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Neuroprotective and antiapoptotic activity of lineage-negative bone marrow cells after intravitreal injection in a mouse model of acute retinal injury.

ABSTRACT: We investigated effects of bone marrow-derived, lineage-negative cell (Lin(-)BMC) transplantation in acute retinal injury. Lin(-)BMCs were intravitreally injected into murine eyes at 24 h after NaIO3-induced injury. Morphology, function, and expression of apoptosis-related genes, including brain-derived neurotrophic factor (BDNF) and its receptor, were assessed in retinas at 7 days, 28 days, and 3 months after transplantation. Moreover, global gene expression at day 7 was analyzed by RNA arrays. We observed that Lin(-)BMCs integrated into outer retinal layers improving morphological retinal structure and induced molecular changes such as downregulation of proapoptotic caspase-3 gene, a decrease in BAX/BCL-2 gene ratio, and significant elevation of BDNF expression. Furthermore, transplanted Lin(-)BMCs differentiated locally into cells with a macrophage-like phenotype. Finally, Lin(-)BMCs treatment was associated with generation of two distinct transcriptomic patterns. The first relates to downregulated genes associated with regulation of neuron cell death and apoptosis, response to oxidative stress/hypoxia and external stimuli, and negative regulation of cell proliferation. The second relates to upregulated genes associated with neurological system processes and sensory perception. Collectively, our data demonstrate that transplanted Lin(-)BMCs exert neuroprotective function against acute retinal injury and this effect may be associated with their antiapoptotic properties and ability to express neurotrophic factors.

SUBMITTER: Machalinska A

PROVIDER: S-EPMC4354968 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundDiabetic retinopathy is a retinal vasculopathy involving all three retinal capillary plexus layers. Since human CD34+ bone marrow stem cells (BMSCs) have the potential to promote revascularization of ischemic tissue, this study tests the hypothesis that intravitreal injection of human CD34+ BMSCs can have protective effects on all layers of the retinal vasculature in eyes with diabetic retinopathy.MethodsStreptozotocin (STZ)-induced diabetic mice were injected intravitreally with 50,000 human CD34+ BMSCs or phosphate-buffered saline (PBS) into the right eye. Systemic immunosuppression with rapamycin and tacrolimus was started 5 days before the injection and maintained for study duration to prevent rejection of human cells. All mice were euthanized 4 weeks after intravitreal injection; both eyes were enucleated for retinal flat mount immunohistochemistry. The retinal vasculature was stained with Isolectin-GS-IB4. Confocal microscopy was used to image four circular areas of interest of retina, 1-mm diameter around the optic disc. Images of superficial, intermediate, and deep retinal capillary plexus layers within the areas of interest were obtained and analyzed using ImageJ software with the Vessel Analysis plugin to quantitate the retinal vascular density and vascular length density in the three plexus layers.ResultsThree distinct retinal capillary plexus layers were visualized and imaged using confocal microscopy. Eyes that received intravitreal injection of CD34+ BMSCs (N=9) had significantly higher vascular density and vascular length density in the superficial retinal capillary plexus when compared to the untreated contralateral eyes (N=9) or PBS treated control eyes (N=12; P values <0.05 using ANOVA followed by post-hoc tests). For the intermediate and deep plexus layers, the difference was not statistically significant.ConclusionsThe protective effect of intravitreal injection of the human CD34+ BMSCs on the superficial retinal capillary plexus layers is demonstrated using confocal microscopy in this murine model of diabetic retinopathy.

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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Neuroprotective and antiapoptotic activity of lineage-negative bone marrow cells after intravitreal injection in a mouse model of acute retinal injury.

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