| Summary: | 博士 === 國立陽明大學 === 生理學研究所 === 104 === We evaluated the therapeutic benefits and underlying mechanisms of human umbilical mesenchymal stem cells (HUMSCs) on epilepsy. At first, we transplanted HUMSCs into bilateral hippocampus of pilocarpine-treated. The rats were divided into the following three groups: (1) a normal group of rats receiving only PBS, (2) a status epilepticus (SE) group of rats with pilocarpine-induced SE and PBS injected into the hippocampi, and (3) a SE+HUMSC group of SE rats with HUMSC transplantation. To examine the effects of HUMSC transplantation on the onset and the duration of spontaneous recurrent motor seizures (SRMS), pilocarpine-treated rats with PBS injection or HUMSCs implantation were monitored by simultaneous video and electroencephalographic recordings at two to four weeks after SE induction. The results showed that the onset of SRMS was delay and found that fewer SRMS occurred in rats in SE+HUMSCs group during all recording period. Furthermore, both number and duration of SRMS within two to four weeks after SE were significantly decreased in SE+HUMSCs rats compared with SE rats. All of the rats were sacrificed for the structural and cellular studies on Day 29 after SE. Brain edema, hippocampal morphology changes and hippocampal volume were evaluated by magnetic resonance imaging, a non-invasive imaging technology, and Nissl staining, respectively. The results showed that the brain edema and total volume of the dorsal hippocampus was smaller in SE rats compared with normal and SE+HUMSCs rats. Following, histochemical studies show the severe pyramidal neuron loss in CA1 and CA3 regions in the SE rats compared with the neuron number in normal and SE+HUMSCs rat. Interestingly, there were no significant differences in the number of hippocampal neurons and GABAergic neurons between normal and SE+HUMSCs rats. Compared with the SE rats, the SE+HUMSCs rats exhibited a suppression of astrocyte activity, microglia activation and aberrant mossy fiber sprouting. The implanted HUMSCs survived in the hippocampus at Day 29. In addition, we found the expression of human trophic factors and cytokines, including amphiregulin (AREG), fibroblast growth factor-6 (FGF-6), glucocorticoid-induced tumor necrosis factors receptor (GITR), macrophage inflammatory protein-3β (MIP-3β), and osteoprotegerin (OPG), in rat hippocampus with HUMSCs transplantation by human cytokine arrays. Accordingly, the further neuroprotective effects of these cytokines were examined through an in vitro study. Glutamate excitotoxicity was associated with many acute or chronic neurodegenerative diseases. Exposure of cortical neurons to glutamate showed a significant decrease in cell viability, and increase of cell apoptosis and oxidative stress. These glutamate-induced neuronal damages were prevented by co-culturing with HUMSCs. In fact, HUMSCs secreted abundant human AREG, OPG and transforming growth factor-β1 (TGF-β1) in the culture media when HUMSCs co-cultured with neurons. TGF-β1 is an injury-related peptide and is involved in tissue repair. Exposure of AREG, FGF-6, MIP-3β and OPG in cultured medium could protect rat cortical neurons from glutamate-induced cell death. The neuroprotective effects of these trophic factors or cytokines verified the paracrine effects of HUMSCs in neurologic diseases. Taken together, our results indicate that grafted HUMSC suppress the development of epilepsy and protect neurons from glutamate-induced excitotoxicity. Moreover, the above therapeutic effects are likely due to the ability of the cells to produce neuroprotective and anti-inflammatory cytokines. This finding provides a potential effective therapy for epilepsy or glutamate-associated neurologic diseases in the future.
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