July, 2013

Our present study demonstrated that melatonin pretreatment enhanced MSC survival under H2O2 oxidative stress or OGD in vitro. Furthermore, transplantation of MT-MSCs into the ischemic rat brain significantly increased the survival of transplanted MSCs and improved its therapeutic efficacy. Compared to the untreated MSC transplantation group, infarct volume was reduced and neurobehavioral outcomes were improved in MT-MSC treated rats.

Mechanistic study demonstrated that melatonin improved MSC survival via the Erk1/2 signaling pathway. MT-MSCs demonstrated higher therapeutic potential through upregulating VEGF, reducing ischemia-induced cell death including apoptosis, and promoting
focal angiogenesis and neurogenesis. Our results provide strong evidence that increasing MSC survival is an important step to enhancing the benefits of MSC therapy.

Stem cell therapy has shown great potential to restore neurological function after ischemic brain injury (10,17,23,29,37,44). However, one of the main hurdles to significant functional improvement after stem cell transplantation is that more than 80% of transplanted stem cells die within 3 days after transplantation in ischemic organs (26,35). Therefore, it is crucial to improve stem cell viability under ischemic conditions. Other studies have shown that the survival of grafted stem cells is improved by preconditioning with hypoxia after transplantation into ischemic rats brain (36,38). Hypoxia or melatonin pretreatment greatly
increased MSCs survival and therapeutic efficiency in ischemic brain.

However, the mechanisms of the two strategies are different. Hypoxia up-regulates growth factors including BDNF, VEGF and down-regulates pro-inflammatory cytokines in MSCs, while melatonin pretreatment actives ERK1/2 signaling pathway. In addition, melatonin is widely used as a diet complement and antioxidant agent with no reported side effects (33). It is readily available be used in clinical trials to improve the efficiency of MSC therapy.

The results of our study indicate that melatonin pretreatment enhanced MSC survival under oxidative stimulation via the Erk1/2 pathway. In previous studies, it has been noted that MSCs express melatonin receptors MT1 and MT2 (45). Melatonin receptor antagonist luzindole completely reversed the protective effect of melatonin, suggesting that the protective effect is receptor-mediated. Our results showed that melatonin pretreatment greatly increased Erk1/2 phosphorylation in MSCs under H2O2 stimulation. Luzindole blocked Erk1/2 phosphorylation, further indicating that the function of melatonin is achieved via Erk1/2 pathway activation. In addition, Erk1/2 inhibitor U0126 could also completely reverse the protective effect of melatonin on MSCs under oxidative stimulation. Taken together, these results demonstrate that melatonin improved MSC survival mainly through activating the Erk1/2 pathway.

We pre-labeled MSCs with Dil dye, which is a carbocyanine membrane probe with strong photo-stable fluorescence, long cellular retention and minimal cytotoxicity (13). Previous studies have demonstrated that it is useful for long-term MSC labeling (9). In
addition, Dil is retained in cells throughout fixation, permeabilization and paraffin-embedding procedures (16). MSCs are able to differentiate into neurons, endothelial cells and osteoblasts in vitro (40). MSCs can also secrete nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF) and other factors that may enhance cell survival in the ischemic microenvironment (22). Consequently, remodeling processes occur, with endogenous stem cells now surviving and migrating towards to the ischemic region, leading to new neurovascular unit formation (46). Our study demonstrated that increased survival of transplanted MSCs reduced infarct volume and improved neurobehavioral outcomes for at least 14 days. It is notable that though melatonin promoted MSCs survival under ischemic conditions, it did not affect MSC differentiation. Neither melatonin-treated nor untreated MSCs injected into ischemic brain differentiated into mature neuron or astrocyte in vivo (data not shown), suggesting that the therapeutic effects of MSCs may be mediated by the secretion of favorable paracrine factors. Indeed, we measured the expression of VEGF and found that VEGF was mainly secreted by astrocyte 14 days after stroke, and transplantation of melatonin treated MSCs could enhance VEGF secretion. Increased expression of VEGF in rats transplanted with MT-MSC subsequently induced angiogenesis and neurogenesis in the ischemic perifocal region, which are beneficial to the repair and remodeling after ischemic brain injury. In our study, we injected MSCs into the striatum, however, some MSCs were migrated to the cortex, enhanced VEGF expression and increased angiogenesis in cortex.

In summary, our study demonstrated that melatonin pretreatment promoted MSC survival under ischemia-related conditions in vitro and augmented the therapeutic efficiency of MSC in transient focal ischemia in vivo. The protective effect of melatonin is achieved through activation of the Erk1/2 pathway. This strategy of pretreating stem cells may represent a safe approach for improving the beneficial effects of stem cell therapy for cerebral ischemia.