Purpose:
Delayed treatment after ischemia is often unsatisfactory. However, we recently demonstrated that "post-conditioning" using a 24 h-delayed, transient ischemic stimulus was neuroprotective in a rat model of retinal ischemia. We hypothesized that delayed injection of bone marrow stem cells (BMSCs) after ischemia could mimick the post-conditioning neuroprotection, and in this study we characterized the functional and histological outcomes, differentiation and migration of the BMSCs, and molecular mechanisms of neuroprotection.
Methods:
Retinal ischemia was produced in adult Wistar rats by increasing IOP for 55 min. BMSCs were harvested from rat femur and injected into the vitreous 24 h after the end of ischemia. Recovery was assessed one week after ischemia using electroretinography (ERG). Scotopic a- and b waves, oscillatory potentials, and STR were measured. We examined 4 micron thick paraffin-embedded sections at 7 d after ischemia. Immunohistochemistry was performed on 7 micron thick cryosections. Double-labeling was performed with anti-neurofilament as a neuronal marker, thy-1 for RGCs, PKCa for bipolar cells, syntaxin for amacrine cells, and anti-GFAP and anti-vimentin as glial markers; TUNEL indentifed apoptotic cells. Western blotting quantitated apoptosis and autophagy markers.
Results:
BMSCs significantly improved recovery of the b-wave, OP, and P2 amplitudes after ischemia (Figure), but not STR. RGC cell loss after ischemia was unchanged, but cell density in the inner and outer nuclear layers was preserved. There was no co-localization with neurofilament or any other neuronal marker, but some cells double-labeled for GFAP and vimentin. Injection of BMSCs significantly reduced post-ischemic TUNEL and caspase-3 and -6 and maintained autophagy vs controls.
Conclusions:
Functional protection after ischemia by BMSCs is suggested to be occurring in inner retinal cells other than RGCs since both STR and RGC numbers were not altered by BMSCs. Neuronal differentiation is not required for BMSC mediated neuroprotection, but suppression of apoptosis/maintenance of autophagy are mechanisms uncovered by this study.
Keywords: ischemia • regeneration • retina