Eye Opener in Stroke

Abstract

Background and Purpose- Retinal ischemia is a major cause of visual impairment in stroke patients, but our incomplete understanding of its pathology may contribute to a lack of effective treatment. Here, we investigated the role of mitochondrial dysfunction in retinal ischemia and probed the potential of mesenchymal stem cells (MSCs) in mitochondrial repair under such pathological condition. Methods- In vivo, rats were subjected to middle cerebral artery occlusion then randomly treated with intravenous MSCs or vehicle. Laser Doppler was used to evaluate the blood flow in the brain and the eye, while immunohistochemical staining assessed cellular degeneration at days 3 and 14 poststroke. In vitro, retinal pigmented epithelium cells were exposed to either oxygen-glucose deprivation or oxygen-glucose deprivation and coculture with MSCs, and subsequently, cell death and mitochondrial function were examined immunocytochemically and with Seahorse analyzer, respectively. Results- Middle cerebral artery occlusion significantly reduced blood flow in the brain and the eye accompanied by mitochondrial dysfunction and ganglion cell death at days 3 and 14 poststroke. Intravenous MSCs elicited mitochondrial repair and improved ganglion cell survival at day 14 poststroke. Oxygen-glucose deprivation similarly induced mitochondrial dysfunction and cell death in retinal pigmented epithelium cells; coculture with MSCs restored mitochondrial respiration, mitochondrial network morphology, and mitochondrial dynamics, which likely attenuated oxygen-glucose deprivation-mediated retinal pigmented epithelium cell death. Conclusions- Retinal ischemia is closely associated with mitochondrial dysfunction, which can be remedied by stem cell-mediated mitochondrial repair.

Keywords: cell survival; endothelial cells; glucose; mitochondrial dynamics; oxygen.

Figure 1.

Middle cerebral artery occlusion (MCAO) reduces blood flow to brain and eye and induced ganglion cell loss in the retina and transplantation of mesenchymal stem cells (MSCs) rescued ganglion cell death at day 14 poststroke. A, Laser Doppler was used to measure blood flow to brain and eye at baseline, during MCAO, and 5-minute after reperfusion. MCAO caused a significant reduction in blood flow to the contralateral (Contra) hemisphere, ipsilateral (Ipsi) hemisphere, and Ipsi eye compared with control. B, Representative images and quantification of immunohistochemical staining of NeuN. Transplantation of MSC rescued ganglion cell loss at day 14 poststroke. ANOVA with Bonferroni post hoc test *P<0.05; **P<0.01; and ***P<0.001. Scale bar 50 µm. FOV indicates field of view.

Figure 2.

Mesenchymal stem cells (MSCs) rescue against retinal pigmented epithelium (RPE) cells loss caused by oxygen-glucose deprivation (OGD) by promoting cell proliferation. A, Representative images of immunocytochemical staining of Ki67 (marker for cell proliferation). OGD produced a significant decrease in Ki67 expression. Coculture with MSCs restored cell Ki67 expression after OGD. B, Representative images of Calcein AM cell viability test. OGD induced a significant decrease in cell viability. Coculture with MSCs rescued RPE cell death after OGD. C, Quantification graphs of Ki67 intensity and cell viability. ANOVA with Bonferroni post hoc test *P<0.05; **P<0.01; and ***P<0.001. Scale bar 50 µm.

Figure 3.

Mesenchymal stem cells (MSCs) ameliorate retinal pigmented epithelium (RPE) cells’ mitochondrial respiration deficits caused by oxygen-glucose deprivation (OGD). RPE cells’ mitochondrial respiration were analyzed using Seahorse XFe96 extracellular flux analyzer with sequential injection of various compounds (1 µmol/L oligomycin [Oligo], 1 µmol/L carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone [FCCP], 0.5 µmol/L rotenone and Antimycin A [Rot/AA]). Coculture with MSCs restored RPE cells’ mitochondrial basal respiration, spare respiratory capacity, proton leak, and ATP production compared with OGD. ANOVA with Bonferroni post hoc test *P<0.05; **P<0.01; ***P<0.001.

Figure 4.

Mesenchymal stem cells (MSCs) restore retinal pigmented epithelium (RPE) cells’ mitochondrial networks altered by oxygen-glucose deprivation (OGD). A, Representative images of RPE cells stained with MitoTracker. B, Analysis and quantification of RPE cells’ mitochondrial network morphology. Coculture with MSCs increased RPE cells’ number of mitochondrial networks, number of individual mitochondria, and number of branches but not average length of the branches compared with OGD. In addition, coculture with MSCs decreased the circularity of RPE cells’ mitochondria compared with OGD. ANOVA with Bonferroni post hoc test *P<0.05; **P<0.01; and ***P<0.001. Mean±SEM. Scale bar 10 µm.

Figure 5.

Mesenchymal stem cells (MSCs) normalize retinal pigmented epithelium (RPE) cells’ mitochondrial dynamics via Mfn2 (mitofusin-2) after oxygen-glucose deprivation (OGD). Representative images of Mfn2 expression (left columns), DAPI (middle columns), and merged (right columns). OGD caused a significant decrease in Mfn2 expression. Coculture with MSC significantly increased the Mfn2 expression compared to OGD. ANOVA with Bonferroni post hoc test *P<0.05; **P<0.01; ***P<0.001. Scale bar 50 µm.

Figure 6.

Mesenchymal stem cells (MSCs) reduce retinal pigmented epithelium (RPE) cells’ mitochondrial membrane depolarization caused by oxygen-glucose deprivation (OGD). RPE cells’ mitochondrial membrane potential was analyzed using JC-1 staining. A, Representative images of JC-1 dye and transferred MSC’s mitochondria. B, Bar graph represents red/green (healthy/unhealthy) intensity ratio of JC-1 staining and correlational analysis between mitochondrial transfer and cell viability. OGD-RPE cells displayed a significant decrease in the JC-1 red/green intensity ratio compared with the control RPE cells. Coculture with MSCs significantly increased JC-1 red/green intensity ratio compared to OGD. C, Confocal imaging revealed colocalization between MSCs mitochondria (blue) and JC-1 (red, arrows) indicating the transfer of functional mitochondria from MSCs to RPE cells. ANOVA with Bonferroni post hoc test *P<0.05; **P<0.01; ***P<0.001. Scale bar 10 µm.