Abstract
Purpose::
To determine if sub-visible laser (SVL) can recruit bone marrow-derived stem cells to the retina.
Methods::
A 810 nm laser was used to apply laser to to the retina of one eye of C57B wild-type mice. Because the laser burns were not visible, retinal vasculature landmarks were used to guide spot placement. Fundus photographs and fluorescein angiography were performed at various times to assess for possible damage. Post-laser, the mice retinas and RPE layers were isolated at fixed time intervals. For half the mice, RNA was isolated by standard methods while proteins lysates were made of the other lasered mice eyes. Quantitative RT-PCR and immunoblot studies were performed on the samples for hsp70, hsp90, crystallins and SDF-1. Similar RNA and protein analysis was performed on primary human RPE cells in culture that were heat shocked at 42°C for various lengths of time. For the stem cell studies, bone marrow derived stem cells were harvested from homozygous GFP transgenic mice and the cells isolated by FACS. For some studies, the stem cells were stably engrafted into irradiated C57B wild-type mice or injected into the retro-orbital sinus of mice. After SVL, flat mounts of the retina and RPE layers were prepared. The GFP positive cells were quantitated by image J analysis. Other lasered retinas were prepared for routine histological sectioning and staining with H&E.
Results::
Histological examination of mice eyes treated with SVL revealed minimal to no damage of the retina. At the pre-determined energy settings, there was a time-dependent increase in the expression of hsp70, hsp90 and other heat shock proteins as well as the stem cell chemoattractant, SDF-1. In primary human RPE cells, classic heat shock conditioning also induced a similar expression changes in hsp and SDF-1 expression. Using both adoptive transfer of GFP-labeled stem cells and GFP chimeric animals, SVL caused these cells to be recruited to the RPE layer.
Conclusions::
Our studies demonstrate that SVL induces the heat shock response and the expression of SDF-1 in mice retinas. Further, SVL recruits bone marrow derived stem cells to the RPE layer. This method may be useful in the regeneration of the retina in diseases of the RPE like dry ARMD.
Keywords: regeneration • retinal pigment epithelium • laser