V G Ruggieri

Q Wang

S Esneault

R Madeleine

L Luo

A Leguerrier

J P Verhoye

P Haigron

Analyse de bioprothèse valvulaire aortique dégénérée par segmentation d'images TDM préopératoires

Keywords: Aortic valve bioprosthesis analysis, valve-in-valve, stick based segmentation, aortic valve implantation Mots-clés: Analyse de bioprothèse de valve aortique, valve dans valve, segmentation basée "stick", implantation de valve aortique

In the next future, transcatheter aortic valve implantation could represent a minimally invasive option in case of bioprosthesis failure for patients at high surgical risk. CT based preoperative planning of this procedure could be useful to optimize valve-in-valve implantation. In this context, bioprosthesis 3D analysis seems to be necessary, particularly for leaflets. The goal of this study was to propose different methods to segment and characterize a degenerated bioprosthesis using standard preoperative CT scan images in order to map structural injury of bioprosthesis and, ultimately, to plan the best positioning for valve-in-valve implantation. We report our preliminary results on segmentation of a degenerated bioprosthesis in aortic position. Three different methods have been tested and all allowed obtaining segmentation of the different bioprosthesis components. Results were compared by means of quantitative criteria. Explanted bioprosthesis CT images were used as reference. Semi-automatic segmentation seems to provide an interesting approach for the morphological characterization of degenerated bioprosthesis.

Résumé

Dans le future prochain, l'implantation percutanée de prothèse valvulaire aortique pourrait représenter une option mini-invasive dans le cadre de la dégénérescence de bioprothèse chez les sujets à haut risque chirurgical. La planification préopératoire basée sur les images scanner pourrait être utile pour optimiser l'implantation de la valve dans la valve. Dans ce contexte, une analyse tridimensionnelle de la bioprothèse dégénérée parait nécessaire, surtout en ce qui concerne les feuillets prothétiques. Le but de l'étude est de proposer différentes méthodes pour segmenter et caractériser une bioprothèse dégénérée en utilisant les images scanner préopératoires à fin de mapper l'endommagement structurel de la bioprothèse et, en dernier hypothèse (dans une optique future), de planifier le positionnement adapté de la valve percutanée dans la bioprothèse. Nous reportons les résultats préliminaires de la segmentation de bioprothèse valvulaire aortique. Trois différentes méthodes ont été testées et toutes ont permis d'obtenir une segmentation des différentes composantes de la bioprothèse. Les résultats ont été comparés par des critères quantitatifs. Les images scanner des bioprothèses explantées lors du ré-intervention ont servi de référence. La segmentation semi-automatique représente une approche intéressante pour la caractérisation morphologique des bioprothèses dégénérées.

I. INTRODUCTION

Aortic valve stenosis remains the most common valvular disease [START_REF] Lindroos | Prevalence of aortic valve abnormalities in the elderly: an echocardiographic study of a random population sample[END_REF][START_REF] Iung | A prospective survey of patients with valvular heart disease, in Europe: The Euro Heart Survey on Valvular Heart Disease[END_REF]. The gold standard for treatment is surgical aortic valve replacement [START_REF] Kojodjojo | Outcomes of elderly patients aged 80 and over with symptomatic, severe aortic stenosis: impact of patient's choice of refusing aortic valve replacement on survival[END_REF]. The combined effects of an aging population and the prevalence of aortic stenosis in this cohort have led to increasing numbers of elderly patients being referred for valvular surgery. The valve of choice in these patients is usually a bioprosthetic device, to avoid the need for anticoagulation with its associated complications [START_REF] Accola | Surgical management of aortic valve disease in the elderly: a retrospective comparative study of valve choice using propensity score analysis[END_REF]. Moreover, with the good long term results in durability of the new generation devices, bioprosthesis implantation is going to increase also in younger patients in the next years.

As a result, there is an increasing prevalence of failing bioprosthetic aortic valve replacements. Concurrently, the spectrum of comorbidities in these patients grows more complex, and therefore the risks of re-do surgery increase. Absolute surgical risk for re-do aortic valve operations might be raised by up to 15% compared with initial implants [START_REF] Eitz | Reoperation of the aortic valve in octogenarians[END_REF][START_REF] Jamieson W R | Re-operation for bioprosthetic aortic structural failure-risk assessment[END_REF].

Trans-catheter aortic valve implantation (TAVI) has entered the mainstream as a viable treatment option for patients with symptomatic, severe aortic stenosis, who are at prohibitively high surgical risk. The initial success in 2002 [START_REF] Cribier | Percutaneous transcatheter implantation of an aortic valve prosthesis for calcific aortic stenosis: first human case description[END_REF] has been followed by a surge of interest, strengthened by encouraging medium-term results with both the self-expanding CoreValve (Medtronic, Luxembourg) [START_REF] Grube | Percutaneous implantation of the CoreValve self-expanding valve prosthesis in high-risk patients with aortic valve disease: the Siegburg first-in-man study[END_REF][START_REF] Grube | Percutaneous aortic valve replacement for severe aortic stenosis in high-risk patients using the second-and current third-generation self-expanding CoreValve prosthesis: device success and 30-day clinical outcome[END_REF] and balloon-expandable Edwards Sapien (Edwards Lifesciences, Irvine, California) systems [START_REF] Cribier | Early experience with percutaneous transcatheter implantation of heart valve prosthesis for the treatment of end-stage inoperable patients with calcific aortic stenosis[END_REF][START_REF] Cribier | Treatment of calcific aortic stenosis with the percutaneous heart valve: mid-term follow-up from the initial feasibility studies: the French experience[END_REF].

Although not initially designed for this purpose, TAVI is also an option for patients with a degenerative aortic valve bioprosthesis who would be at high surgical risk from repeat thoracotomy. The feasability of implanting a trans-catheter valve into a surgical bioprosthesis was first demonstrated in a pig model in 2007 [START_REF] Walther | Valve-in-a-valve concept for transcatheter minimally invasive repeat xenograft implantation[END_REF]. The investigators used the Cribier-Edwards system to transapically implant 23-mm devices into 5 Carpentier Edwards porcine aortic valve prostheses to good effect. A case report of successful treatment of a severely regurgitant aortic valve bioprosthesis with the CoreValve (Medtronic) system has also been described-with retention of good function and no complications at 1-year follow-up [START_REF] Wenaweser | Percutaneous aortic valve replacement for severe aortic regurgitation in degenerated bioprosthesis: the first valve in valve procedure using the Corevalve Revalving system[END_REF].

Until now, some reports about valve-in-valve implantation are available [START_REF] Webb | Transcatheter Valve-in-Valve Implantation for Failed Bioprosthetic Heart Valves[END_REF], but the implanting technique remains empiric and modified for each different case and prosthesis. In next future, preoperative patient-specific simulation of endovascular procedure could be useful to optimize valve-in-valve implantation in order to predict intra-operative fitting and to reach the best positioning of the new prosthesis. In order to realize a preoperative planning of valve-in-valve implantation, 3D analysis and characterization of the structures of interest are necessary.

Nowadays, automatic processing segmentation of preoperative vascular CT images is feasible and provides relatively accurate results. This is no more the case for valvular prosthesis, specifically for prosthetic leaflets and their degeneration. To our knowledge there are some methods reported in the literature dealing with coronary ostia and valve leaflets detection [START_REF] Zheng | Automatic Aorta Segmentation and Valve Landmark Detection in C-Arm CT: Application to Aortic Valve Implantation[END_REF][START_REF] Waechter | Patient Specific Models for Planning and Guidance of Minimally Invasive Aortic Valve Implantation[END_REF]. None of them deals with the issue of CT analysis of failing aortic bioprosthesis. The difficulties come from the combination of different factor: inhomogeneities of contrast product distribution used for the acquisition of CT images of cardiovascular structures, low contrast between the structures of interest (leaflets, calcifications, contrast) and image resolution compared with structure thickness (leaflets).

The goal of this study is to propose a new segmentation-based approach to characterize a degenerated bioprosthesis. We show and discuss our preliminary results. Results of the preoperative segmentation could, ultimately, be useful to discuss about the valve-in-valve procedure indication and to plan the best positioning for trans-catheter implantation.

II. ANALYSIS OF DEGENERATED VALVE

A. Degenerated valve leaflets analysis

Multi Slice Computed Tomography (MSCT) shows a high spatial resolution and can also be used to acquire spatio-temporal data during the cardiac cycle. In this work we are interested in using MSCT data to assess pathological features of cardiovascular structures such as aortic valve leaflets. Figure 1 The overall framework (Fig. 2) proposed to analyze thin cardiovascular structures as valve leaflets from MSCT data, is composed of : (i) the definition of a Region of Interest (ROI) depending on the type of bioprosthesis stent implanted in the patient, (ii) a preprocessing step to denoise the original image, (iii) the segmentation of valve bioprosthesis components mainly based on a region growing process and (iv) the visualization process. Two approaches were considered for the preprocessing and three for the segmentation. In the preprocessing step anisotropic Diffusion curvature filter and Stick filter were implemented. In the segmentation process, the first approach was based on a classical isotropic region growing approach, the second on a directional (stick) region growing process and the third on a stick exhaustive search. In order to evaluate the performance of the segmentation of "in-vivo" images (preoperative images), the proposed approach was also applied to the "ex-vivo" images (explanted bioprosthesis) after patient reoperation.

B. Stent defined ROI

In order to deal with the difficulties of segmentation of the CT images of the bioprosthesis mentioned above (metal artifacts, thickness of leaflets compared with image resolution, density of valve components compared to stent) we considered a region of interest (ROI) defined by the stent. This ROI can have different shapes according to the type of stent implanted in the patient. We considered two kinds of shape for the ROI: a cylindrical shape preferentially for incomplete metallic stents and a conformational shape for complete metallic stents (Fig. 3).

A B

C. Preprocessing

In order to reduce the noise in CT images a preprocessing step is implemented. The preprocessing goal is to increase the signal to noise ratio (SNR) as well as to increase the contrast of the structures of interest, especially the valve leaflets. We considered methods based on Diffusion filters (curvature and anisotropic Diffusion filter) as well as an adaptation of Stick filters to reduce noise and keep details in thin structures. To select the most appropriate approach before the segmentation step, we considered the SNR as the performance criterion. To compute the SNR we used the definition given in [START_REF] González | Digital image processing[END_REF]:

-Anisotropic Diffusion filter

The Diffusion filters are non-linear filters based on partial differential equations. They have been proposed for noise reduction and segmentation, particularly for vascular images.

In classical Perona-Malik (PM) model [START_REF] Perona | Scale space and edge detection using anisotropic Diffusion[END_REF], the values of resulting voxels

1 t p I + are calculated as: 1 ,, () || p tt t t p p pq pq q p I I cI I ! " ! + # =+ $$ % , (2)
where p ! is the neighborhood of the current voxel and

|| p ! is the number of voxels in p ! . t p I is the current value of the voxel. , t pq I !
is the discrete gradient along the q th direction (defined by the current voxel and a voxel of the neighborhood). c(x) is the classifier between edge and noise. c(x) can take different expressions such as: Classical Anisotropic Diffusion operators such as PM model can enhance edge while denoising the image. Nevertheless, they may enhance both edge and noise in low SNR conditions. Anisotropic Curvature Diffusion, which is based on a Modified Curvature Diffusion Equation [START_REF] Yezzi | Modified Curvature Motion for Image Smoothing and Enhancement[END_REF][START_REF] Qian | A comparison of medical image segmentation methods for cerebral aneurysm computational hemodynamics[END_REF][START_REF] Brassel | A modified phase field approximation for mean curvature flow with conservation of the volume[END_REF], has been introduced to overcome this problem and to be less sensitive to noise.

Discrete Gradient

p tt t t p p pq pq q I I c Curv P Curv P N ! " + # = $ % , ( ( () ) ()
) 4
where , t pq P is the plane defined by the current voxel and a direction given by a voxel of the neighborhood p ! .

, () t pq Curv P

is the Gaussian Curvature at the voxel. Higher values of p ! may smooth edge and increase computational complexity too much. Values of ! and k were set at 1 for a low level of noise (high SNR). In the case of images showing a high level of noise (SNR<=8), ! were set at 0.5.

-Stick filter

Stick filter is also a non-linear filter. It was originally introduced to deal with strips-like noise in US images [START_REF] Czerwinski | Line and boundary detection in speckle images[END_REF][START_REF] Xiao | A Diffusion stick method for speckle suppression in ultrasonic images[END_REF]. With Stick filtering approach, the neighborhood of the considered voxel is divided into a set of asymmetric sticks to perform a non-linear filtering of the image (Fig. 4). Because of its asymmetric stick, the filter could get better performance in terms of keeping details of thin structure, as valve leaflets, while denoising the image. The filter output I' at the current voxel was originally defined as:

1 1 ' N ii i I gI W = =• !
, and

1 N i i Wg = = ! , !5"
where i I denotes the local mean value along the th stick, is the number of sticks, and g i is the reciprocal of local variance along the i th stick, used to classify edge and noise. This filter was adapted to preprocess CT images of degenerative aortic valve bioprosthesis. In order to improve the results of the original stick filter applied to CT images, we tested different parameters (including local median). At the end of the tests, we found that local median could keep more details of thin structures both visually and in terms of SNR.

In our approach, we used 3D asymmetric sticks set and the resulting value of the filtering process was considered as the sum of voxel median values normalized by the standard deviation along 4 voxels length sticks defined in the neighborhood (Fig. 5). The values of resulting voxels I' were more precisely defined as: Besides, the number of iteration is not a critical issue unlike Diffusion filter. Stick filter shows a convergent behavior after several iterations. Typically, in a low SNR condition (SNR<=8), the result converged after 5 iterations (after 3 iterations when SNR>10). It was not necessary to limit the number of iterations to avoid an over-smoothing of edges.

Although both Stick filter and Diffusion filter are "anisotropic" filter, Diffusion often use a small mask (3*3*3) or add a median filter process between 2 iterations to avoid making impulse noise. Stick filter can use much larger mask to get a better result.

The drawback of Stick filter is its high computation complexity. A 5 voxels-length Stick filter (i.e. a 9*9*9 cubic neighborhood), requires for each voxel and at each iteration, the calculation of 386 local medians and local variances. The time of calculation can be decreased by the stent-defined ROI. The number of voxels located in the ROI is much less than in the cubic volume encompassing the aortic valve bioprosthesis. Cylindrical ROI typically decreases the number of voxels to 40% and conformational ROI to 16%.

D. Valve segmentation

According to surgical expertise, four main classes of tissues were identified. They were related to HU measured by CT. We considered, from high to low HU, the following classes: stent, calcification, vascular lumen and leaflets. The first two elements, showing high CT values, can be segmented by thresholding easily; the issue of segmentation is more particularly focused on bioprosthetic leaflets.

Three kinds of region-based segmentation processes were implemented, with a decreasing level of user interactivity.

1-Isotropic Region Growing

The first one was an interactive process in order to provide a reference approach for the assessment of the results. It was based on classical Isotropic Region Growing (IRG) method. Following a preprocessing based on Diffusion filtering, as described above, it was applied using 3D Slicer software [START_REF] Ibáñez | The ITK Software Guide -Second Edition November 21[END_REF].

The bioprosthesis volume was selected using three different CT slices. Multiple seed points were interactively selected for each component of the prosthesis. The region growing algorithm was started (26-neighbour connectivity) with automatic calculation of the criterion used for the aggregation of voxels. This similarity criterion was based on mean and standard deviation of the voxel values within the considered cubic neighborhood [START_REF] Boskamp | New vessel analysis tool for morphometric quantification and visualization of vessels in ct and mr imaging data sets[END_REF][START_REF] Adams | Seeded region growing[END_REF][START_REF] Fan | Automatic image segmentation by integrating color-edge extraction and seeded region growing[END_REF]. The extraction and visualization of the resulting 3D surface meshes delineating the regions were performed by using the Marching Cube algorithm [START_REF] González | Digital image processing[END_REF][START_REF] Ibáñez | The ITK Software Guide -Second Edition November 21[END_REF].

-Stick Region Growing

To better deal with the segmentation of thin structures, like the components of a valve bioprosthesis and especially valve leaflets, we developed a directional region growing process based on stick neighborhood. The segmentation was thus performed by the stick region growing algorithm applied to a previously selected region of interest (ROI), either cylindrical or conformational, passing through the metallic stent. Some seeds were interactively selected inside the leaflets at different locations (typically 4 to 6 seeds). A set of N voxels inside the leaflets was by considering for each seed the voxels belonging to their neighborhood. The similarity criterion was locally based on mean and standard deviation of the CT voxel values along the directions defined by the sticks. This approach was implemented using MatLab development tools.

In order to examine each voxel in the cubic neighborhood of the seed, we specifically computed the stick variances in the set of sticks defined by each of these voxels:

2 , 1 () L ij i j II Var L = ! = "
, where , ij I is the grey level of j th voxel in i th stick.

L was chosen to be less than the thickness of the leaflets. Typically L was between 3 and 5 voxels (about 1mm).

To ensure region growth is not early stopped, or in other words to limit the number of seed points selection in some critical cases (highly noisy data), the size of the cubic neighborhood was chosen to be large enough to include space between homogeneous fragment of leaflets. We typically used a 342-connected neighborhood (7x7x7).

We also defined The voxels were classified as belonging to the region of the seed voxel (i.e. as a voxel of normal leaflet tissue) when: (i) m I belongs to the grey level range of normal leaflets and (ii) minimum local variance along stick is lower than a variance threshold (VTH). VTH was determined by the difference of local cubic neighborhood variances of leaflets voxels and boundary voxels. These variances have been previously calculated in SNR computation (for image preprocessing). When this voxel belongs to ROI, it constitutes new seed voxels. This process was iterated until no new voxel can be marked as a seed. This segmentation referenced as Stick Region Growing (Stick RG) in the following was preceded by a Stick filtering preprocessing step.

-Stick Exhaustive Search

To further reduce the level of interactivity in the segmentation process, a systematic exploration of the ROI was implemented with no need for seed point definition. This segmentation referenced as Stick Exhaustive Search (SES) in the following was preceded by a Stick filtering preprocessing step.

Looking at the grey level of leaflets/vascular lumen in the preselected and filtered ROI, we manually set the range of grey level thresholds about mean and variance. We divide neighborhood (Radius=3 pixels) of all voxels inside ROI into an asymmetric sticks sets and we automatically calculate local means and local variances along sticks. If the stick values were inside the range we set, the neighborhood voxel was classified as leaflets or vascular lumen. The exploration of the ROI is completely automatic.

III. RESULTS

A. Experimental data

As preoperative assessment, we obtained CT scan images (General ElectricTM Lightspeed® VCT 64) from four patients (HER, MAG, BOU, DAV) presenting a degeneration of aortic bioprosthesis. Depending on the case, the reoperation was performed from 7 to 19 years after implantation. Acquisition protocol was a multi-slice coronary angio-CT-scan ECG gated in 3D + T mode.

We analyzed 4 databases of aortic bioprostheses, explanted for different pathological reasons. In one case it was a pericardial bioprosthesis (HER), in which bovine pericardium is cut to reproduce aortic valve leaflets that are sewn on a complete metallic stent. Three more cases were represented by porcine bioprostheses, in which a porcine aortic valve is mounted on a complete metallic stent (2 cases: MAG and BOU) or incomplete metallic stent (1 case: DAV).

CT-scan sequences were preprocessed and segmented (as described in Fig. 2) to describe the vascular structure as well as the bioprosthesis metallic stent. After reoperation, CT-scan images of isolated explanted aortic bioprosthesis were obtained using the highest image resolution protocol. The main parameters derived from the CT acquisitions of in-vivo (IV) and ex-vivo (EV) aortic bioprosthesis, for each of the four cases, are summarized in Table I.

TABLE I: MAIN PARAMETERS OF IN-VIVO (IV) AND EX-VIVO (EV)

ACQUISITIONS.

B. Preprocessing

The preprocessing based either on Diffusion filter or on Stick filter was applied to the in-vivo CT volumes of the four patients. The bioprosthesis voxels were first isolated by considering the ROI defined by the stent. Ex-vivo images do not need preprocessing. Differences in grey level between stent, leaflets and calcification/pannus are very clear in ex-vivo data.

Examples of original images and resulting images obtained with the two different filters are reported in Figure 5. A first qualitative appreciation of the results let appears that Stick filter based approach seems to better enhance the contrast between valve leaflets and surrounding structures.

Original image Diffusion filter

Stick filter In addition to this qualitative assessment of the results, SNR was computed on original images and resulting images (Fig. 6). This objective assessment let appear that Stick filtering shows the best SNR for the four patients, and this is consistent with the subjective appreciation.

H E R M A G B O U D A V

C. Segmentation

Following the ad hoc preprocessing step, the different segmentation methods (IRG, Stick RG, and SES) were applied to in-vivo (IV) and ex-vivo (EV) images. A qualitative analysis is reported in Fig. 89comparing the methods used in-and ex-vivo to enhance bioprosthetic leaflets and the explanted bioprosthesis.

The first example is reported in the case of the pericardial bioprosthesis (HER). The visualization was performed by surface rendering. Some anatomical similarity as the shape of the leaflets and the presence of minimal leaflet degeneration can be easily appreciated (Fig. 7). The leaflet prolapse resulting in no perfect coaptation appears as commissure lost (indicated in blue circle) in the segmented images.

Figure 7: Visualization of the segmentation results obtained in case HER: explanted aortic bioprosthesis (EAB), in-vivo bioprosthesis (IV) and ex-vivo bioprosthesis (EV), segmented with methods IRG and Stick RG. The commissure lost is indicated with blue circle and good leaflets coaptation with white arrows.

The second example illustrates the results obtained in the case of a porcine bioprothesis (MAG). The visualization of front and back side of bioprosthesis was performed by volume rendering with transparency effect (Fig. 8). A calcified pannus (indicated by black arrows) is characteristic in this case and responsible for valve obstruction. Leaflet tear (indicated by green arrows) was also observed and found in reconstructions.

The results on the back side of EV_IRG and EV_SES are not exactly the same. In fact, in this case, when the valve is closed (closed position of leaflets), the pannus developed under the bioprosthesis is in contact with a part of the leaflets (partially calcified). Automatic enhancement of these 2 tissues in contact is quiet difficult. This is the reason why the results show some differences between the methods. A quantitative analysis was then conducted to objectively compare the results obtained respectively by IRG, Stick RG, and SES on in-vivo (IV) and ex-vivo (EV) images. It was performed as following: the voxel volume was computed for each database (considering CT acquisition parameters showed in Tab. I) and multiplied for the number of voxels of each bioprosthestic component.

Front Back

The criterion used to evaluate the results was the volume of bioprothesis components. Results of this analysis are shown in Fig. 9 (volumes are expressed in mm 3 ). This analysis wants to show that these 3 different methods more or less automatic, allow to enhance bioprosthetic components including leaflets with similar results. In fact, looking at volumes values, we can't conclude that the 3 methods provide so different results. In-vivo volumes are generally bigger than ex-vivo and it can be explained mostly by the moving artifacts. For sure, ex-vivo results are more reliable than in-vivo and represent the reference to compare the in-vivo results. But, when we compare the results between in-and ex-vivo for each method, the differences in voxels volumes are always less than 20%. In recent years, multi-detector computed tomography has emerged as a promising tool to provide precise diagnostic information in different clinical situations, such as noninvasive evaluation of coronary disease [START_REF] Vanhoenacker | Diagnostic performance of multidetector CT angiography for assessment of coronary artery disease: metaanalysis[END_REF][START_REF] Miller | Diagnostic performance of coronary angiography by 64-row CT[END_REF][START_REF] Sun | Diagnostic value of multislice computed tomography angiography in coronary artery disease: a meta-analysis[END_REF][START_REF] Hausleiter | Non-invasive coronary computed tomographic angiography for patients with suspected coronary artery disease: the Coronary Angiography by Computed Tomography with the Use of a Submillimeter resolution (CACTUS) trial[END_REF][START_REF] Budoff | Diagnostic performance of 64-multidetector row coronary computed tomographic angiography for evaluation of coronary artery stenosis in individuals without known coronary artery disease: results from the prospective multicenter ACCURACY (Assessment by Coronary Computed Tomographic Angiography of Individuals Undergoing Invasive Coronary Angiography) trial[END_REF][START_REF] Mowatt | 64-slice computed tomography angiography in the diagnosis and assessment of coronary artery disease: systematic review and metaanalysis[END_REF], ventricular morphology and function [START_REF] Juergens | Left ventricular function studied with MDCT[END_REF], and myocardial viability [START_REF] Mahnken | Assessment of myocardial viability in reperfused acute myocardial infarction using 16-slice computed tomography in comparison to magnetic resonance imaging[END_REF][START_REF] Gerber | Characterization of acute and chronic myocardial infarcts by multidetector computed tomography: comparison with contrastenhanced magnetic resonance[END_REF][START_REF] Lardo | Contrast-enhanced multidetector computed tomography viability imaging after myocardial infarction: characterization of myocyte death, microvascular obstruction, and chronic scar[END_REF]. CT images can also help visualize the morphology and motion of native and diseased stenotic aortic valves and precisely measure aortic valve opening areas. Several studies [START_REF] Teshima | Dysfunction of an ATS valve in the aortic position: the first reported case caused by pannus formation[END_REF][START_REF] Teshima | Usefulness of a multidetector-row computed tomography scanner for detecting pannus formation[END_REF][START_REF] Teshima | Detection of pannus by multidetector-row computed tomography[END_REF][START_REF] Aoyagi | Prosthetic valve obstruction: diagnostic usefulness of cineradiography and multidetector-row computed tomography[END_REF][START_REF] Kim | Evaluation of prosthetic valve endocarditis by 64-row multidetector computed tomography[END_REF][START_REF] Tsai | Correctness of multi-detector-row computed tomography for diagnosing mechanical prosthetic heart valve disorders using operative findings as a gold standard[END_REF][START_REF] Konen | The role of ECG-gated MDCT in the evaluation of aortic and mitral mechanical valves: initial experience[END_REF] suggest that multi-detector CT can help assess mechanism of dysfunction in mechanical prosthetic heart valve disorders.

An aortic bioprosthesis can have functional degradation and prosthesis dysfunction over time. In more recent bioprostheses, the rate of structural failure appears to be somewhat lower than with the first generation [START_REF] Ruggieri | Long-Term Results of the Carpentier-Edwards Supraannular Aortic Valve Prosthesis[END_REF], but it is still occurring. Understanding the precise mechanism of bioprosthesis dysfunction would thus be important to direct clinical management. Yet, it can often be difficult to identify these entities in clinical practice. Indeed, both TTE and transesophageal echocardiography have limited usefulness for detection of the mechanism of stenotic bioprostheses, because of the acoustic shadowing from valve stents or annulus.

CT images would potentially allow better visualization of the bioprosthetic valve leaflets after aortic valve replacement, but at the moment CT scan softwares don't allow automatic leaflets enhancement. So, additional methods have to be considered to improve leaflets characterization and visualization.

In this study we have tested three different types of segmentation applied to CT images of degenerated aortic bioprosthesis. Considered methods, even if different in their approach, seem to be similar in terms of results. They are considered as semiautomatic segmentation methods.

The mean difference among these methods is that the Isotropic Region Growing method is performed using software for image analysis (3D Slicer), relatively easy to use, but needing heavy interaction for the user (manual selection of different seed points for the different components of the bioprosthesis) . On the other hand, Stick Region Growing and Stick Exhaustive Search methods need, at this time, to be performed by professionals of Matlab development tools, but their interactivity is lower.

The reduced interactivity level results in some differences in terms of voxels volumes. In fact it is difficult to replace the user ability to recognize bioprosthesis different structures in CT transverse sections (IRG) by the automatic criteria based on grey level density (Stick RG and SES).

Quantitative analysis shows these differences among the methods, but we can't consider them significant because of the inability to perform a statistical analysis on these few databases. Moreover, we don't know at the moment if these small differences (always less than 20%) could have a real interest for clinical application. In fact, the goal of segmentation is to approximately reconstruct leaflet morphology and to detect some failing mechanism as pannus formation that could represent a contraindication to the endovascular procedure.

A key point of the analysis is represented by the preprocessing. In fact, bioprostheses CT images need to be denoised before to be segmented because of metallic stent artifacts. Different filters have been tested in order to improve images with the best leaflets enhancement. Stick filtering, modified in order to be applied to CT images, showed the best visual results and this was confirmed by SNR analyses. This filtering process was performed by MatLab software and it takes time to be finalized. To reduce this computing time it is important to select a ROI that takes into account only the bioprosthesis stent region with its leaflets.

Concerning segmentations results, some considerations have to be done. Among analyzed databases the best results have been obtained in the case of the pericardial bioprosthesis (HER). The reason is attributed to the thickness of valve tissue. In fact pericardial leaflets are largely thicker than porcine with consequent better results for CT images.

The higher bioprosthesis components' volumes observed in some cases for in-vivo images comparing to ex-vivo can be explained by different factors. Firstly, image resolution is not the same. In-vivo acquisition protocol represents the standard protocol used for coronary ECG gated angio-CT-scan. Acquisition parameters cannot be changed and specific acquisition for bioprosthesis analysis doesn't exist at the moment. For ex-vivo we chose the highest resolution protocol to be close to reality and to use these images as reference. Other than image resolution, we can consider heart motion and blood superposition as important factor of volumes differences.

Computing of leaflets volumes can be altered by the non-metallic part of bioprosthesis stent (DAV). Only the metallic stent can be used as spatial limitation for the leaflets because the non metallic part has the same density of the leaflets. For this reason leaflet volumes are higher than normal, especially in the case of incomplete metallic stent.

Another factor that plays a fundamental role for the quality of in-vivo images (preoperative images) is the heart rate during the exam. It should be as low as possible and not more than 80 bpm. Patient preoperative clinical conditions can be responsible for higher heart rate and contra-indicate medical therapy as beta-blockers to reduce it. In all cases, we observed also in-vivo underestimation of leaflets degeneration. This is only partially true. In fact, in the study cases, leaflets are not so calcified and most part of degeneration consists in leaflets thickening. These leaflets modifications do not appear with the same density of calcifications and are difficult to detect in in-vivo CT images. Only actual calcifications can be detected with consequent underestimation compared to ex-vivo images, in which smaller differences in density can be easily appreciated.

Our study suggests that segmentation of CT images could play a role in clarifying the mechanism of bioprosthesis dysfunction. Indeed, we demonstrated that segmentation can help directly visualize leaflet morphology. Furthermore, we demonstrated the ability of these methods to identify bioprosthesis leaflet thickening, calcification as well as presence of pannus under bioprosthetic leaflets.

V. CONCLUSION

Echocardiography remains the gold standard for functional analysis of bioprosthesis failure. In the last years an increasing number of groups are looking with particular interest at CT images for anatomo-morphological analysis of the aortic valve.

Segmentation could be useful in the next future to better understand the morphology of the failing bioprosthesis. Moreover segmentation allows to better visualize some non structural valve dysfunction like subvalvular pannus, difficult to visualize by echocardiography and to recognize by transverse CT scan sections. Finally, it could be interesting to integrate 3D reconstructions into planning tools to optimize the valve-in-valve procedure.

In order to further validate the preliminary results, we are testing the three presented methods for different patients datasets.

Moreover additional types of segmentation approaches are going to be evaluated. Results among different techniques will be compared in order to determine the best method for a relevant 3D bioprosthesis segmentation.

  illustrates original noisy MSCT images showing stent with high density and possible artifacts (A), low contrasted leaflets (B), calcifications on leaflets (C) and pannus under stent (D). The range of grey level in vessel lumen was typically about [1300 HU, 1550 HU], and in leaflets about [1150 HU, 1420 HU].

Figure1:

  Figure1: Two noisy MSCT slices of aortic valve prosthesis showing stent (A), leaflets (B), calcification (C) and pannus (D).

Figure 2 :

 2 Figure 2: Framework of bioprosthetic aortic valve analysis from MSCT data.

Figure 3 :

 3 Figure 3: Shapes (A cylindrical, and B conformational) of stent defined ROI and illustration of the corresponding CT observations (within one of the original CT slices).

  the local variance in the neighborhood of leaflets and vascular lumen voxels located in the ROI. Voxels located at the boundary between leaflets and vascular lumen are used to obtain the maximum variance whereas voxels located in the vascular lumen are used to obtain minimum variance. Due to their high value, voxels representing stent, calcification and pannus are not considered in the computation of SNR.

!

  and k are two parameters to control Diffusion intensity.

  value[START_REF] Black | Robust anisotropic Diffusion[END_REF] of discrete gradient of all voxels inside ROI.

  model was replaced by Gaussian Curvature of the voxel in the Anisotropic Curvature Diffusion: 1 ,,

  + , L is the length of side of cubic neighborhood.In the proposed CT data preprocessing approach, we chose | | 26 p ! = and N=13 (3*3*3 cubic neighborhood).

  its simple computational complexity.

Figure 4 :

 4 Figure 4 : A, 4 voxels-length 2D asymmetric sticks set [18] and B, 5 voxel-length sticks (white arrows) which give the higher output in 4 positions inside leaflets (SNR of this image is 7.54).

  where I Mi denotes the local median value along the th stick, is the number of sticks ( L is the number of voxels along the stick.L, the length of stick, is the main parameter of this filter. It is determined by SNR of original CT images. When SNR<=8, L=5; 8<SNR<=10, L=4; SNR>10, L=3.The process of filtering is applied to the ROI. When part of stick is outside of the ROI, we use original image voxels to complete it.

  the local mean of the stick with minimum local variance.

Figure 5 :

 5 Figure 5: Results of CT images preprocessing by Diffusion and Stick filters.

Figure 6 :

 6 Figure 6: Results of preprocessing (SNR) with Stick filter and Curvature Diffusion filter for each of the four cases (HER, MAG, BOU and DAV).

Figure 8 :

 8 Figure 8: Visualization of the segmentation results obtained in case MAG (Front and Back side) explanted aortic bioprosthesis (EAB), in-vivo bioprosthesis (IV) and ex-vivo bioprosthesis (EV), segmented with methods IRG and SES. Calcified pannus (indicated by black arrows) and leaflet tears (indicated by green arrows) are observed and found.

Figure 9 :

 9 Figure 9: Volume analysis of bioprosthesis components


Yet another short proof of Bourgain's distorsion estimate Benoît R. Kloeckner 1, * 1 Institut Fourier, Université de Grenoble I We use a self-improvement argument to give a very short and elementary proof of the result of Bourgain saying that regular trees do not admit bi-Lipschitz embeddings into uniformly convex Banach spaces.

Let T n be the binary rooted tree of depth n and let c B (A) denote the distorsion of the metric space A in B, that is to say the infimum of all numbers D such that there is a number s and a map ϕ : A → B such that sd(x, y) d(ϕ(x), ϕ(y)) sDd(x, y) for all x, y ∈ A.

The modulus of (uniform) convexity δ X (ε) of a Banach space X with norm | • | is defined as

inf 1 - x + y 2 |x| = |y| = 1 and |x -y| ε for ε ∈ (0, 2]
. The space X is said to be uniformly convex of type p 2 if δ X (ε) cε p for some c > 0. Note that in particular, for p ∈ (1, ∞) the L p spaces are uniformly convex of type max(2, p).

Our main goal is to prove the following as simply as possible.

Theorem 1 (Bourgain). If X is uniformly convex of type p then c X (T n ) (ln n) 1 p .
Several proofs of this result have been given over the years, see notably [START_REF] Bourgain | The metrical interpretation of superreflexivity in Banach spaces[END_REF][START_REF] Matoušek | On embedding trees into uniformly convex Banach spaces[END_REF][START_REF] Mendel | Markov convexity and local rigidity of distorted metrics[END_REF]. As we discovered after writing a first draft of this paper, the method we use is very close to Johnson and Schechtman's proof of the distorsion estimate for diamond graphs [START_REF] Johnson | Diamond graphs and super-reflexivity[END_REF]. However, it seems not to have been noticed before that this method gives such an effective proof of Bourgain's estimate.

Proof. The first step is similar to previous proofs, notably the one by Matoušek's [START_REF] Matoušek | On embedding trees into uniformly convex Banach spaces[END_REF]. Let Y be the four-vertices tree with one root a 0 which has one child a 1 and two grandchildren a 2 , a ′ 2 . Lemma 2. There is a constant

K = K(X) such that if ϕ : Y → X is D-Lipschitz and distance non-decreasing, then either |ϕ(a 0 ) -ϕ(a 2 )| 2(D - K D p-1 ) or |ϕ(a 0 ) -ϕ(a ′ 2 )| 2(D - K D p-1
) * benoit.kloeckner@ujf-grenoble.fr

We provide the proof below for the sake of completeness.

Let now ϕ : T n → X a D-Lipschitz, distance nondecreasing map. By the lemma, the root a 0 has at least two grand-children

a i 2 (i = 1, 2) such that 2 |ϕ(a 0 ) -ϕ(a i 2 )| 2f (D)
where f (D) = D -K/D p-1 . Applying the lemma again, each of a i 2 also has two grand-children satisfying similar inequalities, and we can apply he same reasoning every other generation. Restricting ϕ to these vertices, we get an embedding of T ⌊ n 2 ⌋ whose distorsion is at most f (D). We can iterate these restrictions ⌊log 2 (n)⌋ times to get an embedding of T 1 whose distorsion is f ⌊log 2 (n)⌋ (D). This must be at least 1 and f D p /K (D) < 1, so that log 2 (n) D p which is Theorem 1.

Remark 3. Working out the constants gives the more precise result that

c X (T n ) pc 2 1 p (log 2 n) 1 p + l.o.t. (1)
where c can be replaced by lim inf δ X (ε)ε -p . In particular

c ℓ 2 (T n ) 1 2 √ 2 (log 2 n) 1 2 + l.o.t.
Proof of Lemma 2. Assume ϕ(a 0 ) = 0 and let x 1 = ϕ(a 1 ), x 2 = ϕ(a 2 ) and x ′ 2 = ϕ(a ′ 2 ). Suppose that |x 2 | 2(D -η) for some η to be chosen afterward; then by the triangle inequality, |x 1 | and |x 2 - Now we can choose η = K/D p-1 with K small enough to ensure that the above inequality reads |x 2 -x ′ 2 | < 2. This contradicts the hypothesis that ϕ is distance non-decreasing, therefore as desired |x 2 | or |x ′ 2 | must be smaller than 2(D -η).

x 1 | are at least D -2η. Define v = |x1| |x2-x1| (x 2 -x 1 ); then |x 1 + v -x 2 | = ||x 1 | -|x 2 -x 1 || 2η and |x 1 + v| |x 2 | -|x 1 + v -x 2 | 2D -4η.

The vectors x 1 /|x 1 | 1 p

 111 and v/|x 1 | have unit norm and their average has norm at least 1 -2η/D; letting ε = (2η/cD) the convexity assumption therefore yields |x 1 -v| εD. It follows that|2x 1 -x 2 | |x 1 + v -x 2 | + |x 1 -v| 2η + εD.Suppose that also |x ′ 2 | 2(D -η); then the same reasoning yields the same estimate on |x′ 2 -2x 1 | so that |x 2 -x ′ 2 | 4η + 2D 2η cD 1 p

Acknowledgments

I am grateful to Assaf Naor for interesting comments


Karim Chekroud

Laurent Guillou

Stephane Gre ´goire

Gilles Ducharme

Emilie Brun

Chantal Cazevieille

Lionel Bretillon

Christian P Hamel

Philippe Brabet
email: philippe.brabet@inserm.fr

Marie O Pequignot
email: marie.pequignot@inserm.fr

Fatp1 Deficiency Affects Retinal Light Response and Dark Adaptation, and Induces Age-Related Alterations

FATP1 is involved in lipid transport into cells and in intracellular lipid metabolism. We showed previously that this protein interacts with and inhibits the limiting-step isomerase of the visual cycle RPE65. Here, we aimed to analyze the effect of Fatp1-deficiency in vivo on the visual cycle, structure and function, and on retinal aging. Among the Fatp family members, we observed that only Fatp1 and 4 are expressed in the control retina, in both the neuroretina and the retinal pigment epithelium. In the neuroretina, Fatp1 is mostly expressed in photoreceptors. In young adult Fatp1 2/2 mice, Fatp4 expression was unchanged in retinal pigment epithelium and reduced two-fold in the neuroretina as compared to Fatp1 +/+ mice. The Fatp1 2/2 mice had a preserved retinal structure but a decreased electroretinogram response to light. These mice also displayed a delayed recovery of the b-wave amplitude after bleaching, however, visual cycle speed was unchanged, and both retinal pigment epithelium and photoreceptors presented the same fatty acid pattern compared to controls. In 2 yearold Fatp1 2/2 mice, transmission electron microscopy studies showed specific abnormalities in the retinas comprising choroid vascularization anomalies and thickening of the Bruch membrane with material deposits, and sometimes local disorganization of the photoreceptor outer segments. These anomalies lead us to speculate that the absence of FATP1 accelerates the aging process.

Introduction

In mammals, the cone and rod photoreceptors (PRs) are responsible for the transformation of light into an electrical signal that is transmitted to the brain. Adjacent to the PRs is the retinal pigment epithelium (RPE), which is essential for nutrition and detoxification of the PR among many other functions [START_REF] Simo | The retinal pigment epithelium: something more than a constituent of the bloodretinal barrier-implications for the pathogenesis of diabetic retinopathy[END_REF]. These two tissues are interdependent for their survival and the degeneration of one causes the degeneration of the other, leading to various forms of pigmentary retinopathies and often blindness [START_REF] Simo | The retinal pigment epithelium: something more than a constituent of the bloodretinal barrier-implications for the pathogenesis of diabetic retinopathy[END_REF][START_REF] Martinez-Morales | Eye development: a view from the retina pigmented epithelium[END_REF][START_REF] Xiao | Bestrophins and retinopathies[END_REF]. PRs comprise a cellular extension called an outer segment containing the visual pigment in membranous disks. These disks are renewed in a cyclical way from the base while the RPE phagocytes the oldest apical disks. If the phagocytosis process is blocked, membranous debris accumulate and PRs degenerate [START_REF] Kevany | Phagocytosis of retinal rod and cone photoreceptors[END_REF]. Due to the permanent renewal of their disks, PRs need a high supply in fatty acids, in particular in docosahexaenoic acid (DHA, 22:6n-3), and are mainly dependent on the RPE for this supply [START_REF] Bazan | Cell survival matters: docosahexaenoic acid signaling, neuroprotection and photoreceptors[END_REF].

In the rod PRs, the visual pigment rhodopsin is comprised of a protein, opsin, attached to a chromophore, 11-cis retinal. When rhodopsin is activated by photon absorption, the retinal molecule is isomerized into all-trans retinal and secondarily detached from the protein. The retinoid is then exported toward the RPE. There, a lecithin-retinol-acyl transferase (LRAT) esterifies the retinoid into all-trans retinyl ester by addition of a long chain fatty acid (often a palmitate). The resulting retinyl ester is the substrate of RPE65, the RPE isomerase that frees the fatty acid and transforms the retinoid back into 11-cis retinol. After oxidation, the formed 11-cis retinal is transported back to the PRs to form fresh rhodopsin. This metabolism is called the visual cycle, and many actors participate in this process of which the rate-limiting step is the isomerization catalyzed by RPE65 [START_REF] Simo | The retinal pigment epithelium: something more than a constituent of the bloodretinal barrier-implications for the pathogenesis of diabetic retinopathy[END_REF][START_REF] Jin | Rpe65 is the retinoid isomerase in bovine retinal pigment epithelium[END_REF][START_REF] Moiseyev | RPE65 is the isomerohydrolase in the retinoid visual cycle[END_REF]. Mutations in many of the genes encoding visual cycle proteins alter the visual process, leading to various forms of retinal degenerations or stationary disorders [START_REF] Thompson | Vitamin A metabolism in the retinal pigment epithelium: genes, mutations, and diseases[END_REF][START_REF] Sparrow | The retinal pigment epithelium in health and disease[END_REF][START_REF] Wu | Photoreceptor cell degeneration in Abcr (2/2) mice[END_REF]. In addition, the visual cycle generates retinoid by-products such as N-retinylidene-N-retinylethanolamine (A2E), which interfere with the normal function, in particular lysosomal function, of the RPE. With age, this can lead to the accumulation of lipofuscin, composed of residues generated by incomplete lysosomal digestion in the RPE, which leads to retinal degeneration.

In order to identify novel regulators of the visual cycle, using two-hybrid experiments, we previously showed that the visual cycle enzymes RPE65 and LRAT physically interact with Fatty Acid Transport Protein 1 (FATP1), an acyl-coA synthase with broad specificity for both long (such as palmitate) and very long chain fatty acids. Systemically, FATP1 is expressed in white adipose tissue, skeletal muscle, heart, brain and skin [START_REF] Doege | Protein-mediated fatty acid uptake: novel insights from in vivo models[END_REF]. FATP1 prevents fatty acid efflux from the cell and as a consequence promotes further transport into the cell. In the retina, the protein is expressed in both RPE and neuroretina (NR) [START_REF] Guignard | FATP1 inhibits 11-cis retinol formation via interaction with the visual cycle retinoid isomerase RPE65 and lecithin:retinol acyltransferase[END_REF]. Our previous studies showed that, in vitro, the interaction between FATP1 and RPE65 and LRAT inhibits 11-cis retinol. In vivo, this inhibitory mechanism could be exploited to regulate the visual cycle, with an increase in FATP1 activity being used to slow the cycle down. This property may decrease the A2E visual cycle byproduct that abnormally accumulates in certain retinal diseases.

In this study, we used Fatp1-deficient mice to test the role of the protein in the visual cycle and on visual function. We found that PR of young Fatp1 2/2 mice had a weaker response to light and that recovery of the visual sensitivity after bleaching was slightly delayed. A thorough analysis of the Fatp1 2/2 mouse phenotype also showed that old mice had PR outer segment disorganization, irregular Bruch membrane (BM) and abnormal choroid vascularization. These observations suggest that Fatp1 deficiency could induce an accelerated aging of the retina.

Materials and Methods

Mice

All animals were handled in strict accordance with the ARVO Statement for the Use of Animals in Ophthalmic Research and with EU directives. Approval from an Ethical Committee is not obligatory in France until 2013. The animal house and the project head must have a permit from the "Direction De ´partementale des Services Ve ´te ´rinaires" and all persons handling the mice must be specifically trained. In this work, all protocols were performed under the personal permit number 34-331 (MOP) and the animal house permit number B34-17236. Moreover, all efforts have been made to avoid suffering. Fatp1 +/2 mice, generated on a mixed C57BL/6J x 129SvEv genetic background, were kindly provided by Andreas Stahl (Berkeley, California, USA). These mice carried a leucine residue at aa position 450 in Rpe65. They were bred (heterozygous x heterozygous) in the INM animal house in clear plastic cages, subjected to standard light cycles (12 h to 90 lux light, 12 h dark) and were fed ad libitum with a standard rodent diet. The genotyping was conducted as previously described [START_REF] Kim | Inactivation of fatty acid transport protein 1 prevents fat-induced insulin resistance in skeletal muscle[END_REF].

Quantitative PCR (qPCR) assay

Five mo-old Fatp1 +/+ and Fatp1 2/2 mice were euthanised by cervical dislocation, the eyes were enucleated and dissected between 9 and 11 a.m. The NR was separated from the RPEchoroid, and then the RPE-choroid was scraped off from the sclera. We chose to scrape off both tissues rather than to enzymatically separate the RPE alone, to minimize the time and the manipulation between the death of the mice and the removal of the tissue. To obtain sufficient quantities of RNA, the tissues from all mice were pooled for each tissue type and total RNA was isolated by the RNeasy mini kit (Qiagen) according to the manufacturer's protocol. Equivalent amounts of total RNA were used for first strand cDNA synthesis using the Verso(TM) cDNA Kit (ThermoScientific). qPCR was performed using Light Cycler FastStart DNA MasterPLUS SYBR Green I kit (Roche). Amplifications from NR were normalized to actin levels, and those from RPE-choroid were normalized to the RPE-specific gene Mertk, in order to avoid an incorrect estimation due to the presence of choroid RNA. Primers used for amplification are summarized in Table 1.

Histology

All animals were sacrificed by vertebral dislocation. Eyes were rapidly enucleated and fixed in 4% PFA, 24 h at 4uC. Eye cups were i) embedded in paraffin and cut into 5 mm sagittal sections, ii) embedded in 5% agarose and cut into 50 mm sagittal sections, or iii) soaked in 40% sucrose, embedded in freezing medium (OCT) and cut into 10 mm sagittal sections. For HES coloration, sections were deparaffined, labeled with hematoxilin/eosin/safran, rinsed and mounted in Moeviol. For betagalactosidase staining, agarose sections were transferred into the freshly prepared staining solution (200 mM MgCl2, 500 mM K4Fe(CN)6, 500 mM K3Fe(CN)6, 4% X-gal, 2% NP-40, in 1X PBS), kept in the dark and incubated at 30uC for 1 hour. Staining was stopped by several washing in PBS. Oil red O coloration was performed as previously described [START_REF] Provost | Morphologic and electroretinographic phenotype of SR-BI knockout mice after a long-term atherogenic diet[END_REF]. Eye sections with demonstrated lipid accumulation were used as positive controls and kindly provided by Alexandra Provost (CERTO, Paris, France) [START_REF] Provost | Morphologic and electroretinographic phenotype of SR-BI knockout mice after a long-term atherogenic diet[END_REF].

Transmission electron microscopy (TEM)

Eyes were rapidly enucleated and the corneas were removed prior to immersion in a solution of 3.5% glutaraldehyde in Sorensen's buffer (0.1 M, pH 7.4) overnight at 4uC. The tissues were then rinsed in Sorensen's buffer and post-fixed in a 1% osmic acid for 2 h in the dark at room temperature. After two rinses, the tissues were dehydrated in a graded series of ethanol solutions (30-100%) and embedded in EmBed 812 using an Automated Microwave Tissue Processor for Electronic Microscopy, Leica EM AMW. All samples were processed the same way: a part of the eye was cut in a triangle with its summit at the optic nerve and the base at the periphery. Cutting was always initiated from the summit. The sections observed were thus all near the optic nerve. Sections (60 nm thickness; Leica-Reichert Ultracut E) were counterstained with uranyl acetate and observed using a Hitachi 7100 TEM (Centre de Ressources en Imagerie Cellulaire de Montpellier, France). This study was not quantitative as we observed few sections from one part of each eye. However, we evaluated the thickness of the Bruch's membrane by measuring both the thickest and the thinnest parts in 6 fields throughout the retinal section. We then calculated the median value for each eye.

Electrophysiology

All electrophysiological examinations were conducted using the Visiosystem (SIEM, France). Animals were prepared and electro-retinograms (ERGs) were performed with cotton electrodes as previously described [START_REF] Chekroud | Simple and efficient: validation of a cotton wick electrode for animal electroretinography[END_REF]. For adaptation-ERG, the mice were subjected to seven repetitions of a 1.59 cd.s 21 .m 22 blue flash, the seven b-wave amplitudes were averaged and considered as darkadapted control. The mice were then bleached 2 min at 300 lux, placed again in the dark and subjected to the same series of flashes to verify the abolition of the b-wave. This series of flashes was repeated every 4 min from 0 to 32 min, to observe the recovery of the b-wave.

Rhodopsin quantification

All manipulations were performed under dim red light. Immediately after cervical dislocation of the mice, eyes were enucleated, and homogenized in 1 ml of PBS, pH 7.2 and centrifuged at 140,000 g, 4uC for 20 min. The pellet was resuspended in 1 ml of PBS pH 7.2 with 1% Triton X-100 and then incubated at 4uC for 1 h. The homogenate was centrifuged at 140,000 g, 4uC for 20 min. Ten ml of hydroxylamine was added to the supernatant to achieve a final concentration of 20 mM, and absorption spectra of the samples were obtained before and after complete bleaching with a 60-W incandescent bulb. The concentration of rhodopsin was determined by the decrease in absorption at 500 nm and the molar extinction of rhodopsin (e = 42,000 liter/mol/cm).

Retinoid quantification

Pupillary dilatation was obtained with one instillation of 0.5% tropicamide (Mydriaticum, The ´a, France) in each eye. Animals were sacrificed by cervical dislocation before bleaching (5000 lux, 20 min) or at different times thereafter (0, 5, 15, 30, 60 and 120 min). The eyes were rapidly enucleated, frozen in liquid nitrogen and conserved at 280uC until use. The extraction was performed as described [START_REF] Suzuki | A simple procedure for the extraction of the native chromophore of visual pigments: the formaldehyde method[END_REF] with a few modifications: the eyes were homogenized in 800 ml of 3 M formaldehyde; after incubation, 1.5 ml of dichloromethane was added; the volume of hexane added was 3 ml; the extracts were dissolved at the end in 20 ml of ethanol for HPLC. The analyses were performed with a Varian HPLC system equipped with a C18 Isis column (4.66250 mm) (Macherey-Nagel) and a Prostar 330 diode array detector. The retinoids were quantified from the peak areas using calibration curves determined with established standards. Fatty acid profile analysis of the retina At the age of 4 and 8 months, the mice were euthanised, the eyes enucleated, and the NR removed. RPE-choroid was scraped and collected in 1X PBS, then centrifuged to remove the PBS. Tissues were stored dry at 280uC. Lipids were extracted [START_REF] Folch | A simple method for the isolation and purification of total lipides from animal tissues[END_REF] and fatty acids were transmethylated [START_REF] Morrison | Preparation of Fatty Acid Methyl Esters and Dimethylacetals from Lipids with Boron Fluoride-Methanol[END_REF]. Fatty acid methyl esters were analyzed by gas chromatograph (5890 series II; Hewlett-Packard, Palo Alto, CA) equipped with a split/splitless injector, a flame-ionization detector, and a CPSil88-silica capillary column (100 m60.25 mm ID, film thickness 0.20 mm; Varian). The injector and the detector were maintained at 250uC and 280uC, respectively. Hydrogen was used as a carrier gas (inlet pressure, 210 kPa). The oven temperature was fixed at 60uC for 1 minute, increased to 85uC at a rate of 3uC/min and then to 190uC at a rate of 20uC/min and left at this temperature for 65 minutes.

Fatty acid methyl esters were identified by comparison with commercial standards.

Autofluorescence measurements

For each mouse, one eye was homogenized two times 10 minutes in 1X PBS buffer without MgCl 2 using bead-filled tubes (MagNA Lyser, Roche). Tubes were centrifuged 10 minutes at 100 g to separate the scleral debris. The fluorescence in the supernatant was measured using a Safire II micro plate reader with an excitation of 405 nm and an emission of 520 to 640 nm. The values are expressed in arbitrary fluorescent units from the machine. For each mouse, the result is obtained by summing up all values between 520 and 640 nm taken every 10 nm.

Statistics

All data, except ERGs, were analyzed using the non parametric Mann & Whitney test due to the sample size, with a significant threshold level set at #5%. For ERGs, due to the complexity of the data, a Hotelling T-square test was performed to assess the overall difference between each pair of mean vectors from the longitudinal data. In those cases where significance was #5%, a Welch 2 sample T-test, corrected for multiple comparisons by the Bonferroni method, was performed at each time point to assess the difference between the two means. All computations where performed using the R package (http://www.r-project/org) [START_REF] Johnson | Applied Multivariate Statistical Analysis[END_REF].

Results

Fatp expression in the retina

We first examined the expression of all Fatp members in the RPE and NR of Fatp1 +/+ mice by qPCR. Only Fatp1 and Fatp4 were expressed in both tissues, with high levels detected in the RPE relative to Mertk, and in the NR, relative to Actin mRNA. Fatp4 predominated in the NR and Fatp1 in the RPE (Figure 1A).

To precisely determine the cells expressing Fatp1 among the neuroretinal cells, we took advantage of the fact that Fatp1 2/2 mice were generated by replacing the first coding exon of Fatp1, which encodes amino acids 1 to 56, with a targeting cassette containing an nlsLacZ sequence [START_REF] Kim | Inactivation of fatty acid transport protein 1 prevents fat-induced insulin resistance in skeletal muscle[END_REF]. We thus studied Fatp1 expression with a beta-galactosidase staining. Due to pigmentation, it was not possible to observe the labeling in RPE. In NR, the strongest labeling was observed in the PR cells (Figure 1B). The inner nuclear layer (INL) and ganglion cell layer (GCL) nuclei were also slightly labeled.

In Fatp1 2/2 mice, Kim et al. [START_REF] Kim | Inactivation of fatty acid transport protein 1 prevents fat-induced insulin resistance in skeletal muscle[END_REF] previously verified that there was no compensatory up-regulation of other members of the Fatp family (Fatp2 to 5, as Fatp6 was not yet discovered) in skeletal muscle and white adipose tissue, the two major sites of Fatp1 expression known at this time. We analyzed the expression of the 6 Fatp family members in the Fatp1 2/2 retina. Accordingly, Fatp1 expression was effectively at background levels in Fatp1 2/2 eyes (Figure 2A) and Fatp4 was the only member expressed consistent with our results in wt retina. However, Fatp4 expression was decreased in the Fatp1 2/2 NR (2-fold) as compared to wt whereas expression levels were unchanged in the RPE (Figure 2B). Fatp4 is the closest member of the family to Fatp1 but does not seem to compensate the absence of Fatp1 at the mRNA level.

Visual phenotype of young Fatp1 2/2 mice

We studied the visual phenotype of young 5 to 8 mo-old adult mice. The retinal histology of Fatp1 2/2 was similar to that of wt mice (Figure 3A) and we observed no differences with TEM (Figure 3B). PRs appeared similar in morphology and were present in the same number in both genotypes. We then assessed the functionality of the wt and Fatp1 2/2 retinas by ERG recordings. The light stimulus increased gradually from an only-rod stimulation to a mixed rod-cone stimulation. Latencies were similar in wt and Fatp1 2/2 mice. In contrast, the amplitudes of the ERG responses were weaker in Fatp1 2/2 mice than in wt, with the amplitude differences Fatp1 2/2 vs Fatp1 +/+ being more pronounced for the highest light intensities (maximum b-wave amplitude = 88.9% of the wt; Figure 3C). This difference appears to be due to a dysfunction of the PRs (a-wave) and not of the interneurons (b-wave) as the ratio of b-wave over a-wave was not decreased in Fatp1 2/2 mice (Figure 3D). It was not related to a decrease in photon catch by Fatp1 2/2 PRs as active rhodopsin levels were the same in both genotypes (Figure 3E).

To examine whether the decrease in ERG responses could be related to a dysfunction of the visual cycle, we first verified whether the absence of Fatp1 deregulated the expression of three of the main genes involved in the visual cycle, i.e. Rpe65, Lrat and Rdh5 (Figure 4A). In the absence of Fatp1, the expression of the 3 genes was only slightly (but significantly) increased by 9.6%, 12.5% and 19.5% respectively. We then studied the effect of the absence of Fatp1 on the recovery of the b-wave after bleaching. In the Fatp1 2/2 mice, we observed a delay in the first minutes of recovery followed by normalization at 16 min (Figure 4B). This could be explained by a delay in the regeneration of the chromophore 11-cis retinal in the RPE. Therefore, we measured this regeneration directly by measuring the level of each retinoid before and after bleaching during re-adaptation in the dark and observed no differences between Fatp1 +/+ and Fatp1 2/2 retinal extracts at any timepoint (Figure 4C). These results suggested that the lack of Fatp1 does not have a strong impact on chromophore regeneration. Thus, the difference in b-wave recovery observed in vivo between wt and Fatp1 2/2 mice did not seem to be due to a major deregulation of the visual cycle.

Fatty acid composition in the retina of Fatp1 2/2 mice

We observed an approximately 10% decrease in the ERG amplitudes and a delayed ERG b-wave amplitude recovery after saturating flash in Fatp1 2/2 mice as compared to wt. These abnormalities were reminiscent of observations made on n-3 fatty acid-deficient animals. The fatty acid docosahexaenoic acid (DHA, 22:6n-3) is highly enriched in membrane phospholipids of the brain and retina, and in particular PR outer segments. Reduced ERG amplitudes have been reported in animals raised in DHA precursor-deficient diets, and a delayed recovery of ERG bwave amplitude after a b-wave saturating flash has been observed [START_REF] Bush | Dietary deficiency of N-3 fatty acids alters rhodopsin content and function in the rat retina[END_REF][START_REF] Neuringer | Biochemical and functional effects of prenatal and postnatal omega 3 fatty acid deficiency on retina and brain in rhesus monkeys[END_REF][START_REF] Pawlosky | Retinal and brain accretion of long-chain polyunsaturated fatty acids in developing felines: the effects of corn oil-based maternal diets[END_REF]. This could be explained by reduced rhodopsin efficiency due to lower protein diffusion in the membrane of the discs. As Fatp1 is absent and Fatp4 less expressed in the NR of Fatp1 2/2 mice, it was possible that the lipid composition of the PR outer segments was abnormal, leading to photoreceptor dysfunction. We therefore analyzed the fatty acid composition of the Fatp1 2/2 and Fatp1 +/+ NR and RPE for 32 fatty acids at the age of 4 and 8 months. No changes of the fatty acid composition were seen in the neural retina, which include PRs, nor in the RPE (Figure 5).

Histological anomalies in the retina of old Fatp1 2/2 mice

We analyzed the retinal structure and function of 22 to 26 moold wt and Fatp1 2/2 mice. Both genotypes mice had lower ERG responses compared to young animals (Figure 3C). However, responses of older Fatp1 2/2 mice remained lower than that of agematched controls (maximum b-wave amplitude = 89.8% of wt; Figure 6A). We found no evidence of abnormal dark adaptation of the Fatp1 2/2 mice after bleaching, but this was possibly due to the small number of old animals available for this protocol (3 Fatp1 +/+ and 4 Fatp1 2/2 ). We assayed for the presence of lipofuscin, an autofluorescent byproduct of the visual cycle that accumulates with age and is often observed in age-related macular degeneration (AMD) retina. However, there was no difference in the amount of autofluorescence between wt and Fatp1 2/2 animals (Figure 6B). As age-dependent lipid accumulation has been previously described in Bruch membrane (BM) [START_REF] Provost | Morphologic and electroretinographic phenotype of SR-BI knockout mice after a long-term atherogenic diet[END_REF][START_REF] Schmidt-Erfurth | Ultrastructural changes in a murine model of graded Bruch membrane lipoidal degeneration and corresponding VEGF164 detection[END_REF], we searched for lipid droplet deposition in aged mice. However, Fatp1 2/2 or wt mice showed no Oil red O labeling in the BM (Figure 6C). We also studied the retinal structure by TEM. In both Fatp1 +/+ (Figure 7A) and Fatp1 2/2 mice, we observed classical hallmarks of retinal aging including a decrease in the number of rows of PR nuclei (varying from 5 to 10 rows, mean of 7.7 in Fatp1 +/+ and 8 in Fatp1 2/2 ), thickening of the BM, RPE thinning, enlarged or absent RPE basal infoldings and accumulation of lipofuscin granules. Furthermore, in addition to these common signs of aging, some Fatp1 2/2 mice (6/15) had PR outer segment disorganization (Figure 7B-F). In the sections analyzed, there were also greater variations in BM thickness in Fatp1 2/2 compared to Fatp1 +/+ mice (p,0.05; Figure 7L). Two thirds of the Fatp1 2/2 mice (10/15) exhibited an irregular BM or a BM containing laminar deposits (Figures 7A and7G-K). Most of the Fatp1 2/2 mice (12/15) showed strikingly modified choroidal vessels. In some cases (6/15), only large vessels were observed suggesting that small vessels had disappeared and in two mice (2/15), there were even no detectable vessels in the sections analyzed (Figure 7M-O). Yet, in other mice (4/15), choroidal vessels had a normal size but were more numerous, forming a continuous layer under the BM. All Fatp1 2/2 mice examined by TEM exhibited at least one of these particular additional abnormalities (Table 2).

Discussion

We previously demonstrated that FATP1 interacts in vitro with RPE65 and inhibits the isomerase activity [START_REF] Guignard | FATP1 inhibits 11-cis retinol formation via interaction with the visual cycle retinoid isomerase RPE65 and lecithin:retinol acyltransferase[END_REF]. As a follow up to this work, we analyzed the expression of every member of the Fatp gene family in the retina, and we studied Fatp1 2/2 mice of various ages to test the effect of Fatp1 deficiency on the visual phenotype and the visual cycle in vivo.

We demonstrated that only Fatp1 and 4 are expressed in NR and RPE, and that, in NR, Fatp1 is mostly expressed in the PR. Similarly, Fatp1 and 4 are the only members of the Fatp family expressed in adipose tissue and skeletal muscle, two tissues with a high lipid metabolism [START_REF] Doege | Protein-mediated fatty acid uptake: novel insights from in vivo models[END_REF]. This similarity is not surprising as the RPE is the major lipid provider for PRs, which need an important supply of lipids to enable the continuous renewal of their outer segment membranous disks. It has been shown that in skeletal muscle and adipose cells (but not in the heart [START_REF] Chabowski | The subcellular compartmentation of fatty acid transporters is regulated differently by insulin and by AICAR[END_REF]), Fatp1 translocates to the plasma membrane during postprandial periods and is responsible for the insulin-induced long chain fatty acid uptake. In contrast, Fatp4 participates in basal uptake [START_REF] Gimeno | Fatty acid transport proteins[END_REF]. In the RPE, we show that Fatp1 is more prominent than Fatp4. This is consistent with the fact that the RPE is in contact with the choroidal blood supply and thus with highly changing concentrations of fatty acids. In contrast, PRs receive their fatty acids from the RPE, probably in more regulated basal levels, thus explaining the higher levels of Fatp4 as compared to Fatp1 in the NR.

In Fatp1-deficient mice, Fatp4 expression is not increased in the retina as compared to controls. This is also the case for muscle and adipose tissues [START_REF] Kim | Inactivation of fatty acid transport protein 1 prevents fat-induced insulin resistance in skeletal muscle[END_REF] indicating that Fatp4 expression does not compensate for the lack of Fatp1 expression. Moreover, Fatp4 expression was, surprisingly lower in the Fatp1 2/2 NR compared to controls. A previous study on a pair of twins (1 obese, 1 lean) reported that Fatp4 expression is up-regulated by environmental factors such as, in this case, acquired obesity [START_REF] Gertow | Expression of fatty-acid-handling proteins in human adipose tissue in relation to obesity and insulin resistance[END_REF]. Therefore, Fatp4 could be less expressed in the Fatp1 2/2 NR, because the supply in fatty acid is reduced from the RPE, due to the absence of Fatp1, so that less Fatp4 is needed to facilitate the fatty acid entrance into the PR. However, we showed that this reduction had no significant effect on the lipid composition of the NR at 4 and 8 months of age.

Although FATP1 has been previously described as an actor of the visual cycle in vitro [START_REF] Guignard | FATP1 inhibits 11-cis retinol formation via interaction with the visual cycle retinoid isomerase RPE65 and lecithin:retinol acyltransferase[END_REF], in the present study, we showed that the Fatp1-deficient mice do not display a decreased formation of 11-cis retinal after bleaching. This could be explained by compensation between Fatp1 and Fatp4 proteins, which are 60% homologous. Phylogenetic analyses reveals that Fatp1 and Fatp4 are both the orthologs of a single Drosophila fatp gene, suggesting a close function for Fatp1 and Fatp4 [START_REF] Hirsch | A family of fatty acid transporters conserved from mycobacterium to man[END_REF]. Interestingly, fatp deficiency causes the loss of photoreceptors by a caspasedependent death, suggesting an important role for fatp in PR survival [START_REF] Gambis | Two-color in vivo imaging of photoreceptor apoptosis and development in Drosophila[END_REF]. Transcomplementation analysis to rescue PR degeneration in Drosophila fatp mutants by expressing mouse Fatp1 or Fatp4 genes would be necessary to address the functional relationship of Fatp1 and/or Fatp4 with fatp.

We observed a decrease in the ERG amplitudes and a slower recovery of the b-wave amplitude after bleach in the Fatp1 2/2 mice. We hypothesized that these ERG anomalies were due to disequilibrium in the fatty acid composition. However, fatty acids were not differentially distributed in NR and RPE of Fatp1 2/2 and Fatp1 +/+ , suggesting that in the absence of Fatp1, the fatty acid supply remains sufficient. In addition, as Fatp1 2/2 mice fed a normal diet do not exhibit anomalies in intramuscular fatty acid metabolites, insulin sensitivity, body composition (lean and fat mass) or plasma parameters (glucose, insulin, fatty acids, triacylglycerol) [START_REF] Kim | Inactivation of fatty acid transport protein 1 prevents fat-induced insulin resistance in skeletal muscle[END_REF], it is likely that these differences were due to a specific effect on the eye. One possibility is that the absence of Fatp1 could modify the mitochondrial metabolism. It was previously shown that a part of the cell's Fatp1 content localizes to the outer mitochondrial membrane and that its overexpression in muscle increases the glucose uptake and the PDH activity [START_REF] Guitart | FATP1 localizes to mitochondria and enhances pyruvate dehydrogenase activity in skeletal myotubes[END_REF][START_REF] Sebastian | Novel role of FATP1 in mitochondrial fatty acid oxidation in skeletal muscle cells[END_REF]. Moreover, Fatp1 directs fatty acids towards oxidation, and not storage, in skeletal muscle, liver and adipose tissue [START_REF] Ellis | Adipose acyl-CoA synthetase-1 directs fatty acids toward beta-oxidation and is required for cold thermogenesis[END_REF][START_REF] Holloway | Increasing skeletal muscle fatty acid transport protein 1 (FATP1) targets fatty acids to oxidation and does not predispose mice to diet-induced insulin resistance[END_REF][START_REF] Li | Liver-specific loss of long chain acyl-CoA synthetase-1 decreases triacylglycerol synthesis and betaoxidation and alters phospholipid fatty acid composition[END_REF], all tissues with a high lipid metabolism as in the retina. Fatp1 also directly increases the fatty acid transport in the mitochondria, where it interacts physically with the carnitine palmitoyltransferase 1 (CPT1) [START_REF] Sebastian | Novel role of FATP1 in mitochondrial fatty acid oxidation in skeletal muscle cells[END_REF], the enzyme that passes the co-A fatty acids towards both membranes of the mitochondria. The absence of Fatp1 could therefore decrease the efficacy of the fatty acid oxidation by the mitochondria and thus the production of energy. As the retina is highly in demand of energy for phototransduction, ionic transport and metabolism, a moderate decrease in its mitochondrial supply could result in functional modifications as observed in the Fatp1 2/2 mice.

Aging retinas of Fatp1-deficient mice showed strikingly abnormal features, with some individual variations that could be explained by the mixed C57BL/6J 6129SvEv genetic background. There were abnormal depositions of membranous materials into the BM, with a rather diffuse accumulation within and along the BM. This is reminiscent of basal laminar deposits observed in human agerelated macular degeneration (AMD) [START_REF] Sarks | Relationship of Basal laminar deposit and membranous debris to the clinical presentation of early agerelated macular degeneration[END_REF]. Similar deposits are also observed in mouse models of AMD fed with high fat diet such as ApoE 2/2 , CD36 2/2 , SR-BI 2/2 , LDLR 2/2 mice [START_REF] Provost | Morphologic and electroretinographic phenotype of SR-BI knockout mice after a long-term atherogenic diet[END_REF][START_REF] Dithmar | Ultrastructural changes in Bruch's membrane of apolipoprotein E-deficient mice[END_REF][START_REF] Houssier | CD36 deficiency leads to choroidal involution via COX2 down-regulation in rodents[END_REF][START_REF] Picard | CD36 plays an important role in the clearance of oxLDL and associated age-dependent subretinal deposits[END_REF][START_REF] Rudolf | Increased expression of vascular endothelial growth factor associated with accumulation of lipids in Bruch's membrane of LDL receptor knockout mice[END_REF][START_REF] Zhao | Age-related retinopathy in NRF2-deficient mice[END_REF]. In LDLR 2/2 and SR-BI 2/2 mouse models, lipid droplets were found in BM that are labeled with oil Red O [START_REF] Provost | Morphologic and electroretinographic phenotype of SR-BI knockout mice after a long-term atherogenic diet[END_REF][START_REF] Schmidt-Erfurth | Ultrastructural changes in a murine model of graded Bruch membrane lipoidal degeneration and corresponding VEGF164 detection[END_REF]. We did not observe such droplets (nor oil Red O labeling) but more diffuse accumulation within and along the BM, the composition of which remains to be elucidated.

In addition to BM deposits, Fatp1 2/2 mice had disorganization of PR and abnormal choroidal vessels. In humans, it is believed that extracellular deposits in BM lead to reduced nutriment transport and to secondary injury of the RPE, choroid and PR. Indeed, the majority of the Fatp1-deficient mice (12/15) presented an abnormal choroidal vascularization. Many of them had only large vessels or no vessels at all. In both cases, the capillaries of the choroid had disappeared, an observation that was also reported in the CD36 2/2 model of AMD [START_REF] Houssier | CD36 deficiency leads to choroidal involution via COX2 down-regulation in rodents[END_REF]. Conversely, a smaller number of mice featured an almost continuous layer of capillaris under the BM. This could represent a first step towards breaking of the BM and neovascularization into the subretinal space, as occurs in wet AMD. In contrast to previously described AMD mouse models involving lipid metabolism such as ApoE 2/2 , CD36 2/2 , SR-BI 2/2 , LDLR 2/2 mice [START_REF] Provost | Morphologic and electroretinographic phenotype of SR-BI knockout mice after a long-term atherogenic diet[END_REF][START_REF] Dithmar | Ultrastructural changes in Bruch's membrane of apolipoprotein E-deficient mice[END_REF][START_REF] Houssier | CD36 deficiency leads to choroidal involution via COX2 down-regulation in rodents[END_REF][START_REF] Picard | CD36 plays an important role in the clearance of oxLDL and associated age-dependent subretinal deposits[END_REF][START_REF] Rudolf | Increased expression of vascular endothelial growth factor associated with accumulation of lipids in Bruch's membrane of LDL receptor knockout mice[END_REF][START_REF] Zhao | Age-related retinopathy in NRF2-deficient mice[END_REF], Fatp1-deficient mice did not show drusen and oil red O labeling or abnormal lipofuscin accumulation. However, it should be stressed that these mouse models require high fat diet (often used to mimic the human alimentation in industrialized countries) for several months in order to display these anomalies [START_REF] Provost | Morphologic and electroretinographic phenotype of SR-BI knockout mice after a long-term atherogenic diet[END_REF][START_REF] Picard | CD36 plays an important role in the clearance of oxLDL and associated age-dependent subretinal deposits[END_REF][START_REF] Rudolf | Increased expression of vascular endothelial growth factor associated with accumulation of lipids in Bruch's membrane of LDL receptor knockout mice[END_REF]. Additional investigations with Fatp1-deficient mice subjected to high fat diet would be necessary to examine whether or not they develop these abnormalities.

In conclusion, we showed that Fatp1-deficient mice exhibit accelerated aging of the outer retina, and that these features were not linked to abnormal visual cycle kinetics or to lipid composition of photoreceptor and RPE.

Figure 1 .Figure 2 .

 12 Figure 1. Expression of the Fatp family in the retina. A. Expression of the Fatp family in Fatp1 +/+ neuroretina (NR) and retinal pigment epithelium (RPE). Only Fatp1 and Fatp4 were expressed in both tissues. Fatp4 predominated in the NR and Fatp1 in the RPE. ND: not detected. Results are expressed as a percentage of Actin mRNA expression for NR and as a percentage of Mertk mRNA expression for RPE. mRNA was extracted from tissue pools of 12 mice aged 5 months. Bars represent the mean of triplicates 6 SEM. B. Fatp1 expression pattern using the LacZ cassette of the Fatp1 2/2 mouse. beta-galactosidase staining of the retinas of Fatp1 +/+ (WT; without LacZ cassette) and Fatp1 2/2 (KO; containing the LacZ cassette) mice. In neuroretina, Fatp1 is strongly expressed in photoreceptors, and slightly expressed in the inner cells. POS: PR outer segments. PIS: PR inner segments. ONL: outer nuclear layer. OPL: outer plexiform layer. INL: inner nuclear layer. IPL: inner plexiform layer. GCL: ganglion cell layer. doi:10.1371/journal.pone.0050231.g001

Figure 3 .

 3 Figure 3. Visual phenotype of the Fatp1 2/2 mouse. A. Histology of the retina. Ch: choroid. RPE: retinal pigment epithelium. POS: PR outer segments. PIS: PR inner segments. ONL: outer nuclear layer. OPL: outer plexiform layer. INL: inner nuclear layer. IPL: inner plexiform layer. GCL: ganglion cell layer. WT: Fatp1 +/+ . KO: Fatp1 2/2 . B. TEM of WT and KO retinas. BM: Bruch membrane. C. Electroretinography of Fatp1 +/+ (n = 29) and Fatp1 2/2 (n = 23) mice. * p,0.05; ** p,0.01; *** p,0.001. The statistical analyses were performed initially for the whole curve (line above the graph) and subsequently for each point. D. b-wave on a-wave ratio of the ERG experiments. Error bars represent SEM. E. Quantification of the retinal coupled-rhodopsin (n = 4 Fatp1 +/+ and n = 4 Fatp1 2/2 ) in mice aged 5 to 8 months. Error bars represent SEM. The retinal histology is unchanged in the Fatp1 2/2 mice. Fatp1 2/2 PRs present a lower response to light flashes and this decrease is not due to a decrease in the level of retinal coupledrhodopsin in these animals. doi:10.1371/journal.pone.0050231.g003

Figure 4 .

 4 Figure 4. The visual cycle in 5 to 8 mo-old Fatp1 2/2 mice. A. Expression levels of the main visual cycle genes, Rpe65, Lrat and Rdh5. Bars represent the mean of triplicates 6 SEM. * p,0.05. The genes were slightly deregulated with a 9.6% increase observed for Rpe65, a 12.5% increase for Lrat and a 19.5% increase for Rdh5. B. Recovery of the b-wave amplitude after bleaching in 5 to 8 mo-old mice (n = 29 Fatp1 +/+ and n = 21 Fatp1 2/2 ). Error bars represent SEM. * p,0.05. C. Quantification of the 11-cis retinal and all-trans retinal levels in the whole eye. Error bars represent SEM. The recovery of the electroretinogram is delayed in Fatp1 2/2 mice but this difference is not linked to a visual cycle kinetic defect. doi:10.1371/journal.pone.0050231.g004

Figure 5 .

 5 Figure 5. Fatty acid composition in the NR and RPE in 8 mo-old Fatp1 +/+ and Fatp1 2/2 mice. Fatp1 +/+ (n = 5) and Fatp1 2/2 (n = 7). 16:0: palmitate. 22:6n-3: DHA. The functional visual difference between Fatp1 2/2 and Fatp1 +/+ mice is not due to a different fatty acid composition. doi:10.1371/journal.pone.0050231.g005

Figure 6 .

 6 Figure 6. Aging of the Fatp1 2/2 retina. A. Electroretinography of Fatp1 +/+ (n = 5) and Fatp1 2/2 (n = 11) mice, * p = 0.021 for the whole curve. B. Quantification of autofluorescence in young (n = 3 Fatp1 +/+ and n = 4 Fatp1 2/2 ) and old (n = 6 Fatp1 +/+ and n = 5 Fatp1 2/2 ) mice. The values are expressed in arbitrary fluorescent units from the machine. For each mouse, the result is obtained by summing up all values between 520 and 640 nm taken every 10 nm. Error bars represent SEM. Fatp1 2/2 PRs remains less responsive to light with age. Fatp1 2/2 mice do not accumulate more autofluorescence, thus presumably more lipofuscin, than Fatp1 +/+ with age. C. Upper panel: neutral lipid labeling of Fatp1 +/+ (WT) and Fatp1 2/2 (KO) retinas. Ch: choroid. RPE: retinal pigment epithelium. POS: PR outer segments. PIS: PR inner segments. ONL: outer nuclear layer. OPL: outer plexiform layer. INL: inner nuclear layer. IPL: inner plexiform layer. GCL: ganglion cell layer. Arrows indicate the BM. Lower panel: positive control at the same magnification (see Material and methods). Arrows indicate the BM as in the upper panel. The asterisk indicates positive ORO labeling. Fatp1 2/2 or Fatp1 +/+ mice showed no Oil red O labeling in the BM. doi:10.1371/journal.pone.0050231.g006

Figure 7 .

 7 Figure 7. Ultrastructural aging of the Fatp1 2/2 retina. A: TEM at low magnification of a Fatp1 +/+ retina. PR outer segments in Fatp1 2/2 at low magnification (B), in Fatp1 +/+ at high magnification (C), in Fatp1 2/2 mice at high magnification (D-F). Certain Fatp1 2/2 mice had PR outer segment disorganization. G: Fatp1 2/2 retina at low magnification showing a thick BM. H: Fatp1 +/+ BM at high magnification. I-K: Fatp1 2/2 retina at high magnification showing a thicker BM than age-matched control. L: Box plot graph showing the thickness of the BM between Fatp1 +/+ and Fatp1 2/2 retinas (solid line indicates the median; p,0.05). M-O: Fatp1 2/2 retinas at low magnification showing numerous vessels along the BM (M), no vessels (N) and large vessels (O). Arrows indicate the thickness of the BM. Asterisks indicate vessels. The majority of Fatp1 2/2 mice exhibited an irregular or laminar thicker BM. Most of the Fatp1 2/2 mice showed strikingly modified choroidal vessels, ranging from presence of only large vessels to absence of vessels. doi:10.1371/journal.pone.0050231.g007

Table 1 .

 1 Sequences of the qPCR primers.

	Gene		Primer sequence
	Fatp1	F	59-AGGTCAATGAGGACACGATGGAG-39
		R	59-CTGGTACATTGAGTTAGGGTCCAAC-39
	Fatp2	F	59-GCCAAGTCTCTGCTGCACTGC-39
		R	59-CTTCAGACCTCCACGACTCCG-39
	Fatp3	F	59-CCAGAACATAGAGTGGGTCAGACA-39
		R	59-CTTCACTTCCACGATCGTACTGG-39
	Fatp4	F	59-CTGAAGCTGCCCTGGACCCA-39
		R	59-AGGGCATCCCGCCTAAGGTTG-39
	Fatp5	F	59-CAAGTTGGGCTGCCCTGTGGC-39
		R	59-GCCCTATCGTATGGTACAGAGG-39
	Fatp6	F	59-CCAACCTTCGCTTCGATTCCCT-39
		R	59-CATCAGAGGCGAGACTCAGCTT-39
	Rpe65	F	59-GTGCCACTGCTCATCCACATATTG-39
		R	59-TGCAGGGGAACTGCACAACAACT-39
	Rhodopsin	F	59-CGCTTCGGGGAGAATCA-39
		R	59-GTAGGGAAGCCAGCAGAT-39
	Lrat	F	59-GAGCAGCAGTTGGGACTGACT-39
		R	59-TCCCAAGACAGCCGAAGCAAGA-39
	Rdh5	F	59-TCACCAGTGTCTTGGGCCGCA-39
		R	59-AGGTTGGTCACAGGGGTTCGAA-39
	Mertk	F	59-CAGTTTTATCCTGATGAGGAAGG-39
		R	59-GAAGGCTGTGTTTCTGGTGAC-39
	Actin	F	59-CTGGGC CTC GTC ACC CAC ATA-39
		R	59-GACCCAGATCATGTTTGAGACCTT-39

doi:10.1371/journal.pone.0050231.t001

Table 2 .

 2 Abnormalities observed in aged mice by TEM.

		Fatp1 +/+ (n = 9)	Fatp1 2/2 (n = 15)
	Abnormal choroidal vessels	0/9	6/15 with only large vessels 2/15 with no vessels 4/15 with continuous
			vascular layer
	Abnormal Bruch membrane	0/9	10/15
	Disorganized PR outer segments	0/9	6/15

doi:10.1371/journal.pone.0050231.t002

PLOS ONE | www.plosone.org

November 2012 | Volume 7 | Issue 11 | e50231

Acknowledgments

The authors are grateful to Ce ´cile Sanchez, Isabelle Sassetti, Marie-Line Loyalle, Chantal Jacquet and David Cia for their technical assistance, to Drs Alexandra Provost and Karine Bigot for material providing. The authors are very grateful to Dr Vasiliki Kalatzis for her repeated critical reading of the manuscript.

This work was supported by private foundations (Fe ´de ´ration des Aveugles et Handicape ´s Visuels de France, IRRP, UNADEV, SOS Re ´tinite), University of Montpellier (UM1 and UM2), the Re ´gion Languedoc Roussillon, the French Ministry for National Education and Inserm. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contributions