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Therapeutic effects of agmatine and neural precursor cells overexpressing arginine decarboxylase in mice following compression spinal cord injury

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 척수 압박손상 시 아그마틴과 알기닌탈탄산효소가 과발현된 신경 전구세포를 이용한 유전자치료의 역할 
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Dept. of Medicine/박사
The current therapeutic strategies for the treatment of spinal cord injuries (SCI) could not fully facilitate the neural regeneration and/or significant functional improvement following SCI. Agmatine (Agm), an endogenous primary amine and a novel neuromodulator synthesized from the decarboxylation of L-arginine catalyzed by arginine decarboxylase (ADC), is a neurotransmitter/ neuromodulator and has been reported to exert neuroprotective effects in central nervous system (CNS) injury models including SCI (PLoS ONE, 2013; 8(1): e53911). Moreover, recently it was demonstrated that retrovirus containing human ADC gene infection to mouse cortical neural precursor cells (hADC-mNPCs) which can synthesize Agm endogenously has shown to prevent the cell death and exerted cytoprotection following H2O2 injury in vitro (Stem Cells Dev. 2011; 20(3):527-37).Basing on the above evidences our study intended to investigate the effects of agmatine treatment and hADC-mNPCs transplantation in mouse subjected to compression SCI.In brief, the vertebra thoracic level 9 of mice was injured with a bilateral micro clamp clip to produce compression model of SCI. The study was divided into two parts; 1) to determine the multifaceted effects of agmatine after SCI, 2) to determine whether the transplantation of (hADC-mNPCs) could encourage neural regeneration and repair following SCI.At first, mice that received an intraperitoneal (i.p.) injection of Agm (100 mg/kg/day) within 1 hour following SCI until 35 days showed significant improvement in functional recovery tests and bladder function. On the other hand Agm treatment significantly inhibited the demyelination events, neuronal loss and glial scar formation around the lesion site following SCI. Recent evidences suggest that the expressions of bone morphogenetic proteins (BMPs) are modulated in the neuronal and glial cell population following SCI. Our computer assisted stereological toolbox analysis (CAST) demonstrates that Agm treatment significantly increased BMP- 2/7 expressions in neurons and oligodendrocytes. On the other hand, BMP- 4 expressions were significantly decreased in astrocytes and oligodendrocytes around the lesion site suggesting that Agm treatment could modulate BMP- 2/4/7 expressions in neurons, astrocytes, oligodendrocytes and play key role in promoting the neuronal and glial cell survival following SCI.Also, studies suggested that transplantation of neural precursor cells (NPCs) encourage neural regeneration and repair following SCI. So, we further investigated to determine the effects of hADC-mNPCs transplantation in an established SCI model for our study. The characterization of hADC-mNPCs was done by checking the stemness in cell and cell fate commitment. These events were investigated by checking the expression levels of SOX2, and nestin for stemness, MAP-2, GFAP and Olig-2 expressions for cell fate commitment. The immunoblotting and western data suggest that the retrovirus containing hADC genes infection to mNPCs induced stemness and favored the commitment of neurospheres cell lineage to neurons and oligodendrocyte earlier than glia cells in vitro. To determine the effects of mNPCs transplantation in SCI the mice were divided into; 1) mice transplanted with control mNPCs, 2) mice transplanted with empty vector (LXSN) infected mNPCs (LXSN-mNPCs), 3) mice transplanted with hADC-mNPCs. The PKH-26 labeled mNPCs, LXSN-mNPCs and hADC-mNPCs were transplanted at 7th day following SCI. Two injections of 0.5μl (1 x 105cells/µl) were done using a micro syringe into bilateral part from the midline at both rostral and caudal parts to the injury epicenter. Then, mice were sacrificed at 1st, 2nd and 5th weeks and spinal cords were isolated for further analysis. We found that hADC-mNPCs transplanted mice demonstrated the improved motor function compared to LXSN-mNPCs or mNPCs alone transplanted mice after SCI. The higher number of PKH-26 and MAP-2 & Olig-2 double positive cells was seen in hADC-mNPCs transplantation group after SCI. On the other hand, hADC-mNPCs transplantation group significantly reduced the number of PKH-26 and GFAP double positive cells after SCI. Collectively, the present data suggest that hADC-mNSCs transplantation significantly induced proliferation and differentiation of neurons and oligodendrocyte, attenuated the glial scar formation and significantly improved the locomotor function following SCI. Taken together, our overall results suggest that Agm treatment and hADC-mNPCs transplantation improved neurological and histological outcomes, induced oligodendrogenesis, protected neurons, and decreased glial scar formation following SCI.
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2. 학위논문 > 1. College of Medicine (의과대학) > 박사
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