Delayed Administration of Bone Marrow Mesenchymal Stem Cell Conditioned Media Significantly Improves Outcome after Retinal Ischemia in Rats

Abstract

 

Purpose

 

Delayed treatment after ischemia is often unsatisfactory. We hypothesized that delayed injection of bone marrow stem cell (BMSC) conditioned media 24 h after ischemia could rescue ischemic retina. In this study we examined the functional and histological outcomes, and the mechanisms of this neuroprotection.

 

Methods

 

Retinal ischemia was produced in adult rats by increasing IOP for 55 min. BMSCs which were harvested from male Wistar rat femurs were cultured and characterized. Conditioned media (CM) or for control, unconditioned media (UnCM) was injected into the vitreous 24 h after the end of ischemia. Recovery was assessed one week later using electroretinography (ERG). Scotopic a- and b waves, oscillatory potentials, and scotopic threshold responses (STR) were measured. We examined paraffin-embedded retinal sections. TUNEL identified apoptotic cells. Western blot studied protein expression. Mass spectrometry was done to find the proteins present in the BMSC CM and UnCM.

 

Results

 

CM significantly improved recovery of the a- and b-waves, and P2 after ischemia, but not the OPs. Neither the positive STR nor the negative STR was significantly changed. (Fig. 1) Retinal ganglion cell loss after ischemia was attenuated by CM. TUNEL showed that the 24 h CM injection attenuated apoptotic RGCs in ischemic retinae (7.2+1.9%; n=6) v. the UnCM ischemic retinae (23.2+2.2%; n=6; p=0.01) and there was reduced expression of proteins in the caspase apoptotic pathway. Western blot analysis suggests enhancement of the autophagic pathway by CM. Mass spectrometry done on samples of both conditioned and unconditioned media show several proteins unique to the CM. (Table 1)

 

Conclusions

 

Delayed administration of BMSC CM in our rat model of retinal ischemia, resulted in functional and histological neuroprotection. Moreover, BMSC CM mediated neuroprotection, in part, by suppression of apoptosis and enhanced autophagy. Mass spectrometry analysis has resulted in several unique proteins in the CM that may take part in the neuroprotection.

 

 

Stimulus-Intensity responses for a-wave (A), b-wave (B), P2 (C), OPRMS (D), pSTR (E) and nSTR (F) from ERGs, over a range of flash intensities, after retinal ischemia and eyes injected 24 h later with CM or UnCM are shown. There was significant improvement with injection of CM 24 h after ischemia on all except for the OPRMS, pSTR and nSTR.

 

 

Keywords: 572 ischemia • 688 retina • 721 stem cells