.3.01 Bios 30.0). The principle of ASE system is the combined use of high pressure and high temperature to extract the target compounds. Some differences have to be pointed out : sample amount, solvent mixture, cell filling as summarised in table 1. The two PAH extracts are mixed then adjusted to 100 mL with acetone. 40mL extract are mixed with 0.5 mL 1% n-dodecane in hexane. The remained volume of extract is discarded. Extracts are rotary concentrated (vacuum pump, 500 mbar, 60°C water bath) to about 2 mL (never to dryness), then to dryness under a gentle nitrogen flow. Extract are diluted in 2 mL methanol, then allowed to stay at least 30 min before further analysis.

The two NPE extracts are soft evaporated under nitrogen flow to dryness. They are dissolved and pooled in 5 ml (sludge) or 2 ml (soil) of hexane. The extract is then agitated vigorously (10 min.), let diffused (30 min.) and stored (-20°C) for further analysis.

The two LAS extracts are soft evaporated under nitrogen flow to approximatively 10 ml each. They are pooled and soft evaporated to a final volume of 10 ml of methanol for sludge sample and 2 ml for soil sample. The extract is then agitated vigorously (10 min.) or sonicated, let diffused (30 min.) and stored (-20°C) for further analysis.

Two ml of the PAE extract is purified on a florisil (magnesium silicate, Prolabo ref 24 279.185) glass column. Silice (silice gel 60, CarloErba RS) or aluminium oxide (ICN Alumina N, Ecochrom) can be used instead. The florisil is dried 4 hours at 400°C before used. 1 g is used in the column. It was conditionned with ethyl acetate and then the 2 ml extract is purified and eluted with ethyl acetate. The purified extract is then soft evaporated and resuspended in 2 ml of ethyl acetate with DaIP at 100 µg/l.

NPE, LAS and PAH are analyzed by HPLC coupled to fluorescence detection. Table 2 summarises the main difference in the HPLC analysis conditions.

Results and discussion

The analysis of sewage sludge (four samples, year 1976, 1981, 1989, 1991) for PAH, NPE , PAE and LAS reveals concentrations varying between mg/kg to g/kg DW (figure 1). These concentrations fit the average values found for sewage sludge in Europe [START_REF] Bennie | Alkylphenol polyethoxylate metabolites in Canadian sewage treatment plant waste streams[END_REF][START_REF] Webber | Organic contaminants in Canadian municipal sludges[END_REF]Icon, 2001;[START_REF] Zurmuhl | Development of a method for the determination of phthalate esters in sewage sludge including chromatographic separation from polychlorinated biphenyls[END_REF] with very high level of LAS (10 g/kg), medium level of NPE and PAE (200-500 mg/kg) and low level of PAH (mg/kg for the sum of 16 PAHs, [START_REF] Lichtfouse | A 25-year record of polycyclic aromatic hydrocarbons in soils amended with sewage sludges[END_REF]. However, these levels are all above the proposed limits of the third draft of the EU sewage sludge directive (EU, 2001), except for the PAHs.

The same evolution with time is observed in the sewage sludge for the NPE, PAE and LAS: the concentrations decrease from 1976 to 1981 and increase from 1981 to 1989. This evolution could be explained by the highest requirements for the wastewater treatment plant during the 80 th , i.e. increment of the collected waters, implementation of nutrient treatment. Levels in the control soil are around 5 (BaP, BkF, NP1EO), 14 (Phe), 30 (NP), 150 (DEHP) and 700 (LAS-C12, 1600 for the sum of the 4 LAS) µg/kg DW (table 3). The DEHP and LAS levels are in accordance with those reported in literature for soil background. [START_REF] Carlsen | Linear alkylbenzene sulfonates (LAS) in the terrestrial environment[END_REF] reported LAS concentrations of 500-1000 µg/kg DW in soils from a preserved area not cultured for 50-100 years. EU ( 2001) reported DEHP values from 249 to 667 µg/kg DW for soils in the area of Stuttgart, Germany. However, Vikelsoe et al. (2002) reported for an uncultured reference soil 2.9 (NP2EO), 0.47 (NP), and 16 (DEHP) µg/kg DW. EU ( 2001) also reported value for PAH in rural or urban soils lower than those we have found. These highest levels could be assumed to come from atmospheric deposition (DEHP and PAH) or from cross contamination from artificial fertiliser used on other soils (NP1EO and NP). During the period of sewage sludge spreading (table 3), levels in soil increase sharply particularly for the compounds with high concentrations in sewage sludge (LAS, DEHP and NP1EO). In the same way, the distribution of the compounds is consistent with the mixing of sewage sludge and control soil underlying the impact of sludge quality onto soil concentrations during the period of spreading. For example, PAH, LAS and NP1EO levels are either higher than or close to the Predicted Non Effect Concentration (PNEC, table 3). This imply that application of sewage sludge with high load (100 tons/ha each 2 years) but with a common EU pattern of xenobiotics may have an impact on the terrestrial environment.

Looking at the most persistent PAH, it is worthily to note that the BaP level is 10 times less in sewage sludge than the French policy level but the sewage sludge application was 10 times higher than the normal quantity which finally corresponds to the application of a standard EU sewage sludge at the French policy level. Unfortunately, the level in the soil increases during the period of application being 100 times higher than the PNEC. Twelve years after the stop of spreading, the concentration profile seems to be always influenced by the period of application with for all compounds, excepted LAS-C12 and NP, a higher level than the control soil levels. This implies that addition of such compounds to soil by the way of sewage sludge may have a long term impact with high residual level in the soil compared to a control soil despite many years of maize cropping and weather exposition. Particularly, the less degradable one (BaP) remains in the soil after 9 years at a level 100 times higher than the PNEC. This means stabilization of BaP : no degradation, no desorption from the bound residues pool. Surprisingly, the BkF concentration increases at the end of the studied period : this can be explained by external contamination or by desorption from the non-extractable fraction. It has already been shown that the kinetics of adsorption-desorption and degradation of one PAH could influence another PAH [START_REF] Cornelissen | Competitive effects on the slow desorption of organic compounds from sediments[END_REF][START_REF] White | Influence of bisolute competition on the desorption kinetics of polycyclic aromatic hydrocarbons in soil[END_REF].

The evolution with time allows to understand and explain better the fate of the different compounds in the sandy soil. The evolution with time is shown on figure 2. Once added to the soil, the xenobiotics may (1) be degraded, (2) form bound residues as demonstrated by many other experiments like microcosms with radiolabelled compounds [START_REF] Mougin | Phthalic acid and benzo[a]pyrene in soil-plant-water systems amended with contaminated sewage sludge[END_REF] or (3) transfer to plant/water/air, this later part being generally little compared to the two other mechanisms. In our experiment, we measure a residual concentration corresponding to the fraction extracted by our analytical methodology: this residual concentration results from either degradation or/and adsorption on the soil organic fraction. Therefore, the evolution with time of the different compounds could be well correlated to the degradability of the molecules. Indeed, for the most degradable one, the LAS, the highest concentration was at the beginning of the studied period.

During the spreading period, the level does not increase whereas the quantity added is the highest, in counter part it stabilizes around 6 mg/kg. After the stop of spreading it decreases rapidly to reach the control soil level. In this case of easily degradable compound (by comparison to PAH, PAE or NP), the degradation may occur simultaneously to the addition of a high contaminated sludge. However, the remaining quantity in the soil is 12 years after stop spreading closed to the PNEC for soil (1.6 mg/kg), implying that even if the compound is well degraded it could have a long term impact on the environment. For the most persistent compound, the BaP, the behavior is completely different with increase of concentration during the spreading period (increase of the extractable pool) and then a level off meaning that once this compound is added to the soil, it remains for very long period. NP1EO and DEHP present an intermediate behavior. During the spreading period, their levels increase with the supply of the sewage sludge meaning that there is an increase of the extractable pool of the molecule but after the end of sewage sludge supply their levels decrease sharply during the 6 first years and then gently during the 7 following years. In 2004, their levels remain higher than in the control soil, particularly the DEHP with only 75% of disappearance. It means that degradation occurs at the beginning but after many years (ageing of the molecules), it may be much more difficult for the microorganisms to degrade the compounds that finally stabilise in the soil. It underlines thus the importance to fix limit for xenobiotics concentration in sewage sludge for spreading onto soil. These experiments underline also the large influence of the treatment on the fate and impact of the compounds in the sludge amended soil. The NPE is the best example : WWTP are known to participate to the production of a more toxic by-product (NP) by the shortening of the ethoxylate chain of the NPE. In our experiment it seems that due to the characteristics of the WW treatment the shortening stops at the NP1EO level. As these compounds is less toxic and more degradable, its impact is lowest.

Conclusion

The field experiment shows that the concentration fluctuations in the sludge amended soil follow the same pattern of those in the sewage sludge showing that there is a real impact of the sewage sludge concentration on the soil concentration particularly for the less degradable compounds like PAH, PAE and NPE. During the spreading period, if there is no limitations, the level of such compounds in the soil can be higher than thresholds like PNEC : in our experiment this is the case for LAS, NP1EO and PAH. After spreading stop, the less degradable compounds (PAH, particularly B(a)P) remain in the soil at levels 100 times higher than the PNEC. This underlines that even though the PAH level in the sludge was low, it stabilises in the soil for long period of time on a human scale. The three other compounds NPE, PAE and LAS decrease in concentration with time. Nonetheless, 12 years after the last addition of SS, the residual concentrations remain from 2 to 10 times higher than the content of the control soils, even though these levels are inferior to the PNEC. These results show that whatever the degradability range of the target compounds, it may have short and long term effects in soil if high quantities are spread underlying the importance to fix maximum level for xenobiotic compounds for sewage sludge spreading. It also underlines the key role of the sewage sludge processes for reducing the concentration of xenobiotic before they reach the environment.

  Two extractions: 100°C; 100 bars; preheat, 0 min; heat, 5 min; 3 cycles of extraction; static time, 5 minutes, cell flush, 60% and purge time, 100 sec. two extractions: 103.4 bar, 100°C with solvent 1, then 103,4 bar, 150°C with solvent 2 Cell filling alumina and hydromatrix hydromatrix 4 to 5 g celite and sand

*

  ml.min -1 3 min. of isocratic elution, 2 min of linear gradient from 75 to 88 %, 15 min. at 88%, 1 min. of gradient from 88% to 75% methanol and 11 min. of isocratic rinsing of the column 1 ml/min 5 min isocratic, then 50/50 to 0Two fluorimetric detectors are coupled in series to avoid peak overlapping. The first detector is used for naphthalene, acenaphthene, fluorene, fluoranthene, benzo(b)fluoranthène, benzo(a)pyrène and indeno(123cd)pyrene. The second detector is used for phenanthrene, anthracene, pyrene, benzo(a)anthracene, chrysene, benzo(k)fluoranthene, dibenzo(ah)anthracene and benzo(ghi)pyrene.PAE are analyzed by GC-MS (Varian GC3400Cx/saturn 3 ion trap). The conditions are as follow : injection volume 1 µl; injector temperature 250°C; injection mode splitless; capillary column Factor Four Varian 30 m*0.25 mm*0.25µm; program of temperature : 3 min at 70°C, gradient to 280°C at 13°C/min, 20 min at 280°C; transfer line temperature 290°C; mass temperature 230°C; helium 5.5; impact electronic 70 mEV; target ion 149 for DBP, DEHP and DaIP. Each sample or standard is injected 2 times.

Figure 1 :

 1 Figure 1: Level of polycyclic aromatic hydrocarbons (PAH), nonylphenol polyethoxylates (NPE), esters of phthalate (PAE) and surfactants (LAS) concentration of the sewage sludge (SEWAGE SLUDGE) used for application on the Bordeaux soil in 1976 (left),1981, 1989 and 1991 (right). EU third draft proposed limits are : 2600 mg/kg for LAS (sum of LAS-C10, C11, C12, C13, longer of the alkyl chain), 50 mg/kg for NPE (NP1EO, NP2EO and NP), 100 mg/kg for DEHP, 6 mg/kg for PAH (sum of 11). DEHP, Di(2-ethylhexyl) phthalate. Phe, phenanthrene. BaP, benzo(a)pyrene. Bkf, benzo(k)fluoranthene.

Figure 2 :

 2 Figure 2 : LAS, DEHP, NP1EO and BaP evolution in sewage sludge (dotted line, closed square), control soil (open square) and sewage sludge amended soil (closed square) along the 30 years experiment. The dotted line represents the Predicted Non Effect Concentration (PNEC) for each compound. PNEC DEHP is 10000µg/kg DW soil. The vertical line represents the year of stop of spreading.

Table 1 :

 1 ASE condition for the extraction of NPE, LAS and PAH from sludge and soil samples.

		NPE		LAS	PAH
	Sample	0.5 g sewage sludge	0.5 g sewage sludge	2.00 ± 0.01 g
	amount	2 g soil		2 g soil	2.00 ± 0.01 g
	Solvent	hexane/acetone	(50/50,	methanol	Solvent 1 : acetone/methylene
	mixture	v/v)			chloride 50-50 v/v, Solvent 2:
					acetone/hexane 50-50 v/v
	ASE	Two extractions: 120°C;
	condition	100 bars; 2 cycles of
		extraction; static time, 5
		minutes, cell flush, 60%
		and purge time, 100 sec.

Table 2 :

 2 HPLC conditions for the analysis of NPE, LAS and PAH

	Conditions	NPE		LAS	PAH
	matrix	sludge	soil	Sludge and soil	Sludge and soil
	column	Purospher	Purospher	Lichrospher 100 RP-18 5	100 x 4.6 ed Hypersil PAH
		STAR	STAR	micron, 4x250 mm	column (5 µm phase)
		greffée NH 2 ,	greffée NH 2 ,
		5 µm, 4×250	5 µm, 4×250
	temperature	30°C	30°C	25°C	40°C
	Elution	98.5% (V/V)	98.5% (V/V)	75% methanol: 25% water	water/acetonitrile	(v/v):
	solvent	hexane and	hexane and	NaClO 4 12 g/l	50/50
		1.5%	1.5%
		isopropanol	isopropanol

Table 3 :

 3 PAH, NPE, PAE and LAS concentration (µg/kg dry weight soil) on control soil, in sludge amended soil during the period of application and 12 years after the stop of spreading. Average and standard deviation values on three extractions. For PAH analysis, the analytical deviation extended from a minimum of 1.

	5% to a

Acknowledgement

This project was supported by EU under the fifth framework programme BIOWASTE project (QLK5-CT-2002-01138) and has contributed to the implementation of the key action "Sustainable Management and Quality of Water".


Olivier Liard

Stéphanie Segura

Emmanuel Sagui

André Nau

Aurélie Pascual

Melissa Cambon

Jean-Luc Darlix

Thierry Fusai

Emmanuel Moyse
email: moyseemmanuel@yahoo.fr

Adult-brain-derived neural stem cells grafting into a vein bridge increases postlesional recovery and regeneration in a peripheral nerve of adult pig

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Nerve injuries which are frequent in civil practice [START_REF] Noble | Analysis of upper and lower extremity peripheral nerve injuries in a population of patients with multiple injuries[END_REF][START_REF] Kouyoumdjian | Peripheral nerve injuries: a retrospective survey of 456 cases[END_REF] and catastrophe-like earthquake [START_REF] Ahrari | Prevalence and distribution of peripheral nerve injuries in victims of Bam earthquake[END_REF][START_REF] Etienne | Disaster relief in Haiti: a perspective from the neurologists on the USNS COMFORT[END_REF] or tsunami [START_REF] Charuluxananan | Anesthesia for Indian Ocean tsunami-affected patients at a southern Thailand provincial hospital[END_REF] combine associative lesion with crush syndrome. War improves the risk of gap and of treatment delay with ballistic wounds [START_REF] Hansen | A prospective evaluation of orthopedic patients evacuated from Operations Desert Shield and Desert Storm: the Walter Reed experience[END_REF] because these ones are less prone to emergency than vascular injuries [START_REF] Nanobashvili | War injuries of major extremity arteries[END_REF]. This risk pertains after war with remanent explosive [START_REF] Kett | Managing the health effects of the explosive remnants of war[END_REF]. The functional pronostic is dependent on the type of wound: transsection, crush, or gap, which has been further categorized according to the length of the gap, the type of nerve (sensitive, motor or mixed), localization, association of injuries, and patient's ageing, time of surgery, and vascular environment [START_REF] Seddon | Three types of nerve injury[END_REF][START_REF] Sunderland | A classification of peripheral nerve injuries producing loss of function[END_REF][START_REF] Sunderland | The anatomy and physiology of nerve injury[END_REF][START_REF] Mackinnon | A comparison of nerve regeneration across a sural nerve graft and a vascularized pseudosheath[END_REF].

Neuroguides and especially vein grafts are regularly used to bridge small nerve gaps shorter than 3 cm; for long nerve gaps, autologous nerve graft has become the gold standard since the second war [START_REF] Cooley | History of vein grafting[END_REF][START_REF] Colen | Nerve grafts and conduits[END_REF]. But nerve repair outcome depends on fascicle concordance [START_REF] Lago | Correlation between target reinnervation and distribution of motor axons in the injured rat sciatic nerve[END_REF] and scar development, so expected alternatives for long nerve gaps remain a challenge to improve recovery after this type of lesion. Surgical trials to improve nerve repair have mainly targeted diversification of grafted neuroguides with resorbable materials [START_REF] Ray | Management of nerve gaps: autografts, allografts, nerve transfers, and end-to-side neurorrhaphy[END_REF]. The outcome has been improved by neuroguide preloading with extracellular matrix components [START_REF] Yoshii | Collagen filaments as a scaffold for nerve regeneration[END_REF][START_REF] Kalbermatten | Fibrin matrix for suspension of regenerative cells in an artificial nerve conduit[END_REF][START_REF] Zhang | A nerve graft constructed with xenogeneic acellular nerve matrix and autologous adipose-derived mesenchymal stem cells[END_REF], growth factors [START_REF] Pfister | Nerve conduits and growth factor delivery in peripheral nerve repair[END_REF], or cultured regeneration-enhancing cells. The latter strategy, or cell therapy, has been attempted with primary cultures of Schwann cells [START_REF] Timmer | Axonal regeneration across long gaps in silicone chambers filled with Schwann cells overexpressing high molecular weight FGF-2[END_REF][START_REF] Hood | Transplantation of autologous Schwann cells for the repair of segmental peripheral nerve defects[END_REF], olfactory bulb enshealting cells [START_REF] Verdu | Olfactory bulb ensheathing cells enhance peripheral nerve regeneration[END_REF], or various types of stem cells [START_REF] Walsh | Use of stem cells to augment nerve injury repair[END_REF]. In these studies, progenies of grafted stem cells mostly proved to be glial cells, which indirectly enhance axonal regeneration [START_REF] Walsh | Use of stem cells to augment nerve injury repair[END_REF].

Neural stem cells from adult brain (ANSC) have been scarcely assayed to help postlesional repair of peripheral nerves, despite their strong potential of integration and development into neurons [START_REF] Moyse | Microenvironmental determinants of adult neural stem cell proliferation and lineage commitment in the healthy and injured central nervous system[END_REF]. The present study is based on the hypothesis that ANSC grafting inside a venous neuroguide might be efficient to bridge a long nerve gap in adult. We tested this hypothesis in the pig, which is the non-primate animal species most closely related to human and is prone to surgery and anesthesia conditions very close to human protocols in clinic [START_REF] Vodicka | The miniature pig as an animal model in biomedical research[END_REF]. To this aim, we have characterized primary cultures of neural stem cells from adult pig brain, by optimizing the neurosphere assay on subventricular zone explants [START_REF] Liard | In vitro isolation of neural precursor cells from the adult pig subventricular zone[END_REF]. In the present study, we labeled such primary expanded ANSC in vitro with either BrdU or lentiviral green fluorescent protein gene transfer, and we grafted labeled neurospheres into an autologous venous bridge after a surgical 30 mm long gap in adult pig nervi cruralis. We compared neurosphere-grafted and control pigs for functional recovery and assessed the fate of grafted cells inside the lesioned nerve at various post-lesion intervals.

Material and Methods

2.1. Animals. Twenty-four adults, 4-month-old Large White Landrace pigs were used in the present study. They were housed with ad libitum access to water and standard food pellets, in lodges that comprised one yard and one hard infrared-heated shelter. All in vivo experimental protocols were approved by the local Ethics Committee on the protection of animals used for scientific activities with the protocol number 31/2006/IMTSSA/UCPE. Personal protocol number for surgery and experimentation with pigs was 2007/ 29/DEF/DCSSA (to O. Liard). All efforts were made to minimize the number of animals used and their suffering.

Anesthesia.

Deep anesthesia was initiated by intramuscular premedication with 30 mg/kg ketamine (Imalgene 1000, Merial, Lyon, France), 0.1 mL/kg acepromazine (Vetranquil, Calmivet, Bayer, Puteaux, France), and 25 μg/kg atropine sulphate (Meram, Melun, France). The animal was then equipped with a peripheral intravenous perfusion delivering Ringer-lactate-5% glucose serum at 5-7 mL/kg/h, with adhesive electrocardiographic electrodes in CM5-SpO2 (ECG monitor S5, Datex Ohmeda, Madison, WI, USA), and with an artificial oxygenator. Anesthesia induction was performed by intravenous injections of 2 mg/kg propofol (Diprivan, Bayer) during 30 sec slowly, and of sufentanil (Sufenta, Bayer) three times at 1.5 μg/kg. Local glottis anesthesia was made with 5% lidocaïne spray (Xylocaine, Bayer), before orotracheal intubation with a monolight cannula (internal diameter 6.5 mm). Ventilation of lung parenchyma was checked with a stethoscope and mechanically assisted. General anesthesia was sustained with electrically driven intravenous infusion of propofol 2 mg/kg/h (Diprivan, DCI), sufentanil 1 μg/kg/h (Sufenta, DCI) and rocuronium bromide 0.15 mg/kg/h (Esmeron, DCI); curarisation was supervised by means of a S5 monitor (Entropie module) but was not released for EMG. Antibioprophylaxy was administered. All along surgery, the cardioventilatory parameters (heart and ventilation rates, electrocardiogram, blood oxygenation, ventilation pressure and volume, MAC, temperature, hemoglucotest) as well as the sleep threshold were constantly watched with the S5 monitor.

Brain Tissue Sampling for Primary Neural Stem

Cell Culture from Adult Pig. Separate animals were dedicated for primary culture and in vitro expansion of neural stem cells from brain subventricular zone (SVZ) as previously described [START_REF] Liard | In vitro isolation of neural precursor cells from the adult pig subventricular zone[END_REF]. On each deeply anesthetized pig, the dorsal face of the skull was cut with an electric saw and removed. A 15 mm thick coronal slice of the brain was performed with a scalpel at the commissural level of midbrain, manually explanted and transferred at 4 • C on ice bed-laid sterile metal plate, beside a Bunsen flame for atmosphere sterilization. The experimental animal was thus alive until the explantation of the brain tissue slice. Each pig was sacrificed immediately after brain tissue sampling by intravenous injection of Dolethal (R) pentobarbital (DCI) at 1.5 mL/kg.

Primary Culture of SVZ Neural Stem Cells ("Neurosphere

Assay"). As previously described [START_REF] Liard | In vitro isolation of neural precursor cells from the adult pig subventricular zone[END_REF], SVZ pieces were quickly microdissected from the thick midbrain slice (above) along the ventrolateral floor of lateral brain ventricles in lowcalcium artificial cerebrospinal fluid (aCSF: 124 mM NaCl, 5 mM KCl, 3.2 mM MgCl 2 , 0.1 mM CaCl 2 , 26 mM NaHCO 3 , 100 mM glucose, and pH 7.38). Tissue samples were rinsed twice with aCSF and digested first in 40 U cystein-EDTAβ-mercaptoethanol-preactivated papain (Sigma, L'Isle D'Abeau, France) for 10 min at 37 • C, then in 250 μL undiluted TrypLE Express solution (heat-resistant, microbially produced, purified trypsinlike enzyme, Gibco # 12604-013, Invitrogen, Cergy-Pontoise, France) for 10 min at 37 • C. After the addition of 750 μL of fresh aCSF and centrifugation for 8 min at 400 g at room temperature, the cell pellet was resuspended in 1 mL culture medium (DMEM (Sigma), 1x B27 (Gibco Invitrogen), 200 U/mL penicillin, and 200 μg/mL streptomycin (Gibco Invitrogen)) containing 20 ng/mL Epidermal Growth Factor (EGF, Gibco Invitrogen) and 20 ng/ mL basic Fibroblast Growth Factor (bFGF, Gibco Invitrogen). The cells were dissociated gently with a 26 G steel needle on a sterile disposable 1 mL syringe, counted on a Malassez slide, and seeded at 20,000 cells per mL in 6-well plates (Falcon, BD Biosciences, Bedford, USA). Cultures were monitored daily to follow the morphological growth of the neurospheres; passage was performed when the majority of spheres were 100-120 μm in diameter. For passage, the primary spheres were collected in sterile tubes, incubated for 45-60 min at 37 • C in 250 μL undiluted TrypLE Express solution (Gibco Invitrogen) per 6 mL culture-derived pellet, and dissociated gently with a 26 G steel needle on a 1 mL sterile syringe; dispersed cells were centrifuged, counted, and seeded as above. All culture media were renewed by the replacement of 2 mL of medium per 4 mL well every 2-3days. For immunocytochemistry, coverslips bearing differentiated spheres were rinsed in PBS, fixed in 4% paraformaldehyde-containing PBS for 20 min at 4 • C, and kept in PBS at 4 • C until the assay.

In Vitro Labelling of Neurosphere Cells.

In the first set of experiments, tertiary neurospheres were incubated with the DNA synthesis precursor bromo-2-deoxy-uridine at 30 μM (BrdU, Sigma) from 2 DIV after passage up to 8-10 DIV, BrdU being added fresh daily from concentrated stock.

In a second set of experiments, lineage labelling was performed by infection of secondary neurospheres with a lentiviral vector of green fluorescent protein gene (LV-GFP) at 4-5 DIV after passage. LV-GFP was freshly synthetized, titrated, and tested as previously described [START_REF] Mangeot | High levels of transduction of human dendritic cells with optimized SIV vectors[END_REF], stored at -80 • C and unfrozen just before use. Neurosphere cultures were preincubated 30 min in the presence of 1 mM polybrene (Sigma) and void lentiviral particles (VLP, 29) at 0.1x M.O.I., and further incubated 2 h with fresh culture medium containing 1 mM polybrene and LV-GFP at 0.3x M.O.I.; incubation medium was then replaced by fresh standard culture medium. LV-GFP-infected neurospheres were allowed 3 days culture in standard conditions for optimal lineage labelling (Figure 1); [START_REF] Mangeot | High levels of transduction of human dendritic cells with optimized SIV vectors[END_REF].

After optimal culture period (5-6 days for BrdU, 3 days for LV-GFP) and just before transplantation on the nerve lesion site (see below), neurospheres were collected, centrifuged 4 min at 800 g, and resuspended in fresh culture medium at 3 × 10 3 spheres per mL.

Postlesional Grafting Surgery.

The surgical approach was realized on the median face of thigh to isolate nervis cruralis. Nerve damage was established in the right N. cruralis by creating a 30 mm gap, which was then bridged with an autologous vein segment sampled from v. mammelian externalis. This venous neuroguide was sutured at both ends over the perinevre of the sectioned nerve. In some animals, once the venous shaft was sutured at the proximal end of the lesioned nerve, 300 μL of freshly prepared neurosphere suspension (10 3 spheres) were gently infused with a micropipet into the opposite end of the neuroguide, which was then sutured onto the distal lesioned nerve end. In other animals (controls), the venous neuroguide was sutured without any additional manipulation.

In the first set of experiments, lesioned pigs were thus grafted with autologous vein shafts which were either empty (controls, N = 2) or filled with BrdU-prelabeled neurospheres (N = 4). These animals were allowed 8 or 45 days survival after lesion.

In the second set of experiments, lesioned pigs were grafted with venous neuroguides which were either empty (controls, N = 3) or filled with LV-GFP-labeled neurospheres (N = 6). These animals were allowed 8 months survival after lesion.

Functional Analysis of Lesioned Animals

. Animal behaviour was evaluated by assessing muscular atrophy, deficit of leg extension over the thigh. Each animal was examined before peripheral nerve lesioning and at days 45, 90, 180, and 240 following surgical bridging.

Motor function of the crural nerve was assayed by electromyography, using pregeled surface electrodes connected to a portable electromyographic recorder (Keypoint (R) v6.01, Medtronics, MN, USA). Bipolar concentric needles (diameter 0.47 mm) were applied on muscle quadriceps femori for stimulodetection analyses along N. cruralis. Stimuli were single 100 mA shocks, ranging from 0.1 to 1 msec durations. Electromyography was performed on animals before and at 45, 90, 180, and 240 days after transection. Values were recorded in terms of voltage, amplitude and time latency of detectable electromyographic activities.

Immunohistochemistry.

At the end of postlesional survival, the operated region of N. cruralis of each experimental pig was dissected out of the thigh under anesthesia and fixed by immersion in a 4% paraformaldehyde solution in 0.05 M, pH 7.4, NaH 2 PO 4 /Na 2 HPO 4 buffer for 24 h at 4 • C, then rinsed for 24 h at 4 • C in 0.1 M, pH 7.4 phosphatebuffered saline (PBS). It was then cryoprotected for 72 h in a 30% sucrose solution, flat-embedded in OCT mounting medium and snap-frozen in liquid isopentane at -40 • C for storage at -80 • C. Longitudinal 20 μm-thick sections were made in a cryostat (Leica 2800), mounted on commercial precoated slides (Superfrost-Plus, Fisher-Scientific, Canada), dried overnight at ambient air, and stored at -20 • C.

For immunohistofluorescent labellings, these tissue sections were brought back to room temperature and permeabilized in 0.1 M PBS with 0.5% Triton-X-100. For nuclear 1). After three 5-minute-rinses in fresh 0.1 M PBS, immunohistochemical labelings were revealed by 2 h incubation at room temperature in the dark with the appropriate Alexa-594-conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA; 1/400). For double-staining experiments, Alexa-594-revealed slides were rinsed three times in fresh 0.1 M PBS and further incubated overnight at 4 • C with another primary antibody, which was revealed with appropriate Alexa-488-conjugated secondary antibody (Molecular Probes; 1/200). After rinsing, fluorescent staining of cell nuclei was performed by 5 min incubation in the darkwith DAPI (0.5 μg/mL, Sigma). Labeled slides were coverslipped with aqueous mounting medium (Vectashield, Vector, Abcys, France) and photographed with a fluorescence microscope. CNPase immunohistochemical labeling was quantified on these photomicrographs by using the ImageJ software.

Results

Clinical Results.

Before surgery, all experimental animals were healthy and displayed no walking deficit. After surgery, none of operated animals displayed any scar infection or host rejection response. By clinical observation, loss of the right leg extension over the thigh was systematically observed after the surgical nerve transsection. This motor defect was maintained up to 240 days in animals which had crural nerve gap repaired by empty autologous venous bridge only. By contrast, lesioned leg extension over the thigh reappeared between 90 and 180 days in animals which had crural nerve gap repaired by the same type of autologous vein trunk filled with exogenous neurospheres.

Electromyography.

Prior to surgery, electromyograms of m. quadriceps vast internal displayed amplitudes of 4.9 to 6.1 mV and poststimulus latencies of 0.89 to 3.7 ms. Immediately after surgical realization of N. cruralis substance loss, electromyograms of m. quadriceps were negative for all experimental animals, whatever their venous bridge had been filled or not with neurosphere suspension.

At 180 days (25 weeks) after lesion, neurosphere-transplanted pigs displayed positive electromyograms of m. quadriceps with 0.6 to 0.9 mV amplitudes and 0.96 to 2 ms latencies, whereas electromyograms of control animals remained negative (Figure 2). At 240 days (34 weeks) after lesion, m. quadriceps electromyograms of neurosphere-transplanted pigs were improved up to 2.8-3.1 mV amplitudes with 2.4-2.5 ms latencies, while electromyograms of controls were still negative (Figure 2).

In Situ Fate of Grafted Pig

Neurosphere Cells. In the first experiment, grafted neurosphere cells had been labeled by in vitro BrdU incorporation prior to graft. By immunohistochemistry on postmortem venous grafts, groups of BrdUimmunoreactive cells were detected inside the venous tube (Figure 3). In grafts sampled at 8 days after surgery, most of these BrdU+ cells were clustered in sphere-like assemblies (Figures 3(a) and 3(c)) while some were sparsed inside the venous tube (Figure 3(b)). Most of BrdU+ cells were also immunoreactive for doublecortin (DCX) which is a specific marker of immature neurons (Figure 4). At 90 days after surgery, BrdU+ cells were still observed inside the venous tube and displayed immunoreactivity for NeuN, that is, a marker of differentiated neurons (Figure 5). In the second experiment, expanded secondary neurospheres at 8 DIV had been stained by LV-GFP transfection just before collection and postlesional transplantation on Interestingly, at 180 and 240 days after nerve lesion and GFP-expressing neurosphere transplantation, CNPase immunohistofluorescent labeling was much higher than in vein-bridged lesioned controls which had not received neurosphere transplantation (Figure 8). Both the number of CNPase-immunoreactive Schwann cell processes and their labeling intensities were higher in neurosphere-grafted nerves than in controls. By densitometric quantification with the ImageJ software, immunohistochemical CNPase labeling of neurosphere-grafted lesioned nerves, at 240 days after lesion, reached 276 ± 2% of the 100 ± 2.9% value of lesioned controls which were devoid of neurospheres; this difference being statistically significant at P < 0.001 by Anova.

Discussion

Degeneration of the distal axon segment is the consequence of axotomy and the challenge of nerve repair is to connect the proximal stump to this distal segment. For long nerve gap, it is a race between axon regeneration and distal degeneration with phenotypic change of Schwann cells [START_REF] Walsh | Use of stem cells to augment nerve injury repair[END_REF]. Autologous nerve graft is the gold standard but results are not satisfactory [START_REF] Lago | Correlation between target reinnervation and distribution of motor axons in the injured rat sciatic nerve[END_REF]. Any alternative is currently believed to require a neuroguide, providing a permissive microenvironment to increase axonal regeneration and to preserve supportive cells and distal axons. Stem cells have already been studied but differentiation was mostly towards supportive cells. Neural stem cells have been reported to become integrated and provide new neurons after transplantation within central nervous system with permissive microenvironment [START_REF] Moyse | Microenvironmental determinants of adult neural stem cell proliferation and lineage commitment in the healthy and injured central nervous system[END_REF]. In order to test the efficiency of this type of graft for postlesional repair of peripheral nerves, we have established a primary culture of adult pig neural stem cells which generate neurons in vitro in our previous report [START_REF] Liard | In vitro isolation of neural precursor cells from the adult pig subventricular zone[END_REF]. In the present study, we show for the first time in adult pig that transplantation of in vitro preexpanded adult neural stem cells into a peripheral nerve gap inside a venous bridge both leads to genesis of longitudinally aligned neurons and improves functional recovery.

Methodological Considerations.

The present experimental protocol of nerve lesion: transsection and 30 mm long nerve substance removal, has been chosen to mimic the common cases of long nerve injury in human clinics. In these cases indeed, whatever be the initial type of injury (section, tearing, crush), the surgical regularization of both nerve stumps corresponds to a model of transsection. Autologous vein segment graft as a nerve conduit has been established as an effective treatment for the repair of nerve gaps less than 3 cm [START_REF] Chiu | Autogenous vein graft as a conduit for nerve regeneration[END_REF][START_REF] Chiu | A prospective clinical evaluation of autogenous vein grafts used as a nerve conduit for distal sensory nerve defects of 3 cm or less[END_REF]; it is used in hand surgery [START_REF] Risitano | Clinical results and thoughts on sensory nerve repair by autologous vein graft in emergency hand reconstruction[END_REF] and in pediatrics [START_REF] Kaufman | Peripheral nerve injuries of the pediatric hand: issues in diagnosis and management[END_REF]. In a recent study [START_REF] Acar | Femoral seating position of the EndoButton in single incision anterior cruciate ligament reconstruction: an anatomical study[END_REF], histomorphological examination of the sections proximal to, from, and distal to the repair zone over three months revealed less epineural scarring, a thinner epineurium, more regenerated axons and fewer inflammatory cells in groups where vein grafting was used, because the vein graft provided additional mechanical and chemical support in the size discrepancy of the nerve regeneration. A small vein graft keeps its property for end-to-side neurorrhaphy [START_REF] Manasseri | Ulnar nerve repair by end-to-side neurorrhaphy on the median nerve with interposition of a vein: an experimental study[END_REF], but size discrepancy between the donor and recipient nerves and length of the bypass are another problem with the risk of collapse. Vein bridging also gives excellent results in secondary repair of neglected injuries [START_REF] Lee | Secondary nerve reconstruction using vein conduit grafts for neglected digital nerve injuries[END_REF]. The present protocol is routinely realized in adult pig, as a convenient training system for human-operating surgeons.

The present postlesional cell therapy attempt used an in vitro expanded primary culture of adult pig brain neurospheres which had been extensively characterized in a previous study [START_REF] Liard | In vitro isolation of neural precursor cells from the adult pig subventricular zone[END_REF]. We had indeed shown that the present protocols of adult pig subventricular zone sampling and neurosphere assay allow to purify and maintain true neural stem cells with unlimited proliferative potential, self-renewal capacity, and ability to generate all neural cell types [START_REF] Liard | In vitro isolation of neural precursor cells from the adult pig subventricular zone[END_REF]. In particular, these neurospheres from adult pig SVZ displayed the same proportions of neurons (25%), astrocytes (70%), and oligodendrocytes (5%) as neurospheres from various adult rodent brain structures [START_REF] Reynolds | Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system[END_REF][START_REF] Louis | Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay[END_REF][START_REF] Bennett | Circumventricular organs: a novel site of neural stem cells in the adult brain[END_REF].

The procedure for fluorescent lineage labeling of grafted neurospheres used a lentiviral vector of GFP which has been validated previously [START_REF] Mangeot | High levels of transduction of human dendritic cells with optimized SIV vectors[END_REF]. Freshly prepared lentiviral particles were quantified by multiplicity of infection (MOI) determination using routine virologist assays, for optimization of the neurosphere labeling. We checked that this in vitro procedure allowed extensive labeling of adult pig neurospheres without alteration of their cell dynamics. It allowed subsequent identification of grafted cell progenies in situ merely by histofluorescence, which easily enabled to perform phenotype characterization by immunohistochemistry as revealed with different fluorophores.

Functional Recovery Enhancement by

Vein-Guided Neurosphere Transplantation. Electromyographic evaluation of the muscle which is innervated by the lesioned nerve demonstrated that our protocol of venous-graft-assisted neurosphere transplantation strongly improved functional recovery at 6 months after lesion, as compared to controls. Our subsequent histofluorescent analyses provided putative cytological substrates for such positive influence of grafted ANSC-derived cells.

In the same time, clinical evaluation was based on a simple observation. The section of the crural nerve pulled a deficient flexion of the thigh on the pond. The only recoveries of normal step were observed on the transplanted animals. The main question was to know if this recovery was bound to accelerated axonal regrowth or to the creation of a functional intermediate neuronal bypass. In fact we have a double observation of neurons and regrowth of the axons of the host.

Neuronal Outcome of Grafted Neurospheres inside the

Venous Bridge. In the present cell therapy attempt, the grafted neurospheres yielded exclusively neuronal progenies whatever be the postlesional delay. This outcome is striking since the neuronal yields from adult SVZ neural stem cells are known to reach only 25% neurons in vitro and 75% in the olfactory pathway in vivo [START_REF] Moyse | Microenvironmental determinants of adult neural stem cell proliferation and lineage commitment in the healthy and injured central nervous system[END_REF][START_REF] Louis | Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay[END_REF][START_REF] Belvindrah | Postnatal neurogenesis: from neuroblast migration to neuronal integration[END_REF]. The present in vivo result indicates that the venous graft provided an efficient proneurogenic environment for the transplanted neurospheres. It is in keeping with the recent demonstrations that vascular walls favor neurogenesis from adult neural stem cells [START_REF] Bennett | Circumventricular organs: a novel site of neural stem cells in the adult brain[END_REF][START_REF] Tavazoie | A specialized vascular niche for adult neural stem cells[END_REF][START_REF] Goldberg | Diverse roles of the vasculature within the neural stem cell niche[END_REF], which is mediated in some rodent models by the vascular endothelium-derived growth factor (VEGF) [START_REF] Schänzer | Direct stimulation of adult neural stem cells in vitro and neurogenesis in vivo by vascular endothelial growth factor[END_REF]. Our results suggest that choosing a venous trunk versus an artificial neuroguide to bridge a nerve gap is an interesting solution because of intrinsic property. Furthermore, all surgeons are able to take a vein trunk on superficial vein network and this is much less expensive than neurotubes in emergency conditions. At the same time, to understand mechanism of interaction between endothelial cells and grafted neural stem cells will allow progress for the development of new artificial neurotubes. More generally, local tissue microenvironment has been formally demonstrated to be determinant on the phenotypic fate of stemcell-derived progenies: for instance, nonneurogenic neural stem cells from the adult spinal cord generated neurons exclusively, once transplanted into the hippocampus [START_REF] Shihabuddin | Adult spinal cord stem cells generate neurons after transplantation in the adult dentate gyrus[END_REF].

These newly formed neurons survived at long delays after the original transplantation, which indicates they have been successfully integrated inside the severed nerve. Further, the linear distribution of these neuronal perikarya, parallel to the longitudinal axis of the lesioned nerve, suggests that the new neurons might be interconnected into multisynaptic nets bridging the lesional gap of preexisting nerve fibers. This multisynaptic nets have been described after neural stem cells graft [START_REF] Huang | Long-term survival and integration of transplanted engineered nervous tissue constructs promotes peripheral nerve regeneration[END_REF][START_REF] Bonner | Grafted neural progenitors integrate and restore synaptic connectivity across the injured spinal cord[END_REF] This interpretation is also supported by the evolution of transplanted cell clusters across time inside the venous graft, that is, from spherical assemblies in the first week after lesion to linear chainlike successions of sparse cells at longer delays. Such outcome arises apart of the strategies for peripheral nerve repair which have been worked out thus far and consist in stimulating either axonal regrowth into the distal nerve end or remyelination with additional Schwann cells [START_REF] Walsh | Use of stem cells to augment nerve injury repair[END_REF].

The present results contrast with previous ones using different kinds of transplanted cells, like foetal neural stem cells, which triggered Schwann cell like proliferation in the lesioned peripheral nerve [START_REF] Murakami | Transplanted neuronal progenitor cells in a peripheral nerve gap promote nerve repair[END_REF], promoting in turn myelinisation and indirectly regeneration [START_REF] Seo | ERK1/2-mediated schwann cell proliferation in the regenerating sciatic nerve by treadmill training[END_REF]. In addition, transplantation of immortalized C17.2 cells from postnatal cerebellum, which were shown to be distinct of neural stem cells [START_REF] Mi | Immortalized neural stem cells differ from nonimmortalized cortical neurospheres and cerebellar granule cell progenitors[END_REF], has been reported to trigger tumor formation in a rat model of injured sciatic nerve [START_REF] Johnson | Tumor formation following murine neural precursor cell transplantation in a rat peripheral nerve injury model[END_REF]. Adult neural stem cells are especially relevant for cell therapy from this viewpoint, because they were demonstrated to display exceptionally high resistance to tumorigenesis and senescence [START_REF] Foroni | Resilience to transformation and inherent genetic and functional stability of adult neural stem cells ex vivo[END_REF].

Possibility to Graft New Neurons into a Peripheral Nerve

Gap. There are two approaches, either to transplant neurons, or to transplant stem cells possessing neuronal differentiation potential. For primary neurons, dorsal root neurons can be prepared in vitro and their axons stretch-growth [START_REF] Pfister | Nerve conduits and growth factor delivery in peripheral nerve repair[END_REF]. Moreover, engineered nervous tissue construct consisted of longitudinally aligned axonal tracts spanning two neuronal populations, embedded in a collagen matrix and inserted in a PGA tube [START_REF] Huang | Long-term survival and integration of transplanted engineered nervous tissue constructs promotes peripheral nerve regeneration[END_REF] demonstrated neuronal survival during 112 days after surgical repair of the sciatic nerve in the rat. But only studies on the big animals allow the study of nerve gaps in good conditions.

High Survival Rate of Allogenic

Transplants. In our study, adult neural stem cells were prepared in vitro after explantation of SVZ cells from another pig, and allogenic grafted cells were integrated without immunosuppressive therapy. The same observation was noted for embryonic rat progenitors for the adult rat sciatic nerve [START_REF] Huang | Long-term survival and integration of transplanted engineered nervous tissue constructs promotes peripheral nerve regeneration[END_REF] and with xenograft of embryonic rat neural stem cells into a collagen conduit for the rabbit facial nerve injury model [START_REF] Zhang | Implantation of neural stem cells embedded in hyaluronic acid and collagen composite conduit promotes regeneration in a rabbit facial nerve injury model[END_REF]. In these attempts, grafted cells were expanded in vitro before transplantation, like in the present study.

Activation of Intrinsic Schwann Cells by Neurosphere

Transplantation. Phenotypic characterization of grafted cell progenies showed that our transplanted neurospheres generated no Schwann cell directly, but they triggered increases of the number and activity of this cell population as compared to controls. CNPase+ cells were observed along axonal regeneration but were always LV-GFP-. This result is in keeping with a previous report showing that venous graft favored Schwann cells proliferation after nerve lesion [START_REF] Tseng | Histologic analysis of Schwann cell migration and peripheral nerve regeneration in the autogenous venous nerve conduit (AVNC)[END_REF]. However, since in the present study controls have received a neurosphere-devoid venous graft, our results indicated that neurosphere cells emit diffusible signals that stimulate Schwann cells. Neurospheres have indeed been shown in vitro to secrete neurotrophic factors [START_REF] Xiao | Human adult olfactory neural progenitors rescue axotomized rodent rubrospinal neurons and promote functional recovery[END_REF][START_REF] Chirasani | Bone morphogenetic protein-7 release from endogenous neural precursor cells suppresses the tumourigenicity of stem-like glioblastoma cells[END_REF].

Conclusion

The positive impact of the present cell therapy protocol can be attributed to genesis and integration of new neurons inside the lesioned nerve and/or to indirect activation of intrinsic Schwann cell population. However, Schwann cell activation was previously shown to occur after grafting a venous segment onto the lesioned nerve without neural stem cells inside. Therefore, the specific benefit of the present procedure is likely caused by the neurosphere transplantation, and hence by the genesis of new neurons inside the lesioned nerve. LV-GFP is confirmed to provide an excellent marker for grafted cells across long-time studies. Both new neuron genesis and host Schwann cell proliferation contributed to regenerate new axon fascicles in the host to bridge the long nerve gap, and the functionality of this histological repair was demonstrated in the same animals by electromyography and clinics. Large animals, and especially the pig, allow repair and study of long nerve gap with clinical conditions close to human. These results were obtained without adjunction of extracellular matrix or extrinsic growth factor. The present study thus provides a hope for improvement in human clinics.

Figure 1 :

 1 Figure 1: LV-GFP-labeled secondary neurosphere from adult pig SVZ, just before transplantation and 3 days after in vitro infection with the lentiviral vector of GFP, as observed under a photonic microscope with natural light (a) or GFP fluorescence (b). Scale bars: 50 μm.

Figure 2 :

 2 Figure 2: Electromyographic evaluation of muscle quadriceps femori at 90, 180, and 240 days following N. cruralis lesion and graft of GFP-labeled neurospheres inside a venous bridge before ("before surg.") or after ("D + 90", "D + 180", "D + 240") surgery.

Figure 3 :

 3 Figure 3: Localization of transplanted BrdU-labeled neurospheres in the bridged nerve at 8 days after lesion, by postmortem immunohistochemistry. BrdU immunoreactivity is revealed by red Alexa-594 fluorescence on longitudinal postfixed graft sections at low (a) and high (b, c) magnifications. Enlarged fields (b, c) are localized as white forms in (a). Transplanted neurosphere cells are found either sparsed (b) or clustered (c) inside the venous bridge. Scale bars: 0.5 mm (a), 100 μm (b, c).

Figure 4 :

 4 Figure 4: Double BrdU-DCX immunohistochemistry on a sagittal section from a neurosphere-grafted bridged nerve at 8 days after lesion. One given field was sequentially photographed with fluorescent wavelengths revealing, respectively, Alexa-594-labeled BrdU immunoreactivity (a) and Alexa-488-DCX one (b). Note the extensive nuclear colocalization of the immature neuron marker DCX with BrdU (b). Scale bars 100 mm.

Figure 5 :Figure 6 :

 56 Figure 5: Double BrdU-NeuN immunohistochemistry on a sagittal section from a neurosphere-grafted bridged nerve at 90 days after lesion. One given field was sequentially photographed with fluorescent wavelengths revealing, respectively, Alexa-594-labeled BrdU immunoreactivity (a) and Alexa-488-NeuN one (b). Note the extensive nuclear colocalization of the mature neuron marker NeuN with BrdU (b). Scale bars 100 mm.

Figure 7 :

 7 Figure 7: GFP detection and phenotypic marker immunohistofluorescence (NeuN on left column, Schwann cells' CNPase on right column) in sagittal sections of venous bridges which had been transplanted with LV-GFP-labeled neurospheres and were sampled at 240 days after lesion. (a, d) green endogenous fluorescence for detection of lineage-infected neurosphere progeny; (b, e) Alexa-594-revealed phenotypic marker immunoreactivities; (c, f) merged fluorescences. Note yellow-labeled colocalization of GFP-expressing neurosphere progeny with NeuN immunoreactivity (c) but not with CNPase (f). Scale bars: 0.5 mm.

Figure 8 :

 8 Figure 8: CNPase immunohistofluorescence on sagittal sections of venous bridges without (a, b) or with (c, d) transplantation of LV-GFPlabeled neurospheres, which had been sampled 240 days after lesion. Alexa-594 fluorescence-revealed CNPase immunoreactivity (a, c) is combined with histofluroescent DAPI counterstaining (b, d). Note that Schwann cell labelling density is strikingly higher in neurospheretransplanted graft (c, d) than in void venous bridge (a, b). Σχαλε βαρσ: 100 μm.

Table 1 :

 1 List of primary antibodies used.

	Antigen	Antibody	Supplier	Cell-type specificity	Dilution
	BrdU	Rat polyclonal	ImmunologicalDirect.com	none	1/100
	CNPase	Mouse monoclonal	Sigma, L'Isle-d'Abeau, France; C-5922, clone 11-5b	Schwann cells	1/200
	GFAP	Rabbit polyclonal	Dako, Trappes, France; Z0334	Astrocytes, neural stem cells	1/1000
	NeuN	Mouse monoclonal	Chemicon, MAB377	Mature neurons	1/100
	NF-68	Mouse monoclonal	BD-Biosciences, Heidelberg, Germany	Mature neurons	1/400
	S100β	Rabbit polyclonal	Dako, Trappes, France	Astrocytes, Schwann cells	1/500
	antigens, tissue sections were also treated 15 min in 10 mM
	sodium citrate solution (pH 5.5) at 95 • C. After a PBS
	rinse, all preparations were incubated in blocking buffer
	(0.1 M PBS, 0.1% Triton, 3% bovine serum albumin, 5%
	normal serum) for 1 h, then overnight at 4 • C with one pri-
	mary antibody (Table

(a) (b)

Acknowledgments

These studies were supported by Délégation Générale pour l'Armement (DGA, France; REPAR Project, Grant 08co205). The authors express their gratitude to Professor Anne Duittoz (INRA Tours-Nouzilly) for precious advise about phenotypic marker immunohistochemistry in adult pig brain.


Mélanie Gunia

Nathalie Mandonnet

Gisèle Alexandre

Michel Naves

Florence Phocas

Genetic parameters of litter size in Creole goat and their implication for a breeding programme including adaptation traits

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Selection Index

The selection index will include traits measured during performance recording. These traits can be the same as in the objective or correlated to them. They will be adapted from the French sheep performance recording. Pre-weaning growth and reproduction parameters such as fertility and litter size will be recorded. In addition to these traits, post-weaning growth and faecal egg count (FEC) will also be recorded. FEC gives an indication of the level of parasitism of the animals.

Genetic parameter estimation

The genetic parameters were estimated to calculate the weighting of the traits in the index (Gunia et al., 2010b). The experimental Creole goat flock at INRA Gardel provided enough data for the calculation of the genetic parameters. More than 20 years of data about growth and reproduction traits were available.

Organisation of the breeding programme

The mating scheme will be designed. A sample of interested farmers (owning 300 does in total) will select the Creole goat. They will sell improved reproducers to a ''user'' group of farmers. These users will rear Creole goats and sell them for meat purpose, either in totally pure breed systems, or by using Creole does as a maternal line for crossbreeding. The different organisations of the programme will be studied with extension services to choose the most adapted solution.

Conclusion

As it happened for other indigenous breeds, the value of the Creole goat was long underestimated [START_REF] Gauly | Animal Genetic Ressources[END_REF]. Technical, economical, and genetic factors were taken into account in this programme to allow a more efficient selection of the Creole goat. The disease resistance characteristic of Creole goat was emphasised along with growth and reproduction traits. All participants (farmers, research, extension services) were integrated in this project.

Introduction

The Creole goat of Guadeloupe is a small-sized breed reared for meat. This breed, well adapted to its environment, has strong reproductive qualities [START_REF] Alexandre | [END_REF]. The prolificacy of this breed is high, around 2.1. Farmers want to maintain this prolificacy, but without increasing it. Studies were needed to determine how to analyse this trait.

-E-mail: Nathalie.Mandonnet@antilles.inra.fr

Advances in Animal Biosciences

Material and methods

The performances were collected in the Creole goat flock of INRA-Gardel in Guadeloupe from the past 30 years. Climate is tropical humid with a marked dry season. Goats grazed all year round. Only does were supplemented during lactation. The reproduction rhythm was of 3 kidding in 2 years. The first kidding happened at 16 months. The litter size (LS) at birth was recorded at each kidding. Data were available for 1962 does born from 273 sires and 903 dams.

Litter size was considered as a character of the mother. Two different models were studied using ASREML software developed by [START_REF] Gilmour | ASReml User Guide Release 2[END_REF]. Model 1 was a multitrait model. Litter size at parity 1, 2 and 3 were considered as three genetically different traits. They were written LS1, LS2 and LS3. Model 2 was a repeatability model. Litter size was considered to be the same trait over time and was written LSt. Random effects were additive direct and residual for both models. Model 2 also included a permanent environmental effect related to the repeated records of does over parities. A combined effect of cohort-sex was found to be significant in both models. Parity of the doe was added as a fixed effect for model 2.

Results

LS1, LS2 and LS3 were 1.7, 2.0 and 2.1 kids respectively with a standard-deviation of 0.6. LSt was 2.1kids 6 0.7.

For model 1, the same genetic variance of 0.04 was found for LS1, LS2, and LS3. Heritabilities and correlations were reported in Table 1. The heritabilities were moderate and very similar for the three parities, with a small decrease at the third parity. The phenotypic correlations were low, whereas the genetic correlations were very high. The genetic parameters for model 2 (table 2) were close to those found in model 1. Heritability was the same and the genetic variance was similar. Repeatability was moderate.

Heritabilities for LS1 were similar to those found by Bagnicka et al. (2007) for Polish and Norwegian goats, where heritabilities were 0.14 and 0.18 at first kidding for each breed respectively. For LS2 heritabilities were also on the same range. LS was within the range of heritabilities reported by [START_REF] Shrestha | [END_REF] for different goat breeds.

Preliminary studies showed that the genetic correlation between litter size and faecal egg counts was positive (and thus unfavorable). The faecal egg counts are a practicable and valuable way to evaluate the genetic resistance to gastrointestinal parasites (Woolaston and Piper, 1996).

Conclusion

Litter size can be considered as the same trait over time, at least for parities 1, 2 and 3. The genetic correlations between LS1, LS2 and LS3 were close to unity. All records had equal genetic variance. If these results were confirmed for the next parities, we could consider repeatability model as a better model to estimate litter size. Increasing litter size would lead to an increase of faecal egg count. When the litter size increased, the infection level and sensitivity to the gastrointestinal parasite increased too. It would also lead to a higher preweaning mortality. Targeting a stabilisation of the variability of the litter size seems to be the most sustainable solution for a selection programme.

Advances in Animal Biosciences

Table 1

 1 Litter size at parity 1, 2 and 3 estimated with multitrait model (Model 1). Heritability (with its standard-error) on the diagonal, phenotypic correlation above and genetic correlation below the diagonal

		LS1	LS2	LS3
	LS1	0.12 6 0.01	0.18	0.16
	LS2	0.95	0.12 6 0.01	0.15
	LS3	0.98	0.97	0.10 6 0.01

Table 2

 2 Estimated genetic parameters for litter size considered as one character with repeatability model (Model 2) direct heritability; c 2 5 permanent environmental effect; R 5 repeatibity 6 standard error.

		s 2 a	s 2 pe	s 2 e	s 2 p	h 2 a	c 2	R
	LSt	0.05	0.02	0.36	0.43	0.12 6 0.02	0.05 6 0.01	0.16
	s 2 a 5 direct additive genetic variance; s 2 pe 5 permanent environmental variance; s 2 e 5 residual variance; s 2 p 5 phenotypic variance; h 2 a 5

Aknowledgements

The authors thank Ranjit Manicom and Marie-He ´le `ne Madassamy (Carbicoop), Regis Alexandre (Chambre d'Agriculture) and Re ´my Arquet (INRA Gardel).