Hypoxic-Preconditioned Bone Marrow Stem Cell Medium Significantly Improves Outcome After Retinal Ischemia in Rats

Abstract

PurposeWe have previously demonstrated the protective effect of bone marrow stem cell (BMSC)-conditioned medium in retinal ischemic injury. We hypothesized here that hypoxic preconditioning of stem cells significantly enhances the neuroprotective effect of the conditioned medium and thereby augments the protective effect in ischemic retina.MethodsRats were subjected to retinal ischemia by increasing intraocular pressure to 130 to 135 mm Hg for 55 minutes. Hypoxic-preconditioned, hypoxic unconditioned, or normoxic medium was injected into the vitreous 24 hours after ischemia ended. Recovery was assessed 7 days after injections by comparing electroretinography measurements, histologic examination, and apoptosis (TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay). To compare proteins secreted into the medium in the groups and the effect of hypoxic exposure, we used rat cytokine arrays.ResultsEyes injected with hypoxic BMSC–conditioned medium 24 hours after ischemia demonstrated significantly enhanced return of retinal function, decreased retinal ganglion cell layer loss, and attenuated apoptosis compared to those administered normoxic or hypoxic unconditioned medium. Hypoxic-preconditioned medium had 21 significantly increased protein levels compared to normoxic medium.ConclusionsThe medium from hypoxic-preconditioned BMSCs robustly restored retinal function and prevented cell loss after ischemia when injected 24 hours after ischemia. The protective effect was even more pronounced than in our previous studies of normoxic conditioned medium. Prosurvival signals triggered by the secretome may play a role in this neuroprotective effect.

Bone-marrow mesenchymal stem-cell administration significantly improves outcome after retinal ischemia in rats

“…This timing was followed in order to be consistent with our previous studies where the post-ischemic follow up was at 7 days; we followed procedures that we previously described [9, 16]. Ag/AgCl electrodes were fashioned from 0.010” Teflon-coated silver wire (Grass Technologies, West Warwick, RI).…”

“…To enable simultaneous pressurization for increasing intraocular pressure (IOP), and measurement of IOP, the needle was connected by a length of plastic tubing via a three-way stopcock to an electronic pressure transducer (Transpac 42661-04-27; Abbott, North Chicago, IL) and to an elevated bag of balanced salt solution (BSS; by sterile technique, BSS was transferred from its bottle [Alcon] to an empty 1000-mL 0.9% saline plastic bag [Baxter, Deerfield, IL, USA]). IOP, continually displayed on a monitor (Hewlett-Packard HP78534C; Palo Alto, CA), was increased to 130–135 mm Hg and maintained constantly for 55 minutes by pressurizing the bag (Smiths Medical Clear-Cuff, Dublin, OH) [9]. The temperature was maintained at 36–37 °C using a servo–controlled heating blanket (Harvard Apparatus, Natick, MA).…”

“…Sham control rats were administered PBS in both ischemic and non-ischemic eyes. Previous studies indicated no adverse effects, e.g., increased IOP or retinal detachment, from injections [9, 16]. …”

“…Post ischemic prosurvival signaling triggered by the media may play an important role in neuroprotection [9]. While BMSC media is promising, a limitation is that there is no accompanying source for ongoing release of the secretome (i.e., the cells).…”

“…Based on our recent work applying ischemic post-conditioning or injection of BMSC conditioned media 24 h after ischemia, we hypothesized that delayed application of BMSCs, 24 h after ischemia, could significantly improve recovery outcomes [9]. Towards this aim, and using autologous BMSCs, we evaluated the underlying mechanisms responsible for the protective effects of BMSCs in retinal ischemia, in our previously characterized rat model.…”