We generated a Long Evans transgenic rat with targeted deletion of the whole Rs1 exon-1 and evaluated the pathological retinal phenotype of this Rs1^-/Y rat model of X-linked retinoschisis (XLRS). The Rs1^-/Y rat exhibited very early onset and rapidly progressive photoreceptor degeneration. The outer limiting membrane (OLM) was disrupted and discontinuous by post-natal day (P15) and allowed photoreceptor nuclei to dislocate from the outer nuclear layers (ONL) into the sub-retinal side of the OLM. Dark-adapted electroretinogram (ERG) a-wave and b-wave amplitudes were considerably reduced to only 20-25% of WT by P17. Microglia and Müller glial showed cell marker activation by P7. Intravitreal application of AAV8-RS1 at P5-6 induced RS1 expression by P15 and rescued the inner nuclear layer (INL) and outer plexiform layer (OPL) cavity formation otherwise present at P15, and the outer-retinal structure was less disrupted. This Rs1^-/Y exon-1-del rat model displays substantially faster rod cell loss compared to the exon-1-del Rs1-KO mouse. Most unexpected was the rapid appearance of schisis cavities between P7 and P15, and then cavities rapidly disappeared by P21/P30. The rat model provides clues on the molecular and cellular mechanisms underlying XLRS pathology in this model and points to a substantial and early changes to normal retinal development.

Purpose

To employ type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease to suppress ocular angiogenesis by genomic disruption of VEGF-A in human RPE cells.

Methods

CRISPR sequences targeting exon 1 of human VEGF-A were computationally identified based on predicted Cas9 on- and off-target probabilities. Single guide RNA (gRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the Streptococcuspyogenes Cas9 endonuclease (SpCas9) gene. The lentiviral vectors were used to infect ARPE-19 cells, a human RPE cell line. Frequency of insertion or deletion (indel) mutations was assessed by T7 endonuclease 1 mismatch detection assay; mRNA levels were assessed with quantitative real-time PCR; and VEGF-A protein levels were determined by ELISA. In vitro angiogenesis was measured using an endothelial cell tube formation assay.

Results

Five gRNAs targeting VEGF-A were selected based on the highest predicted on-target probabilities, lowest off-target probabilities, or combined average of both scores. Lentiviral delivery of the top-scoring gRNAs with SpCas9 resulted in indel formation in the VEGF-A gene at frequencies up to 37.0% ± 4.0% with corresponding decreases in secreted VEGF-A protein up to 41.2% ± 7.4% (P < 0.001), and reduction of endothelial tube formation up to 39.4% ± 9.8% (P = 0.02). No significant indel formation in the top three putative off-target sites tested was detected.

Conclusions

The CRISPR-Cas9 endonuclease system may reduce VEGF-A secretion from human RPE cells and suppress angiogenesis, supporting the possibility of employing gene editing for antiangiogenesis therapy in ocular diseases.

Purpose

To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina.

Methods

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.

Results

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.

Conclusions

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.

Purpose. To identify retinal pigment epithelium (RPE)/choroid genes and their relevant expression pathways affected by intravitreal injections of dexamethasone and triamcinolone acetonide in mice at clinically relevant time points for patient care. Methods. Differential gene expression of over 34,000 well-characterized mouse genes in the RPE/choroid of 6-week-old C57BL/6J mice was analyzed after intravitreal steroid injections at 1 week and 1 month postinjection, using Affymetrix Mouse Genome 430 2.0 microarrays. The data were analyzed using GeneSpring GX 12.5 and Ingenuity Pathway Analysis (IPA) microarray analysis software for biologically relevant changes. Results. Both triamcinolone and dexamethasone caused differential activation of genes involved in "Circadian Rhythm Signaling" pathway at both time points tested. Triamcinolone (TAA) uniquely induced significant changes in gene expression in "Calcium Signaling" (1 week) and "Glutamate Receptor Signaling" pathways (1 month). In contrast, dexamethasone (Dex) affected the "GABA Receptor Signaling" (1 week) and "Serotonin Receptor Signaling" (1 month) pathways. Understanding how intraocular steroids affect the gene expression of RPE/choroid is clinically relevant. Conclusions. This in vivo study has elucidated several genes and pathways that are potentially altering the circadian rhythms and several other neurotransmitter pathways in RPE/choroid during intravitreal steroid injections, which likely has consequences in the dysregulation of RPE function and neurodegeneration of the retina.

The mucosa that lines the respiratory and gastrointestinal (GI) tracts is an important portal of entry for pathogens and provides the first line of innate immune defense against infections. Although an abundance of memory CD4(+) T cells at mucosal sites render them highly susceptible to HIV infection, the gut and not the lung experiences severe and sustained CD4(+) T cell depletion and tissue disruption. We hypothesized that distinct immune responses in the lung and gut during the primary and chronic stages of viral infection contribute to these differences. Using the SIV model of AIDS, we performed a comparative analysis of the molecular and cellular characteristics of host responses in the gut and lung. Our findings showed that both mucosal compartments harbor similar percentages of memory CD4(+) T cells and displayed comparable cytokine (IL-2, IFN-γ, and TNF-α) responses to mitogenic stimulations prior to infection. However, despite similar viral replication and CD4(+) T cell depletion during primary SIV infection, CD4(+) T cell restoration kinetics in the lung and gut diverged during acute viral infection. The CD4(+) T cells rebounded or were preserved in the lung mucosa during chronic viral infection, which correlated with heightened induction of type I IFN signaling molecules and innate viral restriction factors. In contrast, the lack of CD4(+) T cell restoration in the gut was associated with dampened immune responses and diminished expression of viral restriction factors. Thus, unique immune mechanisms contribute to the differential response and protection of pulmonary versus GI mucosa and can be leveraged to enhance mucosal recovery.

Purpose

Intravitreal 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.

Methods

C3H/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.

Results

In 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.

Conclusions

Intravitreal 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.

Human CD34 ⁺ stem cells are mobilized from bone marrow to sites of tissue ischemia and play an important role in tissue revascularization. This study used a murine model to test the hypothesis that intravitreal injection of human CD34 ⁺ stem cells harvested from bone marrow (BMSCs) can have protective effects in eyes with diabetic retinopathy. Streptozotocin-induced diabetic mice (C57BL/6J) were used as a model for diabetic retinopathy. Subcutaneous implantation of Alzet pump, loaded with Tacrolimus and Rapamycin, 5 days prior to intravitreal injection provided continuous systemic immunosuppression for the study duration to avoid rejection of human cells. Human CD34 ⁺ BMSCs were harvested from the mononuclear cell fraction of bone marrow from a healthy donor using magnetic beads. The CD34 ⁺ cells were labeled with enhanced green fluorescent protein (EGFP) using a lentiviral vector. The right eye of each mouse received an intravitreal injection of 50,000 EGFP-labeled CD34 ⁺ BMSCs or phosphate buffered saline (PBS). Simultaneous multimodal in vivo retinal imaging system consisting of fluorescent scanning laser ophthalmoscopy (enabling fluorescein angiography), optical coherence tomography (OCT) and OCT angiography was used to confirm the development of diabetic retinopathy and study the in vivo migration of the EGFP-labeled CD34 ⁺ BMSCs in the vitreous and retina following intravitreal injection. After imaging, the mice were euthanized, and the eyes were removed for immunohistochemistry. In addition, microarray analysis of the retina and retinal flat mount analysis of retinal vasculature were performed. The development of retinal microvascular changes consistent with diabetic retinopathy was visualized using fluorescein angiography and OCT angiography between 5 and 6 months after induction of diabetes in all diabetic mice. These retinal microvascular changes include areas of capillary nonperfusion and late leakage of fluorescein dye. Multimodal in vivo imaging and immunohistochemistry identified EGFP-labeled cells in the superficial retina and along retinal vasculature at 1 and 4 weeks following intravitreal cell injection. Microarray analysis showed changes in expression of 162 murine retinal genes following intravitreal CD34 ⁺ BMSC injection when compared to PBS-injected control. The major molecular pathways affected by intravitreal CD34 ⁺ BMSC injection in the murine retina included pathways implicated in the pathogenesis of diabetic retinopathy including Toll-like receptor, MAP kinase, oxidative stress, cellular development, assembly and organization pathways. At 4 weeks following intravitreal injection, retinal flat mount analysis showed preservation of the retinal vasculature in eyes injected with CD34 ⁺ BMSCs when compared to PBS-injected control. The study findings support the hypothesis that intravitreal injection of human CD34 ⁺ BMSCs results in retinal homing and integration of these human cells with preservation of the retinal vasculature in murine eyes with diabetic retinopathy.

Purpose

Previously, two cytosolic antioxidant enzymes, Glutathione S-transferase Mu 1 (GSTM1) and Mu 5 (GSTM5), were reduced in retinas with age-related macular degeneration (AMD). This study compared genomic copy number variations (gCNV) of these two antioxidant enzymes in AMD versus controls.

Methods

Genomic copy number (gCN) assays were performed using Taqman Gene Copy Number Assays (Applied Biosystems, Darmstadt, Germany) in technical quadruplicate for both GSTM1 and GSTM5. Peripheral leukocyte RNA levels were compared with controls in technical triplicates. Statistical comparisons were performed in SAS v9.2 (SAS Institute Inc., Cary, NC).

Results

A large percentage of patients in both AMD and age-matched control groups had no copies of GSTM1 (0/0). The mean gCN of GSTM1 was 1.40 (range 0-4) and 1.61 (range 0-5) for AMD and control, respectively (p = 0.29). A greater percentage of control patients had > 3 gCNs of GSTM1 compared with AMD, respectively (15.3% versus 3.0%, p = 0.004). The gCN of GSTM5 was 2 in all samples except one control sample. The relative quantification of GSTM1 and GSTM5 mRNA from peripheral blood leukocytes in patients showed significant differences in relative expression in AMD versus control (p < 0.05). Peripheral blood leukocyte mRNA and gCN were not significantly correlated (p = 0.27).

Conclusion

Since high copy numbers of GSTM1 are found more frequently in controls than in AMD, it is possible that high copy number leads to increased retinal antioxidant defense. Genomic polymorphisms of GSTM1 and GSTM5 do not significantly affect the peripheral blood leukocyte mRNA levels.

Purpose

To 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).

Methods

All 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).

Results

The expression of circulatory miRNAs showed statistically significant changes with age and ETS. In the plasma samples, 45 miRNAs were strongly upregulated (fold change >±1.5, p<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.

Conclusions

ETS 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.

Purpose

Foveoschisis involves the pathologic splitting of retinal layers at the fovea, which may occur congenitally in X-linked retinoschisis (XLRS) or as an acquired complication of myopia. XLRS is attributed to functional loss of the retinal adhesion protein retinoschisin 1 (RS1), but the pathophysiology of myopic foveoschisis is unclear due to the lack of animal models. Here, we characterized a novel nonhuman primate model of myopic foveoschisis through clinical examination and multimodal imaging followed by morphologic, cellular, and transcriptional profiling of retinal tissues and genetic analysis.

Methods

We identified a rhesus macaque with behavioral and anatomic features of myopic foveoschisis, and monitored disease progression over 14 months by fundus photography, fluorescein angiography, and optical coherence tomography (OCT). After necropsy, we evaluated anatomic and cellular changes by immunohistochemistry and transcriptomic changes using single-nuclei RNA-sequencing (snRNA-seq). Finally, we performed Sanger and whole exome sequencing with focus on the RS1 gene.

Results

Affected eyes demonstrated posterior hyaloid traction and progressive splitting of the outer plexiform layer on OCT. Immunohistochemistry showed increased GFAP expression in Müller glia and loss of ramified Iba-1+ microglia, suggesting macro- and microglial activation with minimal photoreceptor alterations. SnRNA-seq revealed gene expression changes predominantly in cones and retinal ganglion cells involving chromatin modification, suggestive of cellular stress at the fovea. No defects in the RS1 gene or its expression were detected.

Conclusions

This nonhuman primate model of foveoschisis reveals insights into how acquired myopic traction leads to phenotypically similar morphologic and cellular changes as congenital XLRS without alterations in RS1.