Resumo

Purpose: In adult mammals, the regeneration of the optic nerve is very limited and to date there are no efficient therapies to generate neuroprotection and axon outgrowth after injuries. In previous work, we performed optic nerve crush in adult rats and we analyzed the effect of intravitreal transplantation of syngeneic bone-marrow mononuclear cells (BMMCs) on optic nerve regeneration. We demonstrated an increase of 1.6 fold in retinal-ganglion cell (RGC) survival and an increase of 3.7 fold in the number of axons growing up to 1mm from the crush site 14 days after the injury. In the present work, our purpose is first to investigate if the observed effects in the optic nerve remain for a longer period of time and second to identify how the BMMCs are generating the observed effects. Methods: We used Lister-hooded adult rats and the optic nerve was crushed using frozen tweezers. The transplantation was performed shortly after optic nerve crush and control animals received intravitreal saline injections. Analysis of mRNA lebvels were performed using qRT-PCR and analysis of CD11b+ cells were done using fluorescent activated cell sorting (FACS). Results: We demonstrated that 28 days after injury, there was an increase of 5.2 fold in the number of axons growing up to 1mm from the crush site, showing that in animals that received BMMCs transplantation the optic nerve axons keep growing from 14 to 28 days after crush. Nevertheless, the BMMCs effect on RGC survival was not sustained and 28 days after injury the number of live RGCs was similar in BMMCs and saline injected animals. Analysis by FACS showed that around 15% of transplanted BMMCs were CD11b+, suggesting they were monocytes or monocytes precursors. The next step was the analysis of CD11b+ cells in the vitreous body after BMMC transplantation or saline injection. We demonstrated an increase in the number of CD11b+ cells in the vitreous body one day after BMMCs transplantation. Some of these cells were derived from the transplant but some of them came from the host. Analysis by qRT-PCR demonstrated that the increase in fibroblast growth factor 2 (FGF-2) levels previously verified 14 days after optic nerve crush and BMMCs was also evident 1 day after injury. Conclusions: These results demonstrated that BMMCs effect in axonal outgrowth persists and is intensified 28 days after injury, but the effects on RGC survival are not sustained. Our results also suggest that these effects may be related to FGF-2 release or macrophage/monocyte activation.