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In vivo Gene Expression Profiling of Retina Post-Intravitreal Injections of Dexamethasone and Triamcinolone at Clinically Relevant Time Points for Patient Care

ABSTRACT: PURPOSE To identify retinal 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 retinas of 6 weeks old C57BL/6J mice were analyzed after intravitreal steroid injections at 1 week and 1 month time points, using Affymetrix Mouse Genome 430 2.0 microarrays. The data were analyzed using GeneSpringGX11 and Ingenuity Pathway Analysis (IPA) microarray analysis software for biologically relevant changes. RESULTS A common gene pathway, with differentially activated genes for both steroids and time points was ‘Semaphorin Signaling in Neurons’, a member of ‘Axonal Guidance Signaling System’. At 1 week post-injections a common theme was activation of genes expressed in retinal glial cells, TNF alpha and TGF beta signaling pathways and upregulation of stress response proteins (Serpina3n, Cebpd), as well as neuropeptide signaling somatostatin receptor (Sstr2). Unique for Dex was the upregulation of acute phase proteins (Gfap, Cp, Edn2) as well as Plexna2, a semaphorin signaling receptor, while EphrinB receptor ephexin1 (Argef15) was downregulated. Folate signaling appears to be unique for TAA at 1 week (Folh1, Cubn), while aryl-hydrocarbon receptor (AhR) signaling might be important for both steroids at 1 month. CONCLUSIONS Understanding the molecular and genetic effects of intraocular steroid treatments is of clinical relevance. This in vivo study has elucidated several genes and pathways that are potentially altering the neuroprotective/neurodegenerative balance between glial and retinal ganglion cells during intravitreal steroid treatment.

ORGANISM(S): Mus musculus

PROVIDER: GSE30701 | GEO | 2011/12/08

SECONDARY ACCESSION(S): PRJNA144359

REPOSITORIES: GEO

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Project description:PURPOSE To identify retinal 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 retinas of 6 weeks old C57BL/6J mice were analyzed after intravitreal steroid injections at 1 week and 1 month time points, using Affymetrix Mouse Genome 430 2.0 microarrays. The data were analyzed using GeneSpringGX11 and Ingenuity Pathway Analysis (IPA) microarray analysis software for biologically relevant changes. RESULTS A common gene pathway, with differentially activated genes for both steroids and time points was ‘Semaphorin Signaling in Neurons’, a member of ‘Axonal Guidance Signaling System’. At 1 week post-injections a common theme was activation of genes expressed in retinal glial cells, TNF alpha and TGF beta signaling pathways and upregulation of stress response proteins (Serpina3n, Cebpd), as well as neuropeptide signaling somatostatin receptor (Sstr2). Unique for Dex was the upregulation of acute phase proteins (Gfap, Cp, Edn2) as well as Plexna2, a semaphorin signaling receptor, while EphrinB receptor ephexin1 (Argef15) was downregulated. Folate signaling appears to be unique for TAA at 1 week (Folh1, Cubn), while aryl-hydrocarbon receptor (AhR) signaling might be important for both steroids at 1 month. CONCLUSIONS Understanding the molecular and genetic effects of intraocular steroid treatments is of clinical relevance. This in vivo study has elucidated several genes and pathways that are potentially altering the neuroprotective/neurodegenerative balance between glial and retinal ganglion cells during intravitreal steroid treatment. Total of 18 retinal samples were analysed. There were two time points, day7 and day30. Each time point had three groups: BSS control, DEX treatment and TAA treatment. Each group had three biological replicas.

Project description: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.

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In vivo Gene Expression Profiling of Retina Post-Intravitreal Injections of Dexamethasone and Triamcinolone at Clinically Relevant Time Points for Patient Care

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