Effect of Uveal Melanocytes on Choroidal Morphology in Rhesus Macaques and Humans on Enhanced-Depth Imaging Optical Coherence Tomography.

PurposeTo compare cross-sectional choroidal morphology in rhesus macaque and human eyes using enhanced-depth imaging optical coherence tomography (EDI-OCT) and histologic analysis.MethodsEnhanced-depth imaging-OCT images from 25 rhesus macaque and 30 human eyes were evaluated for choriocapillaris and choroidal-scleral junction (CSJ) visibility in the central macula based on OCT reflectivity profiles, and compared with age-matched histologic sections. Semiautomated segmentation of the choriocapillaris and CSJ was used to measure choriocapillary and choroidal thickness, respectively. Multivariate regression was performed to determine the association of age, refractive error, and race with choriocapillaris and CSJ visibility.ResultsRhesus macaques exhibit a distinct hyporeflective choriocapillaris layer on EDI-OCT, while the CSJ cannot be visualized. In contrast, humans show variable reflectivities of the choriocapillaris, with a distinct CSJ seen in many subjects. Histologic sections demonstrate large, darkly pigmented melanocytes that are densely distributed in the macaque choroid, while melanocytes in humans are smaller, less pigmented, and variably distributed. Optical coherence tomography reflectivity patterns of the choroid appear to correspond to the density, size, and pigmentation of choroidal melanocytes. Mean choriocapillary thickness was similar between the two species (19.3 ± 3.4 vs. 19.8 ± 3.4 μm, P = 0.615), but choroidal thickness may be lower in macaques than in humans (191.2 ± 43.0 vs. 266.8 ± 78.0 μm, P < 0.001). Racial differences in uveal pigmentation also appear to affect the visibility of the choriocapillaris and CSJ on EDI-OCT.ConclusionsPigmented uveal melanocytes affect choroidal morphology on EDI-OCT in rhesus macaque and human eyes. Racial differences in pigmentation may affect choriocapillaris and CSJ visibility, and may influence the accuracy of choroidal thickness measurements

Localization of complement factor H gene expression and protein distribution in the mouse outer retina.

To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina.Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC.Cfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh(-/-) eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh(-/-) mice. Greatly reduced Cfh protein immunohistological signals in the Cfh(-/-) eyes also supported the specificity of the Cfh protein distribution results.Only Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC

Intravitreal Administration of Human Bone Marrow CD34+ Stem Cells in a Murine Model of Retinal Degeneration.

PurposeIntravitreal murine lineage-negative bone marrow (BM) hematopoietic cells slow down retinal degeneration. Because human BM CD34+ hematopoietic cells are not precisely comparable to murine cells, this study examined the effect of intravitreal human BM CD34+ cells on the degenerating retina using a murine model.MethodsC3H/HeJrd1/rd1 mice, immunosuppressed systemically with tacrolimus and rapamycin, were injected intravitreally with PBS (n = 16) or CD34+ cells (n = 16) isolated from human BM using a magnetic cell sorter and labeled with enhanced green fluorescent protein (EGFP). After 1 and 4 weeks, the injected eyes were imaged with scanning laser ophthalmoscopy (SLO)/optical coherence tomography (OCT) and tested with electroretinography (ERG). Eyes were harvested after euthanasia for immunohistochemical and microarray analysis of the retina.ResultsIn vivo SLO fundus imaging visualized EGFP-labeled cells within the eyes following intravitreal injection. Simultaneous OCT analysis localized the EGFP-labeled cells on the retinal surface resulting in a saw-toothed appearance. Immunohistochemical analysis of the retina identified EGFP-labeled cells on the retinal surface and adjacent to ganglion cells. Electroretinography testing showed a flat signal both at 1 and 4 weeks following injection in all eyes. Microarray analysis of the retina following cell injection showed altered expression of more than 300 mouse genes, predominantly those regulating photoreceptor function and maintenance and apoptosis.ConclusionsIntravitreal human BM CD34+ cells rapidly home to the degenerating retinal surface. Although a functional benefit of this cell therapy was not seen on ERG in this rapidly progressive retinal degeneration model, molecular changes in the retina associated with CD34+ cell therapy suggest potential trophic regenerative effects that warrant further exploration

Effect of Uveal Melanocytes on Choroidal Morphology in Rhesus Macaques and Humans on Enhanced-Depth Imaging Optical Coherence Tomography.

PurposeTo compare cross-sectional choroidal morphology in rhesus macaque and human eyes using enhanced-depth imaging optical coherence tomography (EDI-OCT) and histologic analysis.MethodsEnhanced-depth imaging-OCT images from 25 rhesus macaque and 30 human eyes were evaluated for choriocapillaris and choroidal-scleral junction (CSJ) visibility in the central macula based on OCT reflectivity profiles, and compared with age-matched histologic sections. Semiautomated segmentation of the choriocapillaris and CSJ was used to measure choriocapillary and choroidal thickness, respectively. Multivariate regression was performed to determine the association of age, refractive error, and race with choriocapillaris and CSJ visibility.ResultsRhesus macaques exhibit a distinct hyporeflective choriocapillaris layer on EDI-OCT, while the CSJ cannot be visualized. In contrast, humans show variable reflectivities of the choriocapillaris, with a distinct CSJ seen in many subjects. Histologic sections demonstrate large, darkly pigmented melanocytes that are densely distributed in the macaque choroid, while melanocytes in humans are smaller, less pigmented, and variably distributed. Optical coherence tomography reflectivity patterns of the choroid appear to correspond to the density, size, and pigmentation of choroidal melanocytes. Mean choriocapillary thickness was similar between the two species (19.3 ± 3.4 vs. 19.8 ± 3.4 μm, P = 0.615), but choroidal thickness may be lower in macaques than in humans (191.2 ± 43.0 vs. 266.8 ± 78.0 μm, P < 0.001). Racial differences in uveal pigmentation also appear to affect the visibility of the choriocapillaris and CSJ on EDI-OCT.ConclusionsPigmented uveal melanocytes affect choroidal morphology on EDI-OCT in rhesus macaque and human eyes. Racial differences in pigmentation may affect choriocapillaris and CSJ visibility, and may influence the accuracy of choroidal thickness measurements

Localization of complement factor H gene expression and protein distribution in the mouse outer retina.

PurposeTo determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina.MethodsQuantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC.ResultsCfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh(-/-) eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh(-/-) mice. Greatly reduced Cfh protein immunohistological signals in the Cfh(-/-) eyes also supported the specificity of the Cfh protein distribution results.ConclusionsOnly Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC

Protective Effect of Intravitreal Administration of Exosomes Derived from Mesenchymal Stem Cells on Retinal Ischemia

<p><i>Purpose</i>: Exosomes derived from human mesenchymal stem cells (hMSCs) cultured under hypoxic conditions contain proteins and growth factors that promote angiogenesis. This study investigated the effect of intravitreal administration of these exosomes on retinal ischemia using a murine model.</p> <p><i>Methods</i>: Oxygen-induced retinopathy (OIR) was induced by exposing one-week-old male C57BL/6J mice to 5 days of 75% hyperoxic conditioning, and returning to room air. After hyperoxic conditioning, the right eye of each mouse was injected intravitreally with 1 µl saline or exosomes derived from hMSCs and compared to control mice of the same age raised in room air without OIR injected intravitreally with saline. Two weeks post-injection, fluorescein angiography (FA) and phase-variance optical coherence tomography angiography (pvOCTA) were used to assess retinal perfusion. Retinal thickness was determined by OCT. The extent of retinal neovascularization was quantitated histologically by counting vascular nuclei on the retinal surface.</p> <p><i>Results</i>: Among eyes with OIR, intravitreal exosome treatment partially preserved retinal vascular flow <i>in vivo</i> and reduced associated retinal thinning; retinal thickness on OCT was 111.1 ± 7.4µm with saline versus 132.1 ± 11.6µm with exosome, p < 0.001. Retinal neovascularization among OIR eyes was reduced with exosome treatment when compared to saline-treated eyes (7.75 ± 3.68 versus 2.68 ± 1.35 neovascular nuclei per section, p < 0.0001). No immunogenicity or ocular/systemic adverse effect was associated with intravitreal exosome treatment.</p> <p><i>Conclusions</i>: Intravitreal administration of exosomes derived from hMSCs was well tolerated without immunosuppression and decreased the severity of retinal ischemia in this murine model. This appealing novel non-cellular therapeutic approach warrants further exploration.</p

Effects of aging and environmental tobacco smoke exposure on ocular and plasma circulatory microRNAs in the Rhesus macaque.

PurposeTo identify changes induced by environmental tobacco smoke (ETS) in circulatory microRNA (miRNA) in plasma and ocular fluids of the Rhesus macaque and compare these changes to normal age-related changes. Tobacco smoke has been identified as the leading environmental risk factor for age-related macular degeneration (AMD).MethodsAll Rhesus macaques were housed at the California National Primate Research Center (CNPRC), University of California, Davis. Four groups of animals were used: Group 1 (1-3 years old), Group 2 (19-28 years old), Group 3 (10-16 years old), and Group 4 (middle aged, 9-14 years old). Group 4 was exposed to smoke for 1 month. Ocular fluids and plasma samples were collected, miRNAs isolated, and expression data obtained using Affymetrix miRNA GeneTitan Array Plates 4.0. Bioinformatics analysis was done on the Affymetrix Expression Console (EC), Transcriptome Analysis Software (TAS) using ANOVA for candidate miRNA selection, followed by Ingenuity Pathway Analysis (IPA).ResultsThe expression of circulatory miRNAs showed statistically significant changes with age and ETS. In the plasma samples, 45 miRNAs were strongly upregulated (fold change &gt;±1.5, p&lt;0.05) upon ETS exposure. In the vitreous, three miRNAs were statistically significantly downregulated with ETS, and two of them (miR-6794 and miR-6790) were also statistically significantly downregulated with age. Some retinal layers exhibited a thinning trend measured with optical coherence tomography (OCT) imaging. The pathways activated were IL-17A, VEGF, and recruitment of eosinophils, Th2 lymphocytes, and macrophages.ConclusionsETS exposure of Rhesus macaques resulted in statistically significant changes in the expression of the circulatory miRNAs, distinct from those affected by aging. The pathways activated appear to be common for ETS and AMD pathogenesis. These data will be used to develop an animal model of early dry AMD

Effects of aging and environmental tobacco smoke exposure on ocular and plasma circulatory microRNAs in the Rhesus macaque.

PurposeTo identify changes induced by environmental tobacco smoke (ETS) in circulatory microRNA (miRNA) in plasma and ocular fluids of the Rhesus macaque and compare these changes to normal age-related changes. Tobacco smoke has been identified as the leading environmental risk factor for age-related macular degeneration (AMD).MethodsAll Rhesus macaques were housed at the California National Primate Research Center (CNPRC), University of California, Davis. Four groups of animals were used: Group 1 (1-3 years old), Group 2 (19-28 years old), Group 3 (10-16 years old), and Group 4 (middle aged, 9-14 years old). Group 4 was exposed to smoke for 1 month. Ocular fluids and plasma samples were collected, miRNAs isolated, and expression data obtained using Affymetrix miRNA GeneTitan Array Plates 4.0. Bioinformatics analysis was done on the Affymetrix Expression Console (EC), Transcriptome Analysis Software (TAS) using ANOVA for candidate miRNA selection, followed by Ingenuity Pathway Analysis (IPA).ResultsThe expression of circulatory miRNAs showed statistically significant changes with age and ETS. In the plasma samples, 45 miRNAs were strongly upregulated (fold change &gt;±1.5, p&lt;0.05) upon ETS exposure. In the vitreous, three miRNAs were statistically significantly downregulated with ETS, and two of them (miR-6794 and miR-6790) were also statistically significantly downregulated with age. Some retinal layers exhibited a thinning trend measured with optical coherence tomography (OCT) imaging. The pathways activated were IL-17A, VEGF, and recruitment of eosinophils, Th2 lymphocytes, and macrophages.ConclusionsETS exposure of Rhesus macaques resulted in statistically significant changes in the expression of the circulatory miRNAs, distinct from those affected by aging. The pathways activated appear to be common for ETS and AMD pathogenesis. These data will be used to develop an animal model of early dry AMD