Abstract
Purpose :
Retinal ischemia is a leading cause of vision impairment and retinal degeneration, resulting from insufficient oxygen supply to the retina. Mesenchymal-derived (MSC) extracellular vesicles (EVs) have emerged as a potential therapeutic intervention to rescue retinal cells from apoptotic injury. In this study, we exposed Müller cells to hypoxic conditions to simulate ischemic -like conditions. We evaluated the neuroprotective effects of EVs derived from two diverse sources of MSCs - bone marrow and dental pulp cells - in an in vitro Müller cell-based hypoxic model.
Methods :
Müller cells were subjected to hypoxia for 10 hours or 18 hours, followed by exposure to normoxic oxygen levels for 18 hours and 12 hours, respectively. MSC-derived EVs from bone marrow and dental pulp were isolated through precipitation and centrifugation, with size and concentration determined using Nanoparticle Tracking Analysis. After hypoxic exposure, a concentration of 2 * 107 MSC and DPSC EVs were added independently to the cells, and their neuroprotective effects were quantitatively assessed through MTS assays for cell viability and ROS assay. Qualitative evaluations were performed through immunofluorescence
Results :
Results
Both DPSC EVs and MSC EVs demonstrated neuroprotective effect. The Müller cells subjected to hypoxia showed a significant decrease in viability compared to control Müller cells (0.5 vs 1.6, absorbance < 0.0001). However, a significant improvement was observed in both DPSC EVs and MSC EVs (0.5vs1.5, absorbance,p <0.0001). Similarly, the hypoxia induced ROS was significantly decreased in hypoxic Müller cells receiving EV’s compared to hypoxic Müller cells receiving PBS alone (2000 vs 3500 fluorescence intensity, p<0.0001). Immunostaining analysis showed an increased KI67 expression and profuse vimentin cytoplasmic staining in control Müller cells with distorted vimentin and KI67 staining in hypoxic cells. Hypoxic cells treated with EVs showed an improved KI67 staining in nucleus and vimentin staining in cytoplasm.
Conclusions :
The cell viability and immunostaining show the neuroprotective effect of EVs and supports the hypothesis for successful rescue of Müller cells from cell death. However, further studies are needed to account for mechanism of EV rescue.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.