Background: The aim of this project is to develop a new standardized and cost-efficient method to compare optical coherence tomography (OCT) scans to their corresponding paraffin embedded histopathology sections in post-mortem eyes. This correlation will clarify the interpretation of OCT images, and it will also enable direct immunohistochemical characterization of features observed on OCT.

Methods: Study design: donor eyes were obtained from two separate eye banks. In order to minimize post-mortem change like retinal detachment and vitreous opacification, the eyes were fixed in a previously tested fixative solution. Time between death and fixation has been kept under 6 hours. Methods: Using a customized imaging device, nine post-mortem eyes were imaged with a SD-OCT machine. Subsequently, an 8mm trephine was used to isolate a portion of the posterior pole including the macular area and the optic nerve head for histopathological analysis. Paraffin embedded cross sections of the retina were obtained and visually compared to each OCT image (b-scans).

Results: To facilitate the correlation of OCT images to their histopathological sections, three principle aspects were controlled during tissue processing: rotation, tilt and location. Using markings as well as anatomical landmarks, serial histopathological sections in an orientation comparable to OCT b-scans were obtained, thereby facilitating image pairing.

Conclusions: Compared to other well-established methods using resin and electron microscopy, our standardized Methods allowed us to successfully compare OCT b-scans to serial retinal cross sections of a wider macular area at a lower cost. Our novel approach allows us to translate features observed on OCT images into well-established histopathological images, providing the clinician with additional tools to obtain difficult diagnoses with more confidence.

Background: The aim of this project is to develop a new standardized and cost-efficient method to compare optical coherence tomography (OCT) scans to their corresponding paraffin embedded histopathology sections in post-mortem eyes. This correlation will clarify the interpretation of OCT images, and it will also enable direct immunohistochemical characterization of features observed on OCT.

Methods: Study design: donor eyes were obtained from two separate eye banks. In order to minimize post-mortem change like retinal detachment and vitreous opacification, the eyes were fixed in a previously tested fixative solution. Time between death and fixation has been kept under 6 hours. Methods: Using a customized imaging device, nine post-mortem eyes were imaged with a SD-OCT machine. Subsequently, an 8mm trephine was used to isolate a portion of the posterior pole including the macular area and the optic nerve head for histopathological analysis. Paraffin embedded cross sections of the retina were obtained and visually compared to each OCT image (b-scans).

Results: To facilitate the correlation of OCT images to their histopathological sections, three principle aspects were controlled during tissue processing: rotation, tilt and location. Using markings as well as anatomical landmarks, serial histopathological sections in an orientation comparable to OCT b-scans were obtained, thereby facilitating image pairing.

Conclusions: Compared to other well-established methods using resin and electron microscopy, our standardized Methods allowed us to successfully compare OCT b-scans to serial retinal cross sections of a wider macular area at a lower cost. Our novel approach allows us to translate features observed on OCT images into well-established histopathological images, providing the clinician with additional tools to obtain difficult diagnoses with more confidence.

Background: Pericytes are contractile cells that wrap along the walls of capillaries. In the brain, pericytes play a crucial role in the regulation of capillary diameter and vascular blood flow in response to metabolic demand. The contribution of pericytes to microvascular deficits in glaucoma is currently unknown. To address this, we used two-photon excitation microscopy for longitudinal monitoring of retinal pericytes and capillaries in a mouse glaucoma model.

Methods: Ocular hypertension was induced by injection of magnetic microbeads into the anterior chamber of albino mice expressing red fluorescent protein selectively in pericytes (NG2-DsRed). Minimally invasive, multiphoton imaging through the sclera of live NG2-DsRed mice was used to visualize pericytes and capillary diameter at one, two and three weeks after glaucoma induction. In vivo fluctuations in pericyte intracellular calcium were monitored with the calcium indicator Fluo-4. Ex vivo stereological analysis of retinal tissue prior to and after injection of microbeads was used to confirm our in vivo findings.

Results: Live two-photon imaging of NG2-DsRed retinas demonstrated that ocular hypertension induced progressive accumulation of intracellular calcium in pericytes. Calcium uptake correlated directly with the narrowing of capillaries in the superficial, inner, and outer vascular plexuses (capillary diameter: na?ve control =4.7±0.1 μm, glaucoma =4.0±0.1 μm, n=5–6 mice/group, Student’s t-test P<0.05). Frequency distribution analysis showed a substantial increase in the number of small-diameter capillaries (≤3 μm) and a decrease in larger-diameter microvessels (≥5–9 μm) at three weeks after induction of ocular hypertension (n=5–6 mice/group, Student’s t-test P<0.05).

Conclusions: Our data support two main conclusions. First, two-photon excitation microscopy is an effective strategy to monitor longitudinal changes in retinal pericytes and capillaries in live animals at glaucoma onset and progression. Second, ocular hypertension triggers rapid intracellular calcium increase in retinal pericytes leading to substantial capillary constriction. This study identifies retinal pericytes as important mediators of early microvascular dysfunction in glaucoma.

Background: Pericytes are contractile cells that wrap along the walls of capillaries. In the brain, pericytes play a crucial role in the regulation of capillary diameter and vascular blood flow in response to metabolic demand. The contribution of pericytes to microvascular deficits in glaucoma is currently unknown. To address this, we used two-photon excitation microscopy for longitudinal monitoring of retinal pericytes and capillaries in a mouse glaucoma model.

Methods: Ocular hypertension was induced by injection of magnetic microbeads into the anterior chamber of albino mice expressing red fluorescent protein selectively in pericytes (NG2-DsRed). Minimally invasive, multiphoton imaging through the sclera of live NG2-DsRed mice was used to visualize pericytes and capillary diameter at one, two and three weeks after glaucoma induction. In vivo fluctuations in pericyte intracellular calcium were monitored with the calcium indicator Fluo-4. Ex vivo stereological analysis of retinal tissue prior to and after injection of microbeads was used to confirm our in vivo findings.

Results: Live two-photon imaging of NG2-DsRed retinas demonstrated that ocular hypertension induced progressive accumulation of intracellular calcium in pericytes. Calcium uptake correlated directly with the narrowing of capillaries in the superficial, inner, and outer vascular plexuses (capillary diameter: na?ve control =4.7±0.1 μm, glaucoma =4.0±0.1 μm, n=5–6 mice/group, Student’s t-test P<0.05). Frequency distribution analysis showed a substantial increase in the number of small-diameter capillaries (≤3 μm) and a decrease in larger-diameter microvessels (≥5–9 μm) at three weeks after induction of ocular hypertension (n=5–6 mice/group, Student’s t-test P<0.05).

Conclusions: Our data support two main conclusions. First, two-photon excitation microscopy is an effective strategy to monitor longitudinal changes in retinal pericytes and capillaries in live animals at glaucoma onset and progression. Second, ocular hypertension triggers rapid intracellular calcium increase in retinal pericytes leading to substantial capillary constriction. This study identifies retinal pericytes as important mediators of early microvascular dysfunction in glaucoma.

Background: Sight-threatening diabetic macular edema (DME) is caused by increased microvascular permeability. While few direct vascular targeting strategies are available, VEGF pathway inhibition has shown to be effective in reducing retinal vascular leakage but is associated with non-negligible side effects. Thus, more options are needed. Vascular specific Activin-like kinase receptor type I (ALK1) pathway and its circulating ligand Bone morphogenetic protein-9 (BMP9) is known for its potent quiescent and stabilizing effect on the vasculature. However, little is known about this pathway in the context of microvascular permeability associated with diabetes. We hypothesize that BMP9/ALK1 pathway is inhibited in diabetic (DB) retinas leading to vascular destabilization and leakage and that its activation could re-establish proper vascular endothelial barrier functions (EBF).

Methods: The effect of hyperglycemia (i.e., HG >10 mM of D-glucose) on Alk1 signaling was evaluated in vitro by subjecting endothelial cells (EC) to increasing concentrations of D-glucose (5, 11, 25 mM) and in vivo using DB mice (Streptozotocin-induced diabetes). The contribution of Alk1 signaling on EBF was evaluated using Evans Blue permeation in inducible endothelial specific Alk1 KO mice. To evaluate the potential protective effects of BMP9/Alk1 signaling on EBF, BMP9 overexpression was achieved using adenoviral delivery in DB mice. Statistical-One-Way ANOVA or Student’s t-test was used.

Results: Endothelial tissue from DB mice showed a significant inhibition of BMP9/ALK1-canonical Smad1,5,8 quiescence signaling (DB n=5; CTL n=4; P<0.01), which was associated with reduced expression of target genes (JAG1, Id1,3, Hey1,2 & HES). Moreover, we showed that retinal hyperpermeability associated with diabetes was exacerbated in Alk1 heterozygote mice (n=4–9/group; P<0.0001). Finally, we demonstrated that activation of Alk1 signaling in ECs prevented vascular permeability induced by HG, both in vitro (n=3; P=0.009) and in vivo (n=4–9/group; P<0.0001).

Conclusions: Consistent with our hypothesis, vascular stability and quiescence induced by BMP9-ALK1 signaling is inhibited in the DB/HG endothelium which could be an important factor in vascular leakage leading to DME. Our results show that activation of this pathway could offer a therapeutically interesting future option to slow down the onset of DME.

Background: Sight-threatening diabetic macular edema (DME) is caused by increased microvascular permeability. While few direct vascular targeting strategies are available, VEGF pathway inhibition has shown to be effective in reducing retinal vascular leakage but is associated with non-negligible side effects. Thus, more options are needed. Vascular specific Activin-like kinase receptor type I (ALK1) pathway and its circulating ligand Bone morphogenetic protein-9 (BMP9) is known for its potent quiescent and stabilizing effect on the vasculature. However, little is known about this pathway in the context of microvascular permeability associated with diabetes. We hypothesize that BMP9/ALK1 pathway is inhibited in diabetic (DB) retinas leading to vascular destabilization and leakage and that its activation could re-establish proper vascular endothelial barrier functions (EBF).

Methods: The effect of hyperglycemia (i.e., HG >10 mM of D-glucose) on Alk1 signaling was evaluated in vitro by subjecting endothelial cells (EC) to increasing concentrations of D-glucose (5, 11, 25 mM) and in vivo using DB mice (Streptozotocin-induced diabetes). The contribution of Alk1 signaling on EBF was evaluated using Evans Blue permeation in inducible endothelial specific Alk1 KO mice. To evaluate the potential protective effects of BMP9/Alk1 signaling on EBF, BMP9 overexpression was achieved using adenoviral delivery in DB mice. Statistical-One-Way ANOVA or Student’s t-test was used.

Results: Endothelial tissue from DB mice showed a significant inhibition of BMP9/ALK1-canonical Smad1,5,8 quiescence signaling (DB n=5; CTL n=4; P<0.01), which was associated with reduced expression of target genes (JAG1, Id1,3, Hey1,2 & HES). Moreover, we showed that retinal hyperpermeability associated with diabetes was exacerbated in Alk1 heterozygote mice (n=4–9/group; P<0.0001). Finally, we demonstrated that activation of Alk1 signaling in ECs prevented vascular permeability induced by HG, both in vitro (n=3; P=0.009) and in vivo (n=4–9/group; P<0.0001).

Conclusions: Consistent with our hypothesis, vascular stability and quiescence induced by BMP9-ALK1 signaling is inhibited in the DB/HG endothelium which could be an important factor in vascular leakage leading to DME. Our results show that activation of this pathway could offer a therapeutically interesting future option to slow down the onset of DME.

Abstract: Mononuclear phagocytes (MP) comprise a family of cells that include microglial cells (MC), monocytes, and macrophages. The subretinal space, located between the RPE and the photoreceptor outer segments, is physiologically devoid of MPs and a zone of immune privilege mediated, among others, by immunosuppressive RPE signals. Age-related macular degeneration (AMD) is a highly heritable major cause of blindness, characterized by a breakdown of the subretinal immunosuppressive environment and an accumulation of pathogenic inflammatory MPs. Studies in mice and humans suggest that the AMD-associated APOE2 isoform promotes the breakdown of subretinal immunosuppression and increased MP survival. Of all genetic factors, variants of complement factor H (CFH) are associated with greatest linkage to AMD. Using loss of function genetics and orthologous models of AMD, we provide mechanistic evidence that CFH inhibits the elimination of subretinal MPs. Importantly, the AMD-associated CFH402H isoform markedly increased this inhibitory effect on microglial cells, indicating a causal link to disease etiology. Pharmacological acceleration of resolution of subretinal inflammation might be a powerful tool for controlling inflammation and neurodegeneration in late AMD.

Abstract: Mononuclear phagocytes (MP) comprise a family of cells that include microglial cells (MC), monocytes, and macrophages. The subretinal space, located between the RPE and the photoreceptor outer segments, is physiologically devoid of MPs and a zone of immune privilege mediated, among others, by immunosuppressive RPE signals. Age-related macular degeneration (AMD) is a highly heritable major cause of blindness, characterized by a breakdown of the subretinal immunosuppressive environment and an accumulation of pathogenic inflammatory MPs. Studies in mice and humans suggest that the AMD-associated APOE2 isoform promotes the breakdown of subretinal immunosuppression and increased MP survival. Of all genetic factors, variants of complement factor H (CFH) are associated with greatest linkage to AMD. Using loss of function genetics and orthologous models of AMD, we provide mechanistic evidence that CFH inhibits the elimination of subretinal MPs. Importantly, the AMD-associated CFH402H isoform markedly increased this inhibitory effect on microglial cells, indicating a causal link to disease etiology. Pharmacological acceleration of resolution of subretinal inflammation might be a powerful tool for controlling inflammation and neurodegeneration in late AMD.

Abstract: Pathological retinal neovascularization is the hallmark of primary blinding diseases across all age groups, yet surprisingly little is known about the causative factors. These diseases include diabetic retinopathy and retinopathy of prematurity where progressive decay of retinal vasculature yields zones of neural ischemia. These avascular zones and the hypoxic neurons and glia that reside in them are the source of pro-angiogenic factors that mediate destructive pre-retinal angiogenesis. Central neurons such as retinal ganglion cells (RGCs), which are directly apposed to degenerating vasculature in ischemic retinopathies, require stable metabolic supply for proper function. However, we unexpectedly found that RGCs are resilient to hypoxia/ischemia and a generally compromised metabolic supply and instead of degenerating, trigger protective mechanisms of cellular senescence. Paradoxically, while potentially favoring neuronal survival, the senescent state of RGCs is incompatible with vascular repair as they adopt a senescence-associated secretory phenotype (SASP) that provokes release of a secretome of inflammatory cytokines that drives paracrine senescence and further exacerbates pathological angiogenesis. The mechanisms that lead to retinal cellular senescence and dormancy as well as the therapeutic potential of targeting these pathways will be discussed.

Abstract: Pathological retinal neovascularization is the hallmark of primary blinding diseases across all age groups, yet surprisingly little is known about the causative factors. These diseases include diabetic retinopathy and retinopathy of prematurity where progressive decay of retinal vasculature yields zones of neural ischemia. These avascular zones and the hypoxic neurons and glia that reside in them are the source of pro-angiogenic factors that mediate destructive pre-retinal angiogenesis. Central neurons such as retinal ganglion cells (RGCs), which are directly apposed to degenerating vasculature in ischemic retinopathies, require stable metabolic supply for proper function. However, we unexpectedly found that RGCs are resilient to hypoxia/ischemia and a generally compromised metabolic supply and instead of degenerating, trigger protective mechanisms of cellular senescence. Paradoxically, while potentially favoring neuronal survival, the senescent state of RGCs is incompatible with vascular repair as they adopt a senescence-associated secretory phenotype (SASP) that provokes release of a secretome of inflammatory cytokines that drives paracrine senescence and further exacerbates pathological angiogenesis. The mechanisms that lead to retinal cellular senescence and dormancy as well as the therapeutic potential of targeting these pathways will be discussed.

Background: The goal of this study was to engineer an epithelialized and endothelialized pigmented choroidal substitute using the self-assembly approach of tissue engineering.

Methods: Cells from human choroids were isolated and cultured. Culture purity was assessed using immunostaining (CD31, HMB45, vimentin, keratins 8/18). To engineer the choroid, fibroblasts were cultured in the presence of serum and ascorbic acid to promote extracellular matrix (ECM) assembly. Endothelial cells, melanocytes or RPE cells were separately seeded on the stromal substitutes. Choroidal substitutes were further characterized by histology, mass spectrometry, immunostaining, and compared to native human choroids.

Results: The technique used to isolate choroidal cells yielded pure cultures of fibroblasts, melanocytes and vascular endothelial cells. The stromal substitutes engineered using the self-assembly approach were composed of collagen (types I, VI, XII and XIV), proteoglycans (decorin, lumican) and other ECM proteins. Protein expression was confirmed using immunostaining. Endothelial cells spontaneously assembled into capillary-like structures and vascular networks when cocultured with fibroblast-containing ECM sheets.

Conclusions: This study shows that the self-assembly approach of tissue engineering can be used to reconstruct a choroid using native cells. This model represents a unique tool to better understand the crosstalk between the different choroidal cell types and cell-ECM interactions.

Background: The goal of this study was to engineer an epithelialized and endothelialized pigmented choroidal substitute using the self-assembly approach of tissue engineering.

Methods: Cells from human choroids were isolated and cultured. Culture purity was assessed using immunostaining (CD31, HMB45, vimentin, keratins 8/18). To engineer the choroid, fibroblasts were cultured in the presence of serum and ascorbic acid to promote extracellular matrix (ECM) assembly. Endothelial cells, melanocytes or RPE cells were separately seeded on the stromal substitutes. Choroidal substitutes were further characterized by histology, mass spectrometry, immunostaining, and compared to native human choroids.

Results: The technique used to isolate choroidal cells yielded pure cultures of fibroblasts, melanocytes and vascular endothelial cells. The stromal substitutes engineered using the self-assembly approach were composed of collagen (types I, VI, XII and XIV), proteoglycans (decorin, lumican) and other ECM proteins. Protein expression was confirmed using immunostaining. Endothelial cells spontaneously assembled into capillary-like structures and vascular networks when cocultured with fibroblast-containing ECM sheets.

Conclusions: This study shows that the self-assembly approach of tissue engineering can be used to reconstruct a choroid using native cells. This model represents a unique tool to better understand the crosstalk between the different choroidal cell types and cell-ECM interactions.