Dan Mejlachowicz
email: mejlachowicz.dan@hotmail.fr

Patricia Lassiaz
email: patricia.lassiaz@gmail.com

Marta Zola
email: marta.zola9@gmail.com

Bastien Leclercq

Emmanuelle Gélizé
email: emmanuelle.gelize@gmail.com

Seiki Achiedo
email: seiki.achiedo@u-paris.fr

Min Zhao

Antoine Rousseau
email: arousseau1010@gmail.com

Francine Behar-Cohen
email: francine.behar@gmail.com

Identification of structures labeled by indocyanine green (ICG) in the rat choroid and retina can guide interpretation of ICG angiography Running

Keywords: indocyanine green, angiography, retina, choroid, choroidal nerve, mast cells, retinal pigment epithelium Video 2 253 distance: 78 µm, pixel size: 1 µm. Excitation wavelength: DAPI, 405 nm, ICG, 700 nm

Purpose: Indocyanine green (ICG) is an albumin and lipoprotein binding dye absorbing in the far red used in angiography to visualize choroidal vessels (ICGA). To guide interpretation, ICG transport in the choroid, retinal pigment epithelial (RPE) and retina of rats was studied.

Methods: Two conditions were used: RPE/choroid organoculture (OC), incubated for 45 min in DMEM medium, 1% FBS containing 0.25 mg/ml ICG and RPE/choroid and neural retina flat-mounts at 1 and 6 hours after intravenous ICG injection (IV). Early and late sequences of ICGA were recorded until 6 hours. Ultra-deep red confocal microscope was used to localize ICG in flat-mounts and immunohistochemistry was performed for caveolin-1 (CAV1), tryptase (mast cell marker) and tubulin β3 (TUBB3)(nerve marker).

Results: In the OC, ICG penetrated homogeneously in the cytoplasm and stained the membranes of the RPE. At 1h after IV injection, ICG appeared in fine granules in RPE, partly labeled with CAV1 and decreasing at 6h. At 1h and 6h, ICG was found in the retinal vessels, faintly in the inner retina and in photoreceptor outer segments at 6 hrs. In the choroid, ICG co-localized with mast cells, immunostained with tryptase and accumulated along the large TUBB3-labeled nerve bundles. Hypothesis was raised on the interpretation of late ICGA infrared photography in case of transthyretin amyloidosis with neuropathy.

Conclusions: Beside being a vascular dye, ICG is transported from the vessels to the RPE towards the outer retina. It stains mast cells and large choroidal nerves. These observations could help the analysis of ICG-A images.

Introduction

Indocyanine green angiography (ICGA) is used in clinical practice since more than 50 years, although its indication has decreased with the wide use of spectral domain optical coherence tomography (SD-OCT) and OCT angiography 1 since the main indication of ICGA is the visualization of choroidal neovascularization, often well identified by non-invasive imaging methods. Indeed, ICG is an amphiphilic, water soluble, tricarbocyanine dye with a molecular mass around 750 Da, that is >95% bound to HDL and LDL 2 (8) and to albumin after intravenous (IV) injection [START_REF] Moody | Non-covalent labeling of human serum albumin with indocyanine green: a study by capillary electrophoresis with diode laser-induced fluorescence detection[END_REF] . It is excited by wavelengths comprised between 750 and 800 nm and emits fluorescence that peaks at around 830 nm, avoiding interference with autofluorescence from surrounding tissues. Due to these chemical properties and its binding to plasma proteins and lipoproteins, ICG remains into normal choroidal vessels including the choriocapillaris and can be visualized through the pigmented retinal pigment epithelium, making it an ideal dye to image the choroidal vasculature with high contrast and sensitivity.

The clinical significance of ICGA is mostly based on the spatio-temporal localization of the fluorescence emitted by ICG after intravenous injection. The angiographic sequence allows to follow the very rapid filling of the arteries and choriocapillaris, followed by filling of the choroidal veins and drainage through the vorticose veins. Appearance of hyperfluorescence reflects leakage from choroidal vessels, whilst hypofluorescence indicates decreased perfusion or masking by absorbing structures but the kinetics of the hyper or hypofluorescence and their localization differs depending on the pathology and the disease mechanisms. We will not detail here the ICGA semiology that has been well reviewed by others who showed its value for diagnosis and even for prognosis in some chorioretinal diseases [START_REF] Cohen | Is indocyanine green angiography still relevant?[END_REF][START_REF] Kovacs | Quantitative Modeling of Ultra-Widefield Choroidal Indocyanine Green Angiography in Systemic Vascular Diseases[END_REF][START_REF] Borooah | Pachychoroid spectrum disease[END_REF][START_REF] Cheung | CHOROIDAL VASCULAR ALTERATIONS IN AGE-RELATED MACULAR DEGENERATION AND POLYPOIDAL CHOROIDAL VASCULOPATHY[END_REF] . When interpretating ICGA, beside its kinetics, several factors that influence ICG fluorescence should be considered, including its concentration and its binding to different surfaces. Particularly, the fluorescence intensity might increase at low ICG concentrations (1µM), but strikingly decrease at high concentrations (10µM) [START_REF] Cheung | CHOROIDAL VASCULAR ALTERATIONS IN AGE-RELATED MACULAR DEGENERATION AND POLYPOIDAL CHOROIDAL VASCULOPATHY[END_REF] .

Binding to albumin with high affinity might also increases ICG fluorescence [START_REF] Nairat | Investigating the role of human serum albumin protein pocket on the excited state dynamics of indocyanine green using shaped femtosecond laser pulses[END_REF] . This property was used to detect proteins in capillary electrophoresis as ICG is only weakly fluorescent in dilute aqueous solution, but its near infrared fluorescence is highly enhanced when it binds to proteins and specifically albumin [START_REF] Mačianskienė | Spectral characteristics of voltage-sensitive indocyanine green fluorescence in the heart[END_REF] . More recently, Jang et al used plasmon resonance and saturation binding assay to show that ICG fluorescence in vivo is higher for albumin-bound ICG (2.1-fold at 1h post-injection) [START_REF] Jang | Imaging of Indocyanine Green-Human Serum Albumin (ICG-HSA) Complex in Secreted Protein Acidic and Rich in Cysteine (SPARC)-Expressing Glioblastoma[END_REF] .

Beside visualization of the choroidal vasculature, ICGA has the potential to provide additional indirect information on the metabolic capabilities of the retinal pigment epithelium (RPE) and on the intracellular trafficking of albumin and lipoproteins in endothelial and in RPE cells. Chang et al. demonstrated that ICG is slowly internalized by RPE cells in vivo in the primate and in vitro in human RPE cells [START_REF] Chang | Histologic localization of indocyanine green dye in aging primate and human ocular tissues with clinical angiographic correlation[END_REF][START_REF] Chang | The interaction of indocyanine green with human retinal pigment epithelium[END_REF] . Various vesicular transports have been described in RPE cells for HDLs thar are internalized after binding to scavenger receptor class B type I (SR-BI), which is a mechanism to import xantophylls into the retina [START_REF] Thomas | Mechanisms of selective delivery of xanthophylls to retinal pigment epithelial cells by human lipoproteins[END_REF] and, for albumin and LDL through caveolin-mediated transcytosis since caveolin-1 is highly expressed in RPE cells [START_REF] Enyong | Caveolar and non-Caveolar Caveolin-1 in ocular homeostasis and disease[END_REF] . The vesicular transports could explain the kinetics of ICG internalization in RPE cells [START_REF] Chang | Histologic localization of indocyanine green dye in aging primate and human ocular tissues with clinical angiographic correlation[END_REF][START_REF] Chang | The interaction of indocyanine green with human retinal pigment epithelium[END_REF] . In endothelial cells, HDL partially co-localized with LDL, albumin, and transferrin in intracellular vesicles [START_REF] Perisa | Itinerary of high density lipoproteins in endothelial cells[END_REF] suggesting that ICG could also be internalized in endothelial cells through vesicular transporters. In other fields of medicine, ICG is used as an adjuvant to surgery to localize and preserve nerves and to perform lymphography [START_REF] Amara | Intraoperative angiography of the neurovascular bundle using indocyanine green and near-infrared fluorescence improves anatomical dissection during robot-assisted radical prostatectomy: initial clinical experience[END_REF][START_REF] Patel | Endoscopic-Indocyanine Green Angiography Assisted Microtia Reconstruction[END_REF] since ICG labels nerves [START_REF] Géczi | Near-infrared fluorescence guided surgery: State of the evidence from a health technology assessment perspective[END_REF] , which network is very dense in the human choroid [START_REF] Leclercq | The choroidal nervous system: a link between mineralocorticoid receptor and pachychoroid[END_REF] . But whether ICG could label choroidal nerves in normal or pathologic conditions has not yet been questioned.

In summary, understanding the in vivo kinetics of ICG fluorescence requires to take into consideration the vascular and RPE macromolecular transport mechanisms and how they might be impaired in pathogenic conditions, and to take also into account the specificities of the dye, which fluorescence depends on its concentration, binding capacities and pH, that are all susceptible for changes in the different studied diseases.

A more extensive identification of the fate of injected ICG and its interaction with the retina and choroid could guide our interpretation of the clinical images. With this objective in mind, we performed experiments to analyze how ICG is distributed in the retina and choroid in rats, either after intravenous injection or after ex vivo organoculture.

Materials and Methods

Animals

Animals were kept in pathogen-free conditions with food, water and litter, and housed in a 12-hour/12-hour light/dark cycle. Sprague Dawley albinos male and female rats between 3 and 12 months-old were used (n=22). Albinos rats were used to allow a better visualization of choroidal immune-labeled structures.

Indocyanine green angiography

Rats were anesthetized by IP injection of 100 mg/kg of ketamine (Clorkétam 1000, Virbac France) and 4 mg/kg of Xylazine (Rompun 2%, Bayer Healthcare, Loos, France). After pupil dilation, Indocyanine green (ICG, 200µl, 2.5mg/ml INFRACYANINE®, SERB, Paris, France) was injected intravenously in the tail of rats.

ICG angiography was performed using Heidelberg Retina Angiograph II (Heidelberg Engineering, Inc., Dossenheim, Germany) to image choroidal circulation and visualize ICG tissue staining. Pictures were recorded at 1-3 min, 10-15 min, 30 min, 1 hour and 6 hours. Rats were euthanized by Euthasol-VET (300 mg/kg, Dechra, Northwich, United-Kingdom) either at 1 hour (IV-1h, n=6) or 6 hours (IV-6h, n=4). After enucleation, eyes were fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature. After washing with DPBS (ThermoFisherScientific, Illkirch-graffenstaden, France), eyes were dissected, the RPE-choroid-sclera complex and the neural retina were post-fixed with acetone at -20°C for 10 min and proceeded for flat-mounting.

Organoculture (OC) Rats (n=9, 18 eyes) were euthanized by Euthasol-VET. After enucleation and dissection of the anterior segment, the posterior segment of the eyeball including retina, choroid and sclera was incubated immediately for 45 min in DMEM medium (41965039, Thermo Fisher Scientific, Illkirch-graffenstaden, France) with 1% fetal bovine serum (10270106, Thermo Fisher Scientific) and 10% ICG (2,5mg/ml, INFRACYANINE®) at 37°C (5% CO2). After washing with DPBS, the neuroretina was removed. The RPE-choroid-sclera complex was fixed with 4% PFA, then post-fixed with acetone for 10 min at -20°, and proceeded for flat-mounting. We chose a 10-fold higher concentration for the ex vivo experiments as compared to the in vivo experiments, as this experiment was designed as a positive control for RPE internalization of ICG.

Flat mounting and immunofluorescence Four radical incisions were performed on the neuroretina and RPE-choroid-sclera complex. To observe ICG staining, tissues were directly flat-mounted with Dako Omnis Fluorescence Mounting Medium (Agilent, Les Ulis, France) and counter stained with DAPI (1:5000) for nuclei staining. Ultra-deep red confocal microscope (Leica LAS X software, STELLARIS 5, Leica Microsystems, Wetzlar, Germany) with an excitation wavelength at 700 nm was used to visualize and capture ICG staining. The RPE-choroid-sclera complex from 3 control rats without ICG injection was used to check the autofluorescence at this wavelength.

The RPE-choroid-sclera and neural retina were also used for immunohistochemistry. Tissues were permeabilized with 0.01% triton X100 (Merck, Darmstadt, Germany) in DPBS, blocked with 10% normal goat serum (G6767, Merck, Darmstadt, Germany) in DPBS, and then incubated with primary antibodies (Table 1) at appropriate dilution for 7 days at 4°C under gentle agitation. After washing with 0.01% Triton X100/DPBS, tissues were incubated with adequate secondary antibodies respectively: AlexaFluo® 488 goat anti-rabbit IgG (1:500, Thermo Fisher Scientific) and AlexaFluo® 488 donkey anti-mouse IgG (1:500, Thermo Fisher Scientific). The nuclei were counterstained with DAPI (1:5000). RPE-choroid-sclera and neuroretina were flat-mounted with Dako Omnis Fluorescence Mounting Medium and observed with confocal microscope. Negative controls omitting the primary antibody were tested according to the above protocol and did not show non specific labelling at the level of the RPE or at the level of the choroid.

Table 1. List of primary antibodies

Target

ICG stains mast cells and allows to identify large choroidal nerves

When imaging the choroid from the scleral side, we could identify round large cells (15-30µm in diameter) positively stained by ICG, aligned along the vessels, both after direct incubation of the posterior segment in ICG and one hour after intravenous injection (Figure 6A andB). Higher magnification showed intracellular granules positively stained with ICG (Figure 6C). Immunostaining with an antibody that recognizes tryptase (a marker of at least part of the mast cells [START_REF] Atiakshin | Mast cell chymase: morphofunctional characteristics[END_REF] ) confirmed that those cells were mast cells (Figure 6D,E,F) and not monocytic or dendritic cells, since they are not stained with ionized calcium-binding adapter molecule 1 (IBA1) (Figure 6G). Negative controls are shown in Figure 3B and E for IBA1, and in Figure 3C and F for tryptase. The optic nerve head was only stained with ICG after incubation (Figure 7A) and not after IV. But, both after incubation and at one hour after IV of ICG, we identified filamentous ICG-positive bundles (Figure 7A and B) resembling choroidal nerves. Immunostaining with beta-tubulin 3 (TUBB3), a nerve marker [START_REF] Fath | Evaluation of Differentiation Quality of Several Differentiation Inducers of Bone Marrow-derived Mesenchymal Stem Cells to Nerve Cells by Assessing Expression of Beta-tubulin 3 Marker: A Systematic Review[END_REF] indicated that ICG binds the nerve bundle (Figure 7E, inset white arrows), whilst some nerve fibers seemed also stained with ICG (Figure 7H, inset dark star). Negative control for the antibody is shown in Figure 3 (C and F).

Interpretation of ICGA in the rat

At the very early phase of ICGA (≤1min), the long ciliary posterior vessels (LCPV) emerging nearby the optic nerve are filled with ICG with a very rapid filling of the large veins, that can be followed in the mid periphery (Figure 8A, 30sec upper and lower images). Large retinal vessels are filled (Figure 9A) and vorticose veins (VV) are progressively visible (Figure 9A).

Interpretation of late phase ICGA in a case of amyloidosis

Amyloid deposits are visible on color fundus photographs as white-yellowish vascular sheathings (Figure 10A, white arrowheads, magnification in the dark circle) that are not labelled by ICG at any time point and particularly not at the late phase of the ICGA (Figure 10B andC). On the other hand, on the late ICGA image (>20min), typical ICG-positive elongated structures are visible in mid periphery and in the periphery of the fundus (Figure 10C), previously identified as vascular amyloid deposits in choroidal vessels, mostly arteries [START_REF] Rousseau | Angiographic Signatures of the Predominant Form of Familial Transthyretin Amyloidosis (Val30Met Mutation)[END_REF] . The reason why ICG would label the choroidal amyloid vascular deposits but not the retinal vessel amyloid deposits is unclear and we hypothesize that ICG could stain the pathological nerves of this patients presenting neuropathy. Superimposition of the late ICGA image with color fundus and comparison with the ICG stained vessels at the early ICGA phase, do not show clear co-localization between the elongated structures and the choroidal vessels (Figure 10, white circle on A, B, C), although small arteries could be hard to identify. The B-scan OCT, at the cross section with one of the elongated ICG positive structure shows that the ICG-positive structure localizes with a round structure at the vicinity of a large choroidal vein, that does not contain the hyporeflective inside as observed in vessels.

Discussion

We have used albinos rats to follow the fate of ICG after either IV injection or direct incubation of the ocular tissues from the posterior segment of the eye in ICG and we have imaged the fluorescence ocular tissues at late time points as compared to the classical angiographic sequence. The idea behind this experimental setting was to help understand clinical images taken at the late phases of the angiographic sequence, considering that the direct observation of ICG using confocal microscopy on flat-mounted tissues is much more sensitive than in vivo angiography. Indeed, the late phase of ICGA remains difficult to interpret in several clinical conditions. We recently analyzed the mid-phase hyperfluorescent plaques (6-10min) observed on ICGA in patients with central serous chorioretinopathy (CSCR) and discussed the reason for the complete fading of fluorescence at later time point (>20min), which differs from the hyperfluorescence linked to type 1 macular neovascularization in AMD that remains fluorescent on the late phases [START_REF] Bousquet | Mid-Phase Hyperfluorescent Plaques Seen on Indocyanine Green Angiography in Patients with Central Serous Chorioretinopathy[END_REF][START_REF] Zola | TYPE 1 MACULAR NEOVASCULARIZATION IN AGE-RELATED MACULAR DEGENERATION AND CENTRAL SEROUS CHORIORETINOPATHY[END_REF] . The reason for the intriguing ICG kinetics in multiple evanescent white dot syndrome was questioned recently by Gaudric and Mrejen [START_REF] Gaudric | OF THE INDOCYANINE GREEN ANGIOGRAPHY IN MULTIPLE EVANESCENT WHITE DOT SYNDROME[END_REF] .

The choice for albinos rat was made to allow immunolocalization of various structures together with ICG imaging, whilst reducing the absorption due to melanin. Interpretation of our observation must thus consider this important difference with the pigmented human RPE/choroid. The use of a confocal microscope with simultaneous lines from 405 to 685nm and detectors on three spectral channels provided high photon detection efficiency and extremely low dark noise as well as extended detection into the near infrared of up to 850 nm. In addition, we could overlap infrared imaging and other fluorophores to perform immunolocalization.

As previously described by several authors, we observed that either after ICG intravenous injection or after incubation of ocular tissues at 37°C, ICG-bound molecules were internalized into the RPE cells, although ICG accumulated also at the cell membrane after incubation and not after IV injection. After intravenous injection in non-human primate, Chang et al showed that ICG was internalized within 15 to 50 minutes with increasing fluorescent signal in the RPE cells at the later time points [START_REF] Chen | Visualizing lipid behind the retina in aging and age-related macular degeneration, via indocyanine green angiography (ASHS-LIA)[END_REF] . Several studies were also performed in rodents showing not only that ICG was internalized by RPE cells after IV injection, but that it remained detectable for an extended period of time and up to 28 days, particularly when high dose of ICG (5mg/kg) was injected [START_REF] Pankova | Delayed near-infrared analysis permits visualization of rodent retinal pigment epithelium layer in vivo[END_REF] . In our experiments, we used 1.5-2mg/kg which corresponds to high ICG dose, explaining that the ICG staining remained at 6 hours. The precise localization of ICG showed that at least part of it, is transported in vesicles that were positively labeled with caveolin-1 antibodies, suggesting that caveolae-mediated transcytosis is involved in ICG transport, which is a well down mechanism for albumin transport within cells [START_REF] Pankova | Delayed near-infrared analysis permits visualization of rodent retinal pigment epithelium layer in vivo[END_REF] and a mechanism for albumin transcytosis in endothelial microvascular cells [START_REF] Botos | Caveolin-1 is transported to multi-vesicular bodies after albumin-induced endocytosis of caveolae in HepG2 cells[END_REF] .

Transcytosis of LDL is also regulated by caveolin 1-mediated mechanisms in endothelial cells [START_REF] Shang | High Mobility Group Box Protein 1 Boosts Endothelial Albumin Transcytosis through the RAGE/Src/Caveolin-1 Pathway[END_REF] . In the retina, caveolin-1 is expressed in retinal vascular cells, Müller glial cells and RPE cells [START_REF] Gu | Caveolins and caveolae in ocular physiology and pathophysiology[END_REF] , but the exact role of caveolin-1 in the protein and lipoprotein transports between the choroidal circulation and the neural retina

is not yet fully understood. Lipoproteins, to which ICG is highly bound, are also transported from the choroidal blood flow towards the RPE and are responsible for the delivery of vitamin A, carotenoids and of lutein and zeaxanthin towards the inner retinal layers in the macula through the RPE [START_REF] Harrison | Mechanisms of Transport and Delivery of Vitamin A and Carotenoids to the Retinal Pigment Epithelium[END_REF] , demonstrating that transcytosis can occur also to allow the exit of molecules at the apical side of the RPE. Tserentsoodol et al showed that after intravenous injection of fluorescently labeled LDL and HDL, fluorescent signal was observed in the choriocapillaris, the RPE and part of the neural retina at 2 hours, and that signal was detected in the outer segment of photoreceptors at 4 hours [START_REF] Tserentsoodol | Uptake of cholesterol by the retina occurs primarily via a low density lipoprotein receptor-mediated process[END_REF] , which is consistent with our observation of ICG signal in the outer segments at 6 hours and not at one hour after IV injection. Altogether, in the rat retina, the fate of intravenously injected ICG, which binds to lipoproteins and to albumin, reflects well the known kinetics of albumin and lipoprotein transports into the retina.

The reason for ICG accumulation in mast cells can be understood by the fact that mast cells metabolize lipoproteins and rapidly internalizes LDL [START_REF] Kokkonen | The metabolism of low density lipoproteins by rat serosal mast cells[END_REF] , particularly when activated [START_REF] Kokkonen | Accumulation of low density lipoproteins in stimulated rat serosal mast cells during recovery from degranulation[END_REF] . Since not all mast cells were tryptase positive and not all tryptase positive cells were ICG-labeled, it cannot be excluded that several types of mast cells with various activation states are present in the choroid. Mast cells, enriched along large vessels in the choroid play important role in physiopathology and can induce subretinal fluid accumulation and inflammation upon degranulation [START_REF] Bousquet | Choroidal mast cells in retinal pathology: a potential target for intervention[END_REF] . ICG-A should be re-analyzed in cases of choroidal inflammation with a specific focus on hyperfluorescent dots with the idea that such dots (around 50µm) could represent activated mast cells.

Finally, we have identified that ICG accumulates in choroidal nerves, particularly the large bundles, which reflects well the known entry of albumin into the endoneurial space through the endoneurial vasculature at 1.2 mg.g-1.day-1 and with a daily turnover of endoneurial albumin of about 30% [START_REF] Weerasuriya | Blood-nerve transfer of albumin and its implications for the endoneurial microenvironment[END_REF] . Mata et al showed that serum albumin is found within the perineurium and endoneurium but not in the axon, suggesting that axons are exposed to serum proteins in normal nerves [START_REF] Mata | The distribution of serum albumin in rat peripheral nerve[END_REF] . The late phase ICGA hyperfluorescent dots and tracks found in the retinal periphery of eyes with transthyretin amyloidosis angiopathy [START_REF] Fath | Evaluation of Differentiation Quality of Several Differentiation Inducers of Bone Marrow-derived Mesenchymal Stem Cells to Nerve Cells by Assessing Expression of Beta-tubulin 3 Marker: A Systematic Review[END_REF] might correspond, at least in part, to large choroidal nerves running in the walls of choroidal vessels, mostly arteries, and not to vascular amyloid plaques since plaques in retinal vessels, clearly observed on the fundus photography are not stained by ICG at all the angiographic times of the sequence (Figure 8). If such amyloid deposits were stained by ICG, retinal plaques should also be stained as intramural deposits alter the endothelial integrity giving access to ICG [START_REF] Koike | Schwann cell and endothelial cell damage in transthyretin familial amyloid polyneuropathy[END_REF] . In addition, amyloid neuropathy, particularly alteration of nerves from the autonomous nervous system is part of the clinical signs of the disease [START_REF] Koike | Widespread Cardiac and Vasomotor Autonomic Dysfunction in Non-Val30Met Hereditary Transthyretin Amyloidosis[END_REF] and increase of albumin entry in the endoneurium through endothelial alterations is an indicator of pathologic nerves [START_REF] Koike | Schwann cell and endothelial cell damage in transthyretin familial amyloid polyneuropathy[END_REF][START_REF] Koike | Widespread Cardiac and Vasomotor Autonomic Dysfunction in Non-Val30Met Hereditary Transthyretin Amyloidosis[END_REF][START_REF] Mano | ASSOCIATION BETWEEN CHOROIDAL CHARACTERISTICS AND SYSTEMIC SEVERITY IN AMYLOIDOSIS[END_REF] . ICG angiography is not commonly performed in patients with diabetic retinopathy, although it has been used to identify the early occurrence of choroidal pathology [START_REF] Shiragami | Risk factors for diabetic choroidopathy in patients with diabetic retinopathy[END_REF] . Since diabetic patients with retinopathy often suffer from peripheral neuropathy, including autonomic neuropathy [START_REF] Galiero | Peripheral Neuropathy in Diabetes Mellitus: Pathogenetic Mechanisms and Diagnostic Options[END_REF] , late phase ICG might be useful to reveal choroidal neuropathy in diabetic eyes.

The interpretation of clinical ICG angiography images from observations made on albinos rats is far from being straightforward, partly because these are albinos animals and partly because the observation times are longer than those usually used in the clinical examinations. However, the numerous hyper or hypofluorescent ICG images that we observe but whose significance we do not fully understand should be analyzed with a new perspective considering that ICG is a unique tool to follow the metabolism of proteins and lipoproteins in the eye and not only an inert dye. ICGA image interpretation should also consider that the fluorescence properties of ICG depend on the concentration of ICG when bound to proteins and that high local concentrations can induce hypofluorescence by quenching mechanisms.

The observation we report herein open new perspectives in the use of ICG as a metabolic marker but also a marker of the neural and immune components of the choroid. If ICG-angiography could improve the identification of choroidal neuropathy in several retinal disease, it could help our understanding of disease and their treatments.

Similar studies should be replicated in non-human primates for better translation to the clinic.

Figure 1 160

 1 Figure 1 160

172D:

  Six hours after ICG IV, the gran-173 ular hyperfluorescence decreases. 174 E: D image with nuclei DAPI stain-175 ing. 176 Immunolocalization of caveolin-1 on RPE/choroid flat mounted at one hour after IV of ICG (Figure 2A and B), showed that at least part of the ICG-labelled intracellular vesicles were stained with caveolin-1 (Figure 2C, inset white arrows), demonstrating that ICG is partially transported in RPE through-caveola-mediated transcytosis. Specificity of the antibody is shown by the absence of staining on negative controls omitting the primary antibody (Figure 3A and D).

Figure 2 DeepFigure 3 196

 23 Figure 2 Deep infrared confocal imaging of RPE flat-mounts and caveolin-1 immunohistochemistry at one hour after IV injection of ICG A: Deep infrared imaging of RPE showing granular staining with nuclei DAPI staining. Inset shows magnification. B: Caveolin-1 immunohistochemistry on RPE flat mount shows vesicular staining at the membrane and within the cytoplasm, with nuclei DAPI staining. Inset shows magnification. C: Merge of image A and B shows partial co-labelling of ICG with caveolin-1 with nuclei DAPI staining. Inset shows magnification with yellow co-stained vesicles (white arrows), caveolin-1 vesicles (green arrows) and ICG-stained vesicles (red arrows). Scale bar: 20µm

Figure 4 DeepFigure 5 230

 45 Figure 4 Deep infrared confocal imaging of neural retina flat mounts A, B, C, D: one hour after ICG IV injection; outer segments (A), outer plexiform layer (B), ganglion cell layer (C), ganglion cell layer with DAPI nuclei staining (D). E, F, G, H: six hours after ICG IV injection; outer segments (E), outer plexiform layer (F), ganglion cell layer (G), ganglion cell layer with DAPI nuclei staining (H). Scale bar: 50µm

Figure 6 ICG

 6 Figure 6 ICG staining of mast cells A: ICG staining of perivascular cells 45 min after incubation of the RPE/choroid in ICG. B-C: ICG staining of perivascular cells one hour after intravenous ICG injection with intracellular ICG positive granules (C). D, E, F: co-labeling of ICG-stained cells (D) with tryptase (E) showing co-labeled cells (F, arrows). G: Ionized calcium-binding adapter molecule 1 (IBA1) immunohistochemistry shows that ICG-positive cells are not labelled by IBA1.

Figure 7 Large

 7 Figure 7 Large choroidal nerves identification by ICG A: deep infrared of choroid flat-mount at 45 min after ICG incubation showing optic nerve head and large nerve. B, C and F: deep infrared of choroid at one hour after ICG intravenous injection showing that ICG-stains large nerves. D, G: Tubulin β3 (TUBB3) immunohistochemistry showing nerves fibers. E, H: co-staining of ICG with TUBB33 showing ICG around nerve fibers (E, inset white arrow).

  At 10 min, ICG can barely be detected in the long posterior ciliary vessels (LPCV) and in retinal vessels whilst a homogeneous background fluorescence can be detected with a hyperfluorescence around the optic nerve head (Figure8B, upper image and Figure9B). A closer analysis of the peripapillary fluorescence shows hyperfluorescent tracks along the vessels (Figure9B, inset yellow arrows). At this time point, deep focalization probably shows bright fluorescence from the supra choroid and from the RPE. Along the LCPV at the periphery, a hyperfluorescent signal can be detected, and could correspond to branches of the ciliary nerves that travel along the vessels (Figure8Band 9B, lower image yellow arrows).At 1 hour, homogeneous background fluorescence is observed with dark vessels being visible underneath (Figure8C). Higher magnification could detect possibly the RPE layer (Figure8C, inset). There is still hyperfluorescence around the optic nerve and along the large ciliary vessels that could correspond to nearby nerves (Figure8C, yellow arrow). At 6 hours, the low hyperfluorescent background is still visible (Figure9C) corresponding possibly to the RPE but also to the outer segments with still hyperfluoresence around the optic nerve head with tracks (Figure8C, white arrows). Surrounding the choroidal vessels, hyperfluorescence could indicate choroidal nerves being labelled with ICG at this time point (Figure9C, yellow arrows).

Figure 8 Indocyanine

 8 Figure 8 Indocyanine green angiography (ICGA) in albinos rats up to one hour Indocyanine green angiography of Sprague Dawley albinos rats, showing early (30 sec) labelling of the long posterior ciliary vessels (LPCV) emerging at the posterior pole inferiorly to the optic nerve (A. upper image), and continuing towards the mid periphery (A. lower image). Optic nerve hyperfluorescence is seen in the very early phase with a peripapillary hyperfluorescent ring (A. upper image), whose intensity is attenuated, though still present, at 10 minutes and 1 hour (B. upper image and C. upper image, respectively). At 10mins, most of the retinal and choroidal vessels are visualized as hypofluorescent structures against a homogeneous hyperfluorescent background (B. upper and lower image). Along the hypofluorescent LPCV in the mid periphery, a linear hyperfluorescent signal can be observed (B. enlarged image, yellow arrow). One hour after ICG injection (C), the contrast between background fluorescence and vascular hypofluorescence becomes more evident. The granular background appearance in this late phase (C. upper right image and inset) could allow visualization of other, non-vascular components of the fundus. The mid-periphery pictures at 1h show that the hyperfluorescence along the LPCV is maintained (C. lower image left and right, yellow arrow).

Figure 9 ICGA

 9 Figure 9 ICGA in albinos rats up to six hours Indocyanine green angiography of Sprague Dawley albinos rats, showing early (1min) retinal and choroidal vascularization and venous drainage with vorticose veins (VV) progressively visible (A. all images). At 10 minutes, a closer analysis of the peripapillary hyperfluorescence shows linear hyperfluorescent structures (B. upper left image and lower enlarged left image, yellow arrows). The choroidal hypofluorescent vascular component is clearly visible against a hyperfluorescent background (B. upper right image). In the mid periphery, long posterior ciliary vessels are surrounded by a linear hyperfluorescence (B. lower right image). Six hours after ICG injection, a background fluorescence is detected underneath the dark retinal and choroidal vessels (C. upper left). The irregular peripapillary hyperfluorescence is showning detail (C. lower left image) where distinct hyperfluorescent structures are observed adjacent to vessels (white arrows). In the mid periphery (C. upper right image), irregular hyperfluorescence corresponds to linear perivascular structures (C. upper and lower right images, yellow arrows).

Figure 10 Retina

 10 Figure 10 Retina images of a 52-year old female with hTTRA and amyloid neuropathy A: color fundus showing white yellowish vascular sheathings observed along retinal vessels (white arrowheads, dark cercle with magnification). B: 2 min ICGA infrared photography .C: 27 min ICGA infrared photography, showing no staining of the amyloid deposits in retinal vessels (white arrowheads) and the ICG stained elongated dots and tracks. White circle in A, B, and C corresponds to the same area with superimposition of C and D on the magnified image in C. D: B-scan along the ICGA stained elongated structures with the green line indicating the cross section with one of them. Magnification shows that it does not correspond to the typical image of a choroidal vessel.

Acknowledgments:

The authors acknowledge Theano Irinopoulou at the imaging platform of Fer à Moulin Institute (Paris, France) for its technical support.

Funding: This research was funded by the Agence Nationale de la Recherche (ANR-20-CE17-0034). We thank The Abraham J. & Phyllis Katz Foundation for their financial support for conducting research.

Institutional Review Board

Statement: The animal study protocol was performed in accordance with the European Communities Council Directive 86/609/EEC and French national regulations and approved by local ethical committees (#25158, Charles Darwin).

Data Availability Statement:

The data from the study are available from the corresponding author upon reasonable request.

Conflicts of Interest:

The authors declare no conflict of interest.


V Dorval

L Ganjehi

S Chatillon

F Jenson

MODELING ULTRASONIC NOISE AND ATTENUATION IN ELONGATED DUPLEX POLYCRYSTALLINE MATERIALS

Keywords: Ultrasonic, Polycrystalline Material, Grain Noise, Attenuation PACS: 43.20.Fn, 43.35.Cg, 43.35.Zc, 43.50.Ed

The interaction between ultrasonic waves and the polycrystalline microstructure of metals leads to structural noise and attenuation, which can cause significant losses in detection performances. Taking these phenomena into account in UT simulation can help designing suitable inspection procedures. It is possible to model them based on the intrinsic scattering coefficients of a material. Expressions of this coefficient valid for duplex elongated materials and for any mode and scattering direction are given in this communication. Structural noise from a titanium alloy was computed based on these expressions and is compared to measurement.

INTRODUCTION

Structural noise and attenuation can be limiting factors in the ultrasonic non destructive testing of metallic parts. Modeling these phenomena can help designing inspection procedures. Among the possible approaches proposed in the literature, we can mention the computation of structural noise signals by [START_REF] Gustafsson | Studies of split spectrum processing, optimal detection and maximum likelihood amplitude estimation using a simple clutter model[END_REF] and the modeling of statistical properties of signals by [START_REF] Thompson | Use of elastodynamic theories in the stochastic description of the effects of microstructure on ultrasonic flaw and noise signals[END_REF]. A method similar to the one of [START_REF] Gustafsson | Studies of split spectrum processing, optimal detection and maximum likelihood amplitude estimation using a simple clutter model[END_REF] was developed at CEA LIST [START_REF] Chatillon | Simplified modeling of backscattered noise and attenuation phenomena for quantitative performance demonstration of UT methods[END_REF]. In subsequent works, it was modified [START_REF] Dorval | Simulation of structural noise and attenuation occurring in ultrasonic NDT of polycrystalline materials[END_REF] so that the statistical properties of the generated signals match the outputs of a scattering model. This model described the microstructure as a single phase and equiaxed polycrystal. In this communication, we present an extension of the simulation method and of the underlying model to the case of more complex microstructures such as duplex and elongated materials. First, we describe the simulation method. Then, we detail the application of the underlying scattering model for different microstructures. Finally, we compare outputs of the model to experimental results.

SIMULATION OF ULTRASONIC INSPECTIONS WITH ULTRASONIC NOISE AND ATTENUATION

A method [START_REF] Dorval | Simulation of structural noise and attenuation occurring in ultrasonic NDT of polycrystalline materials[END_REF] previously developed at CEA LIST simulates ultrasonic testing with structural noise. It is based on the following assumptions: multiple scattering can be neglected and the noise is a fully developed speckle (which means that the noise is due to a large number of subresolved scatterers). The fully developed speckle assumption is adequate as long as the grains in the material are small compared to the resolution capabilities of the probe. In this assumption, the actual number of scatterers has no impact on the statistical properties of the noise and we can describe the scattering by a volumic coefficient η. η is the same as the one studied by [START_REF] Margetan | Computation of grain noise scattering coeffi-cients for ultrasonic pitch/catch inspections of metals[END_REF]. We define it as the fraction of the incident intensity that is scattered on average by a unit volume of the structure. It depends on the modes and the directions of the incident and scattered waves.

To use this coefficient to simulate structural noise, we generate a random distribution of scatterers. We choose a number N of scatterers per unit volume high enough to obtain fully developed speckle. The η of that distribution of scatterer is related to the mean of the square of their scattering amplitudes A and to the ratio of the velocity of the scattered and incident waves [START_REF] Margetan | Computation of grain noise scattering coeffi-cients for ultrasonic pitch/catch inspections of metals[END_REF]:

.

(

) 1
The distribution of the scattering amplitudes A is set in order to verify the above equation for the theoretical η of the simulated microstructure. That way, we ensure that the distribution of scatterers produces the same structural noise as the theoretical microstructure.

η can also be used to obtain attenuation coefficients, by relating the energy lost due to attenuation to the energy scattered in every direction. These coefficients are used in the simulation for the computation of both structural noise and defect echoes. In the case of longitudinal waves, we obtain the following relation between the attenuation coefficient α L and the integrals over every direction of the scattered energy: [START_REF] Thompson | Use of elastodynamic theories in the stochastic description of the effects of microstructure on ultrasonic flaw and noise signals[END_REF] where the three coefficients η L→... are the scattering coefficients from L waves to each possible wave. Similar equations can be obtained for transverse waves. A detailed development that leads to equivalent expressions is given in [START_REF] Weaver | Diffusivity of ultrasound in polycrystals[END_REF].

η is given by the scattering model described in the next section.

SCATTERING MODEL

We use a scattering model to establish a relation between the properties of the microstructure and the coefficient η. This model is based on the approach proposed by [START_REF] Rose | Ultrasonic backscatter from microstructure[END_REF]. The microstructure is described as an assembly of randomly oriented crystallites. As the crystallites are anisotropic, differences in orientation translate into elastic contrasts that cause scattering. The Born and single scattering approximation are used to derive expressions of η. Assuming that the density of the material is homogeneous, the following expression can be obtained: [START_REF] Chatillon | Simplified modeling of backscattered noise and attenuation phenomena for quantitative performance demonstration of UT methods[END_REF] where the indices inc and scat refer to incident and scattering waves. k is the wave vector, p the polarization vector and v the velocity. ρ is the density of the material. δC is the variation of elastic constants compared to their average: [START_REF] Dorval | Simulation of structural noise and attenuation occurring in ultrasonic NDT of polycrystalline materials[END_REF] where <> x denotes an average over the positions in the material. It is equivalent to an average over the possible realizations of the microstructure.

The spatial correlation of elastic constants depends on the geometrical and elastic properties of the microstructure. The adaptation of the model to different types of microstructures comes down to the development of that particular term.

Single phase equiaxed microstructure

Expressions of this scattering coefficient for any mode and any scattering direction are given in [START_REF] Margetan | Computation of grain noise scattering coeffi-cients for ultrasonic pitch/catch inspections of metals[END_REF] in the case of an equiaxed single phase material. The geometry of the microstructure is described in a statistical manner, by using a spatial correlation function. This function, noted , is defined as the probability that two points separated by are located in the same grains. An exponential form is usually assumed for this function, D being a characteristic grain size: .

(

For a single phase microstructure with this correlation function, equation (3) simplifies to: [START_REF] Weaver | Diffusivity of ultrasound in polycrystals[END_REF] where Δk is the difference between incident and scattered wave vectors. <δC ijkl δC mnpq > is an average in a single crystallite over all its possible orientations. The indices of that covariance that need to be considered depend on the modes and directions of the incident and scattered waves. For example, in the case of the backscattering of L waves propagating along the z axis, only <δC 3333 δC 3333 > needs to be considered. For a material with cubic symmetry, it is equal to: .

For a material with hexagonal symmetry, it is equal to:

. ( 8
)
Expressions for other modes and directions are given in [START_REF] Margetan | Computation of grain noise scattering coeffi-cients for ultrasonic pitch/catch inspections of metals[END_REF].

Description of a duplex microstructure

This scattering model has been applied in literature to compute backscattering coefficient for L wave in the case of duplex titanium [START_REF] Han | Ultrasonic backscattering in duplex microstructures: theory and application to titanium alloys[END_REF]. We describe here a way to obtain scattering coefficients of any wave in any direction and for more types of duplex metals.

An experimental micrographic picture where a duplex structure appears clearly is given by [START_REF] Costa Teixeira | Modeling of the effect of the β phase deformation on the α phase precipitation in near β-titanium alloys[END_REF]. For modeling purposes, we describe that structure as a mix of primary and secondary phase in a manner similar to [START_REF] Han | Ultrasonic backscattering in duplex microstructures: theory and application to titanium alloys[END_REF]. The primary phase is the first to appear during the solidification. It arranges itself in randomly orientated crystallites called macrograins. Later the solidification, a secondary phase appears. It arranges itself in a set of colonies in each macrograin. The orientation of a colony is influenced by the orientation of the macrograin. A colony is composed of parallel needle-like crystallites that all have the same crystalline orientation. We assume that these crystallites are too small to individually affect ultrasonic scattering and we model a colony as a homogeneous mix of secondary and residual primary phases. We assume that the orientation of the secondary phase is related to the orientation of the surrounding primary phase by an alignment of dense planes and directions. Such alignment has consequences on the elastic constants on the materials.

We developed expressions of elastic constants for two types of metal. In the first one, the crystallographic lattices of the primary and secondary phases are respectively body-centered cubic (bcc) and hexagonal and the relation between the two phases is the same as given by [START_REF] Han | Ultrasonic backscattering in duplex microstructures: theory and application to titanium alloys[END_REF]. It can be used to describe a titanium alloy with beta and alpha titanium as primary and secondary phases. In the second type, the crystallographic lattices of the primary and secondary phases are respectively body-centered cubic (bcc) and facecentered cubic (fcc) and they are related by the Kurdjumov-Sachs relationships. It can be used to describe a steel with ferrite and austenite as primary and secondary phases. The orientation relationships of the two types are given in Table 1.

TABLE 1. Orientation relationships between primary and secondary phases. bcc to hexagonal bcc to fcc

These orientation relationships imply that 6 orientations are possible for the hexagonal secondary phase and 24 orientations are possible for the fcc secondary phase. A possible orientation is called a variant. We assume that each of the variant has the same probability of appearing. We obtained analytical expressions for Euler angles that describe all these possible orientations. Listing of all these expressions takes several pages, so they are not given here. However, averages of the elastic constants over these variants are given later, as they are the quantities needed to calculate scattering coefficients.

Scattering coefficients for a duplex microstructure

Based on the description of the microstructure, we obtain this expression for the covariance of elastic constants that appears in the expression of the scattering coefficient: [START_REF] Costa Teixeira | Modeling of the effect of the β phase deformation on the α phase precipitation in near β-titanium alloys[END_REF] where p P and p S are the proportions of the primary and secondary phases.

is the probability that two point separated by are located in the same macrograin.

is the conditional probability that two point separated by and located in the same macrograin are located in the same colony. C P and C S are the elastic constants of the primary and secondary phases. C SAV is the elastic constants of the secondary phase averaged over the variants, and is expressed: [START_REF] Ahmed | Ultrasonic attenuation as influenced by elon-gated grains[END_REF] where N is the number of variants and C SVa is the elastic constants of variant a.

For both the bcc to hexagonal and the bcc to fcc cases, the averaged elastic constants C SAV have a cubic symmetry. Three terms of C SAV are given in table 2, the others can be obtained using cubic symmetry. In the bcc to hexagonal case, C S is expressed in coordinates such that . The three coefficients of this decomposition are the scattering coefficients of three fictitious materials characterized by spatial correlation functions and elastic constants given in table 3. Equations ( 3) to [START_REF] Han | Ultrasonic backscattering in duplex microstructures: theory and application to titanium alloys[END_REF], as well as the equations in [START_REF] Margetan | Computation of grain noise scattering coeffi-cients for ultrasonic pitch/catch inspections of metals[END_REF], can be used to calculate these three coefficients.

The three coefficients can be interpreted as follows: η macrograin_average is the contribution of the macrograins to the scattering, η secondary -η variant_average is the contribution of the colonies (η secondary being the contribution of individual colonies and η variant_average a reduction of the scattering due to orientation relationships).

Elongated material

All the previous results were for the case of non-elongated grains. Taking into account an elongation of the grains means changing the spatial correlation function given in equation [START_REF] Margetan | Computation of grain noise scattering coeffi-cients for ultrasonic pitch/catch inspections of metals[END_REF]. Following [START_REF] Ahmed | Ultrasonic attenuation as influenced by elon-gated grains[END_REF], we generalize to elongated structures using the form: [START_REF] Ganjehi | Modeling of the ultrasonic propagation in polycristalline materials[END_REF] where h and d are the grain sizes respectively in the elongation direction and in the directions perpendicular to it. θ is the angle between and the elongation direction. Using that new function implies modifying the term known as the form factor in equation [START_REF] Weaver | Diffusivity of ultrasound in polycrystals[END_REF]. For an equiaxed material, it is equal to: .

For an elongated microstructure as defined above it becomes:

. ( 14
)
where Δk elong and Δk perp are the components of respectively in the elongation direction and in the directions perpendicular to it. This substitution yields expressions for the scattering coefficient of single phase elongated materials. By applying it to each term of the decomposition of equation ( 11), it is possible to obtain expressions for duplex elongated materials.

VALIDATION

An experimental validation was performed using the measurement setups presented figure 1. The main characteristics of the transducer are a focal length F of 406.2mm, a diameter of 25.4mm and a center frequency f 0 of 10MHz. Measurements were performed in immersion at normal incidence. Structural noise was measured in a sample of duplex titanium, and the echo of a Flat Bottom Hole in a reference block was measured using the same transducer. The waterpath z M was chosen such that the beam was focused in the half depth of the block.

A micrographic study of the titanium alloy sample showed that the grains were elongated. It also allowed estimating the sizes of the macrograins and the colonies. The elastic constants of each phase were assumed to be equal to data found in literature. All these parameters were used as an entry for a simulation of structural noise, though some adjustments were made concerning the size of colonies as they did not appear clearly enough on the micrographs.

The simulation method is based on the models described in this communication. It was integrated in a development version of the CIVA simulation platform [11].

Simulations were run for both the experiments of figure 1. In order to compare measured and simulated structural noise, the envelopes of the noise signal at each position of the probe were computed and averaged. The average structural noise as a function of time was obtained. It is plotted in figure 2, expressed in decibels compared to the echo of the reference defect.

This comparison shows a good agreement: the gap between the two results is smaller than the oscillations of the noise. Both the absolute value and the evolution of the noise are satisfactory. Other simulation results for this configuration, obtained without taking into account the grain elongation, had shown a disagreement of approximately seven decibels [START_REF] Ganjehi | Modeling of the ultrasonic propagation in polycristalline materials[END_REF]. This illustrates that improving the description of the microstructure can help improving the simulation results.

CONCLUSION

The structural noise that occurs in ultrasonic NDT can be simulated based on the coefficient η of the material. We presented a way to compute this coefficient for every mode and in every scattering direction for duplex and elongated materials. These computations are based and the Born and single scattering approximations. We propose decomposing the scattering coefficient of the duplex material into three terms. It leads to relatively simple expressions despite the complexity of the material. Experimental validations on a duplex titanium alloy with elongated grains show a good agreement. It demonstrates the relevance of both the simulation method and the scattering model. However, they are not expected to be valid for every case of ultrasonic structural noise. It is known that in some cases multiple scattering dominates, and it is not taken into account by this method. In future works we aim at including multiple scattering contributions in this method.

FIGURE 1 .

 1 FIGURE 1. Pulse/echo acquisition for structural noise and reference echo.

FIGURE 2 .

 2 FIGURE 2. Measured and simulated averages of the envelopes of structural noise as a function of time.

TABLE 2 .

 2 Elastic constants averaged over variants.

	bcc to hexagonal	bcc to fcc
	By using equation (9) in equation (3), we can obtain a decomposition of the
	scattering coefficient of a duplex microstructure into three scattering coefficients.
		(11)

TABLE 3 .

 3 Parameters for calculating the scattering coefficient of the decomposition.

	Spatial correlation function	Elastic constants
	η macrograin_average
	η secondary
	η variant_average

ACKNOWLEDGEMENTS

The work leading to this publication has received funding from the European Commission Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 234117: PICASSO project.

The conditions of experimental setup and the metallurgical study have been provided by Snecma (Safran group) NDT department of Villaroche and Metallurgical department of Genevilliers and Corbeil.