Spectral-Domain Optical Coherence Tomography (SD-OCT) is a widely used method to observe retinal layers and follow pathological events in human. Recently, this technique has been adapted for animal imaging. This non-invasive technology brings a cross-sectional visualization of the retina, which permits to observe precisely each layer. There is a clear expansion of the use of this imaging modality in rodents, thus, a precise characterization of the different outer retinal layers observed by SD-OCT is now necessary to make the most of this technology. The identification of the inner strata until the outer nuclear layer has already been clearly established, while the attribution of the layers observed by SD-OCT to the structures corresponding to photoreceptors segments and retinal pigment epithelium is much more questionable. To progress in the understanding of experimental SD-OCT imaging, we developed a method for averaging SD-OCT data to generate a mean image allowing to better delineate layers in the retina of pigmented and albino strains of mice and rats. It allowed us to locate precisely the interface between photoreceptors and retinal pigment epithelium and to identify unambiguously four layers corresponding to the inner and outer parts of photoreceptors segments. We show that the thickness of the various layers can be measured as accurately in vivo on SD-OCT images, than post-mortem by a morphometric analysis of histological sections. We applied SD-OCT to different models and demonstrated that it allows analysis of focal or diffuse retinal pathological processes such as mutation-dependent damages or light-driven modification of photoreceptors. Moreover, we report a new method of combined use of SD-OCT and integration to quantify laser-induced choroidal neovascularization. In conclusion, we clearly demonstrated that SD-OCT represents a valuable tool for imaging the rodent retina that is at least as accurate as histology, non-invasive and allows longitudinal follow-up of the same animal.

Inflammatory neovascularization, such as choroidal neovascularization (CNV), occur in the presence of Notch expressing macrophages. DLL4s anti-angiogenic effect on endothelial cells (EC) has been widely recognized, but its influence on Notch signaling on macrophages and its overall effect in inflammatory neovascularization is not well understood. We identified macrophages and ECs as the main Notch 1 and Notch 4 expressing cells in CNV. A soluble fraction spanning Ser28-Pro525 of the murine extracellular DLL4 domain (sDLL4/28-525) activated the Notch pathway, as it induces Notch target genes in macrophages and ECs and inhibited EC proliferation and vascular sprouting in aortic rings. In contrast, sDLL4/28-525 increased pro-angiogenic VEGF, and IL-1β expression in macrophages responsible for increased vascular sprouting observed in aortic rings incubated in conditioned media from sDLL4/28-525 stimulated macrophages. In vivo, Dll4(+/-) mice developed significantly more CNV and sDLL4/28-525 injections inhibited CNV in Dll4(+/-) CD1 mice. Similarly, sDLL4/28-525 inhibited CNV in C57Bl6 and its effect was reversed by a γ-secretase inhibitor that blocks Notch signaling. The inhibition occurred despite increased VEGF, IL-1β expression in infiltrating inflammatory macrophages in sDLL4/28-525 treated mice and might be due to direct inhibition of EC proliferation in laser-induced CNV as demonstrated by EdU labelling in vivo. In conclusion, Notch activation on macrophages and ECs leads to opposing effects in inflammatory neovascularization in situations such as CNV.

To describe the value of integrating phenotype/genotype data, disease staging, and evaluation of functional vision in patient-centered management of retinal dystrophies.

Retinal melanosome/melanolipofuscin-containing cells (MCCs), clinically visible as hyperreflective foci (HRF) and a highly predictive imaging biomarker for the progression of age-related macular degeneration (AMD), are widely believed to be migrating retinal pigment epithelial (RPE) cells. Using human donor tissue, we identify the vast majority of MCCs as melanophages, melanosome/melanolipofuscin-laden mononuclear phagocytes (MPs). Using serial block-face scanning electron microscopy, RPE flatmounts, bone marrow transplantation and in vitro experiments, we show how retinal melanophages form by the transfer of melanosomes from the RPE to subretinal MPs when the "don't eat me" signal CD47 is blocked. These melanophages give rise to hyperreflective foci in Cd47-/--mice in vivo, and are associated with RPE dysmorphia similar to intermediate AMD. Finally, we show that Cd47 expression in human RPE declines with age and in AMD, which likely participates in melanophage formation and RPE decline. Boosting CD47 expression in AMD might protect RPE cells and delay AMD progression.

Physiologically, the retinal pigment epithelium (RPE) expresses immunosuppressive signals such as FAS ligand (FASL), which prevents the accumulation of leukocytes in the subretinal space. Age-related macular degeneration (AMD) is associated with a breakdown of the subretinal immunosuppressive environment and chronic accumulation of mononuclear phagocytes (MPs). We show that subretinal MPs in AMD patients accumulate on the RPE and express high levels of APOE. MPs of Cx3cr1(-/-) mice that develop MP accumulation on the RPE, photoreceptor degeneration, and increased choroidal neovascularization similarly express high levels of APOE. ApoE deletion in Cx3cr1(-/-) mice prevents pathogenic age- and stress-induced subretinal MP accumulation. We demonstrate that increased APOE levels induce IL-6 in MPs via the activation of the TLR2-CD14-dependent innate immunity receptor cluster. IL-6 in turn represses RPE FasL expression and prolongs subretinal MP survival. This mechanism may account, in part, for the MP accumulation observed in Cx3cr1(-/-) mice. Our results underline the inflammatory role of APOE in sterile inflammation in the immunosuppressive subretinal space. They provide rationale for the implication of IL-6 in AMD and open avenues toward therapies inhibiting pathogenic chronic inflammation in late AMD.

Thinning of the RPE and the underlying vascular layer, the choroid, is observed with age in many human eye disorders. The reasons for this thinning are ill-defined. Here, we highlight the possible role of T lymphocyte recruitment in choroidoretinal thinning in aged and light-challenged mice.

In the Western world, a major cause of blindness is age-related macular degeneration (AMD). Recent research in angiogenesis has furthered the understanding of choroidal neovascularization, which occurs in the "wet" form of AMD. In contrast, very little is known about the mechanisms of the predominant, "dry" form of AMD, which is characterized by retinal atrophy and choroidal involution. The aim of this study is to elucidate the possible implication of the scavenger receptor CD36 in retinal degeneration and choroidal involution, the cardinal features of the dry form of AMD.

Background: CD36, a member of the class B scavenger receptor family, participates in Toll-like receptor signaling on mononuclear phagocytes (MP) and can promote sterile pathogenic inflammation. We here analyzed the effect of CD36 deficiency on retinal inflammation and photoreceptor degeneration, the hallmarks of age-related macular degeneration (AMD), that characterize Cx3cr1-/-mice. Methods: We analyzed subretinal MP accumulation, and cone- and rod-degeneration in light-challenged and aged, CD36 competent or deficient, hyper-inflammatory Cx3cr1-/- mice, using histology and immune-stained retinal flatmounts. Monocytes (Mo) were subretinally adoptively transferred to evaluate their elimination rate from the subretinal space and Interleukin 6 (IL-6) secretion from cultured Mo-derived cells (MdCs) of the different mouse strains were analyzed. Results: CD36 deficient Cx3cr1-/- mice were protected against age- and light-induced subretinal inflammation and associated cone and rod degeneration. CD36 deficiency in Cx3cr1-/- MPs inhibited their prolonged survival in the immune-suppressive subretinal space and reduced the exaggerated IL-6 secretion observed in Cx3cr1-/- MPs that we previously showed leads to increased subretinal MP survival. Conclusion:Cd36 deficiency significantly protected hyperinflammatory Cx3cr1-/- mice against subretinal MP accumulation and associated photoreceptor degeneration. The observed CD36-dependent induction of pro-inflammatory IL-6 might be at least partially responsible for the prolonged MP survival in the immune-suppressive environment and its pathological consequences on photoreceptor homeostasis.

Atrophic age-related macular degeneration (AMD) is associated with the subretinal accumulation of mononuclear phagocytes (MPs). Their role in promoting or inhibiting retinal degeneration is unknown. We here show that atrophic AMD is associated with increased intraocular CCL2 levels and subretinal CCR2(+) inflammatory monocyte infiltration in patients. Using age- and light-induced subretinal inflammation and photoreceptor degeneration in Cx3cr1 knockout mice, we show that subretinal Cx3cr1 deficient MPs overexpress CCL2 and that both the genetic deletion of CCL2 or CCR2 and the pharmacological inhibition of CCR2 prevent inflammatory monocyte recruitment, MP accumulation and photoreceptor degeneration in vivo. Our study shows that contrary to CCR2 and CCL2, CX3CR1 is constitutively expressed in the retina where it represses the expression of CCL2 and the recruitment of neurotoxic inflammatory CCR2(+) monocytes. CCL2/CCR2 inhibition might represent a powerful tool for controlling inflammation and neurodegeneration in AMD.

Recent evidence suggests that transient hyperglycemia in extremely low birth weight infants is strongly associated with the occurrence of retinopathy of prematurity (ROP). We propose a new model of Neonatal Hyperglycemia-induced Retinopathy (NHIR) that mimics many aspects of retinopathy of prematurity. Hyperglycemia was induced in newborn rat pups by injection of streptozocine (STZ) at post natal day one (P1). At various time points, animals were assessed for vascular abnormalities, neuronal cell death and accumulation and activation of microglial cells. We here report that streptozotocin induced a rapid and sustained increase of glycemia from P2/3 to P6 without affecting rat pups gain weight or necessitating insulin treatment. Retinal vascular area was significantly reduced in P6 hyperglycemic animals compared to control animals. Hyperglycemia was associated with (i) CCL2 chemokine induction at P6, (ii) a significant recruitment of inflammatory macrophages and an increase in total number of Iba+ macrophages/microglia cells in the inner nuclear layer (INL), and (iii) excessive apoptosis in the INL. NHIR thereby reproduces several aspects of ischemic retinopathies, including ROP and diabetic retinopathies, and might be a useful model to decipher hyperglycemia-induced cellular and molecular mechanisms in the small rodent.

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.

Proviral integration site for Moloney murine leukemia virus (Pim)-1/2 kinase overexpression has been identified in a variety of hematologic (e.g., multiple myeloma or acute myeloid leukemia (AML)) and solid (e.g., colorectal carcinoma) tumors, playing a key role in cancer progression, metastasis, and drug resistance, and is linked to poor prognosis. These kinases are thus considered interesting targets in oncology. We report herein the design, synthesis, structure-activity relationships (SAR) and in vitro evaluations of new quinoxaline derivatives, acting as dual Pim1/2 inhibitors. Two lead compounds (5c and 5e) were then identified, as potent submicromolar Pim-1 and Pim-2 inhibitors. These molecules were also able to inhibit the growth of the two human cell lines, MV4-11 (AML) and HCT-116 (colorectal carcinoma), expressing high endogenous levels of Pim-1/2 kinases.

Background: Colorectal cancer (CRC) is a highly devastating cancer. Ca2+-dependent channels are now considered key regulators of tumor progression. In this study, we aimed to investigate the association of non-voltage gated Ca2+ channels and Ca2+-dependent potassium channels (KCa) with CRC using the transcriptional profile of their genes. Methods: We selected a total of 35 genes covering KCa channels KCNN1-4, KCNMA1 and their subunits KCNMB1-4, endoplasmic reticulum (ER) calcium sensors STIM1 and STIM2, Ca2+ channels ORAI1-3 and the family of cation channels TRP (TRPC1-7, TRPA1, TRPV1/2,4-6 and TRPM1-8). We analyzed their expression in two public CRC datasets from The Cancer Genome Atlas (TCGA) and GSE39582. Results: KCNN4 and TRPM2 were induced while KCNMA1 and TRPM6 were downregulated in tumor tissues comparing to normal tissues. In proximal tumors, STIM2 and KCNN2 were upregulated while ORAI2 and TRPM6 were downregulated. ORAI1 decreased in lymph node metastatic tumors. TRPC1 and ORAI3 predicted poor prognosis in CRC patients. Moreover, we found that ORAI3/ORAI1 ratio is increased in CRC progression and predicted poor prognosis. Conclusions: KCa and Ca2+ channels could be important contributors to CRC initiation and progression. Our results provide new insights on KCa and Ca2+ channels remodeling in CRC.

Age-related macular degeneration (AMD) is invariably associated with the chronic accumulation of activated mononuclear phagocytes in the subretinal space. The mononuclear phagocytes are composed of microglial cells but also of monocyte-derived cells, which promote photoreceptor degeneration and choroidal neovascularization. Infiltrating blood monocytes can originate directly from bone marrow, but also from a splenic reservoir, where bone marrow monocytes develop into angiotensin II receptor (ATR1)+ splenic monocytes. The involvement of splenic monocytes in neurodegenerative diseases such as AMD is not well understood. Using acute inflammatory and well-phenotyped AMD models, we demonstrate that angiotensin II mobilizes ATR1+ splenic monocytes, which we show are defined by a transcriptional signature using single-cell RNA sequencing and differ functionally from bone marrow monocytes. Splenic monocytes participate in the chorio-retinal infiltration and their inhibition by ATR1 antagonist and splenectomy reduces the subretinal mononuclear phagocyte accumulation and pathological choroidal neovascularization formation. In aged AMD-risk ApoE2-expressing mice, a chronic AMD model, ATR1 antagonist and splenectomy also inhibit the chronic retinal inflammation and associated cone degeneration that characterizes these mice. Our observation of elevated levels of plasma angiotensin II in AMD patients, suggests that similar events take place in clinical disease and argue for the therapeutic potential of ATR1 antagonists to inhibit splenic monocytes for the treatment of blinding AMD.

Photo-transduction in cone segments (CS) is crucial for high acuity daytime vision. For ill-defined reasons, CS degenerate in retinitis pigmentosa (RP) and in the transitional zone (TZ) of atrophic zones (AZ), which characterize geographic atrophy (GA). Our experiments confirm the loss of cone segments (CS) in the TZ of patients with GA and show their association with subretinal CD14(+)mononuclear phagocyte (MP) infiltration that is also reported in RP. Using human and mouse MPs in vitro and inflammation-prone Cx3cr1(GFP/GFP) mice in vivo, we demonstrate that MP-derived IL-1β leads to severe CS degeneration. Our results strongly suggest that subretinal MP accumulation participates in the observed pathological photoreceptor changes in these diseases. Inhibiting subretinal MP accumulation or Il-1β might protect the CS and help preserve high acuity daytime vision in conditions characterized by subretinal inflammation, such as AMD and RP.

Branch retinal vein occlusion (BRVO) leads to extensive vascular remodeling and is important cause of visual impairment. Although the vascular morphological changes following experimental vein occlusion have been described in a variety of models using angiography, the underlying cellular events are ill defined.

The purpose of this study was to analyse the dynamics of red blood cells (RBC) and white blood cells (WBC) in the retinal capillaries of C57BL/KS db/db mice, a genetic model of type 2 diabetes, and control mice, at different ages. Modified epifluorescence microscopy was used to analyse the capillary velocity of FITC-labeled RBC and rhodamine-labeled WBC in the retina. C57BL/KS db/db diabetic mice were compared to heterozygous non-diabetic mice at ages 8 and 18 weeks (n=6 in each group). At 8 weeks, when hyperglycemia begins in db/db mice, no significant difference was found between average RBC and WBC velocity of the 2 groups. At 18 weeks, RBC velocity was significantly higher in diabetic mice compared to controls (1.21+/-0.29 versus 1.08+/-0.28 mm sec(-1) p=0.0003). No significant difference was found between WBC velocities (0.87+/-0.3 versus 0.85+/-0.3 mm sec(-1)) even when normalized by RBC velocity values. Temporal and spatial coefficients of variation were significantly higher for WBC than RBC velocities (p<0.0001) but were not significantly different in diabetic and control mice. Direct measurement of RBC velocity with this new method showed that it was higher in the retinal capillaries of diabetic than control mice after 10-12 weeks of hyperglycemia, but not at the onset of hyperglycemia. This suggests that enhanced RBC velocity is not an immediate effect of hyperglycemia but a consequence of persistent hyperglycemia. The above results are in line with the hypothesis that microvascular flow increases in diabetes, as one of the first microvascular alterations. In contrast, WBC velocity was not different in diabetic and control mice.

Recent evidences highlight a role of the mitochondria calcium homeostasis in the development of colorectal cancer (CRC). To overcome treatment resistance, we aimed to evaluate the role of the mitochondrial sodium-calcium-lithium exchanger (NCLX) and its targeting in CRC. We also identified curcumin as a new inhibitor of NCLX.

Drusen are dynamic sub-RPE deposits that are risk factors for late-stage age-related macular degeneration (AMD). Here we show a new imaging method using flood-illumination adaptive optics (FIAO) that reveal drusen with high contrast and resolution.

Bone marrow -derived cells (BMDCs) can either limit or contribute to the process of pulmonary vascular remodeling. Whether the difference in their effects depends on the mechanism of pulmonary hypertension (PH) remains unknown.