Light sheet fluorescence microscopy of cleared human eyes.

We provide here a procedure enabling light sheet fluorescence microscopy (LSFM) of entire human eyes after iDISCO + -based clearing (ClearEye) and immunolabeling. Demonstrated here in four eyes, post-processing of LSFM stacks enables three-dimensional (3D) navigation and customized display, including en face viewing of the fundus similarly to clinical imaging, with resolution of retinal capillaries. This method overcomes several limitations of traditional histology of the eyes. Tracing of spatially complex structures such as anterior ciliary vessels and Schlemm's canal was achieved. We conclude that LSFM of immunolabeled human eyes after iDISCO + -based clearing is a powerful tool for 3D histology of large human ocular samples, including entire eyes, which will be useful in both anatomopathology and in research.

Automatic diagnosis and classification of breast surgical samples with dynamic full-field OCT and machine learning.

Purpose: The adoption of emerging imaging technologies in the medical community is often hampered when they provide a new unfamiliar contrast that requires experience to be interpreted. Dynamic full-field optical coherence tomography (D-FF-OCT) microscopy is such an emerging technique. It provides fast, high-resolution images of excised tissues with a contrast comparable to H&E histology but without any tissue preparation and alteration. Approach: We designed and compared two machine learning approaches to support interpretation of D-FF-OCT images of breast surgical specimens and thus provide tools to facilitate medical adoption. We conducted a pilot study on 51 breast lumpectomy and mastectomy surgical specimens and more than 1000 individual 1.3×1.3 mm2 images and compared with standard H&E histology diagnosis. Results: Using our automatic diagnosis algorithms, we obtained an accuracy above 88% at the image level (1.3×1.3 mm2) and above 96% at the specimen level (above cm2). Conclusions: Altogether, these results demonstrate the high potential of D-FF-OCT coupled to machine learning to provide a rapid, automatic, and accurate histopathology diagnosis with minimal sample alteration.

Confocal microscopy of telangiectatic capillaries (TelCaps) and other features of microvascular remodeling following branch retinal vein occlusion.

Branch retinal vein occlusion (BRVO) is a frequent retinal vascular disease that may cause extensive microvascular remodeling leading to severe visual impairment. Little is known regarding the histology of non-neovascular microvascular remodeling. Here, we examined by confocal microscopy the structure of retinal microvessels of a donor eye with longstanding BRVO. The post-mortem retina of a 91-year-old woman that had superotemporal BRVO for 2 years was examined by confocal microscopy after anti-collagen IV (collIV), alpha-smooth muscle cell (αSMA), and anti-von Willebrand factor (vWf) immunolabeling. In the retinal quadrant affected by BRVO, extensive vascular remodeling affected all vessels, from arterioles to venules, including the foveal avascular zone. Most affected vessels were either irregularly dilated or, on the opposite, reduced to micrometric-size CollIV positive, vWf negative, nuclear-staining negative strings. Telangiectatic capillaries of various sizes and shapes were seen, the largest one (233 µm) being located in the parafoveal area. Some telangiectatic capillaries had a thick, multilayered vWf- and CollIV-positive wall, that often occluded the lumen. Other features included double-channeled arterioles. The majority of microvascular abnormalities were devoid of nuclear staining, suggesting extensive loss of endothelial cells. We describe the spectrum of microvascular abnormalities upstream of a longstanding BRVO. This spectrum comprises a large parafoveal telangiectatic capillary corresponding to what has been previously clinically defined as TelCap. The absence of intraluminal nuclear staining in the majority of abnormal vessels raises the hypothesis that the loss of endothelial cells plays a crucial role in the development of the different manifestations of capillary remodeling. The presence of vWF in de-endothelialized vessels suggests deposition of plasma, hence that they may remain perfused. Our work may help to understand the clinical imaging features of TelCaps.

Three-dimensional characterization of developing and adult ocular vasculature in mice using in toto clearing.

The ocular vasculature is critically involved in many blinding diseases and is also a popular research model for the exploration of developmental and pathological angiogenesis. The development of ocular vessels is a complex, finely orchestrated sequence of events, involving spatial and temporal coordination of hyaloid, choroidal and retinal networks. Comprehensive studies of the tridimensional dynamics of microvascular remodeling are limited by the fact that preserving the spatial disposition of ocular vascular networks is cumbersome using classical histological procedures. Here, we demonstrate that light-sheet fluorescence microscopy (LFSM) of cleared mouse eyes followed by extensive virtual dissection offers a solution to this problem. To the best of our knowledge, this is the first 3D quantification of the evolution of the hyaloid vasculature and of post-occlusive venous remodeling together with the characterization of spatial distribution of various cell populations in ocular compartments, including the vitreous. These techniques will prove interesting to obtain other insights in scientific questions addressing organ-wide cell interactions.

Antagonist of nucleolin, N6L, inhibits neovascularization in mouse models of retinopathies.

Retinal vascular diseases (RVD) have been identified as a major cause of blindness worldwide. These pathologies, including the wet form of age-related macular degeneration, retinopathy of prematurity, and diabetic retinopathy are currently treated by intravitreal delivery of anti-vascular endothelial growth factor (VEGF) agents. However, repeated intravitreal injections can lead to ocular complications and resistance to these treatments. Thus, there is a need to find new targeted therapies. Nucleolin regulates the endothelial cell (EC) activation and angiogenesis. In previous studies, we designed a pseudopeptide, N6L, that binds the nucleolin and blocks the tumor angiogenesis. In this study, the effect of N6L was investigated in two experimental models of retinopathies including oxygen-induced retinopathy (OIR) and choroidal neovascularization (CNV). We found that in mouse OIR, intraperitoneal injection of N6L is delivered to activated ECs and induced a 50% reduction of pathological neovascularization. The anti-angiogenic effect of N6L has been tested in CNV model in which the systemic injection of N6L induced a 33% reduction of angiogenesis. This effect is comparable to those obtained with VEGF-trap, a standard of care drug for RVD. Interestingly, with preventive and curative treatments, neoangiogenesis is inhibited by 59%. Our results have potential interest in the development of new therapies targeting other molecules than VEGF for RVD.

Weibel-Palade Bodies Orchestrate Pericytes During Angiogenesis.

Objective Weibel-Palade bodies (WPBs) are endothelial cell (EC)-specific organelles formed by vWF (von Willebrand factor) polymerization and that contain the proangiogenic factor Ang-2 (angiopoietin-2). WPB exocytosis has been shown to be implicated for vascular repair and inflammatory responses. Here, we investigate the role of WPBs during angiogenesis and vessel stabilization. Approach and Results WPB density in ECs decreased at the angiogenic front of retinal vascular network during development and neovascularization compared with stable vessels. In vitro, VEGF (vascular endothelial growth factor) induced a VEGFR-2 (vascular endothelial growth factor receptor-2)-dependent exocytosis of WPBs that contain Ang-2 and consequently the secretion of vWF and Ang-2. Blocking VEGF-dependant WPB exocytosis and Ang-2 secretion promoted pericyte migration toward ECs. Pericyte migration was inhibited by adding recombinant Ang-2 or by silencing Ang-1 (angiopoietin-1) or Tie2 (angiopoietin-1 receptor) in pericytes. Consistently, in vivo anti-VEGF treatment induced accumulation of WPBs in retinal vessels because of the inhibition of WPB exocytosis and promoted the increase of pericyte coverage of retinal vessels during angiogenesis. In tumor angiogenesis, depletion of WPBs in vWF knockout tumor-bearing mice promoted an increase of tumor angiogenesis and a decrease of pericyte coverage of tumor vessels. By another approach, normalized tumor vessels had higher WPB density. Conclusions We demonstrate that WPB exocytosis and Ang-2 secretion are regulated during angiogenesis to limit pericyte coverage of remodeling vessels by disrupting Ang-1/Tie2 autocrine signaling in pericytes.

A rationally designed NRP1-independent superagonist SEMA3A mutant is an effective anticancer agent.

Vascular normalizing strategies, aimed at ameliorating blood vessel perfusion and lessening tissue hypoxia, are treatments that may improve the outcome of cancer patients. Secreted class 3 semaphorins (SEMA3), which are thought to directly bind neuropilin (NRP) co-receptors that, in turn, associate with and elicit plexin (PLXN) receptor signaling, are effective normalizing agents of the cancer vasculature. Yet, SEMA3A was also reported to trigger adverse side effects via NRP1. We rationally designed and generated a safe, parenterally deliverable, and NRP1-independent SEMA3A point mutant isoform that, unlike its wild-type counterpart, binds PLXNA4 with nanomolar affinity and has much greater biochemical and biological activities in cultured endothelial cells. In vivo, when parenterally administered in mouse models of pancreatic cancer, the NRP1-independent SEMA3A point mutant successfully normalized the vasculature, inhibited tumor growth, curbed metastatic dissemination, and effectively improved the supply and anticancer activity of chemotherapy. Mutant SEMA3A also inhibited retinal neovascularization in a mouse model of age-related macular degeneration. In summary, mutant SEMA3A is a vascular normalizing agent that can be exploited to treat cancer and, potentially, other diseases characterized by pathological angiogenesis.

Lebecetin, a C-type lectin, inhibits choroidal and retinal neovascularization.

Angiogenesis is a cause of visual impairment and blindness in the wet form of age-related macular degeneration and in ischemic retinopathies. Current therapies include use of anti-VEGF agents to reduce choroidal neovascularization (CNV) and edema. These treatments are effective in most cases, but spontaneous or acquired resistance to anti-VEGF and possible adverse effects of long-term VEGF inhibition in the retina and choroid highlight a need for additional alternative therapies. Integrins αvß3 and αvß5, which regulate endothelial cell proliferation and stabilization, have been implicated in ocular angiogenesis. Lebecetin (LCT) is a 30-kDa heterodimeric C-type lectin that is isolated from Macrovipera lebetina venom and interacts with α5ß1- and αv-containing integrins. We previously showed that LCT inhibits human brain microvascular endothelial cell adhesion, migration, proliferation, and tubulogenesis. To evaluate the inhibitory effect of LCT on ocular angiogenesis, we cultured aortic and choroidal explants in the presence of LCT and analyzed the effect of LCT on CNV in the mouse CNV model and on retinal neovascularization in the oxygen-induced retinopathy model. Our data demonstrate that a single injection of LCT efficiently reduced CNV and retinal neovascularization in these models.-Montassar, F., Darche, M., Blaizot, A., Augustin, S., Conart, J.-B., Millet, A., Elayeb, M., Sahel, J.-A., Réaux-Le Goazigo, A., Sennlaub, F., Marrakchi, N., Messadi, E., Guillonneau, X. Lebecetin, a C-type lectin, inhibits choroidal and retinal neovascularization.

AAV-mediated gene therapy in Dystrophin-Dp71 deficient mouse leads to blood-retinal barrier restoration and oedema reabsorption.

Dystrophin-Dp71 being a key membrane cytoskeletal protein, expressed mainly in Müller cells that provide a mechanical link at the Müller cell membrane by direct binding to actin and a transmembrane protein complex. Its absence has been related to blood-retinal barrier (BRB) permeability through delocalization and down-regulation of the AQP4 and Kir4.1 channels (1). We have previously shown that the adeno-associated virus (AAV) variant, ShH10, transduces Müller cells in the Dp71-null mouse retina efficiently and specifically (2,3). Here, we use ShH10 to restore Dp71 expression in Müller cells of Dp71 deficient mouse to study molecular and functional effects of this restoration in an adult mouse displaying retinal permeability. We show that strong and specific expression of exogenous Dp71 in Müller cells leads to correct localization of Dp71 protein restoring all protein interactions in order to re-establish a proper functional BRB and retina homeostasis thus preventing retina from oedema. This study is the basis for the development of new therapeutic strategies in dealing with diseases with BRB breakdown and macular oedema such as diabetic retinopathy (DR).

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease.

Müller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling Müller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms. There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5 has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label Müller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

Lack of dystrophin protein Dp71 results in progressive cataract formation due to loss of fiber cell organization.

PURPOSE: Dp71 is the main product of the Duchenne muscular dystrophy (DMD) gene in the central nervous system. While studying the impact of its absence on retinal functions, we discovered that mice lacking Dp71 also developed a progressive opacification of the crystalline lens. The purpose of this study was to perform a detailed characterization of the cataract formation in Dp71 knockout (KO-Dp71) mice. METHODS: Cataract formations in KO-Dp71 mice and wild-type (wt) littermates were assessed in vivo by slit-lamp examination and ex vivo by histological analysis as a function of aging. The expression and cellular localization of the DMD gene products were monitored by western blot and immunohistochemical analysis. Fiber cell integrity was assessed by analyzing the actin cytoskeleton as well as the expression of aquaporin-0 (AQP0). RESULTS: As expected, a slit-lamp examination revealed that only one of the 20 tested wt animals presented with a mild opacification of the lens and only at the most advanced age. However, a lack of Dp71 was associated with a 40% incidence of cataracts as early as 2 months of age, which progressively increased to full penetrance by 7 months. A subsequent histological analysis revealed an alteration in the structures of the lenses of KO-Dp71 mice that correlated with the severity of the lens opacity. An analysis of the expression of the different dystrophin gene products revealed that Dp71 was the major DMD gene product expressed in the lens, especially in fiber cells. The role of Dp71 in fiber cells was also suggested by the progressive disorganization of the lens fibers, which was observed in the absence of Dp71 and demonstrated by irregular staining of the actin network and the aqueous channel AQP0. CONCLUSIONS: While its role in the retina has been well characterized, this study demonstrates for the first time the role played by Dp71 in a different ocular tissue: the crystalline lens. It primarily demonstrates the role that Dp71 plays in the maintenance of the integrity of the secondary lens fibers.

AAV-mediated gene delivery in Dp71-null mouse model with compromised barriers.

Formation and maintenance of the blood-retinal barrier (BRB) is required for proper vision and breaching of this barrier contributes to the pathology in a wide variety of retinal conditions such as retinal detachment and diabetic retinopathy. Dystrophin Dp71 being a key membrane cytoskeletal protein, expressed mainly in Müller cells, its absence has been related to BRB permeability through delocalization and down-regulation of the AQP4 and Kir4.1 channels. Dp71-null mouse is thus an excellent model to approach the study of retinal pathologies showing blood-retinal barrier permeability. We aimed to investigate the participation of Müller cells in the BRB and in the inner limiting membrane of Dp71-null mice compared with wild-type mice in order to understand how these barriers work in this model of permeable BRB. To this aim, we used an Adeno-associated virus (AAV) variant, ShH10-GFP, engineered to target Müller cells specifically. ShH10 coding GFP was introduced by intravitreal injection and Müller cell transduction was studied in Dp71-null mice in comparison to wild-type animals. We show that Müller cell transduction follows a significantly different pattern in Dp71-null mice indicating changes in viral cell-surface receptors as well as differences in the permeability of the inner limiting membrane in this mouse line. However, the compromised BRB of the Dp71-null mice does not lead to virus leakage into the bloodstream when the virus is injected intravitreally - an important consideration for AAV-mediated retinal gene therapy.